RESUMO
Periosteal cells were isolated from tibiae of adult male rats after collagenase treatment. Northern blot analysis of total cytoplasmic RNA extracted from the isolated periosteal cells was positive for expression of genes encoding the osteoblast marker proteins osteocalcin (BGP) and pre-pro-alpha 2(I) chain of type 1 precollagen. The isolated periosteal cells were incubated with 1 nM [3H]testosterone [( 3H]T) for up to 240 minutes and the reaction products separated by high-performance liquid chromatography. [3H]5 alpha-dihydrotestosterone [( 3H]DHT) was not detected in extracts of periosteal cell incubations. In contrast, [3H]DHT was produced in a time-dependent manner by cells from seminal vesicles. These results suggest that testosterone 5 alpha-reductase activity is not expressed by osteoblasts in rat tibial periosteum and that the anabolic effects of androgens in this tissue are not mediated by locally produced DHT.
Assuntos
Di-Hidrotestosterona/metabolismo , Periósteo/metabolismo , Testosterona/metabolismo , Animais , Northern Blotting , Cromatografia Líquida de Alta Pressão , Técnicas In Vitro , Oxirredução , Periósteo/citologia , Ratos , Tíbia , TrítioRESUMO
To determine if bone cells produce interleukin-1 beta (IL-1 beta), a potent bone resorption-stimulating agent, we studied well-characterized, nearly homogeneous cultures of normal human osteoblast-like (hOB) cells. With four strains of such cells, vehicle-treated cultures produced minimal IL-1 beta (mean +/- SEM, 1.3 +/- 0.3 pg/ml per 10(6) cells per 24 h) and showed dose-dependent (r = 0.99) increases to 2.2 +/- 0.7, 5.0 +/- 0.9, or 17.8 +/- 6.7 pg/ml, respectively, after treatment with lipopolysaccharide (LPS) at 3, 10, or 30 micrograms/ml (for increases after 10 and 30 micrograms/ml treatments, P less than 0.05). After treatment with tumor necrosis factor alpha (TNF-alpha) at 10 U/ml, IL-1 beta increased to 16.2 +/- 3.7 pg/ml (P less than 0.05). Neither 17 beta-estradiol nor bovine parathyroid hormone(1-34) (each at 10 nM), alone or in combination with LPS or TNF-alpha, affected IL-1 beta release. Northern blot analysis of total cellular RNA preparation revealed a single hybridization band at 1.9 kb when probed with a partially deleted cDNA for human IL-1 beta. The steady-state IL-1 beta mRNA levels showed a significant increase with LPS treatment and a lesser increase with TNF-alpha treatment in hOB cells. Moreover, TNF-alpha produced an even greater increase in IL-1 mRNA in HOBIT cells, a well-differentiated clonal cell line derived from normal hOB cells transfected with the SV40 large T antigen. We conclude that human cells of the osteoblast lineage produce IL-1 beta in response to well-recognized stimuli for IL-1 release from responsive tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Interleucina-1/biossíntese , Osteoblastos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Antígenos Transformantes de Poliomavirus/genética , Reabsorção Óssea/fisiopatologia , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estradiol/farmacologia , Humanos , Interleucina-1/genética , Lipopolissacarídeos , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TeriparatidaRESUMO
Silicon in trace amounts enhances bone formation, and the silicon-containing compound zeolite A (ZA) increases eggshell thickness in hens. In the studies reported here, treatment of nearly homogeneous strains of normal human osteoblast-like cells for 48 h with ZA at 0.1-100 micrograms/ml induced a dose-dependent increase (r = 0.35, P < 0.001) in DNA synthesis (n = 31) to 162 +/- 16% (mean +/- SEM) of control and in the proportion of cells in mitosis (n = 4) from 9.1 +/- 1.8 to 27.0 +/- 4.5% (r = 0.69, P < 0.005). ZA treatment also increased alkaline phosphatase activity (P < 0.05) and osteocalcin release (P < 0.05) but did not significantly affect collagen production per individual cell. The mitogenic action of ZA was dependent on cell seeding density over the range of 1250-40,000 cells per cm2, which is consistent with induction of an autocrine factor(s). TGF-beta is a potent mitogen for osteoblasts. ZA treatment increased the steady-state mRNA levels of transforming growth factor beta 1 (TGF-beta 1) and induced the release of the latent form of TGF-beta protein into the conditioned medium within 6 h. We conclude that ZA induces the proliferation and differentiation of cells of the osteoblast lineage.
