Effects of commonly used medications on bone tissue mineralisation in SaOS-2 human bone cell line: an in vitro study.

We analysed the effects of commonly used medications on human osteoblastic cell activity in vitro, specifically proliferation and tissue mineralisation. A list of medications was retrieved from the records of patients aged > 65 years filed in the database of the largest health maintenance organisation in our country (> two million members). Proliferation and mineralisation assays were performed on the following drugs: rosuvastatin (statin), metformin (antidiabetic), metoprolol (ß-blocker), citalopram (selective serotonin reuptake inhibitor [SSRI]), and omeprazole (proton pump inhibitor (PPI)). All tested drugs significantly stimulated DNA synthesis to varying degrees, with rosuvastatin 5 µg/ml being the most effective among them (mean 225% (SD 20)), compared with metformin 10 µg/ml (185% (SD 10)), metoprolol 0.25 µg/ml (190% (SD 20)), citalopram 0.05 µg/ml (150% (sd 10)) and omeprazole 0.001 µg/ml (145% (SD 5)). Metformin and metoprolol (to a small extent) and rosuvastatin (to a much higher extent) inhibited cell mineralisation (85% (SD 5)). Our results indicate the need to evaluate the medications prescribed to patients in terms of their potential action on osteoblasts. Appropriate evaluation and prophylactic treatment (when necessary) might lower the incidence and costs associated with potential medication-induced osteoporosis.

Age-dependent responsiveness of human female bone cells to vitamin D analog and PTH.

Vitamin D less-calcemic analog JKF 1624 F2-2 (JKF) and PTH 1-34 stimulate in human female cultured osteoblasts (Ob) DNA synthesis (DNA), creatine kinase specific activity (CK), 1α, 25 vitamin D hydroxylase mRNA (1OHase) expression and 1,25(OH)2D3 (1,25) production, estrogen receptors (ER) mRNA expression and intracellular and membranal estrogen binding. In the present study, cultured Ob from different ages were subjected to hormonal stimulations and analyzed for different parameters. We found: 1) ERα expression is higher and ERß expression is lower in pre-meno - pausal Ob (prOb), with similar intracellular and membranal binding. 2) JKF and PTH up-regulated ERα and JKF downregulated ERß in both Ob, while PTH stimulated it in post- (poOb) and inhibited it in prOb. 3) There is higher expression of 1OHase mRNA in prOb, but 1,25 production is similar. Both parameters were hormonally stimulated to higher extent in prOb. 4) Ob express 12 and 15 lipoxygenase (LO) mRNA and produce 12- and 15-hydroxyeicosatetraenoic acid (H). 12LO expression is higher and 15LO is lower in prOb, while 12H is higher in prOb and 15H is similar in both. JKF inhibited 12LO expression in prOb and stimulated in poOb, whereas PTH stimulated it to higher extent in prOb. JKF stimulated and PTH inhibited 15LO expression in both; 12 and 15H were stimulated by both hormones in both Ob. 5. PTH and JKF stimulated DNA and CK in both Ob. In conclusion Ob demonstrate some age-dependent response to calciotrophic hormones, but the mechanism and beneficial outcome for human is unclear.

Mutual interaction of special phytoestrogenic compounds, their synthetic carboxy-derivatives and the less-calcemic vitamin D analog activities in human derived female cultured osteoblasts.

Cultured female-derived human bone cells (hObs) responded by different parameters to different phytoestrogenic and vitamin D compounds. Pre- and post-menopausal hObs express ERα and ERß mRNA with higher abundance of ERα. Pre-treatment with the less-calcemic vitamin D analog JKF 1624F(2)-2 (JKF) upregulated responsiveness to estrogens via modulation of ERs expression. These estrogenic compounds induce the expression and activity of 25 hydroxy-vitamin D(3)-1α hydroxylase (1OHase). We now analyzed the effects of carboxy-genistein (cG), carboxy-biocainin A (cBA) and carboxy-daidzein (cD), of BA, D or G and of licorice derived compounds glabridin (Glb) and glabrene (Gla) and estradiol-17ß (E(2)) on DNA synthesis, creatine kinase specific activity (CK), intracellular and membranal E(2) binding and their modulations by JKF in hObs. We also analyzed modulation by phytoestrogenic compounds of 1OHase mRNA expression and activity. We showed that: (1) all compounds stimulated DNA synthesis and CK. (2) JKF and all estrogenic compounds modulated ERα and ERß mRNA expression. (3) Pre-treatment with JKF increased DNA synthesis and CK responses only to E(2), D, G and Gla. (4) JKF increased the intracellular competitive binding only of E(2), D and G. (5) JKF abolished the membranal binding of all protein-bound estrogens. (6) JKF and all estrogenic compounds except the protein-bound ones up-regulated 1OHase expression and activity. In conclusion phytoestrogens and their analogs increase DNA synthesis and CK, and lead to increased production of 1,25(OH)(2)D(3) in hObs, while pre-treatment with JKF modulates the effect of estrogenic compounds via regulation of ERs mRNA expression in a yet unclear mechanism.

