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Heldin, Johan
Publications (10 of 13) Show all publications
Heldin, J., Rubin Sander, M., Leino, M., Thomsson, S., Lennartsson, J. & Söderberg, O. (2019). Dynamin inhibitors impair platelet-derived growth factor beta-receptor dimerization and signaling. Experimental Cell Research, 380(1), 69-79
Open this publication in new window or tab >>Dynamin inhibitors impair platelet-derived growth factor beta-receptor dimerization and signaling
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2019 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 380, no 1, p. 69-79Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
ELSEVIER INC, 2019
Keywords
RTK signaling, EGFR, PDGFR-beta, Dynasore, Dyngo, Dimerization
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-384986 (URN)10.1016/j.yexcr.2019.04.004 (DOI)000468124700008 ()30970237 (PubMedID)
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilSwedish Cancer Society, CAN 2018/425
Available from: 2019-06-14 Created: 2019-06-14 Last updated: 2019-06-14Bibliographically approved
de Oliveira, F. M., Mereiter, S., Lönn, P., Siart, B., Shen, Q., Heldin, J., . . . Kamali-Moghaddam, M. (2018). Detection of post-translational modifications using solid-phase proximity ligation assay. New Biotechnology, 45, 51-59
Open this publication in new window or tab >>Detection of post-translational modifications using solid-phase proximity ligation assay
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2018 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 45, p. 51-59Article in journal (Refereed) Published
Abstract [en]

Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs. The assay depends on the recognition of the target protein molecule and its modification by three affinity binders. Using antibodies and lectins, we applied the method for detection of glycosylated CD44 and E-Cadherin, and phosphorylated p53 and EGFR. The assay was found to have superior dynamic range and limit of detection compared to standard ELISAs. In summary, we have established the use of SP-PLA as an appropriate method for sensitive detection of PTMs in lysates and sera, which may provide a basis for future PTM-based diagnostic and prognostic biomarkers

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-334520 (URN)10.1016/j.nbt.2017.10.005 (DOI)000441913800008 ()29101055 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 316929
Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2018-10-05Bibliographically approved
Klaesson, A., Grannas, K., Ebai, T., Heldin, J., Koos, B., Leino, M., . . . Landegren, U. (2018). Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes. Scientific Reports, 8, Article ID 5400.
Open this publication in new window or tab >>Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 5400Article in journal (Refereed) Published
Abstract [en]

We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes -UnFold probes - where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic "unfolding" step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-340077 (URN)10.1038/s41598-018-23582-1 (DOI)000428618900043 ()29599435 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568 264737 294409Swedish Foundation for Strategic Research Swedish Research Council
Note

Ola Söderberg and Ulf Landegren jointly supervised this work

Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2018-08-06Bibliographically approved
Heldin, J., O'Callaghan, P., Hernández Vera, R., Fredlund Fuchs, P., Gerwins, P. & Kreuger, J. (2017). FGD5 sustains vascular endothelial growth factor A (VEGFA) signaling through inhibition of proteasome-mediated VEGF receptor 2 degradation. Cellular Signalling, 40, 125-132
Open this publication in new window or tab >>FGD5 sustains vascular endothelial growth factor A (VEGFA) signaling through inhibition of proteasome-mediated VEGF receptor 2 degradation
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2017 (English)In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 40, p. 125-132Article in journal (Refereed) Published
Abstract [en]

The complete repertoire of endothelial functions elicited by FGD5, a guanine nucleotide exchange factor activating the Rho GTPase Cdc42, has yet to be elucidated. Here we explore FGD5's importance during vascular endothelial growth factor A (VEGFA) signaling via VEGF receptor 2 (VEGFR2) in human endothelial cells. In microvascular endothelial cells, FGD5 is located at the inner surface of the cell membrane as well as at the outer surface of EEAl-positive endosomes carrying VEGFR2. The latter finding prompted us to explore if FGD5 regulates VEGFR2 dynamics. We found that depletion of FGD5 in microvascular cells inhibited their migration towards a stable VEGFA gradient. Furthermore, depletion of FGD5 resulted in accelerated VEGFR2 degradation, which was reverted by lactacystin-mediated proteasomal inhibition. Our results thus suggest a mechanism whereby FGD5 sustains VEGFA signaling and endothelial cell chemotaxis via inhibition of proteasome-dependent VEGFR2 degradation.

