Effects of forskolin on bone resorption in the absence and presence of parathyroid hormone and calcitonin

Summary Release of previously incorporated⁴⁵Ca from fetal rat long bones was determined with the diterpene forskolin, both in the absence and presence of parathyroid hormone (PTH) and calcitonin (CT). In the absence of hormone, increased bone resorption was observed with 10⁻⁷M forskolin, but biphasic responses, consisting of initial decreases in⁴⁵Ca release that were followed by increased calcium mobilization, were produced with 10⁻⁶M and 10⁻⁵M forskolin. Inhibition of⁴⁵Ca release was pronounced and delayed more with 10⁻⁵M forskolin while the greatest stimulation of bone resorption was elicited by 10⁻⁶M forskolin, a response that was inhibited by 100 mU/ml CT. In the presence of 250 ng/ml PTH, a synergistic enhancement of⁴⁵Ca release occurred with 10⁻⁷M forskolin treatment while, in contrast, calcium mobilization was inhibited by 10⁻⁶M and 10⁻⁵M forskolin. Inhibition by 10⁻⁶M forskolin was characterized by “escape” while that of 10⁻⁵M forskolin was continuous over a 5 day interval. Inhibition throughout the experimental period also was noted when 10⁻⁵M forskolin was combined with 2.5 ng/ml PTH, but no effect on calcium mobilization was observed upon addition of 10⁻⁷M forskolin and, rather than inhibition, an enhancement of⁴⁵Ca release occurred when 10⁻⁶M forskolin was combined with 2.5 ng/ml PTH. Inhibition of 250 ng/ml PTH, but lack of inhibition of 2.5 ng/ml PTH by 10⁻⁶M forskolin suggests a 10⁻⁶M forskolin-sensitive portion of PTH-mediated calcium efflux. Absence of “escape” when 10⁻⁵M forskolin is combined with 250 ng/ml PTH suggests that heterologous desensitization may not play a major role in the “escape” which occurs with 10⁻⁶M forskolin.     

Parathyroid hormone induces RGS-2 expression by a cyclic adenosine 3',5'-monophosphate-mediated pathway in primary neonatal murine osteoblasts

Parathyroid hormone (PTH) is a promising anabolic agent for the treatment of osteoporosis. However, PTH is also potently catabolic. To help delineate the molecular mediators of PTH's opposing effects on skeletal metabolism, we have examined PTH-induced regulator of G-protein signaling-2 (RGS-2) expression and function in murine osteoblasts. RGS proteins are GTPase-activating proteins (GAPs) that regulate GTP-binding protein-coupled receptor (GPCR) signaling by enhancing the intrinsic GTPase activity of Galpha subunits. We found that 10 nmol/L PTH maximally induced RGS-2 mRNA in murine MC3T3-E1 cells, rat Py1a and ROS-17/2.8 cells, primary mouse osteoblasts (MOB cells), and mouse calvariae organ culture at 1-2 h posttreatment. PTH signaling through its receptor, PTHR1, is coupled to cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling pathways. We examined the effect of selective signaling agonists and antagonists on RGS-2 expression in MOB cells to determine which pathway(s) mediates PTH-induced RGS-2 expression. Although selective activation of all three pathways led to RGS-2 expression, cAMP-PKA activation with 10 nmol/L PTH and 10 micromol/L forskolin elicited the strongest induction. Similarly, RGS-2 mRNA expression was most strongly inhibited by the PKA inhibitor, H89 (10-30 micromol/L). The phorbol ester, PMA (1 micromol/L), which activates the PKC pathway, and ionomycin (1 micromol/L), which activates the calcium pathway, produced small but detectable elevations in RGS-2 mRNA levels. Overnight treatment with 1 micromol/L PMA to deplete PKC did not affect subsequent RGS-2 induction by PTH, but significantly inhibited PMA-induced RGS-2 expression. Treatment with 1-100 nmol/L PTH(3-34), which does not activate cAMP-PKA signaling, did not induce RGS-2 expression. MOB cells pretreated with 3 microg/mL cycloheximide produced sustained RGS-2 mRNA levels 2 h after 10 nmol/L PTH treatment. Actinomycin D (5 microg/mL) completely blocked 10 nmol/L PTH-induced RGS-2 expression. Finally, we tested the effect of RGS-2 overexpression on PTH- and fluprostenol-induced interleukin (IL)-6 promoter activity in MOB cells. PTH induces IL-6 through PKA activation, whereas fluprostenol induces IL-6 through PKC activation. We found that RGS-2 overexpression significantly inhibited IL-6 promoter activity following fluprostenol treatment, but not following PTH treatment. We conclude that RGS-2 is a PTH-induced primary response gene in murine osteoblasts that is induced mainly through the cAMP-PKA pathway and specifically inhibits Galphaq-coupled receptors.     

