Proteomic Identification of ADAM12 as a Regulator for TGF-β1-Induced Differentiation of Human Mesenchymal Stem Cells to Smooth Muscle Cells

Background: Transforming growth factor-beta 1 (TGF-beta 1) 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-beta 1-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-beta-cyclodextrin abrogated TGF-beta 1-induced expression of alpha-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-beta 1-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-beta 1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-beta 1 treatment. TGF-beta 1 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-beta 1-induced differentiation of hASCs into smooth muscle cells. Conclusions: These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-beta 1-induced differentiation of hASCs into smooth muscle cells.