First identification and functional analysis of the human xylosyltransferase II promoter.

Recently, we demonstrated that the human xylosyltransferase II (XT-II) has enzymatic activity and is able to catalyze the initial and rate-limiting step in the biosynthesis of glycosaminoglycans (GAGs) like chondroitin and dermatan sulfate, as well as heparan sulfate and heparin. Therefore, this enzyme also very likely assumes a crucial regulatory role in the biosynthesis of proteoglycans (PGs). In this study, we identified and characterized for the first time the XYLT2 gene promoter region and transcription factors involved in its regulation. Several binding sites for members of the Sp1 family of transcription factors were identified as being necessary for transcriptional regulation of the XYLT2 gene. This was determined by mithramycin A treatment, electrophoretic mobility shift and supershift assays, as well as numerous site-directed mutagenesis experiments. Different 5' and 3' deletion constructs of the predicted GC rich promoter region, which lacks a canonical TATA and CAAT box, revealed that a 177 nts proximal promoter element is sufficient and indispensable to drive the constitutive transcription in full strength in HepG2 hepatoma cells. In addition, we also detected the transcriptional start site using 5'-RACE (rapid amplification of cDNA ends). Our results provide an insight into transcriptional regulation of the XYLT2 gene and may contribute to understanding the manifold GAG-involving processes in health and disease.

Cystic fibrosis transmembrane conductance regulator can export hyaluronan.

OBJECTIVES: Hyaluronan, a major water binding component of the extracellular matrix, is synthesised within the cytosol and exported across the plasma membrane by the ABC-transporter MRP5 in fibroblasts. Although its synthesis is vital for embryogenesis, MRP5-deficient mice are without phenotype, suggesting that another transporter had substituted for the MRP5 protein. Thus, we searched for a compensatory exporter in fibroblasts from MRP5 deficient mice and found that cystic fibrosis transmembrane conductance regulator (CFTR) mRNA was upregulated. METHODS: Hyaluronan export was measured in cell culture. The CFTR transporter was knocked out using si-RNA. Blockers of the ABC-transporter family were used to ascertain the hyaluronan transport capabilities functionally. RESULTS: CFTR specific siRNA inhibited hyaluronan export. The tetrasaccharide was exported in undegraded form only from normal human epithelial cells and not from human epithelial cells carrying DeltaF508 CFTR. The CFTR inhibitors GlyH-101 and CFTR(172) reduced hyaluronan export from CFTR-expressing mouse fibroblasts and from human breast cancer cell lines. Bronchial secretions from patients with cystic fibrosis that consist mainly of necrotic epithelia contained at least 40-fold higher concentration of hyaluronan than secretions from patients with acute bronchitis. CONCLUSIONS: CFTR transports hyaluronan across the plasma membrane of epithelial cells and this transport mechanism is defective in cystic fibrosis patients.

Identification and characterization of the human xylosyltransferase I gene promoter region.

Human xylosyltransferase I catalyzes the initial and rate-limiting step in the biosynthesis of glycosaminoglycans and proteoglycans. Furthermore, this enzyme has been shown to play a major role in the physiological development of bone and cartilage as well as in pathophysiological processes such as systemic sclerosis, dilated cardiomyopathy, or fibrosis. Here, we report for the first time the identification and characterization of the XYLT1 gene promoter region and important transcription factors involved in its regulation. Members of the activator protein 1 (AP-1) and specificity protein 1 (Sp1) family of transcription factors are necessary for the transcriptional regulation of the XYLT1 gene, which was proven by curcumin, tanshinone IIA, mithramycin A, and short interference RNA treatment. A stepwise 5' and 3' deletion of the predicted GC-rich promoter region, which lacks a TATA and/or CAAT box, revealed that a 531-bp core promoter element is able to drive the transcription on a basal level. A binding site for transcription factors of the AP-1 family, which is essential for full promoter activity, was identified by site-directed mutagenesis located 730 bp 5' of the translation initiation site. The ability of this site to bind members of the AP-1 family was further verified by electrophoretic mobility shift assays. A promoter element containing this binding site was able to drive the transcription to about 79-fold above control in SW1353 chondrosarcoma cells. Our findings provide a first insight into the regulation of the XYLT1 gene and may contribute to understanding the processes taking place during extracellular matrix formation and remodeling in health and disease.

