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-β1 (TGF-β1) 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-β1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs.


RESULTS
Pretreatment of hASCs with the lipid raft disruptor methyl-β-cyclodextrin abrogated TGF-β1-induced expression of α-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-β1-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-β1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-β1 treatment. TGF-β1 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-β1-induced differentiation of hASCs into smooth muscle cells.


CONCLUSIONS
These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-β1-induced differentiation of hASCs into smooth muscle cells.
Medical Research Center for Ischemic Tissue Regeneration, School of Medicine, Pusan National University, Yangsan, Republic of Korea.
Young Mi
YM
Kim
Jaeyoon
J
Kim
Soon Chul
SC
Heo
Sang Hun
SH
Shin
Eun Kyoung
EK
Do
Dong-Soo
DS
Suh
Ki-Hyung
KH
Kim
Man-Soo
MS
Yoon
Taehoon G
TG
Lee
Jae Ho
JH
Kim
D051722:ADAM Proteins / Q000378:metabolism*; D000072199:ADAM12 Protein; D000199:Actins / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology; D051242:Caveolin 1 / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002784:Cholesterol / Q000172:deficiency / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D051899:Smad2 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
C541116:ACTA2 protein, human; D000199:Actins; D051242:Caveolin 1; D008565:Membrane Proteins; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D053773:Transforming Growth Factor beta1; D002784:Cholesterol; D051722:ADAM Proteins; D000072199:ADAM12 Protein; C000606480:ADAM12 protein, human