Signal pathways involved in the regulation of prostaglandin E synthase-1 in human gingival fibroblasts

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme regulating the synthesis of prostaglandin E2 (PGE2) in inflammatory conditions. In this study we investigated the regulation of mPGES-1 in gingival fibroblasts stimulated with the inflammatory mediators interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNFalpha). The results showed that IL-1beta and TNFalpha induce the expression of mPGES-1 without inducing the expression of early growth response factor-1 (Egr-1). Treatment of the cells with the PLA2 inhibitor 4-bromophenacyl bromide (BPB) decreased the cytokine-induced mPGES-1 expression accompanied by decreased PGE2 production whereas the addition of arachidonic acid (AA) upregulated mPGES-1 expression and PGE2 production. The protein kinase C (PKC) activator PMA did not upregulate the expression of mPGES-1 in contrast to COX-2 expression and PGE2 production. In addition, inhibitors of PKC, tyrosine and p38 MAP kinase markedly decreased the cytokine-induced PGE2 production but not mPGES-1 expression. Moreover, the prostaglandin metabolites PGE2 and PGF2alpha induced mPGES-1 expression as well as upregulated the cytokine-induced mPGES-1 expression indicating positive feedback regulation of mPGES-1 by prostaglandin metabolites. The peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), decreased mPGES-1 expression but not COX-2 expression or PGE2 production. The results indicate that the inflammatory-induced mPGES-1 expression is regulated by PLA2 and 15d-PGJ2 but not by PKC, tyrosine kinase or p38 MAP kinase providing new insights into the regulation of mPGES-1.     

Differing signal requirements for the activation of macrophages from C3H/HeJ and C3H/OuJ mice

Abstract Endotoxin-associated protein (EP) from Salmonella typhi stimulated the release of prostaglandin E2 (PGE2), interleukin-1 (IL-1), and interferon (IFN) activity in macrophages from the lipopolysaccharide (LPS) responder C3H/OuJ mouse strain. However, only PGE2 and IL-1 were stimulated by EP in macrophages from the LPS nonresponder C3H/HeJ mouse strain. LPS stimulated the release of PGE2, IL-1 and IFN activity in C3H/OuJ macrophages, but not in C3H/HeJ macrophages. The protein kinase C (PKC) activator phorbol myristic acid (PMA) stimulated PGE2 production in both strains but not IL-1 production, suggesting that signalling pathways other than PKC may be involved in IL-1 production. The calcium ionophore ionomycin stimulated PGE2 production in C3H/OuJ but not C3H/HeJ macrophages, suggesting a defective calcium-related pathway in the C3H/HeJ macrophages as compared to the C3H/OuJ 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).     

The effect of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 on chorioamnion secretion of prostaglandins (PG)F 2 alpha and E2 in pigs

The aim of the present study was to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on prostaglandin (PG)F(2 alpha) and PGE(2) secretion as well as cyclooxygenase-2 (COX-2) protein expression in chorioamnion collected on days 25, 30 and 40 of pregnancy in pigs. Fetal membrane slices were incubated for 16 h with TNF-alpha, IL-1 beta, IL-6 (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of TNF-alpha, IL-1 beta and/or IL-6 on PGF(2 alpha) and PGE(2) secretion by the porcine fetal membranes. The medium content of these PGs depended on the cytokine type, treatment dose and day of pregnancy. Cytokine stimulation of PGE(2) was more pronounced than that of PGF(2 alpha). In addition, an increase in PGF(2 alpha) and/or PGE(2) secretion was usually associated with an augmentation of COX-2 protein expression. Our results support the notion concerning the possible role of cytokines in modulating production of PGs by fetal membranes during the first trimester of gestation.     