Keywords
Angiogenesis, Cdc42, FGD5, Vascular biology, VEGFR2, Degradation
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-341984 (URN)10.1016/j.cellsig.2017.09.009 (DOI)000414620900013 ()28927665 (PubMedID)
Available from: 2018-02-19 Created: 2018-02-19 Last updated: 2018-02-19Bibliographically approved
Blom, M., Reis, K., Heldin, J., Kreuger, J. & Aspenström, P. (2017). The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration. Experimental Cell Research, 352(2), 255-264
Open this publication in new window or tab >>The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration
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2017 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 352, no 2, p. 255-264Article in journal (Refereed) Published
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.

Keywords
RhoD, Rho GTPase, Actin, Stress fiber, Cell migration
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-319045 (URN)10.1016/j.yexcr.2017.02.013 (DOI)000396964600010 ()28196728 (PubMedID)
Funder
The Karolinska Institutet's Research FoundationSwedish Cancer Society, JK-2014/820 PA-2014/0644
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2018-01-13Bibliographically approved
Koos, B., Cane, G., Grannas, K., Löf, L., Arngården, L., Heldin, J., . . . Söderberg, O. (2015). Proximity-dependent initiation of hybridization chain reaction. Nature Communications, 6, Article ID 7294.
Open this publication in new window or tab >>Proximity-dependent initiation of hybridization chain reaction
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2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7294Article in journal (Refereed) Published
Abstract [en]

Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

National Category
Medical and Health Sciences Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-255596 (URN)10.1038/ncomms8294 (DOI)000357171100008 ()26065580 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568, 316929, 259796Swedish Foundation for Strategic Research Swedish Research Council
Available from: 2015-06-17 Created: 2015-06-17 Last updated: 2018-01-25Bibliographically approved
Heldin, J. (2014). Identification and Characterization of Proteins and MicroRNAs that Modulate Receptor Signaling, Vesicular Trafficking and Cell Migration in Vascular Cells. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Identification and Characterization of Proteins and MicroRNAs that Modulate Receptor Signaling, Vesicular Trafficking and Cell Migration in Vascular Cells
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Blood vessels deliver nutrients and oxygen to tissues. Importantly, the functions and growth of blood vessels are commonly altered in disease. The inside of all blood vessels are lined with endothelium, a thin specialized layer of endothelial cells that separate the blood from other tissues. This thesis deals with the identification and functional characterization of proteins and microRNAs that have key roles as modulators of growth factor signaling and directed cell migration of endothelial cells and other vascular cells.

A previously uncharacterized protein of the exocyst complex, Exocyst complex component 3-like 2 (ExoC3L2) was identified and shown to be highly expressed in endothelial cells of sprouting vessels. Suppression of ExoC3L2 resulted in reduced VEGF-A signaling together with reduced chemotaxis in response to VEGF-A gradients. VEGF-A-signaling via its receptor VEGFR-2 is thus modulated by the exocyst complex and ExoC3L2.

Expression profiling of highly vascularized tissues were used to identify several microRNAs selectively expressed in blood vessels. miR-145, targeting the transcription factor Fli1, was shown to be expressed in pericytes and mural cells. Elevated levels of miR-145 reduced chemotaxis of both endothelial cells and fibroblasts in response to growth factor gradients. miR-145 depletion in fibroblasts was shown to inhibit chemotaxis in response to PDGF-BB.

The guanine nucleotide exchange factor FGD5 was shown to be selectively expressed in endothelial cells and to regulate Cdc42 activity. FGD5 was shown to regulate the turnover of activated VEGF-receptors. Suppression of FGD5 impaired endothelial cell chemotaxis, suggesting that FGD5 is required for efficient and sustained VEGF-A signaling.

