Stimulation of bone formation by prostaglandin E2

We examined the effect of prostaglandin E2 (PGE2), in the presence or absence of cortisol, on bone formation in 21-day fetal rat calvaria maintained in organ culture for 24 to 96 h. [3H]Thymidine and [3H] proline incorporation were used to assess DNA and collagen synthesis, respectively. Changes in dry weight and DNA content were assessed after 96 h. PGE2 (10(-7) M) stimulated both DNA and collagen synthesis in calvaria. The effect on DNA synthesis was early (24 h), transient and limited to the periosteum. Collagen synthesis was stimulated at a later time (96 h), predominantly in the central bone. Cortisol (10(-7) M) inhibited DNA and collagen synthesis. The addition of PGE2 reversed the inhibitory effects of cortisol on DNA synthesis and content and increased collagen synthesis in central bone to levels above control untreated cultures. We conclude that PGE2 has stimulatory effects on bone formation and can reverse the inhibitory effects of cortisol. Hence the effects of cortisol may be mediated in part by their ability to reduce the endogenous production of prostaglandins.     

Stimulation of progesterone production by adrenocorticotropic hormone and prostaglandin E2 in rat luteal cells

The effects of adrenocorticotropic hormone (ACTH), human chorionic gonadotropin (hCG) and prostaglandin E2 (PGE2) on the progesterone secretion of luteal cells from rats were studied. Corpora lutea were harvested on Day 6 of pseudopregnancy and digested by trypsin. Homogeneous suspensions of luteal cells were used for short-term incubation. ACTH, PGE2, and hCG were added to the medium and the changes in progesterone production were measured by radioimmunoassay (RIA). Furthermore, specific ACTH-binding sites of the luteal cell membrane were studied by Scatchard analysis. ACTH, PGE2 and hCG increased synthesis of progesterone, and the combination of hCG with ACTH or PGE2 further increased production of the hormone. The effect of ACTH could be prevented by indomethacin. These effect of ACTH seem to be connected with specific ACTH-binding sites of the luteal cell membrane and with increased production of PGE2.     

Cyclic AMP formation and release by cultured bone cells stimulated with prostaglandin E2.

PGE2 produced a marked and dose-related increase in cAMP content of cultured bone cells and in the release of cAMP into the incubation medium. The amount of cAMP released from the cells by PGE2 was proportional to the cellular concentration, and was dependent upon the time of incubation with PGE2. The cAMP levels released into the media increased slowly at a linear rate during a 60 min treatment with PGE2. This release was blocked by theophylline, probenecid, ouabain and dinitrophenol, suggesting that the release of cAMP was not a simple diffusive process and required energy. SC-19220 reduced the formation of cAMP more than the release, suggesting that the formation and the release may arise from separate events. Inability of D600 to inhibit PGE2-induced release of cAMP indicates that the release does not require calcium.     

Stimulation of creatine kinase BB activity by 1 alpha,25-dihydroxycholecalciferol and 24R,25-dihydroxycholecalciferol in rat tissues.

Vitamin D metabolites stimulate creatine kinase BB activity in organs of vitamin D-deficient rats. In epiphyses of long bones, creatine kinase BB activity increases 2.6-fold 24 h after injection of 24R,25-dihydroxycholecalciferol but not of 1 alpha,25-dihydroxycholecalciferol. Contrariwise, 1 alpha,25-dihydroxycholecalciferol, but not 24R,25-dihydroxycholecalciferol, increases creatine kinase BB activity in diaphyses and in kidney. Neither metabolite affects creatine kinase activity in duodenal mucosa.     

Differential effects of parathyroid hormone responsive cultured human cells on biological activity of parathyroid hormone and parathyroid hormone inhibitory analogues

