Dirk Raddatz

Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin-D3, dexamethasone, and local growth factors in primary human osteoblasts†

Core binding factor alpha 1 (Cbfa1) is an osteoblast‐specific transcription factor essential to develop a mature osteoblast phenotype. However, its exact role in the signaling of various osteotropic‐differentiating agents is still unclear. In this study, we assessed the effects of 1,25‐(OH)2‐D3 (D3), ascorbic acid, bone morphogenetic protein‐2 (BMP‐2), dexamethasone (Dex), and transforming growth factor‐β (TGF‐β) on Cbfa1 and osteocalcin (OCN) mRNA steady state levels (by semiquantitative RT‐PCR) in an in vitro model of osteoblast differentiation. TGF‐β increased Cbfa1 mRNA levels in normal primary human osteoblasts (pHOB) by 2.6‐fold in a time‐dependent fashion with maximum effect on day 28 (P < 0.001). Similarly, the glucocorticoid Dex enhanced Cbfa1 gene expression by pHOB in a time‐dependent fashion by up to 4.6‐fold (P < 0.001). In contrast, Dex inhibited OCN gene expression levels by 68% (P < 0.01). Treatment with BMP‐2 resulted in an earlier enhancement of Cbfa1 and led to a 4.2‐fold increase with a maximum on day 21 (P < 0.001). Ascorbic acid did not modulate Cbfa1 and OCN gene expression. The effect of vitamin D (D3) on Cbfa1 mRNA expression was influenced by the duration of treatment, being inhibitory after 1 h and having a stimulatory effect after 48 h. Time course experiments indicated a stimulatory effect of D3 on Cbfa1 mRNA levels (by 2.5‐fold after 48 h; P < 0.01). Analysis of the late cellular differentiation marker osteocalcin revealed that D3 increased OCN gene expression by 14‐fold (P < 0.001). In conclusion, in normal primary human osteoblasts, the rapid and pronounced increase of OCN after treatment with D3 seems not to be mediated by Cbfa1. These data imply that Cbfa1 gene expression is differentially regulated by various osteoblastic differentiating agents and is dependent on the stage of maturation. J. Cell. Biochem. 86: 348–356, 2002.

Quantitative measurement of cytokine mRNA in inflammatory bowel disease: relation to clinical and endoscopic activity and outcome.

Objective The objective of this study was to quantitatively determine cytokine mRNA expression in inflammatory bowel disease under different clinical conditions including active disease, remission or an impaired response to a glucocorticoid (GC) therapy. Methods Mucosal biopsies were taken from 33 patients with ulcerative colitis (UC), 21 patients with Crohns disease (CD) and 11 controls. Peripheral blood mononuclear cells (PBMNC) were isolated from 24 CU patients, 18 CD patients and 11 controls. Cytokine-mRNA [interleukin (IL)-1&bgr;, IL-2, IL-4, IL-6, IL-10, interferon gamma (IFN-&ggr;), tumour necrosis factor alpha (TNF-&agr;)] expression was measured by quantitative reverse transcriptase-polymerase chain reaction in biopsies and PBMNC, and correlated to endoscopic findings, clinical activity and outcome after 6 months GC therapy. Results IL-1&bgr;, IL-6 and TNF-&agr; were the largely dominating cytokines. In contrast to IL-1&bgr; and TNF-&agr;-, IL-6 expression was restricted to inflamed mucosa and correlated with the clinical activity and C-reactive protein levels in cases of pancolitis ulcerosa. TNF-&agr; was elevated in CD even in endoscopic normal tissue. IL-2 and IFN-&ggr; were downregulated in PBMNC from CD and UC. No Th1 or Th2 specificity could be detected. Cytokine mRNA levels did not correlate with the response to a GC therapy. Conclusion IL-6 sharply distinguishes between inflamed and non-inflamed mucosa, and is therefore a suitable marker of intestinal inflammation. Its selective expression in the inflammatory site makes it an interesting target for future therapeutic strategies. TNF-&agr; overexpression even in remission suggests a key role of this cytokine in CD pathogenesis and is possibly a feature that allows one to differentiate CD from UC.

