Hormonal regulation of [Ca(2+)](i) in periosteal-derived osteoblasts: effects of parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandin E(2)

The effects of hormonal modulators of osteoblast function, parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandins on [Ca(2+)](i) in periosteal-derived osteoblasts from rat femurs have been investigated. Our results show that application of parathyroid hormone PTH (10(-5) M) and prostaglandin E(2) (PGE(2)) (4 microM) result in a rapid heterogeneous elevation in [Ca(2+)](i) that, in the case of PTH, is dependent on both extracellular and intracellular sources of calcium. Variable responses to treatments have been found within populations of cells. The PGE(2) response is dose dependent. Treatment with 1,25(OH)(2)D(3) (10(-8) M) induces a brief (60-90 sec) elevation in [Ca(2+)](i) that is almost totally abolished in EGTA-buffered Ca(2+)-free medium. Interactive effects of multiple hormone treatments have been observed. Pretreatment with 1,25(OH)(2)D(3) results in near-total inhibition of the PTH and PGE(2) responses. In conclusion, modulation of [Ca(2+)](i) appears to play a role not only in the direct effects of osteotropic hormones on osteoblasts but also in the synergistic and antagonistic effects between circulating hormones.     

Ovariectomy worsens secondary hyperparathyroidism in mature rats during low-Ca diet

Estrogen deficiency impairs intestinal Ca absorption and induces bone loss, but its effects on the vitamin D-endocrine system are unclear. In the present study, calciotropic hormones levels, renal vitamin D metabolism, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-dependent intestinal calcium absorption, and bone properties in 3-mo-old sham-operated (sham) or ovariectomized (OVX) rats fed either a normal-Ca (NCD; 0.6% Ca, 0.65% P) or a low-Ca (LCD; 0.1% Ca, 0.65% P) diet for 2 wk were determined. LCD increased serum 1,25(OH)2D3 levels in both sham and OVX rats. Serum parathyroid hormone [PTH(1-84)] levels were highest in OVX rats fed LCD. Renal 25-hydroxyvitamin D1alpha-hydroxylase (1-OHase) protein expression was induced in both sham and OVX rats during LCD, while renal 1-OHase mRNA expression was highest in OVX rats fed LCD. Renal vitamin D receptor (VDR) and mRNA expressions in rats were induced by ovariectomy in rats fed NCD but suppressed by ovariectomy in rats fed LCD. The induction of intestinal calcium transporter-1 and calbindin-D9k mRNA expressions by LCD were not altered by ovariectomy. As expected, bone Ca content, cancellous bone mineral density, and bone strength index in proximal metaphysis of rat tibia were reduced by both ovariectomy and LCD (P<0.05) as analyzed by two-way ANOVA. Taken together, the data demonstrate that ovariectomy alters the responses of circulating PTH levels, renal 1-OHase mRNA expression, and renal VDR expression to LCD. These results suggest that estrogen is necessary for the full adaptive response to LCD mediated by both PTH and 1,25(OH)2D3.     

Formation of mineralized bone nodules by rat calvarial osteoblasts decreases with donor age due to a reduction in signaling through EP(1) subtype of prostaglandin E(2) receptor.

