RESUMEN

Cathepsin K is an osteoclast-derived cysteine protease that has been implicated as playing a major role in bone resorption. A substantial body of evidence indicates that cathepsin K is critical in osteoclast-mediated bone resorption and suggests that its pharmacological inhibition should result in inhibition of bone resorption in vivo. Here we report the pharmacological characterization of SB-462795 (relacatib) as a potent and orally bioavailable small molecule inhibitor of cathepsin K that inhibits bone resorption both in vitro in human tissue and in vivo in cynomolgus monkeys. SB-462795 is a potent inhibitor of human cathepsins K, L, and V (K(i, app)=41, 68, and 53 pM, respectively) that exhibits 39-300-fold selectivity over other cathepsins. SB-462795 inhibited endogenous cathepsin K in situ in human osteoclasts and human osteoclast-mediated bone resorption with IC50 values of approximately 45 nM and approximately 70 nM, respectively. The anti-resorptive potential of SB-462795 was evaluated in normal as well as medically ovariectomized (Ovx) female cynomolgus monkeys. Serum levels of the C- and N-terminal telopeptides of Type I collagen (CTx and NTx, respectively) and urinary levels of NTx were monitored as biomarkers of bone resorption. Administration of SB-462795 to medically ovariectomized or normal monkeys resulted in an acute reduction in both serum and urinary markers of bone resorption within 1.5 h after dosing, and this effect lasted up to 48 h depending on the dose administered. Our data indicate that SB-462795 potently inhibits human cathepsin K in osteoclasts, resulting in a rapid inhibition of bone resorption both in vitro and in vivo in the monkey. These studies also demonstrate the therapeutic potential of relacatib in the treatment of postmenopausal osteoporosis and serves to model the planned clinical trials in human subjects.

Asunto(s)

Azepinas/uso terapéutico , Resorción Ósea/tratamiento farmacológico , Catepsinas/antagonistas & inhibidores , Osteoclastos/efectos de los fármacos , Sulfonas/uso terapéutico , Administración Oral , Animales , Azepinas/administración & dosificación , Azepinas/farmacología , Biomarcadores/sangre , Biomarcadores/orina , Catepsina K , Células Cultivadas , Colágeno Tipo I/sangre , Colágeno Tipo I/orina , Modelos Animales de Enfermedad , Humanos , Macaca fascicularis , Osteoclastos/enzimología , Péptidos/sangre , Péptidos/orina , Sulfonas/administración & dosificación , Sulfonas/farmacología

RESUMEN

Genistein, a soybean isoflavone, has estrogen-like activity in mammals, including the prevention of bone loss. However, whether its mechanism of action on bone turnover is distinct from that of estrogen or raloxifene is unknown. Although genistein has been reported to bind both estrogen receptor (ER) isoforms (alpha and beta), little is known concerning differential activation of gene expression via these ER isoforms. To examine this question, comparison of the responses of normal fetal osteoblast (hFOB) cells stably expressing either ERalpha (hFOB/ERalpha9) or ERbeta (hFOB/ERbeta6), to treatment with genistein, 17beta-estradiol (E(2)) or raloxifene were conducted. In hFOB/ERalpha9 cells, both genistein and E(2) increased the endogenous gene expression of the progesterone receptor (PR), the proteoglycan versican, and alkaline phosphatase (AP), but inhibited osteopontin (OP) gene expression and interleukin-6 (IL-6) protein levels. Raloxifene had no effect on these bone markers. Genistein, but not raloxifene, also mimicked E(2) action in the hFOB/ERbeta6 cells increasing PR gene expression and inhibiting IL-6 production. To determine whether the gene regulatory actions of genistein in human osteoblast cells occur at the level of transcription, its action on the transcriptional activity of a PR-A promoter-reporter construct was assessed. Both genistein and E(2) were found to stimulate the PR promoter in the hFOB cell line when transiently co-transfected with either ERalpha or ERbeta. Whereas hFOB cell proliferation was unaffected by E(2), raloxifene or genistein at low concentrations, higher concentrations of genistein, displayed significant inhibition. Together, these findings demonstrate that genistein behaves as a weak E(2) agonist in osteoblasts and can utilize both ERalpha and ERbeta.

