Differential effects of nonsteroidal anti-inflammatory drugs on constitutive and inducible prostaglandin G/H synthase in cultured bone cells.

The production of prostaglandins by osteoblasts is an important mechanisms for the regulation of bone turnover. Bone cells contain both inducible and constitutive prostaglandin G/H synthase (PGHS-2 and PGHS-1) and these are differentially regulated. Nonsteroidal anti-inflammatory drugs (NSAIDs), which selectively inhibit one of these enzymes, would be useful in assessing their relative roles in bone metabolism. By Northern analysis, only PGHS-2 is expressed by the immortalized rat osteoblastic cell line, Py1a, while only PGHS-1 is expressed by the rat osteosarcoma cell line, ROS 17/2.8. We tested the relative inhibitory potency (IC50) of seven different NSAIDs on these two cell lines. A recently described selective inhibitor of PGHS-2, NS-398, was approximately 30 times more potent in inhibiting PGHS-2 than PGHS-1, and diclofenac was approximately 10 times more potent. Both had IC50's of approximately 3 nM for PGHS-2 in Py1a cells. Indomethacin, flurbiprofen, naproxen, and piroxicam were relatively nonselective with IC50's ranging from 30 nM to 1 microM, while 6-methoxy-2 naphthyl acetic acid, the active metabolite of nabumetone, was inhibitory only at concentrations greater than 1 microM. These results indicate that the presently available NSAIDs are unlikely to distinguish completely between effects mediated by PGHS-2 or PGHS-1. However, the cell systems employed could provide a model for the analysis of new compounds with greater selective activity.

Interleukin-4 inhibits prostaglandin G/H synthase-2 and cytosolic phospholipase A2 induction in neonatal mouse parietal bone cultures.

We have shown previously that prostaglandin (PG) production in 7-day-old neonatal mouse calvarial cultures is regulated largely by changes in prostaglandin G/H synthase-2 (PGHS-2) expression and to a lesser extent by changes in arachidonic acid (AA) release. In this study, we examined the effects of interleukin-4 (IL-4), and its interactions with other cytokines and with parathyroid hormone (PTH), on mRNA levels of PGHS-2, PGHS-1, and cytosolic phospholipase A2 (cPLA2) and on medium protaglandin E2 (PGE2) levels in calvarial cultures. IL-1 and tumor necrosis factor-alpha (TNF-alpha), both at 1-100 ng/ml, and PTH at 0.1-10 nM increased PGHS-2 and cPLA2 mRNA and medium PGE2 levels dose-dependently after 4 h of treatment. IL-6 and IL-11 at 1-100 ng/ml did not affect mRNA or PGE2 levels. IL-4 at 1-100 ng/ml decreased PGHS-2 and cPLA2 mRNA and PGE2 levels in control as well as IL-1, TNF-alpha, and PTH-stimulated cultures. The inhibition of PGHS-2 and cPLA2 mRNA expression by IL-4 (10 ng/ml) was present at 1 h, reached a maximum at 4 h, and persisted for 24 h. The effects were maintained in the presence of cycloheximide. IL-4 also decreased PGHS-2 protein levels in control and IL-1-stimulated cultures. PGHS-1 mRNA levels were not stimulated by any of the factors studied nor inhibited by IL-4. IL-4 partially inhibited control and PTH-stimulated 45Ca release from prelabeled mouse calvariae at 4 days. However, neither the inhibition of resorption by IL-4 nor the stimulation by IL-1 and PTH were altered by indomethacin (1 microM). We conclude that (1) IL-1, TNF-alpha, and PTH, but not IL-6 nor IL-11, can increase the expression of PGHS-2, cPLA2, and PGE2 production in cultured mouse calvariae; (2) IL-4 inhibits PGE2 production in both control and stimulated calvarial cultures by inhibiting PGHS-2 and cPLA2; and (3) IL-4 has an inhibitory effect on bone resorption which is independent of PG production.

Autoregulation of inducible prostaglandin G/H synthase in osteoblastic cells by prostaglandins.

