Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies in murine models of osteoporosis have suggested the hypothesis that ovarian steroids may control osteoclastic bone remodeling by limiting the production of interleukin-6 (IL-6) from osteoblasts and bone marrow stromal cells. To investigate this hypothesis in a human model, we have examined 12 separate strains of normal human osteoblasts (HOB) and 11 separate strains of human bone marrow stromal cells (HBMSC) and determined whether ovarian steroids regulate the induction of IL-6 by interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) or IL-1 + TNF. Treatment with IL-1, TNF or IL-1 + TNF resulted in the induction of IL-6 from both cell types with IL-1 + TNF inducing a synergistic induction of IL-6 in HOB (24- to 324-fold) and HBMSC (35-288 fold). Addition of 17 beta-estradiol or progesterone did not significantly alter IL-6 messenger RNA or protein levels in either HOB or HBMSC cultures stimulated with IL-1, TNF or IL-1 + TNF. Cultures incubated up to 96 h with the steroids did not affect IL-6 expression. Furthermore ovarian steroids did not affect IL-6 production in either HBMSC cultures representative of preosteoblasts or HOB cultures representative of highly differentiated osteoblasts. Specific chloramphenicol acetyl transferase assays and reverse transcriptase-polymerase chain reaction studies also demonstrated that the lack of an estrogen effect was not due to the failure of HOB to express functional estrogen receptors. Therefore, we conclude that the regulation of human osteoclastic bone remodeling by ovarian steroids does not occur through the direct regulation of IL-6 gene transcription or protein secretion in either early stages of osteoblast differentiation or the differentiated osteoblast.
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PMID:Production of interleukin-6 in human osteoblasts and human bone marrow stromal cells: evidence that induction by interleukin-1 and tumor necrosis factor-alpha is not regulated by ovarian steroids. 764 14

Recent reports have suggested that polymorphisms in the gene encoding the vitamin D receptor (VDR) determine a portion of the genetic contribution to bone mineral density (BMD). Individuals homozygous for the allele lacking the Bsm I restriction site in the intron between exons 8 and 9 (BB genotype) have been found to have lower BMD than individuals homozygous for the allele having the Bsm I site (bb genotype). Interestingly, this polymorphism has also been associated with prostate cancer risk. The observed changes in BMD and prostate cancer risk might be due to an alteration in the function or abundance of the VDR leading to differential responsiveness of target cells to the action of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. To test this hypothesis, we cultured dermal fibroblasts from donors with BB, Bb, and bb genotypes and determined the level of VDR expression and the cellular responsiveness to 1,25(OH)2D3 treatment. VDR abundance, affinity for [3H]1,25(OH)2D3, and VDR mRNA levels were not detectably different in BB cells compared to bb cells. Moreover, equal expression of both VDR gene alleles was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) on mRNA from Bb fibroblasts. Fibroblast responsiveness to 1,25(OH)2D3, assessed by induction of 24-hydroxylase mRNA, was similar between BB and bb cell types in dose-response experiments. Although there were individual variations in the parameters we measured, there were no detectable or consistent differences in mean values from our small sample of cultured dermal fibroblasts. In conclusion, we did not detect significant differences in VDR properties or cellular responsiveness to 1,25(OH)2D3 that correlated with VDR genotype. Our findings suggest that these polymorphisms do not affect VDR function, but rather may be a marker for a nearby gene that is responsible for the genotype-associated variation in osteoporosis and prostate cancer risk.
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PMID:Vitamin D receptor gene polymorphisms: analysis of ligand binding and hormone responsiveness in cultured skin fibroblasts. 946 39

