The role of protein O-GlcNAcylation in regulating keratinocyte function in skin fibrosis

项目摘要 局限性硬皮病(LS)，又称吗啡，是一种硬化性疾病，不仅影响皮肤，而且还 邻近组织包括皮下脂肪组织、筋膜、肌肉和骨。LS可能会造成毁灭性的 永久性缺陷，包括脱发、皮肤萎缩、皮下组织和骨丢失、关节痉挛、 儿童视力丧失，患体部位生长受限。LS的发病机制较差 了解和目前的治疗方法不能令人满意。更好地了解疾病并更有效地 治疗策略显然是必要的。LS和系统性硬化症(SSC)具有相同的组织病理学特征 特点，并有两个阶段：早期炎症阶段和后期纤维化阶段。已上调 皮肤中的促炎和促纤维化信号导致成纤维细胞的过度激活，这是细胞 LS和SSC的标志，并导致胶原过度产生和增加沉积 细胞外基质(ECM)。表皮角质形成细胞通过分泌各种细胞因子，在 通过旁分泌作用调节成纤维细胞功能。目前，在知识方面的一个重大差距是 角质形成细胞和成纤维细胞之间的旁分泌作用是启动和调节的。蛋白质O-GlcN酰化是 核苷酸尿苷二磷酸-N-乙酰氨基葡萄糖(UDP-GlcNAc)中GlcNAc部分的加成 胞质蛋白的丝氨酸或苏氨酸残基上。这是由O-GlcNAc转移酶(OGT)催化的，并且 GlcNAc被O-GlcNAcase(OGA)去除。在各种慢性疾病中发现蛋白质O-GlcN酰化异常 疾病包括糖尿病、癌症和阿尔茨海默氏症。然而，蛋白质O-GlcN酰化的作用 在LS的发病机制方面尚未有研究。我们建议检验这样一种假设，即 皮肤细胞内蛋白O-GlcN酰化导致促炎和促纤维化产物的改变 通过角质形成细胞的细胞因子，进而通过旁分泌效应促进成纤维细胞的激活。抑制 蛋白O-GlcN酰化可能为缓解异常皮肤纤维化提供一种新的治疗策略 LS和SSC的病理学。目的1：确定蛋白质O-GlcN酰化在调节蛋白质合成中的作用 角质形成细胞的促炎和促纤维化细胞因子。目的2：确定旁分泌的调节作用 角质形成细胞蛋白O-GlcN酰化对成纤维细胞功能和纤维化发展的影响 活着。我们希望证明：1)角质形成细胞中蛋白质O-GlcN酰化的操作将影响它们的 产生关键的促炎和促纤维化细胞因子；2)角质形成细胞功能的变化将影响 成纤维细胞的活化和促纤维化功能；以及3)OGT或OGA的化学抑制剂治疗将 影响博莱霉素所致皮肤纤维化的发生发展。影响：我们建议的研究取得积极成果 将为进一步研究这一想法提供强有力的理由，并为 角质形成细胞调节成纤维细胞活化的机制在失调性纤维化病理中 作为LS从新的糖生物学角度(蛋白质O-GlcN酰化)，这一点已被严重忽视。

Project Summary Localized scleroderma (LS), also named morphea, is a sclerotic disorder that affects not only skin, but also adjacent tissues including subcutaneous adipose tissue, fascia, muscle, and bone. LS can cause devastating permanent defects including hair loss, atrophy of skin, subcutaneous tissue and bone loss, joint contractures, vision loss, and growth restriction of affected body sites in children. The disease mechanism of LS is poorly understood and current treatments are unsatisfactory. Better understanding of the disease and more effective treatment strategies are clearly needed. LS and systemic sclerosis (SSc) share the same histopathologic features, and have two phases: an early inflammatory stage and a later fibrotic stage. Upregulated proinflammatory and profibrotic signals in the skin lead to hyperactivation of fibroblasts, which is the cellular hallmark for both LS and SSc and results in excessive collagen production and increased deposition of extracellular matrix (ECM). Epidermal keratinocytes, by secreting various cytokines, have a crucial role in regulating fibroblast function via paracrine effects. Currently, a significant gap in knowledge is how the paracrine effects between keratinocytes and fibroblasts are initiated and regulated. Protein O-GlcNAcylation is the addition of the GlcNAc moiety from nucleotide uridine diphosphate-N-acetyl-glucosamine (UDP-GlcNAc) onto serine or threonine residues of cytosolic proteins. This is catalyzed by O-GlcNAc transferase (OGT), and GlcNAc is removed by O-GlcNAcase (OGA). Abnormal O-GlcNAcylation of proteins is found in various chronic diseases including diabetes, cancers, and Alzheimer’s disease. However, the role of protein O-GlcNAcylation in the pathogenesis of LS has not been studied. We propose to test the hypothesis that abnormal levels of intracellular protein O-GlcNAcylation in skin lead to altered production of proinflammatory and profibrotic cytokines by keratinocytes, which in turn promotes fibroblast activation via paracrine effects. Inhibition of protein O-GlcNAcylation may provide a novel therapeutic strategy to alleviate the abnormal skin fibrosis pathology in LS and SSc. Aim 1: Determine the role of protein O-GlcNAcylation in regulating the production of proinflammatory and profibrotic cytokines by keratinocytes. Aim 2: Determine the paracrine regulatory effects of keratinocytes with altered protein O-GlcNAcylation on fibroblast function and the development of fibrosis in vivo. We expect to show that: 1) Manipulation of protein O-GlcNAcylation in keratinocytes will affect their production of key proinflammatory and profibrotic cytokines; 2) changes in keratinocyte function will affect fibroblast activation and profibrotic functions; and 3) Treatment with chemical inhibitors to OGT or OGA will affect the development of skin fibrosis induced by Bleomycin. Impact: Positive results from our proposed study will provide strong justification for further investigation of this idea and provide novel insights into the mechanisms by which keratinocytes regulate activation of fibroblasts in dysregulated fibrosis pathologies such as LS from a new glycobiological perspective (protein O-GlcNAcylation) that has been significantly overlooked.