Estrogen inhibits bone resorption by directly inducing apoptosis of the bone-resorbing osteoclasts.

Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 beta-estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor- mediated mechanism.

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.

Induction of osteoblastic cell differentiation by forskolin. Stimulation of cyclic AMP production and alkaline phosphatase activity.

The effects of forskolin on differentiation of osteoblastic cells (clone MC3T3-E1) cultured in alpha-minimum essential medium containing 0.1% bovine serum albumin were investigated by assays of intracellular cyclic AMP level and alkaline phosphatase activity in the cells. Forskolin increased cyclic AMP production in the cells in a dose-related manner, the maximum increase being 250-fold above that of the controls. Alkaline phosphatase activity in the cells was also elevated as early as 24 h and rose to nearly its maximum at 48 h. The elevation was dose-dependent, with a maximum increase at 5 X 10(-6) M forskolin. Forskolin and prostaglandin E2 showed a supraadditive effect on cyclic AMP production in the cells and had an additive effect on alkaline phosphatase activity, whereas forskolin and dibutyryl cyclic AMP had little additive effect on either cyclic AMP production or enzyme activity. These results suggest that cyclic AMP is closely linked to the differentiation of osteoblastic cells in vivo.

Basic fibroblast growth factor regulates IGF-I binding proteins in the clonal osteoblastic cell line MC3T3-E1.

In previous studies, we reported that basic fibroblast growth factor (bFGF) regulates insulin-like growth factor messenger RNAs and protein levels in the osteoblastic MC3T3-E1 cells. In the present study, we examined the expression of insulin-like growth factor binding proteins (IGFBPs) in MC3T3-E1 cells and determined whether bFGF altered IGFBP mRNAs and protein levels. Since previous studies suggested that IGFBPs can inhibit DNA synthesis stimulated by IGF-I, we wondered whether the mitogenic effect of bFGF was altered by exogenous IGFBP-3. Confluent MC3T3-E1 cells were serum-deprived for 24 h and then treated with bFGF for 6-24 h. In control cultures, MC3T3-E1 cells expressed the mRNAs for IGF-I, IGF-II, and IGFBP-2, 4, 5, and 6 but not IGFBP-1 or 3. A 24 h treatment with bFGF at 10(-8) M decreased IGF-I mRNA by 97%, IGF-II mRNA by 73%, IGFBP-2 by 64%, IGFBP-4 by 73%, IGFBP-5 by 95%, and IGFBP-6 by 65%. The inhibitory effect of bFGF on IGF-I and IGFBP mRNA levels was not altered by aphidicolin, an inhibitor of cell replication. bFGF 10 nM decreased IGF-I levels determined by radioimmunoassay after acidification by 45% and 72% at 24 and 48 h, respectively. Western ligand blot for IGF binding proteins revealed that MC3T3-E1 cells expressed IGFBPs of 24, 30, and 34 kD. Treatment with bFGF 10(-8) M decreased the levels of the 24 and 30 kD band at 24 h but increased the 34 kD band.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct and indirect actions of fibroblast growth factor 2 on osteoclastic bone resorption in cultures.

Fibroblast growth factor 2 (FGF-2 or basic FGF) is known to show variable actions on bone formation and bone resorption. This study was undertaken to elucidate the mechanisms whereby FGF-2 affects bone metabolism, especially bone resorption, using three different culture systems. FGF-2 at 10(-9) M and higher concentrations induced osteoclastic cell formation in the coculture system of mouse osteoblastic cells and bone marrow cells, and this induction was abrogated by nonsteroidal anti-inflammatory drugs (NSAIDs). 45Ca release from prelabeled cultured mouse calvariae stimulated by FGF-2 (10(-8) M) was also inhibited by NSAIDs, and the inhibition was stronger by NSAIDs, which are more selective for inhibition of cyclooxygenase 2 (COX-2) than COX-1, suggesting the mediation of COX-2 induction. COX-2 was highly expressed and its messenger RNA (mRNA) level was stimulated by FGF-2 in osteoblastic cells whereas it was undetectable or not stimulated by FGF-2 in cells of osteoclast lineage. To further investigate the direct actions of FGF-2 on osteoclasts, resorbed pit formation was compared between cultures of purified osteoclasts and unfractionated bone cells from rabbit long bones. FGF-2 (> or = 10(-12) M) stimulated resorbed pit formation by purified osteoclasts with a maximum effect of 2.0-fold at 10(-11) M, and no further stimulation was observed at higher concentrations. However, FGF-2 at 10(-9) M - 10(-8) M stimulated resorbed pit formation by unfractionated bone cells up to 9.7-fold. NS-398, a specific COX-2 inhibitor, did not affect the FGF-2 stimulation on purified osteoclasts but inhibited that on unfractionated bone cells. We conclude that FGF-2 at low concentrations (> or =10(-12) M) acts directly on mature osteoclasts to resorb bone moderately, whereas at high concentrations (> or = 10(-9) M) it acts on osteoblastic cells to induce COX-2 and stimulates bone resorption potently.

