Regulation of mRNA expression of 3 beta-hydroxy-5-ene steroid dehydrogenase in porcine granulosa cells in culture: a role for the protein kinase-C pathway

We studied the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase-C, on porcine granulosa cells in culture. PMA as well as cholera toxin, forskolin, and hCG increased cAMP accumulation. PMA further augmented the elevation in cAMP accumulation induced by cholera toxin, forskolin, and hCG. In the same cell culture model, hCG induced a time-dependent increase in the 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) mRNA levels with a maximal 3-fold stimulation obtained at 8-16 h of incubation with 1 IU hCG/ml. PMA inhibited the increase in 3 beta HSD mRNA levels induced by hCG in a dose-dependent manner. The phorbol ester also inhibited the increase in 3 beta HSD mRNA levels stimulated by LH as well as cholera toxin and forskolin and the cAMP analogs (Bu)2cAMP and 8-bromo-cAMP. Activation of protein kinase-C by mezerein similarly inhibited hCG stimulation of 3 beta HSD mRNA levels. The present data indicate that activation of the protein kinase-C pathway induces generation of cAMP, but causes a near-complete inhibition of the stimulatory effects of hCG, LH, forskolin, cholera toxin, and cAMP analogs on 3 beta HSD mRNA levels in porcine granulosa cells in culture.     

Differential regulation of hCG alpha and beta subunit mRNAs in JEG-3 choriocarcinoma cells by 8-bromo-cAMP

The coordinate regulation of human chorionic gonadotropin (hCG) subunit synthesis by JEG-3 choriocarcinoma cells was studied at the pretranslational level. The responses of the hCG alpha and beta mRNAs were measured during stimulation with the potent cAMP analog 8-bromo-cAMP (8-Br-cAMP) using 32P-labeled hCG alpha and beta cDNA probes. The hCG alpha mRNA (850 bases) and beta mRNA (1050 bases) from JEG-3 cells were identical in size to that of their respective mRNAs from placenta, by Northern blot analysis. After 48 h of stimulation with 2 mM 8-Br-cAMP, production of immunoreactive alpha and beta subunits increased 25- and 52-fold, respectively; corresponding levels of the alpha and beta mRNAs increased 36- and 43-fold, respectively, in a dot blot hybridization assay. Total cellular protein, DNA content, and messenger RNA pools were not altered by treatment with 8-Br-cAMP. The temporal coordination of the expression of the hCG alpha- and beta-subunit genes was examined by comparing the time course of stimulation of the respective mRNAs and the production of immunoreactive subunits. The kinetic responses of the alpha and beta mRNAs differed: the increase in hCG alpha mRNA preceded the increase in hCG beta mRNA, while levels of free alpha subunit and intact hCG increased in parallel with the increase in beta mRNA. hCG alpha mRNA levels increased rapidly between 8 and 24 h after the addition of 8-Br-cAMP, and approached a plateau by 48 h. The levels of hCG beta mRNA increased steadily throughout the 8-48 h period. These results demonstrate that the cAMP analog 8-Br-cAMP differentially regulates hCG subunit biosynthesis in JEG-3 cells at a pretranslational level, and that the stimulation by 8-Br-cAMP in this system appears to be relatively selective for hCG subunits.     

Stimulatory and inhibitory effects of protein kinase C activation and calcium ionophore on cultured pig Leydig cells

