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  • 251.
    Ihse, Elisabet
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Suhr, Ole B.
    Department of Internal Medicine, Umeå University, 901 85, Umeå, Sweden.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Westermark, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Variation in amount of wild-type transthyretin in different fibril and tissue types in ATTR amyloidosis2011In: Journal of Molecular Medicine, ISSN 0946-2716, E-ISSN 1432-1440, Vol. 89, no 2, p. 171-180Article in journal (Refereed)
    Abstract [en]

    Familial transthyretin (TTR) amyloidosis is caused by a mutation in the TTR gene, although wild-type (wt) TTR is also incorporated into the amyloid fibrils. Liver transplantation (LT) is the prevailing treatment of the disease and is performed in order to eliminate the mutant TTR from plasma. The outcome of the procedure is varied; especially problematic is a progressive cardiomyopathy seen in some patients, presumably caused by continued incorporation of wtTTR. What determines the discrepancy in outcome is not clear. We have previously shown that two structurally distinct amyloid fibrils (with or without fragmented ATTR) are found among ATTRV30M patients. In this study, we investigated the proportion of wtATTR in cardiac and adipose amyloid from patients having either fibril type. It was found that cardiac amyloid more easily incorporates wtTTR than adipose amyloid, offering a potential explanation for the vulnerability of cardiac tissue for continued amyloidosis after LT. In cardiac tissue, fibrils with fragmented ATTR contained a higher wt proportion than fibrils without, suggesting that continued incorporation of wtTTR after LT, perhaps, can take place more easily in these patients. In adipose tissue, a rapid increase in wt proportion after LT indicates that a rather fast turnover of the deposits must occur. A difference in wt proportion between the fibril types was seen post-LT but not pre-LT, possibly caused by differences in turnover rate. Conclusively, this study further establishes the basic dissimilarities between the two fibril types and demonstrates that their role in LT outcome needs to be further investigated.

  • 252.
    Ikushima, Hiroaki
    et al.
    University of Tokyo.
    Komuro, Akiyoshi
    University of Tokyo.
    Isogaya, Kazunobu
    University of Tokyo.
    Shinozaki, Masahiko
    University of Tokyo.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Miyazawa, Keiji
    University of Tokyo.
    Miyazono, Kohei
    University of Tokyo.
    An Id-like molecule, HHM, is a synexpression group-restricted regulator of TGF-β signalling2008In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 27, no 22, p. 2955-2965Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor (TGF)-β induces various cellular responses principally through Smad-dependent transcriptional regulation. Activated Smad complexes cooperate with transcription factors in regulating a group of target genes. The target genes controlled by the same Smad-cofactor complexes are denoted a synexpression group. We found that an Id-like helix-loop-helix protein, human homologue of Maid (HHM), is a synexpression group-restricted regulator of TGF-β signalling. HHM suppressed TGF-β-induced growth inhibition and cell migration but not epithelial–mesenchymal transition. In addition, HHM inhibited TGF-β-induced expression of plasminogen activator inhibitor-type 1 (PAI-1), PDGF-B, and p21WAF, but not Snail. We identified a basic-helix-loop-helix protein, Olig1, as one of the Smad-binding transcription factors affected by HHM. Olig1 interacted with Smad2/3 in response to TGF-β stimulation, and was involved in transcriptional activation of PAI-1 and PDGF-B. HHM, but not Id proteins, inhibited TGF-β signalling-dependent association of Olig1 with Smad2/3 through physical interaction with Olig1. HHM thus appears to regulate a subset of TGF-β target genes including the Olig1-Smad synexpression group. HHM is the first example of a cellular response-selective regulator of TGF-β signalling with clearly determined mechanisms.

  • 253.
    Ikushima, Hiroaki
    et al.
    1.Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Japan.
    Miyazono, Kohei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    TGFβ signalling: a complex web in cancer progression2010In: Nature Reviews. Cancer, ISSN 1474-175X, E-ISSN 1474-1768, Vol. 10, no 6, p. 415-424Article in journal (Refereed)
    Abstract [en]

    The distortion of growth factor signalling is the most important prerequisite in tumour progression. Transforming growth factor-beta (TGFbeta) signalling regulates tumour progression by a tumour cell-autonomous mechanism or through tumour-stroma interaction, and has either a tumour-suppressing or tumour-promoting function depending on cellular context. Such inherent complexity of TGFbeta signalling results in arduous, but promising, assignments for developing therapeutic strategies against malignant tumours. As numerous cellular context-dependent factors tightly maintain the balance of TGFbeta signalling and contribute to the regulation of TGFbeta-induced cell responses, in this Review we discuss how they maintain the balance of TGFbeta signalling and how their collapse leads to tumour progression.

  • 254. Inamitsu, Masako
    et al.
    Itoh, Susumu
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    ten Dijke, Peter
    Kato, Mitsuyasu
    Methylation of Smad6 by protein arginine N-methyltransferase 12006In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, no 28-29, p. 6603-6611Article in journal (Refereed)
    Abstract [en]

    Signal transduction pathways utilize posttranslational modifications to regulate the activity of their components in a temporal-spatial and efficient fashion. Arginine methylation is one of the posttranslational modifications that can result in monomethylated-, asymmetric dimethylated- and/or symmetric dimethylated-arginine residues in proteins. Here we demonstrate that inhibitory-Smads (Smad6 and Smad7), but not receptor-regulated- (R-)Smads and the common-partner Smad4, can be methylated by protein arginine N-methyltransferase (PRMT)1. Using mass-spectrometric analysis, we found that PRMT1 dimethylates arginine(74) (Arg(74)) in mouse Smad6. PRMT1 interacts with the N-terminal domain of Smad6 in which Arg(74) residue is located. Assays examined so far have shown no significant differences between the functions of Smad6 and those of methylation-defective Smad6 (Smad6R74A). Both wild-type and Smad6R74A were equally efficient in blocking BMP-induced growth arrest upon their ectopic expression in HS-72 mouse B-cell hybridoma cells.

  • 255.
    Itoh, Fumiko
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    Regulation of TGF-β/Smad Signaling Through Smad Interacting Proteins2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Transforming growth factor-β (TGF-β) superfamily members are multi-functional regulators of cell fate. These factors signal by binding to a limited number of highly conserved transmembrane type I and type II serine/threonine kinase receptors. These receptors initiate signals into the cell via the Smad proteins. Up to date, 8 different mammalian Smads are reported and are divided into three subgroups; receptor regulated Smads (R-Smads), common mediator Smads (Co-Smads) and inhibitory Smads (I-Smads). This thesis investigates the function and regulation of TGF-β/Smad signaling through identification and characterization of Smad interacting proteins.

    I-Smads, i.e. Smad6 and Smad7, are potent antagonists of the TGF-β superfamily signaling. We found that Smad7, but not Smad6, inhibits TGF-β1-induced growth inhibition and expression of immediate early response genes. Interestingly, in the absence of ligand, Smad7 was found to be predominantly localized in the nucleus, whereas Smad7 accumulated in the cytoplasm upon TGF-β receptor activation. Moreover, we found that the MH2 domain is important for nuclear export.