Prostaglandins (PGs) have been postulated to amplify their own production by stimulating cyclic adenosine monophosphate activity, which in turn stimulates PG production. We examined regulation of messenger RNA levels for the inducible and constitutive prostaglandin G/H synthases, PGHS-2 and PGHS-1, in murine osteoblastic MC3T3-E1 cells, which express both PGHS-1 and PGHS-2, and in rat osteoblastic Py1a cells, which express only PGHS-2. Prostaglandins E2, F2 alpha, and D2 induced PGHS-2 mRNA in both cell lines under serum-free conditions and stimulated small increases in PGHS-1 mRNA levels in MC3T3-E1 cells. PGE2 (1 microM) increased the transcription rate of PGHS-2 mRNA 9-fold at 2 h in serum-free cells and also induced PGHS-2 protein. In the presence of arachidonic acid or serum, PGs also increased medium PGE2. Both forskolin, a protein kinase A activator, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, have previously been shown to induce PGHS-2 mRNA in MC3T3-E1 cells, but in the present study only PMA induced PGHS-2 expression in Py1a cells. The induction of PGHS-2 mRNA in Py1a cells by PGs was inhibited by chelerythrine, a PKC inhibitor, and blocked by 24 h of pretreatment with PMA. The 2 h serum stimulation of PGHS-2 mRNA in MC3T3-E1 cells was inhibited 40-50% by three structurally unrelated nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting that endogenous PGs also amplify PG production through induction of PGHS-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the two prostaglandin G/H synthases by parathyroid hormone, interleukin-1, cortisol, and prostaglandin E2 in cultured neonatal mouse calvariae.

A second prostaglandin G/H synthase (PGHS-2), encoded by a gene separate from that for the original PGHS (PGHS-1), has recently been identified. We have shown that PGHS-2 is expressed in cultured mouse calvariae and have compared regulation of PGHS-2 and PGHS-1 messenger RNA (mRNA) levels. PGHS-2 mRNA was not detectable in freshly isolated bones, but was induced during culture and further stimulated by interleukin-1 (IL-1) and PTH. Both factors also increased PGHS-2 protein levels. Changes in medium prostaglandin E2 (PGE2) production correlated with increases in PGHS-2 mRNA levels. However, with IL-1, PGE2 production was increased more than PGHS-2 mRNA levels (treated/control ratio, 3.4 and 1.5, respectively), whereas with PTH there was a closer correspondence (2.0 and 2.1). Cortisol reduced PTH-stimulated PGE2 production (treated/control ratio decreased from 3.1 to 0.2) more than PGHS-2 mRNA levels (2.8 to 0.8). In the presence of exogenous arachidonic acid, changes in PGHS-2 mRNA levels with IL-1, PTH, and cortisol correlated closely with changes in PGE2 production. PGE2 itself increased PGHS-2 mRNA, and nonsteroidal antiinflammatory drugs decreased PGHS-2 mRNA levels by 80%. In contrast, PGHS-1 mRNA was expressed constitutively and was not affected by IL-1, PTH, or cortisol when measured by competitive reverse transcriptase-polymerase chain reaction. We conclude that regulation of PGE2 production is predominantly through PGHS-2, rather than PGHS-1; that IL-1 and cortisol may also regulate arachidonic acid release; and that PGE2 may amplify its own production through stimulation of PGHS-2.

Differential regulation of inducible and constitutive prostaglandin endoperoxide synthase in osteoblastic MC3T3-E1 cells.