Asunto(s)

Genisteína/farmacología , Osteoblastos/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Secuencia de Bases , Línea Celular Transformada , Cartilla de ADN , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Regulación de la Expresión Génica , Humanos , Interleucina-6/biosíntesis , Osteoblastos/metabolismo , Clorhidrato de Raloxifeno/farmacología , Receptores de Estrógenos/agonistas , Receptores de Progesterona/genética

RESUMEN

2-Methoxyestradiol (2-ME), a naturally occurring mammalian metabolite of 17beta-estradiol, has been implicated as a physiological inhibitor of tumor cell proliferation. In this study, the effects of 2-ME on cultured osteosarcomatous cells were investigated. Dose-dependent growth inhibition was observed in MG63 and TE85 human osteosarcoma cells exposed to 2-ME. The cell killing by 2-ME was ligand-specific; the immediate precursor (2-hydroxyestradiol), the parent compound (17beta-estradiol), and the equivalent metabolite of estrone (2-methoxyestrone) exhibited less potency and efficacy. Furthermore, 2-ME was similarly effective at killing immortalized human fetal osteoblastic cells (hFOB) with and without estrogen receptor-alpha and -beta and rat osteosarcoma cells (ROS17/2.8). The cytotoxicity of 2-ME was selective to transformed and immortalized osteoblastic cells; 2-ME (2 microm) had no effect on the proliferation of primary cultures of human osteoblasts. Co-treatment with the potent estrogen receptor ligand, ICI-182,780, did not reduce 2-ME-induced osteosarcoma cell death, implying that this action is not mediated by conventional estrogen receptors. The expression levels of bone matrix protein genes, type 1 collagen and osteonectin, were transiently reduced after 2-ME treatment, suggesting that the surviving cells are capable of producing bone matrix. The 2-ME-mediated killing of osteosarcoma cells was due to the induction of apoptosis; treatment induced expression of interferon genes within 12 h and histological evidence of apoptosis within 48 h of 2-ME treatment. Thus, our results demonstrate that 2-ME is highly cytotoxic to osteosarcoma cells but not normal osteoblasts. These findings suggest that further study of 2-ME as a potential intervention for treatment of osteosarcoma is warranted.

Asunto(s)

Apoptosis/efectos de los fármacos , Neoplasias Óseas , Estradiol/análogos & derivados , Estradiol/farmacología , Interferones/genética , Osteosarcoma , 2-Metoxiestradiol , Animales , Matriz Ósea/citología , Matriz Ósea/efectos de los fármacos , Matriz Ósea/fisiología , Supervivencia Celular/efectos de los fármacos , Citocinas/genética , Antagonistas de Estrógenos/farmacología , Estrógenos/farmacocinética , Fulvestrant , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/genética , Humanos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Ratas , Células Tumorales Cultivadas

RESUMEN

OBJECTIVE: To determine the messenger RNA expression patterns of estrogen receptor (ER)alpha and ER beta in human vaginal tissue. STUDY DESIGN: Reverse transcriptase-polymerase chain reaction was performed on tissue samples of 75 patients having anterior colporrhaphy (25 premenopausal, 25 postmenopausal receiving estrogen replacement therapy [ERT], 25 postmenopausal not receiving ERT). Levels of mRNA were normalized and ratios were calculated to assess relative levels of expression. RESULTS: All samples showed expression of the ER alpha isoform. Significant differences existed in ER alpha expression among the 3 cohorts (P =.023). Greater differences (P <.001) existed in ER beta expression. For both isoforms, the premenopausal group had the highest level, and the postmenopausal group receiving ERT had the lowest level. No significant difference in ER beta expression existed between postmenopausal groups. CONCLUSION: Significant differences exist between premenopausal and postmenopausal women in presence and expression of ER alpha and ER beta in vaginal tissue. Expression of ER beta markedly declines in menopause, regardless of ERT use.