    To investigate further the role of inhibitory Smads, we have identified AMSH as a Smad6 interacting protein using a yeast two-hybrid screening method. AMSH was previously discovered as the associated molecule with the SH3 domain of STAM. AMSH interacts with I-Smads, but not with R- and Co-Smads upon receptor activation and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. AMSH was found to prevent Smad6 from binding to activated type I receptors and/or activated R-Smads. Smad anchor for receptor activation (SARA) is critical for Smad2 and Smad3 activation by TGF-β receptors. The present studies show that the localization of SARA in early endosomes is regulated through its FYVE domain. We have found that the FYVE domain of SARA is sufficient and necessary for the early endosomal localization, probably through its interaction with PtdIns(3)P. Moreover, the localization of SARA in early endosomes is required for efficient TGF-β/Smad signaling.

    Both Notch and BMP signaling pathways are important for vascular development. We have found that Herp2, which is originally known as one of the Notch target genes, is synergistically induced upon activation of Notch and BMP signaling pathways in endothelial cells (ECs). The critical elements for synergistical activation of Herp2 gene by BMP and Notch pathway were identified. Furthermore, the Notch intracellular domain interacts with Smad5 upon BMP receptor and this interaction becomes stronger in the presence of pCAF. Interestingly, Herp2 was found to antagonize BMP receptor- or Id-mediated EC migration.

    List of papers
    1. Transforming Growth Factor β1 Induces Nuclear Export of Inhibitory Smad7
    Open this publication in new window or tab >>Transforming Growth Factor β1 Induces Nuclear Export of Inhibitory Smad7
    Show others...
    1998 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, ., Vol. 273, no 44, p. 29195-29201Article in journal (Refereed) Published
    Abstract [en]

    Transforming growth factor beta (TGF-beta) signals from membrane to nucleus through serine/threonine kinase receptors and their downstream effector molecules, termed Smad proteins. Recently, Smad6 and Smad7 were identified, which antagonize TGF-beta family signaling by preventing the activation of signal-transducing Smad complexes. Here we report that Smad7, but not Smad6, inhibits TGF-beta1-induced growth inhibition and the expression of immediate early response genes, including Smad7. Interestingly, in the absence of ligand, Smad7 was found to be predominantly localized in the nucleus, whereas Smad7 accumulated in the cytoplasm upon TGF-beta receptor activation. The latter is in accordance with the physical association of Smad7 with the ligand-activated TGF-beta receptor complex in the cell membrane. Whereas the ectopically expressed C-terminal domain of Smad7 was also exported from the nucleus to the cytoplasm upon TGF-beta challenge, a Smad7 mutant with a small deletion at the C terminus or only the N-terminal domain of Smad7 was localized mainly in the cytoplasm in the absence or presence of ligand. This suggests that an intact Mad homology 2 domain is important for nuclear localization of Smad7. The nuclear localization of Smad7 suggests a functional role distinct from its antagonistic effect in receptor-mediated Smad activation.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-90978 (URN)10.1074/jbc.273.44.29195 (DOI)9786930 (PubMedID)
    Available from: 2003-10-20 Created: 2003-10-20 Last updated: 2017-12-14Bibliographically approved
    2. Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads
    Open this publication in new window or tab >>Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads
    Show others...
    2001 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 15, p. 4132-4142Article in journal (Refereed) Published
    Abstract [en]

    Inhibitory Smads, i.e. Smad6 and Smad7, are potent antagonists of the BMP-Smad pathway by interacting with activated bone morphogenetic protein (BMP) type I receptors and thereby preventing the activation of receptor-regulated Smads, or by competing with activated R-Smads for heteromeric complex formation with Smad4. The molecular mechanisms that underlie the regulation of I-Smad activity have remained elusive. Here we report the identification of a cytoplasmic protein, previously termed associated molecule with the SH3 domain of STAM (AMSH), as a direct binding partner for Smad6. AMSH interacts with Smad6, but not with R- and Co-Smads, upon BMP receptor activation in cultured cells. Consistent with this finding, stimulation of cells with BMP induces a co-localization of Smad6 with AMSH in the cytoplasm. Ectopic expression of AMSH prolongs BMP-induced Smad1 phosphorylation, and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. The data strongly suggest that the molecular mechanism by which AMSH exerts its action is by inhibiting the binding of Smad6 to activated type I receptors or activated R-Smads.

    Place, publisher, year, edition, pages
    The European Molecular Biology Organization, 2001
    Keywords
    AMSH, BMP, signaling, Smad, TGF-β
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-90979 (URN)10.1093/emboj/20.15.4132 (DOI)11483516 (PubMedID)
    Available from: 2003-10-20 Created: 2003-10-20 Last updated: 2017-12-14Bibliographically approved
    3. The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-β/Smad signaling
    Open this publication in new window or tab >>The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-β/Smad signaling
    Show others...
    2002 In: Genes to Cells, Vol. 7, p. 321-331Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-90980 (URN)
    Available from: 2003-10-20 Created: 2003-10-20Bibliographically approved
    4. The Activation of Notch Antagonizes BMP Receptor-induced Endothelial Cell Migration via Synergistic Induction of Herp2 Expression
    Open this publication in new window or tab >>The Activation of Notch Antagonizes BMP Receptor-induced Endothelial Cell Migration via Synergistic Induction of Herp2 Expression
    Show others...
    Article in journal (Refereed) Submitted
    Identifiers
    urn:nbn:se:uu:diva-90981 (URN)
    Available from: 2003-10-20 Created: 2003-10-20Bibliographically approved
  • 256.
    Itoh, Fumiko
    et al.
    Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
    Asao, Hironobu
    Department of Microbiology and Immunology, Tohoku University of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
    Sugamura, Kazuo
    Department of Microbiology and Immunology, Tohoku University of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    ten Dijke, Peter
    Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
    Itoh, Susumu
    Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
    Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 15, p. 4132-4142Article in journal (Refereed)
    Abstract [en]

    Inhibitory Smads, i.e. Smad6 and Smad7, are potent antagonists of the BMP-Smad pathway by interacting with activated bone morphogenetic protein (BMP) type I receptors and thereby preventing the activation of receptor-regulated Smads, or by competing with activated R-Smads for heteromeric complex formation with Smad4. The molecular mechanisms that underlie the regulation of I-Smad activity have remained elusive. Here we report the identification of a cytoplasmic protein, previously termed associated molecule with the SH3 domain of STAM (AMSH), as a direct binding partner for Smad6. AMSH interacts with Smad6, but not with R- and Co-Smads, upon BMP receptor activation in cultured cells. Consistent with this finding, stimulation of cells with BMP induces a co-localization of Smad6 with AMSH in the cytoplasm. Ectopic expression of AMSH prolongs BMP-induced Smad1 phosphorylation, and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. The data strongly suggest that the molecular mechanism by which AMSH exerts its action is by inhibiting the binding of Smad6 to activated type I receptors or activated R-Smads.

