The Role Of Cyclin D1 In Human Pulmonary Artery Smooth Muscle Cells Proliferation Induced By Cigarette Smoke And Its Relationship With Protein Kinase Cα Pathway

Background and ObjectiveChronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease, its prevalence rate is high, and so does the mortality it caused. Pulmonary vascular remodeling hinders the respiration process and may result in pulmonary hypertension, which is one of the principal factors on reducing the survival rate of COPD patients. Controlling pulmonary vascular remodeling can effectively prevent the formation of pulmonary hypertension, which is of great significance in the clinical treatment of COPD and cor pulmonale. Previous researchers mainly focused on the pathogenesis of hypoxic pulmonary hypertension and deem that hypoxia-induced pulmonary vasoconstriction and pulmonary vascular remodeling lead to the formation of pulmonary hypertension. In recent studies, it has been reported that pulmonary vascular remodeling occurs in smokers with normal lung functions and mild COPD patients without hypoxemia. Exposure to cigarette smoke for 6 months produces pulmonary hypertension in guinea pigs. These findings suggest that cigarette smoke may also promote pulmonary vascular remodeling, leading to pulmonary hypertension. The abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of major morphological features of pulmonary vascular remodeling induced by smoking. Cigarette smoke can result in an abnormal proliferation of pulmonary artery smooth muscle cells. However, the intrinsic mechanism has not been thoroughly understood yet.The proliferation of cells is closely related with cell cycle changes. While in a cell, cyclin plays a very important role in the regulation of cell cycle. In the Gl phase, cyclin D combines with cyclin-dependent kinase 4 (CDK4) or CDK6 to form complexes. These complexes phosphorylate retinoblastoma protein (Rb) and other family members. Phosphorylation of Rb activates the transcription factors and mediates the transcription of genes required for the cell to progress into S phase, which leads to the cell proliferation. Cyclin D1 is one of the major subtypes of cyclin D, and it regulates the G1/S phase transition. Cyclin D1 has been proven to be related to the proliferation of carcinoma cells. So far, few studies have been reported on the role of cyclin D1 in the vascular smooth-muscle cell proliferation. The effect of cyclin D1 on the proliferation of PASMCs remains to be understood.Protein kinase C (PKC), which is one of crucial members in serine-threonine kinase family, is a key intracellular signal transduction molecule. At present, PKC has been found with at least 12 isoforms, and functions of PKC isoforms in different cells vary greatly. Among the PKC isoforms, PKCαis generally reported to be a positive mediator for the vascular smooth muscle cells proliferation. Our previous study has shown that PKCαwas closely associated with the abnormal proliferation of the rat pulmonary arterial smooth muscle cells induced by cigarette smoking extract. However, the downstream mechanism of PKCαhas remained unclear so far. PKCαhas been implicated in the up-regulation of the cyclin D1 expression in several types of cultured cells. In HPASMCs, however, the existence of a similar mechanism needs to be proven.In this study, we investigate the pathobiology of human pulmonary artery smooth muscle cell proliferation induced by cigarette smoke from the perspective of the cell cycle control and the intracellular signal transduction. Our study may provide the basis for a new therapeutic approach for addressing COPD associated with pulmonary hypertension. Part one The changes of protein kinase Ca and cyclin D1 expressions in pulmonary artery from smokers with and without chronic obstructive pulmonary diseaseObjective The purpose of this study was to investigate the changes of protein kinase Ca (PKCa) and cyclin D1 expressions in pulmonary artery from smokers with normal lung function and smokers with mild to moderate chronic obstructive pulmonary disease (COPD). Methods The peripheral lung tissues were obtained from 10 nonsmokers with normal lung function (nonsmoker 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 hematoxylin-eosin staining. The expressions of a-smooth muscle actin (a-SMA), proliferating cell nuclear antigen (PCNA), PKCa and cyclin D1 proteins in pulmonary artery smooth muscle (PASM) were determined by immunohistochemical staining. The percentages of PCNA-positive cells were considered as the smooth muscle cells proliferation index (PI). The mRNA expressions of PKCa and cyclin D1 in PASM were