Asunto(s)

Posmenopausia/metabolismo , Premenopausia/metabolismo , ARN Mensajero/metabolismo , Receptores de Estrógenos/genética , Vagina/metabolismo , Adulto , Estudios de Cohortes , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Terapia de Reemplazo de Estrógeno , Femenino , Procedimientos Quirúrgicos Ginecológicos , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular , Vagina/cirugía

RESUMEN

Although osteoblasts have been shown to respond to estrogens and express both isoforms of the estrogen receptor (ER alpha and ER beta), the role each isoform plays in osteoblast cell function and differentiation is unknown. The two ER isoforms are known to differentially regulate estrogen-inducible promoter-reporter gene constructs, but their individual effects on endogenous gene expression in osteoblasts have not been reported. We compared the effects of 17 beta-estradiol (E) and tamoxifen (TAM) on gene expression and matrix formation during the differentiation of human osteoblast cell lines stably expressing either ER alpha (hFOB/ER alpha 9) or ER beta (hFOB/ER beta 6). Expression of the appropriate ER isoform in these cells was confirmed by northern and western blotting and the responses to E in the hFOB/ER beta 6 line were abolished by an ER beta-specific inhibitor. The data demonstrate that (1) in both the hFOB/ER cell lines, certain responses to E or TAM (including alkaline phosphatase, IL-6 and IL-11 production) are more pronounced at the late mineralization stage of differentiation compared to earlier stages, (2) E exerted a greater regulation of bone nodule formation and matrix protein/cytokine production in the ER alpha cells than in ER beta cells, and (3) the regulated expression of select genes differed between the ER alpha and ER beta cells. TAM had no effect on nodule formation in either cell line and was a less potent regulator of gene/protein expression than E. Thus, both the ER isoform and the stage of differentiation appear to influence the response of osteoblast cells to E and TAM.

Asunto(s)

Estradiol/análogos & derivados , Estrógenos/metabolismo , Estrógenos/fisiología , Osteoblastos/metabolismo , Isoformas de Proteínas , Receptores de Estrógenos/química , Receptores de Estrógenos/metabolismo , Fosfatasa Alcalina/metabolismo , Northern Blotting , Western Blotting , Diferenciación Celular , Línea Celular , Citocinas/biosíntesis , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Estrógenos/farmacología , Matriz Extracelular/metabolismo , Fulvestrant , Genes Reporteros , Humanos , Ligandos , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Tamoxifeno/farmacología , Factores de Tiempo

RESUMEN

Although transforming growth factor-beta (TGF-beta) is a growth factor with many known regulatory activities in many different cell types, its intracellular signaling pathway is still not fully understood. A TGF-beta-inducible early gene (TIEG) was discovered and shown by this laboratory to be a 3-zinc finger transcription factor family member; its expression is rapidly induced in cells treated with TGF-beta. To ascertain whether TIEG plays a major role in the TGF-beta pathway, human osteosarcoma MG-63 cells were stably transfected either with an expression vector containing a TIEG cDNA or with the vector alone. Clones that contain only the vector express normal levels of TIEG mRNA and protein and display the same patterns of gene expression and levels of cell proliferation as the nontransfected, non-TGF-beta-treated parental cells. However, transfected cells that overexpress TIEG mRNA and protein (TIEG-6 and TIEG-7) display changes that mimic those of MG-63 cells treated with TGF-beta, i.e. increased alkaline phosphatase activity, decreased levels of osteocalcin mRNA and protein, and decreased cell proliferation. The degree of these changes correlated with the level of TIEG expressed in the cell lines. TGF-beta treatment of the overexpressed cells showed no added effects. These findings and other published reports support a primary role of TIEG as a transcription factor in the TGF-beta signaling pathway.

