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 (PKC alpha) 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-diphenyltetrazolium bromide (MU) assay and cell counting. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Activation of PKC alpha was measured by detecting the expression of PKC alpha protein in the cytosolic and membrane fractions using Western blotting analysis. Small interfering RNA (siRNA) was used to knockdown PKC alpha and cyclin D1. The cyclin D1 mRNA was assessed by real-time RT-PCR. The PKC alpha and cyclin D1 protein 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 PKC alpha activation. Inhibition of PKC alpha activity using Go 6976 or siRNA-mediated knockdown of PKC alpha 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 cell proliferation and G1/S transition was abolished by cyclin D1 siRNA. Moreover, Go 6976 or PKC alpha siRNA significantly suppressed CSE-induced upregulation of cyclin D1 at both the mRNA and protein levels. Conclusion PKC alpha-cyclin D1 pathway at least partially mediates the CSE-induced proliferation in HPASMCs. Chin Med J 2010;123(24):3663-3670