  • 257.
    Itoh, Fumiko
    et al.
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    Divecha, Nullin
    Brocks, Lenny
    Oomen, Lauran
    Janssen, Hans
    Calafat, Jero
    Itoh, Susumu
    ten Dijke, Peter
    The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-β/Smad signaling2002In: Genes to Cells, Vol. 7, p. 321-331Article in journal (Refereed)
  • 258.
    Itoh, Fumiko
    et al.
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    Itoh, Susumu
    Goumans, Marie-José
    Valdimarsdottir, Gudrun
    Iso, Tatsuya
    Dotto, G. Paolo
    Hamamori, Yasuo
    Kedes, Larry
    ten Dijke, Peter
    The Activation of Notch Antagonizes BMP Receptor-induced Endothelial Cell Migration via Synergistic Induction of Herp2 ExpressionArticle in journal (Refereed)
  • 259.
    Itoh, Susumu
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Landström, Maréne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Hermansson, Annika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Itoh, Fumiko
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Nils-Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    ten Dijke, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Transforming Growth Factor β1 Induces Nuclear Export of Inhibitory Smad71998In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, ., Vol. 273, no 44, p. 29195-29201Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor beta (TGF-beta) signals from membrane to nucleus through serine/threonine kinase receptors and their downstream effector molecules, termed Smad proteins. Recently, Smad6 and Smad7 were identified, which antagonize TGF-beta family signaling by preventing the activation of signal-transducing Smad complexes. Here we report that Smad7, but not Smad6, inhibits TGF-beta1-induced growth inhibition and the expression of immediate early response genes, including Smad7. Interestingly, in the absence of ligand, Smad7 was found to be predominantly localized in the nucleus, whereas Smad7 accumulated in the cytoplasm upon TGF-beta receptor activation. The latter is in accordance with the physical association of Smad7 with the ligand-activated TGF-beta receptor complex in the cell membrane. Whereas the ectopically expressed C-terminal domain of Smad7 was also exported from the nucleus to the cytoplasm upon TGF-beta challenge, a Smad7 mutant with a small deletion at the C terminus or only the N-terminal domain of Smad7 was localized mainly in the cytoplasm in the absence or presence of ligand. This suggests that an intact Mad homology 2 domain is important for nuclear localization of Smad7. The nuclear localization of Smad7 suggests a functional role distinct from its antagonistic effect in receptor-mediated Smad activation.

  • 260. Itoh, Susumu
    et al.
    Thorikay, Midory
    Kowanetz, Marcin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Itoh, Fumiko
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    ten Dijke, Peter
    Elucidation of Smad requirement in transforming growth factor-beta type I receptor-induced responses2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 6, p. 3751-3761Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor-beta (TGF-beta) elicits cellular effects by activating specific Smad proteins that control the transcription of target genes. Whereas there is growing evidence that there are TGF-beta type I receptor-initiated intracellular pathways that are distinct from the pivotal Smad pathway, their physiological importance in TGF-beta signaling is not well understood. Therefore, we generated TGF-beta type I receptors (also termed ALK5s) with mutations in the L45 loop of the kinase domain, termed ALK5(D266A) and ALK5(3A). These mutants showed retained kinase activity but were unable to activate Smads. Characterization of their signaling properties revealed that the two L45 loop mutants did not mediate Smad-dependent transcriptional responses, TGF-beta-induced growth inhibition, and fibronectin and plasminogen activator-1 production in R4-2 mink lung epithelial cells lacking functional ALK5 protein. Mutation in the L45 loop region did not affect the binding of inhibitory Smads but did abrogate the weak binding of X-linked inhibitor of apoptosis protein and Disabled-2 to ALK5. This suggests that the L45 loop in the kinase domain is important for docking of other binding proteins. Interestingly, JNK MAP kinase activity was found to be activated by the ALK5(3A) mutant in various cell types. In addition, TGF-beta-induced inhibition of cyclin D1 expression and stimulation of PMEPA1 (androgen-regulated prostatic mRNA) expression were found to occur, albeit weakly, in an Smad-independent manner in normal murine mammary gland cells. However, the TGF-beta-induced epithelial to mesenchymal transdifferentiation was found to require an intact L45 loop and is likely to be dependent on the Smad pathways.

  • 261.
    Ivarsson, Ylva
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Norrgård, Malena A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications2007In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1770, no 9, p. 1374-1381Article in journal (Refereed)
    Abstract [en]

    Based on the crystal structure of human glutathione transferase M1-1, cysteine residues were introduced in the substrate-binding site of a Cys-free mutant of the enzyme, which were subsequently alkylated with 1-iodoalkanes. By different combinations of site-specific mutations and chemical modifications of the enzyme the enantioselectivity in the conjugation of glutathione with the epoxide-containing substrates 1-phenylpropylene oxide and styrene-7,8-oxide were enhanced up to 9- and 10-fold. The results also demonstrate that the enantioselectivity can be diminished, or even reversed, by suitable modifications, which can be valuable under some conditions. The redesign of the active-site structure for enhanced or diminished enantioselectivities have divergent requirements for different epoxides, calling for a combinatorial approach involving alternative mutations and chemical modifications to optimize the enantioselectivity for a targeted substrate. This approach outlines a general method of great potential for fine-tuning substrate specificity and tailoring stereoselectivity of recombinant enzymes.

  • 262.
    Iwahana, Hiroyuki
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Yakymovych, Ihor
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Dubrovska, Anna
    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.
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Glycoproteome profiling of transforming growth factor-beta (TGF beta) signaling: Nonglycosylated cell death-inducing DFF-like effector A inhibits TGF beta 1-dependent apoptosis2006In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 6, no 23, p. 6168-6180Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor-beta (TGFbeta) is a potent regulator of cell growth, differentiation, and apoptosis. TGFbeta binds to specific serine/threonine kinase receptors, which leads to activation of Smad-dependent and Smad-independent signaling pathways. O-Glycosylation is a dynamic PTM which has been observed in many regulatory proteins, but has not been studied in the context of TGFbeta signaling. To explore the effect of TGFbeta1 on protein O-glycosylation in human breast epithelial cells, we performed analyses of proteins which were affinity purified with Helix pomatia agglutinin (HPA). HPA lectin allowed enrichment of proteins containing GalNAc and GlcNAc linked to serine and threonine residues. Using 2-DE and MALDI-TOF-MS, we identified 21 HPA-precipitated proteins, which were affected by treatment of cells with TGFbeta1. Among these proteins, regulators of cell survival, apoptosis, trafficking, and RNA processing were identified. We found that TGFbeta1 inhibited the appearance of cell death-inducing DFF-like effector A (CIDE-A) in 2-D gels with HPA-precipitated proteins. CIDE-A is a cell death activator which promotes DNA fragmentation. We observed that TGFbeta1 did not affect expression of CIDE-A, but inhibited its glycosylation. We found that deglycosylation of CIDE-A correlated with enhanced nuclear export of the protein, and that high level of nonglycosylated CIDE-A inhibited TGFbeta1-dependent cell death. Thus, inhibition of the glycosylation of CIDE-A may be a mechanism to protect cells from apoptosis.

  • 263. Jaffe, Aron B
    et al.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hall, Alan
    Human CNK1 acts as a scaffold protein, linking Rho and Ras signal transduction pathways2004In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 24, no 4, p. 1736-1746Article in journal (Refereed)
    Abstract [en]

    Rho family GTPases act as molecular switches to control a variety of cellular responses, including cytoskeletal rearrangements, changes in gene expression, and cell transformation. In the active, GTP-bound state, Rho interacts with an ever-growing number of effector molecules, which promote distinct biochemical pathways. Here, we describe the isolation of hCNK1, the human homologue of Drosophila connector enhancer of ksr, as an effector for Rho. hCNK1 contains several protein-protein interaction domains, and Rho interacts with one of these, the PH domain, in a GTP-dependent manner. A mutant hCNK1, which is unable to bind to Rho, or depletion of endogenous hCNK1 by using RNA interference inhibits Rho-induced gene expression via serum response factor but has no apparent effect on Rho-induced stress fiber formation, suggesting that it acts as a specific effector for transcriptional, but not cytoskeletal, activation pathways. Finally, hCNK1 associates with Rhophilin and RalGDS, Rho and Ras effector molecules, respectively, suggesting that it acts as a scaffold protein to mediate cross talk between the two pathways.

