Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway

We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.     

Prostaglandin E2/parathyroid hormone-induced suppression of alkaline phosphatase activity is mediated by protein kinase C

• 1.1. Bone resorptive factors, prostaglandin E2 and parathyroid hormone are shown to suppress alkaline phosphatase activity in a rat osteoblastic cell line.
• 2.2. Phorbol myristate acetate, but not dibutyryl cAMP or calcium ionophore can suppress alkaline phosphatase activity.
• 3.3. The protein kinase C inhibitors (H89, staurosporine) are able to block the suppression of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone.
• 4.4. These data suggest that protein kinase C is involved in the inhibition of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone.

     

Regulation of arachidonate-mobilizing phospholipase A2 by phosphorylation via protein kinase C in macrophages.

Stimulation of 32P-labeled macrophages with phorbol ester caused an increase in phosphorylation of the intracellular, high molecular weight phospholipase A2. This increase in phosphorylation was accompanied by an increase in enzyme activity, but led to no detectable shift in the concentration dependence for Ca(2+)-induced activation. The phosphorylated phospholipase A2 could be dephosphorylated by treatment with acid phosphatase, and such treatment also reduced its catalytic activity. Together with previous data, these results indicate that the arachidonate-mobilizing phospholipase A2 is dually regulated by Ca2+ (membrane interaction) and by phosphorylation (catalytic activity).     

Activation of protein kinase C delta following cerebral ischemia leads to release of cytochrome C from the mitochondria via bad pathway

Background

The release of cytochrome c from the mitochondria following cerebral ischemia is a key event leading to cell death. The goal of the present study was to determine the mechanisms involved in post-ischemic activation of protein kinase c delta (δPKC) that lead to cytochrome c release.

Methods/Findings

We used a rat model of cardiac arrest as an in vivo model, and an in vitro analog, oxygen glucose deprivation (OGD) in rat hippocampal synaptosomes. Cardiac arrest triggered translocation of δPKC to the mitochondrial fraction at 1 h reperfusion. In synaptosomes, the peptide inhibitor of δPKC blocked OGD-induced translocation to the mitochondria. We tested two potential pathways by which δPKC activation could lead to cytochrome c release: phosphorylation of phospholipid scramblase-3 (PLSCR3) and/or protein phosphatase 2A (PP2A). Cardiac arrest increased levels of phosphorlyated PLSCR3; however, inhibition of δPKC translocation failed to affect the OGD-induced increase in PLSCR3 in synaptosomal mitochondria suggesting the post-ischemic phosphorylation of PLSCR3 is not mediated by δPKC. Inhibition of either δPKC or PP2A decreased cytochrome c release from synaptosomal mitochondria. Cardiac arrest results in the dephosphorylation of Bad and Bax, both downstream targets of PP2A promoting apoptosis. Inhibition of δPKC or PP2A prevented OGD-induced Bad, but not Bax, dephosphorylation. To complement these studies, we used proteomics to identify novel mitochondrial substrates of δPKC.

Conclusions

We conclude that δPKC initiates cytochrome c release via phosphorylation of PP2A and subsequent dephosphorylation of Bad and identified δPKC, PP2A and additional mitochondrial proteins as potential therapeutic targets for ischemic neuroprotection.     

Prostaglandin D2 induces interleukin-6 synthesis via Ca2+ mobilization in osteoblasts: regulation by protein kinase C

We previously showed that prostaglandin (PG) D2 stimulates Ca2+ influx from extracellular space and activates phosphoinositidic (PI)-hydrolyzing phospholipase C and phosphatidylcholine (PC)-hydrolyzing phospholipase D independently from PGE2 or PGF2alpha in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of PGD2 on the synthesis of interleukin-6 (IL-6) and its regulatory mechanism in MC3T3-E1 cells. PGD2 significantly stimulated IL-6 synthesis dose-dependently in the range between 10 nM and 10 microM. The depletion of extracellular Ca2+ by EGTA reduced the PGD2-induced IL-6 synthesis. TMB-8, an inhibitor of intracellular Ca2+ mobilization, significantly inhibited the PGD2-induced IL-6 synthesis. On the other hand, calphostin C, a specific inhibitor of protein kinase C (PKC), enhanced the synthesis of IL-6 induced by PGD2. In addition, U-73122, an inhibitor of phospholipase C, and propranolol, a phosphatidic acid phosphohydrolase inhibitor, enhanced the PGD2-induced IL-6 synthesis. These results strongly suggest that PGD2 stimulates IL-6 synthesis through intracellular Ca2+ mobilization in osteoblasts, and that the PKC activation by PGD2 itself regulates the over-synthesis of IL-6.     

