BACKGROUND Idiopathic pulmonary fibrosis (IPF) is a devastating disease with a median survival of only three to 5 years. Fibroblast proliferation is a hallmark of IPF as is secretion of extracellular matrix proteins from fibroblasts. However, it is still uncertain how IPF fibroblasts acquire the ability to progressively proliferate. Periostin is a matricellular protein highly expressed in the lung tissues of IPF patients, playing a critical role in the pathogenesis of pulmonary fibrosis. However, it remains undetermined whether periostin affects lung fibroblast proliferation. METHODS In this study, we first aimed at identifying periostin-dependently expressed genes in lung fibroblasts using DNA microarrays. We then examined whether expression of cyclins and CDKs controlling cell cycle progression occur in a periostin-dependent manner. We next examined whether downregulation of cell proliferation-promoting genes by knockdown of periostin or integrin, a periostin receptor, using siRNA, is reflected in the cell proliferation of lung fibroblasts. We then looked at whether lung fibroblasts derived from IPF patients also require periostin for maximum proliferation. We finally investigated whether CP4715, a potent inhibitor against integrin αVβ3 (a periostin receptor), which we have recently found blocks TGF-β signaling, followed by reduced BLM-induced pulmonary fibrosis in mice, can block proliferation of lung fibroblasts derived from IPF patients. RESULTS Many cell-cycle-related genes are involved in the upregulated or downregulated genes by periostin knockdown. We confirmed that in lung fibroblasts, periostin silencing downregulates expression of several cell-cycle-related molecules, including the cyclin, CDK, and, E2F families, as well as transcription factors such as B-MYB and FOXM1. Periostin or integrin silencing slowed proliferation of lung fibroblasts and periostin silencing increased the distribution of the G0/G1 phase, whereas the distribution of the G2/M phase was decreased. Lung fibroblasts derived from IPF patients also required periostin for maximum proliferation. Moreover, CP4715 downregulated proliferation along with expression of cell-cycle-related genes in IPF lung fibroblasts as well as in normal lung fibroblasts. CONCLUSIONS Periostin plays a critical role in the proliferation of lung fibroblasts and the present results provide us a solid basis for considering inhibitors of the periostin/integrin αVβ3 interaction for the treatment of IPF patients.

中文翻译：

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骨膜素在肺成纤维细胞的细胞周期中起关键作用。

背景技术特发性肺纤维化（IPF）是一种破坏性疾病，中位生存期仅为三至五年。成纤维细胞增殖是IPF的标志，成纤维细胞分泌细胞外基质蛋白也是如此。然而，仍不确定IPF成纤维细胞如何获得逐渐增殖的能力。骨膜素是在IPF患者的肺组织中高度表达的基质细胞蛋白，在肺纤维化的发病机理中起关键作用。但是，骨膜素是否影响肺成纤维细胞增殖仍未确定。方法在这项研究中，我们首先旨在利用DNA芯片鉴定肺成纤维细胞中骨膜素依赖性表达的基因。然后，我们检查了细胞周期蛋白和CDKs的表达是否以细胞周期蛋白依赖性方式发生，从而控制细胞周期进程。接下来，我们检查了通过敲击骨膜素或整联蛋白（一种骨膜素受体），使用siRNA抑制细胞增殖促进基因的下调是否反映在肺成纤维细胞的细胞增殖中。然后，我们研究了来自IPF患者的肺成纤维细胞是否也需要骨膜素才能最大程度地增殖。我们最终研究了CP4715（一种有效的抗整联蛋白αVβ3（骨膜蛋白受体）抑制剂，我们最近发现它可以阻断TGF-β信号转导，然后减少BLM诱导的小鼠肺纤维化）是否可以阻断源自IPF患者的肺成纤维细胞的增殖。结果许多细胞周期相关基因通过骨膜素抑制作用而参与上调或下调的基因。我们证实在肺成纤维细胞中 骨膜素沉默可下调几种细胞周期相关分子的表达，包括细胞周期蛋白，CDK和E2F家族，以及转录因子（例如B-MYB和FOXM1）。骨膜素或整联蛋白沉默减慢了肺成纤维细胞的增殖，骨膜素沉默沉默增加了G0 / G1期的分布，而G2 / M期的分布减少了。源自IPF患者的肺成纤维细胞也需要骨膜素以实现最大增殖。此外，CP4715下调IPF肺成纤维细胞以及正常肺成纤维细胞的增殖以及细胞周期相关基因的表达。