Species-Specific Glucocorticoid and 1,25-Dihydroxyvitamin D Responsiveness in Mouse MC3T3-E1 Osteoblasts: Dexamethasone Inhibits Osteoblast Differentiation and Vitamin D Down-Regulates Osteocalcin Gene Expression*

Parathyroid hormone (PTH) is an important peptide hormone regulator of bone formation and osteoblast activity. However, its mechanism of action in bone cells is largely unknown. This study examined the effect of PTH on mouse osteocalcin gene expression in MC3T3-E1 preosteoblastic cells and primary cultures of bone marrow stromal cells. PTH increased the levels of osteocalcin mRNA 4 -5-fold in both cell types. PTH also stimulated transcriptional activity of a 1.3-kb fragment of the mouse osteocalcin gene 2 (mOG2) promoter. Inhibitor studies revealed a requirement for protein kinase A, protein kinase C, and mitogen-activated protein kinase pathways in the PTH response. Deletion of the mOG2 promoter sequence from ؊1316 to ؊116 caused no loss in PTH responsiveness whereas deletion from ؊116 to ؊34 completely prevented PTH stimulation. Interestingly, this promoter region does not contain the RUNX2 binding site shown to be necessary for PTH responsiveness in other systems. Nuclear extracts from PTH-treated MC3T3-E1 cells exhibited increased binding to OSE1, a previously described osteoblast-specific enhancer in the mOG2 promoter. Furthermore, mutation of OSE1 in DNA transfection assays established the requirement for this element in the PTH response. Collectively, these studies establish that actions of PTH on the osteocalcin gene are mediated by multiple signaling pathways and require OSE1 and associated nuclear proteins.Parathyroid hormone (PTH) 1 is a major regulator of calcium homeostasis. PTH has catabolic and anabolic effects on osteoblasts and bone in vitro and in vivo, which depend on the temporal pattern of administration; continuous administration decreases bone mass whereas intermittent administration increases bone mass (1-4). PTH functions through the PTH-1 receptor, a G protein-coupled receptor that is expressed in osteoblasts (5-7). Binding of PTH to its receptor activates multiple intracellular signaling pathways that involve cyclic cAMP, inositol phosphates, intracellular Ca 2ϩ , protein kinases A and C (2), and the extracellular signal-regulated kinase/ mitogen-activated protein kinase (MAPK) pathway (8, 9).Osteocalcin (OCN), a 5700-dalton ␥-carboxyglutamic acidcontaining noncollagenous protein, is a major component of the bone extracellular matrix. Although the functions of OCN are poorly understood, gene deletion studies suggest possible functions in bone remodeling (10). The expression of OCN is regulated by a number of calcitropic hormones and growth factors including 1,25-dihydroxy vitamin D 3 (11-13), glucocorticoids (14, 15), PTH (16), bone morphogenetic proteins (17), basic fibroblast growth factor 2 (18), tumor necrosis factor-␣ (19), and transforming growth factor ␤ (20). A number of transcription factors that bind to specific regions of the osteocalcin gene promoter have also been identified. These factors include AP-1 family members (21, 22), MSX-2/HOX 8.1 (23, 24), DLX-5 (25), CCAAT/enhancer binding proteins (26), and RUNX2 (27), the osteoblast-specific product of the Cbfa1 gene. These f...

mentioning

confidence: 99%

Parathyroid Hormone Induction of the Osteocalcin Gene

Jiang

Franceschi

Boules

et al. 2004

“…Diverse cellular and molecular mechanisms contribute to GC-mediated inhibition of osteoblastic bone formation (reviewed in Refs. 3 and 4), including: (a) attenuation of osteoblast proliferation (7)(8)(9)(10) and especially of a differentiation-related cell cycle (11,12); (b) promotion of osteoblast apoptosis (2); (c) abrogation of collagen metabolism (13,14); (d) inhibition of growth factors, such as insulin-like growth factor-1 and bone morphogenetic protein (BMP)-2 (15,16); and (e) inhibition of the osteoblast master transcription factor Runx2, also known as core-binding factor ␣1 and AML3 (17).…”

mentioning

confidence: 99%

Brief Bone Morphogenetic Protein 2 Treatment of Glucocorticoid-inhibited MC3T3-E1 Osteoblasts Rescues Commitment-associated Cell Cycle and Mineralization without Alteration of Runx2

