哮喘人气道平滑肌细胞中蛋白激酶Ca对周期蛋白D1的影响及其对细胞增殖调控的研究
|
摘要
哮喘是一种以气道高反应性,气道炎症和气道重建为特征的慢性疾病,目前患病率显著性增高[1],并且被认为是丧失劳动能力、医疗消费和可预防性死亡的主要原因之一[2]。到目前为止许多研究机构已经证实:在慢性哮喘,气道平滑肌在诱导气道重建中起着主要作用[3-6]。气道平滑肌细胞的反常增殖导致了气道重建和气道高反应性,但是其具体的机制目前还不很清楚。
蛋白激酶C(PKC)是一丝氨酸-苏氨酸激酶家族,它包括三种类型的异构体。传统型(α, ?1, ?2,和γ)可被钙、佛波酯和磷脂酰丝氨酸所激活;新型(δ,ε,η,θ, andμ)对钙不敏感,但可被佛波酯和磷脂酰丝氨酸所激活;不典型型(ζandτ/λ)对钙和佛波酯不敏感,但可被磷脂酰丝氨酸激活。人气道平滑肌细胞表达PKCα, ?1, ?2,δ,ε, andζ[7]。不同的异构体的体外激酶活性特征以及组织分布、亚细胞结构是有差别的。这些异构体参与信号通路,调节细胞增殖、分化、凋亡、迁徙和粘附[8-10]。许多研究者已经证明:PKC的激活是ASMCs增殖的必需事件[11-15],并且,不同的PKC异构体在调节细胞增殖中起着不同的作用。在NIH3T3细胞,PKCε是一强有力的生长刺激因素,而PKCα和δ抑制生长[16]。在A7r5血管平滑肌细胞[17],毛细血管内皮细胞[18]和鼠结肠上皮细胞[19],PKCδ也抑制细胞周期进展。在血管平滑肌细胞,PKCδ的过度表达抑制G1期周期蛋白的表达[17],这与PKCδ控制周期蛋白D1的转录的假设一致。在人,牛和鼠气道平滑肌细胞,PKCδ负性调节cyclinD1的转录[20]。我们实验室已经证实:PKCα参与哮喘血清被动致敏的人气道平滑肌细胞增殖[21]。
然而,PKCα调节的下游机制目前还不清楚。细胞周期进展是细胞增殖的的基本事件,目前已经有一些关于ASMCs中调节特殊细胞周期蛋白表达的信号转导通路的研究[22]。cyclinD(D1,D2和D3)是哺乳动物细胞G1期进展的关键调节蛋白,cyclinD1是许多生长刺激信号通路的靶基因[23,24]。
那么,在哮喘人气道平滑肌细胞,cyclinD1是否起关键作用?PKCα是否可通过调节cyclinD1表达来影响ASMC增殖?为此,我们利用哮喘血清被动致敏的细胞模型来研究这些问题。
研究内容
1.用10%哮喘患者血清被动致敏人气道平滑肌细胞(HASMCs)(B组),以10%非哮喘者血清为对照(A组)。构建cyclinD1正、反义真核表达载体质粒并转染干预HASMC。用RT-PCR方法以及Western Blot方法检测cyclinD1的mRNA及蛋白表达水平,用流式细胞术、四甲基偶氮唑盐(MTT)法及增殖细胞核抗原(PCNA)免疫细胞化学技术检测HASMC增殖。
2.生长融合、同步后的HASMCs用哮喘血清致敏后,分为对照组,PMA组和PMA+Go6976组。干预24小时后,用3H-TdR掺入法和MTT法检测细胞增殖;用碘化丙啶单染法流式细胞术分析细胞周期;用RT-PCR和western blotting检测cyclinD1mRNA和蛋白的表达。进一步,我们还通过转染重组的反义cyclinD1质粒(PCDNA3.1(+)/as-cyclinD1)来降低cyclinD1基因的表达后,检测PKCα对HASMCs增殖的影响。
3.用10%哮喘患者血清被动致敏人气道平滑肌细胞(HASMCs),予以PKC激活剂PMA刺激。分别以反义寡核苷酸(PKCα-asODN)和特异性阻断剂U0126抑制PKCα和ERK的表达,用RT-PCR方法及Western Blot方法检测干预前后cyclinD1和P21cip1的mRNA及蛋白表达水平,用流式细胞术和四甲基偶氮唑盐(MTT)法检测HASMCs增殖。
4.用10%哮喘患者血清被动致敏人气道平滑肌细胞(HASMCs),予以PKC激活剂PMA刺激。分别以反义寡核苷酸(PKCα-asODN)和PDTC抑制PKCα表达和NF-κB活化。用凝胶电泳迁移率改变试验(EMSA)检测HASMCs中NF-κB的活性,用RT-PCR方法及Western Blot方法检测干预前后cyclinD1的mRNA及蛋白表达水平,用流式细胞术和四甲基偶氮唑盐(MTT)法检测HASMCs增殖。
结果
1.正常血清对照组细胞S期细胞比例、MTT吸光度值(A值)、PCNA表达阳率分别为(10.52±1.50)%、0.303±0.024和(39.4±8.53)%;哮喘血清组(B0组)细胞S期细胞比例、MTT吸光度值(A值)、PCNA表达阳率分别为(15.94±2.13)%、0.431±0.047和(53.2±7.5)%,均较对照血清组(A组)明显增加(n=5,P<0.05);转染重组cyclinD1正义质粒组(B2组)细胞S期细胞比例、MTT吸光度值(A值)、PCNA表达阳性分别为(26.10±2.16)%、0.591±0.042和(84.2±5.9)%,均较哮喘血清组(B0组)明显增加(n=5,P<0.05);而转染反义质粒组(B3组)上述结果分别为(6.96±1.25)%、0.220±0.027和(29.8±8.2)%,均较哮喘血清组(B0组)明显下降(n=5,P<0.05)。
2. PMA激活PKCα后上调cyclinD1表达和细胞增殖。而用Go6976特异性抑制PKCα活性后,这种促cyclinD1表达和细胞增殖的作用显著性下降。相反, PMA和Go6976对周期蛋白依赖性激酶4(CDK4)的表达无明显影响。另外,转染反义cyclinD1质粒后消除了PMA诱导的G1/S期进展和HASMCs增殖效应。
3. PMA刺激后磷酸化PKCα(p-PKCα)水平增高,ERK1/2,p-ERK1/2蛋白水平增高,cyclin D1、P21cip1表达明显增强, HASMCs的增殖增强;而反义PKCα寡核苷酸转入细胞抑制p-PKCα表达后,ERK1/2,p-ERK1/2表达明显减弱,cyclin D1、P21cip1表达也明显下降,HASMCs的增殖减弱(P<0.05,n=4).;用U0126抑制ERK1/2表达后,PMA刺激下p-PKCα水平仍然明显增高,但cyclin D1、P21cip1的表达明显下降,HASMCs的增殖减弱(P<0.05,n=4).。
4. PMA刺激后磷酸化PKCα(p-PKCα)水平增高,NF-κB-DNA结合活性增强,cyclin D1表达明显增强, HASMCs的增殖增强;而反义PKCα寡核苷酸转入细胞特异性地抑制PΚCα表达后,p-PKCα水平下降,NF-κB-DNA结合活性明显减弱,cyclin D1也明显下降,HASMCs的增殖减弱(P<0.05,n=4);用PDTC抑制NF-κB活性后,PMA刺激下p-PΚCα水平仍然明显增高,但cyclin D1的表达明显下降,HASMCs的增殖减弱(P<0.05,n=4)。
结论
1. cyclinD1促进哮喘血清被动致敏的HASMC的增殖。cyclinD1可能是哮喘HASMC增殖的重要调控蛋白。
2. PKCα可通过上调cyclinD1表达而促进哮喘血清被动致敏的人气道平滑肌细胞增殖。
3. ERK1/2是PKCα的下游信号分子,PKCα-ERK1/2级联参与了PMA所诱导的哮喘血清被动致敏的人气道平滑细胞cyclin D1、P21cip1的表达上调及细胞增殖。
4. NF-κB是PKCα的下游信号分子,PΚCα- NF-κB级联参与了PMA所诱导的哮喘血清被动致敏的人气道平滑细胞cyclin D1的表达上调及细胞增殖。
综上所述,上述结果表明:PKCα、cyclinD1参与了哮喘HASMC的增殖调控,并存在PKCα-ERK1/2-cyclinD1、PΚCα-NF-κB-cyclinD1信号转导途径,从不同环节阻断这一信号途径对于针对性地抑制哮喘气道平滑肌增殖、减轻气道重建可能具有积极意义。这为研究哮喘气道重建的发病机理提供新的理论,也为将来治疗难治性哮喘提供一定的新思路。
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.
Methods
1. 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.
Results
1. 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).
