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.

Vascular endothelial growth factor gene polymorphisms as prognostic markers for ocular manifestations in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable disorder affecting the skin, eyes and cardiovascular system. It is caused by mutations in the ABCC6 gene and its clinical picture is highly variable. PXE often leads to severe visual impairment due to the development of choroidal neovascularisation (CNV). CNV in PXE-associated retinopathy is believed to be mediated by the action of vascular endothelial growth factor (VEGF). The objective of the present study was to evaluate a possible impact of variations in the VEGFA gene on ocular manifestations of PXE. For this purpose, we evaluated the distribution of 10 single nucleotide polymorphisms (SNPs) in the promoter and coding region of the VEGFA gene in DNA samples from 163 German patients affected by PXE and in 163 healthy control subjects. Haplotype analysis of SNPs c.-1540A>C, c.-460C>T, c.-152G>A, c.405C>G, c.674C>T, c.1032C>T, c.4618C>T and c.5092C>A revealed that the haplotype CTGGCCCC was associated with PXE (OR 2.05, 95% CI 1.33-3.15, P(corrected) = 0.01). Furthermore, five SNPs showed significant association with severe retinopathy. The most significant single SNP association was c.-460C>T (OR 3.83, 95% CI 2.01-7.31, P(corrected) = 0.0003). Logistic regression analysis identified the c.-460T and the c.674C alleles as independent risk factors for development of severe retinopathy. Our findings suggest an involvement of VEGF in the pathogenesis of ocular PXE manifestations. VEGF gene polymorphisms might prove useful as prognostic markers for the development of PXE-associated retinopathy and permit earlier therapeutic intervention in order to prevent loss of central vision, one of the most devastating consequences of this disease.

23S rDNA real-time polymerase chain reaction of heart valves: a decisive tool in the diagnosis of infective endocarditis.

AIMS: A new diagnostic strategy to improve the detection of pathogens in heart valves (HVs) from patients with infective endocarditis (IE) was evaluated. METHODS AND RESULTS: Three hundred and fifty seven HVs surgically removed from 326 patients with proven IE or suspicious intra-operative findings, examined by 16S rDNA polymerase chain reaction (PCR) and culture were retrospectively analysed according to the predictive value of various PCR methods. Patients were classified into four categories: active IE, IE with ambiguous infective status, healed IE, and valve diseases but no IE. Retained samples of 200 HVs were analysed by real-time PCR targeting bacterial 23S rDNA, fungal 28S rDNA, and mycoplasmal tuf gene. 16S rDNA PCR revealed 80.6% sensitivity, 100% specificity, 100% positive predictive value, and 71% negative predictive value (NPV), compared with cultivation with 33.4, 96.6, 95.5, and 40.9%, respectively. The use of real-time PCR increased diagnostic sensitivity to 96.4%, and NPV to 92.5%. Bacterial load, C-reactive protein, and white blood cell counts (WBCs) decreased during antibiotic treatment. Bacterial load showed no correlation to C-reactive protein or WBCs, whereas C-reactive protein and WBCs were significantly correlated. CONCLUSION: 23S rDNA real-time PCR of surgically removed HVs improves the diagnosis of IE. Polymerase chain reaction analysis of explanted HVs allow the optimization of the antimicrobial therapy, especially in patients with culture-negative IE.

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.

Differences in gene expression of human xylosyltransferases and determination of acceptor specificities for various proteoglycans.

The xylosyltransferase (XT) isoforms XT-I and XT-II initiate the posttranslational glycosaminoglycan (GAG) synthesis. Here, we determined the relative expression of both isoforms in 33 human cell lines. The majority of tested cell lines showed dominant XYLT2 gene expression, while only in 23132/87, JAR, NCI-H510A and THP-1 was the XT-I mRNA expression higher. Nearly equal expression levels were detected in six cell lines. Additionally, to shed light on putative differences in acceptor specificities the acceptor properties of potential acceptor sequences were determined. Peptides were expressed as glutathione-S-transferase fusion proteins containing putative or known GAG attachment sites of in vivo proteoglycans. Kinetic analysis showed that K(m) and V(max) values for XT-I mediated xylosylation were slightly higher than those for XT-II, and that XT-I showed a lesser stringency concerning the acceptor sequence. Mutagenesis of the bikunin peptide sequence in the G-S-G attachment site and flanking regions generated potential acceptor molecules. Here, mutations on the N-terminal side and the attachment site were found to be more susceptible to a loss of acceptor function than mutations in the C-terminus. Altogether the known consensus sequence a-a-a-a-G-S-G-a-a/G-a ('a' representing Asp or Glu) for XT-I mediated xylosylation could be approved and additionally extended to apply to XT-II as well.

