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...
We investigated the role of Lef1, one of the four transcription factors that transmit Wnt signaling to the genome, in the regulation of bone mass. Microcomputed tomographic analysis of 13- and 17-week-old mice revealed significantly reduced trabecular bone mass in Lef1+/− females compared to littermate wild-type females. This was attributable to decreased osteoblast activity and bone formation as indicated by histomorphometric analysis of bone remodeling. In contrast to females, bone mass was unaffected by Lef1 haploinsufficiency in males. Similarly, females were substantially more responsive than males to haploinsufficiency in Gsk3β, a negative regulator of the Wnt pathway, displaying in this case a high bone mass phenotype. Lef1 haploinsufficiency also led to low bone mass in males lacking functional androgen receptor (AR) (tfm mutants). The protective skeletal effect of AR against Wnt-related low bone mass is not necessarily a result of direct interaction between the AR and Wnt signaling pathways, because Lef1+/− female mice had normal bone mass at the age of 34 weeks. Thus, our results indicate an age- and gender-dependent role for Lef1 in regulating bone formation and bone mass in vivo. The resistance to Lef1 haploinsufficiency in males with active AR and in old females could be due to the reduced bone turnover in these mice.
Objective. Glucocorticoids are widely used for the management of rheumatoid arthritis. Osteoporosis is a major side effect of glucocorticoid therapy and is attributable to inhibition of bone formation. We developed an osteoblast culture system in which glucocorticoids strongly inhibit development of the osteoblast phenotype, including expression of the bone-specific osteocalcin (OC) gene. Using this gene as a model, the goal of this study was to discover glucocorticoid-sensitive transcriptional mechanisms in osteoblasts.Methods. Dexamethasone (DEX; 1 M) was administered to murine MC3T3-E1 osteoblastic cultures under conditions that inhibit mineralized extracellular matrix formation and OC messenger RNA levels by >10-fold. Because standard (short-term) transient transfection assays with OC promoter-reporter constructs did not recapitulate the strong DEX-mediated repression, mapping of OC negative glucocorticoid response elements (GREs) was performed initially by stable transfection and then with long-term transient transfection assays. Transcription factor binding to the OC negative GRE was studied by electrophoretic mobility shift assays.Results. Several-fold repression of OC-luciferase constructs was recapitulated in stable and long-term transient transfection assays, in which the transfected cells were allowed to progress to a sufficiently advanced developmental stage. Analysis of a 5 promoter deletion series mapped an OC negative GRE to a 15-bp G/C-rich motif (؊161/؊147) located just upstream of the binding site for the osteoblast master transcription factor Runx2. Oligonucleotides encompassing this element and MC3T3-E1 cell extracts formed a protein-DNA complex that contained an Egr/Krox family member(s). Complex formation was competed by either an oligonucleotide containing 2 consensus Egr motifs or by anti-Egr2/Krox20 antibodies. Three copies of this Kroxbinding element conferred 20-fold transcriptional activation on the 147-bp basal OC promoter in osteoblasts, and the enhancer activity was inhibited by DEX. Enhancer activity was not observed in 10T1/2 fibroblasts unless these cells were cotransfected with Runx2.Conclusion. An Egr2/Krox20-binding site located immediately upstream of the Runx2 site of the mouse OC promoter was identified as an enhancer in osteoblasts, whose activity is repressed by glucocorticoids. Sequence similarity suggests that such a mechanism is likely operative in both murine and human cells. Because glucocorticoids inhibit Egr2/Krox20 expression in osteoblasts, and because trabecular bone formation is arrested in Egr2/Krox20-knockout mice, the inhibition of Egr2/Krox20 activity likely contributes to glucocorticoid-induced osteoporosis.Since the 1950 Nobel laureates E. Kendall, T. Reichstein, and P. Hench introduced glucocorticoids for the treatment of rheumatoid arthritis, these drugs have been widely used for the treatment of autoimmune and inflammatory diseases. The enthusiasm with which glucocorticoids were initially accepted was quickly dampened upon realization of their deleter...
