NFAT and MEF-2 Choreograph Bladder Wall Remodeling Following Partial Outlet Obstr

描述（由申请人提供）：作为对部分出口梗阻的反应，膀胱能够通过复杂的重塑过程经历肥大，使其能够适应新的工作负荷。这种重塑是由许多信号级联及其相应的转录因子驱动的。该提案的重点是钙调磷酸酶途径及其相关转录因子活化T细胞核因子（NFAT）和肌细胞增强因子2（MEF-2）的作用。初步数据支持我们的中心假设，即部分膀胱出口梗阻与细胞内钙稳态失调和钙调神经磷酸酶激活以及随后的NFAT核输入有关。随着环孢素A（CSA）的管理，所产生的膀胱肥大减少，并有一个逆转的肌球蛋白重链（MHC）mRNA亚型回到正常的表达模式。我们建议进一步研究这些观察结果在小鼠模型的部分膀胱出口梗阻使用转基因小鼠含有NFAT-荧光素酶报告基因构建体的存在和不存在CSA管理的不同时间点。终点将包括膀胱质量、收缩性和缩短速度的体外测定、形态学和MHC亚型表达的分子分析。我们还寻求原位定位，其中膀胱壁细胞群显示钙调磷酸酶激活的证据。我们还试图定位膀胱壁内发生DNA合成的位点，并确定这些DNA合成位点是否代表成纤维细胞、平滑肌细胞或两者。这些体内实验通过使用培养细胞的体外实验来补充，以评估机械变形对NFAT和MEF- 2易位到细胞核的影响。这种方法将允许在体外筛选的化合物，可以防止钙调磷酸酶激活，并将允许NFAT，MEF-2和平滑肌MHC启动子之间的相互作用的研究。使用微阵列方法，我们建议确定两个细胞群内的NFAT和MEF-2响应的靶基因;膀胱平滑肌和膀胱成纤维细胞。当人们考虑到未经治疗的膀胱壁肥大可能导致的尿失禁和肾衰竭的社会破坏性后遗症时，这项拟议的工作与人类疾病的相关性是显而易见的。 公共卫生相关性： 人类相关性-两种情况可导致儿童严重的膀胱壁肥大，后尿道瓣膜和脊柱裂。如果不进行治疗，膀胱壁肥大会导致尿失禁的社会破坏性后遗症，在极端情况下，由于膀胱失去在低压下储存尿液的能力，可能导致肾衰竭。保留和增强天然膀胱功能的新疗法将有助于避免终末期膀胱的发展。

DESCRIPTION (provided by applicant): In response to partial outlet obstruction, the urinary bladder is capable of undergoing hypertrophy via a complex remodeling process that allows it to adapt to its new workload. This remodeling is driven by a number of signaling cascades and their corresponding transcription factors. This proposal focuses on the role of the calcineurin pathway and its associated transcription factors the Nuclear Factor of Activated T Cells (NFAT) and Myocyte Enhancing Factor 2 (MEF-2). Preliminary data support our central hypothesis that partial bladder outlet obstruction is associated with dysregulation of intracellular calcium homeostasis and activation of calcineurin and the subsequent nuclear importation of NFAT. With administration of cyclosporine A (CSA), the resulting bladder hypertrophy is diminished, and there is a reversal of the myosin heavy chain (MHC) mRNA isoforms back towards a normal expression pattern. We propose to further study these observations in a murine model of partial bladder outlet obstruction using a transgenic mouse containing an NFAT-luciferase reporter construct in the presence and absence of CSA administered of varying time points. End points will include bladder mass, in vitro determinations of contractility and shortening velocity, morphology, and a molecular analysis of MHC isoform expression. We also seek to localize in situ, which bladder wall cell population(s) show evidence of calcineurin activation. We also seek to localize the sites where DNA synthesis is taking place within the bladder wall and determine whether these sites of DNA synthesis represent fibroblasts, smooth muscle cells, or both. These in vivo experiments are complemented by in vitro experiments using cultured cells to assess the impact of mechanical deformation upon NFAT and MEF- 2 translocation to the nucleus. This approach will allow for in vitro screening of compounds that can prevent calcineurin activation, and will allow for study of the interaction between NFAT, MEF-2 and the smooth muscle MHC promoter. Using microarray methods we propose to identify the NFAT and MEF-2 responsive target genes within two cell populations; bladder smooth muscle and bladder fibroblasts. The relevance of this proposed work to human disease is apparent when one considers the socially devastating sequelae of urinary incontinence and renal failure that may result from untreated bladder wall hypertrophy. PUBLIC HEALTH RELEVANCE: Human Relevance - Two conditions can result in severe bladder wall hypertrophy in children, posterior urethral valves, and spinal bifida. If left untreated bladder wall hypertrophy leads to the socially devastating sequelae of urinary incontinence and in extreme cases renal failure may result as the bladder loses its ability to store urine at low pressures. New therapy to preserve and enhance native bladder function will help avoid the development of the end stage bladder.