Masanori Kanatani

Dexamethasone stimulates osteoclast-like cell formation by directly acting on hemopoietic blast cells and enhances osteoclast-like cell formation stimulated by parathyroid hormone and prostaglandin E2.

Although an excess of glucocorticoid induces secondary osteoporosis, the mechanism still remains unclear, particularly in regard to glucocorticoid‐stimulated bone resorption. We examined the effects of dexamethasone (Dex) on osteoclast‐like cell formation and bone‐resorbing activity by employing mouse bone and spleen cell cultures and further investigated whether Dex would modulate osteoclast‐like cell formation stimulated by several bone‐resorbing factors. Dex stimulated osteoclast‐like cell formation in stromal cell‐containing mouse bone cell cultures in a concentration‐dependent manner. Also, Dex significantly stimulated osteoclast‐like cell formation from hemopoietic blast cells in spleen cell cultures derived from 5‐fluorouracil‐pretreated mice. In contrast, Dex (10−8 M) did not affect the bone‐resorbing activity of mature osteoclasts. Pretreatment with 10−8 M Dex significantly enhanced osteoclast‐like cell formation in unfractionated mouse bone cell cultures stimulated by 10−8 M human (h) parathyroid hormone (PTH) (1–34), 10−8 M hPTH‐related protein (1–34) and 10−6 M prostaglandin E2, but not by 10−8 M 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3). Moreover, pretreatment with 10−8 M Dex significantly enhanced osteoclast‐like cell formation stimulated by both forskolin and dbcAMP. In contrast, pretreatment with 10−8 M Dex significantly inhibited osteoclast‐like cell formation in mouse spleen cell cultures stimulated by both 10−8 M hPTH(1–34) and 10−8 M 1,25(OH)2D3. These findings suggest that Dex stimulates osteoclast‐like cell formation, at least in part by directly acting on hemopoietic blast cells. They further suggest that Dex enhances osteoclast‐like cell formation stimulated by PTH and prostaglandin E2 through an indirect pathway via cells other than hemopoietic blast cells.

Effect of elevated extracellular calcium on the proliferation of osteoblastic MC3T3-E1 cells:its direct and indirect effects via monocytes.

There has been evidence that elevated calcium concentration at the resorptive site of the bone directly regulates osteoclast function. In the present study, in order to clarify the role of elevated calcium concentration at the resorptive site in the regulation of osteoblast function, not only direct but also indirect effect via human monocytes of the increase in extracellular calcium (Cae) on the proliferation of osteoblastic MC3T3-E1 cells have been investigated in serum-free condition. The increase in Cae enormously stimulated osteoblast proliferation at the concentration of 3 to 20 mM. When human monocytes were cultured at the elevated Cae concentration, monocyte-conditioned medium-induced stimulation of osteoblast proliferation was significantly amplified. Present data demonstrate that elevated Cae has pronounced stimulatory effect on osteoblast proliferation not only directly but also indirectly via monocytes. Calcium released from bone matrix at the resorptive sites might be linked to the coupling of osteoclast and osteoblast functions.

Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity.

Although high inorganic phosphate (Pi) concentration in culture media directly inhibits generation of new osteoclasts and also inhibits bone resorption by mature osteoclasts, its precise mechanism and the physiological role have not been elucidated. The present study was performed to investigate these issues. Increase in extracellular Pi concentration ([Pi]e) (2.5–4 mM) concentration dependently inhibited 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] or parathyroid hormone (PTH)‐(1‐34)‐induced osteoclast‐like cell formation from unfractionated bone cells in the presence of stromal cells. Increase in [Pi]e (2.5–4 mM) concentration dependently inhibited 1,25(OH)2D3‐, PTH‐(1‐34)‐, or receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF)‐induced osteoclast‐like cell formation from hemopoietic blast cells in the absence of stromal cells. Increase in [Pi]e (2.5–4 mM) dose dependently stimulated the expression of osteoprotegerin (OPG) mRNA and increased the expression of OPG mRNA suppressed by PTH‐(1‐34) or 1,25(OH)2D3 in unfractionated bone cells, while it did not affect RANKL mRNA. Increase in [Pi]e (2.5‐4 mM) concentration dependently inhibited the bone‐resorbing activity of isolated rabbit osteoclasts. Increase in [Pi]e (4 mM) induced the apoptosis of isolated rabbit osteoclasts while it did not affect the apoptosis of osteoclast precursor cells and mouse macrophage‐like cell line C7 cells that can differentiate into osteoclasts in the presence of RANKL and M‐CSF. These results indicate that increase in [Pi]e inhibits osteoclast differentiation both by up‐regulating OPG expression and by direct action on osteoclast precursor cells. It is also indicated that increase in [Pi]e inhibits osteoclastic activity at least in part by the direct induction of apoptosis of osteoclasts.

