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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 β1 (TGFβ1). 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 TGFβ1-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 TGFβ1-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.

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.

The molecular processes of aging are very heterogenic and not fully understood. Studies on rare progeria syndromes, which display an accelerated progression of physiological aging, can help to get a better understanding. Pseudoxanthoma elasticum (PXE) caused by mutations in the ATP-binding cassette sub-family C member 6 (ABCC6) gene shares some molecular characteristics with such premature aging diseases. Thus, this is the first study trying to broaden the knowledge of aging processes in PXE patients. In this study, we investigated aging associated biomarkers in primary human dermal fibroblasts and sera from PXE patients compared to healthy controls. Determination of serum concentrations of the aging biomarkers eotaxin-1 (CCL11), growth differentiation factor 11 (GDF11) and insulin-like growth factor 1 (IGF1) showed no significant differences between PXE patients and healthy controls. Insulin-like growth factor binding protein 3 (IGFBP3) showed a significant increase in serum concentrations of PXE patients older than 45 years compared to the appropriate control group. Tissue specific gene expression of GDF11 and IGFBP3 were significantly decreased in fibroblasts from PXE patients compared to normal human dermal fibroblasts (NHDF). IGFBP3 protein concentration in supernatants of fibroblasts from PXE patients were decreased compared to NHDF but did not reach statistical significance due to potential gender specific variations. The minor changes in concentration of circulating aging biomarkers in sera of PXE patients and the significant aberrant tissue specific expression seen for selected factors in PXE fibroblasts, suggests a link between ABCC6 deficiency and accelerated aging processes in affected peripheral tissues of PXE patients.

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.

Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder which is caused by ABCC6 (ATP-binding cassette subfamily C member 6) gene mutations. Characteristic hallmarks of PXE are progressive calcification and degradation of the elastic fibers in skin, cardiovascular system and ocular fundus. Since the underlying pathomechanisms of PXE remain unidentified, the aim of this study was to get new insights into PXE pathophysiology by characterizing dermal myofibroblast differentiation. Fibroblasts are the key cells of extracellular matrix (ECM) remodeling and, therefore, participate not only in physiological processes, such as calcification or wound healing, but also in pathologic events, such as fibrotization. We revealed that human dermal PXE fibroblasts possess exaggerated migration capability in wound healing and attenuated myofibroblast contractility in comparison to controls. Subsequent analyses reinforced these observations and indicated a diminished induction of the myofibroblast differentiation markers α-smooth muscle actin and xylosyltransferase-I as well as poor transforming growth factor-β1 responsiveness in PXE fibroblasts. In summary, we describe pathological deviations of dermal myofibroblast differentiation in PXE which might be mediated by aberrant supramolecular ECM organization. These results not only improve our insights into cellular PXE pathophysiology, but might also qualify us to interfere with ECM remodeling in the future.

Background ATP-binding cassette (ABC) transporters are involved in a huge range of physiological processes. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum, a metabolic disease with progressive soft tissue calcification. Methods The aim of the present study was to analyze gene expression levels of selected ABC transporters associated with cholesterol homeostasis in metabolic active tissues, such as the liver, kidney and white adipose tissue (WAT) of Abcc6−/− mice from an early and late disease stage (six-month-old and 12-month-old mice). Results The strongest regulation of ABC transporter genes was observed in the liver tissue of six-month-old Abcc6−/− mice. Here, we found a significant increase of mRNA expression levels of phospholipid, bile salt and cholesterol/sterol transporters Abcb1b, Abcb11, Abcg1, Abcg5 and Abcg8. Abcd2 mRNA expression was increased by 3.2-fold in the liver tissue. We observed strong upregulation of Abca3 and Abca1 mRNA expression up to 3.3-fold in kidney and WAT, and a 2-fold increase of Abca9 mRNA in the WAT of six-month-old Abcc6 knockout mice. Gene expression levels of Abcb1b and Abcg1 remained increased in the liver tissue after an age-related disease progression, while we observed lower mRNA expression of Abca3 and Abca9 in the kidney and WAT of 12-month-old Abcc6−/− mice. Conclusions These data support previous findings that Abcc6 deficiency leads to an altered gene expression of other ABC transporters depending on the status of disease progression. The increased expression of fatty acid, bile salt and cholesterol/sterol transporters may be linked to an altered cholesterol and lipoprotein metabolism due to a loss of Abcc6 function. Electronic supplementary material The online version of this article (10.1186/s12944-018-0943-x) contains supplementary material, which is available to authorized users.

