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)

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) inhibits osteoclast differentiation, activity, and survival; therefore OPG/OCIF may regulate the resorption of dental hard tissues, such as alveolar bone, cementum, and dentin. To investigate this issue, reverse transcriptase-polymerase chain reaction using specific primers for OPG/OCIF was performed with total RNAs isolated from human gingival keratinocytes (HGKs), human gingival fibroblasts (HGFs), human periodontal ligament cells (HPDLs), and human pulp cells (HPCs) in culture. PCR products were found in HGFs, HPDLs, and HPCs, but not in HGKs, and the DNA sequence of these products was 100% identical to the reported sequence of the OPG gene. Northern blot analyses also showed that HGFs, HPDLs, and HPCs, but not HGKs, expressed OPG/OCIF transcripts of approximately 2.5 kb. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) increased OPG/OCIF mRNA levels in a dose-and time-dependent manner in HPDL. After 12 h of treatment, IL-1beta at 3 ng/ml and TNF-alpha at 3 ng/ml increased OPG/OCIF mRNA expression by 190% and 110%, respectively, with a maximal effect. The stimulatory effects of IL-1beta and TNF-alpha were also seen in HPC. However, IL-6 and transforming growth factor-beta had little effect on OPG/OCIF mRNA levels in HPDL. These findings suggest that OPG/OCIF synthesized by dental mesenchymal cells locally regulates the resorption of dental hard tissues through cytokines.
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PMID:Expression of osteoprotegerin (osteoclastogenesis inhibitory factor) in cultures of human dental mesenchymal cells and epithelial cells. 1046 76

An in vitro culture system to generate human osteoclasts (OC) was recently described in which OC precursors in the human peripheral blood mononuclear cell (PBMC) population differentiate in the presence of murine ST-2 stromal cells. We used this culture system to define the cytokine environment in which human OC form and to determine the separate contributions of the stromal and hematopoietic elements. We designed a panel of reverse transcriptase-polymerase chain reaction (RT-PCR) primers that specifically amplify the respective murine or human mRNA species that correspond to cytokines and their cognate receptors previously shown to promote or inhibit OC differentiation. ST-2 cells were cocultured with human PBMC for up to 21 days in the presence of 1alpha,25(OH)(2) vitamin D(3), dexamethasone, and recombinant human macrophage-colony stimulating factor (M-CSF). OC formation was monitored by the appearance of cells that were positive for tartrate-resistant acid phosphatase (TRAP) and able to form resorption lacunae on slices of dentine. We found that the ST-2 cells in these cultures express messenger RNA (mRNA) encoding a repertoire of many of the reported osteoclastogenic factors (interleukins [IL]-1/IL-1R1, IL-11, IL-6/IL-6R, and IL-17 transforming growth factor [TGF]-beta), as well as the recently described OC differentiation factor (ODF/TRANCE/RANKL). The stromal cells also expressed mRNA encoding two molecules shown to be inhibitory to osteoclastogenesis, osteoprotegerin (OPG) and IL-18. OPG, IL-1, IL-1R1, IL-6, IL-6R, IL-11R, IL-17, IL-18, IL-18R, TGF-beta, and M-CSF were expressed by both the stromal cells and the PBMC. Expression of mRNA encoding RANK, IL-1R2, and c-fms, was specific for the PBMC. In addition, PBMC were found to express sIL-6R, granulocyte macrophage (GM)-CSF, GM-CSFRalpha, and tumor necrosis factor (TNF)-alpha. Whereas this indicated that human OC formation occurs in a complex environment of many positive and negative influences, we identified three apparent features of the cytokine environment that may be a characteristic of normal osteoclast formation. First, the ratio of mouse ODF:OPG mRNA was found to increase during the cocultures, consistent with a key role for ODF in the promotion by stromal cells of OC formation. Second, we found that mRNA encoding IL-1 and IL-17, as well as IL-6 and sIL-6R, were coordinately expressed by the PBMC. Third, analysis of the culture medium showed that the PBMC secreted IL-1, IL-6, and TNF-alpha protein only in coculture with ST-2 cells during the first few days of osteoclast development. We conclude that human OC formation occurs in a complex environment of many positive and negative influences; however, these are likely to be strictly regulated by a coordinated cytokine response of both stromal and hematopoietic cells.
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PMID:Coordinated cytokine expression by stromal and hematopoietic cells during human osteoclast formation. 1083 38