Assuntos
Silicatos de Alumínio/farmacologia , Osteoblastos/efeitos dos fármacos , Fator de Crescimento Transformador beta/biossíntese , Fosfatase Alcalina/metabolismo , Northern Blotting , Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , DNA/biossíntese , Relação Dose-Resposta a Droga , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Análise de Regressão , Fator de Crescimento Transformador beta/farmacologia , ZeolitasRESUMO
A new bone cell line was established by transfecting normal adult human osteoblast-like (hOB) cells, derived from a 68-year-old woman, with the plasmid pSV3 neo. The plasmid included coding sequences and promotors for the large and small T antigens of the SV40 virus as well as resistance to the antibiotics neomycin and G418. A single antibiotic-resistant colony was located and cloned. Large tumor antigen production in the clonal cell line was confirmed by indirect immunofluorescence study. Treatment with 1,25-dihydroxy-vitamin D3 increased steady-state concentrations of protein and mRNA for osteocalcin and for alkaline phosphatase. Northern blot analyses also demonstrated the presence of mRNAs for alpha(I)-procollagen, osteopontin 1a, transforming growth factor beta, and interleukin-1 beta. The plasma membrane calcium pump and osteonectin were identified by immunocytochemical analysis. These cells produced a matrix that mineralized when beta-glycerophosphate was added to their cultures. As assessed by functional receptor assays, both estrogen and androgen receptors were present and functional, although at low concentrations. Treatment with parathyroid hormone did not stimulate adenylate cyclase activity. Thus, these cells are a well-differentiated, steroid-responsive clonal cell line that closely approximates the phenotype of the mature osteoblast. They should serve as an excellent model for the study of osteoblast biology.
Assuntos
Antígenos Virais/genética , Osteoblastos/imunologia , Vírus 40 dos Símios/imunologia , Transfecção/imunologia , Idoso , Northern Blotting , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Núcleo Celular/metabolismo , DNA/genética , Feminino , Hormônios Esteroides Gonadais/metabolismo , Humanos , RNA/isolamento & purificação , Valores de Referência , Fatores de TempoRESUMO
Although osteoblasts contain estrogen receptors, it is unclear whether estrogen has direct effects on osteoblast proliferation and differentiation. We evaluated the effects of 17 beta-estradiol treatment (1 pM to 10 nM) on the proliferation and differentiation of cultured normal adult human cells that expressed many of the phenotypic characteristics and hormonal sensitivities of mature osteoblasts (hOB cells). Treatment of hOB cells with estradiol for as long as 144 h did not affect the rate of DNA synthesis and had minimal, if any, effects on differentiated function. Whereas alkaline phosphatase activity was increased by nearly twofold (P less than 0.01) when the hOB cells were treated with 1 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], treatment with estradiol had no effect when given alone and did not affect the cells' response to 1,25-(OH)2D3. Similarly, the release of bone gla protein (BGP, osteocalcin) was induced by treatment with 1,25-(OH)2D3 (P less than 0.05), but estradiol treatment did not affect this response. Cellular levels of mRNA for alkaline phosphatase and BGP were not altered by estradiol treatment. We conclude that estradiol treatment does not have major effects on the growth or differentiation of cultured hOB cells. These results are consistent with previous observations in vivo that indicate estrogen acts principally to decrease bone resorption, not to modulate its formation.
Assuntos
Estradiol/farmacologia , Osteoblastos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Osteoblastos/citologia , Valores de ReferênciaRESUMO
Rat lens microsomal preparations possess the capability of converting exogenous arachidonic acid into prostaglandins (PG) E2 and F2 alpha. The low, yet measurable prostaglandin biosynthetic capacity of the lens microsomes was demonstrated by radioimmunoassay (RIA) and by separation of radiolabeled products after incubation with high specific activity U-14 [C]-arachidonic acid. Maximal formation of radioimmunoassayable PGE2 and PGF2 alpha was measured after a 15-min incubation at 37 degrees C with approximately 0.5 mg protein. Prostaglandin biosynthesis was inhibited by the nonsteroidal anti-inflammatory agents aspirin (IC50 = 52 microM) and indomethacin (IC50 = 20 microM). These results unequivocally demonstrate prostaglandin biosynthesis in rat lens.
