Palytoxin: an extraordinarily potent stimulator of prostaglandin production and bone resorption in cultured mouse calvariae

Palytoxin, a nonphorbol ester-type tumor promoter, stimulated the production of prostaglandin E2 (PGE2) and bone resorption in neonatal mouse calvariae in organ culture. The action of palytoxin on bone resorption occurred at extraordinarily low concentrations; enhanced resorption was regularly observed at 0.5 pg/ml, and the ED50 was 1-2 pg/ml (approximately 3 X 10(-13) M). Palytoxin-induced formation of PGE2 and bone resorption were inhibited completely by indomethacin (200 ng/ml). Concentrations of palytoxin above 10 pg/ml led to progressively decreasing enhancement of bone resorption; by 100-250 pg/ml no stimulation of resorption was observed despite continued high production of PGE2. Treatment with high concentrations of palytoxin (100 or 250 pg/ml) for 24-72 h inhibited cAMP accumulation stimulated by exogenous PGE2 or PTH and inhibited bone resorption induced by PGE2, PTH, or an analog of cAMP. Thus, palytoxin exhibited a biphasic dose-response curve for enhanced bone resorption, with stimulation at low concentrations (0.5-10 pg/ml) and toxic inhibition at high concentrations (greater than 50 pg/ml). Palytoxin is one of the most potent stimulators of bone resorption yet identified.     

Biphasic effects of prostaglandin E2 on bone formation in cultured fetal rat calvariae: interaction with cortisol

Previous studies have shown that prostaglandin E2 (PGE2) has both inhibitory and stimulatory effects on the incorporation of proline into collagenase-digestible protein (CDP) in cultured fetal rat calvaria. The present studies were undertaken to analyze further these biphasic effects of PGE2. PGE2 increased [3H]thymidine incorporation at 24 h, and this effect was enhanced in the presence of cortisol (10(-8) and 10(-7) M). An inhibitory effect on CDP labeling was observed at 96 h with PGE2 (10(-6) M) in the absence or presence of indomethacin (10(-6) M), but not in the presence of cortisol (10(-8) or 10(-7) M). When the central osteoblast-rich bone and periosteum were analyzed separately, the inhibitory effect of PGE2, with or without indomethacin, was confined to the central bone. Addition of aphidicolin (30 microM), an inhibitor of cell replication, did not prevent the inhibitory effect of PGE2 on CDP labeling. Analysis of labeled collagen by polyacrylamide gel electrophoresis showed a decrease in labeling of type I collagen in central bone. Moreover, mRNA for alpha 1(I)procollagen was decreased, as measured by dot blot hybridization and Northern blot analysis. Cortisol (10(-8)-10(-6) M) decreased the labeling of CDP as well as noncollagen protein (NCP) at 96 h. In the presence of cortisol, PGE2 (10(-8)-10(-5) M) consistently stimulated labeling of CDP and NCP, with a greater increase in CDP, resulting in an increase in the percentage of collagen synthesized. In the presence of low concentrations of cortisol (10(-8) or 3 x 10(-8) M), PGE2 (10(-7) M) increased CDP labeling by 260-480%, and the absolute value was 145-160% of that in control cultures without any hormone addition. The stimulatory effect was seen in both central bone and periosteum, although absolute values for CDP and percentage of collagen synthesized were higher in central bone. PGE2 (10(-7) M) had similar effects on CDP at 24 and 96 h in the presence of cortisol, and the stimulation at 10(-7) M was the same in the presence and absence of aphidicolin, suggesting that it was not dependent on cell replication. Cortisol decreased labeling of type I collagen, determined by polyacrylamide gel electrophoresis, and alpha 1(I)procollagen mRNA levels, determined by both Northern and dot blot analysis. PGE2 reversed these effects, increasing both radiolabeled collagen type I chains and alpha 1(I)procollagen mRNA levels. These results indicate that PGE2 can regulate bone collagen synthesis at a pretranslational site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cortisol modulates the actions of interleukin-1 alpha on bone formation, resorption, and prostaglandin production in cultured mouse parietal bones