The Pan Genera Detection immunoassay: a novel point-of-issue method for detection of bacterial contamination in platelet concentrates.

Bacterial contamination of platelet concentrates (PCs) still represents an ongoing risk in transfusion-transmitted sepsis. Recently the Pan Genera Detection (PGD) system was developed and FDA licensed for screening of bacterial contamination of PCs directly prior to transfusion. The test principle is based on the immunological detection of lipopolysaccharide (for Gram-negative bacteria) or lipoteichoic acid (for Gram-positive bacteria). In the present study we analyzed the applicability of this method with regard to detection limit, practicability, implementation, and performance. PCs were spiked with Staphylococcus aureus, Bacillus subtilis, and five different Klebsiella pneumoniae strains, as well as eight different Escherichia coli strains. The presence of bacteria was assessed by the PGD immunoassay, and bacteria were enumerated by plating cultures. Application of the PGD immunoassay showed that it is a rapid test with a short hands-on time for sample processing and no demand for special technical equipment and instrument operation. The lower detection limits of the assay for Gram-positive bacteria showed a good agreement with the manufacturer's specifications (8.2 × 10(3) to 5.5 × 10(4) CFU/ml). For some strains of K. pneumoniae and E. coli, the PGD test showed analytical sensitivities (>10(6) CFU/ml) that were divergent from the designated values (K. pneumoniae, 2.0 × 10(4) CFU/ml; E. coli, 2.8 × 10(4) CFU/ml). Result interpretation is sometimes difficult due to very faint bands. In conclusion, our study demonstrates that the PGD immunoassay is an easy-to-perform bedside test for the detection of bacterial contamination in PCs. However, to date there are some shortcomings in the interpretation of results and in the detection limits for some strains of Gram-negative bacteria.

Interactions between endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates and human endothelial cells.

BACKGROUND: Streptococcus gallolyticus subsp. gallolyticus is an important causative agent of infective endocarditis (IE) but the knowledge on virulence factors is limited and the pathogenesis of the infection is poorly understood. In the present study, we established an experimental in vitro IE cell culture model using EA.hy926 and HUVEC cells to investigate the adhesion and invasion characteristics of 23 Streptococcus gallolyticus subsp. gallolyticus strains from different origins (human IE-derived isolates, other human clinical isolates, animal isolates). Adhesion to eight components of the extracellular matrix (ECM) and the ability to form biofilms in vitro was examined in order to reveal features of S. gallolyticus subsp. gallolyticus endothelial infection. In addition, the strains were analyzed for the presence of the three virulence factors gtf, pilB, and fimB by PCR. RESULTS: The adherence to and invasion characteristics of the examined S. gallolyticus subsp. gallolyticus strains to the endothelial cell line EA.hy926 differ significantly among themselves. In contrast, the usage of three different in vitro models (EA.hy926 cells, primary endothelial cells (HUVECs), mechanical stretched cells) revealed no differences regarding the adherence to and invasion characteristics of different strains. Adherence to the ECM proteins collagen I, II and IV revealed the highest values, followed by fibrinogen, tenascin and laminin. Moreover, a strong correlation was observed in binding to these proteins by the analyzed strains. All strains show the capability to adhere to polystyrole surfaces and form biofilms. We further confirmed the presence of the genes of two known virulence factors (fimB: all strains, gtf: 19 of 23 strains) and demonstrated the presence of the gene of one new putative virulence factor (pilB: 9 of 23 strains) by PCR. CONCLUSION: Our study provides the first description of S. gallolyticus subsp. gallolyticus adhesion and invasion of human endothelial cells, revealing important initial information of strain variability, behaviour and characteristics of this as yet barely analyzed pathogen.