Assuntos
Ácidos Araquidônicos/metabolismo , Cristalino/metabolismo , Prostaglandinas E/biossíntese , Prostaglandinas F/biossíntese , Animais , Dinoprosta , Dinoprostona , Microssomos/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The possibility that the enzymatic generation of prostaglandin E2 (PGE2) and PGF2 alpha results from the catalytic activity of two distinct forms of the fatty acid cyclooxygenase was studied in microsomes prepared from kidney, lung, and brain of the mouse and rat. Three criteria established previously to detect the dual cyclooxygenase forms in the rabbit brain were used in the present study: (1) different time course profiles of microsomal PGE2 and PGF2 alpha biosynthesis from exogenous arachidonic acid; (2) elimination of the synthesis of one PG in vitro by non-steroidal anti-inflammatory drug concentrations that did not affect the synthesis of the other PG and; (3) selective autocatalytic inactivation of one cyclooxygenase by preincubation with arachidonic acid. Incubations with PGH2 endoperoxide as substrate tested whether the altered PG biosynthesis resulted from an effect on the endoperoxide utilizing enzymes and not on the cyclooxygenase. Of the six tissues examined, only the mouse brain microsomes satisfied all the criteria. The microsomes prepared from the mouse kidney produced mixed results. We conclude that the mouse brain but not the rat brain gives evidence for two distinct forms of the fatty acid cyclooxygenase. Additional distinguishing features of the different cyclooxygenases are required to determine if the cyclooxygenase forms are found in mouse kidney.
Assuntos
Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Encéfalo/enzimologia , Dinoprosta , Dinoprostona , Relação Dose-Resposta a Droga , Indometacina/farmacologia , Rim/enzimologia , Pulmão/enzimologia , Camundongos , Microssomos/enzimologia , Prostaglandinas E/biossíntese , Prostaglandinas F/biossíntese , Ratos , Especificidade da EspécieRESUMO
The mechanisms by which the sex hormones achieve their bone-sparing effects remains unresolved. Interleukin-1 beta (IL-1 beta) is an autocrine/paracrine regulator of bone that may be produced in an estrogen-sensitive manner. The regulation of IL-1 beta production by the gonadal steroids was tested in the human osteoblastic HOBIT cell model. Dose-dependent 4-8-fold increases (P < 0.05) in IL-1 beta mRNA levels followed a 6-48 h treatment with 17 beta-estradiol or testosterone. Receptor mediation of these responses was indicated by experiments using 17 alpha-estradiol or flutamide. Tumor necrosis factor-alpha (TNF) dependent increase IL-1 beta mRNA levels were additive to the effects of the steroids. Testosterone and TNF increased IL-1 beta protein release (P < 0.05) while 17 beta-estradiol had little effect on release. The bone-sparing effects of the gonadal steroids may be accomplished, in part, through their mediation of local IL-1 beta production.
Assuntos
Hormônios Esteroides Gonadais/farmacologia , Interleucina-1/biossíntese , Osteoblastos/metabolismo , Linhagem Celular , Estradiol/farmacologia , Flutamida/farmacologia , Humanos , Interleucina-1/genética , Osteoblastos/efeitos dos fármacos , RNA Mensageiro/metabolismo , Testosterona/antagonistas & inibidores , Testosterona/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Enzymatic production of prostaglandins (PGs) from exogenous arachidonic acid was studied in brain microsomal fractions prepared from mice following pentylenetetrazol (PTZ)-induced convulsions. Prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) measured either by radioimmunoassay or after incubation with [1-14C]arachidonic acid (AA) was significantly increased in microsomes from the convulsed animals. Pretreatment of the mice with the anticonvulsant ethosuximide prevented the enhanced PG production. The increased PG synthesis could not be attributed to an increased substrate availability nor to an activated phospholipase nor to a direct effect of the convulsant on the fatty acid cyclooxygenase. Evidence that a modification of the cyclooxygenase had occurred with seizure activity was obtained from kinetic analysis; the apparent Km for the AA was lowered from 30 +/- 3 microM in the controls to 12 +/- 1 microM in the PTZ-treated mice. Further evidence for a modification of the fatty acid cyclooxygenase was obtained from incubations of the microsomes with catalase to reduce peroxide formation. Limiting peroxide levels did not decrease the microsomal cyclooxygenase activity in the PTZ-treated mice to control levels. Seizure activity induced by picrotoxin and strychnine also increased the microsomal capacity of the convulsed animals to synthesize PGs. The increased brain fatty acid cyclooxygenase activity may result from a biochemical modification of the enzyme induced by seizure activity.
