RESUMO
Sexual selection and the ornaments that inform such choices have been extensively studied, particularly from a phenotypic perspective. Although more is being revealed about the genetic architecture of sexual ornaments, much still remains to be discovered. The comb of the chicken is one of the most widely recognized sexual ornaments, which has been shown to be correlated with both fecundity and bone allocation. In this study, we use a combination of multiple intercrosses between White Leghorn populations and wild-derived Red Junglefowl to, first, map quantitative trait loci (QTL) for bone allocation and, second, to identify expression QTL that correlate and colocalize with comb mass. These candidate quantitative genes were then assessed for potential pleiotropic effects on bone tissue and fecundity traits. We identify genes that correlate with both relative comb mass and bone traits suggesting a combination of both pleiotropy and linkage mediates gene regulatory variation in these traits.
Assuntos
Osso e Ossos/anatomia & histologia , Galinhas/anatomia & histologia , Galinhas/genética , Crista e Barbelas/anatomia & histologia , Ligação Genética , Pleiotropia Genética , Locos de Características Quantitativas , Animais , Mapeamento Cromossômico , Cruzamentos Genéticos , Feminino , Fertilidade/genética , Masculino , FenótipoRESUMO
SUMMARY: We evaluated the relation between serum FGF23 and bone mineral density (BMD) in a community-based cohort of elderly men. There was a weak correlation between FGF23 and BMD, which was primarily dependent on body weight. INTRODUCTION: FGF23 is a hormonal factor produced in bone and regulates serum levels of phosphate (Pi) and vitamin D. FGF23 over-expression is associated with skeletal abnormalities, including rickets/osteomalacia. The relation between FGF23 and Bone Mineral Density (BMD) in the community remains unexplored. METHODS: We employed a large, population-based cohort of 3014 Swedish men aged 69-80 years, without known renal disease. BMD was measured with dual X-ray absorptiometry (DXA) in the hip and lumbar spine. Serum intact FGF23 was analyzed with a two-site monoclonal ELISA. RESULTS: There was a weak but significant correlation between FGF23 and BMD in femoral neck (r = 0.04, p < 0.05), femoral trochanter (r = 0.05, p = 0.004), total hip (r = 0.06, p = 0.0015) and lumbar spine (r = 0.07, p = 0.0004). The correlations remained significant when adjusting for biochemical covariates (Pi, calcium, PTH, 25(OH)D and renal function). However, the association became insignificant in all regions when adjusting for established confounding variables including age, height, weight and smoking. Further analysis confirmed a significant correlation between FGF23 and body weight (r = 0.13, p < 0.0001). CONCLUSIONS: The weak correlation between FGF23 and BMD in elderly male subjects is mainly due to an association between FGF23 and body weight. Therefore, FGF23 may not play a significant role in the hormonal regulation of BMD.