Partial characterization of rat marrow stromal cells

Summary Fibroblast-like rat marrow stromal cell (CFU-F) cultures have been characterized in terms of their responsiveness to calciotropic hormones, metal ions, the nonsteroidal antiinflammatory drug, and by their putative paracrine role in the maintenance of active populations of osteoblasts at the marrow-bone interface. These studies indicate that CFU-Fs lack a complete osteoblast signature. Subconfluent CFU-Fs grown in the presence or absence of 10⁻⁷ M dexamethasone lack receptors for PTH and calcitonin, and fail to show enhanced cAMP or cGMP responses to 10⁻⁷ M 1–34 PTH (rat), or any evidence of osteocalcin production [±10⁻⁹ M 1,25-(OH)₂D₃]. Low concentrations of fluoride [10⁻¹² and 10⁻⁹ M] stimulated CFU-F grownin vitro in serum-free media, though higher levels (10⁻⁷ M) did not alter normal growth patterns, indicating an action on bone cells more differentiated than CFU-Fs. Serum-free conditioned medium (CM) from control and ovariectomized (OVX)/OVX+ dihydrotachysterol-R_x rat CFU-F cultures was mitogenic for neonatal rat calvarial osteoblastsin vitro, but not for ROS 17/2.8 cells. The studies affirm the mesenchymal-like character of CFU-Fs and project their significant role in sustaining functional endosteal osteogenic cell populations.     

Parathyroid Hormone Induces Receptor Activity Modifying Protein-3 (RAMP3) Expression Primarily Via 3′,5′-Cyclic Adenosine Monophosphate Signaling in Osteoblasts

Parathyroid hormone (PTH) has significant anabolic and catabolic effects on bone. We hypothesize that PTH-induced primary response genes are important determinants of osteoblast function. PTH induces osteoblastic gene expression through PTHR1, a heptahelical receptor that triggers cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA), protein kinase C (PKC), and calcium signaling. By using representational difference analysis we found that receptor activity modifying protein-3 (RAMP3) is a PTH-induced primary response gene in osteoblastic cells. RAMP3 is a coactivator that directs calcitonin receptor (CTR) and CTR-like receptor (CRLR) glycosylation, trafficking, and ligand-binding specificity. Our purpose was to characterize PTH-induced RAMP3 messenger ribonucleic acid (mRNA) levels in primary mouse osteoblasts (MOBs) and to determine which signaling pathway mediates this effect. 10 nM PTH maximally induced RAMP3 mRNA levels in MOBs at 4 hours. Protein synthesis inhibition with 3 μg/mL cycloheximide did not affect PTH-induced RAMP3 mRNA levels. Selective activation of cAMP-PKA signaling with, 10 μM forskolin (FSK) and PKC signaling with 1 μM phorbol 12-myristate 13-acetate (PMA) significantly increased RAMP3 mRNA levels, whereas 1 μM ionomycin (a calcium ionophore) had no effect. Pretreatment with 30 μM H89, a PKA inhibitor, significantly blocked PTH- and FSK-induced RAMP3 mRNA levels. Pretreatment with 1 μM PMA, which depletes PKC, had no effect on PTH- and FSK-induced RAMP3 mRNA levels but blocked PMA-induced RAMP3 mRNA levels. 100 nM PTH (3-34), which activates PKC and calcium but not PKA, had no effect on RAMP3 mRNA levels. These findings indicate that RAMP3 is a PTH-induced primary response gene in primary MOBs and that PTH regulates RAMP3 gene expression primarily through the cAMP-PKA pathway.     