Serum Levels of Insulin-Like Growth Factor (IGF); IGF-Binding Proteins-3, -4, and -5; and Their Relationships to Bone Mineral Density and the Risk of Vertebral Fractures in Postmenopausal Women

We previously found that serum levels of insulin-like growth factor I (IGF-I) and IGF-binding protein (IGFBP)-3, but not IFGBP-2, were associated with bone mineral density (BMD) and the risk of vertebral fractures. The aim of the present study was to investigate the roles of IGFBP-4 and -5 in age-dependent bone loss and vertebral fracture risk in postmenopausal Japanese women and to compare them with those of IGF-I and IGFBP-3. One hundred and ninety-three Japanese women aged 46–88 years (mean 62.5) were enrolled in the cross-sectional study. BMD was measured at the lumbar spine, femoral neck, ultradistal radius (UDR), and total body by dual-energy X-ray absorptiometry. Serum levels of IGFBP-4 and -5 as well as IGF-I and IGFBP-3 were measured by radioimmunoassay. Serum levels of IGF-I, IGFBP-3, and IGFBP-5 declined with age, while serum IGFBP-4 increased with age. Multiple regression analysis was performed between BMD at each skeletal site and serum levels of IGF-I and IGFBPs adjusted for age, body weight, height, and serum creatinine. BMD at the UDR was significantly and positively correlated with all serum levels of IGF-I and IGFBPs measured (P < 0.01), while BMD at the femoral neck was correlated with none of them. Serum IGF-I level was significantly and positively correlated with BMD at all sites except the femoral neck (P < 0.01), while serum IGFBP-3 and -4 levels were significantly and positively correlated with only radial BMD (P < 0.01). Serum IGFBP-5 level was positively correlated with UDR BMD (P < 0.001) and negatively correlated with total BMD (P < 0.05). Serum IGF-I, IGFBP-3, and IFGBP-5 levels were significantly lower in women with vertebral fractures than in those without fractures (mean ± SD: 97.1 ± 32.1 vs. 143.9 ± 40.9 ng/dl, P < 0.0001; 2.18 ± 1.02 vs. 3.23 ± 1.07 μg/ml, P < 0.0001; 223.6 ± 63.3 vs. 246.5 ± 71.5 ng/ml, P = 0.0330, respectively). When multivariate logistic regression analysis was performed with the presence of vertebral fractures as a dependent variable and serum levels of IGF-I and IGFBPs adjusted for age, body weight, height, serum creatinine, and serum alubumin as independent variables, IGF-I and IGFBP-3 were selected as indices affecting the presence of vertebral fractures [odds ratio (OR) = 0.29, 95% confidential interval (CI) 0.15–0.57 per SD increase, P = 0.0003 and OR = 0.31, 95% CI 0.16–0.61 per SD increase, P = 0.0007, respectively]. To compare the significance values, IGF-I, IGFBP-3, and age were simultaneously added as independent variables in the analysis. IGFBP-3 was more strongly associated with the presence of vertebral fractures than IGF-I and age (P = 0.0006, P = 0.0148, and P = 0.0013, respectively). Thus, after comprehensive measurements of serum levels of IGF-I and IGFBPs, it seems that serum IGF-I level is most efficiently associated with bone mass and that serum IGFBP-3 level is most strongly associated with the presence of vertebral fractures in postmenopausal women among the IGF system components examined.