Assuntos
Encéfalo/enzimologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas E/metabolismo , Prostaglandinas F/metabolismo , Convulsões/enzimologia , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Encéfalo/efeitos dos fármacos , Dinoprosta , Dinoprostona , Etossuximida/farmacologia , Feminino , Cinética , Camundongos , Microssomos/enzimologia , Pentilenotetrazol , Picrotoxina/farmacologia , Convulsões/induzido quimicamente , Estricnina/farmacologiaRESUMO
Evidence is presented indicating that young rat lens has the capacity to synthesize 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) from exogenous arachidonic acid (AA). A 9,000 xg supernatant prepared from 15 day old rat lenses, when incubated with calcium and U-[14C]-AA, generated a radiolabelled product with a retention time identical to authentic unlabelled 12-HETE in two different HPLC solvent systems. Mass spectral analysis provided evidence that the metabolite was 12-HETE while chiral studies demonstrated the exclusive presence of the 12(S) isomer. Radiolabelled 12-HETE synthesis was inhibited by preincubating the 15 day old rat lens supernatant with 0.2 Mm curcumin, a mixed cyclooxygenase/lipoxygenase inhibitor. Radiolabelled 12(S)-HETE synthetic capacity was highest in the 4 day old rat lens, the earliest time period measured, and rapidly declined with age reaching negligible levels at about 4 months. These results suggest that the young rat lens possess the biosynthetic capacity to generate radiolabelled 12(S)-HETE from U-[14]C-AA. The profile of 12-HETE synthetic activity suggests a possible regulatory function of the hydroxylated AA metabolite in the developing lens.
Assuntos
Ácidos Hidroxieicosatetraenoicos/biossíntese , Cristalino/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Cloreto de Cálcio/farmacologia , Cromatografia Líquida de Alta Pressão , Curcumina/farmacologia , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Isomerismo , Espectrometria de Massas , Ratos , Ratos EndogâmicosRESUMO
The relationship between the development of galactose-induced cataractogenesis and rat lens microsomal prostaglandin (PG) biosynthesis was studied. Within 24 hr of the introduction of 50% galactose to the rat's diet, lens PGF2 alpha production fell dramatically to 31% of control. Following the initial depression of PGF2 alpha biosynthesis, the ability to generate PGF2 alpha in lens microsomes slightly recovered reaching 58- and 53% of controls at day 2 and day 5 on the sugar diet, respectively. Determination of microsomal PGF2 alpha biosynthesis at 9- and 21 days revealed a continued decline in PG synthesis with complete cessation of PGF2 alpha synthesis by day 36 (hypermature cataracts, +5). The decreased PGF2 alpha biosynthetic capacity was a result of decreased cyclo-oxygenase activity since: PGE2 production demonstrated a similar time course for inhibition; and lens microsomes from control and galactose fed rats revealed no difference in the PGs produced from PGH2 endoperoxide. Neither galactose (1 mM) nor galactitol (1 mM), when added to control microsomal preparations inhibited PG biosynthesis, eliminating the possibility of a direct effect of the sugar, or its metabolite, on cyclo-oxygenase activity. While PG biosynthesis was rapidly inhibited by the galactose feeding no changes were observed in basal lens cyclic AMP levels measured during the first 5 days of the feedings. These results demonstrate that depressed PG biosynthesis is an early consequence of galactose feeding.
