Abstract
Purpose :
CUB domain-containing protein 1 (CDCP1) is a transmembrane glycoprotein that activates downstream signaling pathways after the extracellular CUB1 domain is cleaved by serine proteases, including plasmin. It is implicated in various cellular processes and has been extensively studied in neoplastic progression and metastasis. However, potential functions of CDCP1 in normal (non-tumor) cells are largely unexplored. Previously, we found that corneal epithelial cells express a high level of CDCP1, and that CDCP1 deficiency significantly delays corneal epithelial wound closure in vitro and in vivo. This study examined the intrinsic mechanisms by which corneal epithelial CDCP1 contributes to wound healing.
Methods :
Targeted knockdown (KD) of CDCP1 gene expression in telomerase-immortalized human corneal epithelial (hTCEpi) cells was accomplished by siRNA. Anti-CDCP1 monoclonal antibody (41-2) was used to specifically abrogate cleavage activation at the cell surface. In vitro wound healing was assessed by scratch assay. Wound areas were measured by ImageJ analysis of pictures captured at time 0 and 20 h. Growth factors in culture supernatants and phosphorylated kinases in cell lysates were analyzed by antibody arrays, then validated by ELISA and immunoblotting.
Results :
In accordance with CDCP1-KD hTCEpi cells, WT cells treated with cleavage-blocking anti-CDCP1 antibody as compared to IgG exhibited a notable delay in scratch gap closure. Mechanistically, knockdown of CDCP1 reduced secretion of growth factors, including vascular endothelial growth factor-A (VEGF-A) by 3-fold, platelet-derived growth factor-AA (PDGF-AA) by 1.5-fold, amphiregulin by 2-fold, and epidermal growth factor (EGF) by 1.5-fold. Knockdown of CDCP1 also diminished phosphorylation of Src (Tyr419), protein kinase C delta (PKCδ) (Tyr311), and lysine-deficient protein kinase 1 (WNK1) (Thr60).
Conclusions :
The results strongly support a physiological role of CDCP1 cleavage-induced signaling in promoting growth factor release from corneal epithelial cells. As plasmin is one of several serine proteases known to cleave CDCP1, our findings are consistent with the major role of plasminogen activator system in wound healing. Topical application of plasminogen to enhance CDCP1 cleavage may provide benefits for the treatment of corneal ulcers.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.