Asunto(s)

Proteínas de Unión al ADN/genética , Osteoblastos/metabolismo , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/metabolismo , Fosfatasa Alcalina/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción de la Respuesta de Crecimiento Precoz , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Factores de Transcripción de Tipo Kruppel , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteoblastos/efectos de los fármacos , Osteocalcina/genética , Osteocalcina/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Transfección , Células Tumorales Cultivadas , Dedos de Zinc/genética

RESUMEN

The many recent and exciting advances that have taken place in the field of estrogen action on the skeleton are the subjects of this review. Leading these new developments is the discovery of alternative estrogen receptors that exhibit differential mechanisms of transcriptional control of estrogen-responsive promoters, thereby broadening both the ranges of possible target cells and their responses. More potentially important genes under estrogenic control have been identified in vitro, and the skeletal phenotypes caused by disruption of estrogen signaling due to mutations in humans and mice have been described. Lastly, clinical studies in humans have revealed a greater appreciation for the importance of estrogen in bone mass maintenance in both sexes. J. Cell. Biochem. Suppls. 32/33:123-132, 1999.

Asunto(s)

Huesos/metabolismo , Estrógenos/fisiología , Receptores de Estrógenos/metabolismo , Animales , Desarrollo Óseo/efectos de los fármacos , Huesos/citología , Huesos/efectos de los fármacos , Citocinas/biosíntesis , Citocinas/efectos de los fármacos , Citocinas/genética , Estrógenos/deficiencia , Estrógenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoclastos/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Receptores de Estrógenos/genética

RESUMEN

Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34 degrees C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5 degrees C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5 degrees C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10(-8) M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10(-8) M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5 degrees C with ascorbate and beta1-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.

Asunto(s)

Adipocitos/citología , Células de la Médula Ósea/citología , Osteoblastos/citología , Adipocitos/metabolismo , Antígenos Virales de Tumores/genética , Ácido Ascórbico/farmacología , Calcitriol/farmacología , Diferenciación Celular , División Celular , Línea Celular , Dexametasona/farmacología , Vectores Genéticos , Glicerofosfatos/farmacología , Humanos , Inmunohistoquímica , Mutación , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , ARN Mensajero/análisis , Retrovirus de los Simios/genética , Células del Estroma/citología , Transfección

RESUMEN

Bone morphogenetic proteins (BMPs) induce differentiation of osteoblast and chondroblast lineage cells from uncommitted mesenchymal precursors. Because estrogen has potent osteochondrogenic actions, we investigated its effect on BMP production in two estrogen-responsive, human immortalized cell lines (hFOB/ER3 and hFOB/ER9) that display the mature osteoblast phenotype. These cell lines were produced by stable transfection of the estrogen receptor (ER) gene into immortalized fetal osteoblasts at low ( approximately 800 ER/ nucleus) and at high ( approximately 3, 900 ER/nucleus) levels, respectively. As assessed by reverse transcriptase PCR, treatment with 17beta-estradiol (10(-)10 - 10(-)7 M) increased steady-state levels of BMP-6 mRNA dose dependently by twofold in the hFOB/ER3 cells and by over threefold in the hFOB/ER9 cells. Messenger RNA levels for transforming growth factors-beta1 and -beta2 and BMPs-1 through -5 and -7 levels were unchanged. The results were confirmed by sequence determination of the PCR product and by Northern blot analysis for total RNA. 17beta-estradiol increased BMP-6 protein production sixfold by Western analysis. Cotreatment with antiestrogens (ICI 182,780 or 4-hydroxytamoxifen) antagonized the effects of 17beta-estradiol. These data suggest that some of the skeletal effects of estrogen on bone and cartilage may be mediated by increased production of BMP-6 by osteoblasts.

Asunto(s)

Proteínas Morfogenéticas Óseas/biosíntesis , Estrógenos/farmacología , Osteoblastos/metabolismo , Northern Blotting , Western Blotting , Proteína Morfogenética Ósea 6 , Proteínas Morfogenéticas Óseas/genética , Línea Celular , Estradiol/análogos & derivados , Estradiol/farmacología , Fulvestrant , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Factor de Crecimiento Transformador beta/genética