Conclusions
1. 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 expression
3. 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 HASMCs
4. 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 HASMCs
In 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
蛋白激酶C(PKC)是一丝氨酸-苏氨酸激酶家族,它包括三种类型的异构体。传统型(α, ?1, ?2,和γ)可被钙、佛波酯和磷脂酰丝氨酸所激活;新型(δ,ε,η,θ, andμ)对钙不敏感,但可被佛波酯和磷脂酰丝氨酸所激活;不典型型(ζandτ/λ)对钙和佛波酯不敏感,但可被磷脂酰丝氨酸激活。人气道平滑肌细胞表达PKCα, ?1, ?2,δ,ε, andζ[7]。不同的异构体的体外激酶活性特征以及组织分布、亚细胞结构是有差别的。这些异构体参与信号通路,调节细胞增殖、分化、凋亡、迁徙和粘附[8-10]。许多研究者已经证明:PKC的激活是ASMCs增殖的必需事件[11-15],并且,不同的PKC异构体在调节细胞增殖中起着不同的作用。在NIH3T3细胞,PKCε是一强有力的生长刺激因素,而PKCα和δ抑制生长[16]。在A7r5血管平滑肌细胞[17],毛细血管内皮细胞[18]和鼠结肠上皮细胞[19],PKCδ也抑制细胞周期进展。在血管平滑肌细胞,PKCδ的过度表达抑制G1期周期蛋白的表达[17],这与PKCδ控制周期蛋白D1的转录的假设一致。在人,牛和鼠气道平滑肌细胞,PKCδ负性调节cyclinD1的转录[20]。我们实验室已经证实:PKCα参与哮喘血清被动致敏的人气道平滑肌细胞增殖[21]。
然而,PKCα调节的下游机制目前还不清楚。细胞周期进展是细胞增殖的的基本事件,目前已经有一些关于ASMCs中调节特殊细胞周期蛋白表达的信号转导通路的研究[22]。cyclinD(D1,D2和D3)是哺乳动物细胞G1期进展的关键调节蛋白,cyclinD1是许多生长刺激信号通路的靶基因[23,24]。
那么,在哮喘人气道平滑肌细胞,cyclinD1是否起关键作用?PKCα是否可通过调节cyclinD1表达来影响ASMC增殖?为此,我们利用哮喘血清被动致敏的细胞模型来研究这些问题。
研究内容
1.用10%哮喘患者血清被动致敏人气道平滑肌细胞(HASMCs)(B组),以10%非哮喘者血清为对照(A组)。构建cyclinD1正、反义真核表达载体质粒并转染干预HASMC。用RT-PCR方法以及Western Blot方法检测cyclinD1的mRNA及蛋白表达水平,用流式细胞术、四甲基偶氮唑盐(MTT)法及增殖细胞核抗原(PCNA)免疫细胞化学技术检测HASMC增殖。
2.生长融合、同步后的HASMCs用哮喘血清致敏后,分为对照组,PMA组和PMA+Go6976组。干预24小时后,用3H-TdR掺入法和MTT法检测细胞增殖;用碘化丙啶单染法流式细胞术分析细胞周期;用RT-PCR和western blotting检测cyclinD1mRNA和蛋白的表达。进一步,我们还通过转染重组的反义cyclinD1质粒(PCDNA3.1(+)/as-cyclinD1)来降低cyclinD1基因的表达后,检测PKCα对HASMCs增殖的影响。
3.用10%哮喘患者血清被动致敏人气道平滑肌细胞(HASMCs),予以PKC激活剂PMA刺激。分别以反义寡核苷酸(PKCα-asODN)和特异性阻断剂U0126抑制PKCα和ERK的表达,用RT-PCR方法及Western Blot方法检测干预前后cyclinD1和P21cip1的mRNA及蛋白表达水平,用流式细胞术和四甲基偶氮唑盐(MTT)法检测HASMCs增殖。
4.用10%哮喘患者血清被动致敏人气道平滑肌细胞(HASMCs),予以PKC激活剂PMA刺激。分别以反义寡核苷酸(PKCα-asODN)和PDTC抑制PKCα表达和NF-κB活化。用凝胶电泳迁移率改变试验(EMSA)检测HASMCs中NF-κB的活性,用RT-PCR方法及Western Blot方法检测干预前后cyclinD1的mRNA及蛋白表达水平,用流式细胞术和四甲基偶氮唑盐(MTT)法检测HASMCs增殖。
结果
1.正常血清对照组细胞S期细胞比例、MTT吸光度值(A值)、PCNA表达阳率分别为(10.52±1.50)%、0.303±0.024和(39.4±8.53)%;哮喘血清组(B0组)细胞S期细胞比例、MTT吸光度值(A值)、PCNA表达阳率分别为(15.94±2.13)%、0.431±0.047和(53.2±7.5)%,均较对照血清组(A组)明显增加(n=5,P<0.05);转染重组cyclinD1正义质粒组(B2组)细胞S期细胞比例、MTT吸光度值(A值)、PCNA表达阳性分别为(26.10±2.16)%、0.591±0.042和(84.2±5.9)%,均较哮喘血清组(B0组)明显增加(n=5,P<0.05);而转染反义质粒组(B3组)上述结果分别为(6.96±1.25)%、0.220±0.027和(29.8±8.2)%,均较哮喘血清组(B0组)明显下降(n=5,P<0.05)。
2. PMA激活PKCα后上调cyclinD1表达和细胞增殖。而用Go6976特异性抑制PKCα活性后,这种促cyclinD1表达和细胞增殖的作用显著性下降。相反, PMA和Go6976对周期蛋白依赖性激酶4(CDK4)的表达无明显影响。另外,转染反义cyclinD1质粒后消除了PMA诱导的G1/S期进展和HASMCs增殖效应。
3. PMA刺激后磷酸化PKCα(p-PKCα)水平增高,ERK1/2,p-ERK1/2蛋白水平增高,cyclin D1、P21cip1表达明显增强, HASMCs的增殖增强;而反义PKCα寡核苷酸转入细胞抑制p-PKCα表达后,ERK1/2,p-ERK1/2表达明显减弱,cyclin D1、P21cip1表达也明显下降,HASMCs的增殖减弱(P<0.05,n=4).;用U0126抑制ERK1/2表达后,PMA刺激下p-PKCα水平仍然明显增高,但cyclin D1、P21cip1的表达明显下降,HASMCs的增殖减弱(P<0.05,n=4).。
4. PMA刺激后磷酸化PKCα(p-PKCα)水平增高,NF-κB-DNA结合活性增强,cyclin D1表达明显增强, HASMCs的增殖增强;而反义PKCα寡核苷酸转入细胞特异性地抑制PΚCα表达后,p-PKCα水平下降,NF-κB-DNA结合活性明显减弱,cyclin D1也明显下降,HASMCs的增殖减弱(P<0.05,n=4);用PDTC抑制NF-κB活性后,PMA刺激下p-PΚCα水平仍然明显增高,但cyclin D1的表达明显下降,HASMCs的增殖减弱(P<0.05,n=4)。
结论
1. cyclinD1促进哮喘血清被动致敏的HASMC的增殖。cyclinD1可能是哮喘HASMC增殖的重要调控蛋白。
2. PKCα可通过上调cyclinD1表达而促进哮喘血清被动致敏的人气道平滑肌细胞增殖。
3. ERK1/2是PKCα的下游信号分子,PKCα-ERK1/2级联参与了PMA所诱导的哮喘血清被动致敏的人气道平滑细胞cyclin D1、P21cip1的表达上调及细胞增殖。
4. NF-κB是PKCα的下游信号分子,PΚCα- NF-κB级联参与了PMA所诱导的哮喘血清被动致敏的人气道平滑细胞cyclin D1的表达上调及细胞增殖。
综上所述,上述结果表明:PKCα、cyclinD1参与了哮喘HASMC的增殖调控,并存在PKCα-ERK1/2-cyclinD1、PΚCα-NF-κB-cyclinD1信号转导途径,从不同环节阻断这一信号途径对于针对性地抑制哮喘气道平滑肌增殖、减轻气道重建可能具有积极意义。这为研究哮喘气道重建的发病机理提供新的理论,也为将来治疗难治性哮喘提供一定的新思路。
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.
Methods
1. 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.
Results
1. 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).
Conclusions
1. 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 expression
3. 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 HASMCs
4. 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 HASMCs
In 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
引文
1. Beasley R, Crane J, Lai CK, Pearce N. prevalence and etiology of asthma. J Allergy Chin Immunol 2000;10S: S466-S472
2. Masoli M, Fabian D, Holt S, Beasley R. The global burden of asthma: executive summary of the GINA dissemination committee report. Allergy2004; 59: 467-478
3. Chung, KF. Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation? Eur Respir J 2000; 15: 961-968
4. Elias, JA, Zhu Z, Chupp G, and Homer RJ.Airway remodeling in asthma. J Clin Invest 1999;104: 1001-1006
5. Fredberg, JJ. Airway smooth muscle in asthma. Perturbed equilibria of myosin binding. Am J Respir Crit Care Med 2000; 161: 158S-160S
6. Que, CL, Maksym G, and Macklem PT. Deciphering the homeokinetic code of airway smooth muscle. Am J Respir Crit Care Med 2000; 161: 161S-163S
7. Webb BL, Lindsay MA, Barnes PJ, et al. Protein kinase C isoenzymes in airway smooth muscle. Biochem. J 1997;324: 167-175
8. Dempsey EC, Newton AC, Mochly-Rosen D, et al. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 2000;279:L429-38
9. Parker PJ, Murray-Rust J.PKC at a glance. J Cell Sci 2004;117: 131–132
10. Michie AM, Nakagawa R. The link between PKCalpha regulation and cellular transformation. Immunol Lett. 2005; 96:155-62
11. Panettieri RA, Rubinstein NA, Kelly AM, et al.The specificity of c-fos expression in the induction of airway smooth muscle proliferation by contractile agonists. Am Rev Respir Dis 1991;143: A934
12. Lew DB, Brown ER, Dempsey K, et al. Contribution of PKC toβ-hexosaminidase-induced airway smooth muscle proliferation. Am J Physiol 1997; 272: L639-L643.
13. Hirst SJ, Webb BLJ, Giembycz MA, et al. Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of protein kinase C and protein tyrosine kinase. Am J Respir Cell Mol Biol 1995;12: 149-161
14. Hirst SJ, Barnes PJ, Twort CHC. Protein kinase inhibitors (PKC and PTK) inhibit PDGF isoform-stimulated proliferation of human and rabbit airway smooth muscle. Am J Respir Crit Care Med 1995;151: A46
15. Hirst SJ, Barnes PJ, Twort CHC. Differential proliferation of rabbit tracheal smooth muscle by PDGF isoforms: Inhibition by selective inhibitors of protein kinase and protein tyrosine kinase. Br J Pharmacol 1994; 112: 81P
16. Acs P, Wang QJ, Bogi K, et al. Both the catalytic and regulatory domains of protein kinase C chimeras modulate the proliferative properties of NIH3T3 cells. J Biol Chem1997; 272: 28793–28799.
17. Fukumoto S., Nishizawa Y, Hosoi M, et al. Protein kinase C inhibits the proliferation of vascular smooth muscle by suppressing G1 cyclin expression. J Biol Chem1997; 272:13816–13822.