The acute and the long-term (24 h) effects of protein kinase C activators, phorbol 12 myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol, and the calcium ionophore A23187 on cultured pig Leydig cell functions were investigated. None of these drugs modified basal cAMP production, but they induced a small (3-4-fold) increase in testosterone secretion. The stimulatory effects of human choriogonadotropin (hCG; 1 nM) on both cAMP and testosterone productions were inhibited by short-term incubation with these drugs. In addition, they suppressed the stimulation of testosterone output by forskolin and 8-bromo-adenosine 3',5'-monophosphate, whereas the forskolin-dependent cAMP production was unaffected. The inhibitory effects of PMA on hCG stimulation of both cAMP and testosterone were due mainly to a decrease of the Vmax without modification of the ED50. Moreover, PMA did not modify the binding of 125I-hCG. Pretreatment of Leydig cells with the three drugs for 24 h induced more pronounced modifications, such as a reduction in the number of hCG binding sites and a decreased responsiveness to hCG and forskolin, the testosterone production being drastically reduced. The effects of PMA were dose- and time-dependent; however, the concentration of PMA required to induce half-maximal effects on hCG receptors (10 nM) was about one order of magnitude higher than those required to reduce cAMP and testosterone productions. Further, the inhibitory effects on cAMP and testosterone secretions appeared within the first 3 h, whereas the hCG receptor number remained constant for at least 8 h. It appears therefore, that the main alteration responsible for the steroidogenic refractoriness of PMA-treated Leydig cells is located beyond cAMP formation. Moreover, since conversion of exogenous pregnenolone to testosterone by control and PMA-treated cells was similar, the alteration was probably located before pregnenolone formation. Kinetic studies with 125I-hCG showed that the rate of internalization of the hormone-receptor complexes was similar in control cells and in PMA-treated cells, suggesting that the decline in receptor number observed in the latter group after an 8-h delay is not due to an increased rate of internalization nor to sequestration of the internalized receptors inside the cells. Since cycloheximide blocked the effects of PMA on hCG down-regulation, it is likely that the phorbol esters and 1-oleoyl-2-acetyl-sn-glycerol induce the synthesis of some proteins which blocked the recycling of internalized receptors. A similar hypothesis has been put forward recently to explain the hCG-induced down regulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein kinase C and the stimulation of tissue plasminogen activator release from human endothelial cells. Dependence on the elevation of messenger RNA

Tumor-promoting phorbol esters stimulate tissue plasminogen activator (tPA) release from human endothelial cells, and simultaneous elevation of cyclic AMP potentiates this response 5-fold (Santell, L., and Levin, E. G. (1988) J. Biol. Chem. 263, 16802-16808). A similar effect on tPA mRNA was observed, with phorbol myristate acetate inducing a 3.5-fold increase in steady state tPA mRNA levels and forskolin enhancing that increase to 25-fold. Peak levels occurred at 8 h after agonist addition and returned to baseline levels by 16 h. As was found with tPA antigen secretion, delayed addition of forskolin reduced the level of potentiation, and, at 6 h after phorbol 12-myristate 13-acetate (PMA), forskolin was no longer effective. The protein synthesis inhibitor cycloheximide did not inhibit the rise in tPA mRNA levels in response to PMA/forskolin nor the decline in mRNA levels between 8 and 12 h. However, peak levels (8 h) were approximately 1.5-fold higher than in cultures not treated with cycloheximide. The effect of two inhibitors of protein kinases, H-7 and staurosporine, on PMA-induced tPA antigen secretion and tPA mRNA levels were examined. H-7 and staurosporine inhibited PMA, and PMA/forskolin induced tPA secretion in a dose-dependent manner. This effect was time-dependent; the inhibitory effect was reduced with delayed H-7 addition, and, by 6 h after PMA treatment, no inhibition was observed. H-7 and staurosporine also inhibited the PMA/forskolin-induced increase in tPA mRNA levels and were less effective the later they were added. The same time-dependent effect on the potentiation of PMA-induced tPA mRNA levels by forskolin was observed. Again, delayed addition reduced the effect, and, by 6 h, potentiation was absent. The results of this study indicate that changes in mRNA levels in response to PMA and PMA/forskolin precede and determine those that occur to tPA antigen secretion. In addition, the maximal response is dependent upon the prolonged activation of an H-7- and cAMP-sensitive pathway.     

Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles.     