Function of the alpha 1 beta 2 gamma 2S gamma-aminobutyric acid type A receptor is modulated by protein kinase C via multiple phosphorylation sites.

Activation of protein kinase C (PKC) results in down-modulation of the gamma-aminobutyric acid type A (GABAA) receptor. In this study, the recombinant subunit combination alpha 1 beta 2 gamma 2S was expressed in Xenopus oocytes. The resulting channel was shown to be modulated by 2 microM oleoylacetylglycerol or, stereo-specifically, by low concentrations (10 nM) of the phorbol ester 4 beta-phorbol 12-myristate 13-acetate. By site-specific mutagenesis, we altered the serine or threonine residues of consensus phosphorylation sites for PKC in the large, intracellular domain of alpha 1, beta 2, and gamma 2S. Mutant subunits were co-expressed with wild type subunits to yield alpha 1 beta 2 gamma 2S combinations. All of the tested 14 mutations did not affect the level of expression of GABA current. Two of these mutations, Ser-410 in beta 2 and Ser-327 in gamma 2S, resulted in a significant reduction of the effect of the activator of PKC, 4 beta-phorbol 12-myristate 13-acetate, on the GABA current amplitude. Thus, we have identified two single serine residues, Ser-410 in the subunit beta 2 and Ser-327 in gamma 2S, as phosphorylation sites of a PKC endogenous to Xenopus oocytes. Co-expression of the mutant subunits suggests that phosphorylation of both sites is required for a full, PKC-mediated down-regulation of GABA currents.     

Prostaglandin E2 modulates proximal tubule Na-ATPase activity: Cooperative effect between protein kinase A and protein kinase C

Previous data showed that prostaglandin E₂ (PGE₂) mediates the inhibitory effect of bradykinin (BK) on proximal tubule (PT) Na⁺-ATPase activity. The aim of this work was to investigate the molecular mechanisms involved in the effect of PGE₂ on PT Na⁺-ATPase. We used isolated basolateral membrane (BLM) from pig PT, which expresses several components of different signaling pathways. The inhibitory effect of PGE₂ on PT Na⁺-ATPase activity involves G-protein and the activation of protein kinase A (PKA) because: (1) PGE₂ increased [³⁵S]GTPγS binding; (2) GDPβS abolished the inhibitory effect of PGE₂; (3) PGE₂ increased PKA activity; (4) the inhibitory effect of PGE₂ was abolished by PKA inhibitor peptide. We observed that the PKA-mediated inhibitory effect of PGE₂ on PT Na⁺-ATPase activity requires previous activation of protein kinase C. In addition, we observed that PGE₂ stimulates Ca²⁺-independent phospholipase A₂ activity representing an important positive feedback to maintain the inhibition of the enzyme. These results open new perspectives to understanding the mechanism involved in the effect of PGE₂ on proximal tubule sodium reabsorption.     

Antihypertensive action of 2-hydroxyoleic acid in SHRs via modulation of the protein kinase A pathway and Rho kinase

Olive oil consumption leads to high monounsaturated fatty acid intake, especially oleic acid, and has been associated with a reduced risk of hypertension. However, the molecular mechanisms and contribution of its different components to lower blood pressure (BP) require further evaluation. Here, we examined whether a synthetic, non-beta-oxidation-metabolizable derivative of oleic acid, 2-hydroxyoleic acid (2-OHOA), can normalize BP in adult spontaneously hypertensive rats (SHRs) and whether its antihypertensive action involves cAMP-dependent protein kinase A (PKA) and Rho kinase, two major regulators of vascular smooth muscle contraction. Oral administration of 2-OHOA to SHRs induced sustained systolic BP decreases in a time-dependent (1-7 days) and dose-dependent (100-900 mg/kg every 12 h) manner. After 7 days of treatment with 2-OHOA (600 mg/kg), the systolic BP of SHRs was similar to that of normotensive Wistar Kyoto rats, returning to its initial hypertensive level after withdrawal of 2-OHOA. This treatment strongly increased the protein expression of the catalytic and regulatory RIalpha and RIIalpha PKA subunits as well as PKA activity in aortas from SHRs. Consistently, administration of the PKA inhibitor 8-bromo adenosine-3',5'-cyclic monophosphorothioate, Rp isomer, to 2-OHOA-treated SHRs induced a pronounced reversal (up to 59%) of the antihypertensive effect of 2-OHOA. Additionally, 2-OHOA completely reversed the pathological overexpression of aortic Rho kinase found in SHRs, suppressing the vasoconstrictory Rho kinase pathway.     

