Heme Oxygenase-1 Gene Expression by TGF-Beta 1

描述（由申请人提供）：转化生长因子-B（TGF-B）是一种调节性细胞因子，与多种肾脏疾病有关，可促进细胞外基质沉积和纤维化。巧合的是，TGF-β在正常体内平衡中具有关键作用，并通过尚不清楚的机制稳定和减轻组织损伤。我们以前报道，在肾小管上皮细胞，TGF-β 1是一种有效的诱导剂的细胞保护酶，血红素加氧酶-1（HO-1）。该建议的总体假设是HO-1诱导是对TGF-β 1介导的肾脏细胞损伤的细胞保护反应。TGF-β诱导HO-1的潜在机制以及HO-1表达及其产物对肾上皮细胞对TGF-β 1反应的影响尚不清楚。在初步研究中，我们观察到HO-1-/-小鼠（24周龄）的肾脏显示出比年龄匹配的HO-1（）小鼠显著更高的纤连蛋白表达。相反，HO-1诱导与纤连蛋白水平降低相关。此外，胆红素，HO-1催化反应的产物之一，显着降低TGF-β 1介导的肾小管上皮细胞纤维连接蛋白水平的增加。我们的研究还表明，抑制性Smad，Smad 7，阻断HO-1诱导TGF-β 1在肾上皮细胞。在TGF-β过表达小鼠的肾脏中也观察到Smad 7和HO-1之间的负相关性的体内相关性。TGF-β 1介导的HO-1诱导受转录调控，需要人HO-1启动子的-9.1和-9.4kb之间的顺式作用区，可能涉及Sp1样序列。本提案将使用HO-1缺陷细胞（源自HO-1 -/-、+/+小鼠的原代细胞，并在HK-2细胞中使用siRNA敲低HO-1）以及HO-1过表达HK-2细胞（Aim I），评价HO-1诱导在体外响应TGF-β 1中的生物学作用。HO-1 -/-小鼠及其同窝出生的小鼠将用于研究HO-1和HO反应产物在阻塞性尿路病和肾纤维化模型（单侧输尿管梗阻（DUO）模型）中纤维化发病机制中的作用（Aim II）。最后，将通过测试所鉴定的蛋白质/转录因子的过表达或显性负表达对细胞对TGF-β 1的应答的影响来表征TGF-β 1介导的HO-1基因表达的分子调控（Aim III）并将其与Aim 1整合。了解TGF-β 1诱导HO-1的分子机制的研究是及时和相关的努力微调内源性HO-1活性在肾损伤。

DESCRIPTION (provided by applicant): Transforming growth factor-B (TGF-B) is a regulatory cytokine that is implicated in a variety of kidney diseases where it promotes extracellular matrix deposition and fibrosis. Paradoxically, TGF-B has a critical role in normal homeostasis and stabilizes and attenuates tissue injury through mechanisms that are not clearly understood. We previously reported that, in renal tubular epithelial cells, TGF-B1 is a potent inducer of a cytoprotective enzyme, heme oxygenase-1 (HO-1). The overall hypothesis of this proposal is that HO-1 induction is a cytoprotective response to TGF-B1mediated cellular injury in the kidney. The mechanisms underlying the induction of HO-1 by TGF-B as well as the effects of HO-1 expression and its products on the renal epithelial cellular responses to TGF-B1 are not known. In preliminary studies, we have observed that kidneys from HO-1-/- mice (age 24 weeks) show significantly higher fibronectin expression than age matched HO-1 () mice. In contrast, HO-1 induction is associated with decreased levels of fibronectin. Furthermore, bilirubin, 1of the products of the HO-1 catalyzed reaction, significantly lowers TGF-B1-mediated increases in fibronectin levels in renal tubular epithelial cells. Our studies also demonstrate that the inhibitory Smad, Smad7, blocks HO-1 induction by TGF-B1 in renal epithelial cells. The in vivo relevance of the inverse relationship between Smad7 and HO-1 was also observed in kidneys from TGF-p overexpressing mice. TGF-B1-mediated HO-1 induction is transcriptionally regulated and requires a cis-acting region between-9.1 and-9.4kb of the human HO-1 promoter and may involve Sp1-like sequences. This proposal will evaluate the biological role of HO-1 induction in response to TGF-B1 in vitro, using HO-1 deficient cells (primary cells derived from HO-1 -/-, +/+ mice and HO-1 knockdown using siRNA in HK-2 cells) as well as HO-1 overexpressing HK-2 cells (Aim I). HO-1 -/- mice and their littermates will be used to study the role of HO-1 and HO reaction products in the pathogenesis of fibrosis in a model for obstructive uropathy and renal fibrosis, the unilateral ureteral obstruction (DUO) model (Aim II). Finally, the molecular regulation of TGF-B1 -mediated HO-1 gene expression will be characterized (Aim III) and integrated with Aim 1 by testing the effects of overexpression or dominant negative expression of the protein/transcription factor identified on the cellular responses to TGF-B1. Studies to understand the molecular mechanisms involved in the induction of HO-1 by TGF-B1 are timely and relevant for efforts to fine tune endogenous HO-1 activity in renal injury.