Transient recruitment of osteoclasts and expression of their function in osteopetrotic (op/op) mice by a single injection of macrophage colony-stimulating factor.

Severe deficiency of osteoclasts in op/op mice, caused by the absence of functional macrophage colony-stimulating factor (M-CSF), is cured by daily injections of purified recombinant human M-CSF (rhM-CSF). In this study, we found that a single injection of 5 micrograms rhM-CSF is enough for recruitment of osteoclasts in mutant mice. Osteoclast number increased during the period between 2 and 4 days after the single rhM-CSF injection. When YM175, a new derivative of bisphosphonate, was administered to the mice 4 days after rhM-CSF injection or later, osteoclasts disappeared by 3 days after YM175 administration. However, a significant number of osteoclasts were detected even at 3 days after YM175 administration when YM175 was administered 3 days after rhM-CSF injection or earlier. These results indicate that YM175 is cytotoxic only to functioning osteoclasts and that recruitment of osteoclasts is finished 4 days after a single rhM-CSF injection. The osteoclasts actively resorbed bone trabeculae for a prolonged period, demonstrating that M-CSF is not requisite for the functioning of mature osteoclasts.

Tissue inhibitor of metalloproteinases 1 and 2 directly stimulate the bone-resorbing activity of isolated mature osteoclasts.

Tissue inhibitor metalloproteinases 1 (TIMP-1) and 2 have been reported to inhibit bone resorption. However, here, we report the direct action of both TIMP-1 and TIMP-2 on isolated rabbit mature osteoclasts to stimulate their bone-resorbing activity at significantly lower concentrations (approximately ng/ml) than those (approximately microg/ml) required for the inhibition of bone resorption. The cell population used in this study consisted of a mature osteoclast population with >95% purity. TIMP-1 (approximately 50 ng/ml) and TIMP-2 (approximately 8-10 ng/ml) increased the pit area excavated by the isolated mature osteoclasts. The stimulatory effects of TIMPs were abolished by simultaneous addition of anti-TIMP antibodies. At higher concentrations, the stimulation of bone resorption decreased reversely to the control level. The magnitude of the stimulatory effect of TIMP-2 was more than that of TIMP-1. Metalloproteinase inhibitors such as BE16627B and R94138 could not replace TIMPs with respect to the bone-resorbing activity, suggesting that the osteoclast-stimulating activity of TIMPs was independent of the inhibitory activity on matrix metalloproteinases (MMPs). TIMPs stimulated tyrosine phosphorylation of cellular proteins in the isolated mature osteoclasts. Both herbimycin A, an inhibitor of tyrosine kinases, and PD98059 and U0126, inhibitors of mitogen-activated protein kinase (MAPK), completely blocked the TIMP-induced stimulation of osteoclastic bone-resorbing activity. On the plasma membrane of osteoclasts, some TIMP-2-binding proteins were detected by a cross-linking experiment. These findings show that TIMPs directly stimulate the bone-resorbing activity of isolated mature osteoclasts at their physiological concentrations and that the stimulatory action of TIMPs is likely to be independent of their activities as inhibitors of MMPs.

Down-regulation of gap junctional intercellular communication between osteoblastic MC3T3-E1 cells by basic fibroblast growth factor and a phorbol ester (12-O-tetradecanoylphorbol-13-acetate).

