Regulation of gut smooth muscle contraction and relaxation by cytokines

描述(申请人提供)：已知的促炎介质，如IL-1β和TNF-α，部分通过直接作用于平滑肌而抑制平滑肌的收缩能力。我们的假设是，这些介质通过诱导或抑制介导收缩和松弛的信号通路中关键靶点的表达来抑制收缩能力。在对对照和TNBS结肠炎肌条以及新鲜分散和培养的肌细胞的初步研究中，我们发现了六种新的细胞因子(IL-1β)介导的效应，反映了特定信号靶点的表达和/或活性的变化。这包括：(I)上调RGS4和RGS12的表达，分别导致GQ和GI偶联受体激动剂抑制初始的钙依赖性收缩；(Ii)下调内源性MLC磷酸酶抑制剂CPI-17，从而抑制持续的RhoA依赖的收缩；(Iii)上调分别调节钙摄取和钙释放的SERCA2和IP3R-I；(Iv)上调PDE4D5和PDE5A的表达，分别导致cAMP和cGMP的更大降解；(V)通过依赖PKG/JNK的转录后机制下调可溶性鸟苷酸环化酶(SGC)的表达，从而抑制cGMP的合成；(Vi)通过依赖iNOS的亚硝化使腺酰环化酶(AC)失活，从而抑制cAMP的合成。到目前为止，在IL-1β治疗的结肠肌肉中引起的效果也在TNBS结肠炎的结肠肌肉中观察到。其具体目的是：(1)表征介导初始钙依赖和持续非钙依赖性收缩的信号靶标(RGS4/RGS12、CPI-17、SERCA2和IP3R-I)的表达和活性的变化；(2)表征调节松弛的信号靶标(PDE3A、PDE4D5、PDE5A；sGC和AC)的表达和活性的变化；(3)研究核因子-kappa B和调节蛋白激酶(p38 MAP、ERK1/2和PI3-Kinase/Akt/GSK3β途径)对RGS4/RGS12和CPI-17的转录调控，以及通过PKG/JNK/AP-1/Hur途径对sGC表达的转录后调控。这些研究将在体外和已建立的结肠炎症模型中，全面分析炎性细胞因子在细胞水平上抑制收缩的机制。

DESCRIPTION (provided by applicant): Pro-inflammatory mediators, such as IL-1 beta and TNF-alpha, are known to inhibit smooth muscle contractility in part by acting directly on smooth muscle. Our hypothesis is that these mediators inhibit contractility by inducing or suppressing the expression of critical targets in the signaling pathways mediating contraction and relaxation. In preliminary studies on control and TNBS colitis muscle strips, and on freshly dispersed and cultured muscle cells, we have identified six novel cytokine (IL-1 beta)-mediated effects reflecting changes in the expression and/or activity of specific signaling targets. These include: (i) up-regulation of RGS4 and RGS12 expression, which leads to inhibition of initial Ca2+-dependent contraction by Gq and Gi-coupled receptor agonists, respectively; (ii) down-regulation of the endogenous MLC phosphatase inhibitor, CPI-17, which leads to inhibition of sustained RhoA-dependent contraction; (iii) up-regulation of SERCA2 and IP3R-I, which regulate Ca2+ uptake and Ca2+ release, respectively; (iv) up-regulation of PDE4D5 and PDE5A expression, which leads to greater degradation of cAMP and cGMP, respectively; (v) down-regulation of soluble guanylyl cyclase (sGC) expression via a PKG/JNK-dependent post-transcriptional mechanism, which leads to inhibition of cGMP synthesis; and (vi) inactivation of adenylyl cyclase (AC) via iNOS-dependent nitrosylation, which leads to inhibition of cAMP synthesis. Where examined so far, the effects elicited in IL-1 beta-treated colonic muscle were observed also in colonic muscle from TNBS colitis. The specific aims are to: (1) characterize the changes in the expression and activity of signaling targets (RGS4/RGS12, CPI-17, SERCA2 and IP3R-I) mediating initial Ca2+-dependent and sustained Ca2+-independent contraction; (2) characterize the changes in expression and activity of signaling targets (PDE3A, PDE4D5, PDE5A; sGC and AC) that regulate relaxation; and (3) characterize the transcriptional regulation of RGS4/RGS12 and CPI-17 by NF-kappa B and modulatory kinases (p38 MAP kinase, ERK1/2 and the PI 3-kinase/Akt/GSK3beta pathway), and the post-transcriptional regulation of sGC expression via a PKG/JNK/AP-1/HuR pathway. These studies will provide a comprehensive analysis of the mechanisms by which inflammatory cytokines inhibit contractility at cellular level, both in vitro and in an established model of colonic inflammation.