CDCP1 knockdown suppresses PDGFRβ/AKT pathway-mediated vascular smooth muscle cell proliferation by inhibiting PDGFRβ endocytosis.

CUB domain-containing protein 1 (CDCP1) is a type of cell surface glycoprotein that has been identified as being capable of regulating cell anchorage, migration, proliferation, and differentiation. However, the contributions of CDCP1 in intimal hyperplasia, specifically regarding the proliferation and migration of vascular smooth muscle cells (VSMC), are unclear. In this study, we analyzed CDCP1 expression on intimal hyperplasia through a focal carotid stenosis model in vivo. In vitro, we cultured mouse VSMCs and stimulated them with 20 ng/mL platelet-derived growth factor BB (PDGF-BB) for 24 h. Western blot analysis was performed to detect the expression of CDCP1 in the cells. Next, we knocked down the expression of CDCP1 in VSMCs and assessed its effects on cell proliferation and migration using CCK8 assays, EDU(+) assay, and wound healing experiments. We then performed RNA-Seq analysis on the CDCP1-knockdown VSMCs. Based on the sequencing results, we utilized western blotting to investigate the impact of CDCP1 knockdown on the AKT signaling pathway. Additionally, we validated the interactions between Platelet-derived growth factor receptor (PDGFR)β with NEDD4, clathrin, and Rab5 using immunofluorescence and co-immunoprecipitation assays. The results discovered that CDCP1 levels were activated in the intimal hyperplasia tissues in vivo. CDCP1 knockdown significantly attenuated mouse VSMC proliferation and migration induced by PDGF-BB in vitro. Based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed proteins obtained from RNA-sequencing, we found that the knockdown of CDCP1 is related to the "PI3K-AKT signaling pathway", "ubiquitin-mediated proteolysis", and "endocytosis" pathways. The subsequent experiments demonstrated that CDCP1 knockdown inhibited AKT signaling pathway. CDCP1 knockdown promoted the binding of PDGFRβ and NEDD4, and PDGFRβ ubiquitin. Moreover, CDCP1 knockdown attenuated the binding of PDGFRβ to clathrin and Rab5. These data reveal that the absence of CDCP1 may enhance the binding of PDGFR to NEDD4 and promote the ubiquitination of PDGFR, thereby regulating the AKT signaling pathway and intimal hyperplasia.