Integrin/TGF-beta Axis in Tubulointerstitial Fibrosis

描述（由申请人提供）：我们的目标是分析肾小管间质（TI）纤维化形成的分子机制，以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<$1 <$1，并表明它下调胶原合成，其损失导致损伤后肾小球纤维化增加。整合素1¿ 1通过激活酪氨酸磷酸酶TCPTP负性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 <$1是TI纤维化的负调节因子，因为整合素<$1 KO小鼠显示单侧输尿管梗阻诱导的纤维化增加。此外，整合素1 KO集合管（CD）细胞增加了TGF-β受体（T β R）依赖性促纤维化信号传导的激活，如磷酸化Smad 3和胶原蛋白水平。TGF-β通过激活丝氨酸/苏氨酸激酶T <$RI和T <$RII发挥其功能。TGF-β与T <$RII的结合导致T <$RI的磷酸化和随后的主要促纤维化介质Smad 3的活化。有趣的是，T <$RII的胞质尾区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。该提议的新奇在于整合素<$1 KO CD细胞显示酪氨酸磷酸化T <$RII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交叉，它可能通过下调其酪氨酸磷酸化水平来防止其促纤维化作用。有趣的是，CD细胞中TCPTP的抑制导致Smad 3活化和胶原合成增加。这一结果，连同发现在T <$RII尾部的3个酪氨酸可以是TCPTP的潜在底物，形成了整合素<$1 <$1通过激活TCPTP负调节T <$RII酪氨酸磷酸化的假设。因此，我们认为整合素<$1 <$1/TCPTP介导的T <$RII去磷酸化是一种重要的，但以前未描述的机制，选择性地减少T <$RII激活和随后的纤维化进展。为了验证这一假设：目的1将分析整合素1 KO小鼠中T <$RII介导的促纤维化信号传导在TI损伤中的作用。我们将交叉整合素？1 KO小鼠与整体无效或floxed Smad 3小鼠的比较，以确定阻止收集系统中的T κ R/Smad 3轴是否足以改善1 KO小鼠中的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而有益于TI损伤。目的2将确定整合素1负调控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 <$RII去磷酸化; ii）酪氨酸残基是否对控制T <$RII介导的Smad 3活化和胶原合成很重要; iii）哪种酪氨酸控制T <$RII介导的功能。这项研究将导致识别整合素1和T RII之间的新串扰，最重要的是，一种新的机制，使T RII介导的促纤维化信号可以被负调节。

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.