  • 264. Jandt, Enrico
    et al.
    Denner, Karsten
    Kovalenko, Marina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Östman, Arne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Böhmer, Frank-D
    The protein-tyrosine phosphatase DEP-1 modulates growth factor-stimulated cell migration and cell-matrix adhesion2003In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 22, no 27, p. 4175-4185Article in journal (Refereed)
    Abstract [en]

    Density-enhanced protein-tyrosine phosphatase-1 (DEP-1 also CD148) is a transmembrane molecule with a single intracellular PTP domain. It has recently been proposed to function as a tumor suppressor. We have previously shown that DEP-1 dephosphorylates the activated platelet-derived growth factor (PDGF) beta-receptor in a site-selective manner (Kovalenko et al. (2000). J. Biol. Chem. 275, 16219-16226). We analysed cell lines with inducible DEP-1 expression for cellular functions of DEP-1. Several aspects of PDGFbeta-receptor signaling were negatively affected by DEP-1 expression. These include PDGF-stimulated activation of inositol trisphosphate formation, Erk1/2, p21Ras, and Src. Activation of receptor-associated phosphoinositide-3 kinase activity and of Akt/PKB were weakly attenuated at early time points of stimulation. Inhibition of PDGF-stimulated signaling depended on DEP-1 catalytic activity. Importantly, DEP-1 inhibited PDGF-stimulated cell migration. The catalytically inactive DEP-1 C1239S variant enhanced cell migration and PDGF-stimulated Erk1/2 activation, suggesting a dominant negative interference with endogenous DEP-1. In contrast to cell migration, cell-substrate adhesion was promoted by active DEP-1 and delayed or suppressed by DEP-1 C1239S, correlating with positive effects of DEP-1 on adhesion-stimulated Src kinase. We propose that negative regulation of growth-factor stimulated cell migration and promotion of cell-matrix adhesion may be related to the function of DEP-1 as tumor suppressor.

  • 265.
    Jerlström-Hultqvist, Jon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Stadelmann, Britta
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Birkestedt, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome2012In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 11, no 7, p. 864-873Article in journal (Refereed)
    Abstract [en]

    In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide-glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG-tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes.

  • 266. Jia, Min
    et al.
    Souchelnytskyi, Nazariy
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    O'Hare, Michael
    Jat, Parmjit S.
    Souchelnytskyi, Serhiy
    Proteome profiling of immortalization-to-senescence transition of human breast epithelial cells identified MAP2K3 as a senescence-promoting protein which is downregulated in human breast cancer2010In: PROTEOMICS - Clinical Applications, ISSN 1862-8346, Vol. 4, no 10-11, p. 816-828Article in journal (Refereed)
    Abstract [en]

    Purpose: Immortalization is one of the first changes in cells undergoing carcinogenic transformation. Proteome profiling of the immortalization-senescence transition is expected to provide insights into the molecular mechanisms of early tumorigenesis. Experimental design: 2-DE and MALDI-MS were used to identify proteins in primary human breast epithelial cells, relevant to the immortalization-senescence transition. Cell and molecular biology and immunohistochemistry were used to validate involvement of mitogen-activated protein kinase kinase 3 (MAP2K3) in the immortalization-senescence transition. Results: We identified 71 proteins whose expression changed upon induction of senescence. The identified proteins include regulators of cell growth, death, cell assembly and organization. Analysis of the network formed by the identified proteins suggested that the immortalization-to-senescence transition could affect regulators of the cell cycle, protein synthesis, transport, post-translational modifications, DNA recombination and repair, and lipid and amino acid metabolism. We observed that MAP2K3 was downregulated in immortal human breast epithelial cells and that upregulation of MAP2K3 expression promoted cell senescence. Decreased expression of MAP2K3 was observed in human breast infiltrating ductal carcinomas, as compared to non-cancerous human breast tissues. Conclusion and clinical relevance: We described a proteome profile of the immortalization-to-senescence transition for human breast epithelial cells, and identified MAP2K3 as a protein that promotes senescence in these cells.

  • 267.
    Johansson, Ann-Sofi
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    RhoGTPase Signaling in Cell Polarity and Gene Regulation2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    RhoGTPases are proteins working as molecular switches as they bind and hydrolyze GTP. They are in their active conformation when GTP is bound and are then able to interact with their effector proteins, which relay the downstream signaling. When the GTP is hydrolyzed to GDP, the RhoGTPase is inactivated. RhoGTPases have been shown to be activated by a variety of stimuli and they are implicated in regulation of diverse cellular processes, including cell migration, cell cycle progression, establishment of cell polarity and transformation.

    We identified mammalian Par6 as a novel effector protein for the RhoGTPases Cdc42 and Rac1. The Caenorhabditis elegans homologue of Par6 had previously been shown to be essential for cell polarity development in the worm embryo. We found that endogenous Par6 colocalized with the tight junction protein ZO-1 in MDCKII epithelial cells. Par6 also interacted with mammalian Par3, another member of the par (for partitioning defective) gene family, first identified in C.elegans. Endogenous Par3 also localized to tight junctions in epithelial cells. This suggested that Par6 and Par3 are part of a complex regulating cell polarity also in mammalian cells. The interaction between Par6 and activated Cdc42 and Rac1 suggested a role for these RhoGTPases in the regulation of this complex.

    Co-expression of Par6 together with PKCζ, induced a dramatic change in cell morphology. The cells rounded up and long cellular extensions, resembling neurites, were formed. The ability to induce these changes in cell morphology was found to be dependent on the direct interaction between Par6 and PKCζ, as well as on the kinase activity of PKCζ. We observed that cells co-expressing mPar6C and PKCζ contained bundled microtubules and microtubules that hade been acetylated, indicating that the microtubules were stabilized.

    To investigate the roles of RhoGTPases in PDGF-induced gene expression we performed cDNA microarray analyses on AG01518 human foreskin fibroblasts in which we over-expressed the dominant negative forms of Cdc42, Rac1 and RhoA. We found that the expression of 16 genes, out of the 45 up-regulated by PDGF-BB, were inhibited ≥50% in the presence of dominant negative Cdc42, Rac1 or RhoA. 19 other genes were down-regulated by one or two of the dominant RhoGTPases. Our data implied that the expression of many PDGF-BB induced genes can be affected by RhoGTPase signaling.

    In conclusion, the work presented here has increased the knowledge of the involvement of RhoGTPase signaling in establishment of cell polarity and gene regulation.