To address the relation between osteoblast growth and cell-to-cell communication, we examined the effects of basic fibroblast growth factor (bFGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), both potent stimulators of osteoblastic proliferation, on gap junctional intercellular communication between osteoblastic MC3T3-E1 cells. The level of intercellular communication was estimated by a photobleaching method. TPA inhibited the degree of intercellular communication in two different time-dependent manners. The early (< 1 h) inhibition by TPA was consistent with an increase in the phosphorylation of connexin 43 (Cx43). The later inhibition was caused by reduction in the total amount of Cx43 on the plasma membrane, due to the decrease in the level of Cx43 transcripts. These qualitative and quantitative modulations by TPA were inhibited by a selective inhibitor of protein kinase C, GF109203X. bFGF also attenuated the gap junctional intercellular communication. However, short exposure (< 5 h) to bFGF did not affect the communication. The fact that the growth factor immediately stimulated the phosphorylation of Cx43 indicates that the phosphorylation site(s) affected by bFGF was not involved in the inhibition of communication. The decrease in the intercellular communication level was detected by the longer exposure (> 8 h) to bFGF and paralleled the decline in the Cx-mRNA level. This inhibitory effect of bFGF was abolished by the addition of a tyrosine kinase inhibitor, herbimycin A. Thus, gap junctional intercellular communication between osteoblasts was down-regulated by osteoblastic mitogens through different mechanisms of the modulation of Cx43.

Regulation of the two prostaglandin G/H synthases by parathyroid hormone, interleukin-1, cortisol, and prostaglandin E2 in cultured neonatal mouse calvariae.

A second prostaglandin G/H synthase (PGHS-2), encoded by a gene separate from that for the original PGHS (PGHS-1), has recently been identified. We have shown that PGHS-2 is expressed in cultured mouse calvariae and have compared regulation of PGHS-2 and PGHS-1 messenger RNA (mRNA) levels. PGHS-2 mRNA was not detectable in freshly isolated bones, but was induced during culture and further stimulated by interleukin-1 (IL-1) and PTH. Both factors also increased PGHS-2 protein levels. Changes in medium prostaglandin E2 (PGE2) production correlated with increases in PGHS-2 mRNA levels. However, with IL-1, PGE2 production was increased more than PGHS-2 mRNA levels (treated/control ratio, 3.4 and 1.5, respectively), whereas with PTH there was a closer correspondence (2.0 and 2.1). Cortisol reduced PTH-stimulated PGE2 production (treated/control ratio decreased from 3.1 to 0.2) more than PGHS-2 mRNA levels (2.8 to 0.8). In the presence of exogenous arachidonic acid, changes in PGHS-2 mRNA levels with IL-1, PTH, and cortisol correlated closely with changes in PGE2 production. PGE2 itself increased PGHS-2 mRNA, and nonsteroidal antiinflammatory drugs decreased PGHS-2 mRNA levels by 80%. In contrast, PGHS-1 mRNA was expressed constitutively and was not affected by IL-1, PTH, or cortisol when measured by competitive reverse transcriptase-polymerase chain reaction. We conclude that regulation of PGE2 production is predominantly through PGHS-2, rather than PGHS-1; that IL-1 and cortisol may also regulate arachidonic acid release; and that PGE2 may amplify its own production through stimulation of PGHS-2.

Calcitonin inhibits osteopontin mRNA expression in isolated rabbit osteoclasts.

Recent evidence indicates that osteopontin (Opn), one of the bone matrix proteins, plays an important role in the attachment of osteoclasts to bone matrix. Besides being elaborated by osteoblasts, this protein is also produced by osteoclasts. The present study was performed to examine the effect of calcitonin (CT) on Opn mRNA expression of isolated rabbit osteoclasts and to clarify the second messenger signaling of this effect. Eel CT inhibited Opn mRNA expression as well as bone-resorbing activity of isolated rabbit osteoclasts. Eel CT caused a transient increase in intracellular calcium followed by a sustained increase as well as an increase in cAMP production in these cells. Dibutyryl-cAMP (10(-4) M) and Sp-cAMPS (10(-4) M), an activator of cAMP-dependent protein kinase (PKA), as well as A23187 (10(-7) M), a calcium ionophore, and phorbol myristate acetate (10(-7) M), an activator of protein kinase C (PKC), caused a significant inhibition of Opn mRNA expression, and suppressed bone-resorbing activity of isolated osteoclasts. The present study is the first to demonstrate that CT inhibits Opn mRNA expression in isolated rabbit osteoclasts, presumably through the activation of PKA and calcium/PKC pathways, by which the bone-resorbing activity might be attenuated subsequently.