Previous studies have shown that both interleukin-1 (IL-1) and glucocorticoids inhibit collagen synthesis in bone organ and cell cultures. In this study we examined their interactions in cultured neonatal mouse parietal bones. IL-1 alpha stimulated [3H]thymidine incorporation. Cortisol decreased thymidine incorporation, but did not block the effect of IL-1. Both cortisol and IL-1 alpha decreased the incorporation of [3H]proline into collagenase-digestible protein (CDP) and reduced alpha 1(I)procollagen mRNA levels at 72 h. Noncollagen protein (NCP) labeling was increased by IL-1 and decreased by cortisol. In the presence of cortisol, IL-1 alpha (6 pM) increased CDP as well as NCP labeling. The increase in CDP labeling was paralleled by an increase in alpha 1(I)procollagen mRNA, suggesting a pretranslational site for the cortisol-IL-1 alpha interaction. In the same bones, cortisol consistently blocked IL-1 alpha-stimulated 45Ca release and prostaglandin E2 (PGE2) production. The ability of IL-1 alpha to increase CDP in the presence of cortisol was the same in the presence or absence of indomethacin, an inhibitor of PGE2 synthesis, or aphidicholin (30 microM), an inhibitor of DNA synthesis, indicating that the reversal was neither PG mediated nor dependent on cell proliferation. In conclusion, our results demonstrate that IL-1 inhibits collagen, but not NCP or DNA, synthesis and that cortisol inhibits IL-1 alpha-induced bone resorption and PGE2 production and reverses its inhibitory effect on collagen synthesis in cultured neonatal mouse calvariae.     

Initiation of cell proliferation in cultured mouse fibroblasts by prostaglandin F2alpha.

Prostaglandin (PG) F2alpha added to quiescent Swiss mouse 3T3 cell cultures initiates DNA synthesis and cell proliferation in a small proportion of the cells. Insulin, which markedly potentiates the effect of PGF2alpha on the initiation of cell division when added with PGF2alpha, causes a simultaneous reduction in intracellular cyclic AMP and an increase in cyclic GMP concentrations similar to those observed after serum addition. Prostaglandins E1 and E2 are less effective than PGF2alpha in initiating DNA synthesis and this effect is only observed at higher concentrations. Therefore, some prostaglandins can act as extracellular factors to regulate cell proliferation.     

Aminobisphosphonate inhibition of interleukin-1-induced bone resorption in mouse calvariae

Interleukin-1 (IL-1) is probably an important lymphokine mediator of inflammation and bone resorption. IL-1 derived from mononuclear cells, a melanoma cell line (MM96 cells), and recombinant human IL-1 (rHuIL-1 beta) increased in vitro bone resorption, as measured by the release of 45Ca from cultured mouse calvariae. The 50% maximum active resorption was observed with 0.125 ng/ml or approximately 10(-11) M rHuIL-1 beta. The resorptive action of IL-1 was not entirely dependent on prostaglandin mediation, since its effect was evident when prostaglandin synthesis was inhibited in the cultures by indomethacin. IL-1-induced resorption has been shown to be inhibited by 10(-5) M 3-amino-1-hydroxypropylidene-1-1-bisphosphonate (APD). This inhibition was partially reversed by increasing doses of IL-1. In vitro toxicity studies showed that at concentrations of 10(-4) M, APD inhibited the growth of cultured MM96, murine myelomonocytic P388D1, and rat osteosarcoma UMR 106 cells, but not other mast and lymphoid cell lines. These in vitro observations may have relevance to the use of APD in bone and joint diseases in which inflammation and bone resorption are prominent.     

Alpha and beta human transforming growth factors stimulate prostaglandin production and bone resorption in cultured mouse calvaria.

Human alpha and beta transforming growth factors (TGF-alpha and TGF-beta) stimulated the production of prostaglandin E2 (PGE2) and bone resorption in neonatal mouse calvaria in organ culture. Significant stimulation of bone resorption by TGF-alpha was observed with a concentration of 0.2 ng/ml (35 pM) and by TGF-beta with 0.2 ng/ml (8.0 pM). Enhanced production of PGE2 and bone resorption stimulated by either TGF-alpha or TGF-beta were both inhibited by indomethacin. We conclude that TGF-alpha and TGF-beta are potent and powerful stimulators of the resorption of mouse bone by a mechanism that involves the enhanced local production of PGE2.     

Distribution of prostaglandin E2 and prostaglandin F2 alpha receptors in human myometrium

Human myometrium was studied for specific binding of PGE2 and PGF2 alpha. PGF2 alpha-binding was almost undetectable, but specific binding sites for PGE2 with high affinity (KD = 2.7 +/- 0.4 x 10(-9) M were demonstrated. The binding capacity for PGE2 exhibited a topically different distribution pattern with the highest values in the central parts and low to undetectable levels in the cervical region. Binding characteristics were analyzed by receptor kinetics, revealing a homogeneous receptor population. Binding capacity in uteri obtained from post-menopausal women was of the order of 900-940 fmol/mg protein. Oestrogen pre-treatment and pregnancy were associated with a 3-fold reduction of the PGE2-binding capacity.     

Effects of prostaglandin F2alpha and E2 on the production of progesterone by human granulosa cells in tissue culture.

Human granulosa cells with differing steroidogenic potentials were cultured in vitro. The effects of prostaglandin F2alpha (PGF2alpha) and PGE2 on the progesterone output and viability of these cells were investigated. Prostaglandin F2alpha either alone or in combination with LH and FSH inhibited the production of progesterone over a wide range of concentrations (1-8000 ng/ml). However, the inhibitory effect of PGF2alpha was 200 times less effective when the cells were exposed to LH and FSH for 6 days before the addition of the prostaglandin. By contrast PGE2, at concentrations from 1 to 500 ng/ml, markedly stimulated the production of progesterone by granulosa cells, and this was not prevented by the addition of PGF2alpha. The degree of inhibition by PGF2alpha or stimulation by PGE2 was related to the biosynthetic capacity of the cells. These studies suggest that PGF2alpha may act directly on the adenylate cyclase system of human granulosa cells by blocking its activation by LH, and they demonstrate that functional regression of the luteal cell can be induced independently of the blood vascular system.     

Effect of auranofin on resorption, prostaglandin synthesis and ultrastructure of bone cells in cultured mouse calvaria

Auranofin (AF) in concentrations between 3 x 10(-7) and 3 x 10(-6) mol/l stimulated bone resorption in cultured neonatal mouse calvariae significantly with 1 x 10(-6) mol/l being most potent. Complete inhibition by 5 x 10(-7) mol/l indomethacin and increased medium concentrations of prostaglandin (PG) E2 and 6-keto-PGF1 alpha after 72 h indicate a PG mediated mechanism. Morphology revealed active osteoclasts. Cytotoxic effects were observed with 3 x 10(-6) and 1 x 10(-5) mol/l AF with osteocytes and osteoblasts being considerably more sensitive than osteoclasts. The latter concentrations inhibited bone resorption stimulated by parathyroid hormone (PTH) 1,25-dihydroxyvitamin D3, PGE2, thrombin and interleukin 1. The stimulatory effect of AF on PG production and subsequent bone resorption could limit its therapeutic usefulness.     

Human parathyroid hormone-related peptide-(107-111) does not inhibit bone resorption in neonatal mouse calvariae.

Recent analysis of the structure-function relationship of human PTH-related peptide (hPTHrP) has led to the discovery that its direct inhibitory activity on osteoclastic bone resorption resides fully in the 107-111 sequence of the peptide, as assessed by a bone resorption assay using isolated rat osteoclasts. Here we report that hPTHrP-(107-111) is inactive in neonatal mouse calvariae in culture. hPTHrP-(107-111), at doses of 10(-12)-10(-6) M and incubation periods up to 96 h, did not affect either basal or agonist-stimulated 45Ca release from prelabeled neonatal mouse calvariae, while salmon calcitonin was a potent and powerful inhibitor of both basal and stimulated 45Ca release from bone. Moreover, salmon calcitonin, but not hPTHrP-(107-111), inhibited the increase in osteoclast number in hPTHrP-(1-34)-treated bones. Furthermore, hPTHrP-(107-139) also failed to inhibit 45Ca release and the hPTHrP-(1-34)-induced increase in osteoclast number in this organ culture model when tested under conditions identical to those for hPTHrP-(107-111). The addition of indomethacin to hPTHrP-(107-111)- or hPTHrP-(107-139)-treated bones was without effect, excluding the possibility that the direct inhibitory activity of these peptides on osteoclasts is ablated by a prostaglandin-mediated mechanism. Although the mechanism underlying the apparent inability of the carboxyl-terminal PTHrP fragments to inhibit osteoclastic bone resorption in neonatal mouse calvariae is unknown, it may involve the complex microenvironment of osteoclasts in intact bone, which contains a large variety of cell types other than osteoclasts.     

Aminobisphosphonate inhibition of interleukin-1—induced bone resorption in mouse calvariae

Interleukin-1 (IL-1) is probably an important lymphokine mediator of inflammation and bone resorption. IL-1 derived from mononuclear cells, a melanoma cell line (MM96 cells), and recombinant human IL-1 (rHuIL-1β) increased in vitro bone resorption, as measured by the release of ⁴⁵Ca from cultured mouse calvariae. The 50% maximum active resorption was observed with 0.125 ng/ml or approximately 10⁻¹¹M rHuIL-1β. The resorptive action of IL-1 was not entirely dependent on prostaglandin mediation, since its effect was evident when prostaglandin synthesis was inhibited in the cultures by indomethacin. IL-1—induced resorption has been shown to be inhibited by 10⁻⁵M 3-amino-1-hydroxypropylidene-1-1-bisphosphonate (APD). This inhibition was partially reversed by increasing doses of IL-1. In vitro toxicity studies showed that at concentrations of 10⁻⁴M, APD inhibited the growth of cultured MM96, murine myelomonocytic P388D1, and rat osteosarcoma UMR 106 cells, but not other mast and lymphoid cell lines. These in vitro observations may have relevance to the use of APD in bone and joint diseases in which inflammation and bone resorption are prominent.     

Effects of atrial natriuretic factor on cyclic nucleotides, bone resorption, collagen and deoxyribonucleic acid synthesis, and prostaglandin E2 production in fetal rat bone cultures

We examined the effects of synthetic human atrial natriuretic factor (human ANF 99-126) on adenylate cyclase activity, cAMP and cyclic GMP (cGMP) levels, bone resorption, collagen and DNA synthesis, and prostaglandin E2 (PGE2) production in fetal rat bone organ cultures. ANF (100 nM) inhibited PTH- and PGE2-stimulated cAMP production but had no effect on basal cAMP production in 21-day fetal rat calvaria. ANF increased cGMP levels, and this was not affected by PTH. ANF (10 nM) partially inhibited bone resorption stimulated by PGE2 but had no effect on control or PTH-stimulated resorption in 19-day fetal rat long bones. ANF had no effect on collagen and DNA synthesis or PGE2 production and did not alter responses to PTH or PGE2 in the fetal rat calvaria. Thus, ANF has no major direct effect on bone resorption or formation, but it is possible that ANF modulates the local regulatory function of PGE2 in bone.     

Simultaneous infusion of prostaglandin E2 antagonizes the luteolytic action of prostaglandin F2alpha in vivo

Corpora lutea of ewes bearing ovarian autotransplants were infused for 4 h with prostaglandin F2alpha (PGF2alpha) (10 microng/h), PGF2alpha+PGE2 (10microng/h of each), PGE2 (10 microng/h) or saline on day 10 of the cycle. Ovarian venous blood obtained before, during, and up to 12 h after the infusion period, was assayed for progesterone. Prostaglandin F2alpha produced an immediate, rapid and sustained decline in progesterone secretion, but infusion of PGE2 together with PGF2alpha prevented the decline until after the infusion. Progesterone secretion was unaffected by infusion of PGE2 alone. Oestrous behaviour was observed in four out of seven animals infused with PGF2alpha but in only one out of six infused with PGF2Alpha+PGE2. None of the animals infused with PGE2 alone or saline only came into heat.     

