RESUMO
The nuclear oxysterol receptors LXRalpha (NR1H3) and LXRbeta (NR1H2) coordinately regulate the expression of genes involved in the transport and catabolism of cholesterol. In macrophages, LXR stimulates the transcription of genes encoding transporters involved in cholesterol efflux, which may limit the transformation of these cells into foam cells in response to lipid loading. Here, we report that natural and synthetic LXR ligands induce the expression of the LXRalpha gene in primary human macrophages and differentiated THP-1 macrophages. This regulation was not observed in primary human adipocytes or hepatocytes, a human intestinal cell line, or in any mouse tissue or cell line examined. The human LXRalpha gene was isolated, and the transcription initiation site delineated. Analysis of the LXRalpha promoter revealed a functional LXR/RXR binding site approximately 2.9 kb upstream of the transcription initiation site. We conclude that LXRalpha regulates its own expression in human macrophages and that this response is likely to amplify the effects of oxysterols on reverse cholesterol transport. These findings underscore the importance of LXR as a potential therapeutic target for the treatment of atherosclerosis.
Assuntos
Regulação da Expressão Gênica/fisiologia , Macrófagos/metabolismo , Receptores Citoplasmáticos e Nucleares , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/fisiologia , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/fisiologia , Animais , Sequência de Bases , Linhagem Celular , DNA , Proteínas de Ligação a DNA , Humanos , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Receptores Nucleares Órfãos , Receptores do Ácido Retinoico/química , Receptores dos Hormônios Tireóideos/química , Homologia de Sequência de AminoácidosRESUMO
Acute IL-6 secretion from osteosarcoma cells induced by the PI-linked hormones PTH(1-34) and endothelin-1 is potentiated by IL-1 beta. The present findings indicate that this potentiation is accompanied by increased signal transduction capacity. Specifically, IL-1 beta (30 pM) increased the B(max) of InsP(3) receptor binding (2. 7-fold) and immunoblot showed a 2.4-fold increase specifically in the type 1 InsP(3) receptor protein. Northern analyses of IL-1 beta-treated G-292 cells showed an 1.8-fold increase in type 1 InsP3 receptor mRNA and, in IL-1 beta-treated murine MC3T3-E1 osteoblastic cells, an 8.4-fold enhancement of the type 1 InsP(3) receptor gene transcription. Promoter reporter assays confirmed the mRNA measurements and showed the effect of IL-1 beta to be mediated by the major transcriptional regulatory region of the type 1 InsP(3) receptor promoter. The findings support the hypothesis that chronic regulators of osteoblast function, such as IL-1 beta, affect the capacity of cellular signal transduction through changes in InsP(3) receptor levels.
Assuntos
Canais de Cálcio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1/farmacologia , Interleucina-6/metabolismo , Osteoblastos/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Células 3T3 , Animais , Canais de Cálcio/metabolismo , Linhagem Celular , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Células Tumorais CultivadasRESUMO
Bone morphogenetic protein-7 (BMP-7) affects differentiation of preosteoblasts enabling the resultant cells to respond optimally to acutely acting regulators. As the phosphoinositide cascade and, particularly, the calcium-mobilizing inositol 1,4,5-trisphosphate (InsP3) receptor are integral to stimulus-secretion coupling in osteoblasts, the hypothesis that BMP-7 affects InsP3 receptor expression was examined in the G-292 human osteosarcoma cell line and in primary cultures of human osteoblasts. G-292 osteosarcoma cells were found to be a valid experimental model for primary human osteoblasts, expressing osteoblastic mRNAs encoding osteocalcin, bone sialoprotein, alkaline phosphatase, alpha1-collagen, epidermal growth-factor receptor, and BMP type II receptor. When cultured long term in the presence of ascorbic acid and beta-glycerophosphate, G-292 cells underwent further osteoblastic differentiation, forming nodules and exhibiting restricted mineralization. G-292 cells responded to BMP-7 with an increase in InsP3 receptor density. Ligand-binding studies established that BMP-7 (50 ng/ml) treatment of G-292 cells increased InsP3 receptor density 2.4-fold with no apparent change in affinity. Immunoblot analysis with antibodies specific for type I, type II, and type III InsP3 receptors revealed that BMP-7 (50 ng/ml) treatment resulted in a specific increase (206+/-8%) in the type I receptor. Reverse transcription-polymerase chain reaction and Northern blot analyses of G-292 and primary human osteoblasts confirmed an increase in type I InsP3 receptor mRNA upon BMP-7 treatment. These results demonstrate that G-292 cells respond to BMP-7 with an increase InsP3 receptor density, consistent with the enhanced capacity of these cells to respond to Ca2+-mobilizing secretory hormones during osteoblast differentiation.