UNIPROT:P05231 - FACTA Search

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Query: UNIPROT:P05231 (interleukin-6)
23,907 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A variety of studies have shown that osteoclasts originate from bone marrow, but their exact progenitors and differentiation pathway remain unclear. The treatment of mice with a high dose of 5-fluorouracil (5-FU) results in an enrichment for primitive hematopoietic progenitors; using this procedure, we prepared a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro. When spleen cells from mice pretreated in vivo with 5-FU were cultured in the presence of methylcellulose medium containing recombinant interleukin-3 (rIL-3), small colonies consisting of blast cells with little sign of differentiation developed on day 7 of culture. We lifted these blast colonies, pooled them, and replated them as secondary methylcellulose cultures in the presence of rIL-3 and erythropoietin. Approximately 60% of the cells formed colonies comprising various combinations of neutrophils, macrophages, eosinophils, mast cells, megakaryocytes, and erythroblasts. We replated such blast cells into microtiter wells and cultured them in the presence of rIL-3 (100 U/mL) or recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) (100 U/mL) plus 1.25(OH)2D3 (10(-7) mol/L). Multinucleated cells appeared from day 14 of culture and approximately 100 giant cells per well were scored on day 21 of culture. Parathyroid hormone (1 U/mL) also induced the multinucleated cell formation. May-Grunwald-Giemsa staining revealed the large cells containing many nuclei in their cytoplasm, which is characteristic of bone-resorbing cells or osteoclasts. These cells showed a tartrate-resistant acid phosphatase (TRAP) activity. Calcitonin caused a striking shape change in these cells and suppressed the formation of multinucleated cells. Moreover, electron microscopy shows that these cells were able to resorb fetal calvariae. In the presence of r granulocyte-colony stimulating factor, r macrophage-colony stimulating factor, or r interleukin-6 plus 1.25(OH)2D3, formation of TRAP-positive multinucleated cells was lower compared with the support of rIL-3 or rGM-CSF. Mature macrophages collected from colonies did not form the multinucleated cells as described above, even in the presence of rIL-3 and 1.25(OH)2D3. Moreover, to exclude the possibility that osteoclasts generated from non-blast cells, we performed a cloning experiment from one isolated blast cell and demonstrated that single cells differentiate into osteoclasts or macrophages in the presence of rIL-3 with or without 1.25(OH)2D3. This system will provide a useful model for further analysis of osteoclast formation in vitro.
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PMID:Generation of osteoclasts from isolated hematopoietic progenitor cells. 266 99

Parathyroid hormone and other agents that stimulate bone resorption function, at least in part, by inducing osteoblasts to secrete cytokines that stimulate osteoclast differentiation and activity. We previously demonstrated that parathyroid hormone induces expression by osteoblasts of interleukin-6 and leukemia inhibitory factor without affecting the 16 other cytokines that were examined. We also showed that stimulation of osteoclast activity by parathyroid hormone is dependent on activation of the cAMP signal transduction pathway and secretion of interleukin-6 by osteoblasts. In the current study, we demonstrate that the rapid and transient stimulation of interleukin-6 and leukemia inhibitory factor is inhibited by actinomycin D and superinduced by protein synthesis inhibitors, the classical characteristics of an immediate-early gene response. Moreover, activation of cAMP signal transduction by parathyroid hormone and parathyroid hormone-related protein is necessary and sufficient to induce both interleukin-6 and leukemia inhibitory factor. In addition, cAMP analogues as well as vasoactive intestinal peptide and isoproterenol, two neuropeptides that stimulate bone resorption by activating cAMP signal transduction in osteoblasts, also induce interleukin-6 and leukemia inhibitory factor in these cells. Taken together with our previous results, this study suggests that interleukin-6 is crucial for stimulation of bone resorption not only by parathyroid hormone, but also by parathyroid hormone-related protein, vasoactive intestinal peptide, and beta-adrenergic agonists, like isoproterenol.
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PMID:Stimulation by parathyroid hormone of interleukin-6 and leukemia inhibitory factor expression in osteoblasts is an immediate-early gene response induced by cAMP signal transduction. 863 18