Suramin inhibits bone resorption and reduces osteoblast number in a neonatal mouse calvarial bone resorption assay.

The antineoplastic properties of suramin, a polyanionic agent with demonstrated antigrowth factor activity, are under evaluation in vitro, in vivo, and in clinical trials. Suramin has been shown to have antitumor activity in patients with advanced, hormone refractory prostate cancer. During these trials, significant resolution of osseous pain was observed in nearly three quarters of the patients treated with suramin. To evaluate the effect of suramin on bone cells, we studied the effect of suramin on bone resorption in a neonatal mouse calvarial assay. Suramin inhibited bone-resorbing activity in a dose-related fashion and had an additive effect with calcitonin. Calvaria pretreated with suramin had less bone-resorbing activity, fewer attached osteoblasts, and less medium alkaline phosphatase activity than control calvaria. Suramin also inhibited osteoclastic release of tritiated proline from labeled bone in a dose-dependent fashion. The effect of metastatic prostate carcinoma on bone is incompletely understood, but may be moderated by tumor-produced factors and/or cytokines. The effects of several such agents, therefore, were examined in combination with suramin. Bone resorption induced by PTH, epidermal growth factor, tumor necrosis factor, and a tumor-produced factor, PTH related-protein, was blocked by suramin. The ability of suramin to inhibit the bone-resorbing effects of several cytokines suggests that its mechanism may involve direct action on bone metabolism. Autoradiography performed on calvaria treated with labeled suramin demonstrated heavy deposition of suramin on the outer surface of the matrix, adjacent to osteoblasts and osteoclasts lining the outer table, suggesting that bone cells may be subject to high local concentrations of the drug, in keeping with this hypothesis.     

Functional luteolysis in the pseudopregnant rat: effects of prostaglandin F2 alpha and 16-aryloxy prostaglandin F2 alpha in vitro.

The present work concerns the luteolytic effects of prostaglandin (PG) F2 alpha and its analogue, 16-aryloxy PGF2 alpha, upon isolated luteal cells. Varying doses of these two prostaglandins were incubated with cells in the presence or absence of an optimum stimulatory dose of LH (1 microgram/ml). The total contents of progesterone and 20 alpha-dihydroprogesterone in flasks were determined after the incubation periods by radioimmunoassay. Both prostaglandins inhibited basal synthesis of progesterone and 20 alpha-dihydroprogesterone, maximum inhibition occurring at concentrations of either PG of between 250 and 500 ng/ml. In this dose range both prostaglandins were found to abolish LH-stimulated progestogen synthesis completely. These effects were discernible within 5 min of incubation. The studies demonstrated that the onset of PG-induced luteolysis in vitro is characterized by an inhibition of the biosynthesis of both progesterone and its weakly progestogenic metabolite, 20 alpha-dihydroprogesterone; induction of 20 alpha-hydroxysteroid dehydrogenase activity by either PG was not found in incubations extending up to 60 min. In contrast to their relative potencies in vivo, PGF2 alpha and 16-aryloxy PGF2 alpha were essentially equipotent in this in-vitro system.     

Cyclooxygenase-2 and prostaglandin F2 alpha in Syrian hamster Leydig cells: Inhibitory role on luteinizing hormone/human chorionic gonadotropin-stimulated testosterone production