Assuntos
Catarata/induzido quimicamente , Cristalino/metabolismo , Prostaglandinas/biossíntese , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Catarata/metabolismo , AMP Cíclico/metabolismo , Dinoprosta , Dinoprostona , Galactose , Masculino , Microssomos/metabolismo , Prostaglandina D2 , Endoperóxidos Sintéticos de Prostaglandinas/metabolismo , Prostaglandina H2 , Prostaglandinas D/metabolismo , Prostaglandinas E/metabolismo , Prostaglandinas F/biossíntese , Prostaglandinas H/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Experiments were performed using [1-14C]PGH2 endoperoxide as substrate to assess which, if any, of the PGH2 utilizing enzymes were present in rat lens microsomes. After a 15 min incubation, prostaglandins (PGs) were extracted, separated by thin-layer chromatography and labeled PGs located by autoradiography. The bands were cut and counted and product formation expressed as a percentage of the total recovered label. Prostaglandin PGE2 formation accounted for 24% of the label and was not increased (22%) when 1 mM reduced glutathione (GSH), the obligate cofactor of PGH2----PGE2 isomerase, was added. However, the addition of the GSH significantly increased PGF2 alpha generation from 25% to 37%. Pretreatment of the microsomes with p-hydroxymercuribenzoate, a sulfhydryl-blocking agent and an inhibitor of PGH2----PGE2 isomerase, did not limit lens microsomal PGE2 formation (24% vs. 39%) but dramatically inhibited PGF2 alpha production (25% vs. 6%). Boiling the microsomes did not alter PGF2 alpha or PGE2 generation suggesting that these PGs were non-enzymatically produced. No thromboxane B2 or 6-keto-PGF1 alpha (the stable breakdown products of thromboxane A2 and prostacyclin, respectively) were found indicating that their respective synthetases were absent in the lens microsomes. These data suggest that the lens is a unique tissue, generating the primary PGs in the absence of all known PGH2 endoperoxide-utilizing enzymes. It appears that the PGH2 formed by lens cyclo-oxygenase spontaneously breaks down in the lens microsomes into PGE2, PGD2 and PGF2 alpha.
Assuntos
Oxirredutases Intramoleculares , Cristalino/metabolismo , Endoperóxidos Sintéticos de Prostaglandinas/metabolismo , Endoperóxidos de Prostaglandina/metabolismo , Prostaglandinas H/metabolismo , Prostaglandinas/metabolismo , Animais , Dinoprosta , Dinoprostona , Glutationa/metabolismo , Hidroximercuribenzoatos/farmacologia , Isomerases/metabolismo , Cristalino/enzimologia , Masculino , Microssomos/metabolismo , Prostaglandina H2 , Prostaglandina-E Sintases , Prostaglandinas E/metabolismo , Prostaglandinas F/metabolismo , Ratos , Ratos EndogâmicosRESUMO
In humans, glucocorticoids are known to have marked effects on bone metabolism and function, including the significant regulation of osteoblast cells. To aid in the understanding of the mechanism of glucocorticoid action on normal human osteoblasts (hOB), confluent cells were analyzed for the presence of glucocorticoid receptors (GR) as well as for the effects of the glucocorticoid dexamethasone (Dex) on the expression of both the rapid responding nuclear proto-oncogenes and the late responding structural genes for bone matrix proteins. The interactions between Dex and 1,25 dihydroxy vitamin D3 (1,25 D3) on the gene expression in these cells were also examined. Using a functional receptor assay, a mean of 11,600 functional nuclear bound glucocorticoid receptors (range 6,000-22,000) was measured in fifteen separate cell strains. Northern blot analysis with a cDNA probe to the human GR was used to demonstrate the presence of a 7Kb transcript which is a candidate mRNA for GR in these cells. In agreement with previous studies, treatment of the hOB cells with Dex increased the steady state mRNA levels for alkaline phosphatase (AP) but displayed little or no effect on the mRNA levels for osteocalcin (OC) and glyceraldehyde phosphate dehydrogenase (GAPDH). Interestingly, the 1,25 D3 inductions of mRNA levels for OC were blocked by Dex but enhanced for AP. The above effects of Dex on AP and OC gene expression, including the interaction with 1,25 D3, were also shown to occur at the level of protein. The effect of Dex on the mRNA levels of the nuclear proto-oncogenes c-myc, c-fos, and c-jun was also investigated, since the oncoproteins (Fos/Jun) appear to play a role in the delayed glucocorticoid regulation of structural genes. Interestingly, Dex increased the steady state levels of c-myc, c-fos, and c-jun mRNAs in nonproliferating (confluent) hOB cells by 3.5-, 10-, and 2.0-fold, respectively, over control (untreated cells) values within one h of steroid treatment. The Dex-induced mRNA levels were transient and returned to basal values within 24 h of the steroid treatment. A reduced but qualitatively similar pattern of response was found in proliferating hOB cells. The pattern of response of these genes to glucocorticoids in hOB cells mimics the response in avian liver cells but not in reproductive cells. These results support the theory that hOB cells are target cells for glucocorticoids, and that as a primary event glucocorticoids rapidly regulate the expression of the nuclear oncoproteins Fos/Jun in these cells.
