Bone morphogenetic protein 2 (BMP-2) is a potent inducer of differentiation of osteoblasts both in vivo and in vitro. We examined the action of BMP-2 on expression of a helix-oop-helix-type transcription factor, Id (inhibitor of differentiation), in osteoblast-like cells, as well as in osteoblastenriched cells and possible precursor cells. To our surprise, BMP-2 enhanced Id gene expression in the cell types of osteoblastic lineage we examined. The mimal BMP-2 enhancement was observed within 24 hr in early proliferating cultures and the enhancement lasted up to 96 hr. The BMP-2 effect was not blocked by actinomycin D, while it was blocked by cycloheximide, suggesting that BMP-2 regulates Id gene expression at least in part via posttranscriptional events, which require protein synthesis. Other experiments indicated that BMP-2 did not further enhance Id mRNA levels promoted by dexamethasone, while BMP-2 did not resume the Id mRNA levels suppressed by 1,25-dihydroxyvitamin D3. Similar BMP-2 enhancement of Id message expression was also observed in osteoblast-enriched fetal rat calvaria cells as well as C3H1OT1/2 cells. These results indicate that BMP-2 enhances expression of Id in early cultures of osteoblastic cells and suggest that enhancement of Id expression may somehow be involved in the promotion of differentiation by this cytokine in these osteoblastic cells and in their precursor cells. Wozney et al. (7). BMP was first described by Urist (8) as a proteinaceous activity present in demineralized bone matrix, which, as mentioned above, induces ectopic bone formation. So far, at least eight BMPs are reported of which seven are members of the transforming growth factor type /3 (TGF-P3) superfamily (9). Once such molecules have been identified, the next obvious step is to deduce the intracellular mechanism(s) through which BMP-2 induces osteoblastic differentiation. BMPs bind to distinct cell-surface receptors, whose sizes are similar to TGF-p receptors (10). Since receptors for both TGF-l3 (11) (27). Briefly, the cells were rinsed with phosphate-buffered saline three times, followed by extraction in 4 M guanidinium isothiocyanate, homogenized, and then extracted with acid phenol. The aqueous phase was combined with 1-propanol and the RNA was precipitated, rinsed with 75% ethanol, and resuspended in a buffer containing Tris'HCl (pH 7.4), 1 mM EDTA, and 0.1% SDS. The RNA was quantitated by spectrophotometry at 260 and 280 nm. Total RNA (10 or 20 ug) was fractionated by electrophoresis in 1% agarose/formaldehyde gels. Northern blot analysis was carried out as described (21). Id cDNA was kindly provided by Harold Weintraub.Measurements of Alkaline Phosphatase Activity and DNA Content. Cells were plated in 2-cm2 wells, rinsed twice with

It is widely accepted that mechanical loading is necessary to construct the architecture of bone and to maintain bone mass. However, the mechanism of how bone cells respond to mechanical stimuli is not known. To clarify this, we stimulated osteoblast-like MC3T3E1 cells by mechanical shaking of the culture dishes and found that the level of the egr-1 gene, which is an early response gene induced by growth factors or serum and encodes a transcription factor, increased 15-45 min after the shaking, with a peak at 30 min. The egr-1 gene product increased 1 h after the shaking. The egr-1 gene elevation was not blocked by prior exposure to indomethacin, saralasin, Rp-cAMP, A23187, and colchicine, and it was blocked partially by cytochalasin D, H-7, and prolonged exposure to TPA. On the other hand, a prior incubation with cycloheximide, DRB, genistein, herbimycin A, and BAPTA/AM completely blocked the egr-1 gene level enhanced by shaking the culture dishes. Moreover, we found that in serum-deprived cells the egr-1 gene response to shaking was not induced. These results suggested that the egr-1 gene response is regulated at the transcriptional level and that it involves tyrosine kinase as well as labile or de novo protein and requires a particular level of intracellular calcium and serum.

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To investigate how mechanical loading stimulates bone cells, we subjected murine osteoblast-like cells, MC3T3E1, to fluid flow generated by shaking culture dishes. Since we had previously found that egr-1 mRNA is up-regulated by the flow, and that the regulation involves tyrosine kinase, we examined which proteins are tyrosine-phosphorylated by flow. Western blotting and immunoprecipitation of cell lysates showed tyrosine phosphorylation enhancement of many proteins, including ERK2 and Shc, and activation of ERK1/2. Although these responses did not occur in serum-free media, addition of EGF or bFGF recovered the responses. AG1478, an inhibitor of EGF receptor kinase activity, abolished tyrosine phosphorylation enhancement, ERK1/2 activation, and egr-1 mRNA accumulation induced by the flow of EGF-containing serum-free media. These results suggest that growth factor signaling pathways are involved in these responses. Repetition of fluid flow induced repeatedly up-regulation of egr-1 mRNA. Such events may also occur in bone under mechanical loading.

To investigate how mechanical loading stimulates bone cells, we subjected murine osteoblast-like cells, MC3T3E1, to fluid flow generated by shaking culture dishes. Since we had previously found that egr-1 mRNA is up-regulated by the flow, and that the regulation involves tyrosine kinase, we examined which proteins are tyrosine-phosphorylated by flow. Western blotting and immunoprecipitation of cell lysates showed tyrosine phosphorylation enhancement of many proteins, including ERK2 and Shc, and activation of ERK1/2. Although these responses did not occur in serum-free media, addition of EGF or bFGF recovered the responses. AG1478, an inhibitor of EGF receptor kinase activity, abolished tyrosine phosphorylation enhancement, ERK1/2 activation, and egr-1 mRNA accumulation induced by the flow of EGF-containing serum-free media. These results suggest that growth factor signaling pathways are involved in these responses. Repetition of fluid flow induced repeatedly up-regulation of egr-1 mRNA. Such events may also occur in bone under mechanical loading.