Luppen

Leclerc

Noh

et al. 2003

Self Cite

Glucocorticoids (GCs) inhibit bone formation in vivo.In MC3T3-E1 osteoblasts, chronic administration of 1 M dexamethasone (DEX) starting at confluency results in >98% inhibition of bone morphogenetic protein 2 (BMP-2) expression and apatite mineral deposition. Here, it is shown that brief exposure to recombinant human BMP-2 (rhBMP-2), as short as 6 h, is sufficient to induce irreversible commitment to mineralization in DEX-treated cultures. RhBMP-2 dose dependently rescued mineralization but not collagen accumulation in DEX-inhibited cultures. The selective restoration of mineralization was evident in the higher mineral to matrix ratios of DEX/rhBMP-2 co-treated cultures compared with control. We tested the involvement of the runt-related transcription factor 2 (Runx2) in the DEX inhibition and rhBMP-2 rescue of mineralization. Surprisingly, DEX did not decrease Runx2 DNA binding activity, transactivation, or association with the endogenous osteocalcin gene promoter. Furthermore, the rh-BMP-2 rescue did not involve Runx2 stimulation, suggesting an important role for factors other than Runx2 in BMP-2 action. Finally, we studied the differentiationrelated cell cycle, which persists during commitment to mineralization in untreated cultures, but is inhibited along with mineralization in DEX-treated cultures. Although both rhBMP-2 alone and DEX alone were antimitogenic, rhBMP-2 stimulated this cell cycle in DEXinhibited cultures. In conclusion, brief rhBMP-2 treatment restores mineralization in DEX-inhibited MC3T3-E1 osteoblasts via a mechanism different from Runx2 stimulation. This restoration may be functionally related to the accompanying rescue of the differentiation-related cell cycle. The efficacy of short term BMP-2 treatment supports the potential of short-lived BMP vectors for skeletal therapy in both traditional and gene therapeutic approaches. Glucocorticoids (GCs)1 are potent anti-inflammatory agents for the treatment of diseases such as rheumatoid arthritis, systemic lupus erythematosus, asthma, and some types of cancer. A major side effect of GC treatment is rapid bone loss and increased risk for fracture (1). The chief mechanism underlying bone loss during long term GC treatment is impairment of bone formation (2-4). In addition to inducing bone loss, GCs have been shown to impede fracture healing in animal models (5, 6), potentially via molecular mechanisms partly shared with GCinduced osteoporosis. Diverse cellular and molecular mechanisms contribute to GC-mediated inhibition of osteoblastic bone formation (reviewed in Refs. 3 and 4), including: (a) attenuation of osteoblast proliferation (7-10) and especially of a differentiation-related cell cycle (11, 12); (b) promotion of osteoblast apoptosis (2); (c) abrogation of collagen metabolism (13, 14); (d) inhibition of growth factors, such as insulin-like growth factor-1 and bone morphogenetic protein (BMP)-2 (15, 16); and (e) inhibition of the osteoblast master transcription factor Runx2, also known as core-binding factor ␣1 and AML3 (17).Investigation...

“…The expression of osteocalcin is regulated by a variety of factors, including parathyroid hormone (PTH) 1 (6), 1,25-dihydroxy vitamin D 3 (4, 7), estrogens (8), glucocorticoids (9), growth factors (10,11), and cAMP (3,6,10). The osteocalcin promoter region contains a number of potential regulatory sequences that may be responsive to these factors.…”

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confidence: 99%

Activation of Osteocalcin Transcription Involves Interaction of Protein Kinase A- and Protein Kinase C-dependent Pathways

Boguslawski

Hale

et al. 2000

Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, synthesized and secreted exclusively by osteoblastic cells in the late stage of maturation, and is considered indicator of osteoblast differentiation. Osteocalcin expression is modulated by parathyroid hormone (PTH) and a variety of other factors. The cAMP-dependent protein kinase pathway has been shown previously to have an essential role in PTH signaling and regulation of osteocalcin expression. To determine the extent to which other pathways may also participate in osteocalcin expression, we used rat and human osteoblast-like cell lines to generate stably transfected clones in which the osteocalcin promoter was fused to a luciferase reporter gene. These clones were examined for their responsiveness to agents known to activate or interfere with protein kinase A (PKA)-and protein kinase C (PKC)-dependent pathways. We have found that forskolin, cAMP, and PTH, as well as insulinlike growth factor I (IGF-I) and basic fibroblast growth factor, all were effective in activating the osteocalcin promoter. Phorbol 12-myristate 13-acetate (PMA) was also a strong inducer of the promoter, indicating that PKC plays a role in expression of osteocalcin. In combination with PTH or forskolin, the effect of PMA was additive to synergistic. Calphostin C, a selective inhibitor of PKC, decreased the PMA-, PTH-, and IGF-I-induced luciferase activity in a dose-dependent manner; a PKA inhibitor, H-89, also blocked the induction by PTH and IGF-I but not by PMA. We conclude that regulation of osteocalcin transcription is mediated by both PKAdependent and PKC-dependent mechanisms and that the respective kinases reside on a linear or convergent pathway.