Systemic sclerosis (SSc) is considered a prototypic fibrotic disorder that is characterized by a pathological accumulation of myofibroblasts and extracellular matrix (ECM) components including collagens and proteoglycans (PG). The increase of PG biosynthesis in SSc is closely related to an elevated serum activity of human xylosyltransferase (XT), the initial enzyme in PG biosynthesis. As a basis for future development of an antifibrotic therapy for SSc, this work aimed to enhance understanding of XT-I regulation in fibrosis and to identify XT-I inhibitory molecules.
This study revealed activin A, a cytokine of the transforming growth factor β (TGF-β) family, as a potent regulator of XYLT1 mRNA expression and XT activity in normal human dermal fibroblasts (NHDF). Using small molecule inhibitors and small interfering RNAs, the activin A-mediated XT-I increase was shown to involve activin receptor type 1B and the intracellular MAPK and SMAD pathways. In addition to profibrotic proteins, miRNAs play a central role in the development und regulation of fibrotic processes. In this work, the TGF-β1-inducible miRNAs miRNA-145 and 21 were shown to regulate XT-I in NHDF. The direct binding of miRNA-145 and -21 to the 3’ untranslated region of the XYLT1 mRNA was excluded by reporter luciferase assays, indicating an indirect miRNA-mediated XYLT1 regulation through secondary media¬tors. The involvement of the zinc-finger transcription regulator KLF4 in the miRNA-145-mediated XT-I regulation was shown by in silico analysis, targeted gene silencing and quantitative real-time PCR. Regarding the TGF-β1-induced XYLT1 expression regulation by miRNA-21, the inhibitory SMAD7 was identified as secondary mediator. To address the missing association of increased XT serum activity with increased cellular XYLT1 mRNA expression und XT-I activity in SSc, this study characterized the differences in cellular XT-I activity of SSc fibroblasts (SScF) and NHDF. A higher extracellular XT-I activity in SScF was found to be mediated by an enhanced autocrine TGF-β signaling. A dysregulated miRNA-21/TGF-β receptor II axis in SScF was shown to contribute to the enhanced autocrine TGF-β signaling. Both the XYLT1 mRNA expression, and XT-I activity were shown to be sensi¬tive biomarkers of the dysregulated TGF-β signaling in SSc. Natural products have a wide range of biological activities and represent an important source for drug development. Therefore, a natural product-based molecular library was used in the third subproject in order to identify non-substrate-based XT-I inhibitors by mass spectrometric quantification of the XT-I activity. In combination with cell-based experi¬mental approaches and structure-based in silico analyzes, the two active ingredients ampho¬tericin B and celastrol were identified to possess XT-I inhibitory properties. Their XT-I inhibi¬tory effect was not restricted to the inhibition of the catalytic activity of the XT-I protein by an uncompetitive or a competitive inhibition mode, respectively, but was shown to impact cellu¬lar XYLT1 expression level and XT-I activity in NHDF as well. These cellular inhibitor-mediated changes were shown to involve the TGF-β and miRNA-21 signaling pathway.
This work improves the mechanistic understanding of fibrotic remodeling in SSc by identifying hitherto unknown XT-I mediators and regulatory pathways in dermal fibroblasts. With the identification of putative XT-I inhibitors this study provides a strong rational for the future therapeutical application of those in fibroproliferative diseases.