Oxytocin stimulates prostaglandin F2alpha secretion from porcine endometrial cells through activation of calcium-dependent protein kinase C

The mechanism for oxytocin's (OT) stimulation of PGF2alpha secretion from porcine endometrium is not clear, but is thought to involve mobilization of intracellular Ca2+ and subsequent activation of protein kinase C (PKC). This study determined: (1) if mobilization of inositol trisphosphate-sensitive Ca2+ by thapsigargin or activation of PKC by phorbol 12-myristate 13-acetate (PMA) could stimulate PGF2alpha release from luminal epithelial, glandular epithelial and stromal cells of porcine endometrium and (2) if inhibitors of various PKC isotypes could attenuate the ability of OT, thapsigargin and PMA to stimulate PGF2alpha secretion from these cells. Thapsigargin and PMA each stimulated (P < 0.01) PGF2alpha secretion from all three endometrial cell types examined. However, the effects of thapsigargin and PMA were synergistic (P < 0.05) only in stromal cells. Three protein kinase C inhibitors (i.e. G?6976, G?6983 and Ro-31-8220) differentially attenuated (P < 0.05) the ability of OT, thapsigargin and PMA to stimulate PGF2alpha release. These results are consistent with the hypothesis that OT mobilizes Ca2+ to activate a Ca2+-dependent PKC pathway to promote PGF2alpha secretion from porcine endometrial cells. The differing pattern of response to isotype-specific inhibitors of PKC among cell types suggests that distinct PKC isoforms are differentially expressed in luminal epithelial, glandular epithelial and stromal cells.     

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.     

Oxytocin stimulates prostaglandin F(2alpha) secretion from porcine endometrial cells through activation of calcium-dependent protein kinase C*

The mechanism for oxytocin's (OT) stimulation of PGF(2alpha) secretion from porcine endometrium is not clear, but is thought to involve mobilization of intracellular Ca(2+) and subsequent activation of protein kinase C (PKC). This study determined: (1) if mobilization of inositol trisphosphate-sensitive Ca(2+) by thapsigargin or activation of PKC by phorbol 12-myristate 13-acetate (PMA) could stimulate PGF(2alpha) release from luminal epithelial, glandular epithelial and stromal cells of porcine endometrium and (2) if inhibitors of various PKC isotypes could attenuate the ability of OT, thapsigargin and PMA to stimulate PGF(2alpha) secretion from these cells. Thapsigargin and PMA each stimulated (P < 0.01) PGF(2alpha) secretion from all three endometrial cell types examined. However, the effects of thapsigargin and PMA were synergistic (P < 0.05) only in stromal cells. Three protein kinase C inhibitors (i.e. G?6976, G?6983 and Ro-31-8220) differentially attenuated (P < 0.05) the ability of OT, thapsigargin and PMA to stimulate PGF(2alpha) release. These results are consistent with the hypothesis that OT mobilizes Ca(2+) to activate a Ca(2+)-dependent PKC pathway to promote PGF(2alpha) secretion from porcine endometrial cells. The differing pattern of response to isotype-specific inhibitors of PKC among cell types suggests that distinct PKC isoforms are differentially expressed in luminal epithelial, glandular epithelial and stromal cells.     

Down-regulation of oxytocin-induced cyclooxygenase-2 and prostaglandin F synthase expression by interferon-tau in bovine endometrial cells

Oxytocin (OT) is responsible for the episodic release of luteolytic prostaglandin (PG) F2alpha from the uterus in ruminants. The attenuation of OT-stimulated uterine PGF2alpha secretion by interferon-tau (IFN-tau) is essential for prevention of luteolysis during pregnancy in cows. To better understand the mechanisms involved, the effect of recombinant bovine IFN-tau (rbIFN-tau) on OT-induced PG production and cyclooxygenase-2 (COX-2) and PGF synthase (PGFS) expression in cultured endometrial epithelial cells was investigated. Cells were obtained from cows at Days 1-3 of the estrous cycle and cultured to confluence in RPMI medium supplemented with 5% steroid-free fetal calf serum. The cells were then incubated in the presence or absence of either 100 ng/ml OT or OT+100 ng/ml rbIFN-tau for 3, 6, 12, and 24 h. OT significantly increased PGF2alpha and PGE2 secretion at all time points (p < 0.01), while rbIFN-tau inhibited the OT-induced PG production and reduced OT receptor binding in a time-dependent manner. OT increased the steady-state level of COX-2 mRNA, measured by Northern blot, which was maximal at 3 h (9-fold increase) and then decreased with time (p < 0.01). OT also caused an increase in COX-2 protein, which peaked at 12 h (11-fold increase), as measured by Western blot. Addition of rbIFN-tau suppressed the induction of COX-2 mRNA (89%, p < 0.01) and COX-2 protein (50%, p < 0.01) by OT. OT also increased PGFS mRNA, and this stimulation was attenuated by rbIFN-tau (p < 0.01). To ensure that the decrease in COX-2 was not solely due to down-regulation of the OT receptor, cells were stimulated with a phorbol ester (phorbol 12-myristate 13-acetate; PMA) in the presence and absence of rbIFN-tau. The results showed that rbIFN-tau also decreased PMA-stimulated PG production and COX-2 protein. It can be concluded that rbIFN-tau inhibition of OT-stimulated PG production is due to down-regulation of OT receptor, COX-2, and PGFS.     

