Effects of selective prostaglandin EP4 receptor antagonist on osteoclast formation and bone resorption in vitro.

Prostaglandin estradiol (PGE(2)) stimulates bone resorption by a cyclic AMP (cAMP)-dependent mechanism that involves prostaglandin E receptors of the EP2 and EP4 subtypes. We tested a potent selective EP4 antagonist (EP4RA), which blocks PGE(2) binding to EP4 receptors. We examined the effects of EP4RA on osteoclastogenesis in murine marrow cultures, on cAMP production in primary osteoblastic (POb) cell cultures, and on bone resorption in organ cultures. EP4RA (1 micromol/L) decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP(+) MNC) by 46%-48% in cultures treated with 0.1-1.0 micromol/L PGE(2) and by 96% in cultures treated with 0.01 micromol/L PGE(2). EP4RA also decreased TRAP(+) MNC formation by 60% in 1,25-dihydroxyvitamin D (1,25D)-treated cultures and by 62% in parathyroid hormone (PTH)-treated cultures. A chemically related analog of EP4RA that lacks antagonist activity did not inhibit TRAP(+) MNC formation. EP4RA decreased cAMP production in PGE(2)-treated POb by 44% but did not block cAMP response to PTH. EP4RA inhibited the increase in receptor activator of NF-kappaB ligand (RANKL) mRNA levels produced by PGE(2). In fetal rat long bone cultures, EP4RA decreased 45Ca release from control, unstimulated cultures by 12%-25% and from PGE(2)-stimulated cultures by 22%-37%. Because EP4RA partially inhibited osteoclastogenesis not only in response to PGE(2) but also in response to 1,25D and PTH, these results suggest that activation of the EP4 receptor may play a general role in osteoclastic bone resorption. EP4RA showed partial inhibition of PGE(2)-stimulated osteoclastogenesis at 1 micromol/L, but almost complete inhibition at 0.01 micromol/L PGE(2). This could be due to the limited efficacy of the antagonist at high concentrations of PGE(2), or an alternative pathway, such as activation of the EP2 receptor.

Anabolic effects of prostaglandins in cultured fetal rat calvariae: structure-activity relations and signal transduction pathway.

The structure-activity relations and signal transduction pathways for the anabolic effects of prostaglandins were examined in cultured fetal rat calvariae. In the presence of cortisol prostaglandins of the E and F series (10(-9) to 10(-5) M) produced a dose-related increase in [3H]thymidine incorporation up to 4-fold at 24 h. Prostaglandin E2 (PGE2) was also effective in the absence of cortisol. Butaprost (10(-6) M), a selective EP-2 receptor agonist, produced partial stimulation. Prostaglandin D2, prostacyclin, sulprostone, an EP-1 and EP-3 receptor agonist, and fluprostenol, an FP receptor agonist, were ineffective. Forskolin (10(-4) M) increased [3H]thymidine incorporation 3-fold, while phorbol myristate acetate (PMA) (10(-6) M) produced a 1.8-fold increase. Isobutylmethylxanthine (IBMX) increased [3H]thymidine incorporation in control cultures, in the absence of cortisol, and increased the response to PGE2 in control and cortisol-treated cultures. [3H]proline incorporation into collagen and noncollagen protein was measured in the continuous presence of prostaglandins and cortisol for 72-96 h (continuous model) or when prostaglandins and cortisol were applied for 24 h, followed by culture for 48 h in control medium (on/off model). The effects on collagen were greater than on noncollagen proteins, so that the percent of collagen synthesis increased. The effects of prostaglandins and forskolin paralleled their mitogenic effects. PMA increased only noncollagen protein. Indomethacin did not diminish the anabolic response, while aphidicolin produced only partial inhibition. We conclude that the anabolic effects of prostaglandins on replication and differentiation of osteoblasts are likely to be mediated by an EP-2 receptor that stimulates adenylate cyclase.

The short-term effects of conjugated estrogen on bone turnover in older women.