The cAMP pathway promotes sirtuin-1 expression in human granulosa-lutein cells

Sirtuin-1 (SIRT1), a NAD+-dependent deacetylase, is present in the ovarian granulosa cells (GCs) of various species. This study examined the regulation of SIRT1 expression in human granulosa-lutein cells (hGLCs). Two different, structurally unrelated SIRT1 activators, SRT2104 and resveratrol, dose- and time-dependently enhanced SIRT1 (∼2- and 1.5-fold increase at 50 μmol/L for mRNA and protein levels, respectively), whereas EX-527, an inhibitor of SIRT1 deacetylase activity, significantly suppressed SIRT1 protein induced by these activators. Transfecting cells with SIRT1 siRNA molecules efficiently silenced SIRT1 (∼70 % decrease in 48 h post-transfection). Furthermore, the stimulatory effects of SRT2104 on SIRT1 expression observed in non-transfected or in scrambled siRNA-transfected cells were diminished with SIRT1 silencing. The findings described above imply that SIRT1 autoregulates its own expression. Interestingly, SRT2104 elevated cAMP accumulation (1.4-fold) in the culture media of hGLCs which was further augmented in the presence of hCG (2.2-fold); these effects were evident after 12 h of incubation. This additive effect of hCG and SRT2104 on cAMP accumulation may explain the incremental outcome observed on SIRT1 expression (∼3-fold increase from basal level and ∼1.6-fold stimulation for each compound alone) with these two compounds. SIRT1 knockdown diminished SIRT1 induced by forskolin, providing additional evidence that cAMP promotes SIRT1. These findings imply that by activating adenylyl cyclase (hCG or forskolin) and inhibiting phosphodiesterases (SIRT1 activators), these two signals converge to produce an incremental, positive feedback loop on SIRT1 expression. Such a mechanism highlights the importance of maintaining high SIRT1 levels in human luteinized GCs.     

Inhibition of hCG-stimulated adenylate cyclase in purified mouse Leydig cells by the phorbol ester PMA

The tumour-promoting phorbol ester, PMA (phorbol 12-myristate 13-acetate), markedly reduced the steroidogenic response of mouse Leydig cells to stimulation by hCG and cholera toxin. However, 8Br-cAMP-and forskolin-stimulated steroidogenesis was not inhibited by PMA. PMA did not inhibit hCG-induced steroidogenesis in the simultaneous presence of 1 microM forskolin. The analysis of intracellular cAM P indicated that the PMA-induced inhibition of steroidogenesis was the result of an impaired cAMP accumulation. Adenylate cyclase in membranes prepared from PMA-treated cells showed a diminished response to hCG, GTP, guanosine 5'-[beta, gamma-imido]triphosphate [Gpp(NH)p] or to a combination of the stimulants. PMA, however, was unable to inhibit adenylate cyclase when added directly to the membrane preparation from untreated cells. As previous observations have indicated that 125I-hCG binding and phosphodiesterase activity in mouse Leydig cells are not influenced by PMA, it is concluded from the present study that the site of inhibition has to be localised to the regulatory guanine nucleotide binding protein of the adenylate cyclase system.     

Polyamines control human chorionic gonadotropin production in the JEG-3 choriocarcinoma cell

The effect of inhibition of ornithine decarboxylase with difluoromethylornithine (DFMO) and the resultant lowering of polyamine levels upon human chorionic gonadotropin (hCG) production in JEG-3 choriocarcinoma cells was investigated. DFMO (10 mM) totally inhibited ornithine decarboxylase activity. In DFMO-treated cells, cellular spermidine concentrations fell to nondetectable levels (less than 1% of control values) within 24 h and spermine concentrations were reduced to 41.9% of controls over 6 days. DFMO caused a 70-80% inhibition of hCG production. Levels of mRNA for both the alpha and beta subunits of hCG were also inhibited relative to mRNA for tubulin. Exogenous putrescine normalized hCG production in a dose-dependent manner. Other diamines, including cadaverine, 1,3-diaminopropane, 1,6-diaminohexane, and 1,7-diaminoheptane, were ineffective in reestablishing hCG production in DFMO-treated cells. Dibutyryl cAMP (1 mM) stimulated hCG production and increased levels of mRNA for the alpha and beta subunit 5-40-fold in both DFMO-treated and control cells. Polyamines appear to have a fundamental role in hCG production in JEG-3 choriocarcinoma cells. However, dibutyryl cAMP can partially overcome or circumvent the requirement for polyamines in hCG biosynthesis.     