Assuntos
Peso Corporal/fisiologia , Densidade Óssea/fisiologia , Fatores de Crescimento de Fibroblastos/sangue , Absorciometria de Fóton , Idoso , Idoso de 80 Anos ou mais , Fêmur/diagnóstico por imagem , Colo do Fêmur/diagnóstico por imagem , Fator de Crescimento de Fibroblastos 23 , Humanos , Vértebras Lombares/diagnóstico por imagem , Masculino , Estudos Prospectivos , SuéciaRESUMO
The tuberoinfundibular peptide TIP39 [TIP-(1-39)], which exhibits only limited amino acid sequence homology with PTH and PTH-related peptide (PTHrP), stimulates cAMP accumulation in cells expressing the PTH2 receptor (PTH2R), but it is inactive at the PTH/PTHrP receptor (PTH1R). However, when using either (125)I-labeled rat [Nle(8,21),Tyr(34)]PTH-(1-34)amide (rPTH) or (125)I-labeled human [Tyr(36)]PTHrP-(1-36)amide [PTHrP-(1-36)] for radioreceptor studies, TIP-(1-39) bound to LLCPK(1) cells stably expressing the PTH1R (HKrk-B7 cells), albeit with weak apparent affinity (243 +/- 52 and 210 +/- 64 nM, respectively). In comparison to the parent peptide, the apparent binding affinity of TIP-(3-39) was about 3-fold higher, and that of TIP-(9-39) was about 5.5-fold higher. However, despite their improved IC(50) values at the PTH1R, both truncated peptides failed to stimulate cAMP accumulation in HKrk-B7 cells. In contrast, the chimeric peptide PTHrP-(1-20)/TIP-(23-39) bound to HKrk-B7 cells with affinities of 31 +/- 8.2 and 11 +/- 4.0 nM when using radiolabeled rPTH and PTHrP-(1-36), respectively, and it stimulated cAMP accumulation in HKrk-B7 and SaOS-2 cells with potencies (EC(50), 1.40 +/- 0.3 and 0.38 +/- 0.12 nM, respectively) and efficacies (maximum levels, 39 +/- 8 and 31 +/- 3 pmol/well, respectively) similar to those of PTH-(1-34) and PTHrP-(1-36). In both cell lines, TIP(9-39) and, to a lesser extent, TIP-(1-39) inhibited the actions of the three agonists with efficiencies similar to those of [Leu(11),D-Trp(12),Trp(23),Tyr(36)]PTHrP-(7-36)amide, an established PTH1R antagonist. Taken together, the currently available data suggest that the carboxyl-terminal portion of TIP-(1-39) interacts efficiently with the PTH1R, at sites identical to or closely overlapping those used by PTH-(1-34) and PTHrP-(1-36). The amino-terminal residues of TIP-(1-39), however, are unable to interact productively with the PTH1R, thus enabling TIP-(1-39) and some of its truncated analogs to function as an antagonist at this receptor.
Assuntos
Neuropeptídeos/fisiologia , Receptores de Hormônios Paratireóideos/metabolismo , Sequência de Aminoácidos/genética , Animais , Ligação Competitiva , Linhagem Celular , Quimera , AMP Cíclico/metabolismo , Células LLC-PK1 , Dados de Sequência Molecular , Mutação/fisiologia , Neuropeptídeos/química , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Receptor Tipo 1 de Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos/antagonistas & inibidores , Receptores de Hormônios Paratireóideos/química , Receptores de Hormônios Paratireóideos/genética , SuínosRESUMO
Interleukin-13 (IL-13) is a recently identified cytokine that is secreted by activated T cells and regulates inflammatory responses. We have investigated the effects of IL-13 on isolated human osteoblast-like cells (hOB). IL-13 dose-dependently (1-100 pmol/L) reduced the incorporation rate of [3H]thymidine in hOB cells by more than 50%. Using a cell metabolic assay as well as direct cell counting, we found that treatment with IL-13 lead to a decrease in hOB cell number. The effect was both time and dose dependent, and after 12 days of culture, treatment with IL-13 (0.1 nmol/L) caused a 70% decrease in the number of cells. Also, IL-13 increased the levels of IL-6 messenger ribonucleic acid in hOBs, as measured by ribonuclease protection assay, and stimulated secretion of IL-6 into culture supernatants. In conclusion, IL-13 inhibits cell proliferation and increases IL-6 formation in human osteoblasts. Our findings suggest that IL-13 may cause bone loss due to impaired osteoblastic growth and IL-6-induced osteoclast recruitment.