The effects of prostaglandin E(2) (PGE(2)) on the parameters for proliferation and differentiation were studied in calvarial osteoblast-like cells isolated from rats of various ages. In cells not treated with PGE(2), it was found that mineralized bone nodule (BN) formation, alkaline phosphatase (ALP) activity, and the incorporation rate of [(3)H]thymidine into the cells sharply decreased with the age of the cell donor at 6-50 weeks and then remained at a relatively constant level up to 120 weeks. Before studying the effects of PGE(2) on these parameters, we determined the change in the levels of PGE(2) produced by the untreated cells during the culture period and found that the endogenous PGE(2) reached a maximum on the 4th day of the culture, regardless of the cell donor age, followed by a sharp decrease. The endogenous production was blocked by pretreatment with a cyclooxygenase-2 (COX-2) inhibitor, NS-398, indicating the generation of PGE(2) through a COX-2 pathway. The area of BN was effectively suppressed by NS-398 in the cells from 10- to 35-week-old rats, whereas it was enhanced in the cells from 90- to 120-week-old rats. Treatment with PGE(2 )markedly increased the BN formation and the ALP activity in the cells from 4- to 35-week-old rats (defined as young rats). By contrast, PGE(2) decreased [(3)H]thymidine incorporation into the cells from young rats. The area of BN and the ALP activity decreased significantly, whereas [(3)H]thymidine incorporation into the cells increased by 60-80% in the cells of 80- to 120-week-old rats (defined as aged rats). The stimulatory effects on the cell differentiation and the inhibitory effect on the proliferation in the cells from young rats was mimicked by an EP(1) agonist, 17-phenyl-omega-trinor PGE(2), while an EP(2)/EP(4) agonist, 11-deoxy-PGE(1) and an adenylate cyclase activator, forskolin suppressed the differentiation and enhanced the proliferation regardless of the cell donor age. PGE(2), 11-deoxy-PGE(1) and forskolin, but not 17-phenyl-omega-trinor PGE(2) increased cyclic adenosine monophosphate (cAMP) production. Generation of inositol 1, 4,5-triphosphate (IP(3)) was stimulated by 17-phenyl-omega-trinor PGE(2) or PGE(2), but not by 11-deoxy-PGE(1) or forskolin increased cAMP production in the cells from young rats. By contrast, PGE(2 )had little effect on IP(3 )generation in aged rats. From the overall results, we concluded that PGE(2) exerts stimulatory and inhibitory effects on differentiation through the EP(1)-IP(3) pathway and EP(2)/EP(4)-cAMP pathway, respectively, in the cells from young rats. The EP(1)-IP(3) pathway seems to be inactive in the cells from aged rats.     

Bone morphogenetic protein-7 (OP1) and transforming growth factor-beta1 modulate 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts

Bone morphogenetic proteins (BMPs) and transforming growth factor-beta (TGFbeta) are potent regulators of osteoblast differentiation and proliferation, processes that are crucial in bone remodeling. BMPs and TGFbeta act in concert with other local factors and hormones, among them 1,25(OH)2-vitamin D3 and insulin. Here we show that BMP7 inhibits 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts, whereas TGFbeta1 stimulates it, as assessed by assays for alkaline phosphatase (ALP) induction, matrix mineralization, and morphology changes. BMP7 or TGFbeta1 alone affects the differentiation of human osteoblasts. Similar results were obtained in assays for ALP induction using conditionally immortalized human osteoblasts (hFOB) and primary osteoblasts obtained from trabecular bone of the femoral head after hip replacement surgery. BMP7 stimulation led to a decrease of 1,25(OH)2-vitamin D3-induced binding of nuclear proteins to a vitamin D response element, as shown by electrophoretic mobility shift assay. Our results suggest that 1,25(OH)2-vitamin D3 modulates in opposite ways the effects of BMP7 and TGFbeta1 on osteoblast differentiation.     

The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2.

The possibility that the non-osteogenic mouse pluripotent cell line, C3H10T1/2 (10T1/2), could be induced to differentiate into osteogenic cells by various hormones and cytokines was examined in vitro. Of a number of agents tested, recombinant human bone morphogenetic protein-2 (rhBMP-2) and retinoic acid induced alkaline phosphatase (ALP) activity in 10T1/2 cells. rhBMP-2 also induced mRNA expression of ALP in the cells. Dexamethasone, 1 alpha, 25-dihydroxyvitamin D3, transforming growth factor-beta 1 and insulin-like growth factor-I did not stimulate ALP activity. Treatment with rhBMP-2 greatly induced cAMP production in response to parathyroid hormone in 10T1/2 cells. No ALP activity was induced in NIH3T3 fibroblasts treated with rhBMP-2 or retinoic acid. These results indicate that 10T1/2 cells have a potential to differentiate into osteogenic cells under the control of BMP-2.     