18. Harrington EO, Loffler J, Nelson PR, et al. Enhancement of migration by protein kinase C alpha and inhibition of proliferation and cell cycle progression by protein kinase C delta in capillary endothelial cells. J Biol Chem1997; 272:7390–7397
19. Perletti GP, Marras E, Concari P, et al. PKC delta acts as a growth and tumor suppressor in rat colonic epithelial cells. Oncogene 1999;18: 1251–1256
20. Page K, Li J, Corbit KC, et al. Regulation of Airway Smooth Muscle Cyclin D1 Transcription by Protein Kinase C-δ. American Journal of Respiratory Cell and Molecular Biology. 2002;27: 204-213
21. Xu SY, Xu YJ, Zhang ZX,et al. Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation. Chin Med J (Engl) 2004; 117:30-6
22. Hirst, SJ, Walker TR, and Chilvers ER. Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma. Eur Respir J 2000; 16:159-77
23. Albanese C, Johnson J, Watanabe G, et al. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem. 1995;270:23589-97
24. Shtutman M, Zhurinsky J, Simcha I, et al. The cyclin D1 gene is a target of the - ? catenin/LEF-1 pathway. Proc Natl Acad Sci U S A. 1999;96:5522-7
1 Baldin V, Lukas J, Marcote MJ et al. Cyclin D1 is a nuclear protein required for cell cycle progression in G1.Genes Dev, 1993,7:812-821
2 Fernandes D, Guida E, Koutsoubos V, et al. Glucocorticoids Inhibit Proliferation, Cyclin D1 Expression, and Retinoblastoma Protein Phosphorylation, but Not Activity of the Extracellular-Regulated Kinases in Human Cultured Airway Smooth Muscle. Am J Respir Cell Mol Biol, 1999, 21:77- 88.
3 Lee JH,Johnson PR,Roth M,et al.ERK activation and mitogenesis in human airway smooth muscle cells.Am J Physiol Lung Cell Mol Physiol.,2001 ,280:L1019-1029.
4中华医学会呼吸病学分会哮喘学组.支气管哮喘防治指南.中华结核和呼吸杂志,2003,26:132-138.
5 XU Shu-yun, XU Yong-jian, ZHANG Zhen-xiang, et al. Contribution of protein kinase C to passively sensitizedhuman airway smooth muscle cells proliferation.Chin M J, 2004, 117:30-36.
6 Black JL, Johnson PR. What determines asthma phenotype? Is it the interaction between allergy and the smooth muscle? Am J Respir Crit Care Med, 2000, 161:s207-210.
7 Johnson PR, Black JL, Carlin S, et al. The production of extracellular matrix proteins by human passively sensitized airway smooth-muscle cells in culture. Am J Respir Crit Care Med, 2000, 162: 2145-2151.
8 Kato J, Matsushime H, Hiebert SW, et al. Direct binding of cyclinD to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev, 1993, 7: 331-342.
9 Jaschke B, Milz S, Vogeser M, et al. Local cyclin-dependent kinase inhibition by flavopiridol inhibits coronary artery smooth muscle cell proliferation and migration: Implications for the applicability on drug-eluting stents to prevent neointima formation following vascular injury. FASEB J, 2004, 18:1285-1287.
10 John F, Kuemmerle, Huiping Zhou, et al. IGF-I stimulates human intestinal smooth muscle cell growth by regulation of G1 phase cell cycle proteins. Am J Physiol Gastrointest Liver Physiol,.2004, 286: G412-419.
11 Fukami-Kobayashi J,Mitsui y. Cyclin D1 Inhibits Cell Proliferation through Binding to PCNA and Cdk2 .Exp Cell Res,1999, 246:338-347.
12 Ammit AJ, Panettieri RA. Invited review: the circle of life: cell cycle regulation in airway smooth muscle. J Appl Physiol., 2001, 91: 1431-1437.
1. Beasley R, Crane J, Lai CK, et al. prevalence and etiology of asthma. J Allergy Chin Immunol 2000,10S: S466-S472
2. Masoli M, Fabian D, Holt S, et al. The global burden of asthma: executive summary of the GINA dissemination committee report. Allergy2004, 59: 467-478
3. Chung, KF. Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation? Eur Respir J 2000, 15: 961-968
4. Elias JA, Zhu Z, Chupp G, et al. Airway remodeling in asthma. J Clin Invest 1999, 104: 1001-1006
5. Fredberg, JJ. Airway smooth muscle in asthma. Perturbed equilibria of myosin binding. Am J Respir Crit Care Med 2000, 161: 158S-160S
6. Que, CL, Maksym G, and Macklem PT. Deciphering the homeokinetic code of airway smooth muscle. Am J Respir Crit Care Med 2000, 161: 161S-163S
7. Webb BL, Lindsay MA, Barnes PJ. Protein kinase C isoenzymes in airway smooth muscle. Biochem. J 1997, 324: 167-175
8. Dempsey EC, Newton AC, Mochly-Rosen D. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 2000, 279:L429-38
9. Parker PJ, Murray-Rust J. PKC at a glance. J Cell Sci 2004, 117: 131–132
10. Michie AM, Nakagawa R. The link between PKCalpha regulation and cellular transformation. Immunol Lett. 2005, 96:155-62
11. Panettieri RA, Rubinstein NA, Kelly AM, et al. The specificity of c-fos expression in the induction of airway smooth muscle proliferation by contractile agonists. Am Rev Respir Dis 1991,143: A934
12. Lew DB, Brown ER, Dempsey K, et al. Contribution of PKC toβ-hexosaminidase-induced airway smooth muscle proliferation. Am J Physiol 1997; 272: L639-L643.
13. Hirst SJ, Webb BLJ, Giembycz MA, et al. Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of protein kinase C and protein tyrosine kinase. Am J Respir Cell Mol Biol 1995, 12: 149-161
14. Hirst SJ, Barnes PJ, Twort CHC. Protein kinase inhibitors (PKC and PTK) inhibit PDGF isoform-stimulated proliferation of human and rabbit airway smooth muscle. Am J Respir Crit Care Med 1995, 151: A46
15. Hirst SJ, Barnes PJ, Twort CHC. Differential proliferation of rabbit tracheal smooth muscle by PDGF isoforms: Inhibition by selective inhibitors of protein kinase and protein tyrosine kinase. Br J Pharmacol 1994, 112: 81P
16. Acs P, Wang QJ, Bogi K,et al. Both the catalytic and regulatory domains of protein kinase C chimeras modulate the proliferative properties of NIH3T3 cells. J Biol Chem1997, 272: 28793–28799.
17. Fukumoto S., Nishizawa Y, Hosoi M, et al. Protein kinase C inhibits the proliferation of vascular smooth muscle by suppressing G1 cyclin expression. J Biol Chem1997, 272:13816–13822.
18. Harrington EO, Loffler J, Nelson PR, et al. Enhancement of migration by protein kinase C alpha and inhibition of proliferation and cell cycle progression by protein kinase C delta in capillary endothelial cells. J Biol Chem1997, 272:7390–7397
19. Perletti GP, Marras E, Concari P, et al. PKC delta acts as a growth and tumor suppressor in rat colonic epithelial cells. Oncogene 1999, 18: 1251–1256
20. Page K, Li J, Corbit KC, et al. Regulation of Airway Smooth Muscle Cyclin D1 Transcription by Protein Kinase C-δ. American Journal of Respiratory Cell and Molecular Biology. 2002,27: 204-213
21. Xu SY, Xu YJ, Zhang ZX, et al. Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation. Chin Med J (Engl) 2004, 117:30-6
22. Hirst SJ, Walker TR, and Chilvers ER. Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma. Eur Respir J 2000; 16:159-77
23. Albanese C, Johnson J, Watanabe G,et al. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem. 1995, 270:23589-97
24. Shtutman M, Zhurinsky J, Simcha I, et al. The cyclin D1 gene is a target of the - ? catenin/LEF-1 pathway. Proc Natl Acad Sci U S A. 1999,96:5522-7
25. Ammit AJ, Hastie AT, Edsall LC,et al. Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. FASEB J 2001,15: 1212-1214
26. Amrani Y, Tliba O, Choubey D, et al. IFN-gamma inhibits human airway smooth muscle cell proliferation by modulating the E2F-1/Rb pathway. Am J Physiol Lung Cell Mol Physiol 2003, 284:L1063-71
27. Black JL, Johnson PR. What Determines Asthma Phenotype? Is It the Interaction between Allergy and the Smooth Muscle? Am J Respir Crit Care Med 2000,161:S207-10
28. Johnson PR, Black JL, Carlin S, et al. The production of extracellular matrix proteins by human passively sensitized airway smooth-muscle cells in culture: the effect of beclomethasone. Am J Respir Crit Care Med 2000, 162:2145-51
29. Bai J, Liu XS, Xu YJ, et al. Regulation of Cell Proliferation and Ap optosis by ERK Signaling Pathway in Airway Smooth Muscle Cells of Rats. Chinese Journal of Biochemistry and Molecular Biology 2007,23:38-44
30. Bokhari SM, Zhou L, Karasek MA, et al. Regulation of skin microvasculature angiogenesis, cell migration, and permeability by a specific inhibitor of PKCalpha. J Invest Dermatol 2006, 126:460-7
31. Bornancin F, Parker PJ. Phosphorylation of threonine 638 critically controls the dephosphorylation and inactivation of protein kinase Calpha. Curr Biol 1996,6:1114-23
32. Parekh DB, Ziegler W, Parker PJ. Multiple pathways control protein kinase C phosphorylation. EMBO J 2000, 19:496-503
33. Tan M, Li P, Sun M, et al. Upregulation and activation of PKC alpha by ErbB2 through Src promotes breast cancer cell invasion that can be blocked by combined treatment with PKC alpha and Src inhibitors. Oncogene 2006, 25 :3286-95
34. Soh JW, Weinstein IB. Roles of Specific Isoforms of Protein Kinase C in the Transcriptional Control of Cyclin D1 and Related Genes. J Biol Chem 2003, 278:34709-16
35. Ramakrishnan M., Musa NL, Li J, et al. Catalytic activation of extracellular signal-regulated kinases induces cyclin D1 expression in airway smooth muscle. Am J Respir Cell Mol Biol 1998, 18:736–740
36. Lee JH., Johnson PR, Roth M, et al. ERK activation and mitogenesis in human airway smooth muscle cells. Am. J. Physiol. Lung Cell Mol Physiol 2001, 80:L1019–L1029
37. Page K., Li J, Wang Y, et al. Regulation of cyclin D1 expression and DNA synthesis by phosphatidylinositol 3-kinase in airway smooth muscle cells. Am J Respir Cell Mol Biol2000, 23:436–443
38. Thomas CF Jr, Limper AH. Phosphatidylinositol Kinase Regulation of Airway Smooth Muscle Cell Proliferation. Am J Respir Cell Mol Biol 2000, 23:429-30
39. Kolch W, Heidecker G, Kochs G,et al. Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature 1993, 364:249-52
40. Whelchel A, Evans J, Posada J. Inhibition of ERK activation attenuates endothelin-stimulated airway smooth muscle cell proliferation. Am J Respir Cell Mol Biol 1997, 16:589-96
41. Schonwasser DC, Marais RM, Marshall CJ, et al. Activation of the mitogen-activated protein kinase/extracellular signal-regulatedkinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol 1998, 18:790–8
42. Buitrago CG, Pardo VG, de Boland AR, et al. Activation of RAF-1 through Ras and protein kinase Ca mediates 1α,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells. J Biol Chem 2003, 278:2199–205
43. Trushin SA, Pennington KN, Carmona EM,et al. Protein kinase Calpha (PKCalpha) acts upstream of PKCtheta to activate IkappaB kinase and NF-kappaB in T lymphocytes. Mol Cell Biol 2003, 23:7068-81
44. Xiong W, Pestell RG, Watanabe G, et al. Cyclin D1 is required for S phase traversal in bovine tracheal myocytes. Am J Physiol 1997, 272:L1205–L1210
1. Dempsey EC, Newton AC, Mochly-Rosen D, et al. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol, 2000, 279:L429- L438.
