Xylosyltransferase-Deficiency in Human Dermal Fibroblasts Induces Compensatory Myofibroblast Differentiation and Long-Term ECM Reduction.

Desbuquois dysplasia type 2 (DBQD2) and spondylo-ocular syndrome (SOS) are autosomal recessive disorders affecting the extracellular matrix (ECM) and categorized as glycosaminoglycan (GAG) linkeropathies. Linkeropathies result from mutations within glycosyltransferases involved in the synthesis of the tetrasaccharide linker, a linker between the core protein of proteoglycan (PG) and GAG. DBQD2 and SOS are caused by the isolated mutations of the xylosyltransferase (XT) isoforms. In this work, we successfully generated XYLT1- as well as XYLT2-deficient GAG linkeropathy model systems in human dermal fibroblasts using a ribonucleoprotein-based CRISPR/Cas9-system. Furthermore, it was possible to generate a complete XYLT-knockdown. Short- and long-term XT activity deficiency led to the mutual reduction in all linker transferase-encoding genes, suggesting a potential multienzyme complex with mutual regulation. Fibroblasts compensated for ECM misregulation initially by overexpressing ECM through the TGFß1 signaling pathway, akin to myofibroblast differentiation patterns. The long-term reduction in one XT isoform induced a stress response, reducing ECM components. The isolated XYLT1-knockout exhibited α-smooth muscle actin overexpression, possibly partially compensated by unaltered XT-II activity. XYLT2-knockout leads to the reduction in both XT isoforms and a strong stress response with indications of oxidative stress, induced senescence and apoptotic cells. In conclusion, introducing XYLT-deficiency revealed temporal and isoform-specific regulatory differences.

A novel SPE-UPLC-MS/MS-based assay for the selective, simultaneous quantification of xylosyltransferase-I and -II activity.

Xylosyltransferase-I and -II (XT-I, -II) possess a central role during the glycosylation of proteoglycans (PGs). They catalyze the formation of an O-glycosidic bond between the xylosyl residue of uridinediphosphate-xylose and the core protein of a PG. Thereafter, three following glycosyltransferases lead to the generation of a tetrasaccharide linker, which connects the PG core protein to the respective glycosaminoglycan. The selective quantification of XT-I and XT-II activity is of biological and clinical interest due to their association with fibrotic processes and skeletal dysplasia. There is no assay available to date that simultaneously determines the activity of the two XT isoforms. Although an XT-I selective UPLC MS/MS-based assay was published by Fischer et al., in 2021, the determination of XT-II activity can only be performed simultaneously by the improved assay presented here. To establish the assay, two synthetic peptides, selectively xylosylated by the respective isoform, were identified and the associated measurement parameters for the mass spectrometer were optimized. In addition, the quantitative range of the xylosylated peptides were validated, and the incubation time of the enzyme reaction was optimized for cell culture samples and human sera. The specific enzyme kinetics (KM and Vmax) of the respective XT isoform for the two peptides were also determined. Subsequently, a mathematical model was developed, allowing the simultaneous determination of XT-I and XT-II activity from the chromatographic results. Summarized, a mass spectrometric method suitable for the simultaneous analysis of XT-I and XT-II activity in cell culture lysates, supernatants and human sera was successfully developed.

Understanding of arthrofibrosis: New explorative insights into extracellular matrix remodeling of synovial fibroblasts.

Arthrofibrosis following total knee arthroplasty is a fibroproliferative joint disorder marked by dysregulated biosynthesis of extracellular matrix proteins, such as collagens and proteoglycans. The underlying cellular events remain incompletely understood. Myofibroblasts are highly contractile matrix-producing cells characterized by increased alpha-smooth muscle actin expression and xylosyltransferase-I (XT-I) secretion. Human XT-I has been identified as a key mediator of arthrofibrotic remodeling. Primary fibroblasts from patients with arthrofibrosis provide a useful in vitro model to identify and characterize disease regulators and potential therapeutic targets. This study aims at characterizing primary synovial fibroblasts from arthrofibrotic tissues (AFib) regarding their molecular and cellular phenotype by utilizing myofibroblast cell culture models. Compared to synovial control fibroblasts (CF), AFib are marked by enhanced cell contractility and a higher XT secretion rate, demonstrating an increased fibroblast-to-myofibroblast transition rate during arthrofibrosis. Histochemical assays and quantitative gene expression analysis confirmed higher collagen and proteoglycan expression and accumulation in AFib compared to CF. Furthermore, fibrosis-based gene expression profiling identified novel modifier genes in the context of arthrofibrosis remodeling. In summary, this study revealed a unique profibrotic phenotype in AFib that resembles some traits of other fibroproliferative diseases and can be used for the future development of therapeutic interventions.

