Using our new culture system for multinucleate cells (MNCs) that have many characteristics of osteoclasts, we examined the effects of factors produced by osteoblastic cells on osteoclastic cell formation. Conditioned medium (CM) from undifferentiated osteoblastic MC3T3-E1 cells during their growth phase inhibited MNC formation in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Diluted CM (1:81) from differentiated cells obtained after cultivation for more than 20 days stimulated MNC formation, but at lower dilutions inhibited their formation. Dialyzed CM (greater than 2000 mol wt) from the differentiated cells was more stimulatory than undialyzed CM and showed no inhibitory effect on MNC formation. The inhibitory effect was observed with filtered (less than 3000 mol wt) CMs and was specific for osteoblastic cell CM. Prostaglandin E2 (PGE2) was detected in the CM from undifferentiated or differentiated MC3T3-E1 cells at concentrations (317 +/- 66 and 1287 +/- 179 pg/ml, respectively) sufficient to inhibit MNC formation, and this inhibition was partially abolished with CM (at 3-fold dilution) in indomethacin-treated cells (PGE2, less than 20 pg/ml), suggesting PGE2-mediated inhibition of MNC formation and the presence of another factor(s) besides PGE2 that influenced MNC formation. In contrast to day 3 CM plus 1,25-(OH)2D3, day 60 CM plus 1,25-(OH)2D3 induced MNC formation even in the absence of GM-CSF, and this induction was inhibited by an antibody to GM-CSF. Secondary colony formation assays showed the presence of a GM-CSF-like factor in the day 60 CM. These findings indicate that osteoblastic cells are involved in the process of osteoclastic cell formation, with at least two soluble factors produced by osteoblasts, a GM-CSF-like factor, which is stimulatory, and PGE2, which is inhibitory. The effects of CMs also differed depending on the stage of osteoblast differentiation.

The biological activity of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone, and three intermediate metabolites of the lactone in vivo and in vitro was comparatively examined. The three intermediate metabolites, 1 alpha,25(R)26(OH)3D3, 1 alpha,23(S)25(R)26(OH)4D3, and 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactol, stimulated increases, as did 1 alpha,25(OH)2D3, in intestinal calcium transport and serum calcium level in vitamin D-deficient rats fed a low-calcium diet. On the other hand, 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone increased the calcium transport but decreased the serum calcium level. 1 alpha,25(OH)2D3,23(S)25(R)-Lactone and the other three metabolites stimulated multinucleate cell formation from hematopoietic blast cells in a manner correlated with their binding affinities for the 1 alpha,25(OH)2D3 receptor. But 23(S)25(R)-lactone did not show any inhibitory effect on the multinucleate cell formation induced by 1 alpha,25(OH)2D3 in contrast to the results obtained from unfractionated marrow cultures. Conditioned medium obtained from 23(S)25(R)-lactone-treated MC3T3-E1 cells inhibited the formation, probably by the action of some inhibitory factors elaborated by the cells treated with the lactone, whereas conditioned medium obtained from 1 alpha,25(OH)2D3 or other metabolite-treated MC3T3-E1 cells stimulated the formation. These findings suggest that 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone might inhibit bone resorption through an inhibition of osteoclastic cell formation and that other vitamin D3 metabolites stimulate bone resorption by development of new osteoclastic cells in addition to indirect osteoclast activation.

SProstaglandins (PG' s) are well known as an important local regulator of bone metabolism. In this study, we examined to characterize the effects of PGs on osteoblasts, using a clonal osteoblastic MC3T3-E1 cells. Among PG metabolites, PGE2 is a main prostanoid released in bone tissues. MC3T3-E 1 cells also produced predominantly PGE2. PG E2 at low doses (1-100 ng/ml) and PGE1 increased activity of alkaline phosphatase (ALP), an marker enzyme of early differentiation of osteoblasts, with positive correlation of elevating intracellular cAMP content. The stimulatory effects are amplified by the addition of isobutylmethyl xanthine (IBMX) and mimicked those of forskolin, a direct activator of adenylate cyclase, those results suggest that PGE2 at low doses and PGE1 act predominantly on adenylate cyclase to stimulate the early differntiation of the cells. On the other hand, PGE2 at high doses (50.0-2000 ng/ml) and PGF2a stimulated DNA synthesis of the cells in a dose-related manner. In the same range of concentrations, PGE2 and PGF2, augmented the accumulation of inositol triphosphates. Further, the effect of these PGs on the DNA synthesis is negated by addition of H-7, a potent inhibitor of protein kinase C. These date suggest that PGE2 at high doses and PGF2, stimulate the proliferation of the cells via enhance of phosphatidyl inositol (PI) turnover system and following activation of protein kinase C. Since PGE2 reveals diverse effects on the cells dependent on its concentration, it is difficult to clarify the mechanism of PGE2 action. Thus, we chose PGF2. to elucidate the stimulatory effect of PGs on the prolferation of the cells. At least 12h time-lag was present between PG F2.-signal transduction and an increase in DNA synthesis, and a-amanitin and cyclohexamide counteracted the effects, suggesting that some proteins involved in DNA synthesis are produced by the addition of PGF2, in the duration. Further, neutralizing anti IGF-I antibody blocked the stimulation of DNA synthesis by PGF2,. However, PGF2. didn't affect the endogenous production of IGF-I of the cells. On the other hand, PGF2, greatly elevated level of IGF-I binding sites on the cells, and the increase appeared bout 3h earlier than did the stimulation of DNA synthesis, indicating increase in responsiveness of the cells to IGF-I. These results suggest that the proliferation of the cells is stimulated by synergistic action of PGF2, and IGF-I produced endogenously.

Amylase activity detected in thyroid extracts was significantly higher than that of normal sera. A starch film technique revealed the existence of amylase activity in the follicular lumen and on the follicular epithelia. By electrophoretic analysis of thyroid extracts, 4 bands of amylase activity were observed, one being of the same mobility as parotid and the other 3 more anodic. Amylase extracted from the thyroid appeared in the same position as pancreatic or parotid amylase on Sephadex G75 gel filtration. The possibility is discussed that the thyroid may synthesize amylase of salivary type, which is secreted from the follicular epithelia into the follicular lumen, where it may be transformed into anionic forms.

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