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  • 1.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    The mammalian verprolin homologue WIRE participates in receptor-mediated endocytosis and regulation of the actin filament system by distinct mechanisms2004In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 298, no 2, p. 485-498Article in journal (Refereed)
    Abstract [en]

    The mammalian verprolin family consists of three family members: WIP, WIRE and CR16. WIRE was recently found to bind to WASP and N-WASP and to have roles in regulating actin dynamics downstream of the platelet-derived growth factor beta-receptor. In the current study, the WASP-binding domain of WIRE was identified, with the core of the binding motif encompassing amino acid residues 408-412. A stretch of aromatic amino acid residues close to the core motif also participates in WASP binding. Amino acid substitutions in each of these motifs abrogated WASP binding, suggesting that both motifs are involved in the binding of WIRE to WASP. Interestingly, WIRE mutants unable to bind WASP were still able to induce a reorganisation of the actin filament system, indicating that WASP did not participate in the signalling pathway that link WIRE to actin dynamics. In cells ectopically expressing WIRE, the endocytosis of the platelet-derived growth factor beta-receptor was drastically reduced. However, in contrast to the effect on the actin filament system, the WIRE-induced ablation of the receptor endocytosis required an intact WASP-binding domain. Moreover, WIRE was more efficient than WIP in inhibiting the receptor endocytosis, implicating that these two mammalian verprolins have distinct roles in mammalian cells.

  • 2.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    The WASP-binding protein WIRE has a role in the regulation of the actin filament system downstream of the platelet-derived growth factor receptor2002In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 279, no 1, p. 21-33Article in journal (Refereed)
    Abstract [en]

    Activation of growth factor receptors, such as platelet-derived growth factor (PDGF) receptors, has a major impact on the motile behavior of vertebrate cells. The WASP family of proteins has been recognized as important regulators of actin polymerization via the activation of the Arp2/3 complex. The activity of the WASP proteins has, in turn, been shown to be governed by a number of associated proteins, including the WASP interacting protein (WIP). This report presents a novel WIP-like protein, WIRE (for WIP-related). WIRE was shown to bind to the WH1 domain of WASP and N-WASP. WIRE was localized to actin filaments in transiently transfected PAE/PDGFRbeta cells, and in cells simultaneously expressing WIRE and WASP, WIRE relocalized WASP to actin filaments, a relocalization that required direct interaction between the two proteins. In addition, WIRE was able to bind the PDGF receptor substrate Nckbeta. PDGF treatment of cells ectopically expressing WIRE resulted in formation of peripheral protrusions composed of filopodia and lamellipodia-like structures. In cells expressing both WIRE and WASP, PDGF treatment induced a translocation of WASP to the cell margin, an effect that required the presence of WIRE. Taken together, the data presented indicate that WIRE has a role in the WASP-mediated organization of the actin cytoskeleton and that WIRE is a potential link between the activated PDGF receptor and the actin polymerization machinery.

  • 3.
    Aspenström, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Richnau, Ninna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Johansson, Ann-Sofi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    The diaphanous-related formin DAAM1 collaborates with the Rho GTPases RhoA and Cdc42, CIP4 and Src in regulating cell morphogenesis and actin dynamics2006In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 312, no 12, p. 2180-2194Article in journal (Refereed)
    Abstract [en]

    Binding partners for the Cdc42 effector CIP4 were identified by the yeast two-hybrid system, as well as by testing potential CIP4-binding proteins in coimmunoprecipitation experiments. One of the CIP4-binding proteins, DAAM1, was characterised in more detail. DAAM1 is a ubiquitously expressed member of the mammalian diaphanous-related formins, which include proteins such as mDia1 and mDia2. DAAM1 was shown to bind to the SH3 domain of CIP4 in vivo. Ectopically expressed DAAM1 localised in dotted pattern at the dorsal side of transfected cells and the protein was accumulated in the proximity to the microtubule organising centre. Moreover, ectopic expression of DAAM1 induced a marked alteration of the cell morphology, seen as rounding up of the cells, the formation of branched protrusions as well as a reduction of stress-fibres in the transfected cells. Coimmunoprecipitation experiments demonstrated that DAAM1 bound to RhoA and Cdc42 in a GTP-dependent manner. Moreover, DAAM1 was found to interact and collaborate with the non-receptor tyrosine kinase Src in the formation of branched protrusions. Taken together, our data indicate that DAAM1 communicates with Rho GTPases, CIP4 and Src in the regulation of the signalling pathways that co-ordinate the dynamics of the actin filament system.

  • 4.
    Aspenström, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Ruusala, Aino
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Pacholsky, Dirk
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Taking Rho GTPases to the next level: the cellular functions of atypical Rho GTPases2007In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 313, no 17, p. 3673-3679Article, review/survey (Refereed)
    Abstract [en]

    The Rho GTPases are influential regulators of signalling pathways that control vital cellular processes such as cytoskeletal dynamics, gene transcription, cell cycle progression and cell transformation. A vast majority of the studies involving Rho GTPases have been focused to the famous triad, Cdc42, Rac1 and RhoA, but this protein family actually harbours 20 members. Recently, the less known Rho GTPases have received increased attention. Many of the less studied Rho GTPases have structural, as well as, functional features which makes it pertinent to classify them as atypical Rho GTPases. This review article will focus on the critical aspects of the atypical Rho GTPases, RhoH, Wrch-1, Chp and RhoBTB. These proteins are involved in a broad spectre of biological processes, such as cytoskeletal dynamics, T-cell signalling and protein ubiquitinylation. We will also discuss the roles of atypical Rho GTPases as oncogenes or tumour suppressors, as well as their potential involvement in human diseases.

  • 5. Benedito, Rui
    et al.
    Hellström, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Notch as a hub for signaling in angiogenesis2013In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 319, no 9, p. 1281-1288Article, review/survey (Refereed)
  • 6. Berglund, Erik
    et al.
    Akcakaya, Pinar
    Berglund, David
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
    Karlsson, Fredrik
    Vukojevic, Vladana
    Lee, Linkiat
    Bogdanovic, Darko
    Lui, Weng-Onn
    Larsson, Catharina
    Zedenius, Jan
    Frobom, Robin
    Branstrom, Robert
    Functional role of the Ca2+-activated Cl- channel DOG1/TMEM16A in gastrointestinal stromal tumor cells2014In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 326, no 2, p. 315-325Article in journal (Refereed)
    Abstract [en]

    DOG1, a Ca2+-activated Cl- channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl- currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target.

  • 7. Berglund, Erik
    et al.
    Berglund, David
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
    Akcakaya, Pinar
    Ghaderi, Mehran
    Dare, Elisabetta
    Berggren, Per-Olof
    Kohler, Martin
    Aspinwall, Craig A.
    Lui, Weng-Onn
    Zedenius, Jan
    Larsson, Catharina
    Branstrom, Robert
    Evidence for Ca2+-regulated ATP release in gastrointestinal stromal tumors2013In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 319, no 8, p. 1229-1238Article in journal (Refereed)
    Abstract [en]

    Gastrointestinal stromal tumors (GISTs) are thought to originate from the electrically active pacemaker cells of the gastrointestinal tract. Despite the presence of synaptic-like vesicles and proteins involved in cell secretion it remains unclear whether GIST cells possess regulated release mechanisms. The GIST tumor cell line GIST882 was used as a model cell system, and stimulus-release coupling was investigated by confocal microscopy of cytoplasmic free Ca2+ concentration ([Ca(2+)1](i)), flow cytometry, and luminometric measurements of extracellular ATP. We demonstrate that GIST cells have an intact intracellular Ca2+-signaling pathway that regulates ATP release. Cell viability and cell membrane integrity was preserved, excluding ATP leakage due to cell death and suggesting active ATP release. The stimulus-secretion signal transduction is at least partly dependent on Ca2+ influx since exclusion of extracellular Ca2+ diminishes the ATP release. We conclude that measurements of ATP release in GISTs may be a useful tool for dissecting the signal transduction pathway, mapping exocytotic components, and possibly for the development and evaluation of drugs. Additionally, release of ATP from GISTs may have importance for tumor tissue homeostasis and immune surveillance escape.