Determinants of Plasma Parathyroid Hormone Levels in Young Women

While the effects of calcium, phosphorus intake, and vitamin D on parathyroid hormone (PTH) have been well studied, less is known about other factors that impact PTH. Our goal was to delineate associations between demographic, dietary, and plasma factors and PTH. We conducted a cross-sectional study of intact PTH among 1,288 nonblack women in the Nurses Health Study II aged 33–53 with BMI <30 kg/m² and eGFR ≥60 ml/min/1.73 m². Median PTH was 30.7 pg/ml. After adjusting for 25-hydroxyvitamin D and other factors, PTH was 4.1 pg/ml lower (95% CI −7.7 to −0.5) in women who smoked 1–14 cigarettes/day and 6.4 pg/ml lower (95% CI −11.2 to −1.7) in women who smoked >15 cigarettes/day compared to nonsmokers. After multivariate adjustment, women whose BMI was 27–29 kg/m² had PTH levels 2.0 pg/ml higher (95% CI 0.2–3.9) compared to BMI of 21–22 kg/m² and women in the highest quartile of plasma phosphorus had PTH levels 4.1 pg/ml lower (95% CI −5.8 to −2.4) than women in the lowest quartile. Higher vitamin A intake was independently associated with lower PTH, whereas lower calcium intake, lower plasma calcium, lower plasma 25-hydroxyvitamin D, and winter blood draw were associated with higher PTH. Intakes of phosphorus, animal protein, magnesium, alcohol, and caffeine were not associated with PTH. Factors not classically associated with calcium–phosphorus metabolism impact PTH. Additional research is needed to elucidate the mechanisms whereby smoking, vitamin A, and phosphorus affect PTH and to examine how body size and season may affect PTH independent of 25(OH)D.     

Hormonal and nonhormonal desensitization in isolated bone cells

Summary Prior exposure to PTH markedly decreased the responsiveness of isolated, cultured bone cells to the stimulatory effect of the hormone on cyclic AMP formation. This process of desensitization developed within 30 min, persisted during prolonged incubation of the cells in PTH-free medium, and could not be attributed to enhanced excretion of cyclic AMP from the cells, nor to the extracellular accumulation of an inhibitor of PTH action. Adenylate cyclase activity in a subcellular fraction derived from PTH-treated cells was refractory to PTH and to sodium fluoride. These results indicate that PTH-mediated desensitization reflects, at least in part, impaired cyclic AMP formation. Adenosine and PGE₂, known stimulators of bone cell cyclic AMP formation, elicited agonist-specific desensitization, and also desensitized bone cells to the effects of subsequently added PTH. PTH blunted the cellular response to adenosine, but not to PGE₂. Modest refractoriness to PTH was evident in cells that had been treated previously with the cyclic AMP phosphodiesterase inhibitors IBMX, theophylline, and Bt₂cAMP, whereas treatment with sodium butyrate had no effect. The actions of the inhibitors, like that of PTH, were rapid in onset and long-lasting. Desensitization caused by previous treatment with the phosphodiesterase inhibitors, and with PTH itself, was accompanied by enhanced phosphodiesterase activity in bone cell homogenates. Induction of phosphodiesterase activity may well contribute to desensitization in the bone cell system. Abbreviations used in this paper: HBSS, Hank's balanced salt solution; EBSS, Earle's balanced salt solution; IBMX, 3 isobutyl 1 methyl xanthine; PTH, synthetic bovine parathyroid hormone (amino acids 1-34); PGE₁, prostaglandin E₁; PGE₂, prostaglandin E₂; Bt₂cAMP, N⁶,O²′ dibutyryl cyclic 3′,5′ adenosine monophosphate.     

