Biomedical subjects
J Arts
Publications and source records attributed to J Arts.
Estradiol formation by human osteoblasts via multiple pathways: relation with osteoblast function.
The importance of estrogens in bone metabolism is illustrated by the accelerated bone loss and increase in osteoporotic fractures associated with postmenopausal estrogen deficiency. In this study, the expression and activity of the enzymes involved in estrogen metabolism in human osteoblastic cells were investigated in relation to differentiation of these cells. PCR reactions using mRNA from an in vitro differentiating human cell line (SV-HFO) were performed to assess mRNA expression of the enzymes aromatase, different subtypes of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), and steroid sulfatase. Aromatase, sulfatase, and 17beta-HSD type 2 and 4 were found to be expressed throughout differentiation. Expression of 17beta-HSD type 3, however, was relatively weak, except for early time points in differentiation. Type 1 17beta-HSD expression was not detected. Aromatase activity decreased during differentiation, as was demonstrated by the conversion of androstenedione (A) and testosterone (T) into estrone (E(1)) and estradiol (E(2)), respectively. The 17beta-HSD isozymes catalysing a reductive reaction convert androstenedione and estrone into testosterone and estradiol, respectively. Their activity declined with differentiation. Analysis of 17beta-HSD activity indicated both oxidative (E(2) to E(1); T to A) and reductive (E(1) to E(2); A to T) metabolism at all stages of osteoblast differentiation. Both activities declined as cells moved toward a differentiating mineralizing phenotype. However, the oxidative reaction was increasingly in favor of the reductive reaction at all times during differentiation. Sulfatase activity, as demonstrated by the conversion of estrone-sulfate into estrone, was constant during differentiation. In conclusion, we have demonstrated that all enzymes necessary for estrogen metabolism are expressed and biologically active in differentiating human osteoblasts. The activity of aromatase and 17beta-HSD was found to be dependent on the stage of cell differentiation. In addition, human osteoblasts effectively convert estradiol into estrone. The efficacy of osteoblasts to synthesize estradiol may determine the ultimate change in rate of bone turnover after menopause, as well as the development of osteoporosis. Moreover, the enzymes involved in the metabolism of estradiol may form a target for intervention.
On the role of c-Jun in the induction of PAI-1 gene expression by phorbol ester, serum, and IL-1alpha in HepG2 cells.
We have characterized the regulation of plasminogen activator inhibitor-1 (PAI-1) gene expression by phorbol 12-myristate 13-acetate (PMA), serum, and interleukin-1alpha (IL-1alpha) in the human hepatoma cell line HepG2. PMA, serum, and IL-1alpha induced a rapid and transient 28-fold (PMA), 9-fold (serum), and 23-fold (IL-1alpha) increase in PAI-1 mRNA, peaking after approximately 4 hours. These inductions of PAI-1 mRNA accumulation were reduced by pretreatment of the HepG2 cells with the protein tyrosine kinase inhibitor genistein. Conversely, stimulation of tyrosine phosphorylation by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in PAI-1 mRNA levels. The effects of PMA, serum, and IL-1alpha on PAI-1 mRNA expression have been compared with their ability to modulate the expression of a chloramphenicol acetyltransferase (CAT) reporter plasmid, which was under control of the -489 to +75 region of the PAI-1 promoter, and stably transfected into HepG2 cells. This region of the PAI-1 promoter was previously found to contain a tetradecanoyl phorbol acetate-response element (TRE; between -58 and -50) necessary for PMA responsiveness and with a high affinity for c-Jun homodimers. Whereas incubation of these transfected HepG2 cells with PMA and serum showed an induction profile of CAT mRNA similar to that of PAI-1 mRNA, hardly any induction of CAT mRNA was found with IL-1alpha. In line with these findings, IL-1alpha poorly induced c-Jun homodimer binding to the PAI-1 TRE in gel mobility-shift assays. Pretreatment of HepG2 cells with the protein kinase C inhibitor Ro 31-8220 or the mitogen-activated protein kinase kinase (MAPKK)1,2 activity blocker PD98059 selectively suppressed the induction of PAI-1 (and CAT) expression by PMA, but not that by IL-1alpha. In contrast, the protein tyrosine kinase inhibitor herbimycin A blocked PAI-1 mRNA induction by IL-1 alpha only. We propose 2 separate PAI-1 inductory pathways for PMA and IL-1alpha in HepG2, both involving protein tyrosine kinase activation; the serum-induced signaling pathway may (partially) overlap with the PMA-activated protein kinase C/mitogen-activated protein kinase kinase pathway, leading to c-Jun homodimer binding to the PAI-1 TRE.