RESUMEN

Osteoblasts are derived from precursor cells present in low frequency in the stromal element of bone marrow. Because of the lack of a practical procedure to isolate osteoblast precursors from early cultures of plastic adherent cells from bone marrow, previous studies of marrow stromal cells have been made in confluent cultures of bone marrow when the osteoblast (OB) precursors are already differentiated. Also these studies utilized cultures containing mixed populations of cells including hematopoietic cells. Thus we have employed a negative immunoselection procedure to remove contaminating hematopoietic cells and to isolate nearly homogeneous populations of early human stromal cells derived from the plastic-adherent mononuclear marrow cells cultured in the presence of serum. By reverse transcriptase polymerase chain reaction (RT-PCR) analysis for mRNA, and by immunocytochemical study for protein, we studied the sequential expression in culture of multiple markers of the osteoblast phenotype--alkaline phosphatase, osteopontin, parathyroid hormone receptor, types I and III procollagen, and osteocalcin--as well as lipoprotein lipase (LPL), a marker of the adipocyte phenotype. At an early stage of culture (7-9 days), human OB precursors formed colonies of variable sizes that expressed low levels of mRNA and protein concentrations of OB markers, and their concentration increased on growth to a confluent monolayer (approximately 14 days). LPL mRNA was expressed at high levels in the colony stage, and its level decreased upon confluency, suggesting a loss of potential for commitment to the adipocyte lineage. Interestingly, treatment with dexamethasone at 10(-8) M increased the expression for some of the osteoblast markers and for the LPL gene and was required for the deposition of mineralized matrix and for the formation of adipocytes containing cytoplasmic lipid droplets in confluent cultures. Cloned single early colonies were able to coexpress the osteoblast and adipocyte markers (as assessed by RT-PCR). Thus these immunoselected marrow stromal cells have the characteristics of authentic human osteoblast precursor cells which also are capable of differentiating into adipocytes.

Asunto(s)

Células de la Médula Ósea , Osteoblastos/citología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Secuencia de Bases , Médula Ósea/efectos de los fármacos , Calcitriol/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Clonación Molecular , Dexametasona/farmacología , Humanos , Inmunohistoquímica , Lipoproteína Lipasa/genética , Lipoproteína Lipasa/metabolismo , Datos de Secuencia Molecular , Osteoblastos/efectos de los fármacos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteopontina , Fenotipo , Reacción en Cadena de la Polimerasa , Procolágeno/genética , Procolágeno/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Hormona Paratiroidea/genética , Receptores de Hormona Paratiroidea/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Sialoglicoproteínas/farmacología , Células del Estroma/citología , Células del Estroma/efectos de los fármacos

RESUMEN

Although steroid hormones regulate mature osteoblast function, much less is known about their actions on osteoprogenitor cells. The possibility of steroid hormone regulation of early stages in osteoblast differentiation was investigated by measuring the growth and induction of the osteoblast marker enzyme alkaline phosphatase (AP) in rat bone marrow stromal cell cultures. Experiments were performed in charcoal-stripped serum; conditions which markedly impaired stromal cell growth. However, growth could be stimulated by nonadherent marrow cell-derived conditioned medium. 1,25(OH)2D3, but not dexamethasone, 17 beta-estradiol, or retinoic acid, increased both stromal cell proliferation and AP activity. The increased proliferation with 1,25(OH)2D3 was nonadherent cell-dependent. BMP-2 also increased AP levels and acted in synergy with 1,25(OH)2D3. These results suggest that (i) nonadherent marrow cells may support stromal cell development, and (ii) 1,25(OH)2D3 as well as glucocorticoids may regulate osteogenesis from the bone marrow but a similar role for estrogen is not supported.

Asunto(s)

Médula Ósea/efectos de los fármacos , Calcitriol/farmacología , Osteoblastos/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Células de la Médula Ósea , Proteínas Morfogenéticas Óseas , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados , Dexametasona/farmacología , Estradiol/farmacología , Femenino , Osteoblastos/citología , Osteoblastos/enzimología , Proteínas/farmacología , Ratas , Ratas Wistar , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Tretinoina/farmacología

