| 1. |
- Lönn, Peter, et al.
(author)
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High-throughput in situ mapping of phosphorylated protein complexes across the cell cycle and in response to drugs
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Other publication (other academic/artistic)abstract
- Interactions and posttranslational modifications (PTMs) of proteins orchestrate cellular responses to cytokines, drugs or other agents, but it has been difficult to monitor and characterize these dynamic events at high-throughput. Here, we have established a semi-automated system for large-scale in situ proximity ligation assays (isPLA). The protocol combines isPLA in microtiter wells with automated microscopy and computer-based image analysis whereby specific protein phosphorylations and interactions are digitally recorded in cells, along with measurements of morphological features. We demonstrate how this platform can improve analysis of cellular signaling by investigating TGF-b responsive Smad2 linker phosphorylations and complex formations over time and across millions of individual cells. We depict single cell responses in relation to e.g. local cell crowding and cell cycle progression via measurements of DNA content and nuclear size. Finally, we illustrate the application of the protocol for demonstrating drug effects by screening a library of phosphatase inhibitors. In summary, our approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput.
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| 2. |
- Lönn, Peter, et al.
(author)
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Image-based high-throughput mapping of TGF-beta-induced phosphocomplexes at a single-cell level
- 2021
-
In: Communications Biology. - : Springer Nature. - 2399-3642. ; 4
-
Journal article (peer-reviewed)abstract
- To improve our ability to monitor cellular responses to e.g. cytokines or drugs, Lonn et al have developed a semi-automated system for large-scale in situ proximity ligation assays (isPLA) in HaCAT keratinocyte cells. Their approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput. Protein interactions and posttranslational modifications orchestrate cellular responses to e.g. cytokines and drugs, but it has been difficult to monitor these dynamic events in high-throughput. Here, we describe a semi-automated system for large-scale in situ proximity ligation assays (isPLA), combining isPLA in microtiter wells with automated microscopy and computer-based image analysis. Phosphorylations and interactions are digitally recorded along with subcellular morphological features. We investigated TGF-beta-responsive Smad2 linker phosphorylations and complex formations over time and across millions of individual cells, and we relate these events to cell cycle progression and local cell crowding via measurements of DNA content and nuclear size of individual cells, and of their relative positions. We illustrate the suitability of this protocol to screen for drug effects using phosphatase inhibitors. Our approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput.
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| 3. |
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| 4. |
- Barkefors, Irmeli, et al.
(author)
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Exocyst Complex Component 3-like 2 (EXOC3L2) Associates with the Exocyst Complex and Mediates Directional Migration of Endothelial Cells
- 2011
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:27, s. 24189-24199
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Journal article (peer-reviewed)abstract
- The exocyst is a protein complex that ensures spatial targeting of exocytotic vesicles to the plasma membrane. We present microarray data obtained from differentiating mouse embryonic stem cell cultures that identify an up-regulation of exocyst complex component 3-like 2 (exoc3l2) mRNA in sprouting blood vessels. Vascular expression of exoc3l2 is confirmed by qPCR analysis of different mouse tissues and immunofluorescence analyses of mouse brain sections. We detect an up-regulation of exoc3l2 mRNA synthesis in primary human endothelial cells in response to VEGFA, and this response is enhanced when the cells are grown on a three-dimensional collagen I matrix. Myc-tagged EXOC3L2 co-precipitates with the exocyst protein EXOC4, and immunofluorescence detection of EXOC3L2 shows partial subcellular colocalization with EXOC4 and EXOC7. Finally, we show that exoc3l2 silencing inhibits VEGF receptor 2 phosphorylation and VEGFA-directed migration of cultured endothelial cells.
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| 5. |
- Blom, Magdalena, et al.
(author)
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The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration
- 2017
-
In: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 352:2, s. 255-264
-
Journal article (peer-reviewed)abstract
- 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.
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| 6. |
- Heldin, Johan, et al.
(author)
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Dynamin inhibitors impair platelet-derived growth factor beta-receptor dimerization and signaling
- 2019
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In: Experimental Cell Research. - : ELSEVIER INC. - 0014-4827 .- 1090-2422. ; 380:1, s. 69-79
-
Journal article (peer-reviewed)abstract
- 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.
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| 7. |
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| 8. |
- Heldin, Johan, et al.
(author)
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FGD5 sustains vascular endothelial growth factor A (VEGFA) signaling through inhibition of proteasome-mediated VEGF receptor 2 degradation
- 2017
-
In: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 40, s. 125-132
-
Journal article (peer-reviewed)abstract
- 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.
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| 9. |
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| 10. |
- Heldin, Johan
(author)
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Identification and Characterization of Proteins and MicroRNAs that Modulate Receptor Signaling, Vesicular Trafficking and Cell Migration in Vascular Cells
- 2014
-
Doctoral thesis (other academic/artistic)abstract
- 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.
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| 11. |
- Klaesson, Axel, et al.
(author)
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Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
- 2018
-
In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8
-
Journal article (peer-reviewed)abstract
- 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.
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| 12. |
- Larsson, Erik, 1975, et al.