Phorbol ester-induced expression of the common, low-affinity binding site for primary prostanoids in vascular smooth muscle cells

We showed in an earlier study (Hanasaki, K., and Arita, H. (1989) Biochim. Biophys. Acta 1013, 28-35) that there is a common, low-affinity binding site for primary prostanoids in cultured vascular smooth muscle cells (VSMC). This site, called the "primary prostaglandin (PG) site," can be evaluated by radioreceptor assay using [3H]PGF2 alpha and [3H]PGE1. Comparison of the capacity of several PGF2 alpha analogs to displace both radioligand bindings indicated strict requirements of the 15-hydroxy group as well as the 13,14-double bond in the omega-side chain of prostaglandins for recognition of this site. Treatment of VSMC with phorbol 12-myristate 13-acetate (PMA), a known protein kinase C activator, led to concentration- and time-dependent increases in the binding activities of [3H] PGF2 alpha as well as [3H]PGE1, which could be completely suppressed by the addition of protein kinase C inhibitor, H-7. The PMA effects could be mimicked by phorbol 12,13-dibutylate, but not by inactive phorbol ester. Scatchard analyses revealed an approximately 8-fold increase in the binding density with unaltered binding affinity after PMA treatment. This expression of the primary PG site was blocked by the addition of cycloheximide and actinomycin D. In contrast, PMA did not affect the binding activity for the thromboxane A2/prostaglandin H2 receptor in VSMC. These results suggest that the expression of the primary PG site is regulated by a protein kinase C-dependent mechanism in VSMC.     

Involvement of tyrosine kinases on cyclooxygenase expression and prostaglandin E2 production in human gingival fibroblasts stimulated with interleukin-1beta and epidermal growth factor

The purpose of the present study was to investigate the involvement of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and tyrosine kinase on prostaglandin E2 (PGE2) production in human gingival fibroblasts stimulated by interleukin-1beta (IL-1beta) and/or epidermal growth factor (EGF). The cytokine IL-1beta and to a lesser extent EGF, enhanced COX-2 mRNA levels in gingival fibroblasts. Simultaneous treatment with EGF and IL-1beta resulted in enhanced COX-2 mRNA levels accompanied by a synergistic stimulation of PGE2 biosynthesis compared to the cells treated with IL-1beta or EGF alone. Neither IL-1beta EGF nor the combination of IL-1beta and EGF enhanced COX-1 mRNA levels in gingival fibroblasts. The tyrosine kinase inhibitors, Herbimycin A and PD 153035 hydrochloride, reduced COX-2 mRNA levels as well as PGE2 production induced by IL-1beta or the combination of IL-1beta and EGF whereas COX-1 mRNA levels were not affected. Furthermore, the COX-2 specific inhibitor, NS-398, abolished the PGE2 production induced by IL-1beta, EGF, or the combination. These results indicate that the synergy between IL-1beta and EGF on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase(s) are involved in the signal transduction of COX-2 in gingival fibroblasts.     