Involvement of a cysteine protease in the secretion process of human xylosyltransferase I.

Xylosylation of core proteins takes place in the Golgi-apparatus as the transfer of xylose from UDP-xylose to specific serine residues in proteoglycan core proteins. This initial and rate-limiting step in glycosaminoglycan biosynthesis is catalyzed by human xylosyltransferase I (XT-I). XT-I is proteolytically cleaved from the Golgi surface and shed in its active form into the extracellular space. The secreted, circulating glycosyltransferase represents a serum biomarker for various diseases with an altered proteoglycan metabolism, whereas a physiological function of secreted XT-I is still unknown. To shed light on the secretion process of XT-I and on its biological function, the cleavage site was examined and the group of proteases involved in the cleavage was identified in this study. The peptide mass fingerprint from partly purified secreted XT-I revealed the cleavage site to be localized in the aminoterminal 231 amino acids. The addition of a cysteine protease inhibitor cocktail to cells recombinantly expressing XT-I led to a concentration-dependent shift of enzyme activity towards the cell lysates attended by consistent total intracellular and extracellular XT-I activities. In conclusion, our findings provide a first insight into the XT-I secretion process regulated by a cysteine protease and may contribute to understanding the biological and pathological role of this process.

First in-gel detection and purification of human xylosyltransferase II.

Human xylosyltransferases I and II (XylT-I, XylT-II) are key enzymes in glycosaminoglycan biosynthesis. Knowledge about the in vivo molecular weight, oligomeric state or turnover number are essential characteristics which have been addressed in this study. XylT-II was purified from Pichia pastoris by fractionated ammonium sulfate precipitation, heparin affinity and ion exchange chromatography. XylT-II was purified over 7000-fold with a final yield of 2.6%. By utilizing mass spectra analysis we can prove its first in-gel detection showing a migration pattern behavior that confirms its in silico molecular weight of 95.8 kDa. We could determine a turnover number of 2.18 min(-1) or one transferred xylose molecule per one XylT-II molecule each 27.5s. The k(cat)/K(M) ratio was 0.357 min(-1)microM(-1) for XylT-II using the bikunin-homologous acceptor Bio-QEEEGSGGGQKK-F. The comparison to XylT-I derived from the same organism revealed a 2.4-fold higher catalytic efficiency (0.870 min(-1)microM(-1)) for XylT-I.

Analysis of xylosyltransferase II binding to the anticoagulant heparin.

The key enzymes in the biosynthetic pathway of glycosaminoglycan production are represented by the human xylosyltransferase I and its isoform II (XylT-I and XylT-II). The glycosaminoglycan heparin interacts with a variety of proteins, thereby regulating their activities, also those of xylosyltransferases. The identification of unknown amino acids responsible for heparin-binding of XylT-II was addressed in this study. Thus, six XylT-II fragments were designed as fusion proteins with MBP and we received soluble and purified MBP/XylT-II from Escherichia coli. Heparin-binding studies showed that all fragments bound with low affinity to heparin. Prolonging of XylT-II fragments did not account for a cooperative effect of multiple heparin-binding motifs and in turn for a stronger heparin-binding. Sequence alignment and surface polarity plot led to the identification of two highly positively charged Cardin-Weintraub motifs with surface accessibility, resulting in combination with short clusters of basic amino acids for strong heparin-binding of native xylosyltransferases.

Broad-range real-time PCR assay for the rapid identification of cell-line contaminants and clinically important mollicute species.