(author)
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Discovery of microvascular miRNAs using public gene expression data : miR-145 is expressed in pericytes and is a regulator of Fli1
- 2009
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In: Genome Medicine. - : Springer Science and Business Media LLC. - 1756-994X. ; 1:11, s. 108-
-
Journal article (peer-reviewed)abstract
- BACKGROUNDA function for the microRNA (miRNA) pathway in vascular development and angiogenesis has been firmly established. miRNAs with selective expression in the vasculature are attractive as possible targets in miRNA-based therapies. However, little is known about the expression of miRNAs in microvessels in vivo. Here, we identified candidate microvascular-selective miRNAs by screening public miRNA expression datasets.METHODSBioinformatics predictions of microvascular-selective expression were validated with real-time quantitative reverse transcription PCR on purified microvascular fragments from mouse. Pericyte expression was shown with in situ hybridization on tissue sections. Target sites were identified with 3' UTR luciferase assays, and migration was tested in a microfluid chemotaxis chamber.RESULTSmiR-145, miR-126, miR-24, and miR-23a were selectively expressed in microvascular fragments isolated from a range of tissues. In situ hybridization and analysis of Pdgfb retention motif mutant mice demonstrated predominant expression of miR-145 in pericytes. We identified the Ets transcription factor Friend leukemia virus integration 1 (Fli1) as a miR-145 target, and showed that elevated levels of miR-145 reduced migration of microvascular cells in response to growth factor gradients in vitro.CONCLUSIONSmiR-126, miR-24 and miR-23a are selectively expressed in microvascular endothelial cells in vivo, whereas miR-145 is expressed in pericytes. miR-145 targets the hematopoietic transcription factor Fli1 and blocks migration in response to growth factor gradients. Our findings have implications for vascular disease and provide necessary information for future drug design against miRNAs with selective expression in the microvasculature.
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| 13. |
- Le Jan, Sébastien, et al.
(author)
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Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis
- 2012
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In: Arteriosclerosis, Thrombosis and Vascular Biology. - 1079-5642 .- 1524-4636. ; 32:5, s. 1255-1263
-
Journal article (peer-reviewed)abstract
- 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.
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| 14. |
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| 15. |
- Wåhlén, Erik, et al.
(author)
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Activated EGFR and PDGFR internalize in separate vesicles and downstream AKT and ERK1/2 signaling are differentially impacted by cholesterol depletion
- 2023
-
In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier. - 0006-291X .- 1090-2104. ; 665, s. 195-201
-
Journal article (peer-reviewed)abstract
- The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFR-β) internalizations were investigated by immunofluorescent microscopy following simultaneous treatment with EGF and PDGF-BB. We found that the two receptors utilize separate routes of internalization, which merges in a common perinuclear endosomal compartment after 45 min of stimulation. This is further strengthened when contrasting the recruitment of either EGFR or PDGFR-β to either clathrin or caveolin-1: PDGFR-β dissociates from caveolin-1 upon stimulation, and engages clathrin, whilst an increased recruitment of EGFR, to both clathrin and caveolin-1, was observed upon EGF stimulation. The association between EGFR and caveolin-1 is supported by the observation that EGFR was localized in lipid raft associated fractions, whereas PDGFR-β was not. We also found that disruption of lipid rafts using MβCD led to an increased EGFR dimerization and phosphorylation in response to ligand, as well as a dramatic decrease in AKT- and a smaller but robust decrease in ERK1/2 phosphorylation. This suggest that lipid rafts may be important to effectively connect the EGFR with downstream proteins to facilitate signaling. Our data implies that cholesterol depletion of the plasma membrane affect the signaling of EGFR and PDGFRβ differently.
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| 16. |
- Wåhlén, Erik, et al.
(author)
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Differential impact of lipid raft depletion on platelet-derived growth factor (PDGF)-induced ERK1/2 MAP-kinase, SRC and AKT signaling
- 2022
-
In: Cellular Signalling. - : Elsevier. - 0898-6568 .- 1873-3913. ; 96
-
Journal article (peer-reviewed)abstract
- It has become clear that lipid rafts functions as signaling hotspots connecting cell surface receptors to intracellular signaling pathways. However, the exact involvement of lipid rafts in receptor tyrosine kinase signaling is still poorly understood. In this study, we have analyzed platelet-derived growth factor (PDGF) receptor β (PDGFR-β) signaling in two different cell lines depleted of cholesterol, and as a consequence, disruption of lipid rafts. Cholesterol depletion of BJ-hTERT fibroblasts using methyl-β-cyclodextrin (MβCD) did not affect PDGFR-β activation as measured by its tyrosine phosphorylation. However, we did observe a small reduction in AKT phosphorylation and a more robust decrease of ERK1/2 activation. In contrast, in the osteosarcoma cell line U2OS, we noticed a deficient receptor activation. Interestingly, in U2OS cells, the ERK1/2 pathway was unaffected, but instead AKT and SRC signaling was reduced. These results suggest that cell type specific wiring of signaling pathways can lead to differential sensitivity to cholesterol depletion. Furthermore, MβCD treatment had a much more pronounced morphological effect on U2OS compared to BJ-hTERT cells. This is consistent with a previous report claiming that cancer cells are more sensitive to cholesterol depletion than normal cells. Our data supports the possibility that cholesterol lowering drugs may impede tumor growth.
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| 17. |
- Wåhlén, Erik, et al.
(author)
-
Silencing of Rho GTPases Cdc42, Rac1 or RhoA reduces PDGFRα and -β phosphorylation and downstream signaling of STAT1 and STAT3
-
Other publication (other academic/artistic)abstract
- Platelet-derived growth factor receptors (PDGFR) have been implicated in both pathological and physiological signaling and play a major role in the homeostasis of the tumor microenvironment. It has previously been shown that Cdc42, which belongs to the family of Rho GTPases, impairs the degradation of EGFR and VEGFR2. In the current work, we have investigated how Cdc42 as well as Rac1 and RhoA affect PDGFRα and -β signaling and downstream activation of AKT, ERK1/2, STAT1 and STAT3. In response to individual depletion of all Rho GTPases both PDGFRα and -β show a significant reduction in their phosphorylation. We could see a delayed internalization in response to Cdc42 or Rac1 depletion and an increased steady-state amount in response to RhoA depletion for both PDGFRα and -β. Downstream of both receptors, we saw a dramatic effect on STAT signaling, however, AKT and ERK1/2 signaling was unaffected. PDGF-BB-induced STAT1 and STAT3 phosphorylation was severely impaired in response to the depletion of either Cdc42, Rac1 or RhoA. Furthermore, STAT1 protein levels were also decreased in response to depletion of the Rho GTPases. In conclusion, we show that both PDGFRα and PDGFRβ rely on Cdc42, Rac1 and RhoA for proper signaling. Furthermore, we also show that STAT1 and STAT3 depend on Cdc42, Rac1 and RhoA for their signaling downstream of PDGFRs, and STAT1 for its protein stability.
