RESUMO
Studies with antagonists have provided evidence that protein kinase C (PKC) is involved in several of the actions of parathyroid hormone (PTH) on bone. PTH increases total PKC activity in bone and bone cells. The current studies investigated whether PTH can activate specific PKC isozymes, as demonstrated by translocation of these isozymes from cytosolic to membrane fractions. The isozymes selected for study, alpha, betaI, delta, epsilon, and zeta, were shown previously by us to be present in normal osteoblasts and several osteosarcoma-derived osteoblastic cells. UMR-106 cells, a widely used osteoblastic cell line, were selected for the current study. PKC isozymes in whole cell lysates and cell fractions were visualized by western blotting; isozyme distribution was also visualized by immunofluorescence. The total amounts of the isozymes and their relative distribution between membrane and cytosolic fractions in untreated cells were stable over a range of passages (5-20 from initial plating). In untreated cells, the concentrations of PKC alpha, betaI, and zeta were higher in the cytosol, and PKC delta and epsilon were higher in the membrane fraction. Treatment with 1 or 10 nmol/L PTH for 1 or 5 min stimulated translocation of PKC alpha and betaI, with variable effects on the other isozymes. Treatment with phorbol-12,13-dibutyrate (PDBu), 1 micromol/L for 5 min, elicited similar effects to those of PTH on PKC alpha and betaI. Treatment with PDBu for 48 h resulted in a downregulation of PKC alpha, whereas a 48 h treatment with PTH did not cause downregulation. The results indicate that PTH can affect specific PKC isozymes, providing a mechanism for differential regulation of cellular actions through this pathway.
Assuntos
Neoplasias Ósseas , Isoenzimas/metabolismo , Osteoblastos/enzimologia , Osteossarcoma , Proteína Quinase C/metabolismo , Animais , Carcinógenos/farmacologia , Membrana Celular/enzimologia , Citosol/enzimologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Proteína Quinase C beta , Proteína Quinase C-alfa , Proteína Quinase C-delta , Proteína Quinase C-épsilon , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células Tumorais CultivadasAssuntos
Analgésicos não Narcóticos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios/farmacologia , Dexametasona/farmacologia , Osteoblastos/efeitos dos fármacos , Tolmetino/análogos & derivados , Trometamina/análogos & derivados , Osso e Ossos/citologia , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Escherichia coli , Humanos , Interleucina-1/análise , Cetorolaco de Trometamina , Lipopolissacarídeos/farmacologia , Osteoblastos/imunologia , Tolmetino/farmacologia , Trometamina/farmacologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/efeitos dos fármacosAssuntos
Calcitriol/farmacologia , Dexametasona/farmacologia , Glicerofosfatos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Irrigantes do Canal Radicular/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Glucocorticoides/farmacologia , Humanos , Osteoblastos/citologia , Fatores de TempoRESUMO
Although 17 beta-estradiol (E2) replacement therapy has been shown to be effective in treating postmenopausal osteoporosis, the underlying mechanism remains unclear. The presence of low levels of functional endogenous estrogen receptor (ER) in some osteoblastic cells has been demonstrated, and the suggestion that the abundance of ER may be rate-limiting in the action of E2 on these cells has been made. To study the mechanism of ER in regard to E2-mediated effects, we stably transfected a human osteosarcoma cell line, SaOS-2, with an expression vector, pMV-7-ER, containing the human ER gene. We characterized six of the stably transfected clones. One of the stable clones, SaOS-2-ER, expressed extra copies of ER genes integrated into the genome as detected by Southern blot analysis, showed a significantly increased level of ER mRNA by RT-PCR, and contained an increased level of ER cytosolic protein as detected by an ER-specific EIA. The overexpressed ER was functional and sensitive to E2 in a dose-dependent fashion after transient transfection with a vector containing an estrogen response element (ERE) linked to a chloramphenicol acetyltransferase (CAT) reporter gene. Scatchard analysis revealed a single high-affinity binding site with a Kd similar to values obtained for the ER in MCF-7 breast cancer cells. These SaOS-2-ER cells had altered osteoblast phenotypic features including growth inhibition, decreased basal alkaline phosphatase activity, and decreased IL-6 expression and secretion. In response to E2, a greater than 2-fold increase in TGF-beta 1 mRNA was quantitatively measured in these ER-overexpressing osteoblasts. These cells may provide a sensitive and unique model for understanding the mechanism of E2 and ER in overall bone metabolism.