The in vivo effects of IL-18 on bone metabolism were investigated by histopathology in IL-18 transgenic mice. Deformed cortical bone and decreased turnover rate of lumbar trabecular bone are consistent with increased expression of IFN-gamma and IL-18 in the bone marrow. Interleukin (IL)-18 has been demonstrated to inhibit osteoclastogenesis in an in vitro co-culture system. We investigated the effects of IL-18 overexpression on bone metabolism by comparing bone characteristics in male IL-18 transgenic (TG) mice, which secrete mature murine IL-18 from their B- and T-cells, and their wildtype littermates (WT). Histopathological analysis revealed that the cortical bone of the femur was thinner and more deformed in IL-18 TG mice. Bone histomorphometry showed that the cortical bone area of the mid-diaphysis of the femur and the trabecular bone volume of the lumbar vertebrae were significantly reduced in IL-18 TG mice. IL-18 TG mice also exhibited significantly fewer osteoclasts and a reduced bone formation rate in the trabecular bones of their lumbar vertebrae. Real-time reverse transcriptase-polymerase chain reaction amplification of bone marrow cell mRNA revealed that interferon (IFN)-gamma mRNA expression was significantly increased, whereas IL-4 mRNA expression was significantly reduced, in IL-18 TG mice. However, the expression ratio of receptor activator of NFkappaB ligand and osteoprotegerin mRNA was not significantly altered. Thus, deformed cortical bone and a decreased turnover rate of lumbar trabecular bone are characteristic of IL-18 TG mice, and these features might be associated with the increased expression of IFN-gamma and IL-18 in the bone marrow.
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PMID:Bone malformations in interleukin-18 transgenic mice. 1281 49

Apoptosis-inducing tumor necrosis factor (TNF) ligands and receptors have been reported in human placentas, but the expression patterns of family members lacking this function [a proliferation-inducing ligand (APRIL), B lymphocyte stimulator (BLyS), CD30L/CD153, CD40L/CD154, TNF-related activation-induced cytokine, CD27L/CD70, OX40L, activation-inducible TNF receptor ligand (AITRL)] are incompletely documented or unknown. We therefore investigated expression of these eight ligands and nine of their receptors (B cell maturation antigen, transmembrane activator and calcium-modulator and cyclophilin ligand-interactor, CD30, CD40, receptor activator of nuclear factor-kappaB, osteoprotegerin, CD27, OX40/CD134, AITR). Analysis by reverse transcriptase-polymerase chain reaction revealed mRNAs encoding only three of the ligands (APRIL, BLyS, CD30L/CD153). Immunoblots demonstrated all three proteins in first-trimester and term placentas, and immunohistochemical experiments showed that expression was cell-specific and gestation-related. Although mRNAs encoding receptors for the three expressed ligands were absent, those encoding receptors for all of the unexpressed ligands were detectable. Collectively, the results are consistent with the postulate that nonapoptosis-inducing, placenta-derived TNF superfamily cytokines contribute to the T helper cell type 2 bias required for successful pregnancy. Patterns of placental expression of receptors suggest bidirectional maternal-fetal cytokine communication.
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PMID:Cell-specific expression of B lymphocyte (APRIL, BLyS)- and Th2 (CD30L/CD153)-promoting tumor necrosis factor superfamily ligands in human placentas. 1283 45

