Cub domain-containing protein 1 negatively regulates TGF-β signaling and myofibroblast differentiation

Fibroblasts are thought to be the prime cell type for producing and secreting extracellular matrix (ECM) proteins in the connective tissue. The profibrotic cytokine transforming growth factor-beta 1 (TGF-beta 1) activates and transdifferentiates fibroblasts into alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts, which exhibit increased ECM secretion, in particular collagens. Little information, however, exists about cell-surface molecules on fibroblasts that mediate this transdifferentiation process. We recently identified, using unbiased cellsurface proteome analysis, Cub domain-containing protein 1 (CDCP1) to be strongly downregulated by TGF-beta 1. CDCP1 is a transmembrane glycoprotein, the expression and role of which has not been investigated in lung fibroblasts to date. Here, we characterized, in detail, the effect of TGF-beta 1 on CDCP1 expression and function, using immunofluorescence, FACS, immunoblotting, and siRNA-mediated knockdown of CDCP1. CDCP1 is present on interstitial fibroblasts, but not myofibroblasts, in the normal and idiopathic pulmonary fibrosis lung. In vitro, TGF-beta 1 decreased CDCP1 expression in a time-dependent manner by impacting mRNA and protein levels. Knockdown of CDCP1 enhanced a TGF-beta 1-mediated cell adhesion of fibroblasts. Importantly, CDCP1-depleted cells displayed an enhanced expression of profibrotic markers, such as collagen V or alpha-SMA, which was found to be independent of TGF-beta 1. Our data show, for the very first time that loss of CDCP1 contributes to fibroblast to myofibroblast differentiation via a potential negative feedback loop between CDCP1 expression and TGF-beta 1 stimulation.