    List of papers
    1. The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the RhoGTPases Cdc42 and Rac1
    Open this publication in new window or tab >>The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the RhoGTPases Cdc42 and Rac1
    2000 In: Journal of Cell Science, ISSN 0021-9533, Vol. 113, no 18, p. 3267-3275Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-94196 (URN)
    Available from: 2006-04-05 Created: 2006-04-05Bibliographically approved
    2. Ectopic co-expression of mPar6C and PKCζ induce cell rounding and formation of neurite-like extensions
    Open this publication in new window or tab >>Ectopic co-expression of mPar6C and PKCζ induce cell rounding and formation of neurite-like extensions
    Show others...
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-94197 (URN)
    Available from: 2006-04-05 Created: 2006-04-05 Last updated: 2010-01-13Bibliographically approved
    3.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
  • 268.
    Johansson, Ann-Sofi
    et al.
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    Driessens, Mariette
    Aspenström, Pontus
    The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the RhoGTPases Cdc42 and Rac12000In: Journal of Cell Science, ISSN 0021-9533, Vol. 113, no 18, p. 3267-3275Article in journal (Refereed)
  • 269. Jones, Iwan
    et al.
    Lindberg, Christina
    Jakobsson, Staffan
    Hellqvist, Anna
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Borg, Bertil
    Olsson, Per-Erik
    Molecular Cloning and Characterization of Spiggin: An androgen-regulated extraorganismal adhesive with structural similarities to von Willebrand Factor-related proteins2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 21, p. 17857-17863Article in journal (Refereed)
    Abstract [en]

    One of the most definitive examples of a vertebrate extraorganismal structural protein can be found in three-spined sticklebacks (Gasterosteus aculeatus). In the breeding male the kidney hypertrophies and synthesizes an adhesive protein called "spiggin," which is secreted into the urinary bladder from where it is employed as a structural thread for nest building. This paper describes the first molecular characterization of spiggin and demonstrates that this adhesive is a protein complex assembled from a potential of three distinct subunits (alpha, beta, and gamma). These subunits arise by alternative splicing, and 11-ketoandrogens induce their expression in stickleback kidneys. Analysis of the predicted amino acid sequence of each subunit reveals a modular organization whose structural elements display a similarity to the multimerization domains found within von Willebrand Factor-related proteins. These results implicate that spiggin utilizes a conserved multimerization mechanism for the formation of a viscous agglutinate from its constituent subunits in the urinary bladders of male sticklebacks. This novel extraorganismal structural protein is therefore ideally suited to its function as an adhesive thread.

  • 270.
    Jurek, Aleksandra
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Amagasaki, Kenichi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Gembarska, Agnieszka
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Lennartsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Negative and positive regulation of MAPK phosphatase 3 controls platelet-derived growth factor-induced Erk activation2009In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 7, p. 4626-4634Article in journal (Refereed)
    Abstract [en]

    MAPK phosphatases (MKPs) are dual specificity phosphatases that dephosphorylate and thereby inactivate MAPKs. In the present study, we provide evidence that platelet-derived growth factor BB (PDGF-BB) regulates MKP3 (DUSP6), which is considered to be a phosphatase highly selective for Erk. Intriguingly, we observed that Mek is positively regulated by MKP3, whereas Erk itself is negatively regulated. In addition, we found that activation of PDGF receptor alpha or beta leads to a rapid proteasomal degradation of MKP3 in a manner that requires Mek activation; this feed-forward mechanism was found to be essential for efficient Erk phosphorylation. We could also demonstrate that PDGF-BB stimulation induces phosphorylation of MKP3 at Ser-174 and Ser-300; phosphorylation of Ser-174 is involved in PDGF-induced MKP3 degradation, since mutation of this site stabilized MKP3. Moreover, activated Erk induces mkp3 expression, leading to restoration of MKP3 levels after 1-2 h and a concomitant dephosphorylation of Erk in cells with activated PDGFRalpha. Reducing the MKP3 level by small interfering RNA leads to an increased Erk activation and mitogenic response to PDGF-BB. In conclusion, MKP3 is an important regulator of PDGF-induced Erk phosphorylation acting in both a rapid positive feed-forward and a later negative feed-back loop.

  • 271.
    Jurek, Aleksandra
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Lennartsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Platelet-derived growth factor-induced signaling pathways interconnect to regulate the temporal pattern of Erk1/2 phosphorylation2011In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 23, no 1, p. 280-287Article in journal (Refereed)
    Abstract [en]

    The biological outcome of Erk1/2 activation is specified by the duration and magnitude of its phosphorylation, as well as its subcellular localization. In the present study, we investigated how the cross-talk between signaling pathways induced by platelet-derived growth factor receptor beta (PDGFR beta) regulates the temporal pattern of Erk1/2 activation. We demonstrated that Src kinase activity was necessary for rapid Erk1/2 phosphorylation in PDGF-BB-stimulated cells. A delay in the onset of Erk1/2 activation was also observed upon phospholipase C (PLC) inhibition; this effect was found to be mediated by protein kinase C (PKC). In addition, we observed that both the PI3K pathway and RasGAP negatively regulated the strength of Erk1/2 phosphorylation. In contrast, interfering with SHP2 binding to PDGFR beta did not affect the pattern of Erk1/2 activation. Interestingly, changes in the kinetics and amplitude of Erk1/2 activation were transmitted to the transcriptional level and affected c-fos expression. In conclusion, cross-talk with other PDGFR beta-induced signaling pathways is important for fine-tuning of the pattern of Erk1/2 activation.

  • 272.
    Jurek, Aleksandra
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Turyna, Bohdan
    Kubit, Piotr
    Klein, Andrzej
    The ability of HDL to inhibit VCAM-1 expression and oxidized LDL uptake is impaired in renal patients2008In: Clinical Biochemistry, ISSN 0009-9120, E-ISSN 1873-2933, Vol. 41, no 12, p. 1015-1018Article in journal (Refereed)
    Abstract [en]

    Objectives: This study examines the ability of HDL from hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients to suppress the expression of adhesion molecules in endothelial cells (ICAM-1, VCAM-1) and in monocytes (LFA-1, VLA-4) and to inhibit the uptake of oxidized LDL by macrophages. Design and methods: Gene expression and the uptake of oxidized LDL were determined in 12 HD patients, 12 CAPD patients and 14 healthy volunteers. Results: HDL from renal patients were less effective than control lipoproteins in reducing VCAM-1 expression. HDL from CAPD patients inhibited LFA-1 expression to the highest extent. The ability of HDL from renal patients to reduce oxidized LDL uptake was lower compared to control group. Conclusions: Decreased ability of HDL to suppress expression of VCAM-1 in endothelial cells and the uptake of oxidized LDL by macrophages can be one of the risk factors for atherosclerosis development in patients with renal failure.

  • 273.
    Kahata, Kaoru
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dadras, Mahsa Shahidi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    TGF-beta Family Signaling in Epithelial Differentiation and Epithelial-Mesenchymal Transition2018In: Cold Spring Harbor Perspectives in Biology, ISSN 1943-0264, E-ISSN 1943-0264, Vol. 10, no 1, article id a022194Article in journal (Refereed)
    Abstract [en]

    Epithelia exist in the animal body since the onset of embryonic development; they generate tissue barriers and specify organs and glands. Through epithelial-mesenchymal transitions (EMTs), epithelia generate mesenchymal cells that form new tissues and promote healing or disease manifestation when epithelial homeostasis is challenged physiologically or pathologically. Transforming growth factor-beta s (TGF-beta s), activins, bone morphogenetic proteins (BMPs), and growth and differentiation factors (GDFs) have been implicated in the regulation of epithelial differentiation. These TGF-beta family ligands are expressed and secreted at sites where the epithelium interacts with the mesenchyme and provide paracrine queues from the mesenchyme to the neighboring epithelium, helping the specification of differentiated epithelial cell types within an organ. TGF-beta ligands signal via Smads and cooperating kinase pathways and control the expression or activities of key transcription factors that promote either epithelial differentiation or mesenchymal transitions. In this review, we discuss evidence that illustrates how TGF-beta family ligands contribute to epithelial differentiation and induce mesenchymal transitions, by focusing on the embryonic ectoderm and tissues that form the external mammalian body lining.

