Cell signaling in developing epithelia

描述(由申请人提供)：本次竞争性更新继续关注慢性和急性肾脏疾病中转化生长因子-β和Bmp7的调节。鉴于转化生长因子-β促进肾间质纤维化，而Bmp7被认为可以保护肾间质纤维化，开发抑制转化生长因子-β或增强细胞外间隙内Bmp7活性的新型治疗药物的潜力必须被追求。在之前的资助期间，我们克隆了一个新的基因KCP，它编码一个含有18个重复的半胱氨酸富含结构域的大蛋白，并与转化生长因子-β和骨形成蛋白结合。KCP蛋白影响受体-配体亲和力、Smad磷酸化和Smad介导的报告基因表达。此外，还产生了KCP零等位基因，并将KCP-/-小鼠用于两种独立的肾损伤模型。KCP增强细胞外间隙内的Bmp7信号，而抑制转化生长因子-β和激活素信号。与这一解释一致，KCP-/-小鼠在单侧输尿管梗阻模型中被证明对发生肾间质纤维化高度敏感，在急性肾毒性模型中表现出恢复减慢和疤痕增加。目前的应用将测试KCP缓解转化生长因子-β介导的疾病的能力，检测转化生长因子-β信号在肾近端小管细胞中的下游作用，并表征KCP增强BMP信号的分子机制。体内和体外方法将被用来测试外源性应用或转基因KCP是否可以降低肾脏疾病的严重程度。转化生长因子-β作用的机制将通过直接鉴定转录激活或被Smad2/3蛋白抑制的基因来解决。KCP对转化生长因子-β和骨形态发生蛋白靶基因的影响将在近端肾小管细胞培养、全肾培养和整个动物中进行检测。此外，我们还将以一种新的方式探讨KCP介导的信号调节机制。初步数据表明，KCP1存在于细胞内的内吞囊泡中，这些囊泡在Bmp7刺激细胞后表现突出。我们的假设是，囊泡型KCP1增强了受体介导的内吞囊泡的稳定性，允许细胞内BMP受体通过激活Smad1继续传递信号。如果这是正确的，BMP和转化生长因子-β由分泌因子调节的范式将发生戏剧性的转变。这些研究将直接检测KCP1介导的抗慢性肾脏疾病保护作用的可能性，并将为开发新型抗纤维化药物奠定基础。公共卫生相关性：肾间质纤维化是许多慢性肾脏疾病的共同特征，可导致肾功能不全和衰竭。纤维化通常是梗阻、损伤或炎症的结果，由细胞因子刺激，如转化生长因子-β，促进肌成纤维细胞的增殖、迁移和细胞外基质的分泌。其他相关的细胞因子，BMP蛋白，被认为是保护性的。这些胞外蛋白的作用是通过控制配体-受体相互作用的各种机制来调节的。我们已经确定了一种通过与分泌的配体结合蛋白相互作用来抑制转化生长因子-β的新机制。如何操纵这些相互作用以达到治疗效果需要进行调查，这也是本提案的主题。

DESCRIPTION (provided by applicant): This competitive renewal continues to focus on the regulation of TGF-beta and Bmp7 in chronic and acute renal disease. Given that TGF-beta promotes renal interstitial fibrosis, whereas Bmp7 is thought to protect against this effect, the potential for developing novel therapeutic agents that inhibit TGF-beta or enhance Bmp7 activity within the extracellular space must be pursued. During the previous funding period, we cloned a novel gene, kcp, that encodes a large protein with 18 repeated cysteine rich domains and binds to both TGF-beta and BMPs. The KCP protein affects receptor-ligand affinities, Smad phosphorylation, and Smad mediated reporter gene expression. Furthermore, a kcp null allele was generated and the kcp-/- mice used in two independent models of renal injury. KCP enhances Bmp7 signaling within the extracellular space, while inhibiting TGF-beta and Activin signaling. Consistent with this interpretation, kcp-/- mice proved to be hypersensitive to developing renal interstitial fibrosis in the unilateral ureteral obstruction model and showed decreased recovery and increased scarring in an acute nephrotoxicity model. The current application will test the ability of KCP to relieve TGF-beta mediated disease, examine the downstream effects of TGF-beta signaling in renal proximal tubule cells, and characterize the molecular mechanisms of Bmp signal enhancement by KCP. Both in vivo and in vitro methods will be employed to test whether exogenously applied or transgenic KCP can reduce the severity of renal disease. The mechanism of TGF-beta action will be addressed by direct identification of genes transcriptionally activated or repressed by Smad2/3 proteins. The effects of KCP on TGF-beta and BMP target genes will be assayed in proximal tubule cell cultures, whole kidney cultures, and whole animals. In addition, we will address the mechanism of KCP mediated regulation of signaling in a novel way. Preliminary data demonstrates that KCP1 can be found in intracellular, endocytotic vesicles, and that these vesicles are prominent after stimulation of cells with Bmp7. Our hypothesis is that vesicular KCP1 enhances the stability of receptor mediated endocytotic vesicles allowing for continued signaling from intracellular Bmp receptors through activation of Smad1. If this is correct, the paradigm of BMP and TGF-beta regulation by secreted factors will be dramatically shifted. These studies will directly examine the potential for KCP1 mediated protection against chronic renal disease and will lay the foundation for developing novel anti-fibrotic agents. PUBLIC HEALTH RELEVANCE: Renal interstitial fibrosis is a common denominator among many chronic renal diseases and can lead to insufficiency and failure. Often the result of obstruction, injury, or inflammation, fibrosis is stimulated by cytokines, such as TGF-beta, which promote myofibroblast proliferation, migration, and secretion of extracellular matrix. Other related cytokines, the BMP proteins, are thought to be protective. The effects of these extracellular proteins are mediated by a variety of mechanisms that control ligand-receptor interactions. We have identified a novel mechanism of TGF-beta suppression through interactions with secreted ligand binding proteins. How these interactions can be manipulated for therapeutic effects needs to be investigated and is the subject of this proposal.