Many osteoblastic cell lines are currently in use, but these have limitations either in terms of their relevance to adult human biology and disease or in terms of their suitability for biochemical and molecular analyses. Consequently, we undertook the development of conditionally transformed adult human osteoblastic cell lines. Osteoblasts were obtained from a normal explant cancellous bone chip culture. These cells were infected with adenovirus-ori-SV40 tsA 209, which encodes a temperature-sensitive large T-antigen mutant. Cells immortalized with this virus express a transformed phenotype at the permissive temperature of 34 degrees C but revert to a normal phenotype at the nonpermissive temperature of 40 degrees C. Using this approach, we have isolated several cell clones and describe the characterization of one that was designated HOB-02-C1. Immunocytochemistry revealed that > 95% of the cells express the large T-antigen at both temperatures. These cells exponentially proliferate at 34 degrees C with a doubling time of approximately 2 days but irreversibly stop dividing at 40 degrees C. However, cell volume increases > 2-fold when the cells are maintained for 6 days at the higher temperature. This clone expresses alpha 1 type (I) procollagen mRNA and secretes type I procollagen C-peptide at both temperatures, although the levels were slightly elevated at 40 degrees C. The cell line expresses alkaline phosphatase activity at 34 degrees C, and the basal level of this enzyme increases 2- to 6-fold at 40 degrees C. Alkaline phosphatase activity is induced 4- to 8-fold by 1 alpha,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures, but transforming growth factor-beta 1 (TGF-beta 1) suppresses enzyme expression > 90% at 40 degrees C. Vitamin D3 also induces a 10-fold increase in osteocalcin secretion when the clone is maintained at 34 degrees C, and this induction is enhanced > 8-fold at 40 degrees C. Parathyroid hormone and forskolin stimulate a 4- to 6-fold increase in the production of intracellular cyclic AMP (cAMP) by the cells at 34 degrees C, and this stimulation is enhanced 2- to 4-fold at 40 degrees C. In contrast, prostaglandin E2 stimulates a 7- to 8-fold increase in cAMP only when the cells are maintained at 34 degrees C. This cell line secretes TGF-beta 1 and interleukin-6 (IL-6) at 34 degrees C, but only the basal secretion of IL-6 increases 70% at 40 degrees C. Finally, alizarin red-S histochemical staining demonstrates that these cells produce mineralized nodules at both temperatures. In summary, the results of this study indicate that the HOB-02-C1 cells have a mature osteoblastic phenotype. Consequently, this new cell line and others obtained in a similar fashion should be valuable in vitro tools for cellular, biochemical, and molecular studies of adult human osteoblast biology.
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PMID:Development and characterization of a conditionally transformed adult human osteoblastic cell line. 872 78

Parathyroid hormone (PTH) functions in part by regulating osteoblast cytokine expression. We recently demonstrated that PTH induced a rapid and transient increase in interleukin-6 (IL-6) mRNA expression in rat bones in vivo. To determine the molecular basis of this effect, we analyzed the human IL-6 promoter fused (-1,179 to +9) with the chloramphenicol acetyltransferase (CAT) reporter gene in stable transfections into human osteoblast-like osteosarcoma SaOS-2 cells. We compared the effects of PTH on IL-6 expression with adenylate cyclase activator forskolin, PKC activator phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, interleukin-1 alpha (IL-1 alpha), prostaglandin E-2 (PGE-2), RS-66271 (a parathyroid hormone-related peptide analog), and platelet-derived growth factor-BB (PDGF-BB). Analyses of cell clones showed that IL-6 promoter expression was extremely low in the unstimulated state. Exposure to PTH (0.001-100 nM) for 12 h stimulated CAT expression in a dose-dependent manner (200-500% of control). Treatment with IL-1 alpha was more potent than PTH in inducing transcription of the IL-6 promoter (900-1,000%). Activation of the cAMP-PKA pathway by treatment with forskolin induced a comparable level of induction with PTH. Together, the effects of PTH and forskolin were additive. RS-66271, previously shown to have PTH-like effects, induced a comparable level of IL-6 promoter expression. When examined together, PTH+RS-66271 effects were comparable to PTH effects alone. Exposure to PGE-2, PMA, PDGF-BB, or A23187 for 12 h did not significantly alter IL-6 promoter expression. These results demonstrate PTH, forskolin, the PTHrP analog RS-66271, and IL-1 alpha stimulate IL-6 expression by stimulating gene transcription. The response to forskolin suggests that the messenger system mediated by PKA is sufficient to induce IL-6 expression.
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PMID:Parathyroid hormone (1-34)-mediated interleukin-6 induction. 932 32

