Background/Purpose: Ubiquitination of proteins leads to their degradation by the proteasome, and is regulated by a small number of ubiquitin ligases, and a large number of susbtrate-specific ubiquitin-specific peptidases (USP). The ubiquitination process is not only key to protein recycling, and cell survival but plays also important roles in the regulation of cell metabolism and cell cycle. Here, we found that the expression of several USP is increased in systemic sclerosis tenosynovial biopsies, and we demonstrated that USP inhibition decreases TGFβ signaling in primary fibroblast cell lines.

Methods: HGU133 Plus2.0 arrays were hybridized with total RNA obtained from 5 tenosynovial samples obtained in patients with SSc and overt tenosynovial involvement, and compared to disease control synovial biopsies. Primary fibroblast cell lines were obtained from the skin of SSc patients or controls, and cultured in the presence of DMOG, a HIF1α hydroxylase inhibitor or in the presence of TGFβ +/- USP inhibitor NS-632839. HEK293 cells were stably transfected with an USP15-encoding expression plasmid.

Results: Transcriptomic profiles of tenosynovial biopsies from SSc patients were characterized by the expression of genes involved in known pathogenic pathways : fibrosis, Wnt signaling, but also by the over-expression of  several USP (USP15, 16, 18, 25, 31, 36, 37, 45, 48). Immunohistochemistry experiments on the same samples confirmed the presence of USP15 in SSc biopsies.

Culture of primary fibroblast cell lines in the presence of DMOG, a HIF1α-hydroxylase inhibitor that mimics the effects of hypoxia, results in a significant induction of TGFβ, USP25 and USP36 gene expression, but not USP15.

USP15 is known to specifically deubiquinate SMAD3, and we confirmed that over-expression of USP15 in HEK293 cells results in increased SMAD3, and SMAD3 phosphorylation in response to TGFβ stimulation. Conversely, exposure of cultured fibroblasts to NS-632839, a pan-USP inhibitor decreased SMAD3 expression and phosphorylation, and expression of COLIAI, COL3A1 and fibronectin gene expression in response to TGFβ stimulation. The effect of the USP inhibitor resulted in increased SMAD3 ubiquitination (as proven by SMAD3 immunoprecipitation experiments), and was blocked by epoxomicin, a proteasome inhibitor, thereby confirming the specificity of its action.

Conclusion: Over-expression of several USP, including USP15, is observed in tenosynovial biopsies from SSc patients, and is not fully explained by the effects of hypoxia. USP15 amplifies fibrotic responses induced by TGFβ, and is a potential therapeutic target in SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/over-expression-of-ubiquitin-specific-peptidases-in-systemic-sclerosis-fibroblasts-increases-responses-to-tgf-beta/