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| 18. |
- Amagasaki, Kenichi, et al.
(author)
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c-Jun N-terminal kinase is necessary for platelet-derived growth factor-mediated chemotaxis in primary fibroblasts
- 2006
-
In: Journal of Biological Chemistry. - : The American Society for Biochemistry and Molecular Biology, Inc.. - 0021-9258 .- 1083-351X. ; 281:31, s. 22173-22179
-
Journal article (peer-reviewed)abstract
- c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family. It has become clear that JNK does not only have a role in induction of stress responses but also in processes such as cell movement. In this report we demonstrate that JNK activity is necessary for platelet-derived growth factor (PDGF)-BB-induced chemotaxis of primary foreskin fibroblasts and in other cell types. PDGF-BB stimulation was found to lead to activation of JNK with a maximum after 30 min. Inhibition of JNK reduced Ser178 phosphorylation of the focal adhesion component paxillin. Paxillin phosphorylation at this site has been shown to be involved in the dynamics of focal adhesions and consequently cell migration. Moreover, we observed localization of JNK to the actin-dense membrane ruffles induced by PDGF-BB stimulation both using immunofluorescence staining and green fluorescent protein-tagged JNK. This suggests a role for JNK at the leading edge of the cell compatible with a function in cell migration. Furthermore, we show that phosphatidylinositol 3-kinase (PI 3-kinase), which has an established role in PDGF-stimulated cell migration, is necessary for PDGF-induced activation of JNK. In conclusion, JNK is a critical component downstream of PI 3-kinase that may be involved in PDGF-stimulated chemotaxis presumably by modulating the integrity of focal adhesions by phosphorylating its components.
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| 19. |
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| 20. |
- Bivehed, Erik, et al.
(author)
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Visualizing DNA single- and double-strand breaks in the Flash comet assay by DNA polymerase-assisted end-labelling
- 2024
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In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 52:4
-
Journal article (peer-reviewed)abstract
- In the comet assay, tails are formed after single-cell gel electrophoresis if the cells have been exposed to genotoxic agents. These tails include a mixture of both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). However, these two types of strand breaks cannot be distinguished using comet assay protocols with conventional DNA stains. Since DSBs are more problematic for the cells, it would be useful if the SSBs and DSBs could be differentially identified in the same comet. In order to be able to distinguish between SSBs and DSBs, we designed a protocol for polymerase-assisted DNA damage analysis (PADDA) to be used in combination with the Flash comet protocol, or on fixed cells. By using DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides. Herein, TK6-cells or HaCat cells were exposed to either hydrogen peroxide (H2O2), ionising radiation (X-rays) or DNA cutting enzymes, and then subjected to a comet protocol followed by PADDA. PADDA offers a wider detection range, unveiling previously undetected DNA strand breaks. Graphical Abstract
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| 21. |
- Chitu, V., et al.
(author)
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The PDGFR Receptor Family
- 2015
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In: Receptor Tyrosine Kinases: Family and Subfamilies. - Cham : Springer International Publishing. - 9783319118871 - 9783319118888 ; , s. 373-538
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Book chapter (peer-reviewed)
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| 22. |
- Dadras, Mahsa Shahidi, et al.
(author)
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The polarity protein Par3 coordinates positively self-renewal and negatively invasiveness in glioblastoma
- 2021
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In: Cell Death and Disease. - : Springer Nature. - 2041-4889. ; 12:10
-
Journal article (peer-reviewed)abstract
- Glioblastoma (GBM) is a brain malignancy characterized by invasiveness to the surrounding brain tissue and by stem-like cells, which propagate the tumor and may also regulate invasiveness. During brain development, polarity proteins, such as Par3, regulate asymmetric cell division of neuro-glial progenitors and neurite motility. We, therefore, studied the role of the Par3 protein (encoded by PARD3) in GBM. GBM patient transcriptomic data and patient-derived culture analysis indicated diverse levels of expression of PARD3 across and independent from subtypes. Multiplex immunolocalization in GBM tumors identified Par3 protein enrichment in SOX2-, CD133-, and NESTIN-positive (stem-like) cells. Analysis of GBM cultures of the three subtypes (proneural, classical, mesenchymal), revealed decreased gliomasphere forming capacity and enhanced invasiveness upon silencing Par3. GBM cultures with suppressed Par3 showed low expression of stemness (SOX2 and NESTIN) but higher expression of differentiation (GFAP) genes. Moreover, Par3 silencing reduced the expression of a set of genes encoding mitochondrial enzymes that generate ATP. Accordingly, silencing Par3 reduced ATP production and concomitantly increased reactive oxygen species. The latter was required for the enhanced migration observed upon silencing of Par3 as anti-oxidants blocked the enhanced migration. These findings support the notion that Par3 exerts homeostatic redox control, which could limit the tumor cell-derived pool of oxygen radicals, and thereby the tumorigenicity of GBM.
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| 23. |
- de Oliveira, Felipe Marques Souza, et al.
(author)
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Detection of post-translational modifications using solid-phase proximity ligation assay.
- 2018
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In: New biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 45:October, s. 51-59
-
Journal article (peer-reviewed)abstract
- 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.
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| 24. |
- Demoulin, Jean-Baptiste, et al.