Investigation of osteoblast dysfunction in osteoporosis has been hampered by a poor understanding of normal early osteoblast differentiation, due to a relative lack of markers for the earliest cells in the lineage. Attempts to identify such markers have used cultures of animal or immortalized human cells, of uncertain relevance to human biology, or heterogeneous cultures in which genetic variability precludes the isolation of stage-specific genotypic markers. Primary in vitro generation of clonal populations of human bone marrow stromal cells was used in order to overcome these problems. Fibroblast-like stromal cells were isolated from human sternal bone marrow. They showed differentiation to an osteoblastic phenotype when stimulated with dexamethasone (10(-7) M) and fluorescence activated cell analysis demonstrated immunopositivity for STRO-1 (an antibody that recognizes osteoprogenitor stem cells of the colony-forming unit-fibroblastic) in from 8 to 40 per cent of the cells, dependent on time post-harvest. Cells positive for STRO-1 were immunoselected using magnetic activated cell sorting and seeded at low density (10 cells/cm2) to produce clones. Each clone was subpassaged, osteoblastic differentiation stimulated with dexamethasone, and mRMA-extracted at time points post-stimulation (0 h and 1-14 days). A novel poly (A) reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify cDNA representative of all transcripts expressed at each time point. Differential gene expression within the amplified cDNA was assessed using 3' end cDNA probes to osteocalcin, osteopontin, and collagen type I (positive), demonstrating the acquisition of an osteoblastic phenotype. Time-specific gene products for early osteoblast differentiation have been generated from primary human cultures, utilizing very low density seeding and poly (A) RT-PCR. These products overcome the problems associated with animal, immortalized or heterogeneous culture and can be used to study normal and altered early osteoblast differentiation, indicating the possibility of using the same system to study other disease states.
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PMID:Osteoblastic differentiation and mRNA analysis of STRO-1-positive human bone marrow stromal cells using primary in vitro culture and poly (A) PCR. 1039 94

Sex steroids are important regulators of bone cell function and osteoblast-derived matrix metalloproteinases (MMPs) are key mediators of bone resorption during the initial stage of osteoid removal prior to osteoclast attachment. To investigate the mechanism of bone loss following estrogen deficiency, we examined the effects of estrogen on osteoblast synthesis of MMPs and tissue inhibitor of metalloproteinases (TIMPs). Immunolocalization in mouse bone samples ex vivo and primary mouse osteoblast (MOB) cultures was used to document the synthesis of mouse interstitial collagenase (MMP-13), stromelysin-1 (MMP-3), gelatinase-A (MMP-2), and gelatinase-B (MMP-9). Endosteal bone lining cells from distal femoral head and lumbar vertebral body showed an increase in the pattern of synthesis of stromelysin-1 following ovariectomy, compared with sham-operated controls; the synthesis of other MMPs was unaffected. The expression of all classes of MMPs and TIMP-1 and TIMP-2 by MOB in culture was demonstrated by reverse transcriptase-polymerase chain reaction. Following the withdrawal of 17beta-estradiol, MOB cultures showed a significant increase in the number of cells synthesizing stromelysin-1; this effect was enhanced by stimulation with either interleukin-1 or interleukin-6. Northern blot analysis showed only a slight increase in stromelysin-1 mRNA message following the withdrawal of 17beta-estradiol. Our data show an unexpected up-regulation of stromelysin-1 synthesis by osteoblasts both in vivo and in vitro following estrogen withdrawal. Although this effect was not reflected in a significant change in stromelysin-1 mRNA expression in vitro, there is evidence to suggest a role for this enzyme in the early stages of bone loss during the pathogenesis of osteoporosis.
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PMID:Stromelysin (MMP-3) synthesis is up-regulated in estrogen-deficient mouse osteoblasts in vivo and in vitro. 1057 88