Parathyroid hormone (PTH) exerts its regulatory effects on calcium homeostasis in part by stimulating the release of calcium from the skeleton. PTH stimulates bone resorption indirectly, by inducing the production by stromal/osteoblastic cells of paracrine agents which recruit and activate the bone-resorbing cell, the osteoclast. The identity of the stromal cell/osteoblast-derived paracrine factor(s) responsible for mediating the effects of PTH on osteoclasts is uncertain. Recently, it has been demonstrated that the cytokine interleukin-6 (IL-6), which potently induces osteoclastogenesis, is produced by osteoblastic cells in response to PTH. Further, we have reported that circulating levels of IL-6 are elevated in patients with primary hyperparathyroidism, and correlate with biochemical markers of bone resorption. Thus, IL-6 may play a permissive role in PTH-induced bone resorption. In the current studies, we demonstrate that low-dose PTH infusion in rodents increased serum levels of IL-6, coincident with a rise in biochemical markers of bone resorption. In mice, both acute neutralization and chronic deficiency of IL-6 were associated with markedly lower levels of biochemical markers of bone resorption in response to PTH infusion than were observed in animals with normal IL-6 production. Acute neutralization of IL-6 did not affect PTH-induced changes in markers of bone formation. These findings demonstrate that PTH regulates systemic levels of IL-6 in experimental animals, that IL-6 is an important mediator of the bone-resorbing actions of PTH in vivo and suggest that IL-6 plays a role in coupling PTH-induced bone resorption and formation.
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PMID:A role for interleukin-6 in parathyroid hormone-induced bone resorption in vivo. 1049 26

We examined the effect of parathyroid hormone and various signaling molecules on collagen synthesis and chloramphenicol acetyltransferase activity in cultured transgenic mouse calvariae carrying fusion genes of the rat Col1a1 promoter and the chloramphenicol acetyltransferase reporter. After 48 h of culture, parathyroid hormone, forskolin, dibutyryl cAMP, 8-bromo cAMP, and phorbol myristate acetate inhibited transgene activity, while the calcium ionophore ionomycin had no effect. Pretreatment of calvariae with the phosphodiesterase inhibitor isobutylmethylxanthine potentiated the inhibitory effect of 1 nM parathyroid hormone on transgene activity and collagen synthesis. Parathyroid hormone further inhibited transgene activity and collagen synthesis in the presence of phorbol myristate acetate. Parathyroid hormone inhibition of transgene activity and collagen synthesis was not affected by indomethacin or interleukin-6. After 48 h of culture, parathyroid hormone inhibited chloramphenicol acetyltransferase activity by 50-85% in cultured calvariae carrying transgenes having progressive 5' upstream deletions of promoter DNA down to -1683 bp. These data show that the inhibitory effect of parathyroid hormone on Col1a1 expression in mouse calvariae is mediated mainly by the cAMP signaling pathway. Prostaglandins and IL-6 are not local mediators of the parathyroid hormone response in this model. Finally, regions of the Col1a1 promoter downstream of -1683 bp are sufficient for parathyroid hormone inhibition of the Col1a1 promoter.
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PMID:Parathyroid hormone inhibits collagen synthesis and the activity of rat col1a1 transgenes mainly by a cAMP-mediated pathway in mouse calvariae. 1067 25