Polymerase chain reaction assays have become widely used methods of confirming the presence of Mollicutes species in clinical samples and cell cultures. We have developed a broad-range real-time PCR assay using the locked nucleic acid technology to detect mollicute species causing human infection and cell line contamination. Primers and probes specifically for the conserved regions of the mycoplasmal tuf gene (encoding elongation factor Tu) were designed. Cell culture supernatants, clinical specimens (vaginal swabs, sputum, cryopreserved heart valve tissues), and reference strains were tested for mollicute contamination as well as to exclude cross-reaction to human nucleic acids and other bacterial species. Nucleic acids were extracted using magnetic separation technology. The coamplification of the human beta2-microglobulin DNA served as an internal control. The PCR assay was highly specific and obtained an analytical sensitivity of one copy per microl sample. The 95% detection limit was calculated to 10 copies per microl sample for Mycoplasma pneumoniae and M. orale. No false-positive results were observed due to cross-reaction of walled bacterial, fungal, and human nucleic acids. To evaluate the PCR, we compared the results to two commercialized test systems. Moreover, in combination with a previously developed broad-range RT-PCR assay for the detection of bacteria in blood products, both mollicute and walled bacterial contamination can be detected simultaneously using multiplex real-time RT-PCR.

Lipopolysaccharide-binding protein: a new biomarker for infectious endocarditis?

BACKGROUND: Infectious endocarditis (IE) is a bacterial infection of the endocardium. Diagnosis is based on results obtained from echocardiography, blood cultures, and molecular genetic screening for bacteria and on data for inflammatory markers such as the leukocyte (WBC) count and the C-reactive protein (CRP) concentration. The aim of the present study was to evaluate lipopolysaccharide-binding protein (LBP) as a supportive biomarker for the diagnosis and therapeutic monitoring of IE. METHODS: We measured LBP and CRP concentrations and WBC counts in 57 IE patients at hospital admission, 40 patients with noninfectious heart valve diseases (HVDs), and 55 healthy blood donors. The progression of these 3 markers and the influence of cardiac surgery on them were evaluated in 29 IE patients and 21 control patients. RESULTS: Serum LBP concentrations were significantly higher in IE patients [mean (SD), 33.41 (32.10) mg/L] compared with HVD patients [6.67 (1.82) mg/L, P < 0.0001] and healthy control individuals [5.61 (1.20) mg/L]. The progression in the LBP concentration during therapy of IE patients correlated with the changes in the CRP concentration. The 2 markers were equally influenced by antibiotic treatment and surgical intervention. CONCLUSIONS: Serial LBP measurement may provide an effective and useful tool for evaluating the response to therapy in IE patients. We found a strong correlation between LBP and CRP concentrations; LBP has a tendency to increase earlier in cases of reinfection.

Novel flow cytometry-based screening for bacterial contamination of donor platelet preparations compared with other rapid screening methods.

BACKGROUND: Bacterial contamination is the major infectious hazard associated with transfusion of platelet preparations (PLTs). Routine testing for bacterial contamination in PLTs has become common, but transfusion-transmitted bacterial sepsis has not been eliminated. Here, we describe a novel flow cytometry-based method for point-of-issue screening of PLTs for bacterial contamination. METHODS: We used the BactiFlow flow cytometer to detect and count bacteria based on esterase activity in viable cells. We compared the assay to incubation (BacT/Alert culture system) and rapid nucleic acid-based or immunoassay (reverse transcription PCR, Pan Genera Detection) methods. RESULTS: We established a protocol for bacterial screening of PLTs consisting of enzymatic digestion and centrifugal filtration for the elimination of viable platelets and selective labeling of bacteria with fluorescent esterase substrate (ChemChrome V23). Results from the BactiFlow showed an excellent correlation (r = 0.9923 E. coli, r = 0.9736 S. epidermidis) to traditional plate count results. The lower detection limit of the assay was determined to be 150 CFU/mL, and the time to result was <1 h. CONCLUSIONS: Our study demonstrates that BactiFlow flow cytometry is suitable for rapid screening of PLTs for bacterial contamination and fulfils the requirements for a point-of-issue testing of PLTs with acceptable time to result, specificity, sensitivity, and cost.

Circulating calcitriol concentrations and total mortality.