Estrogen replacement therapy (ERT) prevents bone loss and fracture in early postmenopausal women, but its benefit for women over 70 yr of age has not been determined. We have examined the effect of a short course of ERT on biochemical markers of bone turnover in older women. Eleven women (mean age, 77 yr) were given conjugated estrogen (Premarin; 0.625 mg/day) for 6 weeks. Biochemical markers were measured on serum and urine collected at baseline (two samples), after 5 and 6 weeks of ERT, and 5 and 6 weeks post-ERT. Markers of bone formation were osteocalcin, bone alkaline phosphatase, and type I procollagen peptide. Markers of bone resorption were total urinary hydroxyproline, total and free pyridinoline and deoxypyridinoline cross-links, type I collagen cross-linked N-telopeptides, and serum C-terminal cross-linked telopeptide. Data were analyzed by repeated measures multivariate analysis of variance to estimate the overall effect of ERT on the biochemical markers. Markers of bone resorption decreased during ERT and returned to baseline after ERT (P < 0.05). Markers of bone formation declined less during ERT and continued to decline after ERT (P < 0.05). We conclude that ERT reduces bone turnover in older women and that markers of bone turnover may be useful in assessing the response to treatment in this age group.

Comparison of the effects of 3,5,3'-triiodothyroacetic acid and triiodothyronine on bone resorption in cultured fetal rat long bones and neonatal mouse calvariae.

Thyroid hormones can stimulate bone resorption both directly and indirectly by increasing endogenous prostaglandin (PG) synthesis. Because 3,5,3'-triiodothyroacetic acid (Triac, Tiratricol) can bind to nuclear thyroid hormone receptors with high affinity, we compared the effects of Triac and 3,5,3'-triiodothyronine (T3) on bone resorption. 45Ca release was measured at 2, 5, and 8 days from 19-day-old-fetal rat long bones in the presence or absence of calcitonin, indomethacin, aphidicolin (APC), cortisol, or interleukin-1 receptor antagonist (IL-1ra). Both Triac and T3 stimulated 45Ca release in a dose-dependent manner. The maximal treated/control ratio (T/C) for Triac (10(-7) M) was 1.6 +/- 0.1 at 2 days and 2.5 +/- 0.1 at 5 days and, for T3 (10(-7) M), 1.5 +/- 0.1 at 2 days and 2.6 +/- 0.1 at 5 days. These responses were smaller and slower than that to PTH. Significant stimulation of resorption was observed at 10(-8), 10(-9), and 10(-10) M for Triac but only at 10(-8) and 10(-9) M for T3. Indomethacin (10(-6) M) and IL-1ra (1 microgram/ml) did not decrease the effects of Triac or T3. In contrast, calcitonin (10(-9) M), APC (30 microM), and cortisol could all block the response to these hormones. These results indicated that the resorptive responses were not mediated by endogenous PG or IL-1 but were osteoclast mediated and dependent on DNA synthesis, suggesting that these hormones acted by stimulating replication of osteoclast precursors or other supporting cells. Thyroid hormones were less effective in 7-day-old neonatal mouse calvariae.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of two new vitamin D3 derivatives, 22-oxa-1 alpha-25-dihydroxyvitamin D3 (OCT) and 2 beta-(3-hydroxypropoxy)-1 alpha, 25-dihydroxyvitamin D3 (ED-71), on bone metabolism in organ culture.