Intermittent treatment with parathyroid hormone (PTH) as well as a non-peptide small molecule agonist of the PTH1 receptor inhibits adipocyte differentiation in human bone marrow stromal cells

Whereas continuous PTH infusion increases bone resorption and bone loss, intermittent PTH treatment stimulates bone formation, in part, via reactivation of quiescent bone surfaces and reducing osteoblast apoptosis. We investigated the possibility that intermittent and continuous PTH treatment also differentially regulates osteogenic and adipocytic lineage commitment of bone marrow stromal progenitor/mesenchymal stem cells (MSC). The MSC were cultured under mildly adipogenic conditions in medium supplemented with dexamethasone, insulin, isobutyl-methylxanthine and troglitazone (DIIT), and treated with 50 nM human PTH(1–34) for either 1 h/day or continuously (PTH replenished every 48 h). After 6 days, cells treated with PTH for 1 h/day retained their normal fibroblastic appearance whereas those treated continuously adopted a polygonal, irregular morphology. After 12–18 days numerous lipid vacuole and oil red O-positive adipocytes had developed in cultures treated with DIIT alone, or with DIIT and continuous PTH. In contrast, adipocyte number was reduced and alkaline phosphatase staining increased in the cultures treated with DIIT and 1 h/day PTH, indicating suppression of adipogenesis and possible promotion of early osteoblastic differentiation. Furthermore, intermittent but not continuous PTH treatment suppressed markers of differentiated adipocytes such as mRNA expression of lipoprotein lipase and PPARγ as well as glycerol 3-phosphate dehydrogenase activity. All of these effects of intermittent PTH were also produced by a 1 h/day treatment with AH3960 (30 μM), a small molecule, non-peptide agonist of the PTH1 receptor. AH3960, like PTH, activates both the cAMP and calcium signaling pathways. Treatment with the adenylyl cyclase activator forskolin for 1 h/day, mimicked the anti-adipogenic effect of intermittent PTH, whereas pretreatment with the protein kinase-A inhibitor H89 prior to intermittent PTH resulted in almost complete conversion to adipocytes. In contrast, the MAP kinase inhibitor PD 98059 failed to prevent the anti-adipocytic effect of intermittent PTH, suggesting that the inhibitory effect of PTH on adipocyte differentiation is predominantly cAMP-dependent. These results demonstrate a differential effect of PTH1 receptor agonists on the adipocytic commitment and differentiation of adult human bone marrow mesenchymal stem cells. This response may represent an additional mechanism that contributes to the overall bone anabolic action of intermittent PTH.     

Induction of ornithine decarboxylase activity in isolated chicken osteoblasts by parathyroid hormone: The role of cAMP and calcium

Summary We investigated the role of cAMP and Ca²⁺ as mediators in parathyroid hormone (PTH)-induced ornithine decarboxylase (ODC) activity in primary cultures of chicken osteoblasts. We present evidence that the induction of ODC activity by PTH is most likely a receptor-mediated process and that cAMP is a mediator. However, using three different approaches we have strong indications that cAMP is not the exclusive mediator of PTH-induced ODC activity. First, when the dose-response curve of PTH-induced ODC activity is compared with that of PTH-stimulated cAMP production, the ED₅₀ for cAMP production is about five times as high as that for the induction of ODC activity. Second, 1 mM 9-(tetrahydro-2-furanyl) adenise (SQ 22.536) almost completely inhibited PTH-stimulated cAMP production whereas there was only a small inhibitory effect on PTH-induced ODC activity. Third, some PTH fragments unable to stimulate cAMP production were still able to induce ODC activity. We therefore propose that apart from cAMP, an additional messenger, most likely Ca²⁺, must be present. Evidence for this concept are the observations that substances affecting extracellular and intracellular Ca²⁺ levels (EGTA, A23187, CoCl₂, verapamil) or antagonizing calmodulin (Trifluoroperazin, Compound 48/80) also strongly affect PTH-induced ODC activity. These effects could not be explained by a positive interaction of Ca²⁺ with the hormone-stimulated cAMP system as 2 mM EGTA strongly enhanced PTH-stimulated cAMP production but at the same time completely inhibited PTH-induced ODC activity. A similar dissociation between hormone-induced cAMP production and induction of ODC activity was found with the Ca²⁺-ionophore A23187 (10⁻⁷M) which significantly inhibited PTH-stimulated cAMP production but strongly enhanced PTH-induced ODC activity. Our results suggest that intracellular Ca²⁺, and possibly calmodulin, in addition to cAMP, are involved in PTH-induced ODC activity in chicken osteoblasts. Most probably Ca²⁺ is the initial messenger and cAMP acts in a coordinate pattern as a synarchic messenger making the induction of ODC activity by PTH more sensitive to Ca²⁺. Furthermore, the present findings are in agreement with our concept of the existence of two receptors or two receptor-sites for PTH on osteoblasts. One receptor is coupled to the production of cAMP and is presumably activated when the first two amino-acids of the NH2-terminus of the hormone are present and the other, suggested to be responsible for the increase in intracellular Ca²⁺, is thought to be activated by a region of the hormone sequence between amino acid 3 and 34. Activation of both receptors by the intact hormone PTH(1–84) or its biological active fragment PTH(1–34) leads to the maximal induction of ODC activity.     