Anti-thyroid cancer properties of a novel isoflavone derivative, 7-(O)-carboxymethyl daidzein conjugated to N-t-Boc-hexylenediamine in vitro and in vivo.

The incidence of thyroid cancer is up to 3 folds higher in women than in men, suggesting that estrogenic effects may be involved in the pathogenesis of this malignancy. Here, we explore whether or not human thyroid cancer cell growth can be curbed by a novel isoflavone derivative generated in our laboratory, the N-t-Boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc). With the exception of the follicular cancer cell line WRO, estrogen receptor (ER)α mRNA was only marginally expressed in cell lines derived from papillary (NPA), follicular (MRO), anaplastic thyroid carcinoma (ARO) such that the expression of estrogen receptor (ER) ßmRNA was more abundant than that of ERα mRNA in these cell types. Estradiol-17ß (E2; 0.03-300nmol/l) per se increased proliferation in all four cell-types. The ERß-specific agonist DPN increased [(3)H]-thymidine incorporation in all four thyroid cancer cell lines, whereas the ERα-specific agonist PPT increased growth only in NPA and WRO. By contrast, cD-tboc, derived from the weak estrogen daidzein, did not cause cell growth and dose-dependently diminished cell growth in all four cell lines via apoptosis and not necrosis, as detected by the release of histone-DNA fragments. The cytotoxic growth inhibitory effect of cD-tboc in these cells was modulated by E2 and the general caspase inhibitor Z-VAD-FMK, and the magnitude of this salvage was cell type-and dose-dependent. When nude mice carrying ARO thyroid xenografts were treated with cD-tboc, tumor volume decreased significantly, and no apparent toxicity was observed. These results suggest that cD-tboc may be a promising agent for therapy of thyroid carcinoma either alone or in combination with existing cytotoxic drugs.

The effects of estrogen receptors α- and ß-specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line.

In cultured human osteoblasts estradiol-17ß (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERß-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERß-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERß mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERß pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.

DT56a (Femarelle), contrary to estradiol-17ß, is effective in human derived female osteoblasts in hyperglycemic condition.

We have reported previously, that female-derived cultured osteoblasts (hObs) responded to DT56a (Femarelle) measured by the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness and (3)[H] thymidine incorporation into DNA (DNA synthesis). Since the skeletal protective effects of estrogens are not discernable in hyperglycemic diabetic women, we sought to analyze the effect of estrogenic compounds on CK and DNA synthesis in hObs when grown in high glucose concentration (HG). Cells were grown either in normal glucose (NG) (4.5g/L; 22mM) or HG (9.0g/L; 44mM) for 7 days. HG increased constitutive CK but, the response of CK activity and DNA synthesis to estradiol-17ß (E(2)) treatment was reduced. In contrary, DT56a was found to be active (as measured by CK activity and DNA synthesis) in both NG and HG. HG decreases the hormonal responsiveness and might block important effects of estrogenic compounds, most likely contributing to their decreased skeletal preserving properties in hyperglycemic women. In hObs from post-menopausal women grown in HG, ERs mRNA expressions were unchanged. On the other hand, in hObs from pre-menopausal women HG increased ERs mRNA expressions. Since DT56a unlike E(2) is active in HG environment as well as in normal glucose, it may be an effective bone restoring agent in diabetic post-menopausal women.

The effects of native and synthetic estrogenic compounds as well as vitamin D less-calcemic analogs on adipocytes content in rat bone marrow.