We have tested two new vitamin D3 derivatives, 22-oxa-1 alpha, 25-dihydroxyvitamin D3 (OCT) and 2 beta-(3-hydroxypropoxy)-1 alpha, 25-dihydroxyvitamin D3 (ED-71), for their effects on bone metabolism compared with 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) in two organ-culture systems. In a previous study (Abe et al. 1987), it was reported that OCT had weak activity in stimulating bone resorption in vitro. In the present study, however, OCT stimulated bone resorption in cultured fetal rat long bones and inhibited collagen synthesis in cultured neonatal mouse calvariae in a dose-dependent manner with significant effects at 10(-10) M and maximal responses at 10(-8) M. Its potency and effectiveness were identical to 1,25(OH)2D3. On the other hand, ED-71, which has been found to prevent bone loss in vivo (Okano et al. 1989b), was less active in vitro. The activity of ED-71 at 10(-8) M on bone resorption was similar to 1,25(OH)2D3, but it did not stimulate resorption at 10(-10) M. Its inhibitory effect on collagen synthesis was weaker than for OCT of 1,25(OH)2D3. The activity of all three compounds on bone resorption was not inhibited by indomethacin or cortisol. 1,25(OH)2D3 and OCT significantly inhibited [3H]-thymidine incorporation into mouse calvariae at 10(-9) M, while ED-71 inhibited [3H]-thymidine incorporation only at 10(-8) M. These results indicate that OCT and 1,25(OH)2D3 have similar effects on bone in organ culture. Pharmacokinetic differences may explain the marked difference in response to those two agents in vivo. ED-71 is less potent, particularly in inhibiting bone formation. Such differences may have importance in the development of vitamin D analogs for clinical use.

In vitro mineralization of fetal rat parietal bones in defined serum-free medium: effect of beta-glycerol phosphate.

We have developed a bone organ culture system that mineralizes in vitro. Fetal rat parietal bones (20 days old) were cultured in a chemically defined serum-free medium containing physiological 3 mM phosphate. During 5 days in culture, calcium content increased from 26 to 55 micrograms and dry weight increased from 137 to 194 micrograms. After 2 days in vivo, the calcium content of the parietal bone showed a comparable increase to 49 micrograms and dry weight increased to 183 micrograms. During culture, the mineralized bone area in thick sections increased from 11 to 23%, which paralleled the doubling in calcium content. Fluorescent calcein labeling during the 5 day culture period demonstrated that calcification occurs in an ordered pattern. Protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP). The percentage collagen synthesis decreased from 17.5% at 0 time to 5.0% at 2 days and then increased to 9.4% at 5 days of culture. Varying the inorganic phosphate concentration in the medium or adding beta-glycerol phosphate was found to affect mineralization. After 5 days in culture, bones treated with 1 mM phosphate exhibited a large region of unmineralized osteoid with only a 23% increase in calcium content compared with 112% in control (3 mM phosphate) bones and a 28% increase in dry weight compared with a 40% increase in control. Treatment for 5 days with 6 mM phosphate or 1, 3, or 10 mM beta-glycerol phosphate had no significant effect on dry weight compared to control bones. However, bone calcium content increased significantly from 55 +/- 5 micrograms in control cultures to 105 +/- 7 with 6 mM phosphate, 74 +/- 6 with 3 mM beta-glycerol phosphate, and 75 +/- 5 micrograms with 10 mM beta-glycerol phosphate. Calcified area measured by histomorphometry was also significantly greater than in control bones, but this was mainly due to ectopic calcification in the periosteum, representing from 23 to 74% of the total increase in calcified matrix in bones cultured with 6 mM phosphate or 1-10 mM beta-glycerol phosphate. Ultrastructural analysis demonstrated that ectopic calcification was associated with cell death and debris. Therefore, calcification with beta-glycerol phosphate and high concentrations of inorganic phosphate differed from mineralization in vivo or in bones cultured with a physiologically concentration of phosphate.

Effect of alterations in the cyclopentane ring on bone resorptive activity of prostaglandin.

