title: Temporal phosphoproteomics to investigate the mechanotransduction of vascular smooth muscle cells in response to cyclic stretch.
abstract: Vascular smooth muscle cells (VSMCs) are exposed to mechanical cyclic stretch in vivo, which play important roles in maintenance of vascular homeostasis and regulation of pathological vascular remodeling. Reversible protein phosphorylation is crucial for intracellular signaling transduction. However, the dynamic phosphorylated profile induced by cyclic stretch in VSMCs is still unclear. Using the stable isotope labeling by amino acid in cell culture, VSMCs were labeled and exposed to 10% physiological cyclic stretch in vitro at 1.25 Hz for 0 min, 15 min, 30 min, 1 h and 6 h, respectively. Using TiO2 beads and liquid chromatography tandem mass spectrometry, the temporal phosphoproteomic profiles in response to cyclic stretch were then detected. Bioinformatics analysis including fuzzy c-means clustering, functional classifications, and Ingenuity Pathway Analysis were applied to further reveal the potential mechanotranduction networks. The results indicated that protein kinase C (PKCs) family, Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and Akt may participate in cyclic-stretch induced VSMC functions. Cyclic stretch repressed the expression of ROCK1, while it had no significant effect on the phosphorylation of PKCα/βII, PKCζ/λ and PKCδ/θ. PKCθ was activated first at short time-phase (15 min and 30 min), and again at long time-phase (6 h, 12 h and 24 h). The activation of p-PKCμ was immediate and short-term, similar to p-Akt. Our present in vitro work hence revealed that cyclic stretch activates complex mechanotransduction networks, suggesting that novel mechanoresponsive molecules, i.e., PKCθ, PKCμ, and ROCK1, may participate in the mechanotransduction and modulation VSMC functions.
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Yang
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Wang
authors.forename: Kai
authors.initials: K
authors.lastname: Huang
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.forename: Zong-Lai
authors.initials: ZL
authors.lastname: Jiang
authors.forename: Ying-Xin
authors.initials: YX
authors.lastname: Qi
mesh_terms: D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D006706:Homeostasis / Q000502:physiology; D066298:In Vitro Techniques; D008297:Male; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D023421:Models, Animal; D009124:Muscle Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D040901:Proteomics; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*; D013314:Stress, Mechanical; D013997:Time Factors; D054460:rho-Associated Kinases / Q000502:physiology
chemical_list: D009124:Muscle Proteins; D051057:Proto-Oncogene Proteins c-akt; C578418:ROCK1 protein, rat; D054460:rho-Associated Kinases; D011493:Protein Kinase C