The cytokines interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and IL-6 induce osteoclast formation and may contribute to the development of postmenopausal osteoporosis. Cross-sectional studies have suggested that both IL-1 and IL-1ra secretion increase on estrogen withdrawal, and that postmenopausal osteoporosis is associated with an inadequate increase in monocyte IL-1ra secretion with age. We measured cytokine mRNA (IL-1beta, IL-1ra, IL-6, and TNF-alpha) directly in bone biopsies from early postmenopausal women to determine if a lower compensatory increase in IL-1ra mRNA could be demonstrated in women with rapid bone loss after the menopause. Biopsies were obtained from 23 early postmenopausal women (mean age 53.9 years) who participated in a randomized study of hormone replacement therapy (HRT) and risk factors for osteoporosis. Bone mineral density was assessed by duel energy X-ray absorptiometry at 0, 1, 2, and 5 years. Women in the control group were recruited to the biopsy study based on their observed rate of bone loss (upper or lower tertile). Consent was also obtained from 11 participants receiving HRT. Biopsies were taken at 2 years, frozen in nitrogen, and homogenized. Cytokine mRNA was measured by competitive reverse transcriptase polymerase chain reaction. The IL-1ra/IL-1beta mRNA slope for the slow-loss group was steeper (deltaF = 23.3, p < 0.01) than that observed in the fast-loss group, indicating that slower bone loss was associated with higher IL-1ra mRNA levels relative to IL-1beta. During HRT, the IL-1beta mRNA level was inversely correlated with serum estradiol (log r2 = 0.77, p < 0.01), and women with a serum estradiol below 200 pmol/L during HRT had IL-1beta, mRNA levels identical to the control group. In contrast, IL-1ra mRNA was independent of serum estradiol. Histomorphometric analysis revealed weak correlations between IL-1beta mRNA and activation frequency (r2 = 0.26, p = 0.06) and between IL-1ra and volume referent bone resorption rate (r2 = 0.19, p = 0.11). TNF-alpha was not associated with the bone loss rates or with serum estradiol, and only three samples were positive for IL-6 mRNA. The findings support the hypothesis that IL-1beta production within bone increases with declining estrogen levels, and that an increase in II-1ra protects against accelerated bone loss.
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PMID:Cytokine RNA levels in transiliac bone biopsies from healthy early postmenopausal women. 1067 8

The reduced bone mineral density (BMD) observed in osteoporosis results, in part, from reduced activity of bone-forming osteoblasts. We examined the effect of peroxisome proliferator-activated receptor (PPAR) activators on MC3T3-E1 preosteoblast maturation. Activators of PPARalpha, delta and gamma induced alkaline phosphatase activity, matrix calcification and the expression of osteoblast genes as determined by reverse transcriptase-polymerase chain reaction. However, at relatively high concentrations of the specific PPARgamma ligands, ciglitazone and troglitazone, maturation was inhibited. PPARalpha, delta and gamma1 were expressed in MC3T3-E1 cells. PPARgamma1 mRNA and protein levels were induced early during osteoblastic maturation. We speculate that endogenous and pharmacological PPAR activators may affect BMD by modulating osteoblastic maturation.
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PMID:Peroxisome proliferator-activated receptor activators modulate the osteoblastic maturation of MC3T3-E1 preosteoblasts. 1076 May 25

Human osteoblasts were derived in culture from explants of bone from patients who had recently suffered osteoporotic fractures and from patients with no evidence of osteoporosis. The expression of cytokine mRNA in these osteoblasts was subsequently determined by reverse transcriptase-linked polymerase chain reaction (RT-PCR). We have detected mRNA for IL-1beta, IL-3, IL-6, IL-8, TNF-alpha and -beta, and the three TGF-beta isoforms in the cells. The profile of cytokines expressed by osteoblasts derived from patients with osteoporotic fractures was consistent with profiles observed in osteoblasts derived from patients with no evidence of reduced bone mass--the latter included children undergoing corrective surgery and adult subjects ranging from 31 to 80 years undergoing elective surgery for osteoarthritis and other bone pathologies.
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PMID:Cytokine expression by cultured osteoblasts from patients with osteoporotic fractures. 1076 43