Cell-type specific DNA-protein interactions at the tissue-type plasminogen activator promoter in human endothelial and HeLa cells in vivo and in vitro.
Tissue-type plasminogen activator (t-PA) gene expression in human endothelial cells and HeLa cells is stimulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at the level of transcription. To study the mechanism of transcriptional regulation, we have characterized a segment of the t-PA gene extending from -135 to +100 by in vivo footprinting analysis [dimethyl sulphate (DMS) method] and gel mobility shift assay. In vivo footprinting analysis revealed changes in cleavage pattern in five distinct promoter elements in both endothelial cells and HeLa cells, including a PMA-responsive element (TRE), a CTF/NF-1 binding site and three GC-boxes, and an altered cleavage pattern of the TRE and CTF/NF-1 element after PMA treatment of HeLa cells. Although endothelial cells and HeLa cells differed in the exact G residues protected by nuclear proteins,in vitro bandshift analysis showed that nuclear protein binding to the t-PA promoter was qualitatively and quantitatively very similar in both cell types, except for the TRE. Protein binding to the TRE under non- stimulated conditions was much higher in human endothelial cells than in HeLa cells, and this TRE-bound protein showed a lower dissociation rate in the endothelial cells than in HeLa cells. In endothelial cells, the proteins bound to the TRE consisted mainly of the AP-1 family members JunD and Fra-2, while in HeLa cells predominantly JunD, FosB and Fra-2 were bound. The proteins bound to the other protected promoter elements were identified as SP-1 (GC-box II and III) and CTF/NF-1 (CTF/NF-1 binding site). After PMA treatment of the cells, AP-1 and SP-1 binding was increased two-fold in endothelial cell nuclear extracts and >20-fold in HeLa nuclear extracts. In the endothelial cells, all Jun and Fos forms (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) were part of the AP-1 complex after PMA induction. In HeLa cells, the complex consisted predominantly of c-Jun and the Fos family members FosB and Fra-2. In the light of previous studies involving mutational analysis of the human and murine t-PA promoter our results underline an important role of the five identified promoter regions in basal and PMA-stimulated t-PA gene expression in intact human endothelial cells and HeLa cells. The small differences in DMS protection pattern and differences in the individual AP-1 components bound in endothelial cells and HeLa cells point to subtle cell-type specific differences in t-PA gene regulation.
Studies on the mechanism of fibrate-inhibited expression of plasminogen activator inhibitor-1 in cultured hepatocytes from cynomolgus monkey.
Fibrates are widely used drugs in hyperlipidemic disorders. In addition to lowering serum triglyceride levels, fibrates have also been shown to reduce elevated plasma plasminogen activator inhibitor-1 (PAI-1) levels in vivo. We demonstrate that fibrates suppress PAI-1 synthesis in cultured cynomolgus monkey hepatocytes in a concentration-dependent way (0.1 to 1.0 mmol/L) and independent of their lipid-lowering effect. Different fibrates showed different potency in suppressing PAI-1 production: gemfibrozil and clofibric acid, at a concentration of 1 mmol/L, reduced PAI-1 synthesis over 24 hours to 52 +/- 20% and 60 +/- 5%, while clofibrate and bezafibrate lowered PAI-1 synthesis to only 86 +/- 17% and 84 +/- 15% of control values, respectively. These changes in PAI-1 production by fibrates correlated with changes in PAI-1 mRNA levels and were also visible at the level of gene transcription. Fibrates did not lower basal PAI-1 synthesis but attenuated an acceleration of PAI-1 production during culture. The suppressing effect of fibrates on PAI-1 synthesis could not be mimicked with activators or inhibitors of protein kinase C (PKC). Furthermore, fibrates did not inhibit the increase in PAI-1 synthesis induced by epidermal growth factor or transforming growth factor-beta. These results make mechanisms involving PKC modulation or growth factor receptor inactivation as a mode of action of fibrates unlikely. The suppressing effect of fibrates on PAI-1 synthesis could involve the nuclear receptor peroxisome proliferator-activated receptor (PPAR) and its heterodimeric partner, the retinoid X receptor (RXR). The alpha forms of PPAR and RXR were both found to be expressed in cynomolgus monkey hepatocytes. the ligand for RXR alpha, 9-cis retinoic acid, suppressed PAI-1 synthesis to the same extent as gemfibrozil, while a combination of gemfibrozil and 9-cis retinoic acid had no more effect on PAI-1 synthesis than any of these compounds alone at optimal concentrations. In conclusion, fibrates downregulate an induced PAI-1 production in cynomolgus monkey hepatocytes independent of a decrease in triglyceride levels. A possible involvement of PPAR alpha/RXR alpha in this down-regulation is discussed.