Inactivation of RhoD, a regulator of endosomal trafficking, resulted in an increased pool of acetylated and stable microtubules. Knockdown of RhoD in human fibroblasts resulted in a loss of cell polarity. A link between PDGFR-β and RhoD was implicated by the finding that PDGF-BB was shown to trigger formation of GTP-bound RhoD. Chemotaxis towards PDGF-BB was severely inhibited in cells with reduced RhoD expression, suggesting a role for RhoD in chemotaxis via its regulation of microtubule dynamics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. p. 61
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 961
Keywords
Angiogenesis, cell migration, signaling, VEGF, FGD5, ExoC3L2, microRNA, RhoD
National Category
Cell and Molecular Biology Other Basic Medicine
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-212949 (URN)978-91-554-8833-8 (ISBN)
Public defence
2014-02-14, A1:111a, Husargatan 3, BMC, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2014-01-23 Created: 2013-12-16 Last updated: 2018-05-16
Sundström, G., Larsson, T. A., Xu, B., Heldin, J. & Larhammar, D. (2013). Interactions of zebrafish peptide YYb with the neuropeptide Y-family receptors Y4, Y7, Y8a, and Y8b. Frontiers in Neuroscience, 7, Article ID 29.
Open this publication in new window or tab >>Interactions of zebrafish peptide YYb with the neuropeptide Y-family receptors Y4, Y7, Y8a, and Y8b
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2013 (English)In: Frontiers in Neuroscience, ISSN 1662-4548, E-ISSN 1662-453X, Vol. 7, article id 29Article in journal (Refereed) Published
Abstract [en]

The neuropeptide Y (NPY) system influences numerous physiological functions including feeding behavior, endocrine regulation, and cardiovascular regulation. In jawed vertebrates it consists of 3-4 peptides and 4-7 receptors. Teleost fishes have unique duplicates of NPY and PYY as well as the Y8 receptor. In the zebrafish, the NPY system consists of the peptides NPYa, PYYa, and PYYb (NPYb appears to have been lost) and at least seven NPY receptors: Y1, Y2, Y2-2, Y4, Y7, Y8a, and Y8b. Previously PYYb binding has been reported for Y2 and Y2-2. To search for peptide-receptor preferences, we have investigated PYYb binding to four of the remaining receptors and compared with NPYa and PYYa. Taken together, the most striking observations are that PYYa displays reduced affinity for Y2 (3 nM) compared to the other peptides and receptors and that all three peptides have higher affinity for Y4 (0.028-0.034 nM) than for the other five receptors. The strongest peptide preference by any receptor selectivity is the one previously reported for PYYb by the Y2 receptor, as compared to NPY and PYYa. These affinity differences may be helpful to elucidate specific details of peptide-receptor interactions. Also, we have investigated the level of mRNA expression in different organs using qPCR. All peptides and receptors have higher expression in heart, kidney, and brain. These quantitative aspects on receptor affinities and mRNA distribution help provide a more complete picture of the NPY system.

Keywords
Evolution, genome duplication, NPY, elephant shark
National Category
Natural Sciences Neurology
Research subject
Neuroscience
Identifiers
urn:nbn:se:uu:diva-205599 (URN)10.3389/fnins.2013.00029 (DOI)000346567300029 ()23508731 (PubMedID)
Funder
Swedish Research Council
Available from: 2013-08-20 Created: 2013-08-20 Last updated: 2019-01-03Bibliographically approved
Sundström, G., Xu, B., Larsson, T. A., Heldin, J., Bergqvist, C. A., Fredriksson, R., . . . Larhammar, D. (2012). Characterization of the neuropeptide Y system in the frog Silurana tropicalis (Pipidae): three peptides and six receptor subtypes. General and Comparative Endocrinology, 177(3), 322-331
Open this publication in new window or tab >>Characterization of the neuropeptide Y system in the frog Silurana tropicalis (Pipidae): three peptides and six receptor subtypes
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2012 (English)In: General and Comparative Endocrinology, ISSN 0016-6480, E-ISSN 1095-6840, Vol. 177, no 3, p. 322-331Article in journal (Refereed) Published
Abstract [en]