  • 8.
    Bergström, J.Daniel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Westermark, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Heldin, Nils-Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Epidermal growth factor receptor signaling activates Met in human anaplastic thyroid carcinoma cells2000In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 259, no 1, p. 293-299Article in journal (Refereed)
    Abstract [en]

    Overexpression of Met is a common finding in thyroid carcinomas. Recently, we reported on overexpression and ligand-independent constitutive activation of Met in anaplastic thyroid carcinoma cells. In the present study we have investigated a putative mechanism for this phenomenon. Cell lines with constitutively activated Met expressed both TGF-alpha mRNA and protein. Western blot analysis revealed expression of receptors for epidermal growth factor (EGFR) in all carcinoma cell lines; in tumor cells with elevated levels of TGF-alpha mRNA there was a constitutive tyrosine phosphorylation of the EGFRs. Preincubation of carcinoma cells with suramin decreased EGFR activation and downregulated Met expression as well as the ligand-independent phosphorylation of Met. Similar results were obtained with a EGFR tyrosine kinase inhibitor, AG 1478. The MEK inhibitor U0126 had an even more pronounced effect compared to AG 1478, indicating a Ras/MAPK-mediated signal in the regulation of Met expression and activation. Inhibition of EGFR signaling also decreased proliferation of the anaplastic thyroid carcinoma cells. Thus, aberrant activation of EGFRs may lead to an overexpression and activation of Met, which may be of importance for the malignant phenotype of anaplastic thyroid carcinomas.

  • 9.
    Berthold, Jessica
    et al.
    University of Cologne.
    Schenková, Kristína
    University of Cologne.
    Ramos, Sonia
    University of Cologne.
    Miura, Yoshie
    University of Nebraska.
    Furukawa, Manabu
    University of Nebraska.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Rivero, Francisco
    University of Hull.
    Characterization of RhoBTB-dependent Cul3 ubiquitin ligase complexes--evidence for an autoregulatory mechanism2008In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 314, no 19, p. 3453-3465Article in journal (Refereed)
    Abstract [en]

    RhoBTB proteins are atypical members of the Rho family of small GTPases. Two of the three RhoBTB proteins, RhoBTB1 and RhoBTB2, have been proposed as tumor suppressors and might function as adaptors of Cul3-dependent ubiquitin ligase complexes. Using yeast two-hybrid analysis and co-immunoprecipitation we show that all three RhoBTB proteins interact with Cul3. The interaction requires the N-terminal region of Cul3 and the first BTB domain of RhoBTB. RhoBTB3, the only RhoBTB with a prenylation motif, associates with vesicles that are frequently found in the vicinity of microtubules, suggesting a participation in some aspects of vesicle trafficking. We also show that RhoBTB2 and RhoBTB3 are capable of homo and heterodimerizing through the BTB domain region. The GTPase domain, which does not bind GTP, is able to interact with the BTB domain region, thus preventing proteasomal degradation of RhoBTB. This fits into a model in which an intramolecular interaction maintains RhoBTB in an inactive state, preventing the formation or the functionality of Cul3-dependent complexes. We also report a significantly decreased expression of RHOBTB and CUL3 genes in kidney and breast tumor samples and a very good correlation in the expression changes between RHOBTB and CUL3 that suggests that these genes are subject to a common inactivation mechanism in tumors.

  • 10.
    Blom, Magdalena
    et al.
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet.
    Reis, Katarina
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet.
    Heldin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Aspenström, Pontus
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet.
    The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration2017In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 352, no 2, p. 255-264Article in journal (Refereed)
    Abstract [en]

    RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as cortical actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration.

  • 11. Borgenström, M
    et al.
    Tienhaara, A
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Salmivirta, Markku
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jalkanen, M
    Testosterone-induced growth of S115 mouse mammary tumor cells is dependent on heparan sulfate2001In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 264, no 2, p. 307-314Article in journal (Refereed)
    Abstract [en]

    The androgen-induced proliferation of S115 mouse mammary tumor cells has been suggested to involve autocrinic fibroblast growth factor signaling. Heparan sulfate proteoglycans are required for fibroblast growth factor signaling, presumably due to their ability to alter binding of fibroblast growth factors to their receptors. We have investigated the role of heparan sulfate proteoglycans in the testosterone-induced proliferation of S115 cells. We demonstrate that when the cells are treated with sodium chlorate, which inhibits the sulfation of endogenous heparan sulfate proteoglycans, cell growth becomes dependent on exogenous heparin. The shortest heparin oligosaccharides supporting cell growth were octasaccharides, whereas dodecasaccharides were almost as effective as native heparin. The N-, 2-O-, and 6-O-sulfate groups of heparin were all required for full testosterone response. Treatment of S115 cells with chlorate or testosterone did not alter the expression of fibroblast growth factor receptors 1 or 3, whereas the expression of fibroblast growth factor receptor 2 was down-regulated. We have previously shown that overexpression of syndecan-1 heparan sulfate proteoglycan renders S115 cells insensitive to testosterone and now demonstrate that this effect can be overcome by sodium chlorate treatment in combination with exogenous heparin. Our results suggest that heparin-like molecules are intimately involved in the androgen-mediated proliferation of S115 cells.

  • 12.
    Claesson-Welsh, Lena
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Gerhardt, Holger
    Introduction to the ECR special angiogenesis issue2013In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 319, no 9, p. 1239-1239Article in journal (Other academic)
  • 13.
    Dijksterhuis, Jacomijn P.
    et al.
    Karolinska Inst, Sect Receptor Biol & Signaling, Deptartment Physiol & Pharmacol, S-17177 Stockholm, Sweden..
    Arthofer, Elisa
    Karolinska Inst, Sect Receptor Biol & Signaling, Deptartment Physiol & Pharmacol, S-17177 Stockholm, Sweden..
    Marinescu, Voichita D.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Nelander, Sven
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Uhlen, Mathias
    KTH Royal Inst Technol, Sci Life Lab, SE-17121 Stockholm, Sweden..
    Ponten, Frederik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mulder, Jan
    Karolinska Inst, Dept Neurosci, Sci Life Lab, S-17177 Stockholm, Sweden..
    Schulte, Gunnar
    Karolinska Inst, Sect Receptor Biol & Signaling, Deptartment Physiol & Pharmacol, S-17177 Stockholm, Sweden.;Masaryk Univ, Fac Sci, Inst Expt Biol, CS-61137 Brno, Czech Republic..
    High levels of WNT-5A in human glioma correlate with increased presence of tumor-associated microglia/monocytes2015In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 339, no 2, p. 280-288Article in journal (Refereed)
    Abstract [en]

    Malignant gliomas are among the most severe types of cancer, and the most common primary brain tumors. Treatment options are limited and the prognosis is poor. WNT-5A, a member of the WNT family of lipoglycoproteins, plays a role in oncogenesis and tumor progression in various cancers, whereas the role of WNT-5A in glioma remains obscure. Based on the role of WNT-5A as an oncogene, its potential to regulate microglia cells and the glioma-promoting capacities of microglia cells, we hypothesize that WNT-5A has a role in regulation of immune functions in glioma. We investigated WNT-5A expression by in silico analysis of the cancer genome atlas (TCGA) transcript profiling of human glioblastoma samples and immunohistochemistry experiments of human glioma tissue microarrays (TMA). Our results reveal higher WNT-5A protein levels and mRNA expression in a subgroup of gliomas (WNT-5A(high)) compared to non-malignant control brain tissue. Furthermore, we show a significant correlation between WNT-5A in the tumor and presence of major histocompatibility complex Class II-positive microglia/monocytes. Our data pinpoint a positive correlation between WNT-5A and a proinflammatory signature in glioma. We identify increased presence of microglia/monocytes as an important aspect in the inflammatory transformation suggesting a novel role for WNT-5A in human glioma.

