An inhibitory role for the protein kinase C pathway in ovarian steroidogenesis. Studies with cultured swine granulosa cells.

We have used primary cultures of swine granulosa cells to investigate the regulatory role of the protein kinase C pathway in the ovary. In this system, we observed the following. Swine granulosa cells bound [3H]phorbol 12,13-dibutyrate [( 3H]PDB) specifically with high affinity [apparent Ki for 12-O-tetradecanoylphorbol 13-acetate (TPA) = 3.1 (2.1-4.7) nM] and low capacity [0.68 (0.34-0.99) pmol/10(7) cells]. The cytosol of granulosa cells contained functionally active protein kinase C capable of phosphorylating distinct proteins in response to stimulation with active phorbol ester. TPA and PDB induced dose-dependent inhibition (greater than 85%) of follicle-stimulating-hormone (FSH)-stimulated progesterone production. Half-maximally inhibitory concentrations were 0.10 and 0.75 nM for TPA and PDB respectively, whereas phorbol analogues that do not activate protein kinase C were not inhibitory. TPA did not impede cyclic AMP generation in response to FSH, cholera toxin or forskolin acutely (within 48 h), but did inhibit the stimulatory effects of 8-bromo cyclic AMP, insulin and oestradiol on progesterone biosynthesis. In the presence of maximally effective concentrations of 25-hydroxy-, 20 alpha-hydroxy- or 22R-hydroxy-cholesterol as exogenous sterol substrates for cholesterol side-chain cleavage, treatment with TPA suppressed pregnenolone, progesterone and 20 alpha-hydroxypregn-4-en-3-one biosynthesis by more than 80%. The inhibitory effects of phorbol esters were not attributable to non-specific cytotoxicity, since prostaglandin F2 alpha production increased in the same cultures and aromatization of exogenously supplied testosterone to oestradiol was not suppressed. In intact granulosa cells, the effects of phorbol esters were mimicked by a synthetic non-diterpene diacylglycerol, 1-octanoyl-2-acetylglycerol, and the tumour promoter, mezerein, which specifically activates protein kinase C. We conclude that swine granulosa cells contain specific high-affinity receptors for phorbol esters that are functionally coupled to protein phosphorylation. Moreover, treatment with phorbol esters or non-phorbol activators of protein kinase C results in selective inhibition of cholesterol side-chain cleavage activity without impairing cyclic AMP generation or oestrogen biosynthesis.     

Activation of protein kinase C is coupled to prostaglandin E2 synthesis in swine granulosa cells

We have examined the role of phospholipid-sensitive calcium-dependent protein kinase (protein kinase C) in prostaglandin E2 synthesis by monolayer cultures of swine granulosa cells. Specific phorbol ester derivatives known to activate protein kinase C significantly augmented the production of prostaglandin E2. These stimulatory actions were dose and time-dependent, and could be abolished by the cyclooxygenase inhibitor, indomethacin, or the protein synthesis inhibitor, cycloheximide. Moreover, the rank order of potency of phorbol esters in enhancing prostaglandin E2 production was concordant with that demonstrated for activation of protein kinase C. Phorbol ester in conjunction with the divalent cation ionophore, A23187, increased prostaglandin E2 production synergistically. In addition, a non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol, also significantly enhanced prostaglandin E2 biosynthesis. The stimulated synthesis of prostaglandin E2 was confirmed by high-pressure liquid chromatographic purification of this radiolabeled metabolite of 3H-arachidonic acid, and by capillary gas chromatography high-resolution mass spectrometry. Thus, the present studies indicate that the protein kinase C effector pathway is functionally coupled to prostaglandin E2 production in the swine granulosa cell.     

