Activin A-Mediated Regulation of XT-I in Human Skin Fibroblasts

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.

Identification of Putative Non-Substrate-Based XT-I Inhibitors by Natural Product Library Screening

“…Previous studies have shown that the cellular XT-I activity is regulated on the transcriptional level [ 7 ]. After demonstrating that the DMSO content of 0.04% ( v / v ), the celastrol concentrations of 0.5 and 1.0 μM and the amphotericin B usage of 0.5 to 2.0 μM are well tolerated by NHDFs, we wanted to evaluate the effects of the putative XT-I inhibitors on the XYLT1 mRNA expression and cellular XT-I activity.…”

“…The RNA extraction from cell lysates and cDNA synthesis for mRNA and miRNA expression analysis were performed as described previously [ 7 , 42 ].…”

“…Quantitative real-time polymerase chain reaction (qRT-PCR) analysis using a LightCycler 480 Instrument II system (Roche, Basel, Swiss) was performed as previously described [ 7 ]. The qRT-PCR experiments were conducted with three biological and three technical replicates per donor-derived primary cell culture, unless otherwise stated.…”

“…The qRT-PCR experiments were conducted with three biological and three technical replicates per donor-derived primary cell culture, unless otherwise stated. The intron-spanning primer sequences used are listed in [ 7 ] and Table 1 . The geometric mean of the expression levels of the housekeeping genes SDHA , RPL13A and B2M were calculated for expression normalization.…”

“…Both XTs consist of a short amino-terminal region facing the cytosolic side, a single transmembrane helix, a stem region necessary for Golgi retention [ 1 ], a catalytic glycosyltransferase (GT)-A domain facing the Golgi lumen [ 4 , 5 ] and a C-terminal domain named Xylo_C according to the Pfam database [ 5 , 6 ]. XT-I and XT-II are differentially expressed in tissues and cell types due to variations in the promotor region and cellular transcriptional regulations [ 7 , 8 , 9 , 10 ], but the reason for the existence of these two isoforms in all higher organisms remains unknown. In spite of the intracellular localization of XT-I, the enzyme is shed from the Golgi membrane by an unknown mechanism that involves a cysteine protease [ 11 ].…”