Thyroid hormone stimulates osteoclast differentiation by a mechanism independent of RANKL–RANK interaction

It is well known that thyroid hormone excess causes bone loss. However, the precise mechanism of bone loss by thyroid hormone still remains unclear. When T3 was added to unfractionated bone cells after degeneration of pre‐existent osteoclasts, T3 (1 pM–100 nM) dose‐dependently stimulated osteoclast‐like cell formation, irrespective of the presence of indomethacin and IL‐6 Ab. T3 increased the expression of osteoprotegerin (OPG) messenger RNA (mRNA), but not of receptor activator of nuclear factor κB ligand (RANKL) in unfractionated bone cells, suggesting that the stimulatory effect of T3 on osteoclast formation was not mediated by the RANKL/OPG system. We next examined the direct effect of T3 on osteoclast precursors in the absence of osteoblasts, using hemopoietic blast cells derived from spleen cells. T3 (1 pM–100 nM) dose‐dependently stimulated osteoclast‐like cell formation from osteoclast precursors. OPG did not inhibit T3‐induced osteoclast formation from osteoclast precursor cells. The polymerase chain reaction (PCR) product corresponding in size to the mouse T3 receptor α1 cDNA was detected in osteoclast precursors from mouse hemopoietic blast cells as well as mouse heart and mouse osteoblastic cell line MC3T3‐E1 cells, suggesting that T3 directly stimulated osteoclast‐like cell formation from osteoclast precursors in the absence of osteoblasts. Further, T3 increased the expression of c‐Fos mRNA at 15 min and 24 h and Fra‐1 mRNA at 2 and 6 h in osteoclast precursors. Consistent with the increased expression of c‐Fos mRNA observed by RT‐PCR, the activation of c‐Fos occurred in osteoclast precursor cells stimulated by T3, while the activation of neither NF‐κB nor MAPKs was observed by immunoblot analysis. Antisense oligodeoxynucleotides (as‐ODN) complementary to c‐Fos mRNA at 1 μM significantly inhibited T3‐induced osteoclast‐like cell formation from osteoclast precursors in the absence of stromal cells while sense‐ODN did not affect T3‐induced osteoclast‐like cell formation. These results indicate that T3 directly stimulates osteoclast differentiation at least in part by up‐regulation of c‐fos protein in osteoclast precursor cells. J. Cell. Physiol. 201: 17–25, 2004.

Estrogen via the Estrogen Receptor Blocks cAMP-Mediated Parathyroid Hormone (PTH)-Stimulated Osteoclast Formation†

Several lines of evidence indicate that estrogen inhibits parathyroid hormone (PTH)‐induced bone resorption in vivo and in vitro. However, its precise mechanism remains unknown. The present study was performed to investigate whether osteoclast precursor cells possess the receptors for PTH/PTH‐related protein (PTHrP) and/or estrogen and to clarify the mechanism by which estrogen affects PTH‐induced osteoclast‐like cell (Ocl) formation. The polymerase chain reaction (PCR) product corresponding in size to the mouse PTH/PTHrP receptor cDNA was detected in mouse hemopoietic blast cells supported by granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) as well as in osteoblastic MC3T3‐E1 cells. The nucleotide sequence of the PTH/PTHrP receptor PCR product of hemopoietic blast cells was found to be 95.4% identical to that of PTH/PTHrP receptor cDNA of rat osteoblastic ROS cells. The PCR product corresponding in size to the mouse estrogen receptor cDNA was detected in mouse hemopoietic blast cells supported by GM‐CSF as well as in MC3T3‐E1 cells. The nucleotide sequence of the estrogen receptor PCR product of hemopoietic blast cells was completely identical to that of mouse estrogen receptor cDNA. 17β‐estradiol (17β‐E2) but not 17α‐E2 dose dependently antagonized Ocl formation stimulated by human (h) PTH(1–34) at a minimal effective concentration of 10−10 M in the hemopoietic blast cell culture. 17β‐E2 also significantly inhibited Ocl formation stimulated by 10−8 M hPTHrP(1–34), while it did not affect 1,25‐dihydroxyvitamin D3–induced Ocl formation. However, 10−8 M 17β‐E2 significantly inhibited Ocl formation stimulated by dibutyryladenosine cAMP (10−4 M) and Sp‐cAMPS (10−4 M), an activator of cAMP‐dependent protein kinase (PKA) as well as forskolin (10−5 M). In contrast, 17β‐E2 did not affect Ocl formation by either phorbol myristate acetate (10−7 M), an activator of protein kinase C (PKC), or A23187 (10−7 M), a calcium ionophore. The pretreatment with 17β‐E2 significantly inhibited Ocl formation induced by the combined treatment with PTH and PKC inhibitors (H7 or staurosporine), while it did not affect Ocl formation stimulated by the combined treatment with PTH and Rp‐cAMPS, a PKA inhibitor. The present data indicate that estrogen inhibits PTH‐stimulated Ocl formation by directly acting on hemopoietic blast cells, possibly through blocking a PKA pathway but not a calcium/PKC pathway.

