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Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFRβ and affect PDGF-BB-induced STAT3 signalling.

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 β (PDGFRβ) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFRβ 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 PDFGRβ lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.

SUBMITTER: Sarri N

PROVIDER: S-EPMC8782881 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundUSP4, USP15 and USP11 are paralogous deubiquitinating enzymes as evidenced by structural organization and sequence similarity. Based on known interactions and substrates it would appear that they have partially redundant roles in pathways vital to cell proliferation, development and innate immunity, and elevated expression of all three has been reported in various human malignancies. The nature and order of duplication events that gave rise to these extant genes has not been determined, nor has their functional redundancy been established experimentally at the organismal level.MethodsWe have employed phylogenetic and syntenic reconstruction methods to determine the chronology of the duplication events that generated the three paralogs and have performed genetic crosses to evaluate redundancy in mice.ResultsOur analyses indicate that USP4 and USP15 arose from whole genome duplication prior to the emergence of jawed vertebrates. Despite having lower sequence identity USP11 was generated later in vertebrate evolution by small-scale duplication of the USP4-encoding region. While USP11 was subsequently lost in many vertebrate species, all available genomes retain a functional copy of either USP4 or USP15, and through genetic crosses of mice with inactivating mutations we have confirmed that viability is contingent on a functional copy of USP4 or USP15. Loss of ubiquitin-exchange regulation, constitutive skipping of the seventh exon and neural-specific expression patterns are derived states of USP11. Post-translational modification sites differ between USP4, USP15 and USP11 throughout evolution.ConclusionsIn isolation sequence alignments can generate erroneous USP gene phylogenies. Through a combination of methodologies the gene duplication events that gave rise to USP4, USP15, and USP11 have been established. Although it operates in the same molecular pathways as the other USPs, the rapid divergence of the more recently generated USP11 enzyme precludes its functional interchangeability with USP4 and USP15. Given their multiplicity of substrates the emergence (and in some cases subsequent loss) of these USP paralogs would be expected to alter the dynamics of the networks in which they are embedded.

Project description:BackgroundFibrotic scar formation and inflammation are characteristic pathologies of spinal cord injury (SCI) in the injured core, which has been widely regarded as the main barrier to axonal regeneration resulting in permanent functional recovery failure. Pericytes were shown to be the main source of fibroblasts that form fibrotic scar. However, the mechanism of pericyte-fibroblast transition after SCI remains elusive.MethodsFibrotic scarring and microvessels were assessed using immunofluorescence staining after establishing a crush SCI model. To study the process of pericyte-fibroblast transition, we analyzed pericyte marker and fibroblast marker expression using immunofluorescence. The distribution and cellular origin of platelet-derived growth factor (PDGF)-BB were examined with immunofluorescence. Pericyte-fibroblast transition was detected with immunohistochemistry and Western blot assays after PDGF-BB knockdown and blocking PDGF-BB/PDGFRβ signaling in vitro. Intrathecal injection of imatinib was used to selectively inhibit PDGF-BB/PDGFRβ signaling. The Basso mouse scale score and footprint analysis were performed to assess functional recovery. Subsequently, axonal regeneration, fibrotic scarring, fibroblast population, proliferation and apoptosis of PDGFRβ+ cells, microvessel leakage, and the inflammatory response were assessed with immunofluorescence.ResultsPDGFRβ+ pericytes detached from the blood vessel wall and transitioned into fibroblasts to form fibrotic scar after SCI. PDGF-BB was mainly distributed in the periphery of the injured core, and microvascular endothelial cells were one of the sources of PDGF-BB in the acute phase. Microvascular endothelial cells induced pericyte-fibroblast transition through the PDGF-BB/PDGFRβ signaling pathway in vitro. Pharmacologically blocking the PDGF-BB/PDGFRβ pathway promoted motor function recovery and axonal regeneration and inhibited fibrotic scar formation. After fibrotic scar formation, blocking the PDGFRβ receptor inhibited proliferation and promoted apoptosis of PDGFRβ+ cells. Imatinib did not alter pericyte coverage on microvessels, while microvessel leakage and inflammation were significantly decreased after imatinib treatment.ConclusionsWe reveal that the crosstalk between microvascular endothelial cells and pericytes promotes pericyte-fibroblast transition through the PDGF-BB/PDGFRβ signaling pathway. Our finding suggests that blocking the PDGF-BB/PDGFRβ signaling pathway with imatinib contributes to functional recovery, fibrotic scarring, and inflammatory attenuation after SCI and provides a potential target for the treatment of SCI.

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Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFRβ and affect PDGF-BB-induced STAT3 signalling.

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