Integrin/TGF-beta Axis in Tubulointerstitial Fibrosis

肾小管间质纤维化中的整合素/TGF-β轴

• 批准号: 8840580
• 负责人: AMBRA POZZI
• 金额: $ 33.93万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-08 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8840580
• 关键词: Area; Binding; Cell physiology; Cells; Chronic Kidney Failure; Collagen; Collagen Receptors; Cytoplasmic Tail; Duct (organ) structure; ECM receptor; Epidermal Growth Factor Receptor; Fibrosis; Goals; Growth; Growth Factor Receptors; Health; Homeostasis; In Vitro; Injury; Integrins; Kidney; Kidney Diseases; Laboratories; Lead; Ligands; Mediating; Mediator of activation protein; Molecular; Mus; Pathway interactions; Phosphorylation; Physiological; Play; Process; Production; Protein Dephosphorylation; Protein Tyrosine Phosphatase; Protein-Serine-Threonine Kinases; Receptor Activation; Regulation; Role; Signal Transduction; System; Tail; Testing; Transforming Growth Factor beta; Transforming Growth Factors; Tubular formation; Tyrosine; Tyrosine Phosphorylation; Up-Regulation; Ureteral obstruction; adhesion receptor; effective therapy; glomerulosclerosis; in vivo; injured; kidney cell; novel; prevent; receptor

DESCRIPTION (provided by applicant): Our goal is to analyze the molecular mechanisms that underlie the formation of tubulointerstitial (TI) fibrosis, to devise more effective therapies to prevent its progression. We study the collagen receptor integrins ¿1¿1 and showed that it downregulates collagen synthesis, and its loss leads to increased glomerular fibrosis following injury. Integrin ¿1¿1 plays an anti-fibrotic action by negatively regulating the phosphorylation state of pro-fibrotic growth factor receptors via activation of the tyrosine phosphatase TCPTP. We have also found that integrin ¿1¿1 is a negative regulator of TI fibrosis, as integrin ¿1KO mice show increased unilateral ureteral obstruction-induced fibrosis. Moreover, integrin ¿1KO collecting duct (CD) cells have increased activation of TGF-¿ receptor (T¿R)-dependent pro-fibrotic signaling, such as phosphorylated Smad3 and collagen levels. TGF-¿ exerts its functions via activation of the serine/threonine kinases T¿RI and T¿RII. Binding of TGF-¿ to T¿RII leads to phosphorylation of T¿RI and subsequent activation of the major pro-fibrotic mediator Smad3. Interestingly, the cytoplasmic tail of T¿RII can also be phosphorylated on tyrosine residues. However, whether these tyrosines play any physiological or pathological role in renal cells is unknown. The novelty of this proposal is that integrin ¿1KO CD cells show increased basal levels of tyrosine phosphorylated T¿RII. This result suggests that, in renal cells, integrin ¿1¿1 crosstalks with T¿RII and it might prevent its pro-fibrotic action by downregulating its tyrosine phosphorylation levels. Interestingly, inhibition of TCPTP in CD cells leads to increased Smad3 activation and collagen synthesis. This result, together with the finding that 3 tyrosines in the T¿RII tail can be potential substrates of TCPTP, forms the hypothesis that integrin ¿1¿1 negatively regulates T¿RII tyrosine phosphorylation via activation of TCPTP. Thus, we propose that integrin ¿1¿1/TCPTP-mediated dephosphorylation of T¿RII represents an important, but previously undescribed mechanism to selectively reduce T¿RII activation and consequent progression of fibrosis. To test this hypothesis: Aim 1 will analyze the role of T¿RII-mediated pro-fibrotic signaling in TI injury in the integrin ¿1KO mice. We will cross integrin ¿1KO mice with global null or floxed Smad3 mice to determine if preventing T¿R/Smad3 axis in the collecting system is sufficient to ameliorate TI fibrosis in the ¿1KO mice. We will then determine if in vivo activation of TCPTP is beneficial in the setting of TI injury by counteracting T¿R-mediated pro-fibrotic action. Aim 2 will determine the mechanisms whereby integrin ¿1¿1 negatively regulates T¿RII. We will use in vitro approaches to analyze i) if TCPTP directly binds and dephosphorylates T¿RII; ii) if tyrosine residues are important to control T¿RII-mediated Smad3 activation and collagen synthesis; and iii) which tyrosine(s) controls T¿RII-mediated functions. This study will lead to the identification of a novel crosstalk between integrin ¿1¿1 an T¿RII and, most importantly, a novel mechanism whereby T¿RII-mediated pro-fibrotic signaling can be negatively modulated.

描述（由申请人提供）：我们的目标是分析小管间质（TI）纤维化形成的分子机制，设计更有效的治疗方法来预防其进展。我们研究了胶原受体整合素，发现它下调胶原合成，其缺失导致损伤后肾小球纤维化增加。整合素1通过激活酪氨酸磷酸酶TCPTP负向调节促纤维化生长因子受体的磷酸化状态，发挥抗纤维化作用。我们还发现整合素1是TI纤维化的负调节因子，因为整合素1 ko小鼠显示单侧输尿管梗阻诱导的纤维化增加。此外，整合素- 1KO收集管（CD）细胞增加了TGF-受体（T - R）依赖性促纤维化信号的激活，如磷酸化的Smad3和胶原蛋白水平。TGF-¿通过激活丝氨酸/苏氨酸激酶T¿RI和T¿RII来发挥其功能。TGF-与T - RII结合导致T - RI磷酸化，随后激活主要促纤维化介质Smad3。有趣的是，T¿RII的细胞质尾部也可以在酪氨酸残基上磷酸化。然而，这些酪氨酸是否在肾细胞中发挥任何生理或病理作用尚不清楚。这一建议的新颖之处在于，整合素1KO CD细胞显示出酪氨酸磷酸化的trii的基础水平增加。这一结果表明，在肾细胞中，整合素- 1 - 1与T - RII相互作用，并可能通过下调其酪氨酸磷酸化水平来阻止其促纤维化作用。有趣的是，在CD细胞中抑制TCPTP会导致Smad3激活和胶原合成增加。这一结果，再加上发现T¿RII尾部的3个酪氨酸可能是TCPTP的潜在底物，形成了整合素1¿1通过激活TCPTP负性调节T¿RII酪氨酸磷酸化的假设。因此，我们提出整合素1/ tcptp介导的T - RII去磷酸化代表了一个重要的，但先前未描述的机制，可以选择性地减少T - RII激活和随后的纤维化进展。为了验证这一假设：目的1将分析T ø rii介导的促纤维化信号在整合素1KO小鼠TI损伤中的作用。我们将整合素1KO小鼠与全基因缺失的或固定的Smad3小鼠交叉，以确定在收集系统中阻止T¿R/Smad3轴是否足以改善¿1KO小鼠的TI纤维化。然后，我们将确定TCPTP的体内激活是否通过抵消T¿r介导的促纤维化作用，在TI损伤的情况下有益。目标2将确定整合蛋白1负性调节T - RII的机制。我们将使用体外方法来分析i) TCPTP是否直接结合并使T¿RII去磷酸化；ii)如果酪氨酸残基对控制T¿rii介导的Smad3激活和胶原合成很重要；iii)哪些酪氨酸控制T¿iii介导的功能。这项研究将鉴定整合蛋白1 - 1和T - RII之间的新型串扰，最重要的是，鉴定T - RII介导的促纤维化信号可以负调节的新机制。