Cigarette Smoke Extract Induce Pulmonary Smooth Muscle Cells Proliferation And Its Mechanism

Objective:Pulmonary arterial hypertension(PAH)is a complex disease of small pulmonary arteries,which is characterized by sustained vasoconstriction,thickening of pulmonary artery walls,vascular remodeling,and progressive increase in pulmonary vascular resistance,leading to right ventricular failure and finally death.Enhanced pulmonary artery smooth muscle cell(PASMC)proliferation is a major cause of medial hypertrophy,vascular remodeling and vascular lumen narrowing.Among these risk factor,proliferation of PASMCs is thought to be an important pathological change in vascular remodeling that causes pulmonary hypertension.However,the precise mechanisms underlying this process remain unclear.Intracellular calcium plays an significant role in regulating vascular smooth muscle tone.An increase in intracellular Ca2+ concentration([Ca2+]i)activates contractile proteins and results in contraction.[Ca2+]i can be increased through the release of Ca2+from the sarcoplasmic reticulum(SR)and Ca2+ entry from extracellular space through voltage-operated Ca2+ channels(VOCCs),receptor-operated channels(ROCs)or store-operated channels(SOCs).Recently,Ca2+ entry through SOCC has gained considerable attention in vascular smoothmuscle research.SOCC is a highly selective calcium channel,its composition is not entirely clear,and dependent on different cell types,has been found stromal interaction molecule(STIM)ORAI?transient receptor potential non-selective cation channel(TRPC).TRPC channels are non-selective,Ca2+-permeable cation channels that are activated by stimulation of G proteins-coupled and Tyrosine phosphorylated receptors.There is evidence showing that several TRPC channels function as SOCC.However,the majority of the evidence relies on deletion of specific TRPC isoforms or their silencing by antisense or siRNA,rather than indicating directly their function as SOCs.An important showing was that comparable inhibition of Ca2+ influx occurs by KD of several individual TRPC channels.In general,TRPC1,4 and 5 are sensitive to store depletion and function as SOCs,whereas TRPC3,6 and 7 function as ROCs that are gated by G-protein-phospholipase C and diacylglycerol.Studies show that Orail plays an essential role in activation of SOCE in primary cultured proliferating hASMCs,whereas Orai2 and Orai3 do not contribute to SOCE A recent advance in the understanding of the potential molecular composition of SOCs has been the discovery of a transmembrane protein STIM1,which has been shown to mediate a well characterized store-operated current,the so-called calcium release activated calcium current(Icrac)in non-excitable cells.STIM family mainly includes two subtypes,STIM1 and STIM2.Suppression of STIM1 by treatment of HEK293 cells with STIM1-specific siRNAs inhibited thapsigargin-evoked Ca2+ entry and Ca2+influx induced by muscarinic receptor stimulation,without altering resting,Ca2+ release transients,Ca2+ levels or membrane potentials.In the anther hands,suppression of STIM2 had less of an effect on SOCE than did STIM1 knockdown.The signal peptide sequence at the amino terminus and one predicted transmembrane segment shown that the amino(N-)terminal portion of the protein faces the extracellular fluid or the lumen of the ER.The conserved N-terminal EF-hand motif suggested a possible role of Stiml in Ca2+ signaling,and since Stiml did not resemble any known channel we formulated the idea that it may serve as a Ca2+ sensor to trigger the process of SOCC activation.STIM1 was found to act as a sensor within the stores and also may play a role in the plasma membrane.Recently,Ca2+ entry through SOCC has gained considerable attention in vascular smooth muscle research.Although SOCE was observed for more than two decades,the molecular identity of SOCC has been controversial.Early day,the signaling pathway which relays SOCE and proliferation is largely unexplored.SOCE is an important cellular procedure associated with many cellular activities such as proliferation,migration,contraction,etc.previous studies in other cell types showed that blockage of SOCE inhibited cell proliferation,while cell proliferation is associated with an increased SOCE In PASMC,SOCC is mainly composed of the calcium receptor STIM1 