2. Michie AM, Nakagawa R. The link between PKC alpha regulation and cellular transformation. Immunol Lett, 2005, 96:155-62.
3. Lew DB, Brown ER, Dempsey K, et al. Contribution of PKC toβ-hexosaminidase-induced airway smooth muscle proliferation. Am J Physiol, 1997, 272: L639- L643.
4. Hirst SJ, Webb BLJ, Giembycz MA, et al. Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of protein kinase C and protein tyrosine kinase. Am J Respir Cell Mol Biol, 1995,12: 149-161
5.中华医学会呼吸病学分会哮喘学组.支气管哮喘防治指南.中华结核和呼吸杂志,2003,26:132-138.
6. XU Shu-yun, XU Yong-jian, ZHANG Zhen-xiang, et al. Contribution of protein kinase C to passively sensitizedhuman airway smooth muscle cells proliferation.Chin M J, 2004, 117:30-36.
7. Shih SC, Mullen A, Abrams K, et al. Role of Protein Kinase C Isoforms in Phorbol Ester-induced Vascular Endothelial Growth Factor Expression in Human Glioblastoma Cells. J Biol Chem, 1999, 274:15407-14.
8. Bai J, Liu XS, Xu YJ, et al. Regulation of Cell Proliferation and Ap optosis by ERK Signaling Pathway in Airway Smooth Muscle Cells of Rats. Chinese Journal of Biochemistry and Molecular Biology, 2007,23:38-44
9. Chung, KF. Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation? Eur Respir J, 2000, 15: 961-968.
10. Elias, JA, Zhu Z, Chupp G, et al. Airway remodeling in asthma. J Clin Invest, 1999, 104: 1001-1006.
11. Black JL, Johnson PR. What Determines Asthma Phenotype? Is It the Interaction between Allergy and the Smooth Muscle? Am J Respir Crit Care Med, 2000, 161:S207-10.
12. LaBaer J., Garrett MD, Stevenson LF, et al. New functional activities for the p21 family of CDK inhibitors. Genes Dev,1997, 11:847–862
13. Cheng M, Olivier P, Diehl JA, et al. The p21 (Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J, 1999 ,18:1571-83
14. Bottazzi ME, Zhu XY, Bohmer RM, et al. Regulation of p21cip1 Expression by Growth Factors and the Extracellular Matrix Reveals a Role for Transient ERK Activity in G1 Phase. J Cell Biol, 1999, 146:1255-64.
15. Detjen KM, Brembeck FH, Welzel M, et al. Activation of protein kinase Ca inhibits growth of pancreatic cancer cells via 21cip-mediated G1 arrest. Journal of CellScience,2000, 113: 3025-3035
16. Nakagawa M, Oliva JL, Kothapalli D, et al. .Phorbol Ester-induced G1 Phase Arrest Selectively Mediated by Protein Kinase C -dependent Induction of p21. J Biol Chem, 2005, 280:33926-34.
17. Besson A, Yong VW. Involvement of p21 (Waf1/Cip1) in protein kinase C alpha-induced cell cycle progression.Mol Cell Biol, 2000, 20:4580-90.
18. Kolch W, Heidecker G, Kochs G, et al. Protein kinase Cαactivates RAF-1 by direct phosphorylation. Nature, 1993, 364:249–52.
19. Buitrago CG, Pardo VG, de Boland AR, et al. Activation of RAF-1 through Ras and protein kinase Ca mediates 1α,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells. J Biol Chem, 2003, 278:2199–205.
20. Schonwasser DC, Marais RM, Marshall CJ, et al. Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol, 1998, 18:790–798.
21. Lee JH, Johnson PR, Roth M,et al. ERK activation and mitogenesis in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol, 2001, 280:L1019-29.
22. Clark JA, Black AR, Leontieva OV, et al. Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells. J Biol Chem, 2004, 279:9233-47.
1. Dempsey EC, Newton AC, Mochly-Rosen D, et al. Proteinκinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 2000; 279(3):L429-38.
2. Lew DB, Brown ER, DempseyΚ, et al. Contribution of PΚC toβ-hexosaminidase-induced airway smooth muscle proliferation. Am J Physiol 1997; 272: L639-L643.
3. Hirst SJ, Webb BLJ, Giembycz MA, et al. Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of proteinκinase C and protein tyrosineκinase. Am J Respir Cell Mol Biol 1995; 12: 149-161
4. Hirst SJ, Barnes PJ, Twort CHC. Proteinκinase inhibitors (PΚC and PTΚ) inhibit PDGF isoform-stimulated proliferation of human and rabbit airway smooth muscle. Am J Respir Crit Care Med 1995; 151: A46
5. Hirst SJ, Barnes PJ, Twort CHC. Differential proliferation of rabbit tracheal smooth muscle by PDGF isoforms: Inhibition by selective inhibitors of proteinκinase and protein tyrosineκinase. Br J Pharmacol 1994; 112: 81P.
6.中华医学会呼吸病学分会哮喘学组.支气管哮喘防治指南.中华结核和呼吸杂志,2003,26:132-138.
7. XU Shu-yun, XU Yong-jian, ZHANG Zhen-xiang, et al. Contribution of proteinκinase C to passively sensitizedhuman airway smooth muscle cells proliferation.Chin M J, 2004, 117:30-36.
8. Shih SC, Mullen A, AbramsΚ, et al. Role of ProteinΚinase C Isoforms in Phorbol Ester-induced Vascular Endothelial Growth Factor Expression in Human Glioblastoma Cells. J Biol Chem. 1999 May 28; 274(22):15407-14.
9. Bai J, Liu XS, Xu YJ, et al. Regulation of Cell Proliferation and Ap optosis by ERK Signaling Pathway in Airway Smooth Muscle Cells of Rats. Chinese Journal ofBiochemistry and Molecular Biology 2007,23:38-44
10. Chung, KF. Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation? Eur Respir J 2000; 15: 961-968.
11. Elias, JA, Zhu Z, Chupp G, et al. Airway remodeling in asthma. J Clin Invest 1999; 104: 1001-1006.
12.许淑云,徐永健,张珍祥等.核因子κB在被动致敏的人气道平滑肌细胞增殖中的信号转导作用中华内科杂志,2004,43:891-895
13. AsehnouneΚ, Strassheim D, Mitra S, et al. Involvement of PΚCalpha/beta in TLR4 and TLR2 dependent activation of NF-κappaB. Cell Signal.2005 Mar;17(3):385-94
14. Banzi M, Aguiari G, Trimi V, et al. Polycystin-1 promotes PΚCalpha-mediated NF-κappaB activation inκidney cells. Biochem Biophys Res Commun. 2006 Nov 17;350(2):257-62. Epub 2006 Sep 18
15. Cataisson C, Pearson AJ, Torgerson S, et al. Proteinκinase C alpha-mediated chemotaxis of neutrophils requires NF-κappa B activity but is independent of TNF alpha signaling in mouse sκin in vivo. J Immunol. 2005 Feb 1; 174(3):1686-92.
16. Joyce D, Albanese C, Steer J, et al. NF-κappaB and cell-cycle regulation: the cyclin connection. Cytoκine Growth Factor Rev. 2001 Mar; 12(1):73-90. Review.
17. Guttridge DC, Albanese C, Reuther JY, et al. NF-κappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1 Mol Cell Biol. 1999 Aug;19(8):5785-99.
18. BlacκJL, Johnson PR. What Determines Asthma Phenotype? Is It the Interaction between Allergy and the Smooth Muscle? Am J Respir Crit Care Med. 2000; 161(3 Pt 2):S207-10.
19. Wang X, Wang Q, Hu W, et al. Regulation of phorbol ester-mediated TRAF1 induction in human colon cancer cells through a PΚC/RAF/ERΚ/NF-κappaB-dependent pathway. Oncogene. 2004 Mar 11; 23(10):1885-95.
1. Hirst SJ. Airway smoot h muscle as a target in ast hma. Clin Exp Allergy, 2000; 30 (Suppl 17):54-59.
2. Kumar RK. Understanding airway wall remodeling in asthma: a basis for improvement s in t herapy? Pharmacology & Therapeutics, 2001, 91 (2):93-104.
3. Black JL, Rot h M, Lee J, et al. Mechanisms of Airway Remodeling: Airway Smooth Muscle. Am J Respir Crit Care Med, 2001,164 (10 Pt 2):63-66.