BACKGROUND: We demonstrated previously that phytoestrogens and vitamin D analogs like estradiol-17ß (E2) modulate bone morphology in rat female model. AIM: We now analyze the effects of phytoestrogens, E2, selective E2 re ceptor modulators, and the less-calcemic analogs of vitamin D: JKF1624F2-2 (JKF) or QW1624F2-2 (QW) on fat content in bone marrow (BM) from long bones in ovariectomized female rats (OVX). MATERIALS AND METHODS: OVX rats were injected with treatments known to affect bone formation, 5 days per week for 2.5 month for analysis of fat content in BM. RESULTS: In OVX young adults there is a decreased bone formation and a 10-fold increase in fat cells content in BM. Treatment with E2, raloxifene (Ral) or DT56a resulted in almost completely abolishment of fat cells content. Daidzein (D) decreased fat cells content by 80%, genistein (G) or biochainin A (BA) did not change fat cells content and carboxy BA (cBA) had a small but significant effect. JKF or QW did not affect fat cells content, whereas combined treatment of JKF or QW with E2 resulted in complete abolishment of fat cells content. These changes in fat cells content are inversely correlated with changes in bone formation. CONCLUSIONS: Our results demonstrate that adipogenesis induced by OVX is a reversible process which can be corrected by hormonal treatments. The awareness of a relationship between fat and bone at the marrow level might provide a better understanding of the pathophysiology of bone loss as well as a novel approach to diagnosis and treatment of postmenopausal osteoporosis.

Vitamin D metabolites and analogs induce lipoxygenase mRNA expression and activity as well as reactive oxygen species (ROS) production in human bone cell line.

Vitamin D metabolites and its less-calcemic analogs (vitamin D compounds) are beneficial for bone and modulate cell growth and energy metabolism. We now analyze whether 25(OH)D(3) (25D), 1,25(OH)(2)D(3) (1,25D), 24,25(OH)(2)D(3) (24,25D), JKF1624F(2)-2 (JKF) or QW1624F(2)-2 (QW) regulate lipooxygenase (LO) mRNA expression and its products; hydroxyl-eicosatetraenoic acid (12 and 15HETE) formation, as well as reactive oxygen species (ROS) production in human bone cell line (SaOS2) and their interplay with modulation of cell proliferation and energy metabolism. All compounds except 25D increased 12LO mRNA expression and modulated 12 and 15HETE production whereas ROS production was increased by all compounds, and inhibited by NADPH oxidase inhibitors diphenyleneiodonium (DPI) and N-acetylcysteine (NAc). Baicaleine (baic) the inhibitor of 12 and 15LO activity blocked only slightly the stimulation of DNA synthesis by all compounds, whereas DPI inhibited almost completely the stimulation of DNA and CK by all compounds. Treatments of cells with 12 or 15HETE increased DNA synthesis and CK that were only slightly inhibited by DPI. These results indicate that vitamin D compounds increased oxidative stress in osteoblasts in part via induction of LO expression and activity. The increased ROS production mediates partially elevated cell proliferation and energy metabolism, whereas the LO mediation is not essential. This new feature of vitamin D compounds is mediated by intracellular and/or membranal binding sites and its potential hazard could lead to damage due to increased lipid oxidation, although the transient mediation of ROS in cell proliferation is beneficial to bone growth in a yet unknown mechanism.

Bone loss in ovariectomized rats: dominant role for estrogen but apparently not for FSH.

Estrogen deficiency as the sole factor underlying post-menopausal osteoporosis was challenged, in light of reports that both follicular stimulation hormone (FSH) receptor and FSHß knockout mice were resistant to bone loss, suggesting a detrimental role for FSH. We assessed whether lowering FSH levels by gonadotropin realizing (GnRH) analog decapeptyl in ovariectomized female rats (OVX) affects bone. Wistar-derived 25 days old OVX female rats were injected for 10 weeks with estradiol-17ß (E(2)), with GnRH analog (decapeptyl) or with both. FSH and luteinizing hormone (LH) serum levels were markedly increased in OVX rats, with smaller growth plates with disrupted architecture; heavy infiltration of bone marrow with numerous adipocytes and reduced thickness of cortical bone. In OVX rats treated with E(2), FSH, and LH levels were intermediate, the tibia was similar to that of intact rats, but there was reduced thickness of cortical bone. In decapeptyl treated OVX rats, FSH and LH levels were suppressed, the organization of growth plate and the trabecular bone were disrupted, and there were fewer proliferative and chondroblastic cells and a large adipocytes population in bone marrow, but an increased trabecular bone volume (TBV). In the E(2) + decapeptyl treatment, FSH and LH levels were suppressed, with partially restored growth plate architecture and improved TBV. In conclusion, E(2) deficiency is the dominant factor impairing bone loss in OVX and concomitant changes in FSH/LH levels achieved by decapeptyl have some modulating, though complex role in this setting. The role of high FSH levels in post-menopausal bone loss requires further investigation using combined sub-optimal doses of the different hormones.