Pseudoxanthoma elasticum (PXE) is a rare autosomal-recessive disorder that is mainly caused by mutations in the ATP-binding cassette sub-family C member 6 (ABCC6) gene. Clinically PXE is characterized by a loss of skin elasticity, arteriosclerosis or visual impairments. It also shares some molecular characteristics with known premature aging syndromes like the Hutchinson&ndash, Gilford progeria syndrome (HGPS). However, little is known about accelerated aging processes, especially on a cellular level for PXE now. Therefore, this study was performed to reveal a potential connection between premature cellular aging and PXE pathogenesis by analyzing cellular senescence, a corresponding secretory phenotype and relevant factors of the cell cycle control in primary human dermal fibroblasts of PXE patients. Here, we could show an increased senescence-associated &beta, galactosidase (SA-&beta, Gal) activity as well as an increased expression of proinflammatory factors of a senescence-associated secretory phenotype (SASP) like interleukin 6 (IL6) and monocyte chemoattractant protein-1 (MCP1). We further observed an increased gene expression of the cyclin-dependent kinase inhibitor (CDKI) p21, but no simultaneous induction of p53 gene expression. These data indicate that PXE is associated with premature cellular senescence, which is possibly triggered by a p53-independent p21-mediated mechanism leading to a proinflammatory secretory phenotype.

Total knee replacement (TKR) is a common therapeutic option to restore joint functionality in chronic inflammatory joint diseases. Subsequent arthrofibrotic remodeling occurs in 10%, but the underlying pathomechanisms remain unclear. We evaluated the association of xylosyltransferases (XT), fibrotic mediators catalyzing glycosaminoglycan biosynthesis, leading to arthrofibrosis as well as the feasibility of using serum XT activity as a diagnostic marker. For this purpose, synovial fibroblasts (SF) were isolated from arthrofibrotic and control synovial biopsies. Basal α-smooth muscle actin expression revealed a high fibroblast-myofibroblast transition rate in arthrofibrotic fibroblasts. Fibrotic remodeling marked by enhanced XT activity, α-SMA protein expression as well as xylosyltransferase-I, collagen type III-alpha-1 and ACTA2 mRNA expression was stronger in arthrofibrotic than in control fibroblasts treated with transforming growth factor-β1 (TGF-β1). Otherwise, no differences between serum levels of XT-I activity or common fibrosis markers (galectin-3 and growth differentiation factor-15 levels (GDF-15)) were found between 95 patients with arthrofibrosis and 132 controls after TKR. In summary, XT-I was initially investigated as a key cellular mediator of arthrofibrosis and a target for therapeutic intervention. However, the blood-synovial-barrier makes arthrofibrotic molecular changes undetectable in serum. Future studies on monitoring or preventing arthrofibrotic remodeling should therefore rely on local instead of systemic parameters.

In mammals, two active xylosyltransferase isoenzymes (EC 2.4.2.16) exist. Both xylosyltransferases I and II (XT-I and XT-II) catalyze the transfer of xylose from UDP-xylose to select serine residues in the proteoglycan core protein. Altered XT activity in human serum was found to correlate directly with various diseases such as osteoarthritis, systemic sclerosis, liver fibrosis, and pseudoxanthoma elasticum. To interpret the significance of the enzyme activity alteration observed in disease states it is important to know which isoenzyme is responsible for the XT activity in serum. Until now it was impossible for a specific measurement of XT-I or XT-II activity, respectively, because of the absence of a suitable enzyme substrate. This issue has now been solved and the following experimental study demonstrates for the first time, via the enzyme activity that XT-II is the predominant isoenzyme responsible for XT activity in human serum. The proof was performed using natural UDP-xylose as the xylose donor, as well as the artificial compound UDP-4-azido-4-deoxyxylose, which is a selective xylose donor for XT-I.