Assuntos
Divisão Celular , Interleucina-13/farmacologia , Interleucina-6/biossíntese , Osteoblastos/citologia , Osteoblastos/metabolismo , DNA/biossíntese , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Cinética , Osteossarcoma , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
The gene mutated in autosomal dominant hypophosphatemic rickets (ADHR), a phosphate wasting disorder, has been identified as FGF-23, a protein that shares sequence homology with fibroblast growth factors (FGFs). Patients with ADHR display many of the clinical and laboratory characteristics that are observed in patients with oncogenic hypophosphatemic osteomalacia (OHO), a disorder thought to arise by the secretion of a phosphate wasting factor from different mesenchymal tumors. In the present studies, we therefore investigated whether FGF-23 is a secreted factor and whether it is abundantly expressed in OHO tumors. After transient transfection of OK-E, COS-7, and HEK293 cells with the plasmid encoding full-length FGF-23, all three cell lines efficiently secreted two protein species into the medium that were approximately 32 and 12 kDa upon SDS-PAGE and subsequent Western blot analysis using an affinity-purified polyclonal antibody to FGF-23. Furthermore, Northern blot analysis using total RNA from five different OHO tumors revealed extremely high levels of FGF-23 mRNA, and Western blot analysis of extracts from a sixth tumor detected the 32 kDa FGF-23 protein species. In summary, FGF-23, the gene mutated in ADHR, is a secreted protein and its mRNA is abundantly expressed by several different OHO tumors. Our findings indicate that FGF-23 may be a candidate phosphate wasting factor, previously designated "phosphatonin".
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Hipofosfatemia Familiar/genética , Mesenquimoma/fisiopatologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Fator de Crescimento de Fibroblastos 23 , Humanos , Hipofosfatemia Familiar/complicações , Hipofosfatemia Familiar/fisiopatologia , Mesenquimoma/complicações , Dados de Sequência Molecular , Osteomalacia/fisiopatologia , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Transcrição Gênica , TransfecçãoRESUMO
Interleukin-13 (IL-13) inhibits cell proliferation and stimulates interleukin-6 (IL-6) formation in isolated human osteoblasts (hOBs). Because the related cytokine, interleukin-4 (IL-4), is known to exert effects similar to IL-13 in other tissues, and because IL-4 has been implicated as a regulator of bone metabolism, we compared the effects of IL-13 and IL-4 on cell proliferation, IL-6 synthesis, the expression of osteoblastic phenotypic markers in hOB cultures. Also, the receptor proteins mediating these effects in hOBs have been partly characterized. IL-4 and IL-13 dose-dependently inhibited [(3)H]-thymidine incorporation into the DNA of human osteoblasts and stimulated secretion of IL-6 into culture supernatants. IL-13 and IL-4 also increased the mRNA levels of IL-6, as measured by RNAse protection assay. Furthermore, IL-13 and IL-4 dose-dependently enhanced alkaline phosphatase (ALP) activity, but did not affect osteocalcin or collagen type I synthesis. IL-4 was tenfold more potent than IL-13 in inducing both ALP activity and IL-6 secretion, whereas the cytokines were equipotent as inhibitors of cell proliferation. The expression of mRNA for receptor subunits previously implicated in IL-4 and IL-13 signaling was investigated by reverse transcriptase-polymerase chain reaction. IL-13R, IL-13Ralpha, and IL-4Ralpha mRNA were repeatedly detected in hOBs, whereas mRNA for IL-2Rgamma(C) was not detected. Receptor-blocking antibodies to IL-4Ralpha inhibited the induction of IL-6 formation by both IL-4 and IL-13, indicating that both cytokines utilize this receptor subunit in signaling. However, the antibodies did not affect the IL-4/-13-induced inhibition of [(3)H]-thymidine incorporation or the stimulation of alkaline phosphatase (ALP), suggesting that IL-4Ralpha does not mediate these effects of IL-4/-13 in hOBs. We conclude that the cytokines IL-13 and IL-4, through sharing of receptor components, induce similar effects on hOBs, causing inhibition of cell proliferation, stimulation of IL-6, and enhanced ALP activity.
