| 1. |
- Wåhlén, Erik, et al.
(författare)
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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
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier. - 0006-291X .- 1090-2104. ; 665, s. 195-201
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Wåhlén, Erik, et al.
(författare)
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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
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Ingår i: Cellular Signalling. - : Elsevier. - 0898-6568 .- 1873-3913. ; 96
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Tidskriftsartikel (refereegranskat)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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| 3. |
- Wåhlén, Erik, et al.
(författare)
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Silencing of Rho GTPases Cdc42, Rac1 or RhoA reduces PDGFRα and -β phosphorylation and downstream signaling of STAT1 and STAT3
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Annan publikation (övrigt vetenskapligt/konstnärligt)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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| 4. |
- Kermpatsou, Despoina, et al.
(författare)
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Cellular responses to silencing of PDIA3 (protein disulphide-isomerase A3) : Effects on proliferation, migration, and genes in control of active vitamin D
- 2024
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Ingår i: Journal of Steroid Biochemistry and Molecular Biology. - : Elsevier. - 0960-0760 .- 1879-1220. ; 240
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Tidskriftsartikel (refereegranskat)abstract
- The active form of vitamin D, 1,25-dihydroxyvitamin D3, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) has been associated with some of the functions of 1,25-dihydroxyvitamin D3. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D3-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D3-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D3. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D3-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D3-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D3 levels.
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| 5. |
- Olsson, Frida, et al.
(författare)
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Effects of 1a,25-dihydroxyvitamin D-3 and tacalcitol on cell signaling and anchorage-independent growth in T98G and U251 glioblastoma cells
- 2022
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Ingår i: Biochemistry and Biophysics Reports. - : Elsevier. - 2405-5808. ; 31
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Tidskriftsartikel (refereegranskat)abstract
- The active hormonal form of vitamin D, 1 alpha,25-dihydroxyvitamin D-3, is reported to have 1000s of biological targets. The growth-suppressive properties of 1 alpha,25-dihydroxyvitamin D-3 and its synthetic analogs have attracted interest for the development of treatment and/or prevention of cancer. We examined effects of 1 alpha,25-dihydroxyvitamin D-3 and the vitamin D analog tacalcitol on signaling pathways and anchorage-independent growth in T98G and U251 glioblastoma cells. Assay of signaling proteins important for cellular growth indi-cated suppression of p70-S6 kinase levels by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol in T98G cells, whereas the levels of PLC gamma, a target for phospholipid signaling, was slightly increased. Activation of STAT3, an important regulator of malignancy, was suppressed by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol in T98G and U251 cells. However, despite the close structural similarity of these compounds, suppression was stronger by tacalcitol (1 alpha,24-dihydroxyvitamin D-3), indicating that even minor modifications of a vitamin D analog can impact its effects on signaling. Experiments using soft agar colony formation assay in T98G and U251 cells revealed significant suppression by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol on anchorage-independent growth, a property for cancer invasion and metastasis known to correlate with tumor-igenicity. These findings indicate that vitamin D and its analogs may be able to counteract the oncogenic transformation, invasion and metastatic potential of glioblastoma and prompt further study of these compounds in the development of improved therapy for brain cancer.
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| 6. |
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| 7. |
- Sarri, Niki, et al.
(författare)
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Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFR beta and affect PDGF-BB-induced STAT3 signalling
- 2022
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer. - 1420-682X .- 1420-9071. ; 79:2
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Tidskriftsartikel (refereegranskat)abstract
- Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor beta (PDGFR beta) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFR beta did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGR beta lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.
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| 8. |
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| 9. |
- Sarri, Niki, et al.
(författare)
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The E3 Ubiquitin Ligase TRIM21 Regulates Basal Levels of PDGFRβ
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 24:9
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Tidskriftsartikel (refereegranskat)abstract
- Activation of platelet-derived growth factor (PDGF) receptors α and β (PDGFRα and PDGFRβ) at the cell surface by binding of PDGF isoforms leads to internalization of receptors, which affects the amplitude and kinetics of signaling. Ubiquitination of PDGF receptors in response to ligand stimulation is mediated by the Casitas b-lineage lymphoma (Cbl) family of ubiquitin ligases, promoting internalization and serving as a sorting signal for vesicular trafficking of receptors. We report here that another E3 ligase, i.e., tripartite motif-containing protein 21 (TRIM21), contributes to the ubiquitination of PDGFRβ in human primary fibroblasts AG1523 and the osteosarcoma cell line U2OS and regulates basal levels of PDGFRβ. We found that siRNA-mediated depletion of TRIM21 led to decreased ubiquitination of PDGFRβ in response to PDGF-BB stimulation, while internalization from the cell surface and the rate of ligand-induced degradation of the receptor were not affected. Moreover, induction of TRIM21 decreased the levels of PDGFRβ in serum-starved cells, and even more in growing cells, in the absence of PDGF stimulation. Consistently, siRNA knockdown of TRIM21 caused accumulation of the total amount of PDGFRβ, both in the cytoplasm and on the cell surface, without affecting mRNA levels of the receptor. We conclude that TRIM21 acts post-translationally and maintains basal levels of PDGFRβ, thus suggesting that ubiquitination of PDGFRβ by TRIM21 may direct a portion of receptor for degradation in growing cells in a ligand-independent manner.
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| 10. |
- Tsioumpekou, Maria, et al.
(författare)
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Specific targeting of PDGFR beta in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression
- 2020
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Ingår i: Theranostics. - : IVYSPRING INT PUBL. - 1838-7640. ; 10:3, s. 1122-1135
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Tidskriftsartikel (refereegranskat)abstract
- PDGF-BB/PDGFR beta signaling plays an important role during vascularization by mediating pericyte recruitment to the vasculature, promoting the integrity and function of vessels. Until now it has not been possible to assess the specific role of PDGFR beta signaling in tumor progression and angiogenesis due to lack of appropriate animal models and molecular tools. Methods: In the present study, we used a transgenic knock-in mouse strain carrying a silent mutation in the PDGFR beta ATP binding site that allows specific targeting of PDGFR beta using the compound 1-NaPP1. To evaluate the impact of selective PDGFR beta inhibition of stromal cells on tumor growth we investigated four tumor cell lines with no or low PDGFR beta expression, i.e. Lewis lung carcinoma (LLC), EO771 breast carcinoma, B16 melanoma and a version of B16 that had been engineered to overexpress PDGF-BB (B16/PDGF-BB). Results: We found that specific impairment of PDGFR beta kinase activity by 1-NaPP1 treatment efficiently suppressed growth in tumors with high expression of PDGF-BB, i.e. LLC and B16/PDGF-BB, while the clinically used PDGFR beta kinase inhibitor imatinib did not suppress tumor growth. Notably, tumors with low levels of PDGF-BB, i.e. EO771 and B16, neither responded to 1-NaPP1 nor to imatinib treatment. Inhibition of PDGFR beta by either drug impaired tumor vascularization and also affected pericyte coverage; however, specific targeting of PDGFR beta by 1-NaPP1 resulted in a more pronounced decrease in vessel function with increased vessel apoptosis in high PDGF-BB expressing tumors, compared to treatment with imatinib. In vitro analysis of PDGFR beta ASKA mouse embryo fibroblasts and the mesenchymal progenitor cell line 10T1/2 revealed that PDGF-BB induced NG2 expression, consistent with the in vivo data. Conclusion: Specific targeting of PDGFR beta signaling significantly inhibits tumor progression and angiogenesis depending on PDGF-BB expression. Our data suggest that targeting PDGFR beta in the tumor stroma could have therapeutic value in patients with high tumor PDGF-BB expression.
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