Evidence that arachidonic acid influences hen granulosa cell steroidogenesis and plasminogen activator activity by a protein kinase C-independent mechanism

We recently proposed that arachidonic acid serves as a second messenger within granulosa cells from the largest preovulatory follicle of the hen. The present studies were conducted to determine whether the inhibitory effects of arachidonic acid on LH-induced cAMP accumulation and on the ability of cells to convert 25-hydroxycholesterol to progesterone are mediated via the protein kinase C pathway. Furthermore, we determined the effects of arachidonic acid on plasminogen activator activity in granulosa cells. In the first experiment, the putative protein kinase C inhibitor, staurosporine, completely reversed the inhibitory effects of phorbol 12-myristate 13-acetate (PMA) on LH-promoted cAMP formation, but failed to overcome the inhibitory effects of arachidonic acid. Prolonged pretreatment (18 h) with 1.6 microM PMA depleted granulosa cells of both cytosolic and membrane-associated protein kinase C, and subsequently attenuated the inhibitory effects of PMA on LH-induced progesterone production; however, such depletion did not alter the inhibitory effects of phospholipase A2 (PLA2; an agent that increases intracellular levels of arachidonic acid). PMA, but not arachidonic acid, caused a rapid (within 2 min) translocation of protein kinase C from the cytosol to the membrane (a characteristic of agents that activate protein kinase C). Finally, both arachidonic acid and PLA2 inhibit plasminogen activator (PA) activity in a dose-dependent fashion, whereas activation of protein kinase C with PMA stimulates PA activity. Taken together, the data suggest that the effects of arachidonic acid in granulosa cells can occur independently of protein kinase C activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

p38 MAP kinase is involved in the signalling of sphingosine in osteoblasts: sphingosine inhibits prostaglandin F(2alpha)-induced phosphoinositide hydrolysis

We previously showed that sphingosine inhibits prostaglandin F(2alpha) (PGF(2alpha))-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of sphingosine on phospholipase C-catalyzing phosphoinositide hydrolysis induced by PGF(2alpha) in these cells. Sphingosine inhibited the inositol phosphates formation by PGF(2alpha) or NaF, a GTP-binding protein activator. Sphingosine induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase but did not affect the phosphorylation of p42/p44 MAP kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, rescued the inhibitory effect of sphingosine on the formation of inositol phosphates by PGF(2alpha) or NaF. These results indicate that sphingosine inhibits PGF(2alpha)-induced phosphoinositide hydrolysis by phospholipase C via p38 MAP kinase in osteoblasts.     

Activation of protein kinase C is coupled to prostaglandin E2 synthesis in swine granulosa cells

We have examined the role of phospholipid-sensitive calcium-dependent protein kinase (protein kinase C) in prostaglandin E₂ synthesis by monolayer cultures of swine granulosa cells. Specific phorbol ester derivatives known to activate protein kinase C significantly augmented the production of prostaglandin E₂. These stimulatory actions were dose and time-dependent, and could be abolished by the cyclooxygenase inhibitor, indomethacin, or the protein synthesis inhibitor, cycloheximide. Moreover, the rank order of potency of phorbol esters in enhancing prostaglandin E₂ production was concordant with that demonstrated for activation of protein kinase C. Phorbol ester in conjunction with the divalent cation ionophore, A23187, increased prostaglandin E₂ production synergistically. In addition, a non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol, also significantly enhanced prostaglandin E₂ biosynthesis. The stimulated synthesis of prostaglandin E₂ was confirmed by high-pressure liquid chromatographic purification of this radiolabeled metabolite of ³H-arachidonic acid, and by capillary gas chromatography high-resolution mass spectrometry. Thus, the present studies indicate that the protein kinase C effector pathway is functionally coupled to prostaglandin E₂ production in the swine granulosa cell.     

Steroidogenic enzyme content and progesterone induction by cyclic adenosine 3',5'-monophosphate-generating agents and prostaglandin F2 alpha in bovine theca and granulosa cells luteinized in vitro.