  • 274. Kahata, Kaoru
    et al.
    Hayashi, Makoto
    Asaka, Masahiro
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kitagawa, Hirochika
    Yanagisawa, Jun
    Kato, Shigeaki
    Imamura, Takeshi
    Miyazono, Kohei
    Regulation of transforming growth factor-beta and bone morphogenetic protein signalling by transcriptional coactivator GCN52004In: Genes to Cells, ISSN 1356-9597, E-ISSN 1365-2443, Vol. 9, no 2, p. 143-151Article in journal (Refereed)
    Abstract [en]

    Smad proteins are intracellular signalling mediators of transforming growth factor-beta (TGF-beta) superfamily. In the nucleus, activated Smad complexes regulate transcriptional responses of the target genes in cooperation with transcriptional coactivators and corepressors. To identify new components of transcriptional complexes containing Smad proteins, we purified DNA-binding proteins from human breast cancer MCF-7 cell nuclear extract using a Smad-binding DNA element as bait, and identified a coactivator GCN5 as a direct partner of activated Smad complexes. GCN5 is structurally similar to PCAF, which was previously identified as a coactivator for receptor-regulated Smads (R-Smads) for TGF-beta signalling pathways. GCN5 functions like PCAF, in that it binds to TGF-beta-specific R-Smads, and enhances transcriptional activity induced by TGF-beta. In addition, GCN5, but not PCAF, interacts with R-Smads for bone morphogenetic protein (BMP) signalling pathways, and enhances BMP-induced transcriptional activity, suggesting that GCN5 and PCAF have distinct physiological functions in vivo. Moreover, silencing of the GCN5 gene by RNA interference results in repression of transcriptional activities induced by TGF-beta. In conclusion we identified GCN5 as a Smad-binding transcriptional coactivator which positively regulates both TGF-beta and BMP signalling pathways.

  • 275.
    Kahata, Kaoru
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Maturi, Varun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Moustakas, Aristidis
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    TGF-beta Family Signaling in Ductal Differentiation and Branching Morphogenesis2018In: Cold Spring Harbor Perspectives in Biology, ISSN 1943-0264, E-ISSN 1943-0264, Vol. 10, no 3, article id a031997Article in journal (Refereed)
    Abstract [en]

    Epithelial cells contribute to the development of various vital organs by generating tubular and/or glandular architectures. The fully developed forms of ductal organs depend on processes of branching morphogenesis, whereby frequency, total number, and complexity of the branching tissue define the final architecture in the organ. Some ductal tissues, like the mammary gland during pregnancy and lactation, disintegrate and regenerate through periodic cycles. Differentiation of branched epithelia is driven by antagonistic actions of parallel growth factor systems that mediate epithelial-mesenchymal communication. Transforming growth factor-beta (TGF-beta) family members and their extracellular antagonists are prominently involved in both normal and disease-associated (e.g., malignant or fibrotic) ductal tissue patterning. Here, we discuss collective knowledge that permeates the roles of TGF-beta family members in the control of the ductal tissues in the vertebrate body.

  • 276. Kaivo-Oja, Noora
    et al.
    Bondestam, Jonas
    Kämäräinen, Meerit
    Koskimies, Janne
    Vitt, Ursula
    Cranfield, Mark
    Vuojolainen, Kaisa
    Kallio, Janne P.
    Olkkonen, Vesa M.
    Hayashi, Masaru
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Groome, Nigel P.
    ten Dijke, Peter
    Hsueh, Aaron J. W.
    Ritvos, Olli
    Growth differentiation factor-9 induces Smad2 activation and inhibin B production in cultured human granulosa-luteal cells2003In: Journal of Clinical Endocrinology and Metabolism, ISSN 0021-972X, E-ISSN 1945-7197, Vol. 88, no 2, p. 755-762Article in journal (Refereed)
    Abstract [en]

    The TGF beta family member growth differentiation factor-9 (GDF-9) is an oocyte-derived factor that is essential for mammalian ovarian folliculogenesis. GDF-9 mRNAs have been shown to be expressed in the human ovarian follicle from the primary follicle stage onward, and recombinant GDF-9 has been shown to promote human ovarian follicle growth in vitro. In this study with primary cultures of human granulosa-luteal (hGL) cells, we investigated whether recombinant GDF-9 activates components of the Smad signaling pathways known to be differentially activated by TGF beta and the bone morphogenetic proteins (BMPs). As with TGF beta, GDF-9 treatment caused the phosphorylation of endogenous 53-kDa proteins detected in Western blots with antiphospho-Smad2 antibodies (alpha PS2). However, unlike BMP-2, GDF-9 did not activate the phosphorylation of antiphospho-Smad1 antibody (alphaPS1)-immunoreactive proteins in hGL cells. Infection of hGL cells with an adenovirus expressing Smad2 (Ad-Smad2) confirmed that GDF-9 activates specifically phosphorylation of the Smad2 protein. Infection of hGL cells with Ad-Smad7, which expresses the inhibitory Smad7 protein, suppressed the levels of both GDF-9-induced endogenous and adenoviral alpha PS2-reactive proteins. Furthermore, GDF-9 increased the steady state levels of inhibin beta(B)-subunit mRNAs in hGL cells and strongly stimulated the secretion of dimeric inhibin B. Again, Ad-Smad7 blocked GDF-9-stimulated inhibin B production in a concentration-dependent manner. We identify here for the first time distinct molecular components of the GDF-9 signaling pathway in the human ovary. Our data suggest that GDF-9 mediates its effect through the pathway commonly activated by TGF beta and activin, but not that activated by many BMPs. The results are also consistent with the suggestion that in addition to endocrine control of inhibin production by gonadotropins, a local paracrine control of inhibin production is likely to occur via oocyte-derived factors in the human ovary.

  • 277.
    Kallin, Anders
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    Modulation of PDGF Receptor Signaling via the Phosphatase SHP-2 and the Docking Protein Gab12003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Platelet-derived growth factors (PDGF), a family of potent mitogens and chemoattractants for cells of mesenchymal origin, elicit their biological effects through the binding of two related receptor tyrosine kinases, denoted α- and β-receptors. The binding of PDGF to the receptors causes receptor dimerization and autophosphorylation on tyrosine residues. Src homology 2 (SH2) domain-containing proteins then bind the phosphorylated receptors, mediating further propagation of the signal. This thesis describes how the interaction between the PDGF receptors and some of their downstream targets can modify the cellular response to PDGF.

    The tyrosine phosphatase SHP-2 has been implicated in activation of the Ras/MAPK pathway downstream of several receptor tyrosine kinases. We found that SHP-2 binds to phosphorylated Y763 in the PDGF β-receptor, in addition to the already reported binding to Y1009. Cells expressing PDGF β-receptors with Y763 and Y1009 mutated to phenylalanine exhibited decreased Ras-GTP loading and reduced activation of Erk2 in response to PDGF. Whereas these cells did not show any change in the mitogenic response to PDGF, the PDGF-induced chemotaxis was significantly reduced in cells expressing mutant compared to wild-type receptor.