Effects of vitamin D3 metabolites on cytosolic free calcium in confluent mouse osteoblasts

A fluorescent Ca2+ indicator, acetoxymethyl Quin2, was used to quantify changes in the cytosolic free calcium concentration ([Ca2+]i) of confluent mouse osteoblasts. 1,25 - Dihydroxycholecalciferol (1,25 - (OH)2D3, 10-100 pM), 25-hydroxycholecalciferol (25-(OH)D3, 10-100 nM), parathyroid hormone (PTH(1-84), 0.1-10 nM), and prostaglandin E2 (PGE2, 10-1000 nM) all induced immediate (t less than 15 s) transient increases in [Ca2+]i, from a basal level of 135 +/- 8 nM to levels of 179-224 nM. These increases rapidly returned to a plateau approximately 10% higher than the basal level. 24,25-Dihydroxycholecalciferol (24,25-(OH)2D2, 0.1-10 nM) induced a rapid increase in [Ca2+]i which remained elevated for 5 min before decreasing. The 1,25-(OH)2D3- and PTH-induced spikes were abolished by the prior addition of EGTA and Ca2+ entry blockers (verapamil, nifedipine, 1 microM) while the responses to 25-(OH)D3, 24,25-(OH)2D3, and PGE2 were unaffected. Addition of 1,25-(OH)2D3 + EGTA or PTH + EGTA caused enhanced Ca efflux. Addition of drugs which interfere with calcium sequestration by the endoplasmic reticulum (ER) (caffeine, 4 mM; 8-(diethyl-amino)-octyl 3,4,5-trimethoxybenzoate HCl, 0.5 mM) or mitochondria (antimycin, 10 microM; oligomycin, 5 microM) showed that 25-(OH)D3 and PGE2 mainly mobilized Ca2+ from ER. 1,25-(OH)2D3 and bovine PTH caused a transient increase in [Ca2+]i, 70% of which resulted from Ca2+ influx from outside the cells and 30% by release from the ER. The [Ca2+]i increase induced by 24,25-(OH)2D3 included a 30% contribution from the ER and 70% from the mitochondria.     

Differential stimulation by PGE(2) and calcemic hormones of IL-6 in stromal/osteoblastic cells

Formation of osteoclast-like cells in mouse bone marrow cultures induced by either 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), parathyroid hormone (PTH) or prostaglandin E(2) (PGE(2)), respectively, shows partial dependence on interleukin-6 receptor (IL-6R) activation. This suggests that locally produced IL-6 could be relevant for osteoclast formation. Therefore, we evaluated the effects of 1,25-(OH)(2)D(3), PTH, and PGE(2) on IL-6 production in stromal/osteoblastic cell lines. It appeared that these bone resorptive factors differed widely in their ability to modulate IL-6 mRNA expression and, consequently, protein synthesis in each of the cell lines studied. While 1,25-(OH)(2)D(3) was marginally effective only in ST2 cells, and PTH caused a 2- to 20-fold increase in IL-6 levels MC3T3-E1 and UMR-106 cells, PGE(2) enhanced IL-6 production in the ST2 and MC3T3-E1 cell line by two to three orders of magnitude, respectively, and also induced IL-6 in fibroblastic L929 cells. PGE(2)-stimulated IL-6 release from mesenchymal cells seems to be important for autocrine/paracrine control of osteoclast formation in health and disease.     

Individual and combined effects of calciotropic hormones and growth factors on mineral metabolism in embryonic chick tibiae