Vitamin D analogs induce lipoxygenase mRNA expression and activity as well as reactive oxygen species (ROS) production in human bone cells.

Vitamin D metabolites or its less-calcemic analogs (JKF or QW) are beneficial for bone biology. We analyzed whether or not 25(OH)D3 (25), 1,25(OH)2D3 (1,25), JKF or QW regulate lipooxygenase (LO) enzymes expression and their products hydroxyeicosatetraenoic acid (12 and 15 HETE) formation as well as reactive oxygen species (ROS) production in human bone cell lines (SaOS2 and hFOB) and primary cultured human bone cells (Obs) from males or females. All compounds except 25 increased LOs mRNA expression and HETE production in female or male Obs. ROS formation was induced by JKF and QW in both cell lines, and was inhibited by different inhibitors. Baicalein (baic) an inhibitor of 12 and 15 LO activity, inhibited partially ROS formation by JKF or QW in SaSO2 and hFOB. JKF-stimulated DNA synthesis in female Obs was inhibited by baic but unchanged by addition of HETE or HETE with baic. These results indicate that vitamin D increased oxidative stress in bone cells is in part via induction of LO expression and activity. This new feature of vitamin D is probably mediated by intracellular and/or membranal receptors and its potential hazard could lead to potential damage due to increased lipid oxidation.

Daidzein but not other phytoestrogens preserves bone architecture in ovariectomized female rats in vivo.

Ovariectomy of immature female rats, results in significant decrease of trabecular bone volume and in cortical bone thickness. Previously, we found that estradiol-17beta (E(2)) restored bone structure of ovariectomized (Ovx) female rats to values obtained in intact sham-operated female rats. E(2) also selectively stimulated creatine kinase (CK) specific activity a hormonal-genomic activity marker. In the present study, we compared the effects of E(2) and the phytoestrogens: daidzein (D), biochainin A (BA), genistein (G), carboxy-derivative of BA (cBA), and the SERM raloxifene (Ral) in Ovx, on both histological changes of bones and CK, when administered in multiple daily injections for 2.5 months. Bone from Ovx rats, showed significant disrupted architecture of the growth plate, with fewer proliferative cells and less chondroblasts. The metaphysis underneath the growth plate, contained less trabeculae but a significant increased number of adipocytes in the bone marrow. D like E(2) and Ral but not G, BA, or cBA, restored the morphology of the tibiae, similar to that of control sham-operated animals; the bony trabeculeae observed in the primary spongiosa was thicker, with almost no adipocytes in bone marrow. Ovariectomy resulted also in reduced CK, which in both epiphysis and diaphysis was stimulated by all estrogenic compounds tested. In summary, only D stimulated skeletal tissues growth and differentiation as effectively as E(2) or Ral, suggesting that under our experimental conditions, D is more effective in reversing menopausal changes than any of the other isolated phytoestrogens which cannot be considered as one entity.

Systemic treatments with the low-calcemic 1,25(OH)(2)D(3) analogs JKF or QW increase both the morphological and biochemical responses to estradiol-17beta in rat tibiae.

We demonstrated previously that daily injection for 3 days of the less calcemic vitamin D analogs: JK 1624 F(2)-2 (JKF) and QW 1624F(2)-2 (QW) followed by estradiol-17beta (E(2)) in female rats upregulated creatine kinase-specific activity (CK) in skeletal tissues. In this study, we evaluated both histomorphological and biochemical changes due to a regime of 4 days treatment with JKF or QW, followed by injection of E(2) on day 5, repeated for 2.5 months. Ovariectomized female rats (Ovx) were injected 2 weeks after surgery, with JKF or QW at 0.2 ng/g BW followed by injections of E(2) (1 microg/rat) on day 5 of each week for 2.5 months. Rats were sacrificed 24 h after the last injection and bones were analyzed. JKF alone decreased growth plate width, increased % total bone volume (%TBV), with no change in cortical thickness. In contrast, QW restored growth plate width and %TBV with no change in cortical thickness. Combined with E(2), JKF restored %TBV and growth plate width but with no change in cortical thickness, while QW restored significantly all parameters including cortical thickness. Moreover, there was also an increase in the responsiveness of CK to E(2) in epiphyseal cartilage and diaphyseal bone but not in uterus. Thus, vitamin D less calcemic analogs increased responsiveness to E(2) morphologically as well as biochemically. We, therefore, conclude that combined treatment of less calcemic analogs vitamin D and E(2) might be superior for treatment of bone damage caused by ovariectomy in female rats and might be applied for post-menopausal osteoporosis.

Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: new insights based on studies with carboxy-biochanin A.

Estradiol-17beta (E2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E2 on DNA synthesis, intracellular Ca2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERalpha and suppressed ERbeta mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.

Age and gender specific stimulation of creatine kinase specific activity by gonadal steroids in human bone-derived cells in culture.

We previously reported a non-enzymatic method for isolation of human bone cells in culture that display osteoblastic features and respond to 1,25 dihydroxy vitamin D (1,25) and to parathyroid hormone (PTH). The present study was undertaken to analyze the response of cultured human bone cells to 17beta-estradiol (E2) and to dihydrotestosterone (DHT) as a function of gender and age. Cultured human bone cells, obtained from biopsies during orthopedic surgery, were divided into four groups defined by gender and age: pre- and post-menopausal healthy non-osteoporotic women that were not under hormone replacement therapy (HRT) and mature (<55-year-old) and older (>60-year-old) men. We found gender specific responses to gonadal steroids using the specific activity of the brain type (BB) isozyme of creatine kinase (CK) as a response marker. Constitutive levels of CK activity did not change with age or gender and the enzyme extracted from cells from the different sexes and ages did not respond to either progesterone (P) or to 1,25. CK from the different cells responded to gonadal steroids in a gender specific manner, i.e. CK from female derived cells responded to E2 only and the enzyme from male derived cells responded to DHT only. In female derived cells the response to E2 declined significantly with age, while the response to DHT in CK from male derived cells did not vary with age. This may be due to either decreased proportion of mature osteoblasts and/or their differentiation state and/or changes in the levels of estrogen receptor(s), coactivators or corepressors in these cells. These results extend our knowledge of human osteoblast biology (beyond murine cells) and are therefore more relevant for developing models for treatment of human metabolic bone diseases such as post-menopausal osteoporosis.

Isolation and hormonal responsiveness of primary cultures of human bone-derived cells: gender and age differences.

We present a model for isolating human cell culture derived from biopsies obtained during orthopedic surgery. Four donor groups were defined by gender and age: pre- and postmenopausal women (<50 and >55 years, respectively), and younger (30-55 years) and older (>60 years) men. Bone-derived cells were identified as osteoblasts by major osteoblastic characteristics; that is, high alkaline phosphatase (ALP) activity, dose-dependent increase of ALP by 1,25(OH)2D3, high levels of parathyroid hormone (PTH)-induced cyclic AMP, and 1,25-(OH)2D3-induced osteocalcin. In all cells, levels of osteocalcin were significantly elevated (p < 0.05 and 0.01). In cells derived from men, no significant age differences were found in ALP and osteocalcin values of basal activity and in fold stimulation 1,25(OH)2D3. Cells from postmenopausal women showed a nonsignificant lower basal ALP activity than premenopausal cells. In postmenopausal cells, ALP responded less to 1,25(OH)2D3 (33% increase, p < 0.05) than the premenopausal cells (100% increase, p < 0.05). In cells from either age group, ALP did not respond to the gonadal steroids 17beta-estradiol (E2) and dihydrotestosterone (DHT) or progesterone. Basal levels of osteocalcin were higher in cells of premenopausal origin as compared with postmenopausal cells (p = 0.05), but response to 1,25(OH)2D3 was the same. PTH significantly stimulated cAMP (p = 0.001) in all age and gender groups analyzed. In all groups, no differences were found in either basal activity or in PTH response. Unlike men, cells derived from the bone of women were more susceptible to age changes. We postulate that the postmenopausal cell population had a decreased number of osteoblasts, or cells in a lower differentiation stage. These results extend our knowledge of bone biology found in animal models and reveal that human osteoblasts from men do not show the same age-dependent differences observed in women.