We have employed parathyroid hormone (PTH) responsive human cells cultured from dermis or giant cell tumors of bone (GT) to evaluate the biological properties of a newly developed in vivo PTH inhibitor, [Tyr34]bPTH-(7-34)-amide (PTH-Inh). Short periods of incubation of cells from dermis or GT with maximal stimulatory concentrations of PTH in the presence of increasing concentrations of PTH-Inh resulted in a dose-dependent inhibition of the adenosine cyclic 3',5'-phosphate (cAMP) response (Ki = 3 X 10(-7) M and 4.2 X 10(-7) M for GT and dermal cells, respectively). In both cell cultures, PTH-Inh alone did not increase cAMP levels, and in desensitization experiments, preincubation with PTH-Inh alone did not desensitize cells to PTH. Hence, the analogue displayed no agonist properties. Unexpectedly, when PTH-Inh was incubated with dermal cells in the presence of PTH, the PTH-Inh failed to block desensitization, suggesting a loss of biological effectiveness of the inhibitor. When medium containing PTH-Inh alone was removed from dermal cells and tested for inhibition of the acute PTH response in untreated cells, there was apparent loss of inhibitory efficacy (t1/2 = 20 h). In contrast, incubation of native PTH or bPTH-(1-34) with cells did not affect the biological activity of these ligands. Unlike the dermal cells, the PTH-Inh did block desensitization to PTH in GT, and there was no loss of inhibitor efficacy when medium containing PTH-Inh was incubated with GT (48 h) and then tested in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of prostaglandin E2 (PGE2) production by arachidonic acid, oestrogen and parathyroid hormone in MG-63 and MC3T3-E1 osteoblast-like cells

Polyunsaturated fatty acids (PUFAs) as well as oestrogen (E2) and parathyroid hormone (PTH) affect bone cells. The aim of the study was to determine whether arachidonic acid (AA), E2, and PTH increase prostaglandin E₂ (PGE₂) synthesis in MG-63 and MC3T3-E1 osteoblastic cells and the level of mediation by COX-1 and COX-2. PGE₂ levels were determined in the conditioned culture media of MG-63 and MC3T3-E1 osteoblasts after exposure to AA, PTH and E2. Cells were pre-incubated in some experiments with the unselective COX inhibitor indomethacin or the COX-2 specific blocker NS-398. Indirect immunofluorescence was performed on MG-63 cells to detect the presence and location of the two enzymes involved. AA increased PGE₂ secretion in both cell lines; production by MC3T3-E1 cells, however, was significantly higher than that of MG-63 cells. This could be due to autoamplification via the EP₁ subtype of PGE receptors in mouse MC3T3-E1 osteoblasts. Both COX-1 and COX-2 affected the regulation of PGE₂ synthesis in MG-63 cells. E2 had no effect on PGE₂ secretion in both cell lines, while PTH caused a slight increase in PGE₂ synthesis in the MG-63 cell line.     

Stimulation of extracellular signal-regulated kinases and proliferation in rat osteoblastic cells by parathyroid hormone is protein kinase C-dependent

Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10(-12) to 10(-11) m) is sufficient for substantial increases in ERK activity. Treatment with PTH-(1-34) (10(-8) m), PTH-(1-31), or 8-bromo-cAMP failed to stimulate ERKs, whereas treatment with phorbol 12-myristate 13-acetate, serum, or PTH peptides lacking the N-terminal amino acids stimulated activity. Furthermore, the activation of ERKs was prevented by pretreatment of osteoblastic cells with inhibitors of protein kinase C (GF 109203X) and MEK (PD 98059). Treatment of UMR cells with epidermal growth factor (EGF), but not PTH, promoted tyrosine phosphorylation of the EGF receptor. Transient transfection of UMR cells with p21(N17Ras) did not block activation of ERKs following treatment with low concentrations of PTH. Thus, activation of ERKs and proliferation by PTH is protein kinase C-dependent, but stimulation occurs independently of the EGF receptor and Ras activation.     

Biotinylated parathyroid hormone as a probe for the parathyroid hormone receptor. Structure-function analysis and detection of specific binding to cultured bone cells by flow cytometry