Increased levels of xylosyltransferase I correlate with the mineralization of the extracellular matrix during osteogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells capable to differentiate into osteoblasts. Therefore, they represent attractive cell sources for tissue engineering applications, especially for bone replacement. Proteoglycans (PGs) exhibit a crucial role for matrix assembly and remodeling. Nevertheless, since bone development is a highly dynamic and complex process, the regulation of the extracellular matrix (ECM) formation remains elusive. Consequently, the aim of this study was to investigate the mRNA expression levels of genes involved in PG assembly in different stages of osteogenesis. For the rate-limiting enzyme in glycosaminoglycan (GAG) biosynthesis xylosyltransferase I (XT-I), maximal mRNA expression levels (3.89 +/- 0.83-fold increase) and elevated enzyme activities (285 +/- 17 dpm/mug DNA) were observed 10 days after osteogenic induction, simultaneously to the beginning mineralization of the ECM, whereas the highly homologous protein XT-II showed no specific alterations. The differential expression of chondroitin sulfate, dermatan sulfate and heparan sulfate chains was determined by analyzing the mRNA expression of EXTL2 (alpha-1,4-N-acetylhexosaminyltransferase), GalNAcT (beta-1,4-N-acetylgalactosaminyltransferase), and GlcAC5E (glucuronyl C5-epimerase) as they represent crucial enzymes in GAG biosynthesis. Besides GlcAC5E, all key enzymes showed upregulated mRNA contents (up to 3.6-fold) around day 10. Except for decorin, which exhibited heightened mRNA levels even in the early stages of osteogenesis, we found similar upregulated mRNA contents (up to 14.6-fold) for all investigated PG core proteins. The synchronized expression profiles demonstrate the coordinated biosynthesis of the PGs during bone formation and osteogenic stem cell differentiation occurring in parallel to the mineralization of the extracellular matrix.

Proteoglycan biosynthesis during chondrogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells are multipotent progenitor cells that can differentiate into the chondrogenic lineage. To date, only limited knowledge about the formation and remodeling of the cartilaginous extracellular matrix is available. We recently analyzed the coordinated expression of proteins involved in the biosynthesis of proteoglycans and collagens, the two major components of cartilage matrix, to understand matrix formation and to provide potential tools to improve the quality of tissue-engineered cartilage.

Relative quantification of glycosaminoglycan-induced upregulation of TFPI-mRNA expression in vitro.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation and is mainly expressed by endothelial cells. In this study we examined the in vitro effects of heparin and other glycosaminoglycans on TFPI mRNA-expression in cultivated human endothelial (Ea.hy 926) and in chondrosarcoma (SW 1353) cells. METHODS: We used a LightCycler-based method for relative quantification of the TFPI-mRNA expression before and after stimulation. The cells were stimulated with different concentrations of heparin (with and without addition of protamin), heparan sulfate (HS) and chondroitin-6-sulfate (CS). Cells were harvested after incubation times of 4, 8 and 24h, total RNA was isolated, and cDNA was synthesized and quantified relatively to a constantly expressed housekeeping gene. RESULTS: Stimulation of Ea.hy 926 cells with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) caused a time- and dose-dependent upregulation of TFPI-mRNA expression with LMWH showing the stronger effect. In contrast to this, HS led to a strongly and CS to a slightly decreased TFPI-mRNA expression. SW 1353 cells which were stimulated with LMWH/UFH and HS/CS did not show a significant up- or downregulative effect. CONCLUSION: Our results show that we have developed a versatile method for the relative quantification of TFPI-mRNA expression. As a conclusion, the determined heparin-induced upregulation of TFPI-mRNA expression can be considered a major component of the modulation of the anticoagulant properties of the endothelium.