Differential expression of estrogen receptors alpha and beta mRNA during differentiation of human osteoblast SV-HFO cells.
Estrogens have been shown to be essential for maintaining a sufficiently high bone mineral density and ER alpha expression has been demonstrated in bone cells. Recently, a novel estrogen receptor, estrogen receptor beta (ERbeta) has been identified. Here we demonstrate that also ERbeta is expressed in human osteoblasts, and that ER alpha and ERbeta are differentially expressed during human osteoblast differentiation. ERbeta mRNA expression increased gradually during osteoblast culture, resulting in an average increase of 9.9+/-5.3 fold (mean+/-S.D., n=3) at day 21 (mineralization phase) as compared to day 6 (proliferation phase). In contrast, ER alpha mRNA expression levels increased only slightly until day 10 (2.3+/-1.7 fold) and then remained constant. The observed differential regulation of ER alpha and beta is suggestive for an additional functional role of ERbeta to ER alpha in bone metabolism.
Role of c-Jun and proximal phorbol 12-myristate-13-acetate-(PMA)-responsive elements in the regulation of basal and PMA-stimulated plasminogen-activator inhibitor-1 gene expression in HepG2.
Experiments were designed to clarify the role of c-Jun/c-Fos and of putative phorbol 12-myristate-13-acetate-(PMA)-responsive elements (TREs) in the induction of plasminogen-activator inhibitor 1 (PAI-1) gene transcription in the human hepatoma cell line HepG2 by activators of protein kinase C (PKC). Treatment of HepG2 cells with the phorbol ester PMA or serum rapidly and transiently increased c-Jun and c-Fos mRNA and protein levels prior to PAI-1 induction. This induction of PAI-1 gene transcription was found to be dependent on ongoing protein synthesis. An essential role of c-Jun and c-Fos in basal and PMA-stimulated transcription of the PAI-1 gene is demonstrated by our finding that antisense c-jun and c-fos oligodeoxynucleotides both strongly reduced basal and PMA-stimulated PAI-1 synthesis. Since it has already been shown that two TREs between positions -58 and -50 and between -79 and -72 of the PAI-1 promoter are essential for basal and PMA-induced PAI-1 promoter activity ([16]), we examined binding of nuclear proteins to these elements. The protein-binding activity to the TRE between positions -79 and -72 shows very strong PMA induction of an unknown factor, which is not related to c-Jun or c-Fos. The TRE binding between positions -58 and -50 forms two complexes, both containing c-Jun protein. The faster migrating complex primarily contains c-Jun homodimers. The amount of the faster migrating complex is enhanced more than 30-fold in PMA-treated cells, due to a strongly increased binding of c-Jun homodimers and, to a minor extent, to binding of c-Jun/c-Fos heterodimers. Dissociation experiments suggest that the c-Jun/c-Fos heterodimers bind with much lower affinity compared to binding of c-Jun homodimers. Together with the finding that both antisense c-jun and antisense c-fos oligodeoxynucleotides reduced the amount of c-Jun homodimer, we conclude that binding of c-Jun homodimer to the TRE at positions -58 to -50 is important in the basal activity and PMA activation of the PAI-1 promoter in HepG2 cells.
Involvement of retinoic acid receptor alpha in the stimulation of tissue-type plasminogen-activator gene expression in human endothelial cells.
Retinoids stimulate tissue-type plasminogen-activator (t-PA) gene expression in human endothelial cells, and are likely to do so by binding to one or more nuclear retinoid receptors. The present study was initiated to identify the retinoid receptor(s) involved in this process. Expression and regulation of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) were analyzed by Northern-blot analysis of total or poly(A)-rich RNA prepared from cultured human umbilical vein endothelial cells (HUVEC). Prior to any exposure to retinoids, HUVEC express two transcripts for RAR-alpha (3.6 kb and 2.8 kb), and low levels of transcripts for RAR-beta (3.4 kb and 3.2 kb) and RAR-gamma (3.3 kb and 3.1 kb). Two RXR subtypes were identified, RXR-alpha (4.8 kb) and, at a much lower concentration, RXR-beta (2.4 kb); no evidence for the presence of RXR-gamma was found. Furthermore, HUVEC express cellular retinol-binding protein I (CRBP-I) and cellular retinoic-acid-binding protein I (CRABP-I) mRNA. Exposure of HUVEC to 1 microM retinoic acid or the retinobenzoic acid, Ch55, led to the induction of the two RAR-beta mRNAs, RXR-alpha mRNA and CRBP-I mRNA, whereas the expression of the other receptor and CRABP-I transcripts did not change appreciably. Using retinoid analogues that bind preferentially to one of the RAR or RXR subtypes, we found evidence that RAR-alpha is involved in the retinoid-induced t-PA expression in HUVEC. This conclusion was strengthened by experiments in which blocking of RAR-alpha with a specific RAR-alpha antagonist, Ro 41-5253, was demonstrated to suppress the induction of t-PA by retinoids.