NF-κB-dependent synergistic regulation of CXCL10 gene expression by IL-1β and IFN-γ in human intestinal epithelial cell lines

Background and aimsLittle is known about the intestinal epithelial expression and secretion of CXCL10 (IP-10), a chemokine involved in recruiting T cells and monocytes. We aimed to study CXCL10 gene expression and regulation by the pro-inflammatory cytokines interleukin (IL)-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in intestinal epithelial cell lines.Materials and methodsCXCL10 expression and secretion kinetics were assessed in Caco-2, HT-29 and DLD1 human colon epithelial cells, treated with IL-1β, TNF-α, IFN-γ alone or in combination with each other by real-time polymerase chain reaction (PCR), Northern blotting and enzyme-linked immunoabsorbent assay (ELISA). Transient transfections with TGL-IP10 (CXCL10 promoter) and TGL-IP10-κB2 mutant promoter and gelshifts and supershifts for nuclear factor (NF)-κB were also performed.ResultsReal-time PCRs and ELISA experiments revealed that IL-1β was the strongest and earliest inducer of CXCL10 messenger ribonucleic acid (mRNA) expression and protein secretion in Caco-2 cell line, whereas INF-γ had a delayed kinetics. There was a strong synergistic effect of either TNF-α or IL-1β with IFN-γ both on CXCL10 mRNA expression and protein secretion in all three cell lines. Real-time PCR and ELISA experiments using a specific NF-κB inhibitor and transfection experiments with a NF-κB-binding defective CXCL10 promoter construct revealed that the induction of CXCL10 by IL-1β and its synergism with IFN-γ is NF-κB dependent.ConclusionThese data demonstrate that in colonic epithelial cells, depending on the cellular context and utilizing the NF-κB pathway, IL-1β alone and/or in synergism with IFN-γ may play a major role in the induction of CXCL10.

Effect of heparin and liver heparan sulphate on interaction of HepG2-derived transcription factors and their cis-acting elements: altered potential of hepatocellular carcinoma heparan sulphate.

Proteoglycan assembly in malignant tumours is subject to profound changes. The significance of these alterations is not well understood; especially, their role in nuclear regulation is a topic for debate. The capacity of heparin and liver carcinoma heparan sulphate (HS) to alter DNA-transcription factor interactions has been studied to provide further evidence concerning the regulatory potential of glycosaminoglycan (GAG) in the nucleus. Experiments both in vitro and in vivo indicated that heparin and HS are capable of inhibiting the interaction of transcription factors with their consensus oligonucleotide elements. Among five transcription factors studied, AP-1, SP-1, ETS-1 and nuclear factor kappaB proved to be sensitive to heparin and heparan sulphate, whereas TFIID was hardly inhibited in either in vitro or in vivo systems. Interestingly, HS from peritumoral liver was five times more effective than heparin. Liver carcinoma HS was less effective than liver HS, but its activity was comparable with that of heparin. These results indicate that the structural differences of GAG chains strongly influence their biological behaviour. The loss of their recognized functional activity in malignant tumours might promote the development of uncontrolled growth and gene expression favouring the neoplastic process.

Preserved Na(+)/H(+) exchanger isoform 3 expression and localization, but decreased NHE3 function indicate regulatory sodium transport defect in ulcerative colitis.

Background: A major causative factor of diarrhea in ulcerative colitis (UC) patients is the loss of Na+ absorptive capacity of the inflamed colonic mucosa. Potential contributing mechanisms include reduced driving force for active transport, and impaired expression, mislocalization, or defective transport function of Na+ absorptive proteins. We therefore studied the expression, brush border membrane (BBM) localization, and transport capacity of the major intestinal Na+ absorptive protein, the Na+/H+ exchanger isoform 3 (NHE3) in biopsies from UC patients. Methods: In UC and control biopsies, inflammation was graded histologically, NHE3, tumor necrosis factor alpha (TNF‐&agr;), villin, as well as other housekeeping genes were analyzed by quantitative real‐time polymerase chain reaction (PCR), BBM localization of NHE3 determined by immunohistochemistry, and confocal microscopy. Na+ absorptive capacity was assessed by 22Na+ isotope fluxes and NHE3 transport activity measured microfluorometrically in BCECF‐loaded surface colonocytes within isolated crypts. Results: In mildly, moderately, and severely inflamed sigmoid colon of UC patients, neither NHE3 mRNA expression nor the abundance of NHE3 in the BBM was significantly altered compared to other structural components of the BBM. However, Na+ absorption was strongly reduced by ≈80% and acid‐activated NHE3 transport activity was significantly decreased in the surface cells of sigmoid colonic crypts even in moderately inflamed mucosa. Conclusions: In the colonic mucosa of patients with active UC, NHE3 transport capacity was found significantly decreased despite correct NHE3 location and abundance in the brush border, independent of current treatment. These findings suggest functional NHE3 transport as a novel factor for inflammatory diarrhea in UC patients. (Inflamm Bowel Dis 2010)

