POS0425 INCREASED KERATIN 9 EXPRESSION IN SYSTEMIC SCLEROSIS SKIN IS DRIVEN BY THE lncRNA HOTAIR FROM FIBROBLAST DERIVED EXOSOMES

Abstract

Background:Skin fibrosis is the hallmark fibrotic manifestation of systemic sclerosis (SSc). Despite a key role of tissue fibroblasts, skin changes extend to the keratinocyte layer, which contribute to the loss of skin function. RNA seq. analysis of SSc patient forearm skin showed that palmoplantar specific Keratin 9 (CK9) was highly expressed (1). SSc affected skin shares several features with palmoplantar skin including increased keratinocyte layer thickness and lack of hair. Seminal work of last decade has shown that long noncoding RNA in the HOX loci play a crucial role in skin keratinocyte differentiation (2), with the lncRNA HOTAIR being one of the HOX lncRNA mostly expressed in the palmoplantar region.Objectives:Following recent data suggesting a role of HOTAIR in the profibrotic phenotype of dermal fibroblasts in SSc (3), here we set out to determine if HOTAIR expressed in SSc dermal fibroblasts was a contributing factor to the high levels of CK9 found in SSc patient skinMethods:Full-thickness skin biopsies were surgically obtained from the forearms of patients with SSc of recent onset. Fibroblasts were isolated and cultured in monolayers. HOX transcript antisense RNA (HOTAIR) was expressed in healthy dermal fibroblasts by lentiviral induction employing a vector containing the specific sequence. Exosomes were isolated from dermal fibroblast media using the Total exosome isolation reagent (Thermo Fisher). Enhancer of zeste 2 (EZH2) was blocked with GSK126 inhibitor. Skin equivalents were created using scramble and HOTAIR expressing fibroblasts with primary keratinocytesResults:Media from both SSc patient fibroblasts and HOTAIR expressing fibroblasts induced CK9 expression in healthy keratinocytes in vitro. In addition, HOTAIR expressing fibroblasts induces CK9 expression in keratinocytes in 3D skin equivalent models. Media fractionation studies indicated that HOTAIR was present in fibroblasts exosomes and found at a higher concentration (2.7 fold p=0.01) in exosomes from SSc fibroblasts. Importantly, transfection of Exosomal RNAs from SSc fibroblasts could reproduce the increase in CK9 in keratinocytes. Mechanistically, CK9 induction was mediated by changes to the histone methylation profile in the keratinocytes through EZH2.Conclusion:Pro-fibrotic dermal fibroblasts in systemic sclerosis contribute to the overall skin loss of function by inducing CK9 in adjacent keratinocytes through transfer of the long non-coding RNA HOTAIR. Unraveling the crosstalk of activated fibroblasts with adjacent cells may lead to identify therapeutic targets to re-establish tissue homeostasis and function during fibrosis.