Measurement of fibrosis marker xylosyltransferase I activity by HPLC electrospray ionization tandem mass spectrometry.

BACKGROUND: Xylosyltransferase I (XT-I), the key enzyme in the biosynthesis of glycosaminoglycan chains in proteoglycans, has increased activity in the blood serum of patients with connective tissue diseases. Therefore, the measurement of serum XT-I activity is useful to monitor disease activity in these patients. METHODS: We developed an HPLC electrospray ionization tandem mass spectrometry method to assay XT-I activity in serum by use of a synthetic peptide (Bio-BIK-F) as the XT-I substrate. On the basis of XT-I-mediated transfer of D-xylose from UDP-D-xylose to the synthetic peptide to form Bio-BIK-F-Xyl, we determined XT-I activity in human serum samples. RESULTS: Multiple calibration curves for the analysis of Bio-BIK-F-Xyl exhibited consistent linearity and reproducibility in the range of 0.20-20 mg/L, corresponding to XT-I activity of 1.14-114 mU/L under assay conditions. The mean (SD, range) XT-I activity values in 30 blood donor sera were 18.4 (3.0, 8.7-24.8) mU/L. The limit of detection and lower limit of quantification were 8.5 microg/L (0.05 mU/L) and 163 microg/L Bio-BIK-F-Xyl (0.93 mU/L XT-I activity), respectively. Interassay imprecision (CV) was 5.4%-26.1% in the range of 0.64 to 129 mU/L, and mean recovery was 107% (range, 96%-129%). Method comparison with the radiochemical assay showed a moderate correlation (r = 0.79). The Passing-Bablok regression line was: radiochemical assay = 0.045 LC-MS/MS + 0.061 mU/L, S(y/x) = 0.186. CONCLUSIONS: This simple and robust LC-MS/MS assay permits the rapid and accurate determination of XT-I activity in human serum.

The formation of extracellular matrix during chondrogenic differentiation of mesenchymal stem cells correlates with increased levels of xylosyltransferase I.

In vitro differentiation of mesenchymal stem cells (MSCs) into chondrogenic cells and their transplantation is promising as a technique for the treatment of cartilaginous defects. But the regulation of extracellular matrix (ECM) formation remains elusive. Therefore, the objective of this study was to analyze the regulation of proteoglycan (PG) biosynthesis during the chondrogenic differentiation of MSCs. In different stages of chondrogenic differentiation, we analyzed mRNA and protein expression of key enzymes and PG core proteins involved in ECM development. For xylosyltransferase I (XT-I), we found maximum mRNA levels 48 hours after chondrogenic induction with a 5.04 +/- 0.58 (mean +/- SD)-fold increase. This result correlates with significantly elevated levels of enzymatic XT-I activity (0.49 +/- 0.03 muU/1 x 10(6) cells) at this time point. Immunohistochemical staining of XT-I revealed a predominant upregulation in early chondrogenic stages. The highly homologous protein XT-II showed 4.7-fold (SD 0.6) increased mRNA levels on day 7. To determine the differential expression of heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS) chains, we analyzed the mRNA expression of EXTL2 (alpha-4-N-acetylhexosaminyltransferase), GalNAcT (beta-1,4-N-acetylgalactosaminyltransferase), and GlcAC5E (glucuronyl C5 epimerase). All key enzymes showed a similar regulation with temporarily downregulated mRNA levels (up to -87-fold) after chondrogenic induction. In accordance to previous studies, we observed a similar increase in the expression of PG core proteins. In conclusion, we could show that key enzymes for CS, DS, and HS synthesis, especially XT-I, are useful markers for the developmental stages of chondrogenic differentiation.

Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity.

XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure-function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561-->Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.