Junichi Kano

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.

The activation of cAMP-dependent protein kinase is directly linked to the inhibition of osteoblast proliferation (UMR-106) by parathyroid hormone-related protein

The present study was performed to compare the effect of parathyroid hormone-related protein (PTHrP) on the proliferation of osteoblastic osteosarcoma cells (UMR-106) with that of PTH and characterize the direct involvement of cAMP in the change of osteoblast proliferation by PTHrP. Human(h)PTHrP-(1-34) (10(-11)-10(-7)M) dose-dependently inhibited [3H]thymidine incorporation (TdR) in the same manner as hPTH-(1-34). The simultaneous addition of PTHrP and PTH at a maximal effective dose of 10(-7) M did not cause additive suppressive effect on cell proliferation. Rp-cAMPs, which has been recently shown to act directly as antagonist in the activation of cAMP-dependent protein kinase (PKA), dose-dependently (10(-6)-10(-4)M) antagonized PTHrP-induced suppression of TdR in the same manner as PTH. Present study indicated that PTHrP has the same effect on osteoblast proliferation as PTH and that the activation of PKA is directly linked to the change of osteoblast proliferation by PTHrP.

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 direct involvement of cAMP-dependent protein kinase in the regulation of collagen synthesis by parathyroid hormone (PTH) and PTH-related peptide in osteoblast-like osteosarcoma cells (UMR-106)

The present study was performed to characterize the direct involvement of cAMP-dependent protein kinase (PKA) in the regulation of collagen synthesis by parathyroid hormone (PTH) and PTH-related peptide (PTHrP) in osteoblastic osteosarcoma cells, UMR-106. Sp-cAMPS (10(-4)M), a direct activator of PKA, as well as dibutyryl cAMP (dbcAMP, 10(-4)M) significantly inhibited collagen synthesis. Human (h) PTH-(1-34) (10(-7)M) and hPTHrP (10(-7) M) inhibited collagen synthesis to the same degree. Although Rp-cAMPS, which acted directly as an antagonist in the activation of PKA, did not affect collagen synthesis by itself, it significantly antagonized dbcAMP- and Sp-cAMPS-induced inhibition of collagen synthesis. Moreover, Rp-cAMPS antagonized PTH- and PTHrP-induced inhibition of collagen synthesis to the same degree. The present study first indicated that the activation of PKA was directly linked to the regulation of collagen synthesis by PTH in osteoblast and that PTHrP had the same effect on collagen synthesis presumably through the same mechanism as PTH.

Effect of 17 β -estradiol on the proliferation of osteoblastic MC3T3-E1 cells via human monocytes

Effects of human monocyte-conditioned medium on the proliferation of osteoblastic MC3T3-E1 cells were investigated in serum-free cultured condition. Monocyte-conditioned medium significantly stimulated osteoblast proliferation at the concentration between 10 and 30%, compared to that in the absence of monocytes. 17 beta-estradiol directly stimulated osteoblast proliferation at the concentrations of 10(-8) and 10(-10)M. On the contrary, the conditioned medium prepared by monocytes cultured in the presence of 17 beta-estradiol at the concentrations of 10(-8) and 10(-10)M significantly inhibited osteoblast proliferation. Present data indicate that in addition to direct effect on osteoblasts, 17 beta-estradiol affected osteoblast proliferation presumably through modulating the release of several local regulators of bone turnover from monocytes. The effect on osteoblastic activity via monocytes might be linked to the coupling of osteoclast and osteoblast actions.

Involvement ofc-fos gene in the regulation of osteoblast proliferation and osteoclast differentiation by parathyroid hormone and parathyroid hormone-related protein

Although there is a recent evidence that PTH induces c-fos gene expression in osteoblasts, the physiological role of this expression remains unknown. We, therefore, employed c-fos antisense oligodeoxynucleotide (as-ODN) and sought to clarify the role of c-fos gene in the regulation of osteoblast proliferation and differentiation as well as osteoclast differentiation and bone-resorbing activity in the presence of osteoblasts by PTH and PTH-related protein (PTHrP). We employed osteoclastlike cell formation from mouse bone cells for the evaluation of osseoclast differentiation and the pit formation assay on the dentin slice in mouse bone cells for the evaluation of bone-resorbing activity by mature osteoclasts. Northern blot analysis revealed that both human (h)PTH-(1-34) and hPTHrP-(1-34) (10−8M) induced a transient c-fos gene expression to a similar degree in osteoblastic UMR-106 cells. Sp-cAMPS (10−4M), an activator of cAMP-dependent protein kinase (PKA), as well as dbcAMP induced a weak c-fos gene expression and Rp-cAMPS (10−4M), an inhibitor of PKA, almost completely antagonized these expressions. However, Rp-cAMPS only slightly blocked c-fos gene expression by PTH and PTHrP. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC) (10−7 to 10−6M), but not 4 alpha-phorbol 12, 13-didecanoate, incapable of activating PKC induced an intense expression of c-fos gene. Calcium ionophores (A23187 and ionomycin, 10−7 to 10−6M) did not induce the expression of c-fos gene. An inhibitor of PKC (H-7, 50µM) almost completely blocked the c-fos gene expression by PTH and PTHrP as well as PMA. Pretreatment with 1µM as-ODN significantly antagonized the inhibition of [3H] thymidine incorporation into UMR-106 cells and the stimulation of osteoclast-like cell formation by PTH and PTHrP, compared to pretreatment with the control oligodeoxynucleotide consisting of same nucleotides as as-ODN but with a random sequence. On the other hand, as-ODN did not affect an increase in alkaline phosphatase activity of UMR-106 cells and pit formation by PTH and PTHrP. In all experiments so far, the effects of PTHrP were virtually the same as those of PTH. The present study indicates, first, the direct involvement of PKC as well as PKA in PTH- and PTHrP-induced c-fos gene expression and, second, the participation of its expression in the regulation of osteoblast proliferation and osteoclast differentiation by PTH as well as PTHrP.

Effect of ipriflavone on bone mineral content in osteoporotic patients

Although numerous studies have demonstrated that ipriflavone prevents bone loss, its effect on bone mass remains to be quantitatively assessed by dual-energy x-ray absorptiometry (DXA), a recently developed diagnostic technique which enables precise measurement of bone mineral density (BMD). In this study, we investigated the effect of ipriflavone on BMD in female outpatients with osteoporosis by means of DXA and single-photon absorptiometry (SPA). The study group consisted of 23 female patients with postmenopausal or senile osteoporosis (age: 50–80 years, mean 62.4 years; body weight: 39–63 kg, mean 49.2 kg; height: 145–163 cm, mean 150.8 cm). Nineteen untreated female outpatients (age: 61–65 years) served as a control group. BMD of L1-4 in both the study and control groups was measured by DXA before and after 1 year of treatment with and without ipriflavone (600 mg/day per os), but only 19 of the study group patients were evaluated for BMD by SPA at the radius. For DXA, the mean BMD in the study group was 0.651 g/cm2 before ipriflavone treatment and 0.647 g/cm2 after treatment, with a mean percent change in BMD of 99.5%. For SPA, the mean BMD was 0.495 g/cm2 before ipriflavone treatment and 0.504 g/cm2 after treatment, with a mean percent change in BMD of 101.6%. For patients in the study group aged 61–65 years (n=9), the mean percent change in BMD by DXA after 1 year of treatment was 101.6%. In contrast, the mean percent change in BMD for the control patients of the same age bracket (n=19) was 96.5%. The results by DXA confirmed that ipriflavone inhibits loss of bone mass, making it a useful agent for the treatment of postmenopausal and senile osteoporosis.