evaluated by real-time fluorescence PCR. Results Morphometry 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 nonsmoker group (P< 0.01). The PASMCs proliferation index in smoker group and COPD group was obviously higher than that in nonsmoker group (P< 0.01). The protein levels of PKCa and cyclin D1 in PASMCs were markedly increased in smoker group and COPD group as compared with nonsmoker group (P< 0.01). The mRNA expressions of PKCa and cyclin D1 in PASMCs were significantly elevated in smoker group and COPD group as compared with nonsmoker group(P< 0.01). Significant correlations were found between PKCa 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 positively correlated with the expression of cyclin D1 at both protein and mRNA levels (P< 0.01). Conclusions Increased expressions of PKCa and cyclin D1 might be involved in the pathogenesis of abnormal proliferation of PASMCs in smokers with normal lung function and smokers with mild to moderate COPD.Part two The role of cyclin D1 in human pulmonary artery smooth muscle cells proliferation induced by cigarette smoke extractObjective The present study was aimed to investigate the role of cyclin D1 in human pulmonary artery smooth muscle cells (HPASMCs) proliferation induced by cigarette smoke extract (CSE). Methods Synchronized HPASMCs were treated with different concentrations of CSE (1%,2.5%,5%,10%,20%). The antisense eukaryotic expression vector of cyclin D1 gene (pIRES2-EGFP-ascyclin D1) was recombinated. The recombinant and empty vector were separately transfected into normal HPASMCs using liposome. Then the cells were treated with or without 5% CSE. The cells were randomly divided into six groups:control group, vector group, antisense cyclin D1 group,5% CSE treated group, vector+5% CSE treated group and antisense cyclin Dl+5% CSE treated group. The proliferation of HPASMCs was examined by cell cycle analysis, MTT assay and proliferation cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of cyclin D1 mRNA and protein were detected by real-time fluorescence RT-PCR and Western blot, respectively. Results Low concentrations of CSE (1%,2.5%,5%,10%) stimulated proliferation of HPASMCs, with its maximal effect at concentration of 5%. On the contrary, high concentrations of CSE (20%) were the inhibitory of cell proliferation as a result of cytotoxicity. The antisense eukaryotic expression vector of cyclin D1 gene was constructed and transfected into HPASMCs successfully. The cyclin D1 mRNA and protein levels in antisense cyclin D1 group were significantly lower than those in control group (P < 0.05). In 5% CSE group, the cyclin D1 mRNA and protein levels were elevated significantly compared with those in control group (P< 0.05), and the indicators of cell proliferation were increased as well (P<0.05). The cyclin D1 mRNA and protein levels, the indicators of cell proliferation in antisense cyclin D1+5% CSE group were remarkably lower than those in 5% CSE group (P< 0.05). Conclusions CSE could promote HPASMCs proliferation through up-regulation of cyclin D1 expression. PIRES2-EGFP-ascyclin D1 could attenuate CSE-induced proliferation of HPASMCs by suppressing the expression of cyclin D1, which implicates that cyclin D1 might be involved in the process of HPASMCs proliferation stimulated by CSE.Part three Cigarette smoke extract promotes human pulmonary artery smooth muscle cells proliferation through protein kinase Cα-dependent induction of cyclin D1Objective 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 was aimed to investigate 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 5% CSE. Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and cell counting. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Activation of PKCαwas measured by detecting the expression of PKCαprotein in the cytosolic and membrane fractions using Western blot analysis. Small interference RNA (siRNA) was used to knockdown PKCa and cyclin D1. The cyclin D1 mRNA was assessed by real-time RT-PCR. The PKCa and cyclinDl protein levels were detected by Western blotting. Results CSE (5%) led to PKCa activation. Inhibition of PKCa activity by using Go 6976 or siRNA-mediated knockdown of PKCa 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 CSE (5%) at both mRNA and protein levels. CSE-stimulated cell proliferation and G1/S transition was markedly abolished by cyclin Dl siRNA. Moreover, Go 6976 or PKCa siRNA significantly suppressed CSE-induced upregulation of cyclin Dl at both mRNA and protein levels. Conclusions Cigarette smoke extract promotes HPASMCs proliferation through protein kinase C alpha-dependent induction of cyclin D1...