Cytokines, Osteoprotegerin, and RANKL In Vitro and Histomorphometric Indices of Bone Turnover in Patients With Different Bone Diseases†

Cytokines are supposed to play an essential role in the regulation of the bone metabolic unit. However, information on cytokine production of primary human osteoblasts from patients with metabolic bone disease is scarce, and few attempts have been made to correlate such data to histomorphometric parameters of individual patients. We investigated 11 patients with metabolic bone disease referred to our outpatient department for bone biopsy and analyzed interleukin (IL)‐1, IL‐6, and TNF‐α protein release and gene expression in primary osteoblast cultures. Compared with four controls, five patients showed normal cytokine protein release, whereas six patients showed much higher levels of interleukin‐6 (26‐fold) and TNF‐α (84‐fold). All three cytokines were strongly correlated concerning gene expression and/or protein levels (r = 0.72–0.96). Histomorphometric analysis of the bone samples showed that eroded surface (ES/BS) as a parameter of bone resorption was significantly associated with TNF‐α. In addition, RANKL gene expression was positively associated with ES/BS and osteoclast surface (Oc.S/BS). Finally, the formation parameters osteoid volume and osteoid surface were negatively associated with TNF‐α. In conclusion, in an in vitro‐ex vivo model of bone cells obtained from a group of 11 patients with different forms of metabolic bone disease, cytokine release in conditioned medium was significantly associated with bone resorption and bone formation, as quantified by histomorphometry. TNF‐α seemed to be the more important cytokine; its effect on bone resorption could be mediated by RANKL.

Negative Inotropy of the Gastric Proton Pump Inhibitor Pantoprazole in Myocardium From Humans and Rabbits Evaluation of Mechanisms

Background— Proton pump inhibitors are used extensively for acid-related gastrointestinal diseases. Their effect on cardiac contractility has not been assessed directly. Methods and Results— Under physiological conditions (37°C, pH 7.35, 1.25 mmol/L Ca2+), there was a dose-dependent decrease in contractile force in ventricular trabeculae isolated from end-stage failing human hearts superfused with pantoprazole. The concentration leading to 50% maximal response was 17.3±1.3 &mgr;g/mL. Similar observations were made in trabeculae from human atria, normal rabbit ventricles, and isolated rabbit ventricular myocytes. Real-time polymerase chain reaction demonstrated the expression of gastric H+/K+–adenosine triphosphatase in human and rabbit myocardium. However, measurements with BCECF-loaded rabbit trabeculae did not reveal any significant pantoprazole-dependent changes of pHi. Ca2+ transients recorded from field-stimulated fluo 3–loaded myocytes (F/F0) were significantly depressed by 10.4±2.1% at 40 &mgr;g/mL. Intracellular Ca2+ fluxes were assessed in fura 2–loaded, voltage-clamped rabbit ventricular myocytes. Pantoprazole (40 &mgr;g/mL) caused an increase in diastolic [Ca2+]i by 33±12%, but peak systolic [Ca2+]i was unchanged, resulting in a decreased Ca2+ transient amplitude by 25±8%. The amplitude of the L-type Ca2+ current (ICa,L) was reduced by 35±5%, and sarcoplasmic reticulum Ca2+ content was reduced by 18±6%. Measurements of oxalate-supported sarcoplasmic reticulum Ca2+ uptake in permeabilized cardiomyocytes indicated that pantoprazole decreased Ca2+ sensitivity (Kd) of sarcoplasmic reticulum Ca2+ adenosine triphosphatase: control, Kd=358±15 nmol/L; 40 &mgr;g/mL pantoprazole, Kd=395±12 nmol/L (P<0.05). Pantoprazole also acted on cardiac myofilaments to reduced Ca2+-activated force. Conclusions— Pantoprazole depresses cardiac contractility in vitro by depression of Ca2+ signaling and myofilament activity. In view of the extensive use of this agent, the effects should be evaluated in vivo.