Long-term in vivo studies have highlighted smoking as a risk factor in postmenopausal osteoporosis, bone fracture incidence, and increased nonunion rates. In contrast, there are few data postulating the effects of smoking at the cellular level in human skeletal tissue. In this study, we present novel evidence demonstrating that the nicotinic receptor alpha4 subunit is present in human primary bone cells by using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, we demonstrate direct cellular effects of nicotine on primary human bone cells and blockage of these effects with a nicotinic receptor antagonist, D-tubocurarine. Nicotine effects on cell proliferation were biphasic with toxic, antiproliferative effects at high levels of nicotine (>1 mmol/L) and stimulatory effects at very low levels (0.01-10 micromol/L) after 72 h. This nicotine-induced increase in cell proliferation was inhibited in a dose-dependent manner by the addition of D-tubocurarine. In addition, proliferation effects from low-level treatment correlated with an upregulation of expression of the AP-1 transcription factor, c-fos, within 1 h, which was blocked by incubation with D-tubocurarine. To determine in situ bone cell responses within their trabecular matrix, cores of human bone isolated from biopsies were perfused with 0.1 micromol/L nicotine for 24 h. Western analysis of proteins isolated from the cores highlighted an increase in osteopontin, a bone matrix protein implicated in regulating resorption, which was partially inhibited by the addition of D-tubocurarine. To conclude, our results suggest that nicotine has a direct effect on human bone cells in modulating proliferation, upregulation of the c-fos transcription factor, and the synthesis of the bone matrix protein, osteopontin.
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PMID:Nicotinic regulation of c-fos and osteopontin expression in human-derived osteoblast-like cells and human trabecular bone organ culture. 1142 48

Since the adoption of highly active antiretroviral therapy (HAART) in the mid-1990s, certain metabolic toxicities have been increasingly recognized. These include a fat redistribution syndrome (lipohypertrophy, lipoatrophy), hyperlipidaemia, altered glucose metabolism and insulin resistance, mitochondrial toxicity (presenting as anaemia, myopathy, pancreatitis, neuropathy, hepatic steatosis and lactic acidosis), and bone density abnormalities (osteoporosis and osteonecrosis). Metabolic complications are principally reported with protease inhibitors and nucleoside reverse transcriptase inhibitors, but may be seen with all classes of antiretroviral therapy. In this review, we summarize the epidemiology, pathogenesis and management of these various toxicities.
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PMID:The metabolic toxicities of antiretroviral therapy. 1151 63

Conventional hormone replacement therapy acts primarily by preserving bone, but cannot restore lost bone in women with established osteoporosis. Studies in rodents have shown that high doses of estrogens have anabolic skeletal effects, and recent observations in a group of women treated long term with high doses of estrogen indicated that similar effects occur in humans. This study examines the hypothesis that locally produced growth factors, including transforming growth factor-beta (TGF-beta) and platelet-derived growth factors (PDGFs), are involved in mediating the anabolic effects of high-dose estrogen. Transiliac-crest bone biopsies were taken from ten women, aged 52-67 years (mean 58 years), who had been treated with high-dose estrogen for 15 years. Control samples were obtained from four age-matched postmenopausal women not receiving estrogen therapy. TGF-betas and PDGFs were analyzed for mRNA and protein expression by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Results showed both TGF-beta1 and -beta2 mRNA, expressed as a ratio to GAPDH, were increased in the estrogen-treated group with an eightfold increase for TGF-beta1 (0.258 +/- 0.246 [mean +/- SD] vs. 0.032 +/- 0.053 in the control group, p = 0.02) and a twofold increase for TGF-beta2 (p = n.s.). TGF-beta3 analysis showed only negligible amounts in both groups. Protein expression levels for TGF-beta1, -beta2, -betaRI and -RII were higher in the estrogen-treated group than in controls, the most marked effects being seen for TGF-beta1. PDGF-A protein expression was also significantly higher in osteoblasts and osteocytes in women treated with estrogen, whereas PDGF-B showed only modest differences. The percentage of bone surface occupied by osteoclasts, as determined by tartrate-resistant acid phosphatase (TRAP) staining, was significantly reduced in the estrogen-treated group (p = 0.001). These results demonstrate that high-dose estrogen therapy is associated with increased TGF-beta, TGF-betaR, and PDGF synthesis and decreased osteoclast activity, consistent with the hypothesis that these growth factors may mediate the actions of estrogen in bone.
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PMID:Mechanisms by which high-dose estrogen therapy produces anabolic skeletal effects in postmenopausal women: role of locally produced growth factors. 1155 64


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