The synthetic bovine parathyroid hormone (PTH) analog (Nle8, Nle18, Tyr34) bovine PTH(1-34)amide (bPTH(1-34)amide) was reacted with biotinyl-epsilon aminocaproic acid-N-hydroxysuccinimide under conditions which yielded five isoforms which were fractionated by a combination of reversed phase and ion-exchange chromatography. These reaction products were analyzed by automated Edman degradation in a manner which allowed us to specify the location and number of biotin residues on picomole quantities of hormone. The ability of each of these isoforms to induce a rise in intracellular cAMP in the ROS 17/2.8 cell line allowed us to evaluate the effect on function of biotinylation at different residues. Derivatized PTH molecules which contained a single biotin at either lysine 13, lysine 26, or lysine 27 possessed full biological activity. However, bioactivity was significantly reduced when position 13 plus either lysine 26 or 27 were biotinylated. Biological activity was lost when all 3 lysine residues were biotinylated. Biotinylation of the alpha-NH2 group of alanine at the NH2 terminus also resulted in a total loss of activity. Hence, unlike the effect of altering the alanine at position 1, modification of a single lysine residue at positions 13, 26, and 27 has a less critical effect on biological activity of the molecule. However, biotinylation of all three lysines results in a biologically inert PTH derivative and suggests that changes in isoelectric point, hydrophobicity, or tertiary structure may strongly influence hormone function. A fully bioactive-mixture of isoforms was used to detect receptors on ROS 17/2.8 cells by flow cytometry using fluorescein isothiocyanate-avidin as a fluorescent indicator. Binding to cell surface receptors was saturable and could be inhibited by native bPTH(1-34) but not by transforming growth factor beta, calcitonin or insulin. Moreover, PTH receptors could also be detected on primary cultures of human bone cells and human fibroblasts.     

Protein kinase C activation amplifies prostaglandin F2 alpha-induced prostaglandin E2 synthesis in osteoblast-like cells.

In cloned osteoblast-like cells, MC3T3-E1, prostaglandin F2 alpha (PGF2 alpha) stimulated arachidonic acid (AA) release in a dose-dependent manner in the range between 1 nM and 10 microM. 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, which by itself had little effect on AA release, markedly amplified the release of AA stimulated by PGF2 alpha in a dose-dependent manner. 4 alpha-phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect on the PGF2 alpha-induced AA release. 1-oleoyl-2-acetylglycerol (OAG), a specific activator for PKC, mimicked TPA by enhancement of the AA release induced by PGF2 alpha. H-7, a PKC inhibitor, markedly suppressed the effect of OAG on PGF2 alpha-induced AA release. Quinacrine, a phospholipase A2 inhibitor, showed partial inhibitory effect on PGF2 alpha-induced AA release, while it suppressed the amplification by OAG of PGF2 alpha-induced AA release almost to the control level. Furthermore, TPA enhanced the AA release induced by melittin, known as a phospholipase A2 activator. On the other hand, TPA inhibited the formation of inositol trisphosphate stimulated by PGF2 alpha. Under the same condition, PGF2 alpha indeed stimulated prostaglandin E2 (PGE2) synthesis and TPA markedly amplified the PGF2 alpha-induced PGE2 synthesis as well as AA release. These results indicate that the activation of PKC amplifies PGF2 alpha-induced both AA release and PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like cells.     

Stimulation of protein kinase C (PKC) activity in resting Swiss 3T3 cells by prostaglandin F2 alpha.

Prostaglandin F2 alpha (PGF2 alpha), a mitogen for resting Swiss 3T3 cells, rapidly stimulates phosphorylation of an 80 kDa protein (80 K). 1-Oleoyl-2-acetylglycerol (OAG) and 12-O-tetradecanoyl phorbol-13-acetate (TPA) both protein kinase C (PKC) activators, also elicit 80 K phosphorylation. In contrast PGE1, PGE2 or PGF2 beta, which are non-mitogenic in these cells, had little or no action on this event. However PGE1 and PGE2 potentiate the PGF2 alpha proliferative effect but do not enhance its action on 80 K phosphorylation. These results suggest that PGF2 alpha mitogenic induction involves PKC signalling pathway activation while its enhancement by PGE1 or PGE2 occurs through a different mechanism(s).     

Stimulation of prostaglandin formation by vasoactive mediators in cultured human endothelial cells

Human endothelial cells in culture synthesize prostaglandins and release these products into the culture medium. The major products of arachidonic acid metabolism were identified by high pressure liquid chromatography or thin layer chromatography, and release of prostaglandins was measured by radioimmunoassays. Addition of histamine or bradykinin enhanced release of prostaglandins in both arterial and venous endothelial cells. Other vasoactive compunds including angiotensin II, vasopressin, substance P, epinephrine, norepinephrine, or isoproterenol were ineffective. Release of prostaglandins by histamine was concentration-related, and involved H₁ receptors, as determined by addition of histamine antagonists. Incubation of endothelial cells with C-arachidonic acid resulted in a time-dependent uptake into cell lipids, where most of the radioactivity was incorporated into phosphatidyl choline and neutral lipids. Endothelian cells released ¹⁴C_arachidonic acid as well as ¹⁴C-prostaglandins in response to either histamine or bradykinin. The enhanced release of ¹⁴C-prostaglandins was inhibited by either indomethacin or mepacrine, but ¹⁴C-arachidonic acid release was inhibited only by mepacrine. We conclude that the vasoactive compounds, histamine and bradykinin, stimulate formation of prostaglandins in endothelial cells by the release of arachidonic acid from phospholipids of the cell membrane.     

24R,25-dihydroxyvitamin D stimulates creatine kinase BB activity in chick cartilage cells in culture

In chick limb-bud cartilage cell cultures 24R,25-dihydroxycholecalciferol (24R,25(OH)2D3), but not 24S,25(OH)2D3, 1 alpha,25(OH)2D3 or 25(OH)D3, stimulates the activity of the brain type (BB) isozyme of creatine kinase (EC 2.7.3.2), the 'estrogen-induced protein' first identified in rat uterus. Cultures treated with bromodeoxyuridine, in which cartilage formation is inhibited, show no stimulation of creatine kinase BB by 24R,25(OH)2D3.     

Dexamethasone effects on creatine kinase activity and insulin-like growth factor receptors in cultured muscle cells

We examined the effects of dexamethasone on creatine kinase (CK) activity and insulin-like growth factor I (IGF-I) binding in two skeletal muscle-derived cell lines (mouse, C2C12; rat, L6) and in one cardiac muscle-derived cell line (rat, H9c2). Dexamethasone treatment during differentiation of cultured cells caused a dose-dependent increase in CK activity as well as an increase in the degree of myotube formation in C2C12 and L6, whereas H9c2 cells did not exhibit significant CK activities during culture or dexamethasone treatment. Dexamethasone treatment of C2C12 did not stimulate proliferation in differentiating cultures, but a dose-dependent increase in the number of nuclei was observed for L6 concomitant with increased CK activity. In L6 the increased CK activity may therefore reflect a dose-dependent increase in proliferation. Short-term (48 hr) treatment of C2C12 with dexamethasone (20 nM) did not appear to alter myoblast fusion but reversibly increased CK activity. In C2C12 the observed increase in CK, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities with dexamethasone treatment suggest modulation of protein expression and/or turnover. Although the data for dexamethasone effects on CK activities varied in each of the cell lines, consistent behavior was observed in all three cell lines when IGF-I binding was examined. IGF-I binding to dexamethasone-treated cells (50 nM for 24 hr the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities. Affinity cross linking and autoradiography indicated that the increase in IGF-I binding was the result of dexamethasone up-regulation of type I IGF receptors. Our data for all three muscle cell lines suggest that similar heterologous hormone receptor modulation of type I IGF receptor sites occurs with dexamethasone treatment.     

Stimulation of ovine myometrial activity by 6-keto prostaglandin E1

6-keto prostaglandin E1 (6KE) is a metabolite of PGI2, which we have shown previously inhibits spontaneous myometrial activity. In the present study we examined the effects of 6KE on uterine electrical and mechanical activity in non-pregnant ovariectomized sheep. 6KE stimulated uterine activity in a dose-dependent fashion. The effect was enhanced by pre-treatment with estradiol (E2). It was not influenced by pre-treatment with meclofenamic acid and was not associated with significant changes in the concentrations of 13,14 dihydro 15-keto PGF2 alpha in vena cava plasma. After E2 treatment, 6KE had 0.2-0.3 of the stimulatory activity of PGF2 alpha. In the absence of E2, the uterine response to both 6KE and PGF2 alpha was decreased. In animals in which spontaneous myometrial activity was inhibited by PGI2, the uterus remained responsive to 6KE. We conclude that in the ovariectomized non-pregnant sheep 6KE stimulates uterine activity, and that the effect is independent of endogenous PG production.     

Cardiomyocytes cultured in serum-free medium: Growth and creatine kinase activity

Primary cultures of newborn rat heart cells were grown for up to 3 weeks in serum-free medium supplemented by insulin, hydrocortisone, transferrin and fetuin. The cells resumed spontaneous beating at 20 h post plating. Mean rates of beating on the second and third day were 79.5 and 94 beats per min, respectively. Cell proliferation occurred during the first 3 days of culture with maximal rates of DNA and protein synthesis on the second day. The highest values of creatine kinase activity were observed on days 2–5 and the three cytoplasmic isozymes, MM, MB and BB, were present in the cultures in proportions similar to those of the newborn heart, indicating stability of the differentiated state of the cells. The relative amount of each isozyme remained unchanged throughout the experiments, MM constituted 70–90% of enzyme activity, MB contributed up to 30% and BB did not exceed 15% of activity. The very low proportion of BB and the lack of increase in this isozyme with age of culture support our earlier morphological observations that non-myocytes do not overgrow the culture.     

Stimulation of prostaglandin E2 biosynthesis by estradiol in rat kidneys

The effect of estradiol administration on renal prostaglandin (PG) E2 biosynthetic activity in rats was studied. A specific radioimmunoassay for PGE2 was developed and applied in the quantitation of PGE2 biosynthesis in kidney. Conversion of exogenous arachidonic acid into PGE2 by renal microsomal fraction was assayed. Formation of PGE2 was linear in fashion up to 5 min incubation at 37 degrees C, and linear in fashion up to 3.5 mg of microsome used as enzyme source. The renal biosynthesis of PGE2 was significantly increased by estradiol treatment.     

Involvement of protein kinase C in prostaglandin E2-induced catecholamine release from cultured bovine adrenal chromaffin cells

We recently reported that prostaglandin (PG) E2 stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain induced a gradual secretion of catecholamines from the cells (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). Here we examined the involvement of two signal pathways, Ca2+ mobilization and protein kinase C activation resulting from phosphoinositide metabolism, in the PGE2-induced catecholamine release. Either the Ca2+ ionophore ionomycin or 12-O-tetradecanoylphorbol 13-acetate (TPA) could enhance the release in the presence of ouabain, and ionomycin-induced release was additive to PGE2-induced release, but TPA-induced release was not additive. PGE2 dose-dependently stimulated the formation of diacylglycerol and caused the translocation of 4% of the total protein kinase C activity to become membrane-bound within 5 min. These effects were specific for PGE2 and PGE1 among PGs tested (PGE2 = PGE1 greater than PGF2 alpha greater than PGD2). Furthermore, the phosphoinositide-specific phospholipase C inhibitor neomycin inhibited PGE2-induced accumulation of inositol phosphates, diacylglycerol formation, translocation of protein kinase C, and also stimulation of catecholamine release. Both PGE2- and TPA-induced release were inhibited by the depletion of protein kinase C caused by prolonged exposure to TPA, but ionomycin-induced release was not inhibited. We recently found that the amiloride-sensitive Na+, H+-antiport participates in PGE2-evoked catecholamine release (Tanaka, T., Yokohama, H., Negishi, M., Hayashi, H., Ito, S., and Hayaishi, O. (1990) J. Neurochem. 54, 86-95). In agreement with our recent report, PGE2 and TPA induced a sustained increase in intracellular pH that was abolished by the protein kinase C inhibitor staurosporine but not by the calmodulin inhibitor W-7. Ionomycin also induced a marked increase in intracellular pH, but this increase was abolished by W-7 but not by staurosporine. These results demonstrate that PGE2-induced activation of the Na+, H(+)-antiport and catecholamine release in the presence of ouabain are mediated by activation of protein kinase C, rather than by Ca2+ mobilization, resulting from phosphoinositide metabolism.     

Stimulation of prostaglandin production through purinoceptors on cultured porcine endothelial cells.

Potato (Solanum tuberosum) lectin, which is a very highly glycosylated glycoprotein, has been completely deglycosylated by use of the trifluoromethanesulphonic acid reagent described by Edge, Faltnek, Hof, Reichert & Weber [(1981) Analyt. Biochem. 118, 131-137]. This shows that both hydroxyproline-arabinofuranoside and serine-galactopyranoside linkages are hydrolysed. The deglycosylated lectin is still functional and cross-reacts with one component of an anti-(potato lectin) antiserum.