Summary We have investigated single and combined effects of calciotropic hormones and growth factors on the regulation of alkaline phosphatase (ALP) activity and calcium metabolism in an optimized serum-free bone organ culture system of embryonic chick tibiae. Parathyroid hormone PTH(1–34) alone mobilized calcium from bone tissue time- and dose-dependently and inhibited ALP activity. Both the bisphosphonate (BM 21.0955) and to a lesser extent salmon calcitonin alone slightly increased calcium uptake and inhibited the stimulation of bone resorption by PTH(1–34). 1,25(OH)₂D₃ mobilized calcium and inhibited ALP activity in contrast to 24,25(OH)₂D₃ which inhibited ALP activity but had no significant effect on calcium metabolism. Interestingly the combination of PTH(1–34) with 1,25(OH)₂D₃ but not 24,25(OH)₂D₃ reduced calcium mobilization. The combination of the midregional fragment PTH(28–48), which by itself has no effect on calcium metabolism, with 1,25(OH)₂D₃ reduced calcium mobilization more efficiently. Several PTH-regulated mediators have been assayed in this system. Of the tested growth factors, IGF-I at high concentrations caused bone resorption with no effect on ALP activity. TGF-β₁ (transforming growth factor β) and BMP-2 had no significant effect on calcium metabolism; however, ALP activity was inhibited by TGF-β₁ and induced dose dependently by BMP-2. Of the other factors known to be present in bone, platelet-derived growth factor (PDGF_A/B) and epidermal growth factor (EGF) had a small effect on calcium mobilization but had no effect on ALP activity. bFGF reduced ALP activity slightly without an effect on calcium metabolism. Our results show that this in vitro system can mimic some interactions of calciotropic hormones in vivo and allows the assaying of mediators in terms of regulation of ALP activity and of calcium metabolism.     

Quinoline Compound KM11073 Enhances BMP-2-Dependent Osteogenic Differentiation of C2C12 Cells via Activation of p38 Signaling and Exhibits In Vivo Bone Forming Activity

Recombinant human bone morphogenetic protein (rhBMP)-2 has been approved by the FDA for clinical application, but its use is limited due to high cost and a supra-physiological dose for therapeutic efficacy. Therefore, recent studies have focused on the generation of new therapeutic small molecules to induce bone formation or potentiate the osteogenic activity of BMP-2. Here, we show that [4-(7-chloroquinolin-4-yl) piperazino][1-phenyl-5-(trifluoromethyl)-1H-pyrazol-4-yl]methanone (KM11073) strongly enhances the BMP-2-stimulated induction of alkaline phosphatase (ALP), an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. The KM11073-mediated ALP induction was inhibited by the BMP antagonist noggin, suggesting that its osteogenic activity occurs via BMP signaling. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of KM11073 accompanied by enhanced expression of BMP-2, -6, and -7 mRNA. Furthermore, the in vivo osteogenic activity of KM11073 was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its single use for bone formation. In conclusion, the combination of rhBMP-2 with osteogenic small molecules could reduce the use of expensive rhBMP-2, mitigating the undesirable side effects of its supra-physiological dose for therapeutic efficacy. Moreover, due to their inherent physical properties, small molecules could represent the next generation of regenerative medicine.     

Effect of 1,25(OH)2D3 on bone morphogenetic protein-3 mRNA expression

Bone morphogenetic proteins (BMPs) are members to the transforming growth factor-beta superfamily. They induce ectopic bone formation in rat and are pleiotropic initiators of inducible osteogenic precursor cells. A lot of reports have studied the presence of BMPs and their effects on bone marker expression in many different cell lines, however none describe the regulation of BMP3 by different factors and expression conditions. When a human bone marrow stromal cell (HBMSC) culture was treated simultaneously with 1,25(OH)2D3 (10(-8) M) and BMP3 (2.5 ng/ml), the total osteocalcin content in the cell layer and in the culture medium was higher than when the culture was treated with either factor alone (162%). To elucidate this synergistic activity, Northern blot analysis was done to study the effect of 1,25(OH)2D3 on BMP3 mRNA expression. Several human cell lines (MNNG, U-2OS, MG-63, KHOS, TE85, HOS) and HBMSC were treated by 1,25(OH)2D3 (10(-8) M for 24 h). Purified mRNA from treated and untreated cells were denatured using glyoxal and dimethylsulfoxide, and were fractionated on a 1% agarose gel. After electrophoresis, RNA were blotted onto a nylon membrane and incubated with 32P-labeled BMP3 and GAPDH riboprobes. Northern blot analysis revealed that, the BMP3 mRNA level was increased in a few cell lines (MG-63, HBMSC, HOS) after the addition of 1,25(OH)2D3 when compared to the untreated cells (127%+/-1; 130.5%+/-19.5; 207%+/-14). An higher stimulation was observed in HBMSC primary culture when compared to differentiated HBMSC. In view of these results, we now investigate the following hypothesis: does the BMP3 promoter exhibit the vitamin D receptor response like the osteocalcin gene?     

