Regulation of gut smooth muscle contraction and relaxation by cytokines

描述（由申请人提供）：已知促炎介质，如IL-1 β和TNF-α，通过直接作用于平滑肌而部分抑制平滑肌收缩性。我们的假设是，这些介质通过诱导或抑制介导收缩和舒张的信号通路中的关键靶点的表达来抑制收缩性。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，我们已经确定了六种新的细胞因子（IL-1 β）介导的作用，反映了特定信号传导靶点的表达和/或活性的变化。其中包括：（i）RGS 4和RGS 12表达的上调，其分别导致Gq和Gi偶联受体激动剂对初始Ca 2+依赖性收缩的抑制;（iii）SERCA 2和IP 3R-I的上调，其分别调节Ca 2+摄取和Ca 2+释放;（iv）PDE 4D 5和PDE 5A表达的上调，其分别导致cAMP和cGMP的更大降解;（v）通过PKG/JNK依赖性转录后机制下调可溶性鸟苷酸环化酶（sGC）表达，这导致cGMP合成的抑制;和（vi）通过iNOS依赖性亚硝基化使腺苷酸环化酶（AC）失活，这导致cAMP合成的抑制。到目前为止，在TNBS结肠炎的结肠肌肉中也观察到在IL-1 β处理的结肠肌肉中引起的作用。具体目标是：（1）表征信号传导靶点的表达和活性的变化（2）表征信号传导靶点的表达和活性的变化（PDE 3A、PDE 4D 5、PDE 5A; sGC和AC）调节松弛;以及（3）表征NF-κ B和调节激酶对RGS 4/RGS 12和CPI-17的转录调节（p38 MAP激酶、ERK 1/2和PI 3-激酶/Akt/GSK 3 β途径），以及通过PKG/JNK/AP-1/HuR途径对sGC表达的转录后调节。这些研究将提供一个全面的分析机制，炎症细胞因子抑制收缩在细胞水平上，在体外和在一个既定的模型结肠炎症。

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.