  • 14. Eichner, Annegret
    et al.
    Brock, Josef
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Bone morphogenetic protein-7 (OP1) and transforming growth factor-beta1 modulate 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts2002In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 275, no 1, p. 132-142Article in journal (Refereed)
    Abstract [en]

    Bone morphogenetic proteins (BMPs) and transforming growth factor-beta (TGFbeta) are potent regulators of osteoblast differentiation and proliferation, processes that are crucial in bone remodeling. BMPs and TGFbeta act in concert with other local factors and hormones, among them 1,25(OH)2-vitamin D3 and insulin. Here we show that BMP7 inhibits 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts, whereas TGFbeta1 stimulates it, as assessed by assays for alkaline phosphatase (ALP) induction, matrix mineralization, and morphology changes. BMP7 or TGFbeta1 alone affects the differentiation of human osteoblasts. Similar results were obtained in assays for ALP induction using conditionally immortalized human osteoblasts (hFOB) and primary osteoblasts obtained from trabecular bone of the femoral head after hip replacement surgery. BMP7 stimulation led to a decrease of 1,25(OH)2-vitamin D3-induced binding of nuclear proteins to a vitamin D response element, as shown by electrophoretic mobility shift assay. Our results suggest that 1,25(OH)2-vitamin D3 modulates in opposite ways the effects of BMP7 and TGFbeta1 on osteoblast differentiation.

  • 15.
    Erlandsson, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Jimmy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Forsberg-Nilsson, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Stem cell factor is a chemoattractant and a survival factor for CNS stem cells2004In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 301, no 2, p. 201-210Article in journal (Refereed)
    Abstract [en]

    Migration of neural cells to their final positions is crucial for the correct formation of the central nervous system. Several extrinsic factors are known to be involved in the regulation of neural migration. We asked if stem cell factor (SCF), well known as a chemoattractant and survival factor in the hematopoietic lineage, could elicit similar responses in neural stem cells. For that purpose, a microchemotaxis assay was used to study the effect of SCF on migration of neural stem cells from the embryonic rat cortex. Our results show that SCF-induced chemotaxis and that specific antibodies to SCF or tyrosine kinase inhibitors abolished the migratory response. The SCF-receptor, Kit, was expressed in neural stem cells and in their differentiated progeny. We also show that SCF is a survival factor, but not a mitogen or a differentiation factor for neural stem cells. These data suggest a role for SCF in cell migration and survival in the developing cortex.

  • 16.
    Gallini, Radiosa
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Karolinska Inst, Dept Med Biochem & Biophys, Stockholm, Sweden..
    Lindblom, Per
    Gothenburg Univ, Sahlgrenska Acad, Dept Med Biochem, Gothenburg, Sweden.;AstraZeneca R&D, Molndal, Sweden..
    Bondjers, Cecilia
    Gothenburg Univ, Sahlgrenska Acad, Dept Med Biochem, Gothenburg, Sweden.;Sahlgrens Univ Hosp, Gothenburg, Sweden..
    Betsholtz, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. Karolinska Inst, Dept Med Biochem & Biophys, Stockholm, Sweden..
    Andrae, Johanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    PDGF-A and PDGF-B induces cardiac fibrosis in transgenic mice2016In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 349, no 2, p. 282-290Article in journal (Refereed)
    Abstract [en]

    Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) contribute to normal heart development. Deficient or abnormal expression of Pdgf and Pdgfr genes have a negative impact on cardiac development and function. The cellular effects of PDGFs in the hearts of Pdgf/Pdgfr mutants and the pathogenesis of the resulting abnormalities are poorly understood, but different PDGF isoforms induce varying effects. Here, we generated three new transgenic mouse types which complete a set of studies, where all different PDGF ligands have been expressed under the same heart specific alpha-myosin heavy chain promoter. Transgenic expression of the natural isoforms of Pdgfa and Pdgfb resulted in isoform specific fibrotic reactions and cardiac hypertrophy. Pdgfa overexpression resulted in a severe fibrotic reaction with up to 8-fold increase in cardiac size, leading to lethal cardiac failure within a few weeks after birth. In contrast, Pdgfb overexpression led to focal fibrosis and moderate cardiac hypertrophy. As PDGF-A and PDGF-B have different affinity for the two PDGF receptors, we analyzed the expression of the receptors and the histology of the fibrotic hearts. Our data suggest that the stronger fibrotic effect generated by Pdgfa overexpression was mediated by Pdgfra in cardiac interstitial mesenchymal cells, i.e. the likely source of extracellular matrix depostion and fibrotic reaction. The apparent sensitivity of the heart to ectopic PDGFR alpha agonists supports a role for endogenous PDGFRa agonists in the pathogenesis of cardiac fibrosis.

  • 17.
    Grundström, Gunilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mosher, D.F.
    Sakai, T.
    Rubin, Kristoffer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Integrin αvβ3 mediates platelet-derived growth factor-BB-stimulated collagen gel contraction in cells expressing signaling deficient integrin α2β12003In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 291, no 2, p. 463-473Article in journal (Refereed)
    Abstract [en]

    The interplay between the collagen-binding integrin, α2β1, and platelet-derived growth factor (PDGF) receptors in the context of functional interactions with collagen was studied. We expressed either wild-type α2β1 (α2β1A) or α2β1 with a Y783/795F mutation in the cytoplasmic tail of the β1 subunit (α2β1Amut) in the β1-null fibroblastic cell line, GD25. GD25 cells lack endogenous expression of the α1 and α2 integrin subunits and do not adhere to collagen even after transfection with β1A. Cells expressing α2β1Amut contracted three-dimensional collagen lattices less efficiently than those expressing α2β1A. PDGF-BB significantly stimulated lattice contraction by GD25-α2β1Amut cells. Both cell types responded chemotactically to PDGF-BB. Focal adhesion kinase (FAK) and p130Cas were phosphorylated when GD25-α2β1A cells, but not GD25-α2β1Amut cells were seeded on collagen-coated dishes. Subsequent treatment with PDGF-BB further increased phosphorylation of FAK and p130Cas only in GD25-α2β1A cells. However, when cultured within collagen lattices, FAK and p130Cas phosphorylation were stimulated in both α2β1A- and α2β1Amut-expressing cells but further phosphorylation, in response to subsequent treatment with PDGF-BB, was seen only in GD25-α2β1A cells. We show that the stimulatory effects of PDGF-BB on collagen gel contraction and chemotaxis by GD25-α2β1Amut cells were mediated by the αvβ3 integrin. Phosphorylation of p130Cas, but not FAK, in GD25-α2β1Amut cells seeded in collagen lattices also depended on αvβ3. Our results show that PDGF-BB stimulation of fibroblast–collagen interactions is mediated by the αvβ3 integrin when β1 integrin function is impaired.

  • 18.
    Gullberg, Donald
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Tingström, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Thuresson, A C
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Olsson, L
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Zoology.
    Terracio, L
    Borg, T K
    Rubin, Kristofer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Beta 1 integrin-mediated collagen gel contraction is stimulated by PDGF1990In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 186, no 2, p. 264-272Article in journal (Refereed)
  • 19.
    Gustafsson, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Heffner, Garrett
    Wenzel, Pamela L.
    Curran, Matthew
    Grawé, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    McKinney-Freeman, Shannon L.
    Daley, Georg Q.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    The Src Homology 2 Protein Shb Promotes Cell Cycle Progression In Murine Hematopoietic Stem Cells By Regulation Of Focal Adhesion Kinase Activity2013In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 319, no 12, p. 1852-1864Article in journal (Refereed)
    Abstract [en]

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shbdeficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time.

