结缔组织生长因子/CCN2基因缺失的小鼠胚胎成纤维细胞保持完整的转化生长因子-β反应性。
Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-beta responsiveness.
作者信息
Mori Yasuji, Hinchcliff Monique, Wu Minghua, Warner-Blankenship Matthew, M Lyons Karen, Varga John
机构信息
Division of Rheumatology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
出版信息
Exp Cell Res. 2008 Mar 10;314(5):1094-104. doi: 10.1016/j.yexcr.2007.12.010. Epub 2007 Dec 23.
BACKGROUND
The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-beta (TGF-beta) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-beta, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-beta. To explore this notion, we characterized TGF-beta-induced activation of fibroblasts from CCN2-null (CCN2(-/-)) mouse embryos.
METHODS
The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-beta signal transduction and regulation of collagen gene expression were examined in CCN2(-/-) MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays.
RESULTS
Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2(-/-) MEFs was markedly reduced compared to wild type MEFs, TGF-beta-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2(-/-) MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2.
CONCLUSION
Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-beta-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.
背景
基质细胞蛋白结缔组织生长因子（CCN2）与病理性纤维化有关，但其生理作用仍不清楚。在体外，转化生长因子-β（TGF-β）可诱导间充质细胞表达CCN2。由于CCN2可增强TGF-β引发的促纤维化反应，因此有人提出CCN2作为TGF-β重要的下游信号介质发挥作用。为了探究这一观点，我们对来自CCN2基因敲除（CCN2(-/-)）小鼠胚胎的成纤维细胞中TGF-β诱导的激活情况进行了表征。
方法
在博来霉素诱导的纤维化模型中，在体内检测CCN2表达的调控。通过免疫组织化学、Northern、Western和RT-PCR分析、免疫细胞化学和瞬时转染试验，检测CCN2(-/-) 小鼠胚胎成纤维细胞（MEFs）中的细胞TGF-β信号转导和胶原基因表达的调控。
结果
博来霉素诱导的小鼠皮肤纤维化与病变成纤维细胞中CCN2的大量上调有关。与野生型MEFs相比，CCN2(-/-) MEFs的体外增殖率明显降低，而TGF-β诱导的Smad信号通路激活，包括Smad2磷酸化、Smad2/3和Smad4核积累以及Smad依赖的转录反应，不受CCN2缺失的影响。野生型和CCN2(-/-) MEFs中COL1A2和纤连蛋白mRNA表达及启动子活性以及相应蛋白水平的刺激，显示出类似的时间和剂量反应，而α平滑肌肌动蛋白的刺激和肌成纤维细胞转分化在缺乏CCN2的MEFs中显示出轻微损伤。
结论
内源性CCN2在增殖调节和TGF-β诱导的肌成纤维细胞转分化中发挥作用，但对于小鼠胚胎成纤维细胞中Smad依赖的胶原和细胞外基质合成刺激似乎是可有可无的。
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