To investigate the level at which protein kinase C (PKC) regulates expression of interleukin-6 (IL-6) in osteoblastic cells, effects of several PKC antagonists and PKC down-regulation by phorbol ester were studied in UMR-106 osteoblastic cells that had been transiently transfected with a -224/+11-base pair (bp) IL-6 promoter coupled to a luciferase reporter. Parathyroid hormone (PTH) elicited a dose-dependent stimulation of the IL-6 promoter expression, with significant increases produced by 5 h of treatment with concentrations of PTH as low as 10(-14) M. The increase in IL-6 promoter expression was inhibited by the PKC antagonists GF109203X, 30 nM to 1 microM, and calphostin C, 250 nM. Prior down-regulation of PKC with 100 nM phorbol-12,13-dibutyrate (PDBU) for 48 h inhibited the PTH effect as well as the smaller stimulatory effects elicited by tumor necrosis factor alpha (TNF-alpha), 10(-9)-10(-8) M, and by IL-1beta, 1-10 ng/ml. In contrast to these findings, the stimulatory effects of PTH, TNF-alpha, and IL-1beta on the IL-6 promoter expression were enhanced by staurosporine. Treatment with GF109203X or down-regulation of PKC with PDBU prevented the stimulatory effects of staurosporine. PKC activity was increased by staurosporine. The findings with staurosporine are consistent with our earlier observations that this agent enhances the calcium signaling and bone resorption elicited by PTH. The studies support the role of PKC in the stimulatory effects of PTH, TNF-alpha, and IL-1beta on IL-6 expression.
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PMID:Stimulation of interleukin-6 promoter by parathyroid hormone, tumor necrosis factor alpha, and interleukin-1beta in UMR-106 osteoblastic cells is inhibited by protein kinase C antagonists. 1145 Jun 97

Recent studies have shown that stimulation of osteoclastogenesis in cocultures of osteoblasts and spleen cells in response to prostaglandin E2 (PGE2) is markedly decreased when the osteoblasts are derived from cells lacking either the EP2 or the EP4 receptor. Induction of osteoclast formation requires upregulation of receptor activator of nuclear factor-kappaB ligand (RANKL) on cells of the osteoblastic lineage, which then binds to the RANK receptor on cells of the osteoclast lineage. Osteoprotegerin (OPG) is a decoy receptor for RANKL that can block its interaction with RANK. In addition, macrophage-colony stimulating factor (M-CSF) is essential for osteoclast formation. Finally, PGE2 can increase interleukin-6 (IL-6), which may further enhance osteoclastogenesis. To study the relative influence of the EP2 and EP4 receptors on response of these factors to PGE2, we examined mRNA levels for RANKL, OPG, M-CSF, and IL-6 in primary osteoblastic cell cultures derived from two lines of EP2 knockout mice (EP2-/-) and one line of EP4 knockout mice (EP4-/-) and the relevant wild-type controls (EP2+/+ and EP4+/+). The responses of cells from wild-type animals of all three lines were similar. After PGE2 treatment, RANKL mRNA levels were increased at 2 h, and this was sustained over 72 h. Basal RANKL expression was moderately reduced in EP2-/- cells and markedly reduced in EP4-/- cells. PGE2 increased RANKL mRNA in EP2-/- cells and EP4-/- cells, but the levels were significantly reduced compared with wild-type cells. There were no consistent changes in expression of M-CSF or OPG in the different genotypes or with PGE2 treatment. IL-6 mRNA was variably increased by PGE2 in both wild-type and knockout cells, although the absolute levels were somewhat lower in both EP2-/- and EP4 -/- cultures. Parathyroid hormone (PTH) increased RANKL and IL-6 and decreased OPG mRNA levels similarly in both wild-type and EP2-/- or EP4-/- cells. The major defect in the response to PGE2 in animals lacking either EP2 or EP4 receptors is a reduction in basal and stimulated RANKL levels. Loss of EP4 receptor appears to have a greater effect on basal RANKL expression than EP2.
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PMID:Effects of prostaglandin E2 on gene expression in primary osteoblastic cells from prostaglandin receptor knockout mice. 1193 47