  • 20.
    Heldin, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Rubin Sander, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Leino, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Thomsson, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lennartsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Dynamin inhibitors impair platelet-derived growth factor beta-receptor dimerization and signaling2019In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 380, no 1, p. 69-79Article in journal (Refereed)
    Abstract [en]

    The role of plasma membrane composition and dynamics in the activation process of receptor tyrosine kinases (RTKs) is still poorly understood. In this study we have investigated how signaling via the RTK, platelet-derived growth factor beta-receptor (PDGFR-beta) is affected by Dynasore or Dyngo-4a, which are commonly used dynamin inhibitors. PDGFR-beta preferentially internalizes via clathrin-coated pits and in this pathway, Dynamin II has a major role in the formation and release of vesicles from the plasma membrane by performing the membrane scission. We have found that dynamin inhibitors impedes the activation of PDGFR-beta by impairing ligand-induced dimerization of the receptor monomers, which leads to a subsequent lack of phosphorylation and activation both of receptors and downstream effectors, such as ERK1/2 and AKT. In contrast, dynamin inhibitors did not affect epidermal growth factor receptor (EGFR) dimerization and phosphorylation. Our findings suggest that there is a link between plasma membrane dynamics and PDGFR-beta activation, and that this link is not shared with the epidermal growth factor receptor.

  • 21.
    Holmfeldt, Per
    et al.
    Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sellin, Mikael E.
    Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gullberg, Martin
    Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint2010In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 316, no 12, p. 2017-2026Article in journal (Refereed)
    Abstract [en]

    Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18-->E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.

  • 22. Holmvall, K
    et al.
    Camper, L
    Johansson, Staffan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kimura, J.H.
    Lundgren-Åkerlund, E
    Chondrocyte and chondrosarcoma cell integrins with affinity for collagen type II and their response to mechanical stress1995In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 221, no 2, p. 496-503Article in journal (Refereed)
    Abstract [en]

    Mechanical stress is an important regulator of chondrocyte functions but the mechanisms by which chondrocytes sense mechanical signals are unknown. Receptors for matrix molecules are likely involved in the mechanical signaling. In the first part of this study we identified integrins with affinity for the cartilage-specific collagen type II. We report that the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 isolated from bovine chondrocytes or human chondrosarcoma cells bound collagen type II as judged from affinity chromatography. The integrins alpha 3 beta 1 or alpha 9 beta 1 did not bind collagen type II-Sepharose. In the second part of the study we investigated the effect of mechanical stress on expression of matrix molecules and integrin subunits. Chondrocytes and chondrosarcoma cells, cultured on uncoated flexible silicone membranes in the presence of serum, were exposed to mechanical stress by the Flexercell system. Dynamic stimulation of chondrocytes for 3 h increased the mRNA expression of collagen type II and aggrecan as judged by Northern blotting, while the beta 1-integrin subunit was not changed. When chondrosarcoma cells were exposed to mechanical stimulation under the same conditions, mRNA expression of alpha 5 was found to increase while beta 1, alpha 2, and alpha v did not increase to significant levels. In another study the effect of mechanical stress on integrins was investigated when the cells were cultured on collagen type II-coated flex-dishes. Three hours of dynamic stress increased the mRNA expression of alpha 2-integrin subunit while the level of mRNA for integrin subunits beta 1, alpha 1, alpha 5, and alpha v showed no or small changes, indicating that matrix components may modulate the expression of integrins during mechanical stress.

  • 23.
    Hooshmand-Rad, Roya
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Lu, Lingge
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Platelet-Derived Growth Factor-Mediated Signaling through the Shb Adaptor Protein: Effects on Cytoskeletal Organization2000In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 257, no 2, p. 245-254Article in journal (Refereed)
    Abstract [en]

    The Src homology (SH) 2 domain adaptor protein Shb has previously been shown to interact with the platelet-derived growth factor (PDGF)-β receptor. In this study we show an association between Shb and the PDGF-α receptor which is mediated by the SH2 domain of Shb and involves tyrosine residue 720 in the kinase insert domain of the receptor. To assess the role of Shb in PDGF-mediated signaling, we have overexpressed wild-type Shb or Shb carrying a mutation (R522K) which renders the SH2 domain inactive, in Patch mouse (PhB) fibroblasts expressing both PDGF receptors (PhB/Rα). Overexpression of wild-type Shb, but not the R522K Shb mutant, affected PDGF-mediated reorganization of the cytoskeleton by decreasing membrane ruffle formation and stimulating the generation of filopodia relative the parental control cells. In addition, the PDGF-induced receptor-associated phosphatidylinositol 3′-kinase activity and phosphorylation of Akt was similar in both PhB/Rα/Shb and PhB/Rα/ShbR522K cells compared with the parental control, whereas the activation of Rac in response to PDGF-BB was diminished only in the PhB/Rα/Shb cells. We conclude that Shb plays a role in PDGF-dependent regulation of certain cytoskeletal changes by modulating the ability of PDGF to activate Rac.

  • 24.
    Hägerkvist, Robert
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Mokhtari, Dariush
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lindholm, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Farnebo, Filip
    Mostoslavsky, Gustavo
    Mulligan, Richard C.
    Welsh, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Consequences of Shb and c-Abl interactions for cell death in response to various stress stimuli2007In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 313, no 2, p. 284-291Article in journal (Refereed)
    Abstract [en]

    The adaptor protein Shb has previously been shown to regulate apoptosis in response to cytokines and inhibitors of angiogenesis although the mechanisms governing these effects have remained obscure. We currently demonstrate interactions between Shb and c-Abl and that Shb regulates c-Abl kinase activity. The data suggest that c-Abl binds to tyrosine phosphorylated Shb via a concerted effort involving both the c-Abl SH3 and SH2 domains. The biological significance of the Shb/c-Abl interaction was presently tested in overexpression experiments and was found to promote hydrogen peroxide-induced cell death. We also show by Shb knockdown experiments that Shb regulates c-Abl activity and modulates cell death in response to the genotoxic agent cisplatin and the endoplasmic reticulum stress-inducer tunicamycin. The findings are in agreement with the notion of Shb playing a pivotal role in modulating c-Abl pro-apoptotic signaling in response to various stress stimuli.

  • 25.
    Hägg, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wennström, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Activation of hypoxia-induced transcription in normoxia2005In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 306, no 1, p. 180-191Article in journal (Refereed)
    Abstract [en]

    Hypoxia-inducible factor-1 (HIF-1), the master regulator of transcriptional responses to reduced oxygen tension (hypoxia) in mammalian cells, consists of one HIF-1alpha and one HIF-1beta subunit. In normoxia, HIF-1alpha subunits are hydroxylated on specific proline residues; modifications that signal ubiquitination and degradation of HIF-1alpha by the proteasome. To test the effect of saturating HIF-1alpha degradation, we generated a construct, denoted the saturating domain (SD), based on a region surrounding proline 564 (Pro564) in HIF-1alpha. Expression of the SD led to accumulation of endogenous HIF-1alpha proteins in nuclei of normoxic cells. The induced HIF-1alpha was functional as it activated expression from a hypoxia-regulated reporter gene and from the endogenous vascular endothelial growth facor-a (Vegf-a) and carbonic anhydrase 9 (Ca9) genes. The effect of the SD was dependent on Pro564 since a mutated SD, in which Pro564 had been replaced by a glycine residue, failed to bind the von Hippel-Lindau protein (pVHL) and to stabilise HIF-1alpha. Treatment of cells with the prolylhydroxylase inhibitor dimethyloxalylglycine, or the proteasome inhibitor MG-132, mimicked the effect of the SD. In conclusion, we show that blocking HIF-1alpha degradation, either by saturation, or inhibition of prolyl hydroxylases or proteosomal degradation, leads to nuclear localisation of active HIF-1alpha proteins.

  • 26. Iivanainen, Erika
    et al.
    Paatero, Ilkka
    Heikkinen, Satu-Maria
    Junttila, Teemu T.
    Cao, Renhai
    Klint, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jaakkola, Panu M.
    Cao, Yihai
    Elenius, Klaus
    Intra- and extracellular signaling by endothelial neuregulin-12007In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 313, no 13, p. 2896-2909Article in journal (Refereed)
    Abstract [en]

    Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.

