Cell signaling in developing epithelia

发育中的上皮细胞信号传导

• 批准号: 7729884
• 负责人: Gregory R Dressler
• 金额: $ 34.76万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-20 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7729884
• 关键词: Activins; Acute; Acute Kidney Tubular Necrosis; Address; Affect; Affinity; Alleles; Animal Model; Animals; BMP7 gene; Binding; Binding Proteins; Biochemical Genetics; Biological Assay; Biological Models; Cell Culture System; Cell Culture Techniques; Cells; Chronic; Chronic Disease; Chronic Kidney Failure; Cicatrix; Clinical; Complex; Cysteine-Rich Domain; Data; Disease; Disease model; Embryonic Development; Endocytic Vesicle; Epithelium; Exhibits; Extracellular Matrix; Extracellular Protein; Extracellular Space; Failure; Family; Fibrosis; Foundations; Funding; Gene Expression; Gene Targeting; Genes; In Vitro; Inflammation; Inhibin-beta Subunits; Injury; Kidney; Kidney Diseases; Kidney Failure; Lead; Ligands; Mediating; Methods; Modality; Modeling; Molecular; Mouse Protein; Mus; Myofibroblast; Names; Obstruction; Pathway interactions; Peptide Signal Sequences; Phosphorylation; Predisposition; Principal Investigator; Process; Proteins; Proximal Kidney Tubules; Recovery; Regulation; Renal Tissue; Reporter Genes; Severities; Signal Pathway; Signal Transduction; Signaling Protein; Staging; System; Technology; Testing; Therapeutic; Therapeutic Agents; Therapeutic Effect; Transforming Growth Factor beta; Transgenic Animals; Transgenic Organisms; Ureteral obstruction; Vesicle; Work; Xenopus; chordin; cytokine; design; disease phenotype; glomerulosclerosis; in vivo; injured; interstitial; kidney cell; mesangial cell; migration; nephrotoxicity; novel; novel therapeutics; programs; public health relevance; receptor; receptor mediated endocytosis; research study; response; trafficking

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.

描述（由申请人提供）：这种竞争性更新继续关注慢性和急性肾脏疾病中TGF-beta和BMP7的调节。鉴于TGF-beta促进了肾脏间质纤维化，而BMP7被认为可以防止这种作用，因此必须追求抑制TGF-beta或增强细胞外空间内BMP7活性的新型治疗剂的潜力。在上一个资金期间，我们克隆了一个新型的基因KCP，该基因编码了一个大蛋白质，具有18个重复的半胱氨酸富含结构域，并与TGF-beta和BMP结合。 KCP蛋白会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，产生了KCP无效等位基因，并在两个独立的肾脏损伤模型中使用KCP - / - 小鼠。 KCP增强了细胞外空间内的BMP7信号传导，同时抑制TGF-beta和激活素信号传导。与这种解释一致，在单侧输尿管障碍物模型中，KCP - / - 小鼠对发展肾脏间质纤维化是高度敏感的，并且在急性肾毒性模型中显示出恢复和增加的疤痕。当前的应用将测试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效应，并表征KCP BMP信号增强BMP信号的分子机制。体内和体外方法都将用于测试外源应用还是转基因KCP可以降低肾脏疾病的严重程度。 TGF-β作用的机制将通过直接鉴定被SMAD2/3蛋白抑制的基因直接鉴定来解决。 KCP对TGF-β和BMP靶基因的影响将在近端小管细胞培养物，整个肾脏培养物和全动物中分析。此外，我们将以新颖的方式解决KCP介导的信号调节的机理。初步数据表明，KCP1可以在细胞内，内吞囊泡中找到，并且这些囊泡在用BMP7刺激细胞后是突出的。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，允许通过激活SMAD1的细胞内BMP受体的持续信号传导。如果这是正确的，则会通过分泌因子对BMP和TGF-β调节的范例发生巨大变化。这些研究将直接研究KCP1介导的对慢性肾脏疾病的保护潜力，并将为发展新型抗纤维化药物奠定基础。公共卫生相关性：肾脏间质纤维化是许多慢性肾脏疾病的普通分母，可能导致不足和失败。纤维化通常会受到阻塞，损伤或炎症的结果，纤维化会受到细胞因子的刺激，例如TGF-β，这些因子促进了肌纤维细胞增殖，迁移和细胞外基质的分泌。其他相关的细胞因子，即BMP蛋白，被认为具有保护性。这些细胞外蛋白的作用是由控制配体 - 受体相互作用的多种机制介导的。我们通过与分泌的配体结合蛋白相互作用来确定TGF-β抑制的新机理。如何对这些相互作用进行治疗效果，需要研究这些建议，这是该提议的主题。