Effects of aging and dietary antler supplementation on the calcium-regulating hormones and bone status in ovariectomized SAMP8 mice

This study was conducted to investigate the effects of aging and long-term dietary antler supplementation on the calcium-regulating hormones and bone status in ovariectomized (Ovx) SAMP8 mice. The female SAMP8 mice were divided into four groups (in each group n = 6), Ovx or sham operated at the age of 2 months, and fed with 0.2% antler containing diet or control diet from the age of 2.5 months. The samples were collected at the age of 3, 6, 9, 12, and 15 months, respectively, for physicochemical analyses, biochemical analyses, and the determination of hormones by radioimmunoassay. The results showed that plasma calcium (Ca) concentrations were maintained in a narrow range in all groups throughout the whole experimental period. With aging and/or ovariectomy, plasma parathyroid hormone (PTH) and 1,25-dihydroxycholecalciferol (1,25-(OH)2-D3) levels increased, and plasma phosphorus (P) and calcitonin (CT) levels decreased, and the femoral bone densities and Ca contents increased during the earlier stage, and then decreased gradually in all groups. Plasma PTH and 1,25-(OH)2-D3 levels in the Ovx mice were significantly higher than those in the intact mice, and plasma P concentrations, plasma CT levels, femoral bone densities, and femoral Ca contents in the Ovx mice were significantly lower than those in the intact mice. In addition, the decreases of plasma P levels, plasma CT levels, femoral bone densities, and femoral Ca contents, and the increases of plasma PTH levels were moderated by antler administration in both Ovx and intact mice. However, there was no effect of the dietary antler supplementation on the plasma 1,25-(OH)2-D3 levels in the female mice. It is concluded that prolonged dietary antler supplementation has important positive effects on bone loss with age and/ or ovarian function deficiency.     

Stimulation of creatine kinase BB activity by parathyroid hormone and by prostaglandin E2 in cultured bone cells.

Bone cells in culture responded to parathyroid hormone (PTH) and prostaglandin E2 (PGE2) by a 2-fold increase in creatine kinase (CK) activity. Combined treatment resulted in a higher response than with PTH alone. Calcitonin (CT) failed to stimulate CK activity, did not affect the response of CK to PTH, but inhibited slightly the increase in CK activity by PGE2. Bone-cell cultures grown in low [Ca2+] (0.125 mM), enriched in PTH-responsive osteoblast-like cells, responded to PTH, but not to PGE2 or CT, by increased CK activity. In both normal and low-[Ca2+] cultures, 8-bromo cyclic AMP did not affect CK activity, nor did it change the response of the cells to PTH, PGE2 or CT. The increase in CK activity was time- and dose-dependent and inhibited both by cycloheximide and by actinomycin D. The isoenzyme of CK stimulated was the CKBB form, the isoenzyme induced by other hormones. This appears to be the first report of the stimulation of CK activity by a polypeptide hormone or a prostaglandin. We suggest that stimulation of CKBB can serve as a marker for the action of a variety of hormones and growth promoters.     

Optimized procedure for expression and renaturation of recombinant human bone morphogenetic protein-2 at high protein concentrations

A prokaryotic expression system has been used to produce recombinant human bone morphogenetic protein-2 (rhBMP-2). However, low rhBMP-2 yields and protein loss during purification and renaturation are the hurdles in the clinical application. Previous studies have indicated that variables such as temperature, host cell, salt concentration, and culture time affect the final rhBMP-2 yield. The optimization of these conditions in an Escherichia coli culture yielded 28.258 mg of rhBMP-2 per liter of culture. To reduce rhBMP-2 loss during purification and renaturation, we performed purification before renaturation in the prokaryotic expression system instead of using the traditional renaturation-before-purification approach. rhBMP-2 was separated on a Sephacryl S-300 HR column and eluted from a DEAE-Sepharose Fast Flow column. The collected protein was refolded by dialysis with urea buffer, which was followed by dialysis with ultrapure water. The purified rhBMP-2 dimer significantly increased alkaline phosphatase (ALP) activity and osteogenic activity in the femoral muscle and showed the same level of bone-forming activity as natural BMP-2. This optimized procedure for expression and renaturation of rhBMP-2 has potential clinical applications.     

Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro

The in vitro effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on osteogenic and myogenic differentiation was examined in two clonal cell lines of rat osteoblast-like cells at different differentiation stages, ROB-C26 (C26) and ROB-C20 (C20). The C26 is a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes; the C20 is a more differentiated osteoblastic cell line. Proliferation was stimulated by rhBMP-2 in C26 cells, but inhibited in C20 cells. rhBMP-2 greatly increased alkaline phosphate (ALP) activity in C26 cells, but not in C20 cells. The steady-state level of ALP mRNA was also increased by rhBMP-2 in C26 cells, but not in C20 cells. Production of 3',5'-cAMP in response to parathyroid hormone (PTH) was dose-dependently enhanced by adding rhBMP-2 in both C26 and C20 cells, though the stimulatory effect was much greater in the former. There was neither basal expression of osteocalcin mRNA nor its protein synthesis in C26 cells, but they were strikingly induced by rhBMP-2 in the presence of 1 alpha,25-dihydroxyvitamin D3. rhBMP-2 induced no appreciable changes in procollagen mRNA levels of type I and type III in the two cell lines. Differentiation of C26 cells into myotubes was greatly inhibited by adding rhBMP-2. The inhibitory effect of rhBMP-2 on myogenic differentiation was also observed in clonal rat skeletal myoblasts (L6). Like BMP-2, TGF-beta 1 inhibited myogenic differentiation. However, unlike BMP-2, TGF-beta 1 decreased ALP activity in both C26 and C20 cells. TGF-beta 1 induced neither PTH responsiveness nor osteocalcin production in C26 cells, but it increased PTH responsiveness in C20 cells. These results clearly indicate that rhBMP-2 is involved, at least in vitro, not only in inducing differentiation of osteoblast precursor cells into more mature osteoblast-like cells, but also in inhibiting myogenic differentiation.     

Interactive effects of PTH and mechanical stress on nitric oxide and PGE2 production by primary mouse osteoblastic cells

Parathyroid hormone (PTH) and mechanical stress both stimulate bone formation but have opposite effects on bone resorption. PTH increased loading-induced bone formation in a rat model, suggesting that there is an interaction of these stimuli, possibly at the cellular level. To investigate whether PTH can modulate mechanotransduction by bone cells, we examined the effect of 10-9 M human PTH-(1-34) on fluid flow-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production by primary mouse osteoblastic cells in vitro. Mechanical stress applied by means of a pulsating fluid flow (PFF; 0.6 +/- 0.3 Pa at 5 Hz) stimulated both NO and PGE2 production twofold. In the absence of stress, PTH also caused a twofold increase in PGE2 production, but NO release was not affected and remained low. Simultaneous application of PFF and PTH nullified the stimulating effect of PFF on NO production, whereas PGE2 production was again stimulated only twofold. Treatment with PTH alone reduced NO synthase (NOS) enzyme activity to undetectable levels. We speculate that PTH prevents stress-induced NO production via the inhibition of NOS, which will also inhibit the NO-mediated upregulation of PGE2 by stress, leaving only the NO-independent PGE2 upregulation by PTH. These results suggest that mechanical loading and PTH interact at the level of mechanotransduction.     

Early developmental changes in intracellular Ca2+ stores in rat brain.