Assuntos
Divisão Celular/efeitos dos fármacos , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Osteoblastos/metabolismo , Anticorpos Monoclonais/imunologia , Sequência de Bases , Divisão Celular/fisiologia , Primers do DNA , Humanos , Subunidade alfa1 de Receptor de Interleucina-13 , Interleucina-6/metabolismo , Osteoblastos/citologia , Fenótipo , Receptores de Interleucina/fisiologia , Receptores de Interleucina-13 , Receptores de Interleucina-4/antagonistas & inibidores , Receptores de Interleucina-4/fisiologia , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidina/metabolismoRESUMO
Oncogenic osteomalacia is a rare paraneoplastic syndrome that is characterized biochemically by hypophosphatemia and low plasma 1,25-dihydroxyvitamin D3, and clinically by osteomalacia, pseudofractures, bone pain, fatigue, and muscle weakness. We present a patient with a malignant schwannoma as the underlying cause of this disorder. A permanent cell line (HMS-97) derived from this tumor showed evidence of neuroendocrine differentiation by immunohistochemistry and of neurosecretory activity by electron microscopy. The cell line did express PHEX (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome) and FGF-23 (fibroblast growth factor-23) transcripts on northern hybridization; however, none of the known mutations from the related mendelian disorders of X-linked hypophosphatemic rickets or autosomal-dominant hypophosphatemic rickets could be detected. Tumor cell (HMS-97)-derived conditioned medium did not inhibit phosphate transport in a standard opossum kidney cell assay and in animal experiments. The medium also showed no PTH1- or PTH2-receptor-stimulating bioactivity. HMS-97 cells might be useful for further studies that aim to determine the genetic mechanism that leads to the observed PHEX and FGF-23 expression, both of which might have a direct role in the pathogenesis of oncogenic osteomalacia. In addition, these cells might be a useful tool for the investigation of neuroendocrine Schwann cell function and autoimmune peripheral nerve disease.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Neurilemoma/complicações , Tumores Neuroendócrinos/complicações , Osteomalacia/etiologia , Proteínas/genética , Feminino , Fator de Crescimento de Fibroblastos 23 , Regulação Neoplásica da Expressão Gênica , Humanos , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Neurilemoma/diagnóstico por imagem , Neurilemoma/patologia , Tumores Neuroendócrinos/diagnóstico por imagem , Tumores Neuroendócrinos/patologia , Osteomalacia/diagnóstico por imagem , Endopeptidase Neutra Reguladora de Fosfato PHEX , RNA Mensageiro/análise , Cintilografia , Células Tumorais CultivadasRESUMO
Osteoprotegerin (OPG) is a recently cloned member of the tumour necrosis factor receptor family. It has been suggested that this secreted glycoprotein acts as an inhibitor of osteoclastic differentiation. Expression of OPG has previously been demonstrated in a number of tissues. However, it is still unclear whether or not OPG is expressed by human osteoblasts. We have used the RNase protection assay to demonstrate the OPG transcript in primary cultured human osteoblast-like cells, human marrow stroma cells and osteosarcoma cell lines. Furthermore, we have studied the effect of glucocorticoids on OPG mRNA levels in these cells. We demonstrate that glucocorticoids decrease the OPG transcript in a dose- and time-dependent manner. The time-course study reveals that hydrocortisone (10(-6) M) decreases OPG mRNA levels within 2 h. This decrease is transient, reaching control levels again after 24 h. Our findings demonstrate that human osteoblasts express the mRNA corresponding to OPG, an inhibitor of osteoclast differentiation. The finding that OPG mRNA levels are decreased by glucocorticoids indicates that a reduced production of OPG from osteoblasts and/or marrow stroma cells could, in part, explain glucocorticoid-induced bone resorption.