4. Johnson PR, Rot h M, Tamm M. Airway smoot h muscle cell proliferation is increased in ast hma. Am J Respir Crit Care Med, 2001,164 (3):474-477.
5. Panettier: RA J r. Airway smoot h muscle: immunomodulatory cell s t hat modulate airway remodeling? Respir Physiol Neurobiol, 2003,137 (2-3):277-293.
6. Edward T, Naureckas I, Mauricendukwu, et al .Bronchoalveolar Lavage Fluid f rom Ast hmatic Subject s Is Mitogenic for Human Airway Smoot h Muscle. Am J Respir Crit Care Med, 1999,160 (6):2062-2066.
7. Hirst SJ, Walker TR, Chilvers ER. Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma. Eur Respir J, 2000; Jul (1):159-77.
8. Ammit AJ, Pamettieri RA J r. Signal Transduction in Smooth Muscle, Invited Review: The circle of life: cell cycle regulation in airway smoot h muscle. J Appl Physiol, 2001, 91 (3):1431-1437.
9. Ammit AJ , Kane SA , Panettieri RA J r . Activation of K-p21ras and N-p21ras, but not H-p21ras, is necessary for mitogen induced human airway smooth muscle proliferation. Am J Respir Cell Mol Biol, 1999, 21 (6):719-727.
10. Zhou L, Hershenson MB. Mitogenic. Signaling pathways in airway smooth muscle.Respir Physiol Neurobiol, 2003; 137(2-3):295-308.
11. Schaich M, Illmer T. Mdr1 gene expression and mutations in Ras proto-oncogenes in acute myeloid leukemia.Leuk Lymphoma, 2002 Jul; 43(7):1345-54.
12. Lee J H, Johnson PR, Rot h M, et al. ERK activation and mitogenesis in human airwaysmoot h muscle cells. Am J Physiol Lung Cell Mol Physiol, 2001,280 (5):L1019-L1029.
13. Wetzker R, B?hmer FD. Transactivation joins multiple tracks to the ERK/MAPK cascade.Nat Rev Mol Cell Biol, 2003 Aug;4(8):651-657
14. Ramakrishnan M, Musa N, Li J, et al. Catalytic activation of extracellular signal-regulated kinases induces cyclin D1 expression in primary tracheal myocytes. Am J Respir Cell Mol Biol,1998;18: 736-740.
15. Krymskaya VP, Ammit AJ, Hoffman RK, et al. Activation of class IA PI3 K stimulates DNA synt hesis in human airway smoot h muscle cell s. Am J Physiol Lung Cell Mol Physiol, 2001;280 (5):L1009-L1018.
16. Krymskaya VP, Penn RB, Orsini MJ, et al.1999. Phosphatidylinositol 3-kinase mediatesmitogen-induced human airways smooth muscle cell proliferation. Am J Physiol (Lung Cell Mol Physiol), 1999; 277(21): L65-L78.
17. Scott PH, Belham CM, al-Hafidh J et al. A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells. ,1996 Sep 15; 318 (Pt 3):965-71.
18. Bauerfeld CP, Hershenson MB, Page K. Cdc42, but not RhoA, regulates cyclin D1 expression in bovine tracheal myocytes. Am J Physiol Lung Cell Mol Physiol. 2001 May;280(5):L974-982
19. Panettieri RA, Rubinstein NA, Kelly AM, et al.: The specificity of c-fos expression in the induction of airway smooth muscle proliferation by contractile agonists. Am Rev Respir Dis, 1991;143: A934
20. Lew DB, Brown ER, Dempsey K, et al. Contribution of PKC toβ-hexosaminidase- induced airway smooth muscle proliferation. Am J Physiol 1997; 272: L639-L643.
21. Hirst SJ, Webb BLJ, Giembycz MA, et al. Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors ofprotein kinase C and protein tyrosine kinase. Am J Respir Cell Mol Biol ,1995;12: 149-161
22. Hirst SJ, Barnes PJ, Twort CHC. Protein kinase inhibitors (PKC and PTK) inhibit PDGF isoform-stimulated proliferation of human and rabbit airway smooth muscle. Am J Respir Crit Care Med, 1995; 151: A46
23. Hirst SJ, Barnes PJ, Twort CHC. Differential proliferation of rabbit tracheal smooth muscle by PDGF isoforms: Inhibition by selective inhibitors of protein kinase and protein tyrosine kinase. Br J Pharmacol, 1994;112: 81P
24. Page K, Li J, Corbit KC, et al. Regulation of Airway Smooth Muscle Cyclin D1 Transcription by Protein Kinase C-δ. American Journal of Respiratory Cell and Molecular Biology, 2002;27: 204-213
25. Carlin S, Yang KX, Donnelly R,et al.. Proteinkinase C isoforms in human airway smooth muscle cells: activation of PKCζduring proliferation. Am J Physiol Lung Cell Mol Physiol,1999; 276: L506-L512.
26. Zacour ME, Martin JG. Protein kinase C is involved in enhanced airway smooth muscle cell growth in hyperresponsive rats. Am J Physiol Lung Cell Mol Physiol, 2000; 278: L59-L67
27. Xu SY, Xu YJ, Zhang ZX, et al.Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation. Chin Med J (Engl), 2004;117:30-6
28. KolchW, Heidecker G, Kochs G, et al. Protein kinase Ca activates Raf-1 by direct phosphorylation. Nature, 1993; 364: 249-252.
29. Brar SS, Kennedy TP, Sturrock AB, et al. NADPH oxidase promotes NF-kappaB activation and proliferation in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol, 2002; 282:L782-/L795.
30. Thabut G, El-Benna J, Samb Aet al. Tumor necrosis factor-alpha increases airway smooth muscle oxidants production through a NADPH oxidaselike system to enhance myosin light chain phosphorylation and contractility. J Biol Chem, 2002; 277: 22814-22821.
31. Page K, Li J, Wang Y, et al. Regulation of cyclin D1 expression and DNA synthesis by phosphatidylinositol 3-kinase in airway smooth muscle cells. Am J Respir Cell Mol Biol, 2000; 23: 436-/443.
32. Brar SS, Kennedy TP, Sturrock AB, et al. 2002. NADPH oxidase promotes NF-kappaB activation and proliferation in human airway smooth muscle. Am J Physiol. Lung Cell Mol Physiol, 2002; 282:L782-/L795.
33. Simon AR, Takahashi S, Severgnini M, et al. Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol, 2002; 282: L1296-/L1304.
34. Gali-Muhtasib H, Bakkar N. Modulating cell cycle: current applications and prospect s for future drug development. Curr Cancer Drug Targets, 2002;2 (4):309-336.
35. Geisen C , Karsunky H , Yucel R , et al . Loss of p27 (Kip1) cooperates with cyclin E in T2cell lymphomagenesis. Oncogene, 2003; 22 (11):1724-1729.
36. Blagosklonny MV, Pardee AB. The rest riction point of t he cell cycle. Cell Cycle, 2002, 1(2):103-110.
37. Donjerkovic D, Scott DW. Regulation of t he G1 phase of the mammalian cell cycle. Cell Res, 2000; 10 (1):1-16.
38. Xiong W, Pestell RG, Watanabe G, et al. Cyclin D1 is required for S phase t raversal in bovine tracheal myocytes. Ann Rev Physiol, 1997; 272:L1205-L1210.
39. Amrani Y, Tliba O, Choubey D, et al. IFN-gamma inhibits human airway smooth muscle cell proliferation by modulating the E2F-1/ Rb pat hway. Am J Physiol Lung Cell Mol Physiol, 2003; 284(6):L1063-L1071.
40. Fernandes D , Guida E , Kout soubos V , et al . Glucocorticoids inhibit proliferation, cyclin D1 expression, and retinoblastoma protein phosphorylation, but not activity of t he ext racellular2 regulated kinasesin human cultured airway smoot h muscle. Am J Respir Cell Mol Biol, 1999; 21(1):77-88.
41. Stewart AG, Tomlinson PR, Wil son JW.β2-Adrenoceptoragonist-mediated inhibition of human airway smooth musclecell proliferation :importance of t he duration of β2-adrenoceptorstimulation. Br J Pharmacol, 1997; 121 (3):361-368.
42. Musa NL, Ramakrishnan M, Li J ,et al . Forskolin inhibit s cyclinD1 expression in cultured airway smooth muscle cells. Am J Respir Cell Mol Biol, 1999; 20 (2):352-358.
43. Rot h M, Johnson PR, Rudiger JJ. Interaction between glucocorticoids andβ2 agonist s on bronchialairway smooth muscle cells through synchronised cellular signaling. Mechanism of Disease, 2002; 360 (9342):1293-1299.
2. Masoli M, Fabian D, Holt S, Beasley R. The global burden of asthma: executive summary of the GINA dissemination committee report. Allergy2004; 59: 467-478
3. Chung, KF. Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation? Eur Respir J 2000; 15: 961-968
4. Elias, JA, Zhu Z, Chupp G, and Homer RJ.Airway remodeling in asthma. J Clin Invest 1999;104: 1001-1006
5. Fredberg, JJ. Airway smooth muscle in asthma. Perturbed equilibria of myosin binding. Am J Respir Crit Care Med 2000; 161: 158S-160S
6. Que, CL, Maksym G, and Macklem PT. Deciphering the homeokinetic code of airway smooth muscle. Am J Respir Crit Care Med 2000; 161: 161S-163S
7. Webb BL, Lindsay MA, Barnes PJ, et al. Protein kinase C isoenzymes in airway smooth muscle. Biochem. J 1997;324: 167-175
8. Dempsey EC, Newton AC, Mochly-Rosen D, et al. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 2000;279:L429-38
9. Parker PJ, Murray-Rust J.PKC at a glance. J Cell Sci 2004;117: 131–132
10. Michie AM, Nakagawa R. The link between PKCalpha regulation and cellular transformation. Immunol Lett. 2005; 96:155-62
11. Panettieri RA, Rubinstein NA, Kelly AM, et al.The specificity of c-fos expression in the induction of airway smooth muscle proliferation by contractile agonists. Am Rev Respir Dis 1991;143: A934
12. Lew DB, Brown ER, Dempsey K, et al. Contribution of PKC toβ-hexosaminidase-induced airway smooth muscle proliferation. Am J Physiol 1997; 272: L639-L643.