Human Xylosyltransferase I-An Important Linker between Acute Senescence and Fibrogenesis.

The human xylosyltransferase isoform XT-I catalyzes the initial step in proteoglycan biosynthesis and represents a biomarker of myofibroblast differentiation. Furthermore, XT-I overexpression is associated with fibrosis, whereby a fibrotic process initially develops from a dysregulated wound healing. In a physiologically wound healing process, extracellular matrix-producing myofibroblasts enter acute senescence to protect against fibrosis. The aim of this study was to determine the role of XT-I in acute senescent proto-myofibroblasts. Normal human dermal fibroblasts were seeded in a low cell density to promote myofibroblast differentiation and treated with H2O2 to induce acute senescence. Initiation of the acute senescence program in human proto-myofibroblasts resulted in a suppression of XYLT mRNA expression compared to the control, whereby the isoform XYLT1 was more affected than XYLT2. Moreover, the XT-I protein expression and enzyme activity were also reduced in H2O2-treated cells compared to the control. The examination of extracellular matrix remodeling revealed reduced expression of collagen I, fibronectin and decorin. In summary, acute senescent proto-myofibroblasts formed an anti-fibrotic phenotype, and suppression of XT-I during the induction process of acute senescence significantly contributed to subsequent ECM remodeling. XT-I therefore plays an important role in the switch between physiological and pathological wound healing.

The Human Myofibroblast Marker Xylosyltransferase-I: A New Indicator for Macrophage Polarization.

Chronic inflammation and excessive synthesis of extracellular matrix components, such as proteoglycans (PG), by fibroblast- or macrophage-derived myofibroblasts are the hallmarks of fibrotic diseases, including systemic sclerosis (SSc). Human xylosyltransferase-I (XT-I), which is encoded by the gene XYLT1, is the key enzyme that is involved in PG biosynthesis. Increased cellular XYLT1 expression and serum XT-I activity were measured in SSc. Nothing is known so far about the regulation of XT-I in immune cells, and their contribution to the increase in measurable serum XT-I activity. We utilized an in vitro model, with primary human CD14+CD16+ monocyte-derived macrophages (MΦ), in order to investigate the role of macrophage polarization on XT-I regulation. The MΦ generated were polarized towards two macrophage phenotypes that were associated with SSc, which were classified as classical pro-inflammatory (M1-like), and alternative pro-fibrotic (M2-like) MΦ. The fully characterized M1- and M2-like MΦ cultures showed differential XT-I gene and protein expressions. The fibrotic M2-like MΦ cultures exhibited higher XT-I secretion, as well as increased expression of myofibroblast marker α-smooth muscle actin, indicating the onset of macrophage-to-myofibroblast transition (MMT). Thus, we identified XT-I as a novel macrophage polarization marker for in vitro generated M1- and M2-like MΦ subtypes, and broadened the view of XT-I as a myofibroblast marker in the process of MMT.

The Impact of Inflammatory Stimuli on Xylosyltransferase-I Regulation in Primary Human Dermal Fibroblasts.

Inflammation plays a vital role in regulating fibrotic processes. Beside their classical role in extracellular matrix synthesis and remodeling, fibroblasts act as immune sentinel cells participating in regulating immune responses. The human xylosyltransferase-I (XT-I) catalyzes the initial step in proteoglycan biosynthesis and was shown to be upregulated in normal human dermal fibroblasts (NHDF) under fibrotic conditions. Regarding inflammation, the regulation of XT-I remains elusive. This study aims to investigate the effect of lipopolysaccharide (LPS), a prototypical pathogen-associated molecular pattern, and the damage-associated molecular pattern adenosine triphosphate (ATP) on the expression of XYLT1 and XT-I activity of NHDF. We used an in vitro cell culture model and mimicked the inflammatory tissue environment by exogenous LPS and ATP supplementation. Combining gene expression analyses, enzyme activity assays, and targeted gene silencing, we found a hitherto unknown mechanism involving the inflammasome pathway components cathepsin B (CTSB) and caspase-1 in XT-I regulation. The suppressive role of CTSB on the expression of XYLT1 was further validated by the quantification of CTSB expression in fibroblasts from patients with the inflammation-associated disease Pseudoxanthoma elasticum. Altogether, this study further improves the mechanistic understanding of inflammatory XT-I regulation and provides evidence for fibroblast-targeted therapies in inflammatory diseases.