In vitro luteinization of bovine granulosa (LGC) and theca (LTC) cells was achieved by culturing cells with forskolin (10 microM) and insulin (2 micrograms/ml) for 9 days. This treatment induced the presence of cytochrome P450scc and adrenodoxin in both cell types, but to substantially higher levels in LGC than in LTC. Forskolin dose-dependently stimulated the secretion of progesterone and cAMP after 3 h of incubation in both cell types although LGC were less sensitive to this stimulation than were LTC. Only LTC were responsive to LH, in accordance with their higher LH/hCG binding capacity. Both prostaglandin F2 alpha (PGF2 alpha) and phorbol 12-myristate 13-acetate (TPA) increased progesterone production during 3 h incubation of LGC and LTC, and treatment with staurosporine (a protein kinase C inhibitor) reversed this effect. Neither TPA nor PGF2 alpha alone affected cAMP levels but each acted synergistically with forskolin to increase cAMP accumulation. These results indicate that 1) elevated progesterone output from LGC is related to steroidogenic enzyme level; 2) bovine LH (up to 100 ng/ml) does not provoke a response in LGC due to their low LH/hCG binding capacity; 3) cAMP-protein kinase A and protein kinase C pathways are both involved in progesterone production by LGC and LTC, possibly by enhancing cholesterol transport.     

Stimulation of phosphatidylcholine breakdown and diacylglycerol production by growth factors in Swiss-3T3 cells.

The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase.     

Thrombin exerts a dual effect on stimulated adenylate cyclase in hamster fibroblasts, an inhibition via a GTP-binding protein and a potentiation via activation of protein kinase C.

Previous studies in Chinese-hamster fibroblasts (CCL39 line) indicate that an important signalling pathway involved in thrombin's mitogenicity is the activation of a phosphoinositide-specific phospholipase C, mediated by a pertussis-toxin-sensitive GTP-binding protein (Gp). The present studies examine the effects of thrombin on the adenylate cyclase system and the interactions between the two signal transduction pathways. We report that thrombin exerts two opposite effects on cyclic AMP accumulation stimulated by cholera toxin, forskolin or prostaglandin E1. (1) Low thrombin concentrations (below 0.1 nM) decrease cyclic AMP formation. A similar inhibition is induced by A1F4-, and both thrombin- and A1F4- -induced inhibitions are abolished by pertussis toxin. (2) Increasing thrombin concentration from 0.1 to 10 nM results in a progressive suppression of adenylate cyclase inhibition and in a marked enhancement of cyclic AMP formation in pertussis-toxin-treated cells. A similar stimulation is induced by an active phorbol ester, and thrombin-induced potentiation of adenylate cyclase is suppressed by down-regulation of protein kinase C. Therefore, we conclude that (1) the inhibitory effect of thrombin on adenylate cyclase is the direct consequence of the activation of a pertussis-toxin-sensitive inhibitory GTP-binding protein (Gi) possibly identical with Gp, and (2) the potentiating effect of thrombin on cyclic AMP formation is due to stimulation of protein kinase C, as an indirect consequence of Gp activation. Our results suggest that the target of protein kinase C is an element of the adenylate cyclase-stimulatory GTP-binding protein (Gs) complex. At low thrombin concentrations, activation of phospholipase C is greatly attenuated by increased cyclic AMP, leading to predominance of the Gi-mediated inhibition.     

Production of prostaglandins by porcine preovulatory follicular tissues and their roles in intrafollicular function

Prostaglandin production in vitro by theca and granulosa cells isolated from prepubertal pig ovaries was quantified in order to investigate the role of prostaglandins in intrafollicular function. Prepubertal gilts were slaughtered without treatment (O h, control) or treated with 1000 IU pregnant mare's serum gonadotropin (PMSG) and slaughtered at 36 or 72 h, or at 75 h following treatment with 500 IU of hCG at 72 h. Theca and granulosa cells were isolated from preovulatory follicles and cultured for 24 h alone or with follicle-stimulating hormone (FSH) or luteinizing hormone (LH). In vitro accumulation of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) was measured by radioimmunoassay. On a per follicle basis theca produced more of each prostaglandin (approx. 10-fold) than granulosa at each stage of follicular development; production by each tissue type increased with development of the follicle, responding to administration of gonadotropin (PMSG) in vivo. Neither tissue type was generally responsive to further gonadotropin stimulation in vitro. However, production of PGE2 by granulosa cells was increased by addition of gonadotropin, particularly LH, in vitro, with the greatest response observed in tissue obtained at 36 and 72 h after PMSG. There were no functional correlates between prostaglandin production and steroidogenesis by either tissue type and we conclude that prostaglandins do not have an obligatory role in follicular steroidogenesis. However, these data provide additional circumstantial evidence for a role of PGE2 in granulosa cell luteinization, and possibly in ovulation. The data also indicate that prostaglandins derived from thecal tissue in relatively large quantities may play an important role in ovulation.     

