Urothelial sonic hedgehog signaling plays an important role in bladder smooth muscle formation
Urothelial sonic hedgehog signaling plays an important role in bladder smooth muscle formation
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DOI：
10.1111/j.1432-0436.2007.00187.x
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发表时间：
2007-12-01
期刊：
影响因子：
2.9
通讯作者：
Baskin, Laurence S.
中科院分区：
文献类型：
--
作者：
Shiroyanagi, Yoshiyuki;Liu, Benchun;Baskin, Laurence S.
在膀胱发育过程中，原始间充质在尿道扩张的影响下分化为平滑肌（SM）。负责这一过程的基因尚未阐明。我们提出，声波刺猬（Shh）信号通路是至关重要的膀胱SM的形成。在此，我们研究了胚胎小鼠膀胱SM分化过程中Shh信号通路的作用。通过免疫组织化学（IHC）、原位杂交和逆转录聚合酶链反应（RT-PCR）检测小鼠胚胎（E）膀胱（E12.5、13.5和14.5）中Shh通路和SM表达的基因。为了检查破坏Shh信号传导的作用，在E12.5和E14.5时，即在膀胱SM诱导之前和之后分离膀胱组织。将胚胎膀胱培养在漂浮在含有和不含10 μ M环巴胺（一种Shh抑制剂）的培养基上的膜上。3天后，通过评估以下内容来检查SM表达：SM α-肌动蛋白（SMAA）、SM γ-肌动蛋白（SMGA）、SM-肌球蛋白重链（SM-MHC）、Patched、GLI 1、骨形态发生蛋白4（BMP 4）和增殖细胞核抗原（PCNA）（通过IHC和RT-PCR）。SM相关基因和蛋白在SM分化前E12.5小鼠胚胎膀胱中不表达，但在SM分化开始时E13.5小鼠胚胎膀胱中表达。在E12.5膀胱中，Shh在尿道上皮中表达。E12.5时Shh相关基因表达显著高于E14.5时。与对照组相比，暴露于环巴明的E12.5组织培养物中SMAA、SMGA、GLI 1和BMP 4基因表达显著降低，但PCNA基因表达没有变化。在环巴明暴露的E14.5培养物中，SMGA和SM-MHC基因表达与对照组相比没有变化。使用体外胚胎膀胱培养模型，我们能够定义SM和Shh相关基因表达的动力学。环巴胺抑制逼尿肌SM肌动蛋白的诱导，但不抑制SM-MHC的诱导。SMAA和SMGA基因似乎是由Shh信号通路诱导的，但SM-MHC基因不是。基于尿路上皮的Shh表达和Shh抑制对膀胱SM诱导的影响，我们假设尿路上皮来源的Shh在胎鼠膀胱中协调SM的诱导。
During bladder development, primitive mesenchyme differentiates into smooth muscle (SM) under the influence of urothelium. The gene(s) responsible for this process have not been elucidated. We propose that the Sonic hedgehog (Shh) signaling pathway is critical in bladder SM formation. Herein, we examine the role of the Shh-signaling pathway during SM differentiation in the embryonic mouse bladder. Genes in the Shh pathway and SM expression in mouse embryonic (E) bladders (E12.5, 13.5, and 14.5) were examined by immunohistochemistry (IHC), in situ hybridization, and reverse transcription polymerase chain reaction (RT-PCR). To examine the effects of disrupting Shh signaling, bladder tissues were isolated at E12.5 and E14.5, that is, before and after bladder SM induction. The embryonic bladders were cultured on membranes floating on medium with and without 10 mu M of cyclopamine, an Shh inhibitor. After 3 days, SM expression was examined by assessing the following: SM alpha-actin (SMAA), SM gamma-actin (SMGA), SM-myosin heavy chain (SM-MHC), Patched, GLI1, bone morphogenic protein 4 (BMP4), and proliferating cell nuclear antigen (PCNA) by IHC and RT-PCR. SM-related genes and proteins were not expressed in E12.5 mouse embryonic bladder before SM differentiation, but were expressed by E13.5 when SM differentiation was initiated. Shh was expressed in the urothelium in E12.5 bladders. Shh-related gene expression at E12.5 was significantly higher than at E14.5. In cyclopamine-exposed cultures of E12.5 tissue, SMAA, SMGA, GLI1, and BMP4 gene expression was significantly decreased compared with controls, but PCNA gene expression did not change. In cyclopamine-exposed E14.5 cultures, SMGA and SM-MHC gene expression did not change compared with controls. Using an in vitro embryonic bladder culture model, we were able to define the kinetics of SM- and Shh-related gene expression. Cyclopamine inhibited detrusor SM actin induction, but did not inhibit SM-MHC induction. SMAA and SMGA genes appear to be induced by Shh-signaling pathways, but the SM-MHC gene is not. Based on Shh expression by urothelium and the effects of Shh inhibition on bladder SM induction, we hypothesize that urothelial-derived Shh orchestrates induction of SM in the fetal mouse bladder.