13. Hirst SJ, Webb BLJ, Giembycz MA, et al. Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of protein kinase C and protein tyrosine kinase. Am J Respir Cell Mol Biol 1995;12: 149-161
14. Hirst SJ, Barnes PJ, Twort CHC. Protein kinase inhibitors (PKC and PTK) inhibit PDGF isoform-stimulated proliferation of human and rabbit airway smooth muscle. Am J Respir Crit Care Med 1995;151: A46
15. Hirst SJ, Barnes PJ, Twort CHC. Differential proliferation of rabbit tracheal smooth muscle by PDGF isoforms: Inhibition by selective inhibitors of protein kinase and protein tyrosine kinase. Br J Pharmacol 1994; 112: 81P
16. Acs P, Wang QJ, Bogi K, et al. Both the catalytic and regulatory domains of protein kinase C chimeras modulate the proliferative properties of NIH3T3 cells. J Biol Chem1997; 272: 28793–28799.
17. Fukumoto S., Nishizawa Y, Hosoi M, et al. Protein kinase C inhibits the proliferation of vascular smooth muscle by suppressing G1 cyclin expression. J Biol Chem1997; 272:13816–13822.
18. Harrington EO, Loffler J, Nelson PR, et al. Enhancement of migration by protein kinase C alpha and inhibition of proliferation and cell cycle progression by protein kinase C delta in capillary endothelial cells. J Biol Chem1997; 272:7390–7397
19. Perletti GP, Marras E, Concari P, et al. PKC delta acts as a growth and tumor suppressor in rat colonic epithelial cells. Oncogene 1999;18: 1251–1256
20. Page K, Li J, Corbit KC, et al. Regulation of Airway Smooth Muscle Cyclin D1 Transcription by Protein Kinase C-δ. American Journal of Respiratory Cell and Molecular Biology. 2002;27: 204-213
21. Xu SY, Xu YJ, Zhang ZX,et al. Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation. Chin Med J (Engl) 2004; 117:30-6
22. Hirst, SJ, Walker TR, and Chilvers ER. Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma. Eur Respir J 2000; 16:159-77
23. Albanese C, Johnson J, Watanabe G, et al. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem. 1995;270:23589-97
24. Shtutman M, Zhurinsky J, Simcha I, et al. The cyclin D1 gene is a target of the - ? catenin/LEF-1 pathway. Proc Natl Acad Sci U S A. 1999;96:5522-7
1 Baldin V, Lukas J, Marcote MJ et al. Cyclin D1 is a nuclear protein required for cell cycle progression in G1.Genes Dev, 1993,7:812-821
2 Fernandes D, Guida E, Koutsoubos V, et al. Glucocorticoids Inhibit Proliferation, Cyclin D1 Expression, and Retinoblastoma Protein Phosphorylation, but Not Activity of the Extracellular-Regulated Kinases in Human Cultured Airway Smooth Muscle. Am J Respir Cell Mol Biol, 1999, 21:77- 88.
3 Lee JH,Johnson PR,Roth M,et al.ERK activation and mitogenesis in human airway smooth muscle cells.Am J Physiol Lung Cell Mol Physiol.,2001 ,280:L1019-1029.
4中华医学会呼吸病学分会哮喘学组.支气管哮喘防治指南.中华结核和呼吸杂志,2003,26:132-138.
5 XU Shu-yun, XU Yong-jian, ZHANG Zhen-xiang, et al. Contribution of protein kinase C to passively sensitizedhuman airway smooth muscle cells proliferation.Chin M J, 2004, 117:30-36.
6 Black JL, Johnson PR. What determines asthma phenotype? Is it the interaction between allergy and the smooth muscle? Am J Respir Crit Care Med, 2000, 161:s207-210.
7 Johnson PR, Black JL, Carlin S, et al. The production of extracellular matrix proteins by human passively sensitized airway smooth-muscle cells in culture. Am J Respir Crit Care Med, 2000, 162: 2145-2151.
8 Kato J, Matsushime H, Hiebert SW, et al. Direct binding of cyclinD to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev, 1993, 7: 331-342.
9 Jaschke B, Milz S, Vogeser M, et al. Local cyclin-dependent kinase inhibition by flavopiridol inhibits coronary artery smooth muscle cell proliferation and migration: Implications for the applicability on drug-eluting stents to prevent neointima formation following vascular injury. FASEB J, 2004, 18:1285-1287.
10 John F, Kuemmerle, Huiping Zhou, et al. IGF-I stimulates human intestinal smooth muscle cell growth by regulation of G1 phase cell cycle proteins. Am J Physiol Gastrointest Liver Physiol,.2004, 286: G412-419.
11 Fukami-Kobayashi J,Mitsui y. Cyclin D1 Inhibits Cell Proliferation through Binding to PCNA and Cdk2 .Exp Cell Res,1999, 246:338-347.
12 Ammit AJ, Panettieri RA. Invited review: the circle of life: cell cycle regulation in airway smooth muscle. J Appl Physiol., 2001, 91: 1431-1437.
1. Beasley R, Crane J, Lai CK, et al. prevalence and etiology of asthma. J Allergy Chin Immunol 2000,10S: S466-S472
2. Masoli M, Fabian D, Holt S, et al. The global burden of asthma: executive summary of the GINA dissemination committee report. Allergy2004, 59: 467-478
3. Chung, KF. Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation? Eur Respir J 2000, 15: 961-968
4. Elias JA, Zhu Z, Chupp G, et al. Airway remodeling in asthma. J Clin Invest 1999, 104: 1001-1006
5. Fredberg, JJ. Airway smooth muscle in asthma. Perturbed equilibria of myosin binding. Am J Respir Crit Care Med 2000, 161: 158S-160S
6. Que, CL, Maksym G, and Macklem PT. Deciphering the homeokinetic code of airway smooth muscle. Am J Respir Crit Care Med 2000, 161: 161S-163S
7. Webb BL, Lindsay MA, Barnes PJ. Protein kinase C isoenzymes in airway smooth muscle. Biochem. J 1997, 324: 167-175
8. Dempsey EC, Newton AC, Mochly-Rosen D. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 2000, 279:L429-38
9. Parker PJ, Murray-Rust J. PKC at a glance. J Cell Sci 2004, 117: 131–132
10. Michie AM, Nakagawa R. The link between PKCalpha regulation and cellular transformation. Immunol Lett. 2005, 96:155-62
11. Panettieri RA, Rubinstein NA, Kelly AM, et al. The specificity of c-fos expression in the induction of airway smooth muscle proliferation by contractile agonists. Am Rev Respir Dis 1991,143: A934
12. Lew DB, Brown ER, Dempsey K, et al. Contribution of PKC toβ-hexosaminidase-induced airway smooth muscle proliferation. Am J Physiol 1997; 272: L639-L643.
13. Hirst SJ, Webb BLJ, Giembycz MA, et al. Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of protein kinase C and protein tyrosine kinase. Am J Respir Cell Mol Biol 1995, 12: 149-161
14. Hirst SJ, Barnes PJ, Twort CHC. Protein kinase inhibitors (PKC and PTK) inhibit PDGF isoform-stimulated proliferation of human and rabbit airway smooth muscle. Am J Respir Crit Care Med 1995, 151: A46
15. Hirst SJ, Barnes PJ, Twort CHC. Differential proliferation of rabbit tracheal smooth muscle by PDGF isoforms: Inhibition by selective inhibitors of protein kinase and protein tyrosine kinase. Br J Pharmacol 1994, 112: 81P
16. Acs P, Wang QJ, Bogi K,et al. Both the catalytic and regulatory domains of protein kinase C chimeras modulate the proliferative properties of NIH3T3 cells. J Biol Chem1997, 272: 28793–28799.
17. Fukumoto S., Nishizawa Y, Hosoi M, et al. Protein kinase C inhibits the proliferation of vascular smooth muscle by suppressing G1 cyclin expression. J Biol Chem1997, 272:13816–13822.