Assuntos
Anti-Inflamatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicoproteínas/metabolismo , Hidrocortisona/farmacologia , Osteoblastos/metabolismo , Receptores Citoplasmáticos e Nucleares , Células Cultivadas , Densitometria , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Técnicas Genéticas , Glicoproteínas/genética , Humanos , Osteoprotegerina , Receptores do Fator de Necrose Tumoral , Estimulação Química , Testosterona/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
In oncogenic osteomalacia (OOM), a tumor produces an unknown substance that inhibits phosphate reabsorption in the proximal tubules. This causes urinary phosphate wasting and, as a consequence, hypophosphatemic osteomalacia. To characterize this poorly understood biological tumor activity we generated aqueous extracts from several OOM tumors. Extracts from three of four tumors inhibited, dose- and time-dependently, (32)P-orthophosphate uptake by opossum kidney (OK) cells; maximum inhibition was about 45% of untreated control. Further characterization revealed that the factor is resistant to heat and several proteases, and that it has a low molecular weight. The tumor extracts also stimulated cAMP accumulation in OK cells, but not in osteoblastic ROS 17/2.8 and UMR106 cells, or in LLC-PK1 kidney cells expressing the parathyroid hormone (PTH)/PTH-related peptide receptor or the PTH-2 receptor. HPLC separation of low molecular weight fractions of the tumor extracts revealed that the flow-through of all three positive tumor extracts inhibited (32)P uptake and stimulated cAMP accumulation in OK cells. Additionally, a second peak with inhibitory activity on phosphate transport, but without cAMP stimulatory activity, was identified in the most potent tumor extract. We have concluded that several low molecular weight molecules with the ability to inhibit phosphate transport in OK cells can be found in extracts from OOM tumors. It remains uncertain, however, whether these are related to the long-sought phosphaturic factor responsible for the phosphate wasting seen in OOM patients.
Assuntos
Rim/metabolismo , Neoplasias/metabolismo , Osteomalacia/metabolismo , Fosfatos/metabolismo , Adulto , Animais , Cromatografia Líquida de Alta Pressão , AMP Cíclico/metabolismo , Feminino , Temperatura Alta , Humanos , Masculino , Pessoa de Meia-Idade , Peso Molecular , Neoplasias/complicações , Gambás/metabolismo , Osteomalacia/etiologia , Síndromes Paraneoplásicas/etiologia , Síndromes Paraneoplásicas/metabolismo , Extratos de Tecidos/química , Extratos de Tecidos/farmacologia , Células Tumorais CultivadasRESUMO
We have studied the effect of insulin-like growth factor I (IGF-I) on the formation of osteocalcin and type I collagen in isolated human osteoblasts. IGF-I at and above 0.1 nM stimulated the formation of type I collagen as measured by the type I procollagen carboxyterminal peptide (PICP), in human osteoblasts, incubated for 72 hrs in serum free conditions. The secretion of osteocalcin was not affected by IGF-I while 1,25(OH)2vitamin D3 significantly enhanced the formation of osteocalcin. When human osteoblast-like cells were incubated with hydrocortisone (1 microM), a significant decrease in the release of both PICP and osteocalcin was seen. Addition of IGF-I to human osteoblasts also treated with hydrocortisone normalized the PICP-formation but did not affect the suppressed osteocalcin-formation. These data indicate that IGF-I reverses selective effects of hydrocortisone on bone.