Regulation of mRNA levels for the constitutive and inducible prostaglandin endoperoxide synthases, PGHS-1 and PGHS-2, was examined in murine osteoblastic MC3T3-E1 cells. Serum induction of PGHS-2 mRNA levels was rapid, transient, increased by cycloheximide, and inhibited 72% by cortisol. The cortisol inhibition was blocked by cycloheximide. Serum stimulation of PGHS-1 mRNA was slower, decreased by cycloheximide, and inhibited 28% by cortisol. Increased prostaglandin E2 (PGE2) production and induction of PGHS-2 immunoreactive protein paralleled changes in PGHS-2 mRNA. PGHS-2 mRNA was induced at 2 h in serum-free cells by transforming growth factor-beta (TGF-beta), phorbol 12-myristate 13-acetate, and, to a lesser extent, by forskolin. The combination of phorbol 12-myristate 13-acetate and forskolin was synergistic. TGF-beta induction was prolonged compared with serum, inhibited 67% by cortisol, and the inhibition was not blocked by cycloheximide. TGF-alpha had little effect on PGHS-2 mRNA at 2 h, but the combination of TGF-beta and TGF-alpha was synergistic for PGHS-1 and PGHS-2. PGE2 itself induced PGHS-2 mRNA, and inhibition of PGE2 production decreased the serum induction by 55%, suggesting an important role for autoamplification. The rapidity and amplitude of changes in PGHS-2 suggest that it may be involved in bone responses to acute stresses, such as mechanical strain, inflammation, and injury.

Comparison of the effects of various lengths of synthetic human parathyroid hormone-related peptide (hPTHrP) of malignancy on bone resorption and formation in organ culture.

Parathyroid hormone-related peptide (PTHrP) has been shown to be the pathogenic agent in humoral hypercalcemia of malignancy (HHM), but the molecular forms that are secreted have not been fully characterized. PTHrP 1-34 has effects similar to parathyroid hormone (PTH), but C-terminal regions of the peptide, such as the 107-139 fragment found to inhibit resorption in a study by Fenton et al (1991), may have other biological activities not shared with PTH. We have compared the effects of the longer forms of recombinant human PTHrP (hPTHrP 1-84, 1-108, and 1-141) with hPTHrP 1-34 and synthetic bovine PTH (bPTH) 1-34 on bone resorption and formation in cultured neonatal mouse calvariae and fetal rat long bones. hPTHrP 1-84, 1-108, and 1-141 were qualitatively similar to hPTHrP 1-34 and PTH 1-34 in stimulating 45Ca release from both neonatal mouse calvariae and fetal rat long bones and in inhibiting the incorporation of [3H]-proline into collagenase digestible protein (CDP) and stimulating the incorporation of [3H]-thymidine (3H-TdR) in neonatal mouse calvariae. However, hPTHrP 1-108 and 1-141 were less potent at stimulating 45Ca release and inhibiting CDP labeling than hPTHrP 1-34, while hPTHrP 1-84 showed an intermediate potency. Since hPTHrP 1-108 and 1-141 were quite similar in potency, the difference cannot be attributed to an inhibitory effect of the 107-139 fragment. All the peptide lengths tested showed similar potency in stimulating [3H]-TdR incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of potassium peroxydiphosphate on bone resorption.

Potassium peroxydiphosphate (KPDP) is a slowly hydrolyzed pyrophosphate analog that can release hydrogen peroxide during hydrolysis. We tested its effects on the resorption of cultured fetal rat long bones as measured by the release of previously incorporated 45Ca, both by direct addition of KPDP to the medium and after preincubation of KPDP with large-molecular-weight resorbing factors followed by dialysis to reduce the KPDP concentration. With direct addition, KPDP at a concentration of 1 mM could inhibit the resortive response to bacterial lipopolysaccharide (LPS), parathyroid hormone (PTH), prostaglandin E2 (PGE2), and mouse recombinant interleukin-1 (mrIL-1). The response to LPS was partially inhibited at 0.3 mM KPDP. Control resorption in the absence of stimulators was also inhibited. Potassium pyrophosphate at 1 mM was less effective as an inhibitor of bone resorption. The inhibitory effects of KPDP did not appear to be due entirely to nonspecific toxicity since partial recovery occurred after it was removed. There was no significant decrease in [3H]thymidine or [3H]proline incorporation into bones incubated with KPDP at 1 mM for 5 days, but [3H]proline incorporation was decreased at 24 h, suggesting that KPDP may have a general inhibitory effect on bone cells. When media with and without stimulators of resorption were incubated overnight at 4 degrees C with KPDP at 5.8 mM and then dialyzed to bring the concentration to below 0.3 mM, the bone-resorbing activity of PTH, LPS, and mrIL-1 was completely lost. This may have been due to the slow release of hydrogen peroxide; however, preincubation with equimolar concentrations of H2O3 caused only partial inactivation of PTH and LPS. LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prostaglandin F2 alpha on bone formation and resorption in cultured neonatal mouse calvariae: role of prostaglandin E2 production.