  • 27.
    Johansson, Karin C
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Metzendorf, Christoph
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Microarray analysis of immune challenged Drosophila hemocytes2005In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 305, no 1, p. 145-155Article in journal (Refereed)
    Abstract [en]

    nsect hemocytes play multiple roles in immunity and carry out cellular responses like phagocytosis, encapsulation and melanization as well as producing humoral effector proteins in the first line of defense after injury and invasion of microorganisms. In this work, we used the Drosophila melanogaster hemocyte-like cell line mbn-2 and Affymetrix Drosophila GeneChips to investigate the transcriptome of a single type of immune competent tissue exposed to Gram-negative cell wall components (crude LPS) or high dose infection with live Escherichia coli. We found that gene expression profiles of both treatments overlap but show important differences in expression levels of several genes involved in immunity. In addition, cell morphology during infection was monitored and revealed distinct alterations in cell shape and adhesion. Presence of large numbers of bacteria also increased the number of cells taking on crystal cell fate. Taken together, our results indicate that hemocytes sense and respond differently to purified bacterial surface molecules and infection with live and actively growing bacteria both at the level of gene expression and in cell behavior.

  • 28.
    Karlsson, Henning
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Loss of cancer drug activity in colon cancer HCT-116 cells during spheroid formation in a new 3-D spheroid cell culture system2012In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 318, no 13, p. 1577-1585Article in journal (Refereed)
    Abstract [en]

    Clinically relevant in vitro methods are needed to identify new cancer drugs for solid tumors. We report on a new 3-D spheroid cell culture system aimed to mimic the properties of solid tumors in vivo. The colon cancer cell lines HCT-116 wt and HCT-116 wt/GFP were grown as monolayers and for 3 or 6 days on 96-well NanoCulture (R) plates to form spheroids. Expression of surface markers, genes and hypoxia were assessed to characterize the spheroids and drug induced cytotoxicity was evaluated based on fluorescein diacetate (FDA) conversion by viable cells to fluorescent fluorescein or by direct measurement of fluorescence of GFP marked cells after a 72 h drug incubation. The cells reproducibly formed spheroids in the NanoCulture (R) plates with tight cell-attachment after 6 days. Cells in spheroids showed geno- and phenotypical properties reminiscent of hypoxic stem cells. Monolayer cultured cells were sensitive to standard and investigational drugs, whereas the spheroids gradually turned resistant. Similar results for cytotoxicity were observed using simplified direct measurement of fluorescence of GFP marked cells compared with FDA incubation. In conclusion, this new 3-D spheroid cell culture system provides a convenient and clinically relevant model for the identification and characterization of cancer drugs for solid tumors.

  • 29.
    Karlsson, Torbjörn
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Modulation of Src homology 3 proteins by the proline-rich adaptor protein Shb1997In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 231, no 2, p. 269-275Article in journal (Refereed)
    Abstract [en]

    In the present study we have investigated a possible role for the proline-rich SH2 domain protein Shb as a regulator of expression or activity of certain SH3 domain proteins and MAP kinase. The expression of the Shb binding proteins Eps8, Src, and p85 PI3-kinase, PI3-kinase activity, and MAP kinase activation were assessed in wild-type NIH3T3 cells and in NIH3T3 cells overexpressing the Shb cDNA. In addition, the expression of the SH3 domain STAT1 proteins was assessed in wild-type and Shb overexpressing cells. The Eps8 protein content and Eps8 mRNA steady-state levels were downregulated, whereas the protein contents of Src and p85 PI3-kinase were unaffected by Shb overexpression. There was, however, an increased basal PI3-kinase activity in Shb transfected cells after a 3-h serum starvation. Increased steady-state levels of STAT1 mRNA were accompanied by an increased STAT1 protein content in Shb overexpressing cells. Shb overexpression was not associated with an altered activation of p44 or p42 MAP kinases in response to PDGF stimulation. The data presented in this study suggest novel functions for the adaptor protein Shb regulating the expression of certain signal-transducing SH3 domain proteins and modulating PI3-kinase activity.

  • 30. Karpanen, Terhi
    et al.
    Mäkinen, Taija
    Regulation of lymphangiogenesis--from cell fate determination to vessel remodeling.2006In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 312, no 5Article in journal (Refereed)
    Abstract [en]

    Lymphatic vessels are important for the maintenance of normal tissue fluid balance, immune surveillance and adsorption of digested fats. During the past decade, the identification of lymphatic-specific markers and growth factors has enabled detailed studies of the lymphatic system, and gain- and loss-of-function experiments have greatly increased our understanding of the mechanisms of normal lymphatic development. Understanding the basic biology has provided novel insights into the pathologic conditions of the lymphatic system that contribute to lymphedema, inflammation or lymphatic metastasis, and opened possibilities for the development of better therapeutic strategies. Here we review the current knowledge about the molecular mechanisms regulating the development of the lymphatic vasculature; of the differentiation of lymphatic endothelial cells, of the regulation of the growth of lymphatic vessels, and of remodeling of the vasculature into a network consisting of lymphatic capillaries and collecting lymphatic vessels. Furthermore, we will discuss the molecular mechanisms involved in the pathological conditions of the lymphatic vessels.

  • 31.
    Kilarski, Witold
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jura, Natalia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Inactivation of Src family kinases inhibits angiogenesis in vivo. Implications for a mechanism involving organization of the actin cytoskeleton2003In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 292, no 1, p. 70-82Article in journal (Refereed)
  • 32.
    Kiwanuka, Elizabeth
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Plastic Surgery.
    Andersson, Lauren
    Caterson, Edward J.
    Junker, Johan P. E.
    Gerdin, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Plastic Surgery.
    Eriksson, Elof
    CCN2 promotes keratinocyte adhesion and migration via integrin alpha 5 beta 12013In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 319, no 19, p. 2938-2946Article in journal (Refereed)
    Abstract [en]

    Background: CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration. Materials and methods: Adhesion assays were performed by coating wells with 10 mu g/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 mu l arginine-glycine-aspartic acid (RGD), 500 mu M arginine-glycine-glutamate-serine (RGES), and 10 mu g/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 mu M PD98059 (MEN-1 inhibitor) or 20 mu M PF573228 (FAN inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2. Results: CCN2 enhanced keratinocyte adhesion to fibronectin via integrin alpha 5 beta 1. The addition of anti-integrin alpha 5 beta 1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits alpha 5 and beta 1 CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration. Conclusions: Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin alpha 5 beta 1 and activation of the FAK-MAPK signaling cascade.

  • 33.
    Kvissel, Anne-Katrine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ørstavik, Sigurd
    Eikvar, Sissel
    Brede, Gaute
    Jahnsen, Tore
    Collas, Philippe
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Skålhegg, Bjørn Steen
    Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing2007In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 313, no 13, p. 2795-2809Article in journal (Refereed)
    Abstract [en]

    Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Cα and Cβ are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism.

  • 34.
    Lam, Matti
    et al.
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.
    Moslem, Mohsen
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.
    Bryois, Julien
    Karolinska Inst, Dept Med Epidemiol & Biostat, Stockholm, Sweden.
    Pronk, Robin J.
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.
    Uhlin, Elias
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.
    Ellström, Ivar Dehnisch
    Karolinska Inst, Dept Med Biochem & Biophys, Stockholm, Sweden.
    Laan, Loora
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Olive, Jessica
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.
    Morse, Rebecca
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.
    Rönnholm, Harriet
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.
    Louhivuori, Lauri
    Karolinska Inst, Dept Med Biochem & Biophys, Stockholm, Sweden.
    Korol, Sergiy V.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Birnir: Molecular Physiology and Neuroscience.
    Dahl, Niklas
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Uhlén, Per
    Karolinska Inst, Dept Med Biochem & Biophys, Stockholm, Sweden.
    Anderlid, Britt-Marie
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.
    Kele, Malin
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.
    Sullivan, Patrick F.
    Karolinska Inst, Dept Med Epidemiol & Biostat, Stockholm, Sweden.
    Falk, Anna
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.
    Single cell analysis of autism patient with bi-allelic NRXN1-alpha deletion reveals skewed fate choice in neural progenitors and impaired neuronal functionality2019In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 383, no 1, article id UNSP 111469Article in journal (Refereed)
    Abstract [en]

    We generated human iPS derived neural stem cells and differentiated cells from healthy control individuals and an individual with autism spectrum disorder carrying bi-allelic NRXN1-alpha deletion. We investigated the expression of NRXN1-alpha during neural induction and neural differentiation and observed a pivotal role for NRXN1-alpha during early neural induction and neuronal differentiation. Single cell RNA-seq pinpointed neural stem cells carrying NRXN1-alpha deletion shifting towards radial glia-like cell identity and revealed higher proportion of differentiated astroglia. Furthermore, neuronal cells carrying NRXN1-alpha deletion were identified as immature by single cell RNA-seq analysis, displayed significant depression in calcium signaling activity and presented impaired maturation action potential profile in neurons investigated with electrophysiology. Our observations propose NRXN1-alpha plays an important role for the efficient establishment of neural stem cells, in neuronal differentiation and in maturation of functional excitatory neuronal cells.