BACKGROUND: Evidence is accumulating that vitamin D supplementation of patients with low 25-hydroxyvitamin D concentrations is associated with lower cardiovascular morbidity and total mortality during long-term follow-up. Little is known, however, about the effect of low concentrations of the vitamin D hormone calcitriol on total mortality. We therefore evaluated the predictive value of circulating calcitriol for midterm mortality in patients of a specialized heart center. METHODS: This prospective cohort study included 510 patients, 67.7% with heart failure (two-thirds in end stage), 64.3% hypertension, 33.7% coronary heart disease, 20.2% diabetes, and 17.3% renal failure. We followed the patients for up to 1 year after blood collection. For data analysis, the study cohort was stratified into quintiles of circulating calcitriol concentrations. RESULTS: Patients in the lowest calcitriol quintile were more likely to have coronary heart disease, heart failure, hypertension, diabetes, and renal failure compared to other patients. They also had low 25-hydroxyvitamin D concentrations and high concentrations of creatinine, C-reactive protein, and tumor necrosis factor alpha. Eighty-two patients (16.0%) died during follow-up. Probability of 1-year survival was 66.7% in the lowest calcitriol quintile, 82.2% in the second quintile, 86.7% in the intermediate quintile, 88.8% in the fourth quintile, and 96.1% in the highest quintile (P < 0.001). Discrimination between survivors and nonsurvivors was best when a cutoff value of 25 ng/L was applied (area under the ROC curve 0.72; 95% CI 0.66-0.78). CONCLUSIONS: Decreased calcitriol levels are linked to excess midterm mortality in patients of a specialized heart center.

A randomized controlled trial on the efficacy of carbohydrate-reduced or fat-reduced diets in patients attending a telemedically guided weight loss program.

BACKGROUND: We investigated whether macronutrient composition of energy-restricted diets influences the efficacy of a telemedically guided weight loss program. METHODS: Two hundred overweight subjects were randomly assigned to a conventional low-fat diet and a low-carbohydrate diet group (target carbohydrate content: >55% energy and <40% energy, respectively). Both groups attended a weekly nutrition education program and dietary counselling by telephone, and had to transfer actual body weight data to our clinic weekly with added Bluetooth technology by mobile phone. Various fatness and fat distribution parameters, energy and macronutrient intake, and various biochemical risk markers were measured at baseline and after 6, and 12 months. RESULTS: In both groups, energy intake decreased by 400 kcal/d compared to baseline values within the first 6 months and slightly increased again within the second 6 months. Macronutrient composition differed significantly between the groups from the beginning to month 12. At study termination, weight loss was 5.8 kg (SD: 6.1 kg) in the low-carbohydrate group and 4.3 kg (SD: 5.1 kg) in the low-fat group (p = 0.065). In the low-carbohydrate group, triglyceride and HDL-cholesterol levels were lower at month 6 and waist circumference and systolic blood pressure were lower at month 12 compared with the low-fat group (P = 0.005-0.037). Other risk markers improved to a similar extent in both groups. CONCLUSION: Despite favourable effects of both diets on weight loss, the carbohydrate-reduced diet was more beneficial with respect to cardiovascular risk factors compared to the fat-reduced diet. Nevertheless, compliance with a weight loss program appears to be even a more important factor for success in prevention and treatment of obesity than the composition of the diet. TRIAL REGISTRATION: Clinicaltrials.gov as NCT00868387.

High xylosyltransferase activity in children and during mineralization of osteoblast-like SAOS-2 cells.

Skeletal growth and tissue remodelling processes are characterized by an elevated collagen and proteoglycan biosynthesis. The xylosyltransferases I and II are the rate-limiting step enzymes in proteoglycan biosynthesis and serum xylosyltransferase (XT) activity has been shown to be a biomarker for the actual proteoglycan biosynthesis rate. Here, XT, alkaline phosphatase (ALP), bone ALP (BALP) activities were measured in 133 juvenile Caucasians. Serum XT activities in juveniles were elevated and significantly correlated with ALP and BALP. In an osteoblast-like cell model using SAOS-2 cells mineralization and bone nodule formation were induced and XT-I, XT-II and ALP were monitored. Induction of mineralization in SAOS-2 cells resulted in a long-term increase of XT-I mRNA and enzyme activity, which could be paralleled with elevated ALP activity. In addition, HGH and IGF-I treatment of SAOS-2 cells led to an increased expression of XT-I and ALP. These results point to skeletal growth and tissue remodeling as a cause of the high XT activity in children.