Activation of protein kinase C potentiates cyclic AMP production and stimulates steroidogenesis in differentiated ovarian granulosa cells

Gonadotropin-stimulated steroidogenesis in the differentiating ovarian granulosa cell is mediated through the activation of cAMP-dependent protein kinase, and is also modulated by calcium-dependent mechanisms. Granulosa cells contain calcium-activated, phospholipid-dependent protein kinase (C kinase), and show an increase in phosphatidylinositol turnover in response to GnRH agonist analogs. To evaluate the role of C kinase in ovarian steroidogenesis, the potent phorbol ester, TPA, and the permeant diacylglycerol, OAG, were used to activate C kinase in granulosa cells from PMSG-treated immature rats. Both TPA and OAG caused dose-dependent stimulation of progesterone production without affecting intra- or extracellular cAMP levels. However, the maximum steroid responses to these compounds were less than those stimulated by cAMP. The ED50 for TPA-stimulated progesterone production was 3 nM, which is close to the known Km for activation of C kinase. Stimulation of steroidogenesis was only observed with biologically-active phorbol esters and permeant diacylglycerols such as OAG and DOG. Exposure of granulosa cells to phospholipase C also increased progesterone production in a dose-dependent manner without changing the cAMP content. Although TPA and OAG did not increase basal cAMP production, both agents enhanced the cAMP responses stimulated by hCG and forskolin; likewise, phospholipase C alone did not change cAMP production but caused a dose-dependent increase in the cAMP responses to hCG and forskolin. These results demonstrate that activation of C kinase promotes steroidogenesis in ovarian granulosa cells, and potentiates the activation of adenylate cyclase by hCG and forskolin. Such findings support the possibility that the calcium, phospholipid-dependent enzyme could be involved in the regulation of progesterone production by hormonal ligands such as gonadotropins and GnRH.     

Cyclic adenosine monophosphate and phorbol ester, like gonadotropin-releasing hormone, stimulate the biosynthesis of luteinizing hormone polypeptide chains in a nonadditive manner

We have previously reported that GnRH stimulates the synthesis of both the alpha- and beta-polypeptide chains of LH. In the present study, in order to investigate the mechanisms involved in the GnRH regulation of LH subunit synthesis, we have explored the effects of cAMP and a phorbol ester [12-O-tetradecanoyl phorbol 13-acetate (TPA)] using anterior pituitary cells in primary culture incubated in the presence of [35S]methionine. The radioactivity incorporated into alpha and LH beta immunologically related polypeptides was measured after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled material immunoextracted from cells and media with specific antisera. The cAMP analog 8-Br-cAMP (at concentrations 0.25-2 mM), the cholera toxin (6-60 nM), and forskolin (10-100 microM) induced, like GnRH, an increase in the [35S]methionine incorporation into alpha- and LH beta-subunits. On the other hand, TPA (50-100 nM) also enhanced the synthesis of LH subunits. After a 5-h incubation in the presence of GnRH, 8-Br-cAMP, and TPA in different combinations, no cumulative effect was observed. These results demonstrate that intracellular cAMP and TPA are potent activators of both alpha- and LH beta-polypeptide chain synthesis, suggesting that cAMP as well as diacylglycerols may act as intracellular mediators of the GnRH effect on LH subunit synthesis.     

Up-regulation of thrombomodulin in human umbilical vein endothelial cells in vitro

Previous studies have shown that thrombomodulin (TM) on endothelial cells is down-regulated by endotoxin, interleukin-1 beta (IL-1 beta), and tumor necrosis factor (TNF). This loss of anti-coagulant potential is thought to be related to the hypercoagulable state in sepsis, inflammation, and cancer. The current studies describe up-regulation of TM in human umbilical vein endothelial cells (HUVECs) by several compounds as judged by increased surface cofactor activity, surface TM antigen, and TM mRNA levels. Surface TM activity was increased by active phorbol esters (10(-8) M, 24-48 h), analogs of cAMP (1-10 mM, 4 h), and forskolin (10(-5) M, 24-48 h). Up-regulation of TM in HUVECs by 4 beta-phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (dBcAMP) was due to de novo synthesis of TM protein resulting from increased TM mRNA levels. The results suggest that protein kinase C and protein kinase A may be involved in cellular regulatory mechanisms for TM expression. In addition, PMA effects on surface TM activity are biphasic, with an initial reduction followed by a significant enhancement. Hence, we propose that compounds capable of increasing intracellular cAMP concentrations in HUVECs may be useful in preventing thrombosis by increasing the anti-thrombotic properties of endothelial cells.