In osteoblasts, the mitogen-activated protein kinases ERK1/2 and p38 as well as the cAMP-response element-binding protein (CREB) have been implicated in the regulation of proliferation and differentiation. The osteogenic growth peptide (OGP) is a 14-mer bone cell mitogen that increases bone formation and trabecular bone density and stimulates fracture healing. OGP-(10 -14) is the physiologically active form of OGP. Using gene array analysis, real-time reverse transcription-PCR, and immunoblot and DNA synthesis assays we show here that in MC3T3 E1 and newborn mouse calvarial osteoblastic cultures the OGP-(10 -14) mitogenic signaling is critically dependent on de novo synthesis of mitogen-activated protein kinase-activated protein kinase 2 (Mapkapk2) mRNA and protein.The increase in Mapkapk2 occurs following short term (5-60 min) stimulation of ERK1/2 activity by OGP-(10 -14); phosphorylation of p38 remains unaffected. Downstream of Mapkapk2, CREB is phosphorylated on Ser 133 leading to its enhanced transcriptional activity. That these events are critical for the OGP-(10 -14) mitogenic signaling is demonstrated by blocking the effects of OGP-(10 -14) on the ERK1/2 pathway, Mapkapk2, CREB, and DNA synthesis using the MEK inhibitor PD098059. The OGP-(10 -14) stimulation of CREB transcriptional activity and DNA synthesis is also blocked by Mapkapk2 siRNA. These data define a novel mitogenic signaling pathway in osteoblasts whereby ERK1/2 stimulation of CREB phosphorylation and transcriptional activity as well as DNA synthesis are critically dependent on de novo Mapkapk2 synthesis.In mammalian cells, the family of mitogen-activated protein (MAP) 3 kinases provides a key link between membrane-bound receptors and changes in the pattern of gene expression. The MAP kinases are activated downstream of many different types of receptors, including tyrosine kinase receptors, cytokine receptors, and serpentine G-protein coupled receptors (1, 2). The MAP kinases consist of four subfamilies: the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase/stress-activated kinase, p38 MAP kinase, and ERK 5. Further downstream, they regulate a multitude of transcription factors that control cell proliferation, survival, and differentiation (3, 4). In osteoblasts, ERK1/2-dependent phosphorylation cascades have been implicated in the regulation of proliferation and RUNX2 activity (5, 6). Activation of p38 has been demonstrated in osteoblasts undergoing differentiation after stimulation with bone morphogenetic protein-2 and epidermal growth factor (7,8).The osteogenic growth peptide (OGP) is a 14-mer bone cell mitogen that increases bone formation and trabecular bone density and stimulates fracture healing when administered to mice and rats (9 -11). Transgenic mice overexpressing OGP have a markedly increased peak bone mass (12). OGP is present in mammalian serum in micromolar concentrations mainly complexed to ␣ 2 -macroglobulin (13). Upon its dissociation from the complex, it is proteolytically activated yieldin...
Like alternative splicing, leaky ribosomal scanning (LRS), which occurs at suboptimal translational initiation codons, increases the physiological flexibility of the genome by allowing alternative translation. Comprehensive analysis of 22 208 human mRNAs indicates that, although the most important positions relative to the first nucleotide of the initiation codon, −3 and +4, are usually such that support initiation (A−3 = 42%, G−3 = 36% and G+4 = 47%), only 37.4% of the genes adhere to the purine (R)−3/G+4 rule at both positions simultaneously, suggesting that LRS may occur in some of the remaining (62.6%) genes. Moreover, 12.5% of the genes lack both R−3 and G+4, potentially leading to sLRS. Compared with 11 genes known to undergo LRS, 10 genes with experimental evidence for high fidelity A+1T+2G+3 initiation codons adhered much more strongly to the R−3/G+4 rule. Among the intron-less histone genes, only the H3 genes adhere to the R−3/G+4 rule, while the H1, H2A, H2B and H4 genes usually lack either R−3 or G+4. To address in vivo the significance of the previously described LRS of H4 mRNAs, which results in alternative translation of the osteogenic growth peptide, transgenic mice were engineered that ubiquitously and constitutively express a mutant H4 mRNA with an A+1→T+1 mutation. These transgenic mice, in particular the females, have a high bone mass phenotype, attributable to increased bone formation. These data suggest that many genes may fulfill cryptic functions by LRS.
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