Insulin-like growth factor-I mediates osteoclast-like cell formation stimulated by parathyroid hormone.

There have been several lines of evidence that parathyroid hormone (PTH) stimulates production of insulinlike growth factor I (IGF‐I) in bone and that IGF‐I stimulates osteoclast formation. Thus, the present study was performed to clarify the possible role of IGF‐I in PTH‐stimulated osteoclastlike cell formation and the role of PTH‐responsive dual signal transduction systems (cyclic [c] AMP‐dependent protein kinase [PKA] and calcium/protein kinase C [PKC]) in its mechanism. Treatment with anti‐IGF‐I antibody (1–10 μg/ml) partially but significantly blocked hPTH‐(1‐34)‐stimulated osteoclastlike cell formation in unfractionated mouse bone cell cultures, although it did not affect osteoclastlike cell formation stimulated by 1,25‐dihydroxyvitamin D3. Rp‐cAMPS (10‐4 M), a direct PKA inhibitor, as well as two types of PKC inhibitors, H‐7 (10 μM) and staurosporine (3 nM), and dantrolene (10‐5 M), an inhibitor of calcium mobilization from intracellular calcium stores, all significantly blocked PTH‐stimulated osteoclastlike cell formation. Anti‐IGF‐I antibody (3 μg/ml) significantly blocked osteoclastlike cell formation stimulated by 10‐4 M dbcAMP, 10‐4 M Sp‐cAMPS, a direct PKA activator, and 10‐5 M forskolin in mouse bone cell cultures. Dibutyryl cAMP, forskolin, and hPTH‐(1‐34) significantly stimulated mRNA expression of both IGF‐I and IGF‐binding protein 5 (IGFBP‐5) in these cultures, but neither 10‐7 M PMA, a PKC activator, nor 10‐7 M A23187 did. Moreover, anti‐IGF‐I antibody significantly blocked osteoclastlike cell formation stimulated by the conditioned medium from MC3T3‐E1 cells pretreated with 10‐8 PTH‐(1‐34), which induced IGF‐I and IGFBP‐5 mRNA expression in these cells. In conclusion, the present study indicates that IGF‐I mediates osteoclastlike cell formation stimulated by PTH and that the PKA pathway is involved in its mechanism. However, IGF‐I does not seem to be the sole effector molecule to be active in this system. J. Cell. Physiol. 172:55–62, 1997.

Stimulatory Effect of Insulin-Like Growth Factor Binding Protein-5 on Mouse Osteoclast Formation and Osteoclastic Bone-Resorbing Activity