First Characterization of Human Dermal Fibroblasts Showing a Decreased Xylosyltransferase-I Expression Induced by the CRISPR/Cas9 System.

BACKGROUND: Xylosyltransferases-I and II (XT-I and XT-II) catalyze the initial and rate limiting step of the proteoglycan (PG) biosynthesis and therefore have an import impact on the homeostasis of the extracellular matrix (ECM). The reason for the occurrence of two XT-isoforms in all higher organisms remains unknown and targeted genome-editing strategies could shed light on this issue. METHODS: XT-I deficient neonatal normal human dermal fibroblasts were generated by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated proteins (Cas) 9 system. We analyzed if a reduced XT-I activity leads to abnormalities regarding ECM-composition, myofibroblast differentiation, cellular senescence and skeletal and cartilage tissue homeostasis. RESULTS: We successfully introduced compound heterozygous deletions within exon 9 of the XYLT1 gene. Beside XYLT1, we detected altered gene-expression levels of further, inter alia ECM-related, genes. Our data further reveal a dramatically reduced XT-I protein activity. Abnormal myofibroblast-differentiation was demonstrated by elevated alpha-smooth muscle actin expression on both, mRNA- and protein level. In addition, wound-healing capability was slightly delayed. Furthermore, we observed an increased cellular-senescence of knockout cells and an altered expression of target genes knowing to be involved in skeletonization. CONCLUSION: Our data show the tremendous relevance of the XT-I isoform concerning myofibroblast-differentiation and ECM-homeostasis as well as the pathophysiology of skeletal disorders.

Cytokine-mediated induction of human xylosyltransferase-I in systemic sclerosis skin fibroblasts.

Systemic sclerosis (SSc) is an inflammatory fibrotic disease characterized by an excessive extracellular matrix deposition in the skin and internal organs. One fibrotic key event remains the fibroblast-to-myofibroblast differentiation that is controlled by a combination of mechanical and soluble factors, such as transforming growth factor-ß1 (TGF-ß1) and interleukin-1ß (IL-1ß). One important myofibroblast biomarker is human xylosyltransferase-I (XT-I), the initial enzyme in proteoglycan biosynthesis. Increased serum XT activity was quantified in SSc, but the underlying cellular mechanisms remain elusive. This study aims to determine the cellular basis of XT-I induction in SSc by using a myofibroblast cell culture model with SSc fibroblasts (SScF) and healthy control fibroblasts. We found that SScF exhibit a higher extracellular XT-I activity compared to control fibroblasts. This increased XT-I activity in SScF was demonstrated to be mediated by an enhanced autocrine TGF-ß signaling. Upon IL-1ß treatment, SScF showed an increased mRNA expression level of XT-I and TGF-ß receptor II (TGFBR2), while healthy control fibroblasts did not, pointing towards an involvement of IL-1ß in the cytokine-mediated XT-I induction. Performing microRNA (miRNA) inhibition experiments in the presence of TGF-ß1, we showed that the pro-fibrotic effect of IL-1ß may be mediated by a miRNA-21/TGF-ß receptor II axis, enhancing the autocrine TGF-ß signaling in SScF. Taken together, this study improves the mechanistic understanding of fibrotic XT-I induction in SSc by identifying a hitherto unknown IL-1ß-mediated miRNA-21/TGFBR2 regulation contributing to the enhanced XYLT1 expression and XT-I activity in SScF.

Development of a xylosyltransferase-I-selective UPLC MS/MS activity assay using a specific acceptor peptide.

Xylosyltransferases-I and -II (XT-I and -II) play an important role regarding the homeostasis of the extracellular matrix. Both enzymes catalyze the initial step of the proteoglycan (PG) biosynthesis by the transfer of xylose from their natural substrate uridine diphosphate (UDP) -xylose to a PG-core protein. The subsequent addition of further sugars, catalyzed by different glycosyltransferases, leads to the formation of a tetrasaccharide linker, which connects the PG-core protein and glycosaminoglycans. The reason for the appearance of two XT isoforms in all higher organisms is not known and remarkable, as both enzymes are able to initiate PG biosynthesis. The determination of the XT-I activity is of clinical importance because it can be used as a biomarker of several PG-associated fibrotic diseases. Since previous assays did not adequately differentiate between both XT-isoforms, the aim of this study was to develop an XT-I selective mass spectrometric (MS) assay. For this purpose, we initially used isoform-specific supernatants to successfully identify a synthetic acceptor peptide which was xylosylated much more selectively by the XT-I when compared to the XT-II isoform. The assay was further optimized concerning methodical parameters such as the injection volume and the incubation time of the reaction-mixture. By using samples covering a broad XT-activity spectrum, we successfully validated the assay to be used not only for the quantification of cell culture samples but also human serum specimens. Compared to previously used XT-activity assays, our newly developed test is more selective and sensitive, less expensive and easier to perform in high throughput.

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

Fibroproliferative diseases are characterized by excessive accumulation of extracellular matrix (ECM) components leading to organ dysfunction. This process is characterized by an increase in myofibroblast content and enzyme activity of xylosyltransferase-I (XT-I), the initial enzyme in proteoglycan (PG) biosynthesis. Therefore, the inhibition of XT-I could be a promising treatment for fibrosis. We used a natural product-inspired compound library to identify non-substrate-based inhibitors of human XT-I by UPLC-MS/MS. We combined this cell-free approach with virtual and molecular biological analyses to confirm and prioritize the inhibitory potential of the compounds identified. The characterization for compound potency in TGF-ß1-driven XYLT1 transcription regulation in primary dermal human fibroblasts (key cells in ECM remodeling) was addressed by gene expression analysis. Consequently, we identified amphotericin B and celastrol as new non-substrate-based XT-I protein inhibitors. Their XT-I inhibitory effects were mediated by an uncompetitive or a competitive inhibition mode, respectively. Both compounds reduced the cellular XYLT1 expression level and XT-I activity. We showed that these cellular inhibitor-mediated changes involve the TGF-ß and microRNA-21 signaling pathway. The results of our study provide a strong rationale for the further optimization and future usage of the XT-I inhibitors identified as promising therapeutic agents of fibroproliferative diseases.

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

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.

microRNA-145 mediates xylosyltransferase-I induction in myofibroblasts via suppression of transcription factor KLF4.

Remodelling of the extracellular matrix by myofibroblasts is crucial for wound repair, but if deregulated, it might contribute to the development of fibrosis. Fibroblast-to-myofibroblast differentiation is promoted by aberrant microRNA-145-5p (miR-145) expression in response to transforming growth factor ß1 (TGFß1). One of several myofibroblast markers is human xylosyltransferase-I (XT-I), which is the initial and rate-limiting enzyme of proteoglycan biosynthesis. Increased serum XT activity was quantified in patients with systemic sclerosis (SSc), but the underlying cellular mechanism of this disease remains unknown. This study aims to determine the underlying molecular basis of XT-I induction by considering the miR-mediated regulation of XT-I. We found that miR-145 is upregulated in TGFß1-treated dermal fibroblasts and correlates with an increased cellular XYLT1 expression and XT activity. Overexpression of miR-145 in dermal fibroblasts induced XYLT1 expression and XT activity and enhanced TGFß1-promoted XT activity increase. Since direct XYLT1 3'-UTR targeting by miR-145 could be experimentally excluded, an indirect effect of miR-145 on XT-I regulation was indicated. We identified six transcription factor-binding sites for Krueppel-like factor 4 (KLF4), a zinc-finger transcription regulator and putative miR-145 target, in the XYLT1 promoter in silico. A suppressive role of KLF4 on XYLT1 expression was confirmed by targeted gene silencing in dermal fibroblasts and the quantification of KLF4 expression in SSc fibroblasts. Taken together, this study improves the mechanistic understanding of fibrotic remodelling in SSc by identifying a hitherto unknown miR-145/KLF4 pathway mediating the fibrogenic XT-I induction. This knowledge on XYLT1 may lead to the development of novel approaches in the therapy of fibrosis.