Effect of vitamin D3, 25-hydroxyvitamin D3, and 24,25-dihydroxyvitamin D3 on parathyroid hormone secretion

Summary The active vitamin D metabolite 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] causes marked suppression of both pre-proparathyroid hormone messenger RNA (pre-proPTH mRNA) and parathyroid hormone (PTH) secretion. These effects are dose dependent and reversible when tested in anin vitro primary tissue culture cell system using normal bovine parathyroid cells. In the current studies, the precursors of 1,25(OH)₂D₃ and the related metabolite 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], were used in the same culture system to test for possible regulatory effects. The results were compared with identically prepared cells exposed to 1,25(OH)₂D₃. In short-term studies (30–120 minutes), none of the vitamin D-related compounds produced any effect on PTH secretion. In long-term studies (24–48 hours, using primary tissue culture in the presence of test agents), neither vitamin D₃ nor 25(OH)D₃ affected PTH secretion or pre-proPTH mRNA over the concentration range 10⁻¹¹–10⁻⁷M. On the other hand, 24,25(OH)₂D₃ produced significant suppression of both pre-proPTH mRNA (77% of control,P<.01) and PTH secretion (75% of control,P<.005) at 10⁻⁷ M. By comparison, 10⁻¹¹ M 1,25(OH)₂D₃ produced levels of suppression (25–30%) of both pre-proPTH mRNA and PTH secretion comparable to 10⁻⁷ M 24,25(OH)₂D₃, while even greater suppression (40–50%) occurred at 10⁻⁹-10⁻⁷ M 1,25(OH)₂D₃. From these studies, we conclude that vitamin D₃ and 25(OH)D₃ do not have significant effects on PTH synthesis and secretion over the range of doses tested. Compared with 1,25(OH)₂D₃, 24,25(OH)₂D₃ exhibits mild suppression at pharmacologic concentrations. The effect of 24,25(OH)₂D₃ prabably occurs through weak interaction of 24,25(OH)₂D₃ with the 1,25(OH)₂D₃ receptor.     

Effects of tumor necrosis factor alpha on parathyroid hormone-induced increases in osteoblastic cell cyclic AMP

Summary Tumor necrosis factor α (10⁻¹⁰–10⁻⁸M) had no effects on cyclic AMP production by the osteoblastic osteosarcomal cells, Saos-2 and G292, or normal rat calvarial cells. The cytokine did, however, inhibit the parathyroid hormone (PTH)-induced effect on cyclic AMP in the Saos-2 and normal rat osteoblastic cells. This inhibitory effect did not occur on prostaglandin E₂-induced cyclic AMP increases in the osteoblastic cells. Interleukin-1 (10 U/ml −100 U/ml) did not produce any effect on basal levels or PTH-induced cyclic AMP increases in these cells.     

The effects of hydrocortisone,parathyroid hormone and the bisphosphonate,APD, on bone resorption in neonatal mouse calvaria

Summary The effects of hydrocortisone and parathyroid hormone (PTH) upon bone resorption rates in neonatal mouse calvaria have been studied. Bone resorption (measured as⁴⁵Ca release) was significantly increased by hydrocortisone (10⁻⁷ M and 10⁻⁶ M) and there was a dose-dependent rise with PTH (0.3–0.9 μg/liter). When both PTH 0.3 μg/liter and hydrocortisone 10⁻⁸ M were present in the incubating medium, bone resorption did not differ from control, but increasing the hydrocortisone concentration to 10⁻⁷ M augmented⁴⁵Ca release by 25% (P<0.02) and doubling of the PTH level was associated with a 10% increase (nonsignificant). When both PTH and hydrocortisone were present in the higher concentrations (0.6 μg/liter and 10⁻⁷ M, respectively)⁴⁵Ca release increased by 39% (P<0.005) above that resulting from the lower levels of both hormones (0.3 μg/l and 10⁻⁸ M, respectively). (3-Amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) in concentrations of 3×10⁻⁵ M and 10⁻⁴ M, produced inhibition of basal and hydrocortisone/PTH-stimulated bone resorption without evidence of toxicity. These results indicate that hydrocortisone stimulates bone resorption in neonatal mouse calvariain vitro, in contrast to the result found in fetal rat bone culture systems. PTH has a similar effect, which is additive to that of hydrocortisone and the combined stimulation can be overcome by APD. The possible relevance of these results to the development and prevention of glucocorticoid-induced osteoporosis is discussed.     

