Regulation Of CyclinD1 Expression And Cell Proliferation By PKCa In Passively Sensitized Human Airway Smooth Muscle Cells

Asthma, a chronic disease characterized by airway hyperreactivity, inflammation, and remodeling, has increased dramatically in the prevalence [1] and is now recongnized as a major cause of disability, medical expense, and preventable death [2]. So far, many researches have confirmed a prominent role of airway smooth muscle (ASM) in the induction of airway remodeling in chronic asthma [3-6]. The abnormal proliferation of ASMC contributes to airway remodeling and hyper-reactivity characteristic of asthma. However, the underlying mechanism remains unclear.Protein kinase C (PKC) is a family of serine/threonine kinases that include three types of isoenzymes. The conventional isoforms (α, ?1, ?2, andγ) are activated by calcium, phorbol esters, and phosphatidylserine, whereas the novel isoforms (δ,ε,η,θ, andμ) are calcium-insensitive and activated by phorbol esters and phosphatidylserine. Theatypical isoforms (ζandτ/λ) are calcium- and phorbol ester–insensitive and activated by phosphatidylserine. PKCα, ?1, ?2,δ,ε, andζare expressed in human tracheal myocytes [7]. Differences between isoforms have been reported in tissue distribution and subcellular localization as well as in vitro kinase -activity characteristics. These isoforms are involved in the signaling pathways that regulate cell proliferation, differentiation, apoptosis, motility and adhesion [8-10].Many reports have demonstrated that activation of PKC is a necessary event for proliferation of ASMCs [11-15], and also different PKC isoforms may have distinct roles in the regulation of cell proliferation. In NIH3T3 cells, PKCεis a powerful growth stimulus, whereas PKCαandδinhibit growth [16]. PKCδalso inhibits cell cycle progression in A7r5 vascular smooth muscle cells [17], capillary endothelial cells [18], and rat colonic epithelial cells [19]. In vascular smooth muscle cells, overexpression of PKCδsuppressed G1 cyclin expression [17], consistent with the hypothesis that cyclin D1 expression is under the transcriptional control of PKCδ. In human, bovine, and rat tracheal smooth muscle cells, PKCδnegtively regulates cyclinD1 transcription [20]. Our laboratory has demonstrated that PKCαmay contribute to proliferation of asthmatic serum passively sensitized human airway smooth muscle cells [21].However, the downstream mechanism of PKCαregulation is unknown now. Progression of ASMC through the cell cycle is a fundamental event of cell proliferation. Recent studies have examined the signal transduction pathways that regulate specific cell cycle protein expression in ASM cells [22]. D-type cyclins (cyclinD1, D2, and D3) have been found to be the key regulators of G1 progression in mammalian cells. The cyclinD1 is one of the major targets for several growth stimulatory signaling pathways [23, 24].However, whether CyclinD1 play a critical role in regulating the HASMCs proliferation in asthma and whether PKCαregulates the proliferation of ASMC via cyclinD expression remains unknown. Therefore, in the present study we examined the possible roles of cyclin D1 andαisoforms of PKC, using human atopic asthmatic serum passively sensitized HASMCs as a model system.Methods1. HASMCs in culture were passively sensitized with 10% serum from asthmatic patients (group B), with non-asthmatic human serum treated HASMCs as the control (group A). The eukaryotic expression plasmids of sense and antisense cyclinD1 gene were constructed and transfected into HASMCs. The levels of cyclinD1 mRNA and protein were detected by RT-PCR and western blot analysis respectively. The proliferation of HASMCs was examined by cell cycle analysis, MTT colorimetric assay and proliferation cell nuclear antigen (PCNA) immunocytochemistry staining respectively.2. Confluent and synchronized HASMCs were treated with atopic asthmatic serum in the absence and presence of PMA for 24h, or pretreated 30min with Go6976 followed by PMA stimulation for 24h. Thereafter, the proliferation was measured by [3H]-thymidine incorporation and MTT. The cell cycle was analyzed by flow cytometry with propidium iodide staining. The cyclinD1 mRNA and protein expression was measured by RT-PCR and Western blotting. Further, we assessed the role of cyclinD1 in PKCαinduced HASMCs proliferation by cyclinD1 gene knock down using antisense cyclinD1 recombinant (PCDNA3.1(+)/ as-cyclinD1) transfection.3. HASMCs in culture were passively sensitized with 10% serum from asthmatic patients, stimulated with PKC activator PMA. The expression of PKCαand ERK1/2 were inhibited by transfected with PKCαantisense Oligodeoxynucleotides (PKCα-asODN) and treated with MAP Kinase Kinase (MEK) inhibitor U0126 respectively. Before and after this treatment, the