Mouse osteoblastic cells (MC3T3-E1) at different stages of differentiation have opposite effects on osteoclastic cell formation.

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.

Prostaglandin F2alpha stimulates tyrosine phosphorylation and mitogen-activated protein kinase in osteoblastic MC3T3-E1 cells via protein kinase C activation.

PGF2alpha stimulates the proliferation of clonal osteoblastic MC3T3-E1 cells via PGF2alpha receptor linked to phospholipase C activation. To elucidate intracellular events elicited by this receptor, we examined the effects of PGF2alpha on tyrosine phosphorylation and mitogen-activated protein kinase (MAPK) activity in MC3T3-E1 cells. PGF2alpha rapidly raised the level of phosphotyrosine of cellular proteins with Mr values of 62, 68, 72, 76, 82, 125, and 150 kDa. This PGF2alpha-induced tyrosine phosphorylation of proteins (except for pp62) was blocked by down-regulating protein kinase C (PKC) by 12-O-tetradecanoylphorbol 13-acetate pretreatment and by GF 109203X, a potent specific PKC inhibitor. The addition of PGF2alpha also transiently activated MAPK in the same range of concentrations that stimulated tyrosine phosphorylation. In addition, PGF2alpha augmented the MAPK kinase kinase activity of Raf-1, whereas basal activity of MAPK/extracellular signal-regulated protein kinase kinase was less than that of Raf-1 and was little affected by PGF2alpha. Like the tyrosine phosphorylation, these activations of Raf-1 and MAPK activities were reduced by inhibition and down-regulation of PKC. Genistein, a potent inhibitor of tyrosine kinases, did not block the Raf-1 induced by PGF2alpha, indicating a tyrosine kinase-independent pathway for Raf-1 activation. However, the tyrosine kinase inhibitor partially inhibited the MAPK activity, suggesting an involvement of another Raf-1-independent kinase cascade for activation of MAPK by PGF2alpha. Fluprostenol, a specific agonist of PGF2alpha receptor, mimicked the actions of PGF2alpha consistent with a PGF2alpha receptor pathway. Thus, the action of PGF2alpha on osteoblastic MC3T3-E1 cells appears to involve a single receptor that uses diverse interacting signal transduction systems.

Biological activity assessment of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone and its intermediate metabolites in vivo and in vitro.

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.

Prostaglandin E1 and F2 alpha stimulate differentiation and proliferation, respectively, of clonal osteoblastic MC3T3-E1 cells by different second messengers in vitro.

The effect of several prostaglandins (PGs) on osteoblastic cells was investigated using clone MC3T3-E1 under serum-free conditions. PGA1, A2, B1, and B2 had little effect on intracellular cAMP, alkaline phosphatase (ALP) activity, and DNA synthesis in the cells. At 4-2000 ng/ml, PGE1 among PG analogs tested had a dose-dependent stimulatory effect on ALP activity in the cells, and this effect was amplified by isobutyl methylxanthine. Also, PGE1 strongly augmented the amount of intracellular cAMP over the same concentration range. However, PGE1 had little effect on ornithine decarboxylase activity and DNA synthesis, and at high doses it rather depressed DNA synthesis. Furthermore, PGE1 did not affect the intracellular cGMP level. The effect of PGE1 on the cells closely mimics that of forskolin, suggesting that the PG stimulates the differentiation of the osteoblastic cells predominantly via the stimulation of adenylate cyclase. In contrast with PGE1, PGF2 alpha strongly increased ornithine decarboxylase activity and DNA synthesis in the cells in a dose-related fashion at low concentrations (4-100 ng/ml), at which concentrations it had little effect on the intracellular cAMP or cGMP level and depressed ALP activity. Moreover, PGF2 alpha depressed the stimulatory effect of PGE1 on ALP activity but did not affect the elevation of cAMP level by PGE1. The accumulation of inositol phosphates was greatly increased by PGF2 alpha in the concentration range effective in stimulating DNA synthesis, but was increased little by PGE1, suggesting that PGF2 alpha is a potent stimulator of phosphatidyl inositol turnover in the cells. In addition, A23187, a Ca ionophore, alone did not influence the DNA synthesis, but the effects of tetradecanoyl phorbol acetate, a direct activator of protein kinase C, were very similar to those of PGF2 alpha. Moreover, the stimulation of DNA synthesis or the inhibition of ALP activity by PGF2 alpha was partially counteracted by H-7, a strong inhibitor of protein kinase C. These results suggest that PGF2 alpha stimulates the proliferation of osteoblastic cells predominantly through the phosphatidyl inositol turnover system following in part the activation of protein kinase C. Our data presented here indicate that PGE1 and PGF2 alpha are closely involved in the differentiation and proliferation, respectively, of osteoblasts in vitro and that their action may be mediated by second messengers which differ from each other.

