Background Exposure to cigarette smoke stimulates the proliferation of human pulmonary artery smooth muscle cells （HPASMCs） in vivo and in vitro. However, the molecular mechanism remains unclear. This study aimed at investigating the role of signaling pathways involving protein kinase C alpha （PKCα） and cyclin D1 in the cigarette smoke extract （CSE）-induced HPASMCs proliferation.Methods Synchronized HPASMCs were treated with different concentrations of CSE. Cell proliferation was evaluated by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyttetrazolium bromide （MTT） assay and cell counting. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Activation of PKCα was measured by detecting the expression of PKCαprotein in the cytosolic and membrane fractions using Western blotting analysis. Small interfering RNA （siRNA） was used to knockdown PKCα and cyclin D1. The cyclin D1 mRNA was assessed by real-time RT-PCR. The PKCα and cyclin D1protein levels were detected by Western blotting.Results Low concentrations of CSE （1%-10%） stimulated proliferation of HPASMCs, with its maximal effect at 5%.CSE （5%） led to PKCα activation. Inhibition of PKCα activity using G（o） 6976 or siRNA-mediated knockdown of PKCα significantly attenuated CSE-induced cell proliferation and G1/S transition. Cyclin D1, one of key regulators of G1/S transition, was found to be upregulated by 5% CSE at both the mRNA and protein levels. CSE-stimulated cellproliferation and G1/S transition was abolished by cyclin D1 siRNA. Moreover, G（o） 6976 or PKCα siRNA significantlysuppressed CSE-induced upregulation of cyclin D1 at both the mRNA and protein levels.Conclusion PKCα-cyclin D1 pathway at least partially mediates the CSE-induced proliferation in HPASMCs.
XIANG Min XU Yong-jian LIU Xian-sheng ZENG Da-xiong
The purpose of this study was to investigate the changes of protein kinase Ca （PKCα） and cyclin D1 expressions in pulmonary arteries from＇smokers with normal lung function and smokers with mild to moderate chronic obstructive pulmonary disease （COPD）. The peripheral lung tissues were obtained from 10 non-smokers with normal lung function （non-smoker group）, 14 smokers with normal lung function （smoker group）, 11 smokers with mild to moderate COPD （COPD group）. The morphological changes of pulmonary arteries were observed by HE-staining. The expressions of ct-smooth muscle actin （α-SMA）, proliferating cell nuclear antigen （PCNA）, PKCα and cyclin D1 proteins in pulmonary artery smooth muscle cells （PASMCs） were immunohistochemically determined. The percentages of PCNA-positive cells were taken as the smooth muscle cells proliferation index （PI）. The mRNA expressions of PKCα and cyclin D l in PASMCs were evaluated by real-time fluorescence PCR. Morphometrical analysis showed that the ratio of pulmonary artery wall area to total area （WA%） in smoker group and COPD group was significantly greater than that in non-smoker group （P〈0.01）. The PASMCs proliferation index in smoker group and COPD group was significantly higher than that in nonsmoker group （P〈0.01）. The protein levels of PKCct and cyclin D1 inPASMCs were significantly increased in smoker group and COPD group as compared with non-smoker group （P〈0.01）. The mRNA expressions of PKCα and cyclin D1 in PASMCs were significantly elevated in smoker group and COPD group as compared with non-smoker group （P〈0.01）. Significant correlations were found between PKCα protein and WA% or PI （P〈0.01）. Correlations between cyclin D1 protein and WA% or PI also existed （P〈0.01）. The expression of PKCa was positively correlated with the expression of cyclin D 1 at both protein and mRNA levels （P〈0.01）. In conclusion, increased expressions of PKCα and cyclin D1 might be involved in the patho
