Elucidation of the hr-dependent pathway of hair growth

近年来，对毛囊（HF）生物学的形态发生方面的理解取得了很大进展，然而，对头发生长控制的分子和遗传方面仍然知之甚少。其中，氟化氢原转化的分子途径是最不清楚的，尽管在人类中许多常见的头发疾病的基础上有异常的原进展。近年来的研究表明，坏死原是一个细胞凋亡驱动的过程，细胞程序性死亡是坏死原HF转化的主要机制。我们之前的研究表明，HF基质的异位和过早凋亡是导致突变型hr/hr无毛小鼠皮肤完全脱发的主要缺陷。这些发现提示无毛基因在心衰过程中是协调基本细胞过程的关键因素，而程序性细胞死亡可能是无毛基因活性的关键靶点。为了进一步了解hr对HF功能的分子控制，我们建议使用突变体（hrrh/hrrh）脱毛小鼠的天然皮肤和培养角质形成细胞作为模型，阐明hr基因在细胞凋亡驱动原调控中的作用，并确定hr基因活性的下游靶点。我们将通过形态学方法、TUNEL和膜联蛋白V染色以及功能实验（UVB照射、DFMO、ICE和caspase-3抑制剂处理下hr/hr和wt皮肤中凋亡相关基因表达的比较）来验证hr基因参与凋亡调控途径的假设。使用Northern Blot分析和免疫组织化学，我们将测试wt和hr/hr皮肤中hr依赖调节途径的推定下游靶点的表达模式。研究人员将利用从hrrh/hrrh和wt基因型小鼠完整表皮中分离的核酸进行杂交阵列，寻找hr基因活性的未知靶点。确定的候选靶基因将根据其在毛囊生理学中的已知作用以及我们从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，本提案中概述的实验旨在以无毛表型为实验模型，剖析毛囊脱落原转化的细胞和分子机制。阐明hr基因在细胞凋亡调控中的作用，发现hr依赖通路中直接涉及的基因，将对理解头发生长控制的基本机制具有深远的意义，并可能对开发靶向控制脱发的新药具有深远的意义，这是头发生物学中最相关的临床问题。

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.