The induction of cyclooxygenase-2 in IL-1beta-treated endothelial cells is inhibited by prostaglandin E2 through cAMP

Prostaglandins (PGs) have numerous cardiovascular and inflammatory effects. Cyclooxygenase (COX), which exists as COX-1 and COX-2 isoforms, is the first enzyme in the pathway in which arachidonic acid is converted to PGs. Prostaglandin E2 (PGE2) exerts a variety of biological activities for the maintenance of local homeostasis in the body. Elucidation of PGE2 involvement in the signalling molecules such as COX could lead to potential therapeutic interventions. Here, we have investigated the effects of PGE2 on the induction of COX-2 in human umbilical vein endothelial cells (HUVEC) treated with interleukin-1beta (IL-1beta 1 ng/ml). COX activity was measured by the production of 6-keto-PGF1alpha, PGE2, PGF2alpha and thromboxane B2 (TXB2) in the presence of exogenous arachidonic acids (10 microM for 10 min) using enzyme immunoassay (EIA). COX-1 and COX-2 protein was measured by immunoblotting using specific antibody. Untreated HUVEC contained only COX-1 protein while IL-1beta treated HUVEC contained COX-1 and COX-2 protein. PGE2 (3 microM for 24h) did not affect on COX activity and protein in untreated HUVEC. Interestingly, PGE2 (3 microM for 24h) can inhibit COX-2 protein, but not COX-1 protein, expressed in HUVEC treated with IL-1beta. This inhibition was reversed by coincubation with forskolin (100 microM). The increased COX activity in HUVEC treated with IL-1beta was also inhibited by PGE2 (0.03, 0.3 and 3 microM for 24h) in a dose-dependent manner. Similarly, forskolin (10, 50 or 100 microM) can also reverse the inhibition of PGE2 on increased COX activity in IL-1beta treated HUVEC. The results suggested that (i) PGE2 can initiate negative feedback regulation in the induction of COX-2 elicited by IL-1beta in endothelial cells, (ii) the inhibition of PGE2 on COX-2 protein and activity in IL-1beta treated HUVEC is mediated by cAMP and (iii) the therapeutic use of PGE2 in the condition which COX-2 has been involved may have different roles.     

An anti-inflammatory role for N-acetyl aspartate in stimulated human astroglial cells

Although N-acetyl-L-aspartate (NAA) has been shown to be important to myelin synthesis and osmotic regulation, the biological rationale for the high levels of NAA found in the brain remains unknown. Here, a human astroglial cell line (STTG) was treated with NAA and stimulated with ionomycin, ionomycin/PMA, or IL-1beta. PGE(2) levels in ionomycin-stimulated STTG cells decreased by 76% and > 95% at NAA concentrations of 10 and 20mM, respectively. NAA also decreased the levels of COX-2 protein and activated NF-kappaB in IL-1beta-stimulated STTG cells but had little effect on unstimulated cells. Also, NAA significantly decreased intracellular calcium levels in ionomycin/PMA-stimulated cells. NAA had no effect on total COX-2 activity or COX-2 mRNA. Acetylation of IkappaBalpha kinase, an acetylation target of aspirin, was not observed when NAA was present. These results demonstrate that NAA appears to be important in the modulation of inflammation in the human STTG astroglial cell line. The results of these findings are discussed in relation to neuronal pathologies that exhibit abnormal NAA levels within the brain.     

Induction of gene expression by IL-1 in NIH 3T3 cells. Possible requirement of protein kinase C activity and independence from arachidonic acid metabolism

NIH 3T3 fibroblasts were transfected with the chloramphenicol-acetyltransferase (CAT) gene under the control of the SV40 early promoter, which can be stimulated by IL-1. CAT activity in cell lysates and PGE2 release in the supernatants were measured in control and stimulated cell cultures in parallel. Human IL-1 beta (180 pM) and human rTNF-alpha (3 nM) significantly stimulated both CAT activity and PGE2 release. The combined incubation of the two cytokines resulted in a synergistic effect on PGE2 release. The addition of AA (30 microM) greatly stimulated PGE2 release without affecting CAT activity. Similarly, drugs interfering with AA metabolism were without effect on CAT activity although profoundly reducing PGE2 release. Forskolin (0.1 microM) did not modify either parameter. The glucocorticoid fluocinolone (20 nM) was able to decrease both parameters. Protein kinase inhibitors H7 (5-50 microM) and sphingosine (50 microM) inhibited only IL-1-induced CAT activity, whereas H8 (5-50 microM) and HA1004 (50 microM) were ineffective on both parameters. PMA (0.5 microM) and R59 022, a diacylglycerol kinase inhibitor (10 microM), did not modify either control or IL-1-induced CAT activity. IL-1-stimulated PGE2 release was potentiated by PMA, although this effect was not inhibited by H7. The data suggest that: 1) in NIH 3T3 cells the activation of AA metabolism by IL-1 is not involved in IL-1-induced gene expression; 2) protein kinase C activity is required but not sufficient for IL-1-induced gene expression; and 3) PMA may stimulate AA metabolism by a mechanism in part independent of protein kinase activity.     