Neuropeptide Y and its related peptides PYY and PP (pancreatic polypeptide) are involved in feeding behavior, regulation of the pituitary and the gastrointestinal tract, and numerous other functions. The peptides act on a family of G-protein coupled receptors with 4-7 members in jawed vertebrates. We describe here the NPY system of the Western clawed frog Silurana (Xenopus) tropicalis. Three peptides, NPY, PYY and PP, were identified together with six receptors, namely subtypes Y1, Y2, Y4, Y5, Y7 and Y8. Thus, this frog has all but one of the ancestral seven gnathostome NPY-family receptors, in contrast to mammals which have lost 2-3 of the receptors. Expression levels of mRNA for the peptide and receptor genes were analyzed in a panel of 19 frog tissues using reverse transcriptase quantitative PCR. The peptide mRNAs had broad distribution with highest expression in skin, blood and small intestine. NPY mRNA was present in the three brain regions investigated, but PYY and PP mRNAs were not detectable in any of these. All receptor mRNAs had similar expression profiles with high expression in skin, blood, muscle and heart. Three of the receptors, Y5, Y7 and Y8, could be functionally expressed in HEK-293 cells and characterized with binding studies using the three frog peptides. PYY had the highest affinity for all three receptors (K(i) 0.042-0.34 nM). Also NPY and PP bound to the Y8 receptor with high affinity (0.14 and 0.50 nM). The low affinity of NPY for the Y5 receptor (100-fold lower than PYY) differs from mammals and chicken. This may suggest a less important role of NPY on Y5 in appetite stimulation in the frog compared with amniotes. In conclusion, our characterization of the NPY system in S. tropicalis with its six receptors demonstrates not only greater complexity than in mammals but also some interesting differences in ligand-receptor preferences.

Keywords
NPY, PYY, G-protein-coupled receptor, Silurana tropicalis, evolution
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-129517 (URN)10.1016/j.ygcen.2012.04.027 (DOI)000306390100005 ()22565163 (PubMedID)
Note

Erratum in General and Comparative Endocrinology 2015:215, doi:10.1016/j.ygcen.2014.11.014.

Available from: 2010-08-18 Created: 2010-08-18 Last updated: 2019-01-03Bibliographically approved
Le Jan, S., Hayashi, M., Kasza, Z., Eriksson, I., Bishop, J. R., Weibrecht, I., . . . Kreuger, J. (2012). Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis. Arteriosclerosis, Thrombosis and Vascular Biology, 32(5), 1255-1263
Open this publication in new window or tab >>Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis
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2012 (English)In: Arteriosclerosis, Thrombosis and Vascular Biology, ISSN 1079-5642, E-ISSN 1524-4636, Vol. 32, no 5, p. 1255-1263Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: Heparan sulfate proteoglycans regulate key steps of blood vessel formation. The present study was undertaken to investigate if there is a functional overlap between heparan sulfate proteoglycans and chondroitin sulfate proteoglycans during sprouting angiogenesis.

METHODS AND RESULTS: Using cultures of genetically engineered mouse embryonic stem cells, we show that angiogenic sprouting occurs also in the absence of heparan sulfate biosynthesis. Cells unable to produce heparan sulfate instead increase their production of chondroitin sulfate that binds key angiogenic growth factors such as vascular endothelial growth factor A, TGFβ, and platelet-derived growth factor B. Lack of heparan sulfate proteoglycan production however leads to increased pericyte numbers and reduced adhesion of pericytes to nascent sprouts, likely due to dysregulation of TGFβ and platelet-derived growth factor B signal transduction.

CONCLUSIONS: The present study provides direct evidence for a previously undefined functional overlap between chondroitin sulfate proteoglycans and heparan sulfate proteoglycans during sprouting angiogenesis. Our findings provide information relevant for potential future drug design efforts that involve targeting of proteoglycans in the vasculature.

Keywords
Angiogenesis, proteoglycan, heparan sulfate, chondroitin sulfate, ext1
National Category
Basic Medicine
Research subject
Medicine; Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-169136 (URN)10.1161/ATVBAHA.111.240622 (DOI)000303195100031 ()22345168 (PubMedID)
Funder
Swedish Research Council, K2011-67X-21868-01-3
Available from: 2012-02-23 Created: 2012-02-23 Last updated: 2018-01-12Bibliographically approved
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