Assuntos
Fosfatase Alcalina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Osteoblastos/metabolismo , Osteocalcina/genética , Proto-Oncogenes/genética , Ligação Competitiva , Calcitriol/farmacologia , Núcleo Celular/metabolismo , Células Cultivadas , Dexametasona/metabolismo , Dexametasona/farmacologia , Humanos , Osteoblastos/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/metabolismo , Triancinolona Acetonida/metabolismoRESUMO
The effects of treatment with the osteotropic steroids 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 17 beta-estradiol, or dexamethasone on [1-14C]arachidonic acid (AA) uptake and distribution into glycerophospholipid classes by normal adult human osteoblast-like (hOB) cells were investigated. Total uptake of [1-14C]AA was decreased in cells treated with dexamethasone when assayed after a 24-, 48-, or 96-h exposure to the hormone. Specific radiolabel incorporation into phosphatidylcholine was reduced by a 48-h treatment with dexamethasone with a concurrent increase in the radiolabeling of phosphatidylethanolamine. However, these changes were transient, and by 96 h of dexamethasone treatment the distribution of the radiolabeled fatty acid had reequilibrated to resemble the pattern found for vehicle treated samples. Total uptake of [1-14C]AA was diminished by 96-h treatment with 1,25(OH)2D3 (79 +/- 3% of control, P < 0.01); at that time point, a significant decrease in the proportional radiolabeling of the phosphatidylinositol pool was identified (92 +/- 2% of control, P < 0.05). The 1,25(OH)2D3-dependent decrease in total uptake and in phosphatidylinositol incorporation of [1-14C]AA were found to be hormone dose dependent. Treatment with 24,25(OH)2D3 was without effect on either total [1-14C]AA uptake or the specific [1-14C]AA radiolabeling of the phosphatidylinositol pool. 1,25(OH)2D3 treatment decreased hOB cell uptake of [1-14C]oleic acid and decreased its proportional incorporation into the phosphatidylinositol pool. Gas chromatographic analyses revealed no 1,25(OH)2D3-dependent effects on total phosphatidylinositol lipid mass or on the mole percent of arachidonic acid within the phosphatidylinositol pool, leaving the mechanism of the effects of the secosteroid on hOB cell AA metabolism unexplained. 17 beta-Estradiol had no effects on the parameters of AA metabolism measured. As a consequence of their modulation of arachidonic acid uptake and its distribution into hOB cellular phospholipids, steroids might alter the biological effects of other hormones whose actions include the stimulated production of bioactive AA metabolites, such as prostaglandins or the various lipoxygenase products.