Stimulatory effect of prostaglandin F(2alpha) on Na-dependent phosphate transport in osteoblast-like cells

Prostaglandin F(2alpha) (PGF(2alpha)) has been reported to activate protein kinase C (PKC) through both phospholipase (PL) C and D, resulting in the proliferation of osteoblast-like cells. In addition, it has also been reported that Erk mitogen-activated protein kinase is also involved in the mechanism of PGF(2alpha)-induced proliferation of these cells. Recently, we have reported that several growth factors stimulate Na-dependent phosphate transport (Pi transport) activity of osteoblast-like cells, which has been recognized to play an important role in their mineralization. In the present study, we investigated the effect of PGF(2alpha) on Pi transport in MC3T3-E1 osteoblast-like cells. PGF(2alpha) stimulated Na-dependent Pi transport dose dependently in the range between 1nM and 10 micro M in MC3T3-E1 cells. The effect was time dependent up to 24h. Kinetic analysis revealed that PGF(2alpha) induces newly synthesized Pi transporter. Pretreatment with actinomycin D and cycloheximide suppressed PGF(2alpha)-induced enhancement of Pi transport. Combined effect of PMA and PGF(2alpha) was not additive in Pi transport. Calphostin C, a PKC inhibitor, dose-dependently suppressed Pi transport induced by PGF(2alpha). On the contrary, U0126, which inhibits an upstream kinase of Erk (MEK), did not affect PGF(2alpha)-induced enhancement of Pi transport. In conclusion, PGF(2alpha) stimulates Pi transport through activation of PKC in osteoblast-like cells.     

Sphingosine potentiates IL-1-mediated prostaglandin E2 production in human fibroblasts

IL-1 stimulates PGE2 production in human fibroblasts by stimulating arachidonic acid (AA) mobilization and cyclooxygenase synthesis. Cyclooxygenase is the first enzyme in the pathway that converts AA to PGE2. To examine the role of protein kinase C (PKC) in IL-1-mediated PGE2 production, we treated cells with PMA, which stimulated PGE2 production suggesting a positive role for PKC activation in the regulation of PGE2 synthesis. Therefore, we tested the effect of sphingosine, a PKC inhibitor, on IL-1-induced PGE2 production. Alone, sphingosine had little effect on PGE2 production. However, when sphingosine was added with IL-1, or IL-1 was added to sphingosine-pretreated cells, PGE2 production increased severalfold, suggesting that the inhibition of PKC results in enhanced IL-1-mediated PGE2 production; structural analogs of sphingosine did not potentiate the IL-1 effect. In cells made deficient in PKC by prolonged exposure to PMA, IL-1-mediated PGE2 production was enhanced compared with normal cells, further suggesting that functional PKC is not required for, and may down-modulate, IL-1-mediated PGE2 production. These findings also suggest that PMA and IL-1 stimulate PGE2 synthesis via fundamentally different pathways. In separate studies on the effect of IL-1 on AA mobilization, we found that IL-1 induced an increase in phospholipase A2 (PLA2) activity and that cycloheximide blocked the increase, suggesting the requirement for new protein synthesis. We also found that the PLA2 activity increased as a result of IL-1 exposure was further stimulated by sphingosine. Thus, in addition to its primary effects on the cell, which are likely mediated via PKC, we present evidence suggesting that sphingosine may also play a role in potentiating an IL-1-induced PLA2 activity, resulting in increased availability of AA for conversion to PGE2.     

Contrasting effects of the protein kinase C inhibitor staurosporine on the interleukin-1 and phorbol ester activation pathways in the EL4-6.1 thymoma cell line.