Although important roles of receptor activator of NF-kappaB ligand (RANKL) and its receptor (RANK) have been established for osteoclastogenesis and bone resorption, their expression and roles during physiological root resorption remain uncertain. Physiological root resorption for shedding of human deciduous teeth is mediated by osteoclast-like cells (odontoclasts). In this study, we examined the expression of RANKL and osteoprotegerin (OPG), a decoy receptor that prevents RANKL from binding to RANK in human periodontal ligament (PDL) cells during physiological root resorption using immunocytochemistry and reverse transcriptase polymerase chain reaction. The effect of RANKL on root resorbing activity of odontoclasts was evaluated by measuring the size of dissolved area on calcium phosphate-coated coverslips. The PDL cells isolated from either non-resorbing deciduous teeth or permanent teeth abundantly expressed OPG, but not RANKL. In contrast, PDL cells derived from resorbing deciduous teeth dominantly expressed RANKL. Human odontoclasts derived from resorbing deciduous teeth expressed both RANKL and RANK. It was observed that RANKL increased odontoclast actin ring formation and resorbing activity in a dose-dependent manner. These results indicate that PDL cells during the root-resorbing state express RANKL but decrease OPG expression. Expression of RANKL likely participates in odontoclastogenesis and activates physiological root resorption.
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PMID:Expression and role of RANKL in periodontal ligament cells during physiological root-resorption in human deciduous teeth. 1288 1

The maintenance of endothelial integrity is important for prevention of vascular diseases. Several growth factors, such as bFGF and angiopoietin-1, have been shown to suppress endothelial cell apoptosis and thus help to maintain endothelial integrity. Several studies suggested that receptor activator of NF-kappaB (RANK) and its ligand (RANKL) could be involved in endothelial physiology. Using immunofluorescence and reverse transcriptase-polymerse chain reaction, we found that RANK was expressed by endothelial cells, and RANKL was expressed by arterial smooth muscle cells. Furthermore, RANKL suppressed apoptosis of primary cultured endothelial cells. The RANKL-induced survival appeared to be dependent on PI 3'-kinase activity, because wortmannin and LY294002, PI 3'-kinase-specific inhibitors, blocked the RANKL-induced survival effect. RANKL elicited the phosphorylation of the serine-threonine kinase Akt at Ser473 in a PI 3'-kinase-dependent manner. The expression of a dominant-negative form of Akt or pretreatment of Akt-specific inhibitor in endothelial cells reversed the RANKL-induced survival effect. Tumor necrosis factor-alpha, which causes endothelial cell apoptosis, induced endothelial cells to express osteoprotegerin, a decoy receptor that inhibits RANK-RANKL signaling. These findings indicate that RANK, in response to the paracrine stimulus of RANKL, may play an important role in maintaining endothelial cell integrity through the PI 3'-kinase/Akt signal transduction pathway.
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PMID:RANKL regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. 1450 May 43

We studied the mRNA expression of osteoprotegerin (OPG), receptor activator of NF-kappa B ligand (RANKL), tissue inhibitor of matrix metalloprotease (TIMP)-1 and -2, and matrix metalloprotease (MMP)-1 and -2 by human periodontal ligament (PDL) cells under intermittent tensile stress using a Flexercell Strain Unit. Analysis by reverse transcriptase-polymerase chain reaction showed that mechanical force upregulated OPG mRNA. We also demonstrated that the protein concentration of OPG in conditioned medium increased upon loading with tensile stress, as determined by enzyme-linked immunosorbent assay. TIMP-1 and -2 mRNA levels also increased, whereas levels of RANKL, MMP-1, and MMP-2 mRNA were barely affected. We further examined the effect of loading with tensile stress and addition of Salmonella abortus equi lipopolysaccharide (LPS) on the mRNA expression of PDL cells. The amount of OPG mRNA induced by mechanical strain was found to decrease with the addition of LPS to cultures. The induction of OPG mRNA expression by stretching was inhibited in the presence of indomethacin or genistein, whereas TIMP-1 mRNA expression induced by stretching was inhibited by the addition of cycloheximide, suggesting that tensile stress regulates cyclooxygenase activities, tyrosine phosphorylation, and de novo protein synthesis in PDL cells through the induction of OPG and TIMP-1 mRNA expression. These results provide evidence that the mechanical stimulus of stretching is responsible for the observed regulation of bone resorption and tissue degradation in PDL tissue.
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PMID:Periodontal ligament cells under intermittent tensile stress regulate mRNA expression of osteoprotegerin and tissue inhibitor of matrix metalloprotease-1 and -2. 1499 19

The present study was designed to evaluate the effects of neuropeptide substance P (SP) on the formation of osteoclasts via synovial fibroblastic cells. Synovial fibroblastic cells derived from rat knee joint expressed the SP receptor, neurokinin-1 receptor (NK(1)-R). The addition of SP stimulated the proliferation of synovial fibroblastic cells and this effect was inhibited by SP or NK(1)-R antagonists. Increased expression of the receptor activator of nuclear factor kappaB ligand (RANKL) in synovial fibroblastic cells after the addition of SP was demonstrated by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Osteoprotegerin expression in synovial fibroblastic cells was decreased after incubation with SP. In co-cultures of synovial fibroblastic cells and rat peripheral blood monocytes, SP stimulated osteoclastogenesis. These results suggest that SP in the joint cavity may cause both hypertrophy of the synovium and induction of increased osteoclast formation through the increased expression of RANKL in the synovium.
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PMID:Neuropeptide substance P stimulates the formation of osteoclasts via synovial fibroblastic cells. 1564 11

Giant cell tumor of bone (GCT) is a generally benign, osteolytic neoplasm comprising stromal cells and osteoclast-like giant cells. The osteoclastic cells, which cause bony destruction, are thought to be recruited from normal monocytic pre-osteoclasts by stromal cell expression of the ligand for receptor activator of nuclear factor kappaB (RANKL). This model forms the foundation for clinical trials in GCTs of novel cancer therapeutics targeting RANKL. Using expression profiling, we identified both osteoblast and osteoclast signatures within GCTs, including key regulators of osteoclast differentiation and function such as RANKL, a C-type lectin, osteoprotegerin, and the wnt inhibitor SFRP4. After ex vivo generation of stromal- and osteoclast-enriched cultures, we unexpectedly found that RANKL mRNA and protein were more highly expressed in osteoclasts than in stromal cells, as determined by expression profiling, flow cytometry, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. The expression patterns of molecules implicated in signaling between stromal cells and monocytic osteoclast precursors were analyzed in both primary and fractionated GCTs. Finally, using array-based comparative genomic hybridization, neither GCTs nor the derived stromal cells demonstrated significant genomic gains or losses. These data raise questions regarding the role of RANKL in GCTs that may be relevant to the development of molecularly targeted therapeutics for this disease.
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PMID:Molecular profiling of giant cell tumor of bone and the osteoclastic localization of ligand for receptor activator of nuclear factor kappaB. 1597 58

Human osteoblast cell line (MG63) cells were treated with long wave (45 kHz, intensity 30 mW/cm(2)) continuous ultrasound (US) for 5 min and incubated for various time periods following the treatment. The reverse transcriptase polymerase chain reaction (RT-PCR) technique was used for observing the genetic expression and real-time PCR for quantitative analysis of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) along with alkaline phosphatase (ALP), an early bone marker, and osteocalcin (OCN) a late marker. ELISA was performed to estimate the amount of the cytokine released into the culture media. The osteoblasts responded to US by significantly upregulating both the OPG mRNA and protein levels. There was no RANKL mRNA expression observed in both the US and control groups and the protein levels were also very low in both groups. There was also no TNF-alpha expression and the TNF-alpha protein levels were insignificant. ALP and OCN mRNA were significantly upregulated in the US group. To our knowledge, this is the first study that shows the effect of US on OPG, RANKL and TNF-alpha expression. US appears to upregulate OPG and may downregulate RANKL production. From these findings, we conclude that therapeutic ultrasound may increase bone regeneration by altering the OPG/RANKL ratio in the bone microenvironment.
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PMID:Long wave ultrasound may enhance bone regeneration by altering OPG/RANKL ratio in human osteoblast-like cells. 1656 38


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