(author)
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Platelet-derived growth factor stimulates membrane lipid synthesis through activation of phosphatidylinositol 3-kinase and sterol regulatory element-binding proteins
- 2004
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 279:34, s. 35392-35402
-
Journal article (peer-reviewed)abstract
- We analyzed the transcriptional program elicited by stimulation of normal human fibroblasts with platelet-derived growth factor (PDGF) using cDNA microarrays. 103 significantly regulated transcripts that had not been previously linked to PDGF signaling were identified. Among them, a cluster of genes involved in fatty acid and cholesterol biosynthesis, including stearoyl-CoA desaturase (SCD), fatty acid synthase, and hydroxymethylglutaryl-CoA synthase (HMGCS), was up-regulated by PDGF after 24 h of treatment, and their expression correlated with increased membrane lipid production. These genes are known to be controlled by sterol regulatory element-binding proteins (SREBP). PDGF increased the amount of mature SREBP-1 and regulated the promoters of SCD and HMGCS in an SREBP-dependent manner. In line with these results, blocking SREBP processing by addition of 25-hydroxycholesterol blunted the effects of PDGF on lipogenic enzymes. SREBP activation was dependent on the phosphatidylinositol 3-kinase (PI3K) pathway, as judged from the effects of the inhibitor LY294002 and mutation of the PDGFbeta receptor tyrosines that bind the PI3K adaptor subunit p85. Fibroblast growth factors (FGF-2 and FGF-4) and other growth factors mimicked the effects of PDGF on NIH3T3 and human fibroblasts. In conclusion, our results suggest that growth factors induce membrane lipid synthesis via the activation SREBP and PI3K.
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| 25. |
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| 26. |
- Eger, Glenda, et al.
(author)
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NR4A1 Promotes PDGF-BB-Induced Cell Colony Formation in Soft Agar
- 2014
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:9, s. e109047-
-
Journal article (peer-reviewed)abstract
- The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth.
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| 27. |
- Eger, Glenda, 1984-
(author)
-
Regulation and Function of MAP Kinases in PDGF Signaling
- 2016
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Doctoral thesis (other academic/artistic)abstract
- Platelet-derived growth factor (PDGF) is a family of signaling molecules that stimulates cell growth, survival and migration. PDGF is recognized by specific transmembrane proteins, the PDGF receptors, which relay the signals to the cell activating the Mitogen-activated protein (MAP) kinases and other signaling pathways. Aberrant activation of these pathways is frequently detected in cancer. Hence, the study of these processes is essential for identifying potential drug targets or diagnostic markers.In paper I, we identified Receptor Subfamily 4 Group A Member 1 NR4A1 to be regulated by PDGF via MAP kinases, clarifying the role of Extracellular signal–regulated kinases (Erk) 1/2, Erk5 and Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) in its regulation. NR4A1 was found to be important for the tumorigenic potential, measured as anchorage-independent growth, of glioblastoma cells.Since the cellular responses elicited by PDGF result from the balance between phosphorylation and dephosphorylation events, we investigated the role of the dual specificity phosphatases DUSP4/MKP-2 and DUSP6/MKP-3. In paper II, we describe the crucial role of Erk1/2 and p53 in the expression of DUSP4/MKP2. Moreover, we observed that DUSP4/MKP-2 downregulation decreases Erk5 activation and accelerates PDGFRβ internalization and downregulation resulting in a specific inhibition of Signal transducers and activators of transcription (Stat) 3, Src and protein kinase C (PKC), and partially of p38, Stat1/5 and Phoshoplipase Cγ (PLCγ).In paper III, we report that DUSP6/MKP-3 creates a negative cross-talk between Erk1/2 and Erk5 and an auto-inhibitory feedback loop on the PI3-kinase/Akt pathway. In paper IV, we identify a new regulative mechanism of the PDGF pathway. PDGF induces Erk5 expression and activation that modulates the PDGFRβ activity. After Erk5 downregulation, the receptor undergoes to a faster and stronger activation that results in a faster internalization and degradation.In conclusion, we present a mechanism through which the PDGF/MAP kinases support tumor growth, and elucidate different regulatory pathways involved in PDGF signaling.
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| 28. |
- Ekman, Simon, et al.
(author)
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Activation of growth factor receptors in esophageal cancer : implications for therapy
- 2007
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In: The Oncologist. - : Oxford University Press (OUP). - 1083-7159 .- 1549-490X. ; 12:10, s. 1165-1177
-
Research review (peer-reviewed)abstract
- Esophageal cancer is a highly aggressive disease and is the seventh most common cause of cancer-related death in the western world. Worldwide, it ranks as the sixth most frequent cause of cancer death. Despite advances in surgical techniques and treatment, the prognosis of esophageal cancer remains poor, with very few long-term survivors. The need for novel strategies to detect esophageal cancer earlier and to improve current therapy is urgent. It is well established that growth factors and growth factor receptor-mediated signaling pathways are important components of the transformation process in many forms of cancer, including esophageal cancer. With the recent advances in drug development, there are emerging possibilities to use growth factor signal transduction pathways in targeted therapy. This review provides a summary of the role of growth factors and their receptors in esophageal cancer and discusses their potential roles as biomarkers and as targets in therapy.
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| 29. |
- Grönroos, Eva, et al.
(author)
-
Control of Smad7 stability by competition between acetylation and ubiquitination
- 2002
-
In: Molecular Cell. - 1097-2765 .- 1097-4164. ; 10:3, s. 483-493
-
Journal article (peer-reviewed)abstract
- Smad proteins regulate gene expression in response to TGFbeta signaling. Here we present evidence that Smad7 interacts with the transcriptional coactivator p300, resulting in acetylation of Smad7 on two lysine residues in its N terminus. Acetylation or mutation of these lysine residues stabilizes Smad7 and protects it from TGFbeta-induced degradation. Furthermore, we demonstrate that the acetylated residues in Smad7 also are targeted by ubiquitination and that acetylation of these lysine residues prevents subsequent ubiquitination. Specifically, acetylation of Smad7 protects it against ubiquitination and degradation mediated by the ubiquitin ligase Smurf1. Thus, our data suggest that competition between ubiquitination and acetylation of overlapping lysine residues constitutes a novel mechanism to regulate protein stability.