EL 4-6.1 cells, variants of the murine EL4 thymoma cell line, can be activated by interleukin 1 (IL-1) or phorbol 12-myristate-13-acetate (PMA), or PMA+IL-1 to secrete interleukin 2 (IL-2) and interleukin 4 (IL-4) and to express the IL-2 receptor (IL-2R). To compare the different activation pathways, we examined the effects of staurosporine (STAR) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), two protein kinase C (PKC) inhibitors, on the induction of interleukin secretion and IL-2R expression in these cells. We report here that nanomolar concentrations of STAR strongly potentiated (20- to 30-fold) the production of IL-2 or IL-4, when EL 4-6.1 cells were induced by IL-1 alpha (or IL-1 beta) alone. By contrast, at identical concentrations, STAR dose-dependently inhibited the production of IL-2 and IL-4 resulting from PMA or PMA+IL-1 cell treatment. STAR also negatively affected the expression of IL-2R, which was dependent on PMA-sensitive PKC with either IL-1, PMA, or PMA+IL-1 stimulation. The changes in interleukin production and IL-2R expression in EL 4-6.1 activated cells were correlated with changes at the mRNA level measured by quantitative polymerase chain reaction (PCR). This finding suggests a pretranslational effect of the drug. At micromolar concentrations, H7 showed the same effects as STAR, but only increased IL-1-triggered interleukin secretions twofold. We observed that the action of PKC inhibitors did not result from modification of IL-1 receptor (IL-1R) expression in EL 4-6.1 cells. Thus, our data show that PKC inhibitors clearly distinguish between IL-1 and PMA stimulatory pathways. In addition, they suggest that the IL-1 stimulatory pathway involves PKC(s) [or other undefined kinase(s)] which regulate this pathway and differ from PKC(s) activated by PMA.     

Angiotensin II stimulates hypertrophic growth of cultured neonatal rat ventricular myocytes: roles of PKC and PGF2alpha

Angiotensin II (Ang II) has been shown to regulate growth in smooth muscle cells. Protein kinase C (PKC), which mediates Ang II action, has been implicated in myocardial cell hypertrophy. Acute pressure overload in the left ventricles has been demonstrated to produce prostaglandin F2 alpha (PGF2alpha) release. Therefore, we used cultured neonatal rat ventricular myocytes to study Ang II, PKC and PGF2alpha and their relationship to hypertrophy. The amount of PGF2alpha produced was determined by radioimmunoassay, Ang II-induced hypertrophy and PGF2alpha release. Pretreatment with 10(-6) M of PKC inhibitor, 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7), blocked Ang II-induced hypertrophy and PGF2alpha release. In neonatal rat ventricular myocytes that were treated with either Ang II or PKC activator (Phorbol 12, 13, dibutyrate; PDBu), PKC enzyme assay showed PKC was translocated from the cytosol to the membrane which indicates activation. This suggests that PKC mediates, in part, Ang II-induced PGF2alpha release and hypertrophy. In summary, Ang II activates PKC, which causes PGF2alpha release and hypertrophy, and this PGF2alpha release and hypertrophy can be overcome by pretreatment with PKC inhibitor.     

Protein kinase C-dependent and -independent mechanisms of cloned murine T cell proliferation. The role of protein kinase C translocation and protein kinase C activity

PMA can induce the proliferation of several CTL clones but not of several Th clones derived and tested in our laboratory. The PMA-stimulated proliferation of our CTL clones (which do not make IL-2 mRNA or protein) occurs independently of IL-2 and is not accompanied by lymphokine release. We now report, however, that protein kinase C (PKC) translocation is induced by PMA in CTL clones as well as in Th clones, which lack a proliferative response to PMA. These results suggest that PKC translocation itself is not a sufficient regulatory mechanism to account for cloned T cell proliferation. Moreover, IL-2 did not induce PKC translocation in a CTL clone, which proliferates when stimulated with IL-2. Thus, PKC translocation may not be necessary for activation of CTL proliferation. Nonetheless, cellular PKC activity appears to be required for the proliferative response of T cell clones after stimulation by PMA/PMA + calcium ionophore (A23187) or by triggering through the TCR: chronic PMA treatment, which depletes intracellular PKC activity, abrogates the proliferative response of T cell clones stimulated by PMA/PMA + A23187 or triggered through the TCR. T cell clones depleted of PKC activity, however, retain the ability to proliferate when challenged with IL-2. Murine T cell clones, therefore, possess PKC-dependent and PKC-independent pathways of proliferation that are not regulated by PKC translocation alone.