Assuntos
Hidrocortisona/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Osteoblastos/metabolismo , Pró-Colágeno/biossíntese , Calcitriol/farmacologia , Células Cultivadas , Sinergismo Farmacológico , Humanos , Osteoblastos/efeitos dos fármacosRESUMO
Over the last decade, the regulation of phosphate (Pi) homeostasis has been under intense investigation. By utilizing modern biochemical and genetic tools, the pathophysiological mechanisms behind several known hereditary and acquired hypo- and hyperphosphatemic diseases have been clarified. The results of these efforts have opened new insights into the causes of Pi dysregulation and hereby also the physiological mechanisms determining Pi homeostasis. Although several potential Pi-regulating proteins have been discovered and investigated, current data strongly argues for fibroblast growth factor-23 (FGF23), a hormonal factor produced in bone, as a particularly important regulator of Pi homeostasis. In this article, we review the discovery of the FGF23 protein, as well as its biochemistry, localization of production, receptor specificity and mechanisms of action.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Homeostase/fisiologia , Fosfatos/metabolismo , Animais , Fator de Crescimento de Fibroblastos 23 , Humanos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismoRESUMO
Insulin-like growth factor I (IGF-I) has documented anabolic effects on osteoblasts, whereas its influence on osteoclasts and on bone resorption is unclear. We have investigated the effects of IGF-I on osteoclast recruitment and bone resorption in vitro. IGF-I (at and above 1 nM) stimulated the formation of multinucleated tartrate-resistant acid phosphatase positive cells in murine bone marrow cultures, incubated for 9 days. The number of multinucleated cells increased to 540 +/- 160% of control (mean +/- SEM) in cultures treated with 10 nM IGF-I. IGF-I (0.1-100 nM) had no effect by itself on 45Ca-release from prelabelled neonatal mouse calvarial bones. However, IGF-I (100 nM) had an inhibitory effect on bone resorption induced by prostaglandin E2 and 1,25(OH)2D3. These findings indicate that IGF-I enhances the formation of osteoclasts-like cells in long-term bone marrow cultures. In bone organ cultures, however, IGF-I has an inhibitory effect on stimulated bone resorption, suggesting that IGF-I inhibits existing osteoclasts and, alternatively, that IGF-I interferes with the osteoblast-derived factor(s) that stimulate existing osteoclasts.
Assuntos
Reabsorção Óssea , Fator de Crescimento Insulin-Like I/farmacologia , Osteoclastos/patologia , Crânio/patologia , Animais , Contagem de Células/efeitos dos fármacos , Células Cultivadas , Células Gigantes/patologia , Camundongos , Osteoclastos/efeitos dos fármacosRESUMO
We investigated the effects of various pro-inflammatory cytokines on the proliferation rate of isolated human osteoblastic cells in primary cultures. Interleukin-1 beta (IL-1 beta) and tumor necrosis factor-beta (TNF-beta) time- and dose-dependently enhanced the proliferation of human osteoblasts. Both of these cytokines also enhanced endogenous prostaglandin E2 (PGE2) formation. Exogenous PGE2 dose- and time-dependently-stimulated cell proliferation. However, the stimulatory effects of IL-1 beta and TNF-beta on osteoblast proliferation were not abolished by indomethacin, indicating a direct effect by these cytokines on the rate of proliferation. TNF-alpha stimulated proliferation at low doses, while it significantly inhibited proliferation at higher concentrations (at and above 100 pM) and with prolonged incubation times. This biphasic effect was unaffected by indomethacin. Interleukin-6, finally, did not affect the rate of proliferation. Our findings show that inflammatory cytokines may stimulate or inhibit the proliferation of isolated human osteoblasts, depending on concentration and time.
Assuntos
Interleucina-1/fisiologia , Interleucina-6/fisiologia , Linfotoxina-alfa/fisiologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Divisão Celular , Células Cultivadas , Dinoprostona/biossíntese , Relação Dose-Resposta a Droga , Fluorometria , Consolidação da Fratura , Humanos , Fatores de TempoRESUMO