Docetaxel inhibits bone resorption through suppression of osteoclast formation and function in different manners

Osteoclasts are formed from the monocyte-macrophage lineage in response to receptor activator of nuclear factor κB ligand (RANKL) expressed by osteoblasts. Bone is the most common site of breast cancer metastasis, and osteoclasts play roles in the metastasis. The taxane-derived compounds paclitaxel and docetaxel are used for the treatment of malignant diseases, including breast cancer. Here we explored the effects of docetaxel on osteoclastic bone resorption in mouse culture systems. Osteoclasts were formed within 6 days in cocultures of osteoblasts and bone marrow cells treated with 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂. Docetaxel at 10⁻⁸ M inhibited osteoclast formation in the coculture when added for the entire culture period or for the first 3 days. Docetaxel, even at 10⁻⁶ M added for the final 3 days, failed to inhibit osteoclast formation. Osteoprotegerin, a decoy receptor of RANKL, completely inhibited osteoclast formation when added for the final 3 days. Docetaxel at 10⁻⁸ M inhibited the proliferation of osteoblasts and bone marrow cells. RANKL mRNA expression induced by 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂ in osteoblasts was not affected by docetaxel even at 10⁻⁶ M. Docetaxel at 10⁻⁶ M, but not at 10⁻⁸ M, inhibited pit-forming activity of osteoclasts cultured on dentine. Actin ring formation and l-glutamate secretion by osteoclasts were also inhibited by docetaxel at 10⁻⁶ M. Thus, docetaxel inhibits bone resorption in two different manners: inhibition of osteoclast formation at 10⁻⁸ M and of osteoclast function at 10⁻⁶ M. These results suggest that taxanes have beneficial effects in the treatment of bone metastatic cancers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Parathyroid Hormone,Prostaglandin E2, and 1,25-Dihydroxyvitamin D3 Decrease the Level of Na+-Ca2+ Exchange Protein in Osteoblastic Cells

We previously described Na⁺-Ca²⁺ exchange in osteoblastic rat osteosarcoma cells (UMR-106) and demonstrated that Na⁺-dependent Ca²⁺ transport was inhibited by 24-hour treatment of cells with parathyroid hormone (PTH), prostaglandin E₂ (PGE₂), or 1,25(OH)₂D₃. To determine whether this inhibition of Na⁺-Ca²⁺ exchange is at the level of exchanger protein synthesis we have examined exchanger protein levels using immunoblot analysis. UMR-106 cells were treated for 24 hours with or without PTH, PGE₂, or 1,25(OH)₂D₃. Plasma membrane fractions (7500 g) were obtained and proteins were separated by SDS-PAGE, transferred to nylon membranes, and immunoblotted with a polyclonal antibody to the canine cardiac Na⁺-Ca²⁺ exchanger. In rat cardiac membranes, we detected 125 and 75 kD bands, similar to findings for the canine exchanger. In the osteoblastic UMR cell membranes, a specific band was detected at 90 kD that decreased 65% after treatment of cells with PTH. Inhibition by PTH was dose dependent, was maximal with 10⁻⁷ M PTH, and required 16–24 hour treatment time. Similar inhibition was observed after a 24 hour treatment with 10⁻⁶ M PGE₂ or 10⁻⁸ M 1,25(OH)₂D₃. These results demonstrate the presence of a specific protein in UMR cells that cross-reacts with antibody directed against the cardiac Na⁺-Ca²⁺ exchanger. Thus, the previously reported inhibition of Na⁺-Ca²⁺ exchange activity by calcemic agents in osteoblasts appears to be due to regulation of exchanger protein levels in these osteoblastic cells. Received: 5 February 1996 / Accepted: 18 October 1996     