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| 30. |
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| 31. |
- Heldin, Carl-Henrik, 1952-, et al.
(author)
-
Involvement of platelet-derived growth factor ligands and receptors in tumorigenesis
- 2018
-
In: Journal of Internal Medicine. - : Wiley. - 0954-6820 .- 1365-2796. ; 283:1, s. 16-44
-
Research review (peer-reviewed)abstract
- Platelet-derived growth factor (PDGF) isoforms and their receptors have important roles during embryogenesis, particularly in the development of various mesenchymal cell types in different organs. In the adult, PDGF stimulates wound healing and regulates tissue homeostasis. However, overactivity of PDGF signalling is associated with malignancies and other diseases characterized by excessive cell proliferation, such as fibrotic conditions and atherosclerosis. In certain tumours, genetic or epigenetic alterations of the genes for PDGF ligands and receptors drive tumour cell proliferation and survival. Examples include the rare skin tumour dermatofibrosarcoma protuberance, which is driven by autocrine PDGF stimulation due to translocation of a PDGF gene, and certain gastrointestinal stromal tumours and leukaemias, which are driven by constitute activation of PDGF receptors due to point mutations and formation of fusion proteins ofthe receptors, respectively. Moreover, PDGF stimulates cells in tumour stroma and promotes angiogenesis as well as the development of cancer-associated fibroblasts, both of which promote tumour progression. Inhibitors of PDGF signalling may thus be of clinical usefulness in the treatment of certain tumours.
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| 32. |
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| 33. |
- Heldin, Carl-Henrik, et al.
(author)
-
Structural and functional properties of platelet-derived growth factor and stem cell factor receptors
- 2013
-
In: Cold Spring Harbor Perspectives in Biology. - : Cold Spring Harbor Laboratory. - 1943-0264. ; 5:8, s. UNSP a009100-
-
Journal article (peer-reviewed)abstract
- The receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) are members of the type III class of PTK receptors, which are characterized by five Ig-like domains extracellularly and a split kinase domain intracellularly. The receptors are activated by ligand-induced dimerization, leading to autophosphorylation on specific tyrosine residues. Thereby the kinase activities of the receptors are activated and docking sites for downstream SH2 domain signal transduction molecules are created; activation of these pathways promotes cell growth, survival, and migration. These receptors mediate important signals during the embryonal development, and control tissue homeostasis in the adult. Their overactivity is seen in malignancies and other diseases involving excessive cell proliferation, such as atherosclerosis and fibrotic diseases. In cancer, mutations of PDGF and SCF receptors-including gene fusions, point mutations, and amplifications-drive subpopulations of certain malignancies, such as gastrointestinal stromal tumors, chronic myelomonocytic leukemia, hypereosinophilic syndrome, glioblastoma, acute myeloid leukemia, mastocytosis, and melanoma.
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| 34. |
- Jurek, Aleksandra, et al.
(author)
-
Negative and positive regulation of MAPK phosphatase 3 controls platelet-derived growth factor-induced Erk activation
- 2009
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:7, s. 4626-4634
-
Journal article (peer-reviewed)abstract
- 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.
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| 35. |
- Jurek, Aleksandra, et al.
(author)
-
Platelet-derived growth factor-induced signaling pathways interconnect to regulate the temporal pattern of Erk1/2 phosphorylation
- 2011
-
In: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 23:1, s. 280-287
-
Journal article (peer-reviewed)abstract
- 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.
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| 36. |
- Koos, Björn, et al.
(author)
-
Proximity-dependent initiation of hybridization chain reaction
- 2015
-
In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 6
-
Journal article (peer-reviewed)abstract
- 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.
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| 37. |
- Kurisaki, Keiko, et al.
(author)
-
Nuclear factor YY1 inhibits transforming growth factor beta- and bone morphogenetic protein-induced cell differentiation
- 2003
-
In: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 23:13, s. 4494-4510
-
Journal article (peer-reviewed)abstract
- Smad proteins transduce transforming growth factor beta (TGF-beta) and bone morphogenetic protein (BMP) signals that regulate cell growth and differentiation. We have identified YY1, a transcription factor that positively or negatively regulates transcription of many genes, as a novel Smad-interacting protein. YY1 represses the induction of immediate-early genes to TGF-beta and BMP, such as the plasminogen activator inhibitor 1 gene (PAI-1) and the inhibitor of differentiation/inhibitor of DNA binding 1 gene (Id-1). YY1 inhibits binding of Smads to their cognate DNA elements in vitro and blocks Smad recruitment to the Smad-binding element-rich region of the PAI-1 promoter in vivo. YY1 interacts with the conserved N-terminal Mad homology 1 domain of Smad4 and to a lesser extent with Smad1, Smad2, and Smad3. The YY1 zinc finger domain mediates the association with Smads and is necessary for the repressive effect of YY1 on Smad transcriptional activity. Moreover, downregulation of endogenous YY1 by antisense and small interfering RNA strategies results in enhanced transcriptional responses to TGF-beta or BMP. Ectopic expression of YY1 inhibits, while knockdown of endogenous YY1 enhances, TGF-beta- and BMP-induced cell differentiation. In contrast, overexpression or knockdown of YY1 does not affect growth inhibition induced by TGF-beta or BMP. Accordingly, YY1 does not interfere with the regulation of immediate-early genes involved in the TGF-beta growth-inhibitory response, the cell cycle inhibitors p15 and p21, and the proto-oncogene c-myc. In conclusion, YY1 represses Smad transcriptional activities in a gene-specific manner and thus regulates cell differentiation induced by TGF-beta superfamily pathways.
