Abstract
Cells are constantly influenced by their biochemical and physical microenvironments. Chemical ligands binding to their specific receptors under biochemical stimulation and mechanical forces regulating membrane mechanosensors, e.g., integrins, can transduce information into the cell and modulate cell functions. Thus, the modulation of cell signaling, gene expression, structure and function by both chemical and physical factors plays an important role in health and disease. To demonstrate the mechanisms by which chemical stimuli of transforming growth factor-β1 (TGF-β1) regulating the cell cycle and the differentiation of mesenchymal cells into smooth muscle cells (SMCs), we examined the role of TGF-β1-mediated cell cycle control and SMC phenotypic modulation of C3H10T1/2 (10T1/2) mesenchymal cells. Furthermore, peroxisome proliferator-activated receptors (PPARs) and their agonists have recently gained more attention in the study of the SMC function. In this study, the results showed the following: (1) the PI3K/Akt/p70S6K signaling cascade is involved in TGF-β1-induced differentiation of 10T1/2 cells into cells with a SMC phenotype. (2) PPAR-α agonists (i.e., WY14,643 and clofibrate), but not a PPAR-δ/β agonist (GW501516) or PPAR-γ agonist (troglitazone), inhibit TGF-β1-induced SMC markers and the DNA binding activity of serum response factor (SRF) in 10T1/2 cells. (3) WY14,643 and clofibrate inhibit the TGF-β1 activation of the Smad3/Akt/P70S6K signaling cascade. (4) TGF-β1-induced cell cycle arrest at the G0/G1 phases is mediated by Smad3 in 10T1/2 cells. (5) The PPAR-α-mediated 10T1/2 cell cycle arrest at the G0/G1 phases is TGF-β receptor independent. These results suggest that PPAR-α mediates cell cycle control and TGF-β1-induced SMC phenotypic changes in 10T1/2 cells. Mechanical forces, including interstitial fluid flow in and surrounding tissues, can modulate metastasis and invasion of tumor cells, and anticancer drug delivery. To elucidate the shear stress-regulated mechanism on tumor cell survival, four tumor cell lines (i.e., Hep3B, MG63, SCC25 and A549) were exposed to laminar or oscillatory flow at different magnitude of shear stress. We found that laminar shear stress (LSS) ranging from 0.5 to 12 dynes/cm2 induces death of these four tumor cell lines. Only laminar (0.5 dynes/cm2) but not oscillatory shear stress (0.5±4 dynes/cm2) could significantly induce the level of Hep3B hepatocarcinoma cell death. Both shear patterns had no effect on normal hepatocyte Chang liver cell line. LSS increased the percentage of cells positive for annexin V-FITC staining in all four cell lines up to 72 h after flow exposure, with cleaved caspase-8, -9, and -3, and PARP up-regulated by shear stress. In addition, LSS also induced Hep3B cell autophagy, as detected by acidic vesicular organelle formation, LC3B transformation, and p62/SQSTM1 degradation. By transiently transfecting small interfering RNA, we found that the shear-induced apoptosis and autophagy are mediated by bone morphogenetic protein receptor type 1B (BMPRIB), BMPR-specific Smad1 and Smad5, and p38 MAPK in Hep3B cells. In summary, our results indicate that TGF-βsuperfamily signaling may mediate the smooth muscle phenotypic change of mesenchymal cells and the mechanical flow force-induced tumor cell apoptosis and autophagy. Our findings also provide new insights into the mechanisms by which the mechanical microenvironment modulates molecular signaling, gene expression, cell survival, and functions in tumor cells. The modulation of TGF-β-superfamily signaling may be useful in establishing new approaches to the treatment of a variety of cardiovascular or tumor disorders. Moreover, the communication between mechanical-flow forces and BMP signaling may contribute to new research directions for treating tumor patients, and further detailed investigations are needed.