Characterization of the ATP-binding cassette transporter gene expression profile in Y79: a retinoblastoma cell line.

Chemotherapy failure was reported in treatment of retinoblastoma suggesting a role for ATP-binding cassette (ABC) proteins. Little is known about the expression pattern of ABC proteins in this cancer type. We investigated the gene expression profile of 47 ABC proteins in the human retinoblastoma cell line Y79 by TaqMan low-density array. Analysis revealed 31 ABC transporter genes expressed in this tumor cell line. Y79 cells demonstrate high gene expression of ABCA7, ABCA12, ABCB7, ABCB10, ABCC1, ABCC4, ABCD3, ABCE1, ABCF1, ABCF2, and ABCF3 (more than twofold compared to pooled RNA from different tissues). Moreover, we show that Y79 cells exhibit an active calcein efflux pointing to multidrug resistance protein (MRP)-like transporter activity. In summary, we present for the first time an ABC transporter gene expression profile in cells derived from retinoblastoma. Most of the highly expressed ABC transporter genes are typical markers of cancer cells and might exhibit potential targets for medical treatment of retinoblastoma.

Quantitative determination and comparison of the glycosaminoglycan Delta-disaccharide composition in 22 different human cell lines.

Changes in proteoglycan and glycosaminoglycan (GAG) content and distribution may play an important role in the development of many diseases, atherosclerosis, cancer and diabetes. Human cell lines act as models for the underlying pathomechanisms. Despite the importance of proteoglycans for cell functioning, information on the GAG composition of most human cell lines is limited. Comparative analysis of the GAG Deltadisaccharide amount in 22 human cell lines yielded a mean value of 94 +/- 58 pmol/10(6) cells (mean+/-SEM). Total GAG amount and heparan sulfate/heparin Deltadisaccharide composition, but not chondroitin sulfate/dermatan sulfate Deltadisaccharide composition, differed significantly between the investigated adherent and suspension cell lines. We provide a novel overview of GAG Deltadisaccharide composition in 22 different human cell lines.

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.

Gene expression profiling of ABC transporters in dermal fibroblasts of pseudoxanthoma elasticum patients identifies new candidates involved in PXE pathogenesis.

Mutations in the ABCC6 gene, encoding the multidrug resistance-associated protein 6 (MRP6), cause pseudoxanthoma elasticum (PXE). This heritable disorder leads to pathological alterations in connective tissues. The implication of MRP6 deficiency in PXE is still unknown. Moreover, nothing is known about a possible compensatory expression of other ATP binding-cassette (ABC) transporter proteins in MRP6-deficient cells. We investigated the gene expression profile of 47 ABC transporters in human dermal fibroblasts of healthy controls (n=2) and PXE patients (n=4) by TaqMan low-density array. The analysis revealed the expression of 37 ABC transporter genes in dermal fibroblasts. ABCC6 gene expression was not quantifiable in fibroblasts derived from PXE patients. Seven genes (ABCA6, ABCA9, ABCA10, ABCB5, ABCC2, ABCC9 and ABCD2) were induced, whereas the gene expression of one gene (ABCA3) was decreased, comparing controls and PXE patients (with at least twofold changes). We reanalyzed the gene expression of selected ABC transporters in a larger set of dermal fibroblasts from controls and PXE patients (n=6, each). Reanalysis showed high interindividual variability between samples, but confirmed the results obtained in the array analysis. The gene expression of ABC transporter genes, as well as lineage markers of PXE, was further examined after inhibition of ABCC6 gene expression by using specific small-interfering RNA. These experiments corroborated the observed gene expression alterations, most notably in the ABCA subclass (up to fourfold, P<0.05). We therefore conclude that MRP6-deficient dermal fibroblasts exhibit a distinct gene expression profile of ABCA transporters, potentially to compensate for MRP6 deficiency. Moreover, our results point to a function for ABCC6/MRP6 in sterol transport, as sterols are preferential regulators of ABCA transporter activity and expression. Further studies are now required to uncover the role of ABCA transporters in PXE.