Parathyroid hormone (PTH)-related protein (PTHrP) seems to affect bone resorption by interaction with bone cytokines, among them interleukin-6 (IL-6). Recent studies suggest that nuclear factor (NF)-kappaB activation has an important role in bone resorption. We assessed whether the N-terminal fragment of PTHrP, and its C-terminal region, unrelated to PTH, can activate NF-kappaB, and its relationship with IL-6 gene induction in different rat and human osteoblastic cell preparations. Here we present molecular data demonstrating that both PTHrP (1-36) and PTHrP (107-139) activate NF-kappaB, leading to an increase in IL-6 mRNA, in these cells. Using anti-p65 and anti-p50 antibodies, we detected the presence of both proteins in the activated NF-kappaB complex. This effect induced by either the N- or C-terminal PTHrP domain in osteoblastic cells appears to occur by different intracellular mechanisms, involving protein kinase A or intracellular Ca(2+)/protein kinase C activation, respectively. However, the effect of each peptide alone did not increase further when added together. Our findings lend support to the hypothesis that the C-terminal domain of PTHrP, in a manner similar to its N-terminal fragment, might stimulate bone resorption. These studies also provide further insights into the putative role of PTHrP as a modulator of bone remodeling.
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PMID:Both N- and C-terminal domains of parathyroid hormone-related protein increase interleukin-6 by nuclear factor-kappa B activation in osteoblastic cells. 1200 Jul 45

The underlying mechanisms responsible for both cartilage loss and subchondral bone changes in osteoarthritis (OA) remain unknown. It is becoming recognized that the extracellular matrix influences the metabolism of cells both in vivo and in vitro and can modify their responses to external stimuli. Indeed, the glycosaminoglycan/proteoglycan matrix is of major importance for the proliferation and/or differentiation of a number of cells. Here, we determined the potential role of hyaluronic acid (HA) of increasing molecular weight (MW) to alter the expression of metabolic markers and cytokine production by human osteoarthritic (OA) subchondral osteoblasts (Ob). Both 1,25(OH)(2)D(3)-induced alkaline phosphatase activity (ALPase) and osteocalcin release were increased in OA Ob when compared to normal. HA reduced osteocalcin release in OA Ob at MW of 300 and above, whereas HA failed to significantly modify ALPase. Parathyroid hormone (PTH) stimulated cyclic AMP (cAMP) formation by OA Ob. HA had a biphasic effect on this PTH-dependent activity, totally inhibiting cAMP formation at MW of 300 and 800. HA of increasing MW progressively reduced the levels of Prostaglandin E(2) (PGE(2)) and interleukin-6 (IL-6) produced by OA Ob. Interestingly, urokinase plasminogen activator (uPA) and and PA inhibitor-1 (PAI-1) levels were not significantly affected by HA of increasing MW; however, the PAI-1 to uPA ratio showed a slight, yet nonsignificant increase. Surprisingly, uPA activity was increased in OA Ob under the same conditions. Last, HA had no effect on the production of insulin-like growth factor-1 by these cells. Our data suggest that high MW HA can modify cellular parameters in OA Ob that are increased when compared to normal. The effect of HA on inflammatory mediators, such as PGE(2) and IL-6, and on uPA activity is more striking at higher MW as well. Taken together, these results could suggest that HA of increasing MW has positive effects on OA Ob by modifying their biological synthetic capacities.
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PMID:Hyaluronic acid reverses the abnormal synthetic activity of human osteoarthritic subchondral bone osteoblasts. 1455 76

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