RESUMEN

Adult vertebrates require a continuous supply of osteoblasts for both bone remodeling and regeneration during fracture repair. This implies the existence of a reservoir of cells in the body capable of osteogenesis. One source of these osteoprogenitors is the stem cells within the fibroblastic component of bone marrow stroma. Mature osteoblasts are characterized by high alkaline phosphatase and osteopontin levels, combined with expression of the bone-specific matrix proteins osteocalcin and bone sialoprotein and the capacity for matrix mineralization. We have used these markers to define the conditions permitting rapid osteoblast differentiation from cultured bone marrow stromal cells. Osteoblastic differentiation was induced by continuous culture with 10(-8) M dexamethasone (dex) which stimulated alkaline phosphatase (AP) activity and mRNA levels as well as osteopontin, bone sialoprotein, and osteocalcin mRNA by Day 8 of culture; coaddition of 10(-8) M 1,25-dihydroxyvitamin D3 (vitamin D) with dex was essential for high osteocalcin mRNA expression. Recombinant bone morphogenetic protein-2 (BMP-2) exerted similar effects to dex and acted in synergy with dex to yield greatly elevated AP activity as well as increased levels of osteoblastic mRNAs. Using in situ hybridization to detect the presence of mRNAs in individual cells, it was shown that appearance of osteopontin mRNA preceded AP mRNA, and was expressed in dex-treated cell colonies as early as Day 4. Quantitation of cell surface AP protein by flow cytometry indicated that culture with dex or BMP-2 produced a mixed population of cells with low AP (dim cells) and cells with high AP levels, while the combination of dex + BMP-2 yielded very few dim cells and a population of cells containing higher AP levels than with either inducer alone. When the dim population from dex-treated cells was sorted and recultured with inducers, these cultures developed high AP levels and were able to deposit a mineralized matrix. Thus, treatment of marrow stromal cells with inducer results in a population of mature osteoblasts as well as a population of undifferentiated cells which retains the capacity for osteoblastic differentiation with further exposure to inducers. These data demonstrate that stem cells within the stromal compartment of bone marrow are capable of rapidly acquiring osteoblast features and suggest a potential role for glucocorticoids in combination with BMP-2 and vitamin D in stages of osteogenic development.

Asunto(s)

Células de la Médula Ósea , Dexametasona/farmacología , Osteoblastos/efectos de los fármacos , Proteínas/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Proteínas Morfogenéticas Óseas , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Citometría de Flujo , Hibridación in Situ , Osteoblastos/fisiología , Osteopontina , ARN Mensajero/análisis , Ratas , Ratas Wistar , Sialoglicoproteínas/biosíntesis , Sialoglicoproteínas/genética , Células del Estroma/citología

RESUMEN

The use of primary (nontransformed) bone cell cultures is hampered by their cellular heterogeneity. Primary cultures of osteoblast-like cells have been shown to proliferate in response to several osteotropic agents, but because mixed cell populations are present it is uncertain whether a true osteoblastic response was observed. By combining (1) localization of [3H]-thymidine incorporation into the nuclei of actively dividing cells by autoradiography with (2) subsequent induction of osteoblast differentiation by 1,25(OH)2D3 to optimize the number of cells expressing high alkaline phosphatase activity and (3) its localization by histochemical staining, it is possible to measure the proliferation of cells that are capable of expressing a more mature osteoblastic phenotype in heterogeneous human trabecular bone cell cultures. Over a 72-hour incubation period, rhIL-1 alpha (0.2-2 ng/ml) exerted a dose-dependent stimulation of proliferation of cells expressing alkaline phosphatase. Purified human TGF beta 1 produced a biphasic increase in the proliferation of these cells (0.01-1 ng/ml) but 17 beta and 17 alpha-estradiol (10(-12)-10(-8) M) failed to consistently regulate cell growth. Furthermore, 17 beta-estradiol did not reproducibly modulate proliferation induced by IL-1 alpha or TGF beta when added together in cultures. This procedure represents a more accurate method for the assessment of osteoblast proliferation in primary bone cell cultures and demonstrates that estrogen is not mitogenic for human osteoblasts and does not potentiate the actions of putative local stimulators of osteoblast replication.

Asunto(s)

Estradiol/farmacología , Interleucina-1/farmacología , Osteoblastos/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/metabolismo , Autorradiografía , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoblastos/citología , Osteoblastos/enzimología