Regulation of hormone-induced cyclic AMP response to parathyroid hormone and prostaglandin E2 in cells cultured from human giant cell tumors of bone

Summary Cells dispersed from human giant cell tumors of bone and grown in monolayer culture increase intracellular cyclic AMP (cAMP) when incubated with parathyroid hormone (PTH) or prostaglandin E₂ (PGE₂). When cells are continuously exposed to PTH, cAMP levels increase acutely but then decrease rapidly to pretreatment values despite continued presence of hormone or addition of new hormone. Preincubation of cells with PTH for periods as short as 10 min results in a decrease in the capacity of cells to increase cAMP content when re-exposed to maximal stimulatory concentrations of PTH. The decrease in the magnitude of the PTH-induced cAMP response observed in cells pretreated with this hormone is dependent on the concentration of PTH present during the pre-incubation. The loss of cAMP response in cells pre-treated with either PGE₂ or PTH is hormone specific in that cells made refractory by pretreatment with one hormone still increase cAMP content when exposed to the other. Although the cells are not releasing measurable amounts of prostaglandins into the medium, pretreatment with indomethacin results in an increase in the magnitude of the cAMP response to PGE₂. The PTH-induced cAMP response is not affected by indomethacin pre-treatment. The loss of PTH responsiveness produced by hormone preincubation is consistent with the phenomenon of “down-regulation” observed with ligand-receptor interactions in a variety of tissues.     

Anabolic effect of phosphogenistein and phosphodaidzein on bone components in rat femoral-metaphyseal tissues in vitro

The effect of phosphogenistein and phosphodaidzein, which are phosphorylated for the hydroxyl group (OH) at the 7-position of genistein and daidzein, on bone components was investigated. Femoral-metaphyseal tissues obtained from male rats (4 weeks old) were cultured for 24–72 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The presence of phosphogenistein (10⁻⁵ M) caused a significant increase in calcium content, alkaline phosphatase activity, and deoxyribonucleic acid (DNA) content in bone tissues cultured for 24 h. Phosphodaidzein (10⁻⁵ M) significantly elevated bone calcium and DNA content. These effects were completely prevented by the presence of cycloheximide (10⁻⁶ M), an inhibitor of protein synthesis. When femoral-metaphyseal tissues were cultured for 48 h in the presence of parathyroid hormone (1–34) (PTH; 10⁻⁸ M) or prostaglandin E₂ (PGE₂; 10⁻⁶ M), bone calcium content was significantly decreased. This decrease was significantly blocked by the presence of phosphogenistein (10⁻⁶ and 10⁻⁵ M) or phosphodaidzein (10⁻⁶ and 10⁻⁵ M). The presence of PTH (10⁻⁸ M) or PGE₂ (10⁻⁶ M) caused a significant increase in glucose consumption and lactic acid production by bone tissues. These increases were significantly inhibited by the presence of phosphogenistein (10⁻⁵ M) or phosphodaidzein (10⁻⁵ M), indicating their inhibitory effect on bone resorption. The present study has demonstrated that both phosphogenistein and phosphodaidzein have an anabolic effect on bone metabolism in rat femoral-metaphyseal tissues in vitro. Received: June 13, 2001 / Accepted: November 22, 2001     

β-arrestin2 regulates parathyroid hormone effects on a p38 MAPK and NFκB gene expression network in osteoblasts