Stabilization of Mdm2 via decreased ubiquitination is mediated by protein kinase B/Akt-dependent phosphorylation

The tumor suppressor p53 is commonly inhibited under conditions in which the phosphatidylinositide 3'-OH kinase/protein kinase B (PKB)Akt pathway is activated. Intracellular levels of p53 are controlled by the E3 ubiquitin ligase Mdm2. Here we show that PKB inhibits Mdm2 self-ubiquitination via phosphorylation of Mdm2 on Ser(166) and Ser(188). Stimulation of human embryonic kidney 293 cells with insulin-like growth factor-1 increased Mdm2 phosphorylation on Ser(166) and Ser(188) in a phosphatidylinositide 3'-OH kinase-dependent manner, and the treatment of both human embryonic kidney 293 and COS-1 cells with phosphatidylinositide 3'-OH kinase inhibitor LY-294002 led to proteasome-mediated Mdm2 degradation. Introduction of a constitutively active form of PKB together with Mdm2 into cells induced phosphorylation of Mdm2 at Ser(166) and Ser(188) and stabilized Mdm2 protein. Moreover, mouse embryonic fibroblasts lacking PKBalpha displayed reduced Mdm2 protein levels with a concomitant increase of p53 and p21(Cip1), resulting in strongly elevated apoptosis after UV irradiation. In addition, activation of PKB correlated with Mdm2 phosphorylation and stability in a variety of human tumor cells. These findings suggest that PKB plays a critical role in controlling of the Mdm2.p53 signaling pathway by regulating Mdm2 stability.     

Stimulation of A(2A) adenosine receptor phosphorylation by protein kinase C activation: evidence for regulation by multiple protein kinase C isoforms.

Activation of the A(2A) adenosine receptor (A(2A)AR) contributes to the neuromodulatory and neuroprotective effects of adenosine in the central nervous system. Here we demonstrate that, in rat C6 glioma cells stably expressing an epitope-tagged canine A(2A)AR, receptor phosphorylation on serine and threonine residues can be increased by pretreatment with either the synthetic protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or endothelin 1, which increases PKC activity via binding to endogenous endothelin(A) receptors. Under conditions in which PMA was maximally effective, activation of other second messenger-regulated kinases was without effect. While basal and PMA-stimulated phosphorylation were unaffected by the A(2A)AR-selective antagonist ZM241385, they were both blocked by GF109203X (a selective inhibitor of conventional and novel PKC isoforms) and rottlerin (a PKCdelta-selective inhibitor) but not Go6976 (selective for conventional PKC isoforms). However, coexpression of the A(2A)AR with each of the alpha, betaI, and betaII isoforms of PKC increased basal and PMA-stimulated phosphorylation. Mutation of the three consensus PKC phosphorylation sites within the receptor (Thr298, Ser320, and Ser335) to Ala failed to inhibit either basal or PMA-stimulated phosphorylation. In addition, phosphorylation of the receptor was not associated with detectable changes in either its signaling capacity or cell surface expression. These observations suggest that multiple PKC isoforms can stimulate A(2A)AR phosphorylation via activation of one or more downstream kinases which then phosphorylate the receptor directly. In addition, it is likely that phosphorylation controls interactions with regulatory proteins distinct from those involved in the classical cAMP signaling pathway utilized by this receptor.     

Arachidonic acid promotes phosphorylation of 5-lipoxygenase at Ser-271 by MAPK-activated protein kinase 2 (MK2)

We demonstrated previously that 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, can be phosphorylated by p38 MAPK-regulated MAPKAP kinases (MKs). Here we show that mutation of Ser-271 to Ala in 5-LO abolished MK2 catalyzed phosphorylation and clearly reduced phosphorylation by kinases prepared from stimulated polymorphonuclear leukocytes and Mono Mac 6 cells. Compