Cell signaling in developing epithelia

描述（由申请人提供）：该竞争性更新继续关注慢性和急性肾脏疾病中tgf - β和Bmp7的调节。鉴于tgf - β促进肾间质纤维化，而Bmp7被认为可以防止这种作用，因此必须寻求开发抑制tgf - β或增强细胞外空间Bmp7活性的新型治疗药物的潜力。在之前的资助期间，我们克隆了一个新的基因kcp，该基因编码一个具有18个重复的富含半胱氨酸的结构域的大蛋白，并与tgf - β和bmp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，生成了kcp空等位基因，并将kcp-/-小鼠用于两种独立的肾损伤模型。KCP增强细胞外空间Bmp7信号，同时抑制tgf - 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肾间质纤维化过敏，在急性肾毒性模型中表现出恢复减少和疤痕增加。目前的应用将测试KCP缓解tgf - β介导的疾病的能力，检查tgf - 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。将采用体内和体外两种方法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。tgf - β的作用机制将通过直接鉴定被Smad2/3蛋白激活或抑制的基因来解决。KCP对tgf - β和BMP靶基因的影响将在近端小管细胞培养、全肾培养和全动物中进行检测。此外，我们将以一种新的方式解决KCP介导的信号调节机制。初步数据表明，KCP1可以在细胞内、内吞囊泡中发现，这些囊泡在Bmp7刺激细胞后突出。我们的假设是，囊泡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继续发送信号。如果这是正确的，那么BMP和tgf - β由分泌因子调节的模式将会发生巨大的变化。这些研究将直接研究KCP1介导的慢性肾脏疾病保护的潜力，并为开发新的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。通常是梗阻、损伤或炎症的结果，纤维化是由细胞因子刺激的，如tgf - β，它促进肌成纤维细胞增殖、迁移和细胞外基质的分泌。其他相关的细胞因子，BMP蛋白，被认为具有保护作用。这些细胞外蛋白的作用是由多种控制配体-受体相互作用的机制介导的。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抑制tgf - 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研究，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.