    The phosphorylation of Y771 of the PDGF β-receptor had been shown to be significantly lower in the αβ-heterodimeric receptor compared to in the ββ-homodimer, causing reduced binding of RasGAP to the heterodimer and increased Ras/MAPK activation. We could demonstrate that the reduced phosphorylation of Y771 is due to dephosphorylation by tyrosine phosphatases, including SHP-2.

    SHP-2 had been shown to associate with the docking protein Gab1 after growth factor stimulation. We showed that the adaptor protein Grb2 was required for PDGF mediated phosphorylation of Gab1, and that phosphorylated Gab1, Grb2 and SHP-2 create a complex upon PDGF stimulation. Using a cell system with an inducible Gab1 expression, we further demonstrated that Gab1 increased SHP-2 activity in response to PDGF, without affecting the interaction between SHP-2 and the b-receptor. Induction of Gab1 correlated with an increase in both PDGF-induced Erk and p38 MAPK activation, whereas Akt activation was unaffected. The latter finding was in line with our observation that PDGF had no effect on the interaction between Gab1 and p85 of PI3’-kinase. The increase in MAPK activity after Gab1 induction and PDGF treatment did not correlate with an increase in PDGF-induced mitogenicity; instead these cells displayed more pronounced actin reorganization in response to PDGF.

    In conclusion, our data indicate that SHP-2 regulates the PDGF response both through direct dephosphorylation of the receptor and through its interaction with Gab1. PDGF stimulated activation of SHP-2 seems to be correlated not only with mitogenesis, but also with reorganization of the actin cytoskeleton and cell migration.

    List of papers
    1. SHP-2 binds to Tyr763 and Tyr1009 in the PDGF β-receptor and mediates PDGF-induced activation of the Ras/MAP kinase pathway and chemotaxis
    Open this publication in new window or tab >>SHP-2 binds to Tyr763 and Tyr1009 in the PDGF β-receptor and mediates PDGF-induced activation of the Ras/MAP kinase pathway and chemotaxis
    Show others...
    1999 (English)In: Oncogene, ISSN 0950-9232, Vol. 18, no 25, p. 3696-3702Article in journal (Refereed) Published
    Abstract [en]

    Activation of the beta-receptor for platelet-derived growth factor (PDGF) by its ligand leads to autophosphorylation on a number of tyrosine residues. Here we show that Tyr763 in the kinase insert region is a novel autophosphorylation site, which after phosphorylation binds the protein tyrosine phosphatase SHP-2. SHP-2 has also previously been shown to bind to phosphorylated Tyr1009 in the PDGF beta-receptor. Porcine aortic endothelial (PAE) cells transfected with a PDGF beta-receptor in which Tyr763 and Tyr1009 were mutated to phenylalanine residues failed to associate with SHP-2 after ligand stimulation. Moreover, PDGF-BB-induced Ras GTP-loading and Erk2 activation were severely compromised in the receptor mutant. Whereas the mitogenic response to PDGF-BB remained at the same level as in cells expressing wild-type PDGF beta-receptor, chemotaxis induced by PDGF-BB was significantly decreased in the case of the Y763F/Y1009F mutant cells, suggesting an important role for SHP-2 in chemotactic signaling.

    Place, publisher, year, edition, pages
    Stockton Press, 1999
    Keywords
    PDGF beta-receptor; SHP-2; chemotaxis; Ras/MAP kinase pathway
    Identifiers
    urn:nbn:se:uu:diva-91007 (URN)10391677 (PubMedID)
    Available from: 2003-10-23 Created: 2003-10-23 Last updated: 2009-09-23Bibliographically approved
    2. SHP-2 is involved in heterodimer specific loss of phosphorylation of Tyr771 in the PDGF β-receptor
    Open this publication in new window or tab >>SHP-2 is involved in heterodimer specific loss of phosphorylation of Tyr771 in the PDGF β-receptor
    Show others...
    2002 (English)In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 21, no 12, p. 1870-1875Article in journal (Refereed) Published
    Abstract [en]

    We have previously shown that the binding site for GTPase activating protein of Ras (RasGAP) in the PDGF beta-receptor, Tyr771, is phosphorylated to a much lower extent in the heterodimeric configuration of PDGF alpha- and beta-receptors, compared to the PDGF beta-receptor homodimer. The decreased recruitment of the RasGAP to the receptor leads to prolonged activation of the Ras/MAP kinase pathway, which could explain the increase in mitogenicity seen upon induction of heterodimers. The molecular mechanism underlying these differences was investigated. We could show that the loss of phosphorylation of Tyr771 was dependent on presence of intact binding sites for the protein tyrosine phosphatase SHP-2 on the PDGF beta-receptor. Thus, in PDGF receptor mutants in which binding of SHP-2 was lost, a higher degree of phosphorylation of Tyr771 was seen, while other phosphorylation sites in the receptor remained virtually unaffected. Thus, SHP-2 appears to play an important role in modulating phosphorylation of Y771, thereby controlling RasGAP recruitment and Ras/MAP kinase signaling in the heterodimeric configuration of the PDGF receptors.

    Place, publisher, year, edition, pages
    Nature Publishing Group, 2002
    Keywords
    Platelet-derived growth factor; receptor tyrosine kinase; SHP-2; heterodimer; dephosphorylation
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-91008 (URN)10.1038/sj/ onc/1205210 (DOI)11896619 (PubMedID)
    Available from: 2003-10-23 Created: 2003-10-23 Last updated: 2017-12-14Bibliographically approved
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  • 278.
    Kallin, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Demoulin, Jean-Baptiste
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Nishida, Keigo
    Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Kanagawa 230-0045, Japan.
    Hirano, Toshio
    Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Kanagawa 230-0045, Japan.
    Rönnstrand, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Gab1 contributes to cytoskeletal reorganization and chemotaxis in response to platelet-derived growth factor2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 17, p. 17897-17904Article in journal (Refereed)
    Abstract [en]

    Gab1 is a scaffolding/docking protein that has been suggested to play a role in signal transduction downstream of certain plasma membrane receptors, including platelet-derived growth factor (PDGF) receptors. We found that PDGF induced a rapid Gab1 phosphorylation, which depended on the recruitment of Grb2, indicating that Grb2 acts as a bridge between Gab1 and the PDGF beta-receptor. PDGF also enhanced the binding of Gab1 to the phosphatase SHP-2, but not to p85. To further study the role of Gab1 in PDGF signaling, we transfected porcine aortic endothelial cells with a doxycycline-inducible Gab1 construct. Increased Gab1 expression enhanced the recruitment and activation of SHP-2, as well as the phosphorylation of the mitogen-activated protein kinases Erk and p38 by PDGF. Gab1 expression also enhanced the formation of lamellipodia and cellular protrusions. In Gab1-deficient mouse embryonic fibroblasts, the same phenotype was induced by restoring the expression of wild-type Gab1, but not a mutant Gab1 that was unable to associate with SHP-2. These effects of PDGF on the actin cytoskeleton were not altered by the inhibition of p38 or Erk, but could be blocked by a dominant-negative form of Rac (Asn(17)). Finally, Gab1-deficient fibroblasts showed a decreased chemotactic response toward gradients of PDGF as compared with wild-type cells. In conclusion, Gab1 plays a selective role in the regulation of the mitogen-activated protein kinases Erk and p38 downstream of the PDGF beta-receptor, and contributes to cytoskeletal reorganization and chemotaxis in response to PDGF.