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| 38. |
- Leino, Mattias, et al.
(author)
-
Optimization of proximity-dependent initiation of hybridization chain reaction for improved performance
- 2019
-
In: Molecular Systems Design & Engineering. - : Royal Society of Chemistry (RSC). - 2058-9689. ; 4:5, s. 1058-1065
-
Journal article (peer-reviewed)abstract
- Proximity based detection methods are invaluable tools in the field of molecular biology, increasing selectivity and allowing for analysis of protein interactions. ProxHCR utilizes pairs of antibodies labelled with oligonucleotides to probe for proximal binding and to initiate a hybridization chain reaction (HCR) to generate an amplified detection signal. As HCR is based upon hybridization of DNA hairpins, the performance is dependent on salt concentrations and temperature. Herein we have redesigned the proxHCR system to increase the performance and to reduce dependency on temperature and salt concentrations. The new oligonucleotides provide an increased signal when performed at physiological salt concentrations and in room temperature.
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| 39. |
- Lennartsson, Johan, et al.
(author)
-
Alix Facilitates the Interaction between c-Cbl and Platelet-derived Growth Factor beta-Receptor and Thereby Modulates Receptor Down-regulation
- 2006
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:51, s. 39152-39158
-
Journal article (peer-reviewed)abstract
- Alix (ALG-2-interacting protein X) is an adaptor protein involved in down-regulation and sorting of cell surface receptors through the endosomal compartments toward the lysosome. In this study, we show that Alix interacts with the C-terminal region of the platelet-derived growth factor (PDGF) β-receptor (PDGFRβ) and becomes transiently tyrosine-phosphorylated in response to PDGF-BB stimulation. Increased expression levels of Alix resulted in a reduced rate of PDGFRβ removal from the cell surface following receptor activation, and this was associated with decreased receptor degradation. Furthermore, Alix was found to co-immunoprecipitate with the ubiquitin ligase c-Cbl, and elevated Alix levels increased the interaction between c-Cbl and PDGFRβ. Interestingly, Alix interacted constitutively with both c-Cbl and PDGFRβ. Moreover, c-Cbl was found to be hyperphosphorylated in cells engineered to overexpress Alix compared with control cells. The increased c-Cbl phosphorylation correlated with enhanced proteasomal degradation of c-Cbl, which in turn correlated with a decreased ubiquitination of PDGFRβ. Our data suggest that Alix inhibits down-regulation of PDGFRβ by modulating the interaction between c-Cbl and the receptor, thereby affecting the ubiquitination of the receptor.
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| 40. |
- Lennartsson, Johan, et al.
(author)
-
Erk 5 is necessary for sustained PDGF-induced Akt phosphorylation and inhibition of apoptosis
- 2010
-
In: Cellular Signalling. - : Elsevier Inc.. - 0898-6568 .- 1873-3913. ; 22:6, s. 955-960
-
Journal article (peer-reviewed)abstract
- Extracellular regulated kinase (Erk) 5 is a member of the mitogen activated protein (MAP) kinase family that has been implicated in both cell proliferation and survival. In the present study, we found that stimulation with platelet-derived growth factor (PDGF)-BB leads to a transient activation of Erk5, which was shown to be dependent on recruitment of both Src kinases and the tyrosine phosphatase Shp2 to the activated PDGF receptor beta (PDGFRbeta). We could also demonstrate that Shp2 docking to the receptor is critical for Src kinase activation, suggesting that Shp2 may contribute to Erk5 activation through its involvement in Src kinase activation. Under control conditions, PDGF-BB promoted a sustained Akt phosphorylation. However, reduction of the expression of Erk5 by siRNA resulted in only a transient Akt phosphorylation, and an inability of PDGF-BB to suppress caspase 3 activation and inhibit apoptotic nuclear morphological changes such as condensed or fragmented chromatin under serum-free conditions.
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| 41. |
- Lennartsson, Johan, et al.
(author)
-
The Fer Tyrosine Kinase Is Important for Platelet-derived Growth Factor-BB-induced Signal Transducer and Activator of Transcription 3 (STAT3) Protein Phosphorylation, Colony Formation in Soft Agar, and Tumor Growth in Vivo.
- 2013
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:22, s. 15736-15744
-
Journal article (peer-reviewed)abstract
- Fer is a cytoplasmic tyrosine kinase that is activated in response to platelet-derived growth factor (PDGF) stimulation. In the present report, we show that Fer associates with the activated PDGF β-receptor (PDGFRβ) through multiple autophosphorylation sites, i.e. Tyr-579, Tyr-581, Tyr-740, and Tyr-1021. Using low molecular weight inhibitors, we found that PDGF-BB-induced Fer activation is dependent on PDGFRβ kinase activity, but not on the enzymatic activity of Src or Jak kinases. In cells in which Fer was down-regulated using siRNA, PDGF-BB was unable to induce phosphorylation of STAT3, whereas phosphorylations of STAT5, ERK1/2, and Akt were unaffected. PDGF-BB-induced activation of STAT3 occurred also in cells expressing kinase-dead Fer, suggesting a kinase-independent adaptor role of Fer. Expression of Fer was dispensable for PDGF-BB-induced proliferation and migration but essential for colony formation in soft agar. Tumor growth in vivo was delayed in cells depleted of Fer expression. Our data suggest a critical role of Fer in PDGF-BB-induced STAT3 activation and cell transformation.
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| 42. |
- Lennartsson, Johan, et al.