Assuntos
Ácido Araquidônico/farmacocinética , Calcitriol/farmacologia , Dexametasona/farmacologia , Osteoblastos/efeitos dos fármacos , Ácidos Fosfatídicos/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Radioisótopos de Carbono , Células Cultivadas , Cromatografia Gasosa , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ácido Oleico , Ácidos Oleicos/farmacocinética , Osteoblastos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositóis/metabolismoRESUMO
Eicosanoids are multifunctional autocrine/paracrine regulators of bone that are enzymatically derived from arachidonic acid (AA). The rate-limiting step in the eicosanoid biosynthetic pathways may be the release of AA from membrane glycerophospholipids by activated phospholipases. Free AA can serve as the substrate for cyclooxygenase(s) or lipoxygenases that catalyze the commitive steps in eicosanoid synthesis; alternatively, free AA may be used in reacylation processes, resulting in its reincorporation into cellular lipids. The hormones 17 beta-estradiol (17 beta-E2), dexamethasone (a synthetic glucocorticoid), and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) have been identified as regulators of AA metabolism, at various levels, in several tissues including bone. The possibility that these osteotropic steroids modulate the availability of free AA in bone cells was studied in the human osteoblast-like (hOB) cell model system. Following a 48-h steroid pretreatment, bradykinin or the calcium ionophore A23187 were used as agonists to stimulate hOB cell release of AA. The principal findings from these investigations were that (1) 17 beta-E2 pretreatment potentiated the appearance of free AA following bradykinin stimulation of the cells but, did not alter their response to A23187 stimulation; (2) dexamethasone pretreatment limited bradykinin-induced increases in free AA levels but did not alter cell response to A23187 stimulation; (3) hOB cells derived from different trabecular bone compartments (manubrium of the sternum, femoral head) differed quantitatively in their responses to bradykinin stimulation of AA release; and (4) 1,25(OH)2D3 did not effect AA release stimulated by either agonist. The ability of the steroids to modulate AA release by hOB cells suggests that these hormones may indirectly mediate bone cell responses to other osteotropic hormones that act through eicosanoid-dependent processes.
Assuntos
Ácido Araquidônico/metabolismo , Bradicinina/farmacologia , Estradiol/farmacologia , Osteoblastos/efeitos dos fármacos , Reabsorção Óssea/induzido quimicamente , Calcimicina/farmacologia , Calcitriol/farmacologia , Células Cultivadas , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Humanos , Ionóforos/farmacologia , Valores de Referência , Estimulação QuímicaRESUMO
Reports that estrogen treatment modulates arachidonic acid metabolism by bone and bone cells are found in the literature. However, conflicting indications of the relationship that exists between estrogen and arachidonic acid metabolism emerge from the analysis of those reports. The present studies were undertaken to determine if estrogen effected the production of prostaglandins (PG) in human osteoblast-like (hOB) cell cultures derived from adults, under basal or cytokine-stimulated conditions. A 48-hour estrogen pretreatment did not modify hOB cell PG biosynthesis on a qualitative basis, and PGE2 formation predominated under all tested conditions. Estrogen pretreatment did lead to increased PGE2 production in specimens stimulated conjointly with transforming growth factor-beta1 and tumor necrosis factor-alpha ( p < 0.001). No changes in PGE2 production were observed in estrogen pretreated specimens stimulated singly with either tested cytokine, nor in samples in which either TGFbeta or TNF was replaced by interleukin-1beta. Anti-estrogen (ICI 164,384) inclusion prevented the estrogen-dependent increase in PGE2 production in the TGFbeta plus TNF-stimulated samples. These results suggest that an estrogen effect on bone cell prostaglandin biosynthesis may be most evident and significant under conditions in which the cells are exposed to multiple osteotropic cytokines, a condition that applies during the bone remodeling process.
Assuntos
Dinoprostona/biossíntese , Estradiol/análogos & derivados , Estradiol/farmacologia , Osteoblastos/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Dinoprostona/genética , Combinação de Medicamentos , Antagonistas de Estrogênios/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoblastos/metabolismo , Alcamidas Poli-Insaturadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The sex steroids, androgens and estrogens, are major regulators of bone metabolism. However, whether these hormones act on bone cells through direct or indirect mechanisms has remained unclear. A nuclear binding assay recently used to demonstrate estrogen receptors in bone [Eriksen, E.F., Colvard, D.S., Berg, N.J., Graham, M.L., Mann, K.G., Spelsberg, T.C. & Riggs, B.L. (1988) Science 241, 84-86] was used to identify specific nuclear binding of a tritiated synthetic androgen, [3H]R1881 (methyltrienolone), in 21 of 25 (84%) human osteoblast-like cell strains and a concentration of bound steroid receptors of 821 +/- 140 (mean +/- SEM) molecules per cell nucleus. Binding was saturable and steroid-specific. Androgen receptor gene expression in osteoblasts was confirmed by RNA blot analysis. Relative concentrations of androgen and estrogen receptors were compared by measuring specific nuclear estrogen binding. Nuclear binding of [3H]estradiol was observed in 27 of 30 (90%) cell strains; the concentration of bound estradiol receptor was 1537 +/- 221 molecules per cell nucleus. The concentrations of nuclear binding sites were similar in males and females for both [3H]R1881 and [3H]estradiol. We conclude that both androgens and estrogens act directly on human bone cells through their respective receptor-mediated mechanisms.