expressed in the endoplasmic reticulum and the nonselective protein TRPC1 expressed in the cell membrane.SOCE formation of intracellular signal transduction pathways are as follows:when the intracellular,especially the sarcoplasmic reticulum storage of Ca2+ depletion,the endoplasmic reticulum transmembrane protein that calcium receptor STIM 1 clustered and separated from the endoplasmic reticulum binding site,Moving to the cell membrane,activation of the porogen TRPC1,and the formation of STIM1-TRPC1 complex,leading to open of Ca2+ channel,and thus the formation of SOCE.SOCE plays an important role in the proliferation and migration of PASMCs.SOCE blockers such as Ni2+ and SKF96365 can significantly slow the proliferation of PASMC.Cyclin D1 is an significant regulatory substance in the cell proliferation process.Being a member of D-type cyclin family,cyclin D1 promotes cell proliferation by means of encoding the rate-limiting step in transitions from the G1 to S phase During cell proliferation,cyclin D1 can act as a receptor for mitosis,and can also promote cell proliferation by driving the target cell through G1 phase.As a collaborative oncogene,the cyclin D1 gene is frequent over-expressed in human malignancy and is thought to play critical role in the development and maintenance of tumor.One model suggests that the overexpression of cyclin D1 may serve as a drive oncogene through its cell-cycle regulating function.Cyclin D1 is amplified and/or overexpressed in a significant proportion of different human tumors.Enhanced cyclin D1 abundance occurs relatively early during tumorigenesis And more and more recent reports indicate that cyclin D1 is an important downstream medium associated with pulmonary hypertension and can be involved in the remodeling of pulmonary arteries by meas of promoting the proliferation of PASMCs.Based on the above evidence,we hypothesized that the mechanism by which smoking promotes the proliferation of PASMCs is:CSE-induced up-regulation of SOCC expression in PASMC(enhancement of SOCE),followed by enhanced SOCE expression leading to increased proliferation of downstream cyclin D1 to promote cell proliferation,CSE)on the proliferation of human pulmonary artery smooth muscle cells(HPASMCs).In this study,we will research on the molecular mechanism of cell proliferation induced by smoking.Method:Part one:To study the effect of CSE on the proliferation of HPASMCs.HPASMCs were divided into 7 groups,0%(control group),0.5%,1%,2%,3%,5%,10%concentration of CSE treatment,HPASMCs proliferation will be observaed after 24 hours.Part two:To study the mRNA and protein expression levels of STIM1,TRPC1 and cyclinD1,and calcium ion concentration after CSE treatment.According to the experimental results of the first part,HPASMCs were divided into four groups:0(control group),0.5%CSE group,1%CSE group and 3%CSE group.STIM1,TRPC1 and cyclinD1 were detected by western blot and RT-PCR,and their mRNA and protein expression levels were observed for more intuitive observation of intracellular calcium concentration.intracellular calcium concentration was detected by the using of calcium probe on each group.Part three:Study the relationship between SOCC and cyclinDl.The proliferation of HPASMCs and the expression of cyclinDl mRNA and protein were detected by siRNA(TRPC1)and negative control.Result:Part one:0.5%and 1%CSE had the effect of promoting the proliferation of HPASMCs(P<0.05),and 0.5%CSE group was more effective(P<0.05),while 3%and above CSE had inhibitory effect on HPASMCs(P<0.05).Part two:the mRNA and protein expression levels of STIM1,TRPC1 and cyclin D1 in 0.5%and 1%CSE groups were significantly higher than those in the control group(P<0.05).The mRNA and protein expression levels of STIIM1,TRPC1 and cyclin D1 in 3%CSE group were decreased(P<0.05).Part three:The proliferation of HPASMCs and the expression of cyclinDl mRNA and protein in siRNA(TRPC1)transfection group were lower than those in negative control group(P<0.05).Conclusion:Part one:Low concentration of CSE can promote the proliferation of HPASMCs,while high concentrations of CSE inhibit HPASMCs proliferation.Part two:CSE enhances the proliferation of HPASMCs by SOCE enhancement(SOCC upturn).Part three:CSE induced HPASMCs proliferation at least partly via SOCE-mediated cyclin D1 expression.