(author)
-
The Fer tyrosine kinase is necessary for platelet-derived growth factor-BB-induced Stat3 phosphorylation and colony formation in soft agar
-
Other publication (other academic/artistic)abstract
- Fer is a cytoplasmic tyrosine kinase that is activated in response to platelet-derived growth factor (PDGF) stimulation. In the present report, we show that Fer associates with the activated PDGF b-receptor (PDGFRb) through multiple autophosphorylation sites, i.e. Tyr579, Tyr581, Tyr740 and Tyr1021. Using low molecular weight inhibitors we found that PDGF-BB-induced Fer activation is dependent on PDGFRb kinase activity, but not on the enzymatic activity of Src or Jak kinases. To elucidate the function of Fer downstream of PDGFRb, we downregulated the expression using siRNA; under conditions with reduced Fer expression, PDGF-BB was unable to induce phosphorylation of Stat3, whereas Stat5, Erk1/2 and Akt were unaffected. In addition, PDGFRb autophosphorylation was partially dependent on Fer expression. On a functional level, we could demonstrate that expression of Fer is dispensable for PDGF-BB-induced proliferation and migration. However, Fer was found to be essential for colony formation in soft agar, consistent with its critical role in Stat3 activation, which has frequently been implicated in cell transformation.
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| 43. |
- Li, Xuri, et al.
(author)
-
Revascularization of ischemic tissues by PDGF-CC via effects on endothelial cells and their progenitors
- 2005
-
In: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 115:1, s. 118-127
-
Journal article (peer-reviewed)abstract
- The angiogenic mechanism and therapeutic potential of PDGF-CC, a recently discovered member of the VEGF/PDGF superfamily, remain incompletely characterized. Here we report that PDGF-CC mobilized endothelial progenitor cells in ischemic conditions; induced differentiation of bone marrow cells into ECs; and stimulated migration of ECs. Furthermore, PDGF-CC induced the differentiation of bone marrow cells into smooth muscle cells and stimulated their growth during vessel sprouting. Moreover, delivery of PDGF-CC enhanced postischemic revascularization of the heart and limb. Modulating the activity of PDGF-CC may provide novel opportunities for treating ischemic diseases.
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| 44. |
- Li, Yang, et al.
(author)
-
VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats.
- 2008
-
In: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 118:3, s. 913-923
-
Journal article (peer-reviewed)abstract
- Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. Using gene expression profiling of mouse primary aortic smooth muscle cells, and confirming the results by real-time PCR using mouse and rat cell lines, we showed that VEGF-B inhibited the expression of genes encoding the proapoptotic BH3-only proteins and other apoptosis- and cell death-related proteins, including p53 and members of the caspase family, via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Interestingly, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization, suggesting that VEGF-B is the first member of the VEGF family that has a potent antiapoptotic effect while lacking a general angiogenic activity. These findings indicate that VEGF-B may potentially offer a new therapeutic option for the treatment of neurodegenerative diseases.
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| 45. |
- Ma, Haisha, et al.
(author)
-
Histidine-domain-containing protein tyrosine phosphatase regulates platelet-derived growth factor receptor intracellular sorting and degradation
- 2015
-
In: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 27:11, s. 2209-2219
-
Journal article (peer-reviewed)abstract
- Histidine domain-containing protein tyrosine phosphatase (HD-PTP) is a putative phosphatase that has been shown to affect the signaling and downregulation of certain receptor tyrosine kinases. To investigate if HD-PTP affects platelet-derived growth factor receptor beta (PDGFR beta) signaling, we employed the overexpression of HA-tagged HD-PTP, as well as siRNA-mediated and lentivirus shRNA-mediated silencing of HD-PTP in NIH3T3 cells. We found that HD-PTP was recruited to the PDGFR beta in a ligand-dependent manner. Depletion of HD-PTP resulted in an inability of PDGF-BB to promote tyrosine phosphorylation of the ubiquitin ligases c-Cbl and Cbl-b, with a concomitant missorting and reduction of the degradation of activated PDGFRS. In contrast, ligand-induced internalization of PDGFR beta was unaffected by HD-FTP silencing. Furthermore, the levels of STAM and Hrs of the ESCRT0 machinery were decreased, and immunofluorescence staining showed that in HD-PTP-depleted cells, PDGFR beta accumulated in large aberrant intracellular structures. After the reduction of HD-PTP expression, an NIH3T3-derived cell line that has autocrine PDGF-BB signaling (sis-3 T3) showed increased ability of anchorage-independent growth. However, exogenously added PDGF-BB promoted efficient additional colony formation in control cells, but was not able to do so in HD-PTP-depleted cells. Furthermore, cells depleted of HD-PTP migrated faster than control cells. In summary, HD-PTP affects the intracellular sorting of activated PDGFRS and the migration, proliferation and tumorigenicity of cells stimulated by PDGF.
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| 46. |
- Micke, Patrick, et al.
(author)
-
In situ identification of genes regulated specifically in fibroblasts of human basal cell carcinoma.
- 2007
-
In: The Journal of investigative dermatology. - : Elsevier BV. - 1523-1747 .- 0022-202X. ; 127:6, s. 1516-23
-
Journal article (peer-reviewed)abstract
- Basal cell carcinoma (BCC) is characterized by slow growth, virtual absence of metastases, and strong stroma-dependency. Cancer-associated fibroblasts (CAFs) in the tumor stroma influence tumor growth, invasion, and metastasis. To comprehensively characterize CAFs of BCC in their in situ cancer environment, laser capture microdissection, linear gene amplification, microarray analysis, and quantitative real-time PCR (qRT-PCR) were combined. Pair-wise comparison of gene expression of microdissected CAFs and corresponding normal perifollicular fibroblasts identified 65 genes that were significantly upregulated in at least two of three different patients. Among the annotated genes, as many as 13 genes encoded secreted proteins, of which six were previously implicated as CAF-associated proteins in various tumor types. Four of the seven novel CAF genes--matrix Gla-protein, secreted frizzled-related protein 2, angiopoietin-related protein-2, and platelet-derived growth factor receptor-like protein--were selected for further analyses by qRT-PCR and were found to be frequently upregulated in CAFs of three independent BCC tissues. Analyses of CAFs from squamous cell cancer, prostate cancer, and colon cancer did not indicate that these genes were upregulated in these cancers. This study thus validates a novel approach for comprehensive characterization CAFs in their in situ environment of BCC. The results suggest a specific expression profile of CAFs in BCC possibly accounting for disease-specific pathological roles.
