Proteomic identification of ADAM12 as a regulator for TGF-ß1-induced differentiation of human mesenchymal stem cells to smooth muscle cells.

ABSTRACT

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) induces the differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle cells. Lipid rafts are cholesterol-rich microdomains in cell membranes that reportedly play a key role in receptor-mediated signal transduction and cellular responses. In order to clarify whether lipid rafts are involved in TGF-ß1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs. METHODS AND RESULTS: Pretreatment of hASCs with the lipid raft disruptor methyl-ß-cyclodextrin abrogated TGF-ß1-induced expression of α-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-ß1-induced differentiation of hASCs to smooth muscle cells. Sucrose density gradient centrifugation along with a shotgun proteomic strategy using liquid chromatography-tandem mass spectrometry identified 1002 individual proteins as the lipid raft proteome, and 242 of these were induced by TGF-ß1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-ß1 treatment. TGF-ß1 treatment of hASCs stimulated the production of both ADAM12 protein and mRNA. Silencing of endogenous ADAM12 expression using lentiviral small hairpin RNA or small interfering RNA abrogated the TGF-ß1-induced differentiation of hASCs into smooth muscle cells. CONCLUSIONS: These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-ß1-induced differentiation of hASCs into smooth muscle cells.