Interaction of the cytoplasmic adaptor molecule β-arrestin2 with the activated parathyroid hormone (PTH)/PTHrP receptor inhibits G protein mediated signaling and triggers MAPKs signaling. In turn, the effects of both intermittent (i.) and continuous (c.) PTH on bone are altered in β-arrestin2-deficient (Arrb2^−/−) mice. To elucidate the expression profile of bone genes responsive to PTH and targeted for regulation by β-arrestin2, we performed microarray analysis using total RNA from primary osteoblastic cells isolated from wild-type (WT) and Arrb2^−/− mice. By comparing gene expression profiles in cells exposed to i.PTH, c.PTH or vehicle (Veh) for 2 weeks, we found that i.PTH specifically up-regulated 215 sequences (including β-arrestin2) and down-regulated 200 sequences in WT cells, about two-thirds of them being under the control of β-arrestin2. In addition, β-arrestin2 appeared necessary to the down-regulation of a genomic cluster coding for small leucin-rich proteins (SLRPs) including osteoglycin, osteomodulin and asporin. Pathway analyses identified a main gene network centered on p38 MAPK and NFκB that requires β-arrestin2 for up- or down-regulation by i.PTH, and a smaller network of PTH-regulated genes centered on TGFB1, that is normally repressed by β-arrestin2. In contrast the expression of some known PTH gene targets regulated by the cAMP/PKA pathway was not affected by the presence or absence of β-arrestin2 in osteoblasts. These results indicate that β-arrestin2 targets prominently p38 MAPK- and NFκB-dependent expression in osteoblasts exposed to i.PTH, and delineates new molecular mechanisms to explain the anabolic and catabolic effects of PTH on bone.     

Inhibition of parathyroid hormone-stimulated bone resorption by monovalent cation ionophores

Summary The actions of divalent cation ionophores on bone resorption in vitro are complex; both enhancement of resorption and inhibition of stimulated resorption have been observed with the ionophore A23187. We have found in neonatal mouse calvaria, in which divalent ionophores were only inhibitory, that monovalent cation ionophores were even more potent inhibitors of stimulated bone resorption. Nigericin, monensin, and X206 each inhibited the release of calcium (Ca) from calvaria that were stimulated to resorb by 0.1 U/ml parathyroid hormone (PTH). Actions of the three ionophores were dose dependent and were maximal at 10⁻⁷M, 3×10⁻⁷M, and 1.2×10⁻⁷M, respectively, compared to A23187, which was maximally inhibitory at 2×10⁻⁶M. After pretreatment with nigericin alone or together with PTH for 24 h, inhibition of stimulated resorption was partially reversible. Prolonged (48 h) treatment, either with ionophore alone or together with PTH, caused irreversible inhibition of stimulated Ca release. However, the ionophore was only partially inhibitory if it was added to calvaria stimulated by pretreatment with PTH alone for 24 or 48 h. Resorption stimulated by prostaglandin E₂, 1,25-dihydroxyvitamin D₃, and epidermal growth factor was also inhibited by monovalent ionophores, indicating that the inhibition was not at the level of the PTH receptor. In addition, ionophores did not lower basal or PTH-stimulated production of cyclic AMP by calvaria. Submaximal doses of nigericin were synergistic with calcitonin or ouabain in inhibiting PTH-stimulated resorption. These results are consistent with the hypothesis that stimulated release of Ca from bone occurs by a Na/Ca exchange mechanism. Thus monovalent cation ionophores would increase intracellular Na⁺, thereby decreasing the Na gradient across bone cell membranes, leading to conditions unfavorable for Ca efflux coupled to further Na influx.     

Calcium and protein kinase C enhance parathyroid hormone- and forskolin-stimulated adenylate cyclase in ROS 17/2.8 cells

Summary Both parathyroid hormone (PTH)- and forskolin-stimulated adenylate cyclase activities in ROS 17/2.8 cells are enhanced by increasing the medium concentrations of CaCl₂ from 10⁻⁵ M to 3 ×10⁻³ M. The ED₅₀ for CaCl₂ for both PTH- and forskolin-stimulated activities are similar. The tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, also enhanced both PTH- and forskolin-stimulated adenylate cyclase. This action of PMA is specific for protein kinase C as phorbol esters that are not activators of protein kinase C had no effect on the system. The combined effects of PMA and CaCl₂ were more than additive. The separate and combined effects of PMA and CaCl₂ changed the rate of activation of the enzyme (V_max) but did not modify the ED₅₀ for PTH or for forskolin. PMA and CaCl₂ both enhanced the potentiating effect of submaximal dose of forskolin on PTH-stimulated adenylate cyclase. It is concluded that calcium and PMA enhance PTH-sensitive adenylate cyclase and increase the production of cAMP by a mechanism that appears to involve the catalytic subunit of the enzyme and probably its interaction with a guanine nucleotide regulatory protein.