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| 47. |
- Morikawa, Masato, et al.
(author)
-
The ALK-1/SMAD/ATOH8 axis attenuates hypoxic responses and protects against the development of pulmonary arterial hypertension
- 2019
-
In: Science Signaling. - : American Association for the Advancement of Science. - 1945-0877 .- 1937-9145. ; 12:607
-
Journal article (peer-reviewed)abstract
- Dysregulated bone morphogenetic protein (BMP) signaling in endothelial cells (ECs) is implicated in vascular diseases such as pulmonary arterial hypertension (PAH). Here, we showed that the transcription factor ATOH8 was a direct target of SMAD1/5 and was induced in a manner dependent on BMP but independent of Notch, another critical signaling pathway in ECs. In zebrafish and mice, inactivation of Atoh8 did not cause an arteriovenous malformation-like phenotype, which may arise because of dysregulated Notch signaling. In contrast, Atoh8-deficient mice exhibited a phenotype mimicking PAH, which included increased pulmonary arterial pressure and right ventricular hypertrophy. Moreover, ATOH8 expression was decreased in PAH patient lungs. We showed that in cells, ATOH8 interacted with hypoxia-inducible factor 2 alpha (HIF-2 alpha) and decreased its abundance, leading to reduced induction of HIF-2 alpha target genes in response to hypoxia. Together, these findings suggest that the BMP receptor type II/ALK-1/SMAD/ATOH8 axis may attenuate hypoxic responses in ECs in the pulmonary circulation and may help prevent the development of PAH.
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| 48. |
- Mu, Yabing, et al.
(author)
-
TRAF6 ubiquitinates TGFβ type I receptor to promote its cleavage and nuclear translocation in cancer
- 2011
-
In: Nature Communications. - London : Macmillan Publishers Limited. - 2041-1723. ; 2:330
-
Journal article (peer-reviewed)abstract
- Transforming growth factor β (TGFβ) is a pluripotent cytokine promoting epithelial cell plasticity during morphogenesis and tumour progression. TGFβ binding to type II and type I serine/threonine kinase receptors (TβRII and TβRI) causes activation of different intracellular signaling pathways. TβRI is associated with the ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here we show that TGFβ, via TRAF6, causes Lys63-linked polyubiquitination of TβRI, promoting cleavage of TβRI by TNF-alpha converting enzyme (TACE), in a PKCζ-dependent manner. The liberated intracellular domain (ICD) of TβRI associates with the transcriptional regulator p300 to activate genes involved in tumour cell invasiveness, such as Snail and MMP2. Moreover, TGFβ-induced invasion of cancer cells is TACE- and PKCζ- dependent and the TβRI ICD is localized in the nuclei of different kinds of tumour cells in tissue sections. Thus, our data reveal a specific role for TβRI in TGFβ mediated tumour invasion.
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| 49. |
- Papadopoulos, Natalia, et al.
(author)
-
PDGFR beta translocates to the nucleus and regulates chromatin remodeling via TATA element-modifying factor 1
- 2018
-
In: Journal of Cell Biology. - : ROCKEFELLER UNIV PRESS. - 0021-9525 .- 1540-8140. ; 217:5, s. 1701-1717
-
Journal article (peer-reviewed)abstract
- Translocation of full-length or fragments of receptors to the nucleus has been reported for several tyrosine kinase receptors. In this paper, we show that a fraction of full-length cell surface platelet-derived growth factor (PDGF) receptor beta (PDG FR beta) accumulates in the nucleus at the chromatin and the nuclear matrix after ligand stimulation. Nuclear translocation of PDG FR beta was dependent on PDGF-BB-induced receptor dimerization, clathrin-mediated endocytosis, beta-importin, and intact Golgi, occurring in both normal and cancer cells. In the nucleus, PDG FR beta formed ligand-inducible complexes with the tyrosine kinase Fer and its substrate, TATA element-modifying factor 1 (TMF-1). PDGF-BB stimulation decreased TMF-1 binding to the transcriptional regulator Brahma-related gene 1 (Brg-1) and released Brg-1 from the SWI-SNF chromatin remodeling complex. Moreover, knockdown of TMF-1 by small interfering RNA decreased nuclear translocation of PDG FR beta and caused significant up-regulation of the Brg-1/p53-regulated cell cycle inhibitor CDKN1A (encoding p21) without affecting PDG FR beta-inducible immediate-early genes. In conclusion, nuclear interactions of PDG FR beta control proliferation by chromatin remodeling and regulation of p21 levels.
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| 50. |
- Pielberg, Gerli Rosengren, et al.
(author)
-
A cis-acting regulatory mutation causes premature hair graying and susceptibility to melanoma in the horse
- 2008
-
In: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718. ; 40:8, s. 1004-1009
-
Journal article (peer-reviewed)abstract
- In horses, graying with age is an autosomal dominant trait associated with a high incidence of melanoma and vitiligo-like depigmentation. Here we show that the Gray phenotype is caused by a 4.6-kb duplication in intron 6 of STX17 (syntaxin-17) that constitutes a cis-acting regulatory mutation. Both STX17 and the neighboring NR4A3 gene are overexpressed in melanomas from Gray horses. Gray horses carrying a loss-of-function mutation in ASIP (agouti signaling protein) had a higher incidence of melanoma, implying that increased melanocortin-1 receptor signaling promotes melanoma development in Gray horses. The Gray horse provides a notable example of how humans have cherry-picked mutations with favorable phenotypic effects in domestic animals.
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