A novel fluorometric proliferation assay, AlamarBlue (AB), was used to study the proliferative capacity of isolated human osteoblasts (hOBs). AB is an oxidation-reduction indicator that yields a fluorescent signal in response to metabolic activity. The assay was performed by replacing the experiment media in a microtiter plate with a 10% AB solution and measuring fluorescence after a 3-8-hour incubation. The assay was optimized with respect to incubation time, cell density, and AB concentration. When the results of the AB assay were compared with cell counting in a Bürker chamber there were consistently good correlations (r > 0.9), regardless of the agonist with which the cells were treated. The mean intraassay coefficient of variance (CV) values were 9.9-11.8% in experiments where osteoblasts were treated for 12 days with insulin-like growth factor-I (IGF-I; 100 nM), or dexamethasone (1 micro;M). IGF-I dose dependently, at and above 1 nM, stimulated proliferation of hOBs. This effect was detectable after 3 days and reached 130-140% of untreated controls after 12 days in culture. The effects of dexamethasone (DEX) on the proliferation rate of hOBs were more complex. In short-term cultures, 3 days, DEX dose dependently stimulated proliferation. However, at and above 6 days, DEX exerted a biphasic effect, with stimulation seen at 1-10 nM and a marked inhibition of cell proliferation at and above 100 nM. dexamethasone, hydrocortisone, prednisolone, and deflazacort had almost identical biphasic effects on osteoblastic proliferation in 12 day cultures with a stimulation seen at 1-10 nM, and a marked inhibition down to 50-60% of untreated controls at and above 100 nM. When IGF-I (0. 1-100 nM; 12 day culture) was combined with different doses of DEX, IGF-I still dose dependently stimulated the proliferation rate in hOBs regardless of the amount of DEX added. The stimulatory effect of DEX (10 nM, 12 days culture) was additive to the effect of 100 nM IGF-I. We conclude that AB is an easy and reliable assay for osteoblastic cell proliferation, well suited for large scale studies of cell growth using small amounts of cells, and that IGF-I partly reverses the glucocorticoid-induced inhibition of osteoblastic proliferation.
Assuntos
Fluorometria/métodos , Glucocorticoides/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Osteoblastos/citologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Osteoblastos/efeitos dos fármacosRESUMO
Osteolysis or osteosclerosis often occurs in bone tissue adjacent to chronic inflammatory processes. Numerous cytokines and inflammatory mediators have been implicated as osteoclast-activating agents, explaining inflammation-induced bone resorption. In many cases, the cause of the sclerosis seen in these lesions is less thoroughly investigated. We have studied the effects of thrombin and bradykinin, 2 inflammatory mediators, on the rate of proliferation in isolated human osteoblasts (hOBs). Thrombin, at and above 1 U/mL, stimulated the rate of thymidine incorporation into hOBs. The absolute cell number also increased, as measured by an assay based on the detection of cell metabolism. A synthetic peptide ligand for the thrombin receptor enhanced the rate of [3H]thymidine incorporation in hOBs, indicating that thrombin-induced proliferation is mediated via the tetheric thrombin receptor. The thrombin-induced proliferation was not affected by indomethacin, excluding prostanoids as mediators of this effect. Bradykinin did not affect either the rate of thymidine incorporation, or number of cells in long-term cultures of hOBs. In conclusion, the inflammatory mediator, thrombin, stimulates proliferation in isolated human osteoblasts probably via the recently described G-protein-coupled tetheric thrombin receptor. Thrombin may therefore be involved as a mediator of inflammation-induced sclerosis and bone formation.
Assuntos
Bradicinina/imunologia , Bradicinina/farmacologia , Osteoblastos/fisiologia , Osteólise/imunologia , Osteosclerose/imunologia , Prostaglandinas/biossíntese , Trombina/imunologia , Trombina/farmacologia , Anti-Inflamatórios não Esteroides/imunologia , Anti-Inflamatórios não Esteroides/farmacologia , Contagem de Células , Divisão Celular , Células Cultivadas , Doença Crônica , DNA/biossíntese , Replicação do DNA/imunologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Indometacina/imunologia , Indometacina/farmacologia , Inflamação , Osteoblastos/efeitos dos fármacos , Receptores da Bradicinina/análise , Receptores de Trombina/análiseRESUMO