18. Harrington EO, Loffler J, Nelson PR, et al. Enhancement of migration by protein kinase C alpha and inhibition of proliferation and cell cycle progression by protein kinase C delta in capillary endothelial cells. J Biol Chem1997, 272:7390–7397
19. Perletti GP, Marras E, Concari P, et al. PKC delta acts as a growth and tumor suppressor in rat colonic epithelial cells. Oncogene 1999, 18: 1251–1256
20. Page K, Li J, Corbit KC, et al. Regulation of Airway Smooth Muscle Cyclin D1 Transcription by Protein Kinase C-δ. American Journal of Respiratory Cell and Molecular Biology. 2002,27: 204-213
21. Xu SY, Xu YJ, Zhang ZX, et al. Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation. Chin Med J (Engl) 2004, 117:30-6
22. Hirst SJ, Walker TR, and Chilvers ER. Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma. Eur Respir J 2000; 16:159-77
23. Albanese C, Johnson J, Watanabe G,et al. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem. 1995, 270:23589-97
24. Shtutman M, Zhurinsky J, Simcha I, et al. The cyclin D1 gene is a target of the - ? catenin/LEF-1 pathway. Proc Natl Acad Sci U S A. 1999,96:5522-7
25. Ammit AJ, Hastie AT, Edsall LC,et al. Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. FASEB J 2001,15: 1212-1214
26. Amrani Y, Tliba O, Choubey D, et al. IFN-gamma inhibits human airway smooth muscle cell proliferation by modulating the E2F-1/Rb pathway. Am J Physiol Lung Cell Mol Physiol 2003, 284:L1063-71
27. Black JL, Johnson PR. What Determines Asthma Phenotype? Is It the Interaction between Allergy and the Smooth Muscle? Am J Respir Crit Care Med 2000,161:S207-10
28. Johnson PR, Black JL, Carlin S, et al. The production of extracellular matrix proteins by human passively sensitized airway smooth-muscle cells in culture: the effect of beclomethasone. Am J Respir Crit Care Med 2000, 162:2145-51
29. Bai J, Liu XS, Xu YJ, et al. Regulation of Cell Proliferation and Ap optosis by ERK Signaling Pathway in Airway Smooth Muscle Cells of Rats. Chinese Journal of Biochemistry and Molecular Biology 2007,23:38-44
30. Bokhari SM, Zhou L, Karasek MA, et al. Regulation of skin microvasculature angiogenesis, cell migration, and permeability by a specific inhibitor of PKCalpha. J Invest Dermatol 2006, 126:460-7
31. Bornancin F, Parker PJ. Phosphorylation of threonine 638 critically controls the dephosphorylation and inactivation of protein kinase Calpha. Curr Biol 1996,6:1114-23
32. Parekh DB, Ziegler W, Parker PJ. Multiple pathways control protein kinase C phosphorylation. EMBO J 2000, 19:496-503
33. Tan M, Li P, Sun M, et al. Upregulation and activation of PKC alpha by ErbB2 through Src promotes breast cancer cell invasion that can be blocked by combined treatment with PKC alpha and Src inhibitors. Oncogene 2006, 25 :3286-95
34. Soh JW, Weinstein IB. Roles of Specific Isoforms of Protein Kinase C in the Transcriptional Control of Cyclin D1 and Related Genes. J Biol Chem 2003, 278:34709-16
35. Ramakrishnan M., Musa NL, Li J, et al. Catalytic activation of extracellular signal-regulated kinases induces cyclin D1 expression in airway smooth muscle. Am J Respir Cell Mol Biol 1998, 18:736–740
36. Lee JH., Johnson PR, Roth M, et al. ERK activation and mitogenesis in human airway smooth muscle cells. Am. J. Physiol. Lung Cell Mol Physiol 2001, 80:L1019–L1029
37. Page K., Li J, Wang Y, et al. Regulation of cyclin D1 expression and DNA synthesis by phosphatidylinositol 3-kinase in airway smooth muscle cells. Am J Respir Cell Mol Biol2000, 23:436–443
38. Thomas CF Jr, Limper AH. Phosphatidylinositol Kinase Regulation of Airway Smooth Muscle Cell Proliferation. Am J Respir Cell Mol Biol 2000, 23:429-30
39. Kolch W, Heidecker G, Kochs G,et al. Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature 1993, 364:249-52
40. Whelchel A, Evans J, Posada J. Inhibition of ERK activation attenuates endothelin-stimulated airway smooth muscle cell proliferation. Am J Respir Cell Mol Biol 1997, 16:589-96
41. Schonwasser DC, Marais RM, Marshall CJ, et al. Activation of the mitogen-activated protein kinase/extracellular signal-regulatedkinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol 1998, 18:790–8
42. Buitrago CG, Pardo VG, de Boland AR, et al. Activation of RAF-1 through Ras and protein kinase Ca mediates 1α,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells. J Biol Chem 2003, 278:2199–205
43. Trushin SA, Pennington KN, Carmona EM,et al. Protein kinase Calpha (PKCalpha) acts upstream of PKCtheta to activate IkappaB kinase and NF-kappaB in T lymphocytes. Mol Cell Biol 2003, 23:7068-81
44. Xiong W, Pestell RG, Watanabe G, et al. Cyclin D1 is required for S phase traversal in bovine tracheal myocytes. Am J Physiol 1997, 272:L1205–L1210
1. Dempsey EC, Newton AC, Mochly-Rosen D, et al. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol, 2000, 279:L429- L438.
2. Michie AM, Nakagawa R. The link between PKC alpha regulation and cellular transformation. Immunol Lett, 2005, 96:155-62.
3. Lew DB, Brown ER, Dempsey K, et al. Contribution of PKC toβ-hexosaminidase-induced airway smooth muscle proliferation. Am J Physiol, 1997, 272: L639- L643.
4. Hirst SJ, Webb BLJ, Giembycz MA, et al. Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of protein kinase C and protein tyrosine kinase. Am J Respir Cell Mol Biol, 1995,12: 149-161
5.中华医学会呼吸病学分会哮喘学组.支气管哮喘防治指南.中华结核和呼吸杂志,2003,26:132-138.
6. XU Shu-yun, XU Yong-jian, ZHANG Zhen-xiang, et al. Contribution of protein kinase C to passively sensitizedhuman airway smooth muscle cells proliferation.Chin M J, 2004, 117:30-36.
7. Shih SC, Mullen A, Abrams K, et al. Role of Protein Kinase C Isoforms in Phorbol Ester-induced Vascular Endothelial Growth Factor Expression in Human Glioblastoma Cells. J Biol Chem, 1999, 274:15407-14.
8. Bai J, Liu XS, Xu YJ, et al. Regulation of Cell Proliferation and Ap optosis by ERK Signaling Pathway in Airway Smooth Muscle Cells of Rats. Chinese Journal of Biochemistry and Molecular Biology, 2007,23:38-44
9. Chung, KF. Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation? Eur Respir J, 2000, 15: 961-968.
10. Elias, JA, Zhu Z, Chupp G, et al. Airway remodeling in asthma. J Clin Invest, 1999, 104: 1001-1006.
11. Black JL, Johnson PR. What Determines Asthma Phenotype? Is It the Interaction between Allergy and the Smooth Muscle? Am J Respir Crit Care Med, 2000, 161:S207-10.
12. LaBaer J., Garrett MD, Stevenson LF, et al. New functional activities for the p21 family of CDK inhibitors. Genes Dev,1997, 11:847–862
13. Cheng M, Olivier P, Diehl JA, et al. The p21 (Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J, 1999 ,18:1571-83
14. Bottazzi ME, Zhu XY, Bohmer RM, et al. Regulation of p21cip1 Expression by Growth Factors and the Extracellular Matrix Reveals a Role for Transient ERK Activity in G1 Phase. J Cell Biol, 1999, 146:1255-64.
15. Detjen KM, Brembeck FH, Welzel M, et al. Activation of protein kinase Ca inhibits growth of pancreatic cancer cells via 21cip-mediated G1 arrest. Journal of CellScience,2000, 113: 3025-3035
16. Nakagawa M, Oliva JL, Kothapalli D, et al. .Phorbol Ester-induced G1 Phase Arrest Selectively Mediated by Protein Kinase C -dependent Induction of p21. J Biol Chem, 2005, 280:33926-34.
17. Besson A, Yong VW. Involvement of p21 (Waf1/Cip1) in protein kinase C alpha-induced cell cycle progression.Mol Cell Biol, 2000, 20:4580-90.
18. Kolch W, Heidecker G, Kochs G, et al. Protein kinase Cαactivates RAF-1 by direct phosphorylation. Nature, 1993, 364:249–52.
19. Buitrago CG, Pardo VG, de Boland AR, et al. Activation of RAF-1 through Ras and protein kinase Ca mediates 1α,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells. J Biol Chem, 2003, 278:2199–205.
20. Schonwasser DC, Marais RM, Marshall CJ, et al. Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol, 1998, 18:790–798.
21. Lee JH, Johnson PR, Roth M,et al. ERK activation and mitogenesis in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol, 2001, 280:L1019-29.
22. Clark JA, Black AR, Leontieva OV, et al. Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells. J Biol Chem, 2004, 279:9233-47.
1. Dempsey EC, Newton AC, Mochly-Rosen D, et al. Proteinκinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 2000; 279(3):L429-38.
2. Lew DB, Brown ER, DempseyΚ, et al. Contribution of PΚC toβ-hexosaminidase-induced airway smooth muscle proliferation. Am J Physiol 1997; 272: L639-L643.
3. Hirst SJ, Webb BLJ, Giembycz MA, et al. Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of proteinκinase C and protein tyrosineκinase. Am J Respir Cell Mol Biol 1995; 12: 149-161
4. Hirst SJ, Barnes PJ, Twort CHC. Proteinκinase inhibitors (PΚC and PTΚ) inhibit PDGF isoform-stimulated proliferation of human and rabbit airway smooth muscle. Am J Respir Crit Care Med 1995; 151: A46
5. Hirst SJ, Barnes PJ, Twort CHC. Differential proliferation of rabbit tracheal smooth muscle by PDGF isoforms: Inhibition by selective inhibitors of proteinκinase and protein tyrosineκinase. Br J Pharmacol 1994; 112: 81P.
6.中华医学会呼吸病学分会哮喘学组.支气管哮喘防治指南.中华结核和呼吸杂志,2003,26:132-138.
7. XU Shu-yun, XU Yong-jian, ZHANG Zhen-xiang, et al. Contribution of proteinκinase C to passively sensitizedhuman airway smooth muscle cells proliferation.Chin M J, 2004, 117:30-36.
8. Shih SC, Mullen A, AbramsΚ, et al. Role of ProteinΚinase C Isoforms in Phorbol Ester-induced Vascular Endothelial Growth Factor Expression in Human Glioblastoma Cells. J Biol Chem. 1999 May 28; 274(22):15407-14.
9. Bai J, Liu XS, Xu YJ, et al. Regulation of Cell Proliferation and Ap optosis by ERK Signaling Pathway in Airway Smooth Muscle Cells of Rats. Chinese Journal ofBiochemistry and Molecular Biology 2007,23:38-44
10. Chung, KF. Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation? Eur Respir J 2000; 15: 961-968.
11. Elias, JA, Zhu Z, Chupp G, et al. Airway remodeling in asthma. J Clin Invest 1999; 104: 1001-1006.
12.许淑云,徐永健,张珍祥等.核因子κB在被动致敏的人气道平滑肌细胞增殖中的信号转导作用中华内科杂志,2004,43:891-895
13. AsehnouneΚ, Strassheim D, Mitra S, et al. Involvement of PΚCalpha/beta in TLR4 and TLR2 dependent activation of NF-κappaB. Cell Signal.2005 Mar;17(3):385-94
14. Banzi M, Aguiari G, Trimi V, et al. Polycystin-1 promotes PΚCalpha-mediated NF-κappaB activation inκidney cells. Biochem Biophys Res Commun. 2006 Nov 17;350(2):257-62. Epub 2006 Sep 18
15. Cataisson C, Pearson AJ, Torgerson S, et al. Proteinκinase C alpha-mediated chemotaxis of neutrophils requires NF-κappa B activity but is independent of TNF alpha signaling in mouse sκin in vivo. J Immunol. 2005 Feb 1; 174(3):1686-92.