We have previously found that cyclooxygenase-2 (COX-2), a key enzyme in the biosynthesis of prostaglandins (PGs), is present in the testicular interstitial cells of infertile men, whereas it is absent in human testes with no evident morphological changes or abnormalities. To find an animal model for further investigating COX-2 and its role in testicular steroidogenesis, we screened testes from adult species ranging from mice to monkeys. By using immunohistochemical assays, we found COX-2 expression only in Leydig cells of the reproductively active (peripubertal, pubertal, and adult) seasonal breeder Syrian hamster. COX-2 expression in hamster Leydig cells was confirmed by RT-PCR. In contrast, COX-1 expression was not detected in hamster testes. Because COX-2 expression implies PG synthesis, we investigated the effect of various PGs on testosterone production and found that PGF2 alpha stood out because it significantly reduced human chorionic gonadotropin-stimulated testosterone release from isolated hamster Leydig cells in a dose-dependent manner. This mechanism involves a decreased expression of testicular steroidogenic acute regulatory protein and 17beta-hydroxysteroid dehydrogenase. Testicular concentration and content of PGF2 alpha in reproductively active hamsters as well as production of PGF2 alpha from isolated hamster Leydig cells were also determined. Moreover, PGF2 alpha receptors were localized in Leydig cells of hamsters and testicular biopsies from patients with Sertoli cell only and germ arrest syndromes. Thus, in this study, we described a COX-2-initiated pathway that via PGF2 alpha production, PGF2 alpha receptors, steroidogenic acute regulatory protein, and 17beta-hydroxysteroid dehydrogenase represents a physiological local inhibitory system of human chorionic gonadotropin-stimulated testosterone production in the Syrian hamster testes.     

Lack of specific binding of prostaglandin E2, prostaglandin F2 alpha, and 6-keto prostaglandin F1 alpha to serum in patients with peptic ulcer disease and in healthy subjects

Rabbits immunized against prostaglandins develop antibodies to prostaglandins and peptic ulcer disease (duodenal and gastric ulcers). We have evaluated the hypothesis that idiopathic gastric or duodenal ulcer disease in humans may be associated with the spontaneous occurrence of serum antibodies directed against endogenous prostaglandins. We found that serum from 45 ulcer patients (34 duodenal, 11 gastric) had a low degree of binding of radiolabeled prostaglandin E2, prostaglandin F2 alpha, or 6-keto prostaglandin F1 alpha. The extent to which prostaglandins were bound to serum of ulcer patients was not statistically different from prostaglandin binding to serum from 25 normal subjects. Therefore, we conclude that spontaneous occurrence of circulating antibodies against endogenous prostaglandins is an unlikely cause of gastroduodenal ulceration in humans.     

Papaverine stimulation of prostaglandin E2 production by cultured rabbit gastric mucosa

Prostaglandins (PGs) are synthesised by gastric mucosa, and have been shown to inhibit gastric acid secretion and ulcer formation in man and experimental animals. Recently exogenous PGs, mainly of the E group, have been used for the treatment of peptic ulcer disease. We therefore searched for a drug that would stimulate endogenous gastric prostaglandin E₂ (PGE₂) synthesis. Rabbit gastric mucosa slices were cultured for 22 hours at 77°C. PGE₂, measured by radioimmunoassay, was found to be linearly secreted into the culture medium. PGE₂ accumulation in the medium during 22 hours of culture was 7·9±0·5 (SE) ng/mg tissue (N=20). Addition of papaverine (100 μ/ml), a cyclic nucleotide phosphodiesterase inhibitor, resulted in a significant increase (250% of control) in PGE₂ accumulation in the medium: 24·3±1·8 ng/mg tissue (N=25). Isobutylmethylxanthine (IBMX 100 μg/ml), another phosphodiesterase inhibitor, only slightly increased PGE₂ accumulation, while 8 bromo-cyclic AMP (1 mM) had no effect. Under these conditions IBMX increased by 20-fold mucosal cyclic AMP levels: 3·9±0·3 pmol/mg tissue (N=8) as compared with control levels: 0·2±0·03 pmol/mg tissue (N=8). Papaverine, however, did not alter mucosal cyclic AMP accumulation. These results indicate that papaverine stimulates PGE₂ production by cultured rabbit gastric mucosa and that this stimulation is not related to the inhibition of phosphodiesterase activity and accumulation of mucosal cyclic AMP. Papaverine induced stimulation of PGE₂ production should be further evaluated regarding its possible beneficial effects in protecting gastric mucosa and in reducing acid secretion in peptic ulcer patients.