Insulin-like growth factor-I supports formation and activation of osteoclasts.

Although the action of insulin-like growth factor-I (IGF-I) on bone formation has been extensively investigated, the effect of the factor on bone resorption is little known. We first examined the effect of IGF-I on bone resorption by preexistent osteoclasts by using unfractionated bone cells cultured on dentin slices. IGF-I had a dose-related effect of stimulating bone resorption by preexistent osteoclasts, whereas IGF-II did not. When IGF-I was added to cultures of bone cells after preexistent osteoclasts had degenerated on the dentin slices, IGF-I increased the number of osteoclastic multinucleate cells (MNCs) with tartrate-resistant acid phosphatase activity. Moreover, IGF-I augmented the area of pits produced by newly formed osteoclasts. These results suggest that IGF-I directly or indirectly stimulates osteoclast recruitment and activation. Therefore, we next examined the direct effect of IGF-I on osteoclastic MNC formation by using hemopoietic blast cells. In the presence of 1,25-dihydroxyvitamin D3, IGF-I, like granulocyte-macrophage colony-stimulating factor (GM-CSF), dose-dependently increased the number of TRAP-positive MNCs. This stimulatory effect of IGF-I was additive with that of GM-CSF. Both IGF-I and GM-CSF supported the survival of the blast cells, indicating that IGF-I as well as GM-CSF are supporting factors for osteoclast differentiation. In addition, the blast cells possessed high affinity binding sites for IGF-I, with a Kd of 0.8 nM. These data, thus, indicate that IGF-I stimulates osteoclastic bone resorption through its direct or indirect action of supporting the generation and activation of osteoclasts.

Effect of a highly potent fluoro analog of 1,25-dihydroxyvitamin D3 on human bone-derived cells.

The fluorine introduced analog of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 [26,27-F6-1,25-(OH)2D3] is 5-10 times more potent than 1,25-(OH)2D3 in vitamin D-deficient rats and chicks. In this study we established cultures of human bone cells in order to elucidate the mechanisms responsible for the higher activity of this compound. The effects of 26,27-F6-1,25-(OH)2D3 and 26,26,26,27,27,27-hexafluoro-1,23(S),25-trihydroxyvitamin D3[26,27-F6-1,23(S),25-(OH)3D3], the postulated main metabolite of 26,27-F6-1,25-(OH)2D3, were assessed by the response of alkaline phosphatase (ALP) activity. 26,27-F6-1,25-(OH)2D3 increased ALP activity in a dose-related fashion, from a concentration of 10(-11) M and caused a 3-fold elevation at a concentration of 10(-9) M. To achieve the same stimulating effect on ALP activity, the required dose of 26,27-F6-1,25-(OH)2D3 was 100 times less than that of 1,25-(OH)2D3. Analysis of the receptors of these cells revealed that they have specific receptors for 1,25-(OH)2D3, which have a dissociation constant of 0.9 x 10(-10) M. The competitive binding assays of 26,27-F6-1,25-(OH)2D3 on these receptors showed that binding ability of 26,27-F6-1,25-(OH)2D3 is almost the same as that of 1,25-(OH)2D3. Therefore, receptor binding affinity does not account for the higher potency of 26,27-F6-1,25-(OH)2D3. The trihydroxylated compound, 26,27-F6-1,23(S),25-(OH)3D3 revealed almost the same stimulatory activity on ALP activity in these cells. The most likely explanation for the higher activity of 26,27-F6-1,25-(OH)2D3 than 1,25-(OH)2D3 is that 26,27-F6-1,25-(OH)2D3 is metabolized to 26,27-F6-1,23(S),25-(OH)3D3, which has almost the same activity as 26,27-F6-1,25-(OH)2D3 in target tissues, whereas 1,25-(OH)2D3 is degraded to less active metabolites such as 1,24,25-(OH)3D3.