Previous studies have shown that the natural prostanoids, PGE2, PGE1 and PGF2 alpha are potent stimulators of bone resorption. In this study, we have examined the effects of alterations in the cyclopentane ring of these prostanoids for their effect on the resorptive response of cultured long bones from 19-day fetal rats as measured by the release of previously incorporated 45Ca. Indomethacin (10(-6)M) was added to minimize endogenous prostaglandin production. In this system PGE2 and PGE1, the 9 keto, 11 alpha hydroxy compounds, were approximately equally effective at concentrations of 10(-8) to 10(-6) M. The 9 alpha hydroxy, 11 alpha hydroxy compound, PGF2 alpha, was active at 10(-7) to 10(-5) M. In contrast, the 9 alpha hydroxy, 11-keto compound, PGD2, showed only a minimal stimulation of bone resorption at 10(-5) M. While these data suggested that the 11 alpha hydroxy group was important for bone resorbing activity, 11 beta PGE2 and 11-deoxy PGE1 were only slightly less potent than their physiologic counterparts. Both 9 beta, 11 alpha PGF2 and 9 alpha, 11 beta PGF2 were less potent than PGF2 alpha but did cause substantial stimulation of bone resorption and were equally effective at 10(-6) to 10(-5) M. 9 alpha, 11 beta PGF2 alpha is of particular interest since it is major metabolite of PGD2. These results suggest that the binding of prostanoids to the receptor which mediates bone resorption is affected by changes at the 9 and 11 positions of the pentane ring but do not support the hypothesis that the 11 alpha OH function is essential for this biological activity.

Comparative study of invertases of Streptococcus mutans.

Sucrase activity was studied in 13 strains of Streptococcus mutans representing the five Bratthall serotypes. Sucrose-adapted cells have sucrase activity in the 37,000 x g-soluble fraction of all strains. The enzyme was identified as invertase (beta-d-fructofuranoside fructohydrolase; EC 3.2.1.26) because it hydrolyzed the beta-fructofuranoside trisaccharide raffinose, giving fructose and melibiose as its products, and because it hydrolyzed the beta-fructofuranoside dissacharide sucrose, giving equimolar glucose and fructose as its products. Invertases of c and e strains exhibit two activity peaks by molecular exclusion chromatography with molecular weights of 45,000 to 50,000 and about 180,000; those of serotypes a, b, and d strains exhibit only a single component of 45,000 to 50,000 molecular weight. The electrophoretic mobility of invertases is different between the serotypes and the same within them. Inorganic orthophosphate (P(i)) has a weak positive effect on the V(max) of invertases of serotypes c and e cells but a strong positive effect on the invertases of serotype b cells; P(i) has a strong positive effect on the apparent K(m) of the invertases of serotype d cells, but has no effect on the V(max); P(i) has a strong positive effect on both the apparent K(m) and V(max) of the invertases of serotype a cells. Thus, the invertases were different between all of the serotypes but similar within the serotypes. These findings support the taxonomic schemes of Coykendall and of Bratthall. It was additionally noted that 37,000 x g-soluble fractions of only serotypes b and c but not serotypes a, d, and e cells have melibiase activity, and it could be deduced that serotype d cells lack an intact raffinose permease system.

Identification, separation, and preliminary characterization of invertase and beta-galactosidase in Actinomyces viscosus.

The initial step of disaccharide dissimilation by Actinomyces viscosus serotype 2 strain M-100 was studied. Sucrase activity was found in the 3,000 X g particulate fraction and the 37,000 X g soluble fraction of the cells, whereas lactase activity was found almost exclusively in the 37,000 X g soluble fraction. Neither sucrase nor lactase activity was appreciable in the culture liquor. Sucrose phosphorylase, alpha-glucosidase, and polysaccharide synthesis activities were not observed in the soluble cell fraction. The sucrase was identified as invertase (EC 3.2.1.26; beta-D-fructofuranoside fructohydrolase). The lactase was identified as beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase). The enzymes in the 37,000 X g soluble fraction were separable by diethylamino-ethyl-cellulose chromatography, giving one beta-galactosidase peak and one major and one minor invertase peak. Acrylamide gel electrophoresis showed different electrophoretic mobilities of the enzymes. The molecular weight of the beta-galactosidase is about 4.2 X 10(5) and that of invertase is about 8.6 X 10(4). The beta-galactosidase has a Km for lactose of about 6 mM and a pH optimum between pH 6.0 and 6.5. The major invertase component has a Km for sucrose of about 71 mM and a pH optimum between pH 5.8 and 6.3.