Deubiquitinase USP13 promotes extracellular matrix expression by stabilizing Smad4 in lung fibroblast cells

ABSTRACT Smad4 plays a central role in the regulation of extracellular matrix (ECM) protein expression and cell differentiation; however, the molecular regulation of Smad4 protein stability by a deubiquitinase has not been reported. In the current study, we reveal that a deubiquitinase USP13 stabilizes Smad4, ultimately modulating ECM protein expression in lung fibroblast cells. USP13 was increased in primary adult lung fibroblasts isolated from bleomycin-challenged mice and transforming growth factor (TGF)-β1-treated primary mouse lung fibroblasts. In a bleomycin-induced murine model of lung fibrosis, USP13 deficient mice showed reduced ECM levels such as fibronectin (FN) and collagen compared with wild type mice. The reductions in both protein levels and mRNA expression of ECM were observed in the isolated lung fibroblasts from USP13 deficient mice, suggesting that downregulation of USP13 reduces ECM levels through inhibiting its transcription. To investigate the molecular mechanisms by which USP13 modulates ECM expression, we focused on the role of USP13 on Smad4 expression. Overexpression of USP13 increased FN and Smad4 protein levels in lung fibroblasts, while downregulation of USP13 reduced Smad4 protein levels, without altering Smad4 mRNA expression, suggesting that USP13 regulates Smad4 protein stability. Knockdown of USP13 decreased Smad4 half-life and promoted Smad4 ubiquitination. Both Smad4 and USP13 were co-localized in the cytoplasm in treated cell, and co-translocated into the nucleus in response to TGF-β1. The results indicate that USP13 promotes ECM expression by stabilizing Smad4 in lung fibroblasts and plays a role in the maintenance of the extracellular matrix in lungs.