Integrin/TGF-beta Axis in Tubulointerstitial Fibrosis

描述(申请人提供)：我们的目标是分析形成肾小管间质(TI)纤维化的分子机制，设计更有效的治疗方法来防止其进展。我们研究了胶原受体整合素1，发现它下调胶原的合成，它的丢失导致损伤后肾小球纤维化的增加。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，从而发挥抗纤维化作用。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1KO小鼠表现出单侧输尿管梗阻诱导的纤维化增加。此外，整合素？1KO集合管(CD)细胞可增加依赖于转化生长因子受体(T？R)的促纤维化信号的激活，如磷酸化的Smad3和胶原水平。转化生长因子-β通过激活丝氨酸/苏氨酸激酶T？RI和T？RII发挥其功能。转化生长因子-β与T？RII的结合导致T？RI的磷酸化，进而激活主要的促纤维化介质Smad3。有趣的是，T？RII的细胞质尾部也可以在酪氨酸残基上被磷酸化。然而，这些酪氨酸是否在肾脏细胞中发挥任何生理或病理作用尚不清楚。这一建议的新奇之处在于，整合素1KO CD细胞显示出基础水平升高的酪氨酸磷酸化T？RII。这一结果表明，在肾细胞中，整合素1与T？RII有串扰作用，它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，抑制CD细胞中的TCPTP会导致Smad3的激活和胶原合成的增加。这一结果，再加上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负向调节T？RII酪氨酸磷酸化的假说。因此，我们认为整合素1/TCPTP介导的T？RII去磷酸化是选择性减少T？RII激活和随后的纤维化进展的一个重要但先前未被描述的机制。为了验证这一假设：目的1将分析T？RII介导的促纤维化信号在整合素？1KO小鼠TI损伤中的作用。我们将把整合素？1KO小鼠与全球空白或有牙线的SMAD3小鼠杂交，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，我们将确定体内激活TCPTP是否有助于通过对抗T？R介导的促纤维化作用而在TI损伤中发挥作用。目的2将确定整合素1负向调节T？RII的机制。我们将使用体外方法来分析：i)TCPTP是否直接结合并去磷酸化T？RII；ii)酪氨酸残基是否对控制T？RII介导的Smad3激活和胶原合成具有重要作用；iii)哪种酪氨酸(S)控制T？RII介导的功能。这项研究将导致在整合素和T？RII之间发现一种新的串扰，最重要的是，一种新的机制，即T？RII介导的促纤维化信号可以被负调制。

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.