  • 35.
    Lee, Chunsik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Dixelius, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Thulin, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Kawamura, Harukiyo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Olsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Signal transduction in endothelial cells by the angiogenesis inhibitor histidine-rich glycoprotein targets focal adhesions2006In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 312, no 13, p. 2547-2556Article in journal (Refereed)
    Abstract [en]

    Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein. We have shown that a fragment released from the central histidine/proline-rich (His/Pro-rich) domain of HRGP blocks endothelial cell migration in vitro and vascularization and growth of murine fibrosarcoma in vivo. The minimal active HRGP domain exerting the anti-angiogenic effect was recently narrowed down to a 35 amino acid peptide, HRGP330, derived from the His/Pro-rich domain of HRGP. By use of a signal transduction antibody array representing 400 different signal transduction molecules, we now show that HRGP and the synthetic peptide HRGP330 specifically induce tyrosine phosphorylation of focal adhesion kinase and its downstream substrate paxillin in endothelial cells. HRGP/HRGP330 treatment of endothelial cells induced disruption of actin stress fibers, a process reversed by treatment of cells with the FAK inhibitor geldanamycin. In addition, VEGF-mediated endothelial cell tubular morphogenesis in a three-dimensional collagen matrix was inhibited by HRGP and HRGP330. In contrast, VEGF-induced proliferation was not affected by HRGP or HRGP330, demonstrating the central role of cell migration during tube formation. In conclusion, our data show that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures.

  • 36.
    Lennartsson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Rönnstrand, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk2003In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 288, no 1, p. 110-118Article in journal (Refereed)
    Abstract [en]

    We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.

  • 37.
    Li, Wei
    et al.
    Southeast Univ, Sch Med, Inst Diabet, Zhongda Hosp,Dept Endocrinol, Nanjing, Jiangsu, Peoples R China.
    Zhou, Yunting
    Southeast Univ, Sch Med, Inst Diabet, Zhongda Hosp,Dept Endocrinol, Nanjing, Jiangsu, Peoples R China.
    Wang, Xiaohang
    Southeast Univ, Sch Med, Inst Diabet, Zhongda Hosp,Dept Endocrinol, Nanjing, Jiangsu, Peoples R China.
    Cai, Min
    Southeast Univ, Sch Med, Inst Diabet, Zhongda Hosp,Dept Endocrinol, Nanjing, Jiangsu, Peoples R China.
    Gao, Feng
    Southeast Univ, Grad Innovat Platform, Nanjing, Jiangsu, Peoples R China.
    Carlsson, Per-Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sun, Zilin
    Southeast Univ, Sch Med, Inst Diabet, Zhongda Hosp,Dept Endocrinol, Nanjing, Jiangsu, Peoples R China.
    A modified in vitro tool for isolation and characterization of rat quiescent islet stellate cells2019In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 384, no 1, article id 111617Article in journal (Refereed)
    Abstract [en]

    Background: Islet stellate cells (ISCs) play a critical role in islet fibrosis, contributing to the progression of pancreatic diseases. Previous studies have focused on fibrosis-associated activated ISCs obtained by standard islet explant techniques. However, in vitro models of quiescent ISCs (qISCs) are lacking. This study aims to develop a method to isolate qISCs and analyze their phenotype during activation.

    Methods: Immunofluorescence staining was applied to localize ISCs in normal human, rat, and mouse islets. qISCs were isolated from rat islets using density gradient centrifugation (DGC) method. qRT-PCR, immunoblotting, proliferation, and migration assays were employed for their characterization.

    Results: Desmin-positive ISCs were detected in normal human, rat, and mouse islets. Freshly isolated qISCs, obtained by density gradient centrifugation, displayed a polygonal appearance with refringent cytoplasmic lipid droplets and expressed transcriptional markers indicating a low activation/quiescent state. With increasing culture time, the marker expression pattern changed, reflecting ISC activation. qISCs contained more lipid droplets and exhibited lower proliferation and migration abilities compared to spindle-shaped ISCs obtained by traditional explant techniques.

    Conclusions: This study describes a new method for efficient isolation of qISCs from rat islets, representing a useful in vitro tool to study the biology of ISCs in more physiological conditions.

  • 38.
    Lin, Xionghui
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Kim, Young-A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Lee, Bok Luel
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Identification and properties of a receptor for the invertebrate cytokine astakine, involved in hematopoiesis2009In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 315, no 7, p. 1171-1180Article in journal (Refereed)
    Abstract [en]

    We have recently isolated an invertebrate cytokine from a freshwater crayfish, which we named astakine 1. Interestingly this protein is expressed exclusively in hemocytes and hematopoietic tissue and is essential for the release of new hemocytes into the open circulatory system of these animals. This astakine has a prokineticin (PK) domain but lacks the N-terminal AVIT amino acids and hence receptor binding may differ from vertebrate PKs. Accordingly, here we report that a receptor for astakine 1 on hematopoietic tissue (Hpt) cells is identical to the beta-subunit of F1ATP synthase. In this study we have used several different methods to clearly demonstrate that ATP-synthase is located on the plasma membrane of a subpopulation of Hpt cells and there may function as a receptor for astakine, whereas mature blood cells (hemocytes) do not have any ATP-synthase on the outside of their plasma membranes. Our results clearly show that ATP synthase beta subunits are present on the cell surface of Hpt cells and highlight the need for more detailed studies on intracellular traffic connections between mitochondria and other membrane compartments.

  • 39.
    Martinez-Corral, Ines
    et al.
    Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.
    Mäkinen, Taija
    Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.
    Regulation of lymphatic vascular morphogenesis: Implications for pathological (tumor) lymphangiogenesis2013In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 319, no 11, p. 1618-1625Article, review/survey (Refereed)
    Abstract [en]

    Lymphatic vasculature forms the second part of our circulatory system that plays a critical role in tissue fluid homeostasis. Failure of the lymphatic system can lead to excessive accumulation of fluid within the tissue, a condition called lymphedema. Lymphatic dysfunction has also been implicated in cancer metastasis as well as pathogenesis of obesity, atherosclerosis and cardiovascular disease. Since the identification of the first lymphatic marker VEGFR-3 and growth factor VEGF-C almost 20 years ago, a great progress has been made in understanding the mechanisms of lymphangiogenesis. This has been achieved largely through characterization of animal models with specific lymphatic defects and identification of genes causative of human hereditary lymphedema syndromes. In this review we will summarize the current understanding of the regulation of lymphatic vascular morphogenesis, focusing on mechanisms that have been implicated in both developmental and pathological (tumor) lymphangiogenesis.

