Abstract
Fibrosis is a fundamental feature of systemic sclerosis (SSc) and is characterized by excessive accumulation of extracellular matrix components like proteoglycans (PG) or collagens in skin and internal organs. Serum analysis from SSc patients showed an increase in the enzyme activity of xylosyltransferase (XT), the initial enzyme in PG biosynthesis. There are two distinct XT isoforms—XT-I and XT-II—in humans, but until now only XT-I is associated with fibrotic remodelling for an unknown reason. The aim of this study was to identify new XT mediators and clarify the underlying mechanisms, in view of developing putative therapeutic anti-fibrotic interventions in the future. Therefore, we used different cytokines and growth factors, small molecule inhibitors as well as small interfering RNAs, and assessed the cellular XT activity and XYLT1 expression in primary human dermal fibroblasts by radiochemical activity assays and qRT-PCR. We identified a new function of activin A as a regulator of XYLT1 mRNA expression and XT activity. While the activin A-induced XT-I increase was found to be mediated by activin A receptor type 1B, MAPK and Smad pathways, the activin A treatment did not alter the XYLT2 expression. Furthermore, we observed a reciprocal regulation of XYLT1 and XYLT2 transcription after inhibition of the activin A pathway components. These results improve the understanding of the differential expression regulation of XYLT isoforms under pathological fibroproliferative conditions.
Highlights
The skin is the largest organ in the human body and provides a protective barrier against microorganisms, injuries and water loss [1]
After 48 h of incubation we found significant increased activity and mRNA levels of myofibroblast marker XT-I (5.1 ± 0.9-fold and 4.2 ± 0.9-fold, both p < 0.0001; Figure S1A,B) and ACTA2 mRNA level (2.4 ± 0.9, p < 0.0001; Figure S1C) in activin A-treated cells compared to the untreated controls, whereas no changes in XYLT2 mRNA expression were observed (p = 0.06; Figure S1D)
While we demonstrated the contribution of the three mitogen-activated protein kinases (MAPK) Jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK) in activin A-driven XYLT1 expression increase in normal human dermal fibroblasts (NHDF) by the usage of small-molecule inhibitors, we were unable to detect a more pronounced decrease of activin A-mediated effects by simultaneous blocking of MAPK JNK and p38 compared to single p38, JNK or ERK inhibition
Summary
The skin is the largest organ in the human body and provides a protective barrier against microorganisms, injuries and water loss [1]. Disturbances of these processes can result in severe pathological diseases, such as hypertrophic scarring, scleroderma and fibrosis induced by surgery, radiotherapy or medication [3]. The hallmark of these fibroproliferative conditions is the activation and differentiation of fibroblasts into myofibroblasts due to physical or inflammatory insults. These cells secrete vast amounts of ECM proteins that accumulate in the tissue and impair proper organ function [4]. Myofibroblasts are engaged in paracrine and autocrine interactions with their surrounding environment by secretion of growth factors and cytokines like activin A and transforming growth factor β1 (TGFβ1), which are increased in many tissues during fibrosis [5,6,7]
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