Vascular endothelial growth factor (VEGF) directly enhances osteoclastic bone resorption and survival of mature osteoclasts.

In bone development and regeneration, angiogenesis and bone/cartilage resorption are essential processes and are closely associated with each other, suggesting a common mediator for these two biological events. To address this interrelationship, we examined the effect of vascular endothelial growth factor (VEGF), the most critical growth factor for angiogenesis, on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. VEGF caused a dose- and time-dependent increase in the area of bone resorption pits excavated by the isolated osteoclasts, partially by enhancing the survival of the cells. Two distinct VEGF receptors, KDR/Flk-1 and Flt-1, were detectable in osteoclasts at the gene and protein levels, and VEGF induced tyrosine phosphorylation of proteins in osteoclasts. Thus, osteoclastic function and angiogenesis are up-regulated by a common mediator such as VEGF.

Stimulation of osteoblast proliferation by the cartilage-derived growth promoting factors chondromodulin-I and -II.

We previously reported the isolation of the cartilage-derived growth promoting factors chondromodulin-I (ChM-I) and chondromodulin-II (ChM-II) from fetal bovine epiphyseal cartilage. Both of these factors stimulate the growth and matrix formation of chondrocytes in vitro. In the present study, we found that ChM-I and ChM-II stimulated the proliferation of clonal mouse osteoblastic MC3T3-E1 cells as well as primary mouse osteoblasts in culture. Unlike other known growth factors, these factors did not support the proliferation of fibroblasts. Concomitantly with growth stimulation of osteoblasts, there was a reduction of alkaline phosphatase (ALP) activity in the cells, the expression of the differentiated phenotype. These results suggest that epiphyseal cartilage may play a functional role in longitudinal bone growth by production of these unique growth-promoting factors.

Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts.

Bone morphogenetic proteins (BMPs) play an important role in various kinds of pattern formation and organogenesis during vertebrate development. In the skeleton, BMPs induce the differentiation of cells of chondrocytic and osteoblastic cell lineage and enhance their function. However, the action of BMPs on osteoclastic bone resorption, a process essential for pathophysiological bone development and regeneration, is still controversial. In this study, we examine the direct effect of BMPs on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. BMP-2 caused a dose- and time-dependent increase in bone resorption pits excavated by the isolated osteoclasts. BMP-4 also stimulated osteoclastic bone resorption. The increase in osteoclastic bone resorption induced by BMP-2 was abolished by the simultaneous addition of follistatin, a BMP/activin binding protein that negates their biological activity. Just as it increased bone resorption, BMP-2 also elevated the messenger RNA expressions of cathepsin K and carbonic anhydrase II, which are key enzymes for the degradation of organic and inorganic bone matrices, respectively. Type IA and II BMP receptors (BMPRs), and their downstream signal transduction molecules, Smad1 and Smad5, were expressed in isolated osteoclasts as well as in osteoblastic cells, whereas type IB BMPR was undetectable. BMPs directly stimulate mature osteoclast function probably mediated by BMPR-IA and BMPR-II and their downstream molecules expressed in osteoclasts. The results presented here expand our understanding of the multifunctional roles of BMPs in bone development.

Immunocytochemical study of collagen in epidermal growth factor (EGF)-treated osteoblastic cells.

The alteration of collagen components in clone MC3T3-E1 cells by epidermal growth factor (EGF) was investigated immunocytochemically, using antibodies to type I and type III collagens. EGF transformed those cells that had become more slender than those of control cultures. Type I and type III collagens were observed in the same cells in both EGF-treated and control cultures. Type I collagen was decreased by EGF, whereas type III collagen appeared to be increased. However, no cells with only type III collagen were observed, suggesting that EGF influences collagen metabolism in clone MC3T3-E1 cells.