  • 40.
    Melhus, Håkan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Nilsson, Tommy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Peterson, Per A.
    Rask, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Retinol-binding protein and transthyretin expressed in HeLa cells form a complex in the endoplasmic reticulum in both the absence and the presence of retinol1991In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 197, no 1, p. 119-124Article in journal (Refereed)
    Abstract [en]

    To establish a suitable experimental system for studies of the interaction of retinol-binding protein (RBP) with transthyretin (TTR) we have expressed the corresponding cDNAs in HeLa cells. To investigate whether complex formation might occur already in the endoplasmic reticulum (ER), the C-terminal ER retention signal, KDEL, was attached to TTR. The tetrameric TTR-KDEL fusion protein was retained in the ER of HeLa cells. When RBP was co-expressed with TTR-KDEL, RBP was retained intracellularly. A cDNA-encoding purpurin, a protein which is 50% identical to RBP, was then expressed together with TTR-KDEL. Purpurin was not retained intracellularly and did not bind to TTR coupled to Sepharose. The effect of the vitamin A status on the secretion of TTR and RBP was examined. While TTR expressed alone was not retained intracellularly, TTR was retained in vitamin A-deficient cells when co-expressed with RBP. Addition of retinol stimulated rapid secretion of both proteins. These results demonstrate that TTR can form a complex with RBP in the ER. The data suggest that RBP and TTR are secreted as a complex.

  • 41.
    Nazir, Madiha
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Senkowski, Wojciech
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nyberg, Frida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Blom, Kristin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Edqvist, Per-Henrik D
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Andersson, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats G
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Targeting tumor cells based on Phosphodiesterase 3A expression2017In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 361, no 2, p. 308-315Article in journal (Refereed)
    Abstract [en]

    We and others have previously reported a correlation between high phosphodiesterase 3 A (PDE3A) expression and selective sensitivity to phosphodiesterase (PDE) inhibitors. This indicates that PDE3A could serve both as a drug target and a biomarker of sensitivity to PDE3 inhibition. In this report, we explored publicly available mRNA gene expression data to identify cell lines with different PDE3A expression. Cell lines with high PDE3A expression showed marked in vitro sensitivity to PDE inhibitors zardaverine and quazinone, when compared with those having low PDE3A expression. Immunofluorescence and immunohistochemical stainings were in agreement with PDE3A mRNA expression, providing suitable alternatives for biomarker analysis of clinical tissue specimens. Moreover, we here demonstrate that tumor cells from patients with ovarian carcinoma show great variability in PDE3A protein expression and that level of PDE3A expression is correlated with sensitivity to PDE inhibition. Finally, we demonstrate that PDE3A is highly expressed in subsets of patient tumor cell samples from different solid cancer diagnoses and expressed at exceptional levels in gastrointestinal stromal tumor (GIST) specimens. Importantly, vulnerability to PDE3 inhibitors has recently been associated with co-expression of PDE3A and Schlafen family member 12 (SLFN12). We here demonstrate that high expression of PDE3A in clinical specimens, at least on the mRNA level, seems to be frequently associated with high SLFIV12 expression. In conclusion, PDE3A seems to be both a promising biomarker and drug target for individualized drug treatment of various cancers.

  • 42.
    Niklasson, Mia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Bergström, Tobias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Cancer and Vascular Biology.
    Zhang, X-Q
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gustafsdottir, Sigrun M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sjögren, Maria
    Edqvist, Per-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Vennström, Björn
    Forsberg, Maud
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Forsberg-Nilsson, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Enlarged lateral ventricles and aberrant behavior in mice overexpressing PDGF-B in embryonic neural stem cells2010In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 316, no 17, p. 2779-2789Article in journal (Refereed)
    Abstract [en]

    Platelet-derived growth factor (PDGF) is important in central nervous system (CNS) development, and aberrant expression of PDGF and its receptors has been linked to developmental defects and brain tumorigenesis. We previously found that neural stem and progenitor cells in culture produce PDGF and respond to it by autocrine and/or paracrine signaling. We therefore aimed to examine CNS development after PDGF overexpression in neural stem cells in vivo. Transgenic mice were generated with PDGF-B under control of a minimal nestin enhancer element, which is specific for embryonic expression and will not drive adult expression in mice. The resulting mouse showed increased apoptosis in the developing striatum, which suggests a disturbed regulation of progenitor cells. Later in neurodevelopment, in early postnatal life, mice displayed enlarged lateral ventricles. This enlargement remained into adulthood and it was more pronounced in male mice than in transgenic female mice. Nevertheless, there was an overall normal composition of cell types and numbers in the brain and the transgenic mice were viable and fertile. Adult transgenic males, however, showed behavioral aberrations and locomotor dysfunction. Thus, a tightly regulated expression of PDGF during embryogenesis is required for normal brain development and function in mice.

  • 43. Parmryd, Ingela
    et al.
    Adler, Jeremy
    Patel, Roopal
    Magee, Anthony I
    Imaging metabolism of phosphatidylinositol 4,5-bisphosphate in T-cell GM1-enriched domains containing Ras proteins.2003In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 285, no 1, p. 27-38Article in journal (Refereed)
    Abstract [en]

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and Ras proteins are involved in signalling pathways originating at the plasma membrane. The localisation and metabolism of PI(4,5)P(2) was studied in Jurkat T cells using fluorescence microscopic imaging with EGFP-tagged and antibody probes. Software was developed to objectively quantitate colocalisation and was used to show that plasma membrane PI(4,5)P(2) was enriched in lipid raft-containing patches of GM1 ganglioside, formed by crosslinking cholera toxin B-subunit (CT-B). The PI(4,5)P(2) metabolites phosphatidylinositol 3,4,5-trisphosphate and diacylglycerol appeared in plasma membrane CT-B-GM1 patches upon induction of signalling. Transferrin receptor and the CD45 tyrosine phosphatase did not colocalise with CT-B-GM1 patches, whereas the tyrosine kinase Lck, the scaffolding protein LAT, and endogenous Ras proteins did partially colocalise with CT-B-GM1 patches as did transfected EGFP-K-Ras(4B) and EGFP-H-Ras. The results demonstrate that T-cell PI(4,5)P(2) metabolism is occurring in GM1-enriched domains and that Ras proteins are present in these domains in vivo.

  • 44. Petrova, T V
    et al.
    Makinen, T
    Alitalo, K
    Signaling via vascular endothelial growth factor receptors.1999In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 253, no 1Article in journal (Refereed)
    Abstract [en]

    Angiogenesis, or development of blood vessels from preexisting vasculature, has important functions under both normal and pathophysiological conditions. Vascular endothelial growth factor receptors 1-3, also known as flt-1, KDR, and flt-4, are endothelial cell-specific receptor tyrosine kinases which serve as key mediators of the angiogenic responses. The review focuses on the signaling pathways that are initiated from these receptors and the recently identified VEGF coreceptor neuroplilin-1.

  • 45. Reijonen, Sami
    et al.
    Putkonen, Noora
    Nørremølle, Anne
    Lindholm, Dan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Korhonen, Laura
    Inhibition of endoplasmic reticulum stress counteracts neuronal cell death and protein aggregation caused by N-terminal mutant huntingtin proteins2008In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 314, no 5, p. 950-960Article in journal (Refereed)
    Abstract [en]

    Accumulation of abnormal proteins occurs in many neuro degenerative diseases including Huntington's disease (HD). However, the precise role of protein aggregation in neuronal cell death remains unclear. We show here that the expression of N-terminal huntingtin proteins with expanded polyglutamine (polyQ) repeats causes cell death in neuronal PC6.3 cell that involves endoplasmic reticulum (ER) stress. These mutant huntingtin fragment proteins elevated Bip, an ER chaperone, and increased Chop and the phosphorylation of c-jun-N-terminal kinase (JNK) that are involved in cell death regulation. Caspase-12, residing in the ER, was cleaved in mutant huntingtin expressing cells, as was caspase-3 mediating cell death. In contrast, cytochrome-c or apoptosis inducing factor (AIF) was not released from mitochondria after the expression of these proteins. Treatment with salubrinal that inhibits ER stress counteracted cell death and reduced protein aggregations in the PC6.3 cells caused by the mutant huntingtin fragment proteins. Salubrinal upregulated Bip, reduced cleavage of caspase-12 and increased the phosphorylation of eukaryotic translation initiation factor-2 subunit-alpha (eIF2 alpha) that are neuroprotective. These results show that N-terminal mutant huntingtin proteins activate cellular pathways linked to ER stress, and that inhibition of ER stress by salubrinal increases cell survival. The data suggests that compounds targeting ER stress may be considered in designing novel approaches for treatment of HD and possibly other polyQ diseases.