The human isoenzymes xylosyltransferase-I and -II (XT-I, XT-II) catalyze the rate-limiting step in proteoglycan biosynthesis. Therefore, serum XT activity, mainly representing XT-II activity, displays a powerful biomarker to quantify the actual proteoglycan synthesis rate. Serum XT activity is increased up to 44% in disorders which are characterized by an altered proteoglycan metabolism, whereby underlying regulatory mechanisms remain unclear. The aim of this study was to investigate new regulatory pathways by identifying and characterizing naturally occurring XYLT2 promoter sequence variants as well as their potential influence on promoter activity and serum XT activity. XYLT2 promoter single nucleotide variants (SNVs) were identified and genotyped in the genomic DNA of 100 healthy blood donors by promoter amplification and sequencing or restriction fragment length polymorphism analysis. The SNVs were characterized by an in silico analysis considering genetic linkage and transcription factor binding sites (TBSs). The influence of SNVs on promoter activity and serum XT activity was determined by dual luciferase reporter assay and HPLC-ESI mass spectrometry. Allele frequencies of seven XYLT2 promoter sequence variants identified were investigated. In silico analyses revealed a strong genetic linkage of SNVs c.-80delG and c.-188G > A, c.-80delG and c.-1443G > A, as well as c.-188G > A and c.-1443G > A. However, despite the generation of several SNV-associated changes in TBSs in silico, XYLT2 promoter SNVs did not significantly affect promoter activity. Serum XT activities of SNV carriers deviated up to 8% from the wild-type, whereby the differences were also not statistically significant. This is the first study which identifies, genotypes and characterizes XYLT2 promoter SNVs. Our results reveal a weak genetic heterogeneity and a strong conservation of the human XYLT2 promoter region. Since the SNVs detected could be excluded as causatives for strong interindividual variabilities in serum XT activity, our data provide increasing evidence that XT-II activity is obviously regulated by hitherto unknown complex genetic pathways, such as cis- or trans-acting enhancers, silencers or miRNAs.

Background Human xylosyltransferase-I (XT-I) catalyzes the rate-limiting step in proteoglycan glycosylation. An increase in XYLT1 mRNA expression and serum XT activity is associated with diseases characterized by abnormal extracellular matrix accumulation like, for instance, fibrosis. Nevertheless, physiological and pathological mechanisms of transcriptional XT regulation remain elusive. Results To elucidate whether promoter variations might affect the naturally occurring variability in serum XT activity, a complete sequence analysis of the XYLT1 promoter was performed in genomic DNA of healthy blood donors. Based on promoter amplification by a specialized PCR technique, sequence analysis revealed a fragment of 238 bp, termed XYLT1238*, which has never been described in the human XYLT1 reference sequence so far. In silico characterization of this unconsidered fragment depicted an evolutionary conservation between sequences of Homo sapiens and Pan troglodytes (chimpanzee) or Mus musculus (mouse), respectively. Promoter activity studies indicated that XYLT1238* harbors various transcription factor binding sites affecting basal XYLT1 expression and inducibility by transforming growth factor-β1, the key fibrotic mediator. A microsatellite and two single nucleotide variants (SNV), c.-403C>T and c.-1088C>A, were identified and genotyped in 100 healthy blood donors. Construct associated changes in XYLT1 promoter activity were detected for several sequence variants, whereas serum XT activity was only marginally affected. Conclusions Our findings describe for the first time the entire XYLT1 promoter sequence and provide new insights into transcriptional regulation of XT-I. Future studies should analyze the impact of regulatory XYLT1 promoter variations on XT-associated diseases.

Mutations in the ABC transporter ABCC6 were recently identified as cause of Pseudoxanthoma elasticum (PXE), a rare genetic disorder characterized by progressive mineralization of elastic fibers. We used an untargeted metabolic approach to identify biochemical differences between human dermal fibroblasts from healthy controls and PXE patients in an attempt to find a link between ABCC6 deficiency, cellular metabolic alterations and disease pathogenesis. 358 compounds were identified by mass spectrometry covering lipids, amino acids, peptides, carbohydrates, nucleotides, vitamins and cofactors, xenobiotics and energy metabolites. We found substantial differences in glycerophospholipid composition, leucine dipeptides, and polypeptides as well as alterations in pantothenate and guanine metabolism to be significantly associated with PXE pathogenesis. These findings can be linked to extracellular matrix remodeling and increased oxidative stress, which reflect characteristic hallmarks of PXE. Our study could facilitate a better understanding of biochemical pathways involved in soft tissue mineralization.

Hydroxymethylglutaryl coenzyme A reductase (HMGCR) catalyzes the rate limiting step in cholesterol biosynthesis converting HMG-CoA into mevalonic acid (MVA), which equilibrates with mevalonic acid lactone (MVL) under neutral pH conditions. We developed a fast, sensitive, and efficient method to determine HMGCR activity in human cell lines measuring MVL levels by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Convenient prepared samples containing MVL-D7 as an internal standard were injected, separated, and eluted from an ACQUITY HSS PFP column. Measurement of MVL was performed by electrospray ionization mass spectrometry with multiple reaction monitoring. Calibration curves were linear and reproducible in the range of 0.15-165 μg/l (r>0.99). Lower limit of quantification was 0.12 μg/l. Intra- and interassay imprecision were