Developmental changes in intracellular Ca2+ stores in brain was studied by examining: (1) IP3- and cADPR-induced increase in [Ca2+]i in synaptosomes; (2) Ca(2+)-ATPase activity and ATP-dependent 45Ca2+ uptake into Ca2+ store in ER microsomes; (3) TG-induced inhibition of Ca(2+)-ATPase activity and ATP-dependent 45Ca2+ uptake into Ca2+ store in ER microsomes; and (4) gene expression of Ca(2+)-ATPase pump in neurons obtained from brains of the new-born and the 3-week-old rats. IP3 (EC50 310 +/- 8 nM, 200% maximum increase in [Ca2+]i) and cADPR (EC50 25 +/- 3 nM, greater than 170% maximum increase in [Ca2+]i) both were potent agonist of Ca2+ release from internal stores in synaptosomes obtained from the 3-week-old rats. However, IP3 (EC50 250 +/- 10 nM, 175 maximum increase in [Ca2+]i) was a potent, but cADPR (EC50 300 +/- 20 nM, 75% maximum increase) was a poor agonist of Ca2+ release from intracellular stores in synaptosomes obtained from the new-born rats. [3H]IP3, [32P]cADPR and [3H]Ry binding in the new-born samples were significantly less than that in the 3-week-old samples. [3H]Ry binding to its receptor was more sensitive to cADPR in microsomes from the 3-week-old rats than those from the new-born rats. Microsomes from the new-born rats exhibited TG-sensitive (IC50 30 +/- 4 nM) and TG-insensitive forms of Ca(2+)-ATPase, while microsomes from the 3-week-old rats exhibited only the TG-sensitive form of Ca(2+)-ATPase (5 +/- 1 nM IC50). Microsomes from the 3-week-old rats were more sensitive to TG but less sensitive to IP3, while microsomes from the new-born rats were more sensitive to IP3 but less sensitive to TG. The lower TG sensitivity of the new-born Ca2+ store may be because they poorly express a 45 amino acid C-terminal tail of Ca(2+)-ATPase that contains the TG regulatory sites. This site is adequately expressed in the older brain. This suggests that: (1) the new-born brain contains fully operational IP3 pathway but poorly developed cADPR pathway, while the older brain contains both IP3 and cADPR pathways; and (2) a developmental switch occurs in the new-born Ca(2+)-ATPase as a function of maturity.     

Prostaglandin E2 stimulates the formation of mineralized bone nodules by a cAMP-independent mechanism in the culture of adult rat calvarial osteoblasts

The effects of prostaglandin E2 (PGE2) on the proliferation and differentiation of osteoblastic cells were studied in osteoblast-like cells isolated from adult rat calvaria. Treatment of the cells with PGE2 within the concentration range 10(-8)-10(-5) M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [3H]proline incorporation into collagenase-digestible protein, and mineralized bone nodule (BN) formation, as well as a dose-dependent decrease in [3H]thymidine incorporation into the cells. PGE2 also caused a dose-dependent increase in the intracellular cyclic adenosine monophosphate (cAMP) content, with a maximal effective concentration of 10(-5) M; this effect of PGE2 was mimicked by forskolin, an adenylate cyclase activator. The treatment of adult calvarial cells with forskolin decreased BN formation, ALP activity, and collagen synthesis. These results suggested that cAMP does not have a stimulatory, but rather a suppressive, effect on the differentiation of adult rat calvarial cells. A time-course study of cAMP accumulation showed that both PGE2- and forskolin-induced cAMP reached a maximum at 5 min after the treatment, but the former rapidly returned to the basal level by 40 min, while the latter declined slowly and was still at 70% of the maximal level at 60 min, suggesting that PGE2 activates phosphodiesterase as well as adenylate cyclase. The presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, reduced the rate of degradation of cAMP formed after PGE2 treatment, suggesting the involvement of calmodulin in the activation of phosphodiesterase. However, PGE2 also caused the production of inositol 1,4,5-triphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ([Ca2+]i), both of which peaked at 15 s and returned to the basal level within 1 min. Submaximal responses of the IP3 production and the [Ca2+]i elevation to PGE2 were obtained at 10(-5) M. W-7 decreased both basal and PGE2-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE2-induced differentiation of calvarial cells. From these results, we concluded that PGE2 inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+]i through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyclase. We also found that BN formation varies, depending on the time of PGE2 addition, suggesting that responsiveness of the cells to PGE2 may change during the culture period.     