Stimulation of tissue-type plasminogen activator gene expression by sodium butyrate and trichostatin A in human endothelial cells involves histone acetylation.
We have previously shown that the pleiotropic agent sodium butyrate strongly stimulates tissue-type plasminogen activator (t-PA) expression in human umbilical vein endothelial cells (HUVEC). Here we provide the following evidence that the butyrate-induced t-PA expression in HUVEC involves histone H4 acetylation. (1) t-PA induction by butyrate occurs at the transcriptional level and does not require new protein synthesis, indicating a direct effect. (2) t-PA induction by butyrate can be fully mimicked by a specific, structurally unrelated, histone deacetylase inhibitor, trichostatin A. (3) At optimally stimulatory conditions, a combination of butyrate and trichostatin A does not enhance t-PA production more than each of the compounds alone, indicating that both compounds act through a common regulatory mechanism. (4) Induction of t-PA transcription by butyrate and trichostatin A was found to be preceded by histone H4 acetylation; at suboptimal inducing concentrations of butyrate and trichostatin A, the degree of acetylation of histone H4 caused by each agent was similarly reduced. These results are consistent with a role for histone H4 acetylation in t-PA induction by butyrate in HUVEC.
Regulation of endothelial cell t-PA synthesis and release.
The fibrinolytic activity of blood is to a large extent determined by the plasma level of tissue-type plasminogen activator (t-PA). Changes in the plasma level of t-PA are mainly achieved by the endothelium by two mechanisms: (a) a rapid, short-term release of t-PA, which occurs within minutes (acute release, regulated secretion) and (b) a long-term change in the rate of synthesis and constitutive secretion of t-PA. The rapid t-PA release response of the endothelium upon stimulation may play an important role in the dissolution of fibrin at the initial event of fibrin formation, and hence in the prevention of thrombus formation. The rate of constitutive t-PA secretion is an important determinant of the actual levels of t-PA under basal and stimulated conditions. Our insight into the regulation of the synthesis and release of t-PA has extended markedly in the last decade and still adapts by the continuous stream of new experimental data. In this review we summarize our present knowledge about the factors, including intracellular signalling pathways, promoter elements and transcription factors involved in the modulation of t-PA gene expression, with particular reference to the regulation in human endothelial cells. We also discuss the mechanisms underlying acute release of t-PA from the endothelium, and provide evidence that the regulated and constitutive secretion of t-PA are interrelated in several aspects.
On the interaction of IgG subclasses with the low affinity Fc gamma RIIa (CD32) on human monocytes, neutrophils, and platelets. Analysis of a functional polymorphism to human IgG2.
An allotypic form of the low affinity IgG Fc receptor Fc gamma RIIa (CD32), termed low responder (LR) because of its weak reactivity with mouse (m) IgG1, interacts efficiently with human (h) IgG2. Fc gamma RIIaLR is the first known human FcR that binds this IgG subclass. In this study, we analyzed the role of Fc gamma RIIa in binding of stable hIgG-subclass dimers, and in induction of T cell mitogenesis using chimeric anti-CD3 mAb. We demonstrate that the functional polymorphism to hIgG2 is expressed on the majority of Fc gamma R-bearing peripheral blood cells: monocytes, neutrophils, and platelets. We were able to assess Fc gamma RII-mediated IgG-binding without interference of other Fc gamma R-classes, by blockade of Fc gamma RI on monocytes, and by using neutrophils of an individual deficient for the Fc gamma RIIIB gene. This study indicates as subclass specificity: hIgG3 >hIgG1,hIgG2 >> hIgG4 for Fc gamma RIIaLR and hIgG3,hIgG1 >> hIgG2 > hIgG4 for Fc gamma RIIaHR. Comparing the serum hIgG levels of individuals homozygous for the two fc gamma RIIa allotypic forms, we observed significantly lower hIgG2 serum levels in individuals expressing the hIgG2-binding LR allotypic form. This observation may implicate that Fc gamma RIIa regulates hIgG subclass production or turnover in man.
Acute renal homotransplant insufficiency.
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