16. Joyce D, Albanese C, Steer J, et al. NF-κappaB and cell-cycle regulation: the cyclin connection. Cytoκine Growth Factor Rev. 2001 Mar; 12(1):73-90. Review.
17. Guttridge DC, Albanese C, Reuther JY, et al. NF-κappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1 Mol Cell Biol. 1999 Aug;19(8):5785-99.
18. BlacκJL, Johnson PR. What Determines Asthma Phenotype? Is It the Interaction between Allergy and the Smooth Muscle? Am J Respir Crit Care Med. 2000; 161(3 Pt 2):S207-10.
19. Wang X, Wang Q, Hu W, et al. Regulation of phorbol ester-mediated TRAF1 induction in human colon cancer cells through a PΚC/RAF/ERΚ/NF-κappaB-dependent pathway. Oncogene. 2004 Mar 11; 23(10):1885-95.
1. Hirst SJ. Airway smoot h muscle as a target in ast hma. Clin Exp Allergy, 2000; 30 (Suppl 17):54-59.
2. Kumar RK. Understanding airway wall remodeling in asthma: a basis for improvement s in t herapy? Pharmacology & Therapeutics, 2001, 91 (2):93-104.
3. Black JL, Rot h M, Lee J, et al. Mechanisms of Airway Remodeling: Airway Smooth Muscle. Am J Respir Crit Care Med, 2001,164 (10 Pt 2):63-66.
4. Johnson PR, Rot h M, Tamm M. Airway smoot h muscle cell proliferation is increased in ast hma. Am J Respir Crit Care Med, 2001,164 (3):474-477.
5. Panettier: RA J r. Airway smoot h muscle: immunomodulatory cell s t hat modulate airway remodeling? Respir Physiol Neurobiol, 2003,137 (2-3):277-293.
6. Edward T, Naureckas I, Mauricendukwu, et al .Bronchoalveolar Lavage Fluid f rom Ast hmatic Subject s Is Mitogenic for Human Airway Smoot h Muscle. Am J Respir Crit Care Med, 1999,160 (6):2062-2066.
7. Hirst SJ, Walker TR, Chilvers ER. Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma. Eur Respir J, 2000; Jul (1):159-77.
8. Ammit AJ, Pamettieri RA J r. Signal Transduction in Smooth Muscle, Invited Review: The circle of life: cell cycle regulation in airway smoot h muscle. J Appl Physiol, 2001, 91 (3):1431-1437.
9. Ammit AJ , Kane SA , Panettieri RA J r . Activation of K-p21ras and N-p21ras, but not H-p21ras, is necessary for mitogen induced human airway smooth muscle proliferation. Am J Respir Cell Mol Biol, 1999, 21 (6):719-727.
10. Zhou L, Hershenson MB. Mitogenic. Signaling pathways in airway smooth muscle.Respir Physiol Neurobiol, 2003; 137(2-3):295-308.
11. Schaich M, Illmer T. Mdr1 gene expression and mutations in Ras proto-oncogenes in acute myeloid leukemia.Leuk Lymphoma, 2002 Jul; 43(7):1345-54.
12. Lee J H, Johnson PR, Rot h M, et al. ERK activation and mitogenesis in human airwaysmoot h muscle cells. Am J Physiol Lung Cell Mol Physiol, 2001,280 (5):L1019-L1029.
13. Wetzker R, B?hmer FD. Transactivation joins multiple tracks to the ERK/MAPK cascade.Nat Rev Mol Cell Biol, 2003 Aug;4(8):651-657
14. Ramakrishnan M, Musa N, Li J, et al. Catalytic activation of extracellular signal-regulated kinases induces cyclin D1 expression in primary tracheal myocytes. Am J Respir Cell Mol Biol,1998;18: 736-740.
15. Krymskaya VP, Ammit AJ, Hoffman RK, et al. Activation of class IA PI3 K stimulates DNA synt hesis in human airway smoot h muscle cell s. Am J Physiol Lung Cell Mol Physiol, 2001;280 (5):L1009-L1018.
16. Krymskaya VP, Penn RB, Orsini MJ, et al.1999. Phosphatidylinositol 3-kinase mediatesmitogen-induced human airways smooth muscle cell proliferation. Am J Physiol (Lung Cell Mol Physiol), 1999; 277(21): L65-L78.
17. Scott PH, Belham CM, al-Hafidh J et al. A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells. ,1996 Sep 15; 318 (Pt 3):965-71.
18. Bauerfeld CP, Hershenson MB, Page K. Cdc42, but not RhoA, regulates cyclin D1 expression in bovine tracheal myocytes. Am J Physiol Lung Cell Mol Physiol. 2001 May;280(5):L974-982
19. Panettieri RA, Rubinstein NA, Kelly AM, et al.: The specificity of c-fos expression in the induction of airway smooth muscle proliferation by contractile agonists. Am Rev Respir Dis, 1991;143: A934
20. Lew DB, Brown ER, Dempsey K, et al. Contribution of PKC toβ-hexosaminidase- induced airway smooth muscle proliferation. Am J Physiol 1997; 272: L639-L643.
21. Hirst SJ, Webb BLJ, Giembycz MA, et al. Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors ofprotein kinase C and protein tyrosine kinase. Am J Respir Cell Mol Biol ,1995;12: 149-161
22. Hirst SJ, Barnes PJ, Twort CHC. Protein kinase inhibitors (PKC and PTK) inhibit PDGF isoform-stimulated proliferation of human and rabbit airway smooth muscle. Am J Respir Crit Care Med, 1995; 151: A46
23. Hirst SJ, Barnes PJ, Twort CHC. Differential proliferation of rabbit tracheal smooth muscle by PDGF isoforms: Inhibition by selective inhibitors of protein kinase and protein tyrosine kinase. Br J Pharmacol, 1994;112: 81P
24. Page K, Li J, Corbit KC, et al. Regulation of Airway Smooth Muscle Cyclin D1 Transcription by Protein Kinase C-δ. American Journal of Respiratory Cell and Molecular Biology, 2002;27: 204-213
25. Carlin S, Yang KX, Donnelly R,et al.. Proteinkinase C isoforms in human airway smooth muscle cells: activation of PKCζduring proliferation. Am J Physiol Lung Cell Mol Physiol,1999; 276: L506-L512.
26. Zacour ME, Martin JG. Protein kinase C is involved in enhanced airway smooth muscle cell growth in hyperresponsive rats. Am J Physiol Lung Cell Mol Physiol, 2000; 278: L59-L67
27. Xu SY, Xu YJ, Zhang ZX, et al.Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation. Chin Med J (Engl), 2004;117:30-6
28. KolchW, Heidecker G, Kochs G, et al. Protein kinase Ca activates Raf-1 by direct phosphorylation. Nature, 1993; 364: 249-252.
29. Brar SS, Kennedy TP, Sturrock AB, et al. NADPH oxidase promotes NF-kappaB activation and proliferation in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol, 2002; 282:L782-/L795.
30. Thabut G, El-Benna J, Samb Aet al. Tumor necrosis factor-alpha increases airway smooth muscle oxidants production through a NADPH oxidaselike system to enhance myosin light chain phosphorylation and contractility. J Biol Chem, 2002; 277: 22814-22821.
31. Page K, Li J, Wang Y, et al. Regulation of cyclin D1 expression and DNA synthesis by phosphatidylinositol 3-kinase in airway smooth muscle cells. Am J Respir Cell Mol Biol, 2000; 23: 436-/443.
32. Brar SS, Kennedy TP, Sturrock AB, et al. 2002. NADPH oxidase promotes NF-kappaB activation and proliferation in human airway smooth muscle. Am J Physiol. Lung Cell Mol Physiol, 2002; 282:L782-/L795.
33. Simon AR, Takahashi S, Severgnini M, et al. Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol, 2002; 282: L1296-/L1304.
34. Gali-Muhtasib H, Bakkar N. Modulating cell cycle: current applications and prospect s for future drug development. Curr Cancer Drug Targets, 2002;2 (4):309-336.
35. Geisen C , Karsunky H , Yucel R , et al . Loss of p27 (Kip1) cooperates with cyclin E in T2cell lymphomagenesis. Oncogene, 2003; 22 (11):1724-1729.
36. Blagosklonny MV, Pardee AB. The rest riction point of t he cell cycle. Cell Cycle, 2002, 1(2):103-110.
37. Donjerkovic D, Scott DW. Regulation of t he G1 phase of the mammalian cell cycle. Cell Res, 2000; 10 (1):1-16.
38. Xiong W, Pestell RG, Watanabe G, et al. Cyclin D1 is required for S phase t raversal in bovine tracheal myocytes. Ann Rev Physiol, 1997; 272:L1205-L1210.
39. Amrani Y, Tliba O, Choubey D, et al. IFN-gamma inhibits human airway smooth muscle cell proliferation by modulating the E2F-1/ Rb pat hway. Am J Physiol Lung Cell Mol Physiol, 2003; 284(6):L1063-L1071.
40. Fernandes D , Guida E , Kout soubos V , et al . Glucocorticoids inhibit proliferation, cyclin D1 expression, and retinoblastoma protein phosphorylation, but not activity of t he ext racellular2 regulated kinasesin human cultured airway smoot h muscle. Am J Respir Cell Mol Biol, 1999; 21(1):77-88.
41. Stewart AG, Tomlinson PR, Wil son JW.β2-Adrenoceptoragonist-mediated inhibition of human airway smooth musclecell proliferation :importance of t he duration of β2-adrenoceptorstimulation. Br J Pharmacol, 1997; 121 (3):361-368.
42. Musa NL, Ramakrishnan M, Li J ,et al . Forskolin inhibit s cyclinD1 expression in cultured airway smooth muscle cells. Am J Respir Cell Mol Biol, 1999; 20 (2):352-358.
43. Rot h M, Johnson PR, Rudiger JJ. Interaction between glucocorticoids andβ2 agonist s on bronchialairway smooth muscle cells through synchronised cellular signaling. Mechanism of Disease, 2002; 360 (9342):1293-1299.