Regulation of the corpus luteum by protein kinase C. II. Inhibition of lipoprotein-stimulated steroidogenesis by prostaglandin F2 alpha

This study was designed to examine the antisteroidogenic action of prostaglandin (PG) F2 alpha on ovine luteal cells in vitro. Purified populations of large and small steroidogenic luteal cells were treated with lipoproteins, luteinizing hormone (LH), and/or PGF2 alpha. To investigate the involvement of the protein kinase C (PKC) pathway in hormone action, luteal cells were made PKC-deficient by treatment for 12 h with 1 microM phorbol-12-myristate-13-acetate. Progesterone production by nonstimulated large and LH-stimulated small luteal cells was significantly increased by treatment with high- and low-density lipoprotein (HDL, 5-fold increase; LDL, 2-fold increase). PGF2 alpha inhibited (p less than 0.0001) progesterone production by HDL-stimulated large luteal cells in a dose-dependent manner, with 60 nM causing maximal inhibition. No effect of PGF2 alpha (20nM-20 microM) was found on production of progesterone by HDL-stimulated, PKC-deficient large cells or by LH- and HDL-stimulated small luteal cells. These results suggest that PGF2 alpha has a direct antisteroidogenic effect on the large luteal cell that is mediated through the PKC second messenger pathway.     

Tumor necrosis factor-alpha stimulates prostaglandin F2alpha secretion by bovine luteal cells via activation of mitogen-activated protein kinase and phospholipase A2 pathways

It has been well demonstrated that tumor necrosis factor-alpha (TNFalpha) stimulates prostaglandin (PG) F2alpha secretion by bovine corpus luteum (CL) in vitro. The objective of the present study was to clarify the intracellular signaling pathway of TNFalpha to stimulate PGF2alpha production in cultured bovine luteal cells. Bovine luteal cells that were obtained from mid- (days 8-12 after ovulation) CL were incubated with TNFalpha (0.6 nM) and/or various compounds as follows: U-73122 (an inhibitor of phospholipase [PL] C), ACA (an inhibitor of PL-A2), H-89 (an inhibitor of protein kinase [PK] A), calphostin C (an inhibitor of PK-C), L-NAME/L-NORG (inhibitors of nitric oxide synthase), and PD98059 (an inhibitor of mitogen-activated protein kinase [MAPK] kinase). Although U-73122 (0. 1-10 microM), H-89 (0.1-10 microM), calphostin C (0.01-1 microM) and L-NAME/L-NORG (1-100 microM) did not affect TNFalpha-induced PGF2alpha secretion by the cultured cells, ACA (1-100 microM) and PD98059 (0.1-100 microM) inhibited TNFalpha-stimulated PGF2alpha secretion by the cells in a dose-dependent fashion (P < 0.05 or lower). These findings suggest that TNFalpha activates the MAPK and PL-A2 pathways in bovine luteal cells to stimulate PGF2alpha secretion.     

Prostaglandin production by the largest preovulatory follicles in the domestic hen (Gallus domesticus)

An injection of 5 micrograms of gonadotropin-releasing hormone (GnRH) into hens 8 h prior to oviposition advanced the expected time of oviposition by approximately 1 h. The plasma concentration of progesterone increased approximately 1 h earlier in GnRH-injected hens in comparison to saline-injected hens. The plasma concentration of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) increased significantly (p less than 0.05) at the time of oviposition in both the GnRH- and saline-injected hens. Significantly (p less than 0.05) greater concentrations of prostaglandin F2 alpha (PGF2 alpha) were assayed in media containing the largest preovulatory follicles collected at oviposition than in media containing the second and fifth largest preovulatory follicles collected at the same time. No prostaglandin was detected in media containing small, nonhierarchial follicles. The concentration of PGF2 alpha in media containing granulosa cells from the largest preovulatory follicle was significantly greater (p less than 0.05) than in media containing 4 times as many theca cells. Ovine luteinizing hormone (oLH) alone or in combination with arachidonic acid had no effect on PGF2 alpha output from granulosa cells collected 6 h before oviposition, whereas A23187 caused a small stimulation of PGF2 alpha output. However, treating cells first with oLH and then with A23187 stimulated a 15- to 20-fold increase in PGF2 alpha. None of these stimuli enhanced the already high output of PGF2 alpha when added to incubations of granulosa cells collected within 5 min after oviposition. These data suggest that the granulosa cells of the largest preovulatory follicle are the major intraovarian source of prostaglandin and that production of PGF2 alpha is associated with the preovulatory surges of gonadotropins and steroid hormones preceding oviposition.     