Xylosyltransferase-deficient human HEK293 cells show a strongly reduced proliferation capacity and viability.

Human xylosyltransferases-I and -II (XT-I and XT-II) catalyze the initial and rate-limiting step in proteoglycan (PG)-biosynthesis. Because PG are major components of the extracellular matrix (ECM), an alternated XT expression is associated with the manifestation of ECM-related diseases. While Drosophila melanogaster and Caenorhabditis elegans only harbor one XT-isoform, all higher organisms contain two isoforms, which are expressed in a tissue-specific manner. The reason for the appearance of two isoenzymes remains unexplained and remarkable, as all other enzymes involved in the synthesis of the tetrasaccharid linker, which connects the PG core protein with attached glycosaminoglycans, only show one isoform. In human, mutations in the XYLT genes cause diseases affecting the homeostasis of the ECM, such as skeletal dysplasias. We investigated for the first time whether already XT-I-deficient human embryonic kidney (HEK293) cells can compensate for decreased expression levels of both XT-isoforms. A siRNA-mediated XYLT2 mRNA knockdown led to reduced cellular proliferation rates and a partially increased cellular senescence of treated HEK293 cells. These results were verified by conducting a stable CRISPR/Cas9-mediated XYLT2 knockout, which revealed that only cells expressing at least partially functional XT-II proteins remain proliferative. Our study, therefore, shows for the first time that cells lacking both XT-isoforms are not viable and clearly indicates the importance of the XT concerning the cellular metabolism.

First description of a compensatory xylosyltransferase I induction observed after an antifibrotic UDP-treatment of normal human dermal fibroblasts.

Fibrosis is a serious health problem often leading to accompanying organ failure. During the manifestation of the disease, an accumulation of different extracellular matrix (ECM) molecules, such as proteoglycans, takes place. There is no appropriate therapeutic option available to heal fibrosis to date. Current research focuses primarily on targets such as the cytokine transforming growth factor-ß1 (TGF-ß1), which is assumed to be one of the key mediators of fibrosis. Both xylosyltransferase isoforms, XT-I and XT-II, catalyze the rate-limiting step of the proteoglycan biosynthesis. Consequently, inhibiting XT activity could be a promising approach to treat fibrosis. It was shown in earlier studies that nucleotides and nucleosides have anti-fibrotic properties and decrease XT activity in cell-free systems. In contrast, we evaluated the mechanisms beyond an UDP-mediated induction of intracellular XT-activity in normal human dermal fibroblasts (NHDF). The observed pseudo-fibrotic XT increasement could be attributed to a compensation of decreased UDP-glucuronate decarboxylase 1 (UXS1) mRNA expression as well as a diminished intracellular UDP-xylose concentration. In summary, our results describe a so far unknown XT-inductive pathway and show that UDP could be a promising molecule for the development of an anti-fibrotic therapy. Nevertheless, XT activity has to be inhibited in parallel intracellularly.

microRNA-29b mediates fibrotic induction of human xylosyltransferase-I in human dermal fibroblasts via the Sp1 pathway.

Diminished microRNA-29b levels have recently been revealed to provoke increased expression and accumulation of extracellular matrix molecules, such as collagens in fibrotic remodeling. Subsequently, the aim of this study was to find out whether microRNA-29b might also regulate human xylosyltransferase (XT)-I expression. XT-I has been characterized previously as a fibrosis biomarker catalyzing the key step of proteoglycan biosynthesis. While we demonstrate that XYLT1 is neither a target of microRNA-29b identified in silico nor a direct 3' untranslated region binding partner of microRNA-29b, transfection of normal human dermal fibroblasts with microRNA-29b inhibitor strongly increased XYLT1 mRNA expression and XT activity. Combined results of the target prediction analysis and additional transfection experiments pointed out that microRNA-29b exerts indirect influence on XT-I by targeting the transcription factor specificity protein 1 (Sp1). We could confirm our hypothesis due to the decrease in XYLT1 promoter activity after Sp1 binding site mutation and the approval of occupancy of these binding sites by Sp1 in vitro. Taken together, a hitherto unidentified pathway of XT-I regulation via microRNA-29b/Sp1 was determined in this study. Our observations will facilitate the understanding of complex molecular fibrotic pathways and provide new opportunities to investigate microRNA-based antifibrotic tools.