  • 279. Kallin, Anders
    et al.
    Johannessen, Lene E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Cani, Patrice D.
    Marbehant, Catherine Y.
    Essaghir, Ahmed
    Foufelle, Fabienne
    Ferré, Pascal
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Delzenne, Nathalie M.
    Demoulin, Jean-Baptiste
    SREBP-1 regulates the expression of heme oxygenase 1 and the phosphatidylinositol-3 kinase regulatory subunit p55 gamma2007In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 48, no 7, p. 1628-1636Article in journal (Refereed)
    Abstract [en]

    Sterol-regulatory element binding proteins (SREBPs) control the expression of genes involved in fatty acid and cholesterol biosynthesis. Using microarrays, we observed that mature SREBP-1 also induced the expression of genes unrelated to lipid metabolism, such as heme oxygenase 1 (HMOX1), plasma glutathione peroxidase, the phosphatidylinositol-3 kinase regulatory subunit p55 gamma, synaptic vesicle glycoprotein 2A, and COTE1. The expression of these genes was repressed upon addition of sterols, which block endogenous SREBP cleavage, and was induced by the statin drug mevinolin. Stimulation of fibroblasts with platelet-derived growth factor, which activates SREBP-1, had a similar effect. Fasted mice that were refed with a high-carbohydrate diet presented an increased expression of HMOX1 and p55 gamma in the liver. Overall, the transcriptional signature of SREBP-1 in fibroblasts stimulated by growth factors was very similar to that described in liver cells. We analyzed the HMOX1 promoter and found one SREBP binding site of the E-box type, which was required for regulation by SREBP-1a and SREBP-1c but was insensitive to SREBP-2. In conclusion, our data suggest that SREBP-1 regulates the expression of stress response and signaling genes, which could contribute to the metabolic response to insulin and growth factors in various tissues.

  • 280.
    Kanamoto, Takashi
    et al.
    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.
    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.
    Functional proteomics of transforming growth factor-beta1-stimulated Mv1Lu epithelial cells: Rad51 as a target of TGFbeta1-dependent regulation of DNA repair2002In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 21, no 5, p. 1219-1230Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor-beta (TGFbeta) conveys regulatory signals through multiple intracellular pathways, subsequently affecting various cellular functions. To identify new targets for TGFbeta, we studied the changes in the proteome of Mv1Lu lung epithelial cells in response to TGFbeta1 treatment. Thirty-eight non-abundant protein spots, affected by TGFbeta1, were selected, and proteins were identified by peptide mass-fingerprinting (PMF). Among them, proteins involved in regulation of immune response, apoptosis, regulation of TGFbeta signalling, metabolism and DNA repair were identified. Twenty-eight of the 38 proteins are new targets for TGFbeta1, thus suggesting novel ways of integration of TGFbeta signalling in intracellular regulatory processes. We show that TGFbeta1-dependent decrease in expression of one of the new targets, Rad51, correlates with a decrease in DNA repair efficiency. This was evaluated by formation of nuclear Rad51-containing DNA repair complexes in response to DNA damage, by single cell gel electrophoresis and by cell survival assay. The TGFbeta1-dependent inhibition of DNA repair was reversed by ectopic overexpression of Rad51. Therefore, TGFbeta can promote DNA instability through down-regulation of Rad51 and inhibition of DNA repair.

  • 281. Kappert, Kai
    et al.
    Leppänen, Olli
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Vascular Surgery.
    Paulsson, Janna
    Furuhashi, Masao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Carlsson, Mari-Anne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Vascular Surgery.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Fätkenheuer, Gerd
    Rosenkranz, Stephan
    Östman, Arne
    Highly Active Antiretroviral Therapy Attenuates Re-Endothelialization and Alters Neointima Formation in the Rat Carotid Artery After Balloon Injury2006In: Journal of Acquired Immune Deficiency Syndromes, ISSN 1525-4135, E-ISSN 1944-7884, Vol. 43, no 4, p. 383-392Article in journal (Refereed)
    Abstract [en]

    Highly active antiretroviral therapy (HAART) has led to a sustained decline of HIV-associated morbidity and mortality. HAART exhibits significant side effects, however, such as hyperlipidemia and hyperglycemia, which possibly contribute to accelerated atherosclerosis in HAART-treated patients. In addition, direct effects of HAART on vascular cells have been described, which may promote atherosclerotic lesion formation. The effects of HAART on balloon-induced neointima formation have not been studied previously. The rat carotid artery balloon model was used to evaluate the effects of HAART (lopinavir, ritonavir, lamivudine, and zidovudine) on neointima formation and endothelial recovery. Furthermore, the effects of concomitant administration of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor rosuvastatin were investigated. HAART-treated animals displayed an increase in lesion size (neointima/media ratio: 1.14 +/- 0.32 vs. 1.31 +/- 0.20 in control vs. HAART; P < 0.05) and an impaired regenerative capacity of the endothelium, as indicated by reduction in endothelial regrowth from an adjacent undilated vessel segment 14 days after injury (re-endothelialization area: 8.29 +/- 1.45 mm vs. 5.09 +/- 0.53 mm in control vs. HAART; P < 0.05). When rosuvastatin was given in addition to HAART, these effects were not observed. In conclusion, HAART inhibited endothelial cell-mediated healing and promoted neointima formation after angioplasty in rats. These deleterious effects were attenuated by cotreatment with rosuvastatin, however. Our studies suggest that currently used drug regimens against HIV infection may lead to an increased risk for restenosis after percutaneous vascular interventions. Moreover, the findings indicate that the additional treatment with statins might counteract these adverse effects by HAART.

  • 282. Kappert, Kai
    et al.
    Paulsson, Janna
    Sparwel, Jan
    Leppänen, Olli
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Vascular Surgery.
    Hellberg, Carina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Östman, Arne
    Micke, Patrick
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Dynamic changes in the expression of DEP-1 and other PDGF receptor-antagonizing PTPs during onset and termination of neointima formation2007In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 21, no 2, p. 523-534Article in journal (Refereed)
    Abstract [en]

    Growth factor-dependent tissue remodeling, such as restenosis, is believed to be predominantly regulated by changes in expression of receptor-tyrosine-kinases (RTKs) and their ligands. As endogenous antagonists of RTKs, protein-tyrosine-phosphatases (PTPs) are additional candidate regulators of these processes. Using laser-capture-microdissection and quantitative RT-polymerase chain reaction (qRT-PCR), we investigated the layer-specific expression of the four platelet-derived growth factor (PDGF) isoforms, the PDGF-alpha and beta receptors, and five PTPs implied in control of PDGF-receptor signaling 8 and 14 days after balloon injury of the rat carotid. Results were correlated with analyses of PDGF-beta receptor phosphorylation and vascular smooth muscle cell (VSMC) proliferation in vivo. The expression levels of all components, as well as receptor activation and VSMC proliferation, showed specific changes, which varied between media and neointima. Interestingly, PTP expression--particularly, DEP-1 levels--appeared to be the dominating factor determining receptor-phosphorylation and VSMC proliferation. In support of these findings, cultured DEP-1(-/-) cells displayed in