Isopropanolic Extract of Black Cohosh Stimulates Osteoprotegerin Production by Human Osteoblasts

An isopropanolic extract (iCR) from the rhizomes of Cimicifuga racemosa (black cohosh) is used an alternative in the treatment of menopausal symptoms, and animal studies suggest positive skeletal effects. iCR stimulated osteoblastic OPG protein secretion by 3‐ to 5‐fold as early as 12 h without affecting RANKL expression. The iCR effect, abrogated by the pure estrogen receptor antagonist ICI 182,780, also enhanced ALP activity (4‐fold) and osteocalcin expression (3‐fold), possibly contributing to the skeletal effects of black cohosh.

Combination of Alcohol and Fructose Exacerbates Metabolic Imbalance in Terms of Hepatic Damage, Dyslipidemia, and Insulin Resistance in Rats

Although both alcohol and fructose are particularly steatogenic, their long-term effect in the development of a metabolic syndrome has not been studied in vivo. Consumption of fructose generally leads to obesity, whereas ethanol can induce liver damage in the absence of overweight. Here, Sprague-Dawley rats were fed ad libitum for 28 days on five diets: chow (control), liquid Lieber-DeCarli (LDC) diet, LDC +30%J of ethanol (L-Et) or fructose (L-Fr), and LDC combined with 30%J ethanol and 30%J fructose (L-EF). Body weight (BW) and liver weight (LW) were measured. Blood and liver samples were harvested and subjected to biochemical tests, histopathological examinations, and RT-PCR. Alcohol-containing diets substantially reduced the food intake and BW (≤3rd week), whereas fructose-fed animals had higher LW than controls (P<0.05). Additionally, leukocytes, plasma AST and leptin levels were the highest in the fructose-administered rats. Compared to the chow and LDC diets, the L-EF diet significantly elevated blood glucose, insulin, and total-cholesterol levels (also vs. the L-Et group). The albumin and Quick-test levels were the lowest, whereas ALT activity was the highest in the L-EF group. Moreover, the L-EF diet aggravated plasma triglyceride and reduced HDL-cholesterol levels more than 2.7-fold compared to the sum of the effects of the L-Et and L-Fr diets. The decreased hepatic insulin clearance in the L-EF group vs. control and LDC groups was reflected by a significantly decreased C-peptide:insulin ratio. All diets except the control caused hepatosteatosis, as evidenced by Nile red and H&E staining. Hepatic transcription of insulin receptor substrate-1/2 was mainly suppressed by the L-Fr and L-EF diets. The L-EF diet did not enhance the mitochondrial β-oxidation of fatty acids (Cpt1α and Ppar-α expressions) compared to the L-Et or L-Fr diet. Together, our data provide evidence for the coaction of ethanol and fructose with a high-fat-diet on dyslipidemia and insulin resistance-accompanied liver damage.

A TNF-Regulated Recombinatorial Macrophage Immune Receptor Implicated in Granuloma Formation in Tuberculosis

Macrophages play a central role in host defense against mycobacterial infection and anti- TNF therapy is associated with granuloma disorganization and reactivation of tuberculosis in humans. Here, we provide evidence for the presence of a T cell receptor (TCR) αβ based recombinatorial immune receptor in subpopulations of human and mouse monocytes and macrophages. In vitro, we find that the macrophage-TCRαβ induces the release of CCL2 and modulates phagocytosis. TNF blockade suppresses macrophage-TCRαβ expression. Infection of macrophages from healthy individuals with mycobacteria triggers formation of clusters that express restricted TCR Vβ repertoires. In vivo, TCRαβ bearing macrophages abundantly accumulate at the inner host-pathogen contact zone of caseous granulomas from patients with lung tuberculosis. In chimeric mouse models, deletion of the variable macrophage-TCRαβ or TNF is associated with structurally compromised granulomas of pulmonary tuberculosis even in the presence of intact T cells. These results uncover a TNF-regulated recombinatorial immune receptor in monocytes/macrophages and demonstrate its implication in granuloma formation in tuberculosis.