Elucidation of the hr-dependent pathway of hair growth

近年来，对毛囊生物学的形态发生方面的了解已经取得了很大的进展，然而，关于毛发生长控制的分子和遗传学方面仍然知之甚少。其中，尽管退行性变的异常是人类许多常见的毛发疾病的基础，但对HF退行性变的分子途径了解最少。最近的研究表明，卡他林是一个由细胞凋亡驱动的过程，程序性细胞死亡是卡他命原HF转化的主要机制。我们以前的研究表明，突变的hr/hr无毛小鼠皮肤中的异位和过早的细胞凋亡是导致完全脱发的主要缺陷。这些发现表明，无毛(Hr)基因是协调HF退变过程中基本细胞过程的关键因素，而程序性细胞死亡可能是hr基因活性的关键靶点。为了更深入地了解HR对HF功能的分子调控，我们建议使用来自突变(hrrh/hrrh)无毛小鼠的天然皮肤和培养的角质形成细胞作为模型，以阐明hr基因在调节细胞凋亡驱动的成熟剂中的作用，并用于鉴定hr基因活性的下游靶点。通过形态学方法、原位末端标记法和膜联蛋白V染色以及功能实验(比较紫外线照射、DFMO、ICE和caspase-3抑制剂治疗下的皮肤细胞凋亡相关基因的表达)，验证hr基因参与细胞凋亡调控途径的假说。利用Northern Blot分析和免疫组织化学，我们将测试wt和hr/hr皮肤中hr依赖调控通路的假定下游靶点的表达模式。利用从完整的hrrh/hrrh和wt两种类型的小鼠表皮中提取的核酸，将使用杂交阵列来搜索hr基因活性的未知目标。确定的候选目标基因将根据它们在毛囊生理学中的已知作用以及从我们的功能实验和免疫组织化学研究中获得的数据进行优先排序。因此，本方案中概述的实验旨在以无毛表型为实验模型来剖析毛囊退行性变的细胞和分子机制。阐明hr基因在细胞凋亡调控中的作用，发现与hr依赖途径直接相关的基因，将对理解毛发生长控制的基本机制具有深远的意义，并有可能开发针对毛发生物学最相关的临床问题的新药。

Recently, great progress has been achieved in the understanding of the morphogenic aspects of hair follicle (HF) biology, nevertheless, the molecular and genetic aspects of hair growth control remain largely unknown. Among these, the molecular pathways of HF catagen transformation are the least well understood despite the abnormalities of catagen progression underlie many common hair disorders in humans. Recent studies showed that catagen is an apoptosis-driven process and programmed cell death is the major mechanism of catagen HF transformation. Our previous studies indicated that ectopic and premature apoptosis in HF matrix is the major defect in the mutant hr/hr hairless mouse skin that causes the complete hair loss. These findings implicate the hairless (hr) gene as a key factor in coordinating basic cellular processes during HF catagen and programmed cell death as a possible key target of hr gene activity. In order to gain the further insights into the molecular control of HF functions by hr, we propose the use of the native skin and cultured keratinocytes derived from the mutant (hrrh/hrrh) hairless mice as a model for elucidation the role of hr gene in regulation of apoptosis-driven catagen and for the identification of the downstream targets of hr gene activity. The hypothesized involvement of hr gene into apoptotic regulatory pathway will be tested using morphological methods, TUNEL and annexin V staining along with functional experiments (comparison of apoptosis-associated gene expression in hr/hr and wt skin under UVB irradiation, treatment with DFMO and ICE and caspase-3 inhibitors). Using Northern Blot analysis and immunohistochemistry, we will test the expression patterns of presumptive downstream targets of the hr-dependent regulatory pathway in wt and hr/hr skin. The search for unknown targets of hr gene activity will be performed using hybridization arrays with nucleic acids isolated from intact mouse epidermis of both hrrh/hrrh and wt genotypes. Identified candidate target genes will be prioritized according to their known role in hair follicle physiology, and also according to data obtained from our functional experiments and immunohistochemical studies. Thus, the experiments outlined in this proposal are designed to dissect the cellular and molecular mechanisms of hair follicle catagen transformation using the hairless phenotype as an experimental model. Elucidation the role of hr gene in apoptosis regulation and discovery of the genes directly implicated in a hr-dependent pathway would have far-reaching implications for the understanding of basic mechanisms of hair growth control and potentially, for the development of novel drugs that target the control of catagen as the most relevant clinical problem of hair biology.