Long-term systemic administration of human recombinant interleukin-1beta induces a dose-dependent fall in circulating parathyroid hormone in rats.

The synergism/antagonism between interleukin (IL)-1beta and parathyroid hormone (PTH) has been the subject of in vitro and in vivo work, but a possible direct action of the cytokine on PTH release has not been reported. We have investigated the effect of a continuous infusion of human recombinant IL-1beta (rIL-1beta) on circulating PTH during a 14-day period in 7-week-old female rats. This time interval was chosen in order to exclude initial hypercalcemia and to enable data collection under steady-state conditions. Five groups of 20 animals each had miniosmotic pumps (Alzet 2002, 200 microl) implanted subcutaneously and primed to release either distilled water (controls) or 100, 500, 1,000 and 2, 000 ng/24 h of rIL-1beta. Blood was drawn on days 1 and 14 for PTH, corticosterone and Ca2+ determinations. Adequate biological activity of the infused rIL-1beta was supported by elevated rectal temperature records and significant elevations of plasma corticosterone on day 14. The 100-ng dose had no effect but 500-2, 000 ng rIL-1beta/24 h significantly reduced plasma PTH in a dose-dependent manner down to 54% of basal value (20.4 +/- 1.1 vs. 15.3 +/- 1.4 pg/ml for 500 ng, p < 0.005; 20.5 +/- 1.3 vs 12.3 +/- 1.1 for 1,000 ng, p < 0.001, and 19.5 +/- 2.0 vs. 10.6 +/- 1.1 pg/ml for 2,000 ng, p < 0.0008). Despite these findings, no differences in blood Ca2+ could be detected between treated animals and controls. The following conclusions can be inferred from the foregoing: Systemic administration of rIL-1beta to rats induced a dose-dependent fall in circulating PTH without altering calcemia, calling into question the biological relevance of the former finding. Although the recorded PTH depression may indeed not have been severe enough to cause hypocalcemia, it can be hypothesized that osteoclast activation by rIL-1beta would enhance bone mineral release into the pool compensating for depressed PTH activity.     

Prostaglandins enhance parathyroid hormone-evoked increase in free cytosolic calcium concentration in osteoblast-like cells.

Prostaglandins (PGs) are autocrine or paracrine hormones that may interact with circulating hormones such as parathyroid hormone (PTH) in bone. We examined the interaction of the PGs, PGF2 alpha, PGE2, and 6-keto-PGF1 alpha with PTH to enhance the rapid, initial transient rise in free cytosolic calcium ([Ca2+]i) and cAMP levels stimulated by PTH. Pretreatment of UMR-106, MC3T3-E1, and neonatal rat calvarial osteoblast-like cells by PGs resulted in an enhancement of the early transient rise in [Ca2+]i stimulated by PTH. PGF2 alpha was approximately 100 times more potent than PGE2. PGE2 itself was more potent than 6-keto-PGF1 alpha in enhancing PTH-stimulated rise in [Ca2+]i. Near-maximal augmentation was achieved at PGF2 alpha doses of 10 nM and PGE2 of 1 microM. The degree of augmentation in [Ca2+]i by PGF2 alpha was independent of preincubation time. PGF2 alpha pretreatment did not alter the EC50 for the PTH-induced [Ca2+]i increase but only the extent of rise in [Ca2+]i at each dose of PTH. The augmented increase in [Ca2+]i was mostly due to enhanced PTH-mediated release of Ca2+ from intracellular stores. PGF2 alpha did not stimulate an increase in PTH receptor number as assessed by [125I]-PTH-related peptide binding. PG pretreatment partially reversed PTH inhibition of cell proliferation, suggesting that an increase in [Ca2+]i may play a role in tempering the anti-proliferative effect of PTH mediated by cAMP. These studies suggest a new mode by which PGs can affect cellular activity.