Background/Purpose: Systemic sclerosis (SSc) is a complex multi-system autoimmune disease characterized by immune dysregulation, vasculopathy, and organ fibrosis. Skin fibrosis causes high morbidity and impaired quality of life in affected individuals. In this study, we identified genes that may be involved in the development of dermal fibrosis mediated by TGF-β in human skin maintained in organ culture.

Methods: To identify new genes regulated by TGF-β in human skin, we performed high-throughput RNA sequencing using ex vivo human skin samples treated with TGF-β or a vehicle control. We identified genes whose expression is altered in human skin treated with TGF-β. Of these genes, COL22A1 showed the highest level of differential expression. The expression levels of COL22A1 and its time-dependent changes were evaluated using real-time PCR and immunoblotting (IB) in ex vivo human skin tissues and in vitro in normal human dermal fibroblasts. To investigate which TGF-β signaling cascades are involved in the induction of COL22A1 expression, normal skin fibroblasts were cultured with TGF-β in combination with specific inhibitors. Since COL22A1 was an early response gene, we sought to assess its role in the TGF-β-mediated response, thus normal skin fibroblasts were transfected with COL22A1 siRNA and then stimulated with TGF-β. Furthermore, we compared mRNA and protein levels of COL22A1 in skin fibroblasts from control donors and patients with the diffuse cutaneous form of SSc by using real-time PCR and IB.

Results: COL22A1 was most differentially expressed and its levels were significantly increased by TGF-β ex vivo and in vitro. TGF-β caused a significant increase in the expression levels of COL22A1 at earlier time points than other fibrosis associated genes. Further, TGF-β-induced COL22A1 expression was abrogated by ALK5 and MEK inhibition. Silencing of COL22A1 significantly reduced TGF-β-induced ACTA2 expression. Dermal fibroblasts from patients with diffuse cutaneous SSc showed significantly increased expression levels of COL22A1 compared with normal skin fibroblasts.

Conclusion: In conclusion, our findings suggest that the increased expression of COL22A1 is associated with the pathogenesis of fibrosis in SSc, and that the regulation of COL22A1 expression may have important implications for skin fibrosis. These results contribute novel insights into genes regulated under pro-fibrotic conditions in human skin, providing direct relevance to human dermal fibrosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rnaseq-analysis-of-human-skin-in-organ-culture-identifies-collagen-22a1-as-a-tgf-%ce%b2-early-response-gene/