  • 46.
    Rennel, Emma
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Cross, Michael J.
    Klint, Peter
    Bai, Xianhe
    Arbiser, Jack L.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Regulation of endothelial cell differentiation and transformation by H-Ras2003In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 291, no 1, p. 189-200Article in journal (Refereed)
    Abstract [en]

    Angiogenesis is regulated by growth factors which activate tyrosine kinase receptors leading to the activation of a number of intracellular signaling pathways. The specific function of H-Ras during FGF-2 stimulated endothelial cell differentiation, defined as invasive growth and formation of branching networks in fibrin gels, was investigated by using conditionally immortalized endothelial cell lines induced to express H-Ras mutants. Expression of inhibitory N17Ras did not impair differentiation in response to FGF-2 and TNF-alpha. The farnesyltransferase inhibitor FTI-277 inhibited farnesylation of Ras but did not inhibit differentiation of human microvascular endothelial cells or mouse brain endothelial cells. In contrast, activated V12Ras inhibited endothelial cell differentiation and cells displayed a transformed phenotype with an increased rate of proliferation and loss of contact inhibited growth. Furthermore, V12Ras expressing endothelial cells grew as solid tumors when injected subcutaneously into mice. Our data suggest that, in endothelial cells, H-Ras activity is not required for differentiation. However, this activity must be tightly regulated as aberrant activity can disturb the ability of endothelial cells to undergo differentiation.

  • 47.
    Rennel, Emma
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Mellberg, Sofie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Dimberg, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Petersson, Ludvig
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Botling, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ameur, Adam
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Westholm, Jakub Orzechowski
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Lassalle, Philippe
    Cross, Michael J.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Radiology.
    Endocan is a VEGF-A and PI3K regulated gene with increased expression in human renal cancer2007In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 313, no 7, p. 1285-1294Article in journal (Refereed)
    Abstract [en]

    An in vitro model of VEGF-A-induced angiogenesis was used to generate transcription profiles of human microvascular endothelial cells. Microarray analysis showed increased transcription of genes known to regulate angiogenesis, but also genes that previously have not been firmly associated with angiogenesis such as endocan, pinin, plakophilin, phosphodiesterase 4B and gelsolin. Increased endocan mRNA levels in response to VEGF-A in endothelial cells and in human renal cancer have previously been reported. We now show increased endocan protein levels in VEGF-A treated endothelial cells and in human renal clear cell carcinoma. Increased protein expression was observed both in tumor cells and in a subset of tumor vessels, while expression in normal kidney tissue was low. VEGF-A seemed to be a specific inducer of endocan transcription since FGF-2, PDGF-BB, HGF/SF and EGF did not alter expression levels. Inhibition of PI3K with LY294002 caused a 12-fold increase in endocan transcription suggesting a repressive function of PI3K. In contrast inhibition of Src or MEK, which are signaling pathways activated by VEGF-A, did not influence basal or VEGF-A-induced endocan levels. In conclusion our study shows that, among angiogenic growth factors, VEGF-A is a specific inducer of endocan transcription which is translated into increased protein levels in VEGF-A treated endothelial cells. Increased endocan protein expression in human renal cancer suggests a role in tumor growth.

  • 48.
    Rolny, Charlotte
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lu, Lingge
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Ågren, Nina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Nilsson, Ingrid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Roe, Cheryl
    Webb, Gene C
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Shb promotes blood vessel formation in embryoid bodies by augmenting vascular endothelial growth factor receptor-2 and platelet-derived growth factor receptor-beta signaling2005In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 308, no 2, p. 381-393Article in journal (Other academic)
    Abstract [en]

    The mechanisms controlling blood vessel formation during early embryonal development have only partly been elucidated. Shb is an adaptor protein previously implicated in the angiogenic response to vascular endothelial growth factor (VEGF). To elucidate a possible role of Shb in embryonic vascular development, wild-type and SH2 domain mutated (R522K) Shb were overexpressed in murine embryonic stem (ES) cells. Embryoid bodies (EBs) differentiating from Shb-overexpressing ES cells in vitro were stained for CD31 or VEGFR-2 to visualize the formation of vascular structures. We found that Shb promotes the outgrowth of blood vessels in EBs both in the absence and presence of growth factors. This response may be the consequence of an increased number of VEGFR-2 positive cells at an early stage of EB development, a finding corroborated by both immunostaining and real-time RT-PCR. In addition, Shb overexpression upregulated the expression of PDGFR-beta, CD31, CD41 and Tal1. Cells co-expressing VEGFR-2 and PDGFR-beta were commonly observed when Shb was overexpressed and inhibition of PDGF-BB signaling reduced the amount of VEGFR-2 mRNA under these conditions. EBs expressing the Shb R522K-mutant did not form vascular structures. Microarray analysis of VEGFR-2/CD31 positive cells after 6 days of differentiation revealed numerous changes of expression of genes relating to an endothelial/hematopoietic phenotype in response to Shb overexpression. The findings suggest that Shb may play a crucial role during early ES cell differentiation to vascular structures by transducing VEGFR-2 and PDGFR-beta signals.

  • 49.
    Sakwe, Amos M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Rask, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Involvement of protein kinase C-alpha and -epsilon in extracellular Ca(2+) signalling mediated by the calcium sensing receptor2004In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 297, no 2, p. 560-573Article in journal (Refereed)
    Abstract [en]

    The sensing of extracellular Ca(2+) concentration ([Ca(2+)](o)) and modulation of cellular processes associated with acute or sustained changes in [Ca(2+)](o) are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca(2+)](o) signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca(2+)](o) activated PKC-alpha and PKC- in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca(2+)](o) required influx of Ca(2+)through Ni(2+)-sensitive Ca(2+)channels and phosphatidylinositol-dependent phospholipase C-beta activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-alpha or - with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca(2+)](o). Activation of ERK1/2 by high [Ca(2+)](o) was not necessary for the [Ca(2+)](o)-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca(2+)](o) signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.

  • 50.
    Sellin, Mikael E.
    et al.
    Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Holmfeldt, Per
    Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Stenmark, Sonja
    Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gullberg, Martin
    Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Op18/Stathmin counteracts the activity of overexpressed tubulin-disrupting proteins in a human leukemia cell line2008In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 314, no 6, p. 1367-77Article in journal (Refereed)
    Abstract [en]

    Op18/stathmin (Op18) is a phosphorylation-regulated and differentially expressed microtubule-destabilizing protein in animal cells. Op18 regulates tubulin monomer-polymer partitioning of the interphase microtubule system and forms complexes with tubulin heterodimers. Recent reports have shown that specific tubulin-folding cofactors and related proteins may disrupt tubulin heterodimers. We therefore investigated whether Op18 protects unpolymerized tubulin from such disruptive activities. Our approach was based on inducible overexpression of two tubulin-disrupting proteins, namely TBCE, which is required for tubulin biogenesis, and E-like, which has been proposed to regulate tubulin turnover and microtubule stability. Expression of either of these proteins was found to cause a rapid degradation of both alpha-tubulin and beta-tubulin subunits of unpolymerized, but not polymeric, tubulin heterodimers. We found that depletion of Op18 by means of RNA interference increased the susceptibility of tubulin to TBCE or E-like mediated disruption, while overexpressed Op18 exerted a tubulin-protective effect. Tubulin protection was shown to depend on Op18 levels, binding affinity, and the partitioning between tubulin monomers and polymers. Hence, the present study reveals that Op18 at physiologically relevant levels functions to preserve the integrity of tubulin heterodimers, which may serve to regulate tubulin turnover rates.

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