Although most studies show that prostaglandin E2 (PGE2) is the most potent and effective of the prostanoids in bone, recent data in cell culture suggest that PGF2 alpha may have unique effects, particularly on cell replication. The present study was undertaken to compare the effects of PGF2 alpha and PGE2 in cultured neonatal mouse parietal bones by simultaneous measurement of bone resorption as release of previously incorporated 45Ca, bone formation as incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein, and DNA synthesis as incorporation of [3H]thymidine. PGF2 alpha was less effective than PGE2 as a stimulator of bone resorption, and its effects were partially inhibited by indomethacin and markedly inhibited by glucocorticoids. In contrast, the resorptive response to PGE2 was unaffected by indomethacin and only partially inhibited by cortisol. PGF2 alpha had little effect on bone formation, in contrast to the biphasic effect of PGE2, which inhibited labeling of CDP in the absence of cortisol and stimulated CDP labeling in the presence of cortisol. PGF2 alpha increased thymidine incorporation into DNA, but the effect was smaller than that of PGE2 and was inhibited by indomethacin. These observations suggested that PGF2 alpha might act in part by stimulating PGE2 production. By RIA, PGE2 concentrations were increased in the medium of bones treated with PGF2 alpha, and this increase was blocked by indomethacin. By HPLC, bones prelabeled with [3H]arachidonic acid showed an increase in labeled PGE2 release, and RIA showed an increase in PGE2 after PGF2 alpha treatment. These results indicate that PGF2 alpha is a relatively weak agonist in bone compared to PGE2 and that some of the effects of PGF2 alpha on bone resorption, formation, and cell replication may be mediated by an increase in endogenous PGE2 production.

Effects of prostaglandin E3 and eicosapentaenoic acid on rat bone in organ culture.

To assess the possibility that diets rich in eicosapentaenoic acid (EPA) could have adverse effects on the skeleton, we examined the resorptive response to its major project, PGE3, and the effects and metabolism of EPA itself in cultured fetal rat long bones and neonatal rat calvaria. PGE3 stimulated bone resorption with a potency similar to that of PGE2. However, EPA was a much less effective precursor for PGE3 than was arachidonic acid (AA) for PGE2. In bones cultured with complement sufficient rabbit serum, which stimulates endogenous PGE release, addition of EPA had little effect on bone resorption while AA produced a substantial increase. Bones labeled with [3H]-AA and incubated with transforming growth factor-alpha (TGF-alpha), which stimulates endogenous PGE production, produced substantial amounts of PGE2, while bones labeled with [3H]-EPA and treated similarly produced less than 1/10th as much labeled PGE3. Thus, EPA appears to be a less effective precursor for the production of bone resorbing prostanoids than AA in cultured rat bone. However, since PGE3 is a potent stimulator of bone resorption, the possibility that dietary EPA can effect the production of bone resorbing prostanoids in man requires further study.

Evaluation of the in vivo and in vitro calcium-regulating actions of noncalcitonin peptides produced via calcitonin gene expression.

Recent studies of rat and human calcitonin (CT) gene expression have uncovered a diversity of secretory peptides. Here we report the results of testing two such non-CT secretory peptides for CT-like action in live rats and in cultured fetal rat long bone. One peptide, the carboxyl-terminal CT-adjacent peptide that is cosynthesized with CT, has no hypocalcemic effect and no inhibitory action on bone resorption in vitro. The other peptide, CT gene-related peptide, lowers blood calcium and inhibits bone resorption. In vitro experiments are consistent with the idea that CT gene-related peptide is acting at CT receptors in bone.