Assuntos
Osteoblastos/metabolismo , Receptores Androgênicos/metabolismo , Ligação Competitiva , Northern Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Humanos , Cinética , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Receptores Androgênicos/genética , Receptores de Estrogênio/metabolismoRESUMO
Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE2 was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE2, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGF beta and tumor necrosis factor-alpha(TNF). The production of 6-keto-PGF1 alpha (the stable metabolite of prostacyclin) and of PGF2 alpha were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGF beta and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE2 biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6-12 h, and levels had appreciably diminished by 24 h after treatment. Both TGF beta and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE2 resulting from TNF stimulation was blocked by the addition of an interleukin-1 beta (IL-1 beta) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1 beta production. TGF beta regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines.
Assuntos
Osso e Ossos/metabolismo , Citocinas/farmacologia , Dinoprostona/biossíntese , Osteoblastos/metabolismo , Adulto , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Humanos , ImunoensaioRESUMO
The steroid derivative 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a regulator of bone biology, and there is evidence that 1,25(OH)2D3 modulates arachidonic acid metabolism in osteoblastic cell model systems and in bone organ cultures. In the present studies, 1,25(OH)2D3 decreased prostaglandin (PG) biosynthesis by normal adult human osteoblast-like (hOB) cell cultures by about 30%. The decrease was observed under basal incubation conditions, or in specimens stimulated by transforming growth factor-beta 1 (TGF-beta) or by tumor necrosis factor-alpha (TNF). The inhibition of the TGF-beta-stimulated PG production appeared to reflect a diminished efficiency of arachidonic acid conversion into PGs by the cells, while the efficiency of substrate utilization for PG biosynthesis was unaffected by 1,25(OH)2D3 pretreatment in the unstimulated samples, or in samples stimulated with TNF or with TNF plus TGF-beta. Free arachidonic acid levels were decreased following 1,25(OH)2D3 pretreatment in the TNF stimulated samples. hOB cell phospholipase A2 activity was measured in subcellular fractions, and this activity was decreased by 20-25% in the 1,25(OH)2D3 pretreated samples. The addition of the selective inhibitor AACOCF3 to the phospholipase A2 assays provided evidence that it was the cytoplasmic isoform of the enzyme that was affected by the 1,25(OH)2D3 pretreatment of the hOB cells. Thus, 1,25(OH)2D3 regulation of hOB cell biology includes significant effects on arachidonic acid metabolism. In turn, this could influence the effects of other hormones and cytokines whose actions include the stimulated production of bioactive arachidonic acid metabolites.
Assuntos
Calcitriol/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Prostaglandinas/biossíntese , Adulto , Idoso , Ácido Araquidônico/metabolismo , Ácidos Araquidônicos/farmacologia , Radioisótopos de Carbono , Cromatografia em Camada Fina , Citocinas/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoblastos/citologia , Fosfatidiletanolaminas/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/efeitos dos fármacos , Fosfolipases A/metabolismo , Fosfolipases A2 , Fatores de Tempo , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The eicosanoids, including prostaglandin E2 (PGE2) and other bioactive arachidonic acid metabolites, are important local mediators of bone remodeling. Presumably, the limited or excessive synthesis of the eicosanoids could compromise bone homeostasis. We have noted that the stimulated release of arachidonic acid by adult male donor derived human osteoblast-like (hOB) cells exceeded the stimulated release measured for female-derived hOB cells by 1.5-fold. Assays of PGE2 biosynthesis by cytokine-stimulated hOB cells also demonstrated a sex-linked difference, such that male hOB cell PGE2 production exceeded female cell production by 1.6-2.2-fold. The calcium-dependent cytoplasmic phospholipase A2 activity in subcellular fractions prepared from hOB cell homogenates was higher in both the cytosolic (1.6-fold) and particulate (1.5-fold) fractions from the male cells than in those prepared from female hOB cells, suggesting a molecular basis for the observed sexually dimorphic characteristics related to arachidonic acid metabolism by hOB cells. The relatively limited capacity of the female cells may limit needed intracellular and intercellular signaling during bone remodeling, thereby contributing to the development of bone pathology.