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

Cleveland Clinic Lerner Com-Cwru, Cleveland OH

Investigators

Abstract

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.