levels of cyclinD1 and P21cip1 mRNA and protein were detected by RT-PCR and western blot analysis respectively. The proliferation of HASMCs was examined by cell cycle analysis and MTT colorimetric assay.4. HASMCs in culture were passively sensitized with 10% serum from asthmatic patients, stimulated with PΚC activator PMA. The expression of PΚCαand NF-κB activity were inhibited by transfected with PΚCαantisense Oligodeoxynucleotides (PΚCα-asODN) and treated with PDTC respectively. Before and after this treatment, the NF-κB activity of HASMCs was analyzed by Electrophoretic mobility gel shift assay (EMSA). The levels of cyclinD1 mRNA and protein were detected by RT-PCR and western blot analysis respectively. The proliferation of HASMCs was examined by cell cycle analysis and MTT colorimetric assay.Results1. In non-asthmatic serum treated HASMCs (group A), the percentage of S phase, absorbance value (A value) and the positive expression rate of PCNA were (10.52±1.50)%,0.303±0.024 and (39.4±8.53)% respectively. In asthmatic serum treated HASMCs (group B0), the percentage of S phase, absorbance value (A value) and the positive expression rate of PCNA were (15.94±2.13)%, 0.431±0.047and (53.2±7.5)% respectively. They were significantly increased compared with those of group A (n=5, P<0.05). In HASMCs transfected with sense cyclinD1 recombinant plasmid (group B2), the above figures were (26.10±2.16)%, 0.591±0.042 and(84.2±5.9)% respectively. They were significantly increased compared with those of group B0 (n=5, P<0.05). In HASMCs transfected with antisense cyclinD1 recombinant plasmid (group B3),the above figures were (6.96±1.25)%,0.220±0.027and (29.8±8.2)% respectively. They were significantly decreased compared with those of group B0 (n=5, P<0.05).2. Activation of PKCαwith PMA up-regulated cyclinD1 expression and increased the proliferation of passively sensitized HASMCs. This effect was significantly inhibited by specific inhibition of PKCαwith Go6976. In contrast, administration of PMA and Go6976 had no effect on cyclin-dependent kinase 4(CDK4) expressions. In addition, we showed that transfection with PCDNA3.1(+)/antisense-cyclinD1 abolished PMA-induced G1/S progression and HASMCs proliferation.3. After stimulated with PMA, the levels of p- PKCαand ERK1/2,p-ERK1/2 increased, the expression of cyclin D1,P21cip1 and cells proliferation enhanced compared with those of the control group(P<0.05,n=4). After transfected with PKCα-asODN, the level of p-PKCαdecreased, the levels of ERK1/2,p-ERK1/2 decreased correspondingly, and the expression of cyclin D1,P21cip1 and cells proliferation reduced compared with those of the PMA treated alone group (P<0.05,n=4). After administration of U0126 , the level of p-PKCαincreased but the levels of ERK1/2,p-ERK1/2 decreased, the expression of cyclin D1,P21cip1 and cells proliferation reduced compared with those of the PMA treated alone group (P<0.05,n=4).4. After stimulated with PMA, the levels of phosphorylated (p-PΚCα) and NF-κB activity increased, the expression of cyclin D1 and cells proliferation enhanced compared with those of the control group(P<0.05,n=4). After transfected with PΚCα-asODN, the level of p-PΚCαdecreased, the NF-κB activity decreased correspondingly, and the expression of cyclin D1 and cells proliferation reduced compared with those of the PMA treated alone group (P<0.05,n=4). After administration of PDTC , the level of p-PΚCα increased but the NF-κB activity decreased, the expression of cyclin D1 and cells proliferation reduced compared with those of the PMA treated alone group (P<0.05,n=4).Conclusions1. CyclinD1 contributes to the proliferation in in passively sensitized HASMCs. CyclinD1 may play a critical role in regulating the HASMCs proliferation in asthma.2. Protein kinase Cαpromoted the proliferation of atopic asthmatic sensitized human airway smooth muscle cells by up-regulating cyclin D1 expression3. ERK1/2 is one of downstream regulator of PKCα. PKCα-ERK1/2 cascade is involved in the PMA induced up-regulation of cyclinD1 and P21cip1 and proliferation in atopic asthmatic sensitized HASMCs4. NF-κB is one of downstream regulator of PΚCα. PΚCα-NF-κB cascade is involved in the PMA induced up-regulation of cyclinD1 and proliferation in atopic asthmatic sensitized HASMCsIn summary, all these findings indicated that PKC and CyclinD1 might take part in the proliferation of asthmatic HASMCs, in which there is PKCα-ERK1/2-cyclinD1,PΚCα-NF-κB-cyclinD1 signal pathway. To block this signal transduction pathway on various points is significant to suppress asthmatic HASMCs proliferation and relieve airway remodeling.The results should be helpful for further providing the novel clue for further elucidating the pathogenesis in asthma and finding the treatment in severe asthma...