The recently cloned osteoclastogenesis inhibitory factor, or osteoprotegerin (OPG), has been shown to be a potent inhibitor of osteoclast formation. The inhibition is believed to be mediated through specific binding of OPG to a cell surface ligand on osteoblastic stromal cells. In this report we have studied the effect of the bone resorbing agent prostaglandin E2 (PGE2) on OPG mRNA levels in primary cultures of human bone marrow stroma cells (hBMSC). PGE2 dose- and time-dependently down-regulated the mRNA levels of OPG, as measured by RNAse protection assay. After 4 hours of stimulation with 1 microM PGE2, OPG mRNA levels were significantly decreased. The inhibitory effect was seen at and above 1 nM of PGE2. To elucidate whether the OPG mRNA levels are regulated via the proteinkinase A and/or the proteinkinase C pathways we stimulated cells with either forskolin (FSK) or phorbolic ester (PDbu) respectively. FSK (10 microM) decreased OPG mRNA levels to 50 % of control, whereas PE (10 nM) upregulated the mRNA levels to 250 % of control. These data show that PGE2 down-regulates the expression of OPG mRNA in hBMSC, probably via an increase in cAMP. This mechanism might be involved in PGE2-induced bone resorption.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Dinoprostona/farmacologia , Glicoproteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares , Sequência de Bases , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/genética , Remodelação Óssea/fisiologia , Reabsorção Óssea/etiologia , Reabsorção Óssea/genética , Reabsorção Óssea/fisiopatologia , Células Cultivadas , Primers do DNA/genética , Regulação para Baixo/efeitos dos fármacos , Humanos , Osteoprotegerina , Receptores do Fator de Necrose Tumoral , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
Osteoprotegerin (OPG) is a recently cloned soluble member of the tumor necrosis factor receptor family. OPG has been shown to inhibit osteoclast recruitment by binding to OPG-ligand, an osteoclast differentiating factor on osteoblastic stromal cells, thereby blocking osteoclastogenesis. In this report we have examined the effect of tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta) on OPG mRNA levels in the human osteosarcoma cell line MG-63. We demonstrate that both TNF-alpha and TNF-beta dose- and time-dependently upregulate the mRNA levels of OPG. The effect is significant at and above 5 pM of TNF-alpha and 1 pM of TNF-beta. The stimulatory effect on OPG mRNA levels in MG-63 cells was detected after 2 hrs of incubation with TNF-alpha or TNF-beta. These data demonstrate that the expression of OPG in osteoblasts, with subsequent effects on osteoclastogenesis, is regulated by TNFs.
Assuntos
Glicoproteínas/biossíntese , Linfotoxina-alfa/biossíntese , Receptores Citoplasmáticos e Nucleares , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Neoplasias Ósseas , Humanos , Cinética , Osteoprotegerina , Osteossarcoma , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Receptores do Fator de Necrose Tumoral/biossíntese , Fatores de Tempo , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
The culture of osteoblast-like cells of human origin has become an important experimental model in bone biology. We report here a comparison and evaluation of three of the most widely used systems available today: bone marrow stroma cell cultures (BMSC), human osteoblast explant cultures (hOB) and osteoblast explant cultures from collagenase-treated bone (hOBcol). Cultures from 16 bone specimens obtained from various donors were established and their expression of the osteoblast phenotype were then compared in secondary cultures by use of biochemical markers. BMSC had the highest basal and 1,25-dihydroxyvitaminD3 (1,25(OH)2D3)-induced alkaline phosphatase activities in all cell isolations, with levels approximately twice those in explant cultures. Basal osteocalcin secretion was low-to-undetectable in all cell cultures but was detected in 1,25(OH)2D3-stimulated cultures. BMSC produced half of the amount of osteocalcin synthesized in explant cultures. The BMSC cultures also synthesized the lowest amounts of type I collagen, whereas collagen type III synthesis did not differ significantly among the various cultures. When secondary cultures were treated with 100 nM dexamethasone in the presence of ascorbic acid (50 microg/mL) and beta-glycerophosphate (10 mM), cultures deposited calcium mineral into the cell layer within 2-4 weeks. PTH-induced cAMP formation was detected in only 5 of 15 isolations and no consistent isolation-dependent response pattern was seen. We conclude that BMSC cultures differ significantly from explant cultures obtained from the same bone specimen. However, all cultures represent cells which can differentiate further and induce mineralization of the extracellular matrix in response to osteoinductive drugs.