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）：本次竞争性更新继续关注慢性和急性肾病中 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效应，因此必须探索开发抑制 TGF-β 或增强细胞外空间内 Bmp7 活性的新型治疗剂的潜力。在之前的资助期间，我们克隆了一个新基因 kcp，它编码具有 18 个重复的半胱氨酸丰富结构域的大蛋白，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 无效等位基因，并将 kcp-/- 小鼠用于两个独立的肾损伤模型。 KCP 增强细胞外空间内的 Bmp7 信号传导，同时抑制 TGF-β 和激活素信号传导。与这种解释一致，kcp-/-小鼠在单侧输尿管梗阻模型中被证明对肾间质纤维化的发生高度敏感，并且在急性肾毒性模型中表现出恢复下降和疤痕增加。目前的应用将测试KCP缓解TGF-β介导的疾病的能力，检查肾近曲小管细胞中TGF-β信号传导的下游效应，并表征KCP增强Bmp信号的分子机制。将采用体内和体外方法来测试外源应用或转基因KCP是否可以减轻肾脏疾病的严重程度。 TGF-β 作用机制将通过直接鉴定 Smad2/3 蛋白转录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物、全肾培养物和整个动物中进行测定。此外，我们将以一种新颖的方式解决 KCP 介导的信号传导调节机制。初步数据表明，KCP1 可以在细胞内的内吞囊泡中找到，并且这些囊泡在用 Bmp7 刺激细胞后会变得突出。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，从而允许细胞内 Bmp 受体通过激活 Smad1 持续发出信号。如果这是正确的，分泌因子调节 BMP 和 TGF-β 的模式将发生巨大转变。这些研究将直接检验 KCP1 介导的针对慢性肾病的保护潜力，并将为开发新型抗纤维化药物奠定基础。公众健康相关性：肾间质纤维化是许多慢性肾脏疾病的共同点，可导致肾功能不全和衰竭。纤维化通常是阻塞、损伤或炎症的结果，受到细胞因子（例如 TGF-β）的刺激，促进肌成纤维细胞增殖、迁移和细胞外基质的分泌。其他相关细胞因子（BMP 蛋白）被认为具有保护作用。这些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来抑制 TGF-β 的新机制。如何操纵这些相互作用以获得治疗效果需要研究，这也是本提案的主题。