Insulin‐like growth factor binding protein‐5 (IGFBP‐5) stimulates osteoblast proliferation directly or indirectly through IGF‐I action, but its effects on osteoclast formation and osteoclastic activity are unknown. We tested the effects of IGFBP‐5 on osteoclastic activity and osteoclast formation. IGFBP‐5 significantly stimulated pit formation by pre‐existent osteoclasts in mouse bone cell cultures and its stimulatory effect was completely blocked by IGF‐I antibody (Ab). However, IGFBP‐5 did not affect the bone‐resorbing activity of isolated rabbit osteoclasts. When IGFBP‐5 was added to unfractionated bone cells after degeneration of pre‐existent osteoclasts, IGFBP‐5 (77 pM–7.7 nM) dose‐dependently stimulated osteoclast‐like cell formation, irrespective of the presence of IGF‐I Ab. Moreover, osteoclast‐like cells newly formed by IGFBP‐5 from unfractionated bone cells possessed the ability to form pits on dentine slices. We next examined the direct effect of IGFBP‐5 on osteoclast precursors in the absence of stromal cells, using hemopoietic blast cells derived from spleen cells. IGFBP‐5 dose‐dependently stimulated osteoclast‐like cell formation from osteoclast precursors, irrespective of the presence of IGF‐I Ab. Growth hormone (GH) as well as IGF‐I significantly stimulated bone resorption by pre‐existent osteoclasts in mouse bone cell cultures and these stimulatory effects were completely blocked by IGF‐I Ab. GH as well as IGF‐I stimulated osteoclast‐like cell formation from unfractionated bone cells and this stimulatory effect of GH was significantly but partially blocked by IGF‐I Ab. The direct stimulatory effect of GH on osteoclast‐like cell formation from hemopoietic blast cells was not affected by IGF‐I Ab. The present data indicate that IGFBP‐5 stimulates bone resorption both by stimulation of osteoclast formation in an IGF‐I–independent fashion and by IGF‐I–dependent activation of mature osteoclasts, possibly via osteoblasts, in vitro. (J Bone Miner Res 2000;15:902–910)

IGF-I mediates the stimulatory effect of high phosphate concentration on osteoblastic cell proliferation

Although high concentrations of inorganic phosphate (Pi) are known to have a distinct anabolic effect on bone structure and metabolism, the precise mechanism by which phosphate possesses anabolic effect on bone formation has not been elucidated. The present study was performed to examine the effects of an increase in extracellular Pi concentration ([Pi]e) on the proliferation of osteoblastic MC3T3‐E1 cells. Increase in [Pi]e(2–4 mM) dose‐dependently stimulated DNA synthesis. Indomethacin, an inhibitor of prostaglandin synthesis, did not affect high [Pi]e‐induced DNA synthesis. DNA synthesis first increased affer a 3 h exposure to 4 mM [Pi]e and its stimulatory effect was observed in a time‐dependent manner up to 24 h. On the other hand, DNA synthesis was significantly but partially blocked by cycloheximide, suggesting that this stimulatory effect of high [Pi]e was at least in part dependent on new protein synthesis. There is recent evidence that MG3T3‐E1 cells constitutively produce and secrete insulin‐like growth factor‐I (IGF‐I) and possess IGF‐I receptors. IGF‐I antiserum (1:10,000 to 1:100) significantly but partially blocked the stimulatory effect of [Pi]e (4 mM) on DNA synthesis in a concentration‐dependent manner. A neutralizing IGF‐I antibody as well as IGF‐I receptor antibody also significantly but partially blocked DNA synthesis stimulated by high [Pi]e in a concentration‐dependent manner, indicating that IGF‐I at least in part mediated the high [Pi]e‐induced effect. Actually, high [Pi]e significantly increased the secretion of immunoreactive IGF‐I into the medium as well as the expression of IGF‐I mRNA. Present findings indicate that an increase in [Pi]e stimulated DNA synthesis partly via an increase in IGF‐I action. J. Cell. Physiol. 190: 306–312, 2002.

The activation of cAMP-dependent protein kinase is directly linked to the stimulation of bone resorption by parathyroid hormone

The present study was performed to characterize the direct involvement of cAMP in the stimulation of bone resorption by parathyroid hormone (PTH), using Sp-cAMPS and Rp-cAMPS, which were the direct agonist and antagonist in the activation of cAMP-dependent protein kinase (PKA), respectively. Bone resorbing activity was estimated as the number of pits formed on the dentine slice and total area of pits per slice in bone marrow cells derived from 2 week-old mice. Dibutyryl cAMP (dbcAMP)(10(-4)M) and Sp-cAMPS (10(-4)M) caused the remarkable stimulation of bone resorption. Although Rp-cAMPS (10(-4)M) did not affect bone resorption by itself, it significantly inhibited dbcAMP- and Sp-cAMPS-induced stimulation of bone resorption. Moreover, Rp-cAMPS (10(-4)M) antagonized 10(-7)M human PTH-(1-34)-induced stimulation of bone resorption, although it did not affect 10(-8)M 1,25(OH)2D3-induced stimulation of bone resorption. Present study indicates the direct involvement of PKA in the stimulation of bone resorption by PTH.