Involvement of p42/p44 mitogen-activated protein kinase in prostaglandin f(2alpha)-stimulated induction of heat shock protein 27 in osteoblasts

We previously reported that prostaglandin F(2alpha) (PGF(2alpha)) activates both phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells and then induces the activation of protein kinase C (PKC). In this study, we investigated the effect of PGF(2alpha) on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein, in these cells. PGF(2alpha) significantly induced the accumulation of HSP27 dose-dependently within the range of 10 nM to 10 microM. PGF(2alpha) stimulated the increase in the levels of mRNA for HSP27. A total of 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, induced the accumulation of HSP27. The stimulative effect of PGF(2alpha) was reduced in the PKC down-regulated cells. Calphostin C, a specific inhibitor of PKC, suppressed the PGF(2alpha)-induced HSP27 accumulation as well as that induced by TPA. HSP27 induction by PGF(2alpha) was reduced by U-73122, a phospholipase C inhibitor, or propranolol, a phosphatidic acid phosphohydrolase inhibitor. PGF(2alpha) and TPA stimulated p42/p44 mitogen-activated protein (MAP) kinase. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, suppressed the induction of HSP27 stimulated by PGF(2alpha) or TPA. PD98059 and calphostin C reduced the levels of mRNA for HSP27 increased by PGF(2alpha). These results indicate that PGF(2alpha) stimulates the induction of HSP27 via p42/p44 MAP kinase activation, which depends on upstream PKC activation in osteoblasts.     

Prostacyclin as a potent effector of adipose-cell differentiation.

The terminal differentiation of Ob1771 pre-adipose cells induced by arachidonic acid in serum-free hormone-supplemented medium containing insulin, transferrin, growth hormone, tri-iodothyronine and fetuin (5F medium) was strongly diminished in the presence of inhibitors of prostaglandin synthesis, namely aspirin or indomethacin. Carbaprostacyclin, a stable analogue of prostacyclin (prostaglandin I2) known to be synthesized by pre-adipocytes and adipocytes, behaved as an efficient activator of cyclic AMP production and was able, when added to 5F medium, to mimic the adipogenic effect of arachidonic acid. Prostaglandins E2, F2 alpha and D2, unable to affect the cyclic AMP production, failed to substitute for carbaprostacyclin. However, prostaglandin F2 alpha, which is another metabolite of arachidonic acid in pre-adipose and adipose cells, able to promote inositol phospholipid breakdown and protein kinase C activation, potentiated the adipogenic effect of carbaprostacyclin. In addition, carbaprostacyclin enhanced both a limited proliferation and terminal differentiation of adipose precursor cells isolated from rodent and human adipose tissues maintained in primary culture. These results demonstrate the critical role of prostacyclin and prostaglandin F2 alpha on adipose conversion in vitro and suggest a paracrine/autocrine role of both prostanoids in the development of adipose tissue in vivo.     

The role of inositol lipid metabolism in the ovary

Two major signal transduction systems operate within ovarian cells to control their function. Gonadotropins, such as follicle-stimulating hormone and luteinizing hormone, primarily utilize the cyclic adenosine 3',5'-monophosphate (cAMP) pathway to stimulate steroid hormone biosynthesis. On the other hand, an inositol lipid metabolism pathway is used by other effector molecules such as gonadotropin-releasing hormone or prostaglandin F2 alpha, as well as gonadotropins, to alter ovarian hormone production. Membrane polyphosphoinositides are hydrolyzed to inositol phosphates and diacylglycerol, resulting in alterations of intracellular free calcium concentration, activation of protein kinase C, and liberation of arachidonic acid. Some or all of these intracellular messengers may interact with the gonadotropin-induced cAMP pathway to control ovarian function.     

Involvement of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in prostaglandin F2alpha-induced heat shock protein 27 in osteoblasts

We have reported that prostaglandin F2(alpha) (PGF2(alpha)) activates p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells, and that p44/p42 MAP kinase plays a role in the PGF2(alpha)-induced heat shock protein 27 (HSP27). In the present study, we investigated the involvement of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), a member of the MAP kinase superfamily, in PGF2(alpha)-induced HSP27 in MC3T3-E1 cells. PGF2(alpha) time dependently induced the phosphorylation of SAPK/JNK. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced the PGF2(alpha)-stimulated HSP27 accumulation. The inhibitory effect of SP600125 was dose dependent in the range between 0.1 and 30 microM. SP600125 reduced the PGF2(alpha)-increased level of HSP27 mRNA. SP600125 suppressed the phosphorylation of SAPK/JNK induced by PGF2(alpha), but did not affect the PGF2(alpha)-induced phosphorylation of p44/p42 MAP kinase. On the other hand, PD98059, a specific inhibitor of the upstream kinase of p44/p42 MAP kinase, which reduced the phosphorylation of p44/p42 MAP kinase stimulated by PGF2(alpha), had little effect on the PGF2(alpha)-induced phosphorylation of SAPK/JNK. These results strongly suggest that SAPK/JNK plays a part in PGF2(alpha)-induced HSP27 in addition to p44/p42 MAP kinase in osteoblasts.     

Interleukin (IL)-17 enhances prostaglandin F(2 alpha)-stimulated IL-6 synthesis in osteoblasts

We previously showed that prostaglandin F(2alpha) (PGF(2alpha)) and endothelin-1 (ET-1) induce interleukin (IL)-6 through the activation of protein kinase C-dependent p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. It has recently been reported that tumor necrosis factor-alpha-induced IL-6 synthesis is amplified by IL-17 in these cells. In the present study, we investigated the effect of IL-17 on the IL-6 synthesis stimulated by PGF(2alpha) in MC3T3-E1 cells. IL-17 significantly enhanced the PGF(2alpha)-induced IL-6 synthesis in a dose-dependent manner in the range between 0.1 and 10 ng/ml. IL-17 also enhanced the IL-6 synthesis stimulated by 12- O -tetradecanoylphorbol-13-acetate, a direct activator of protein kinase C. In addition, IL-17 amplified the IL-6 synthesis induced by ET-1. However, IL-17 hardly affected the phosphorylation of p44/p42 MAP kinase induced by PGF(2alpha) or ET-1. These results strongly suggest that IL-17 enhances the IL-6 synthesis stimulated by PGF(2alpha) as well as ET-1 in osteoblasts, and that the effect is exerted at a point downstream from p44/p42 MAP kinase.     

Zinc suppresses IL-6 synthesis by prostaglandin F2alpha in osteoblasts: inhibition of phospholipase C and phospholipase D

We previously reported that prostaglandin F2alpha (PGF2alpha) induces phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein, resulting in the activation of protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells and that PGF2alpha stimulates the synthesis of interleukin-6 (IL-6) via PKC-dependent p44/p42 mitogen-activated protein (MAP) kinase activation. In the present study, we investigated whether zinc affects the PGF2alpha-induced IL-6 synthesis in these cells. Zinc complex of l-carnosine (l-CAZ) dose-dependently suppressed the PGF2alpha-stimulated IL-6 synthesis. In addition, zinc alone reduced the IL-6 synthesis. L-CAZ suppressed the PGF2alpha-induced p44/p42 MAP kinase phosphorylation. However, the p44/p42 MAP kinase phosphorylation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of PKC, or NaF, a direct activator of GTP-binding protein, was not affected by l-CAZ. l-CAZ reduced the PGF2alpha-stimulated formation of inositol phosphates and choline. However, l-CAZ did not affect the formation of inositol phosphates or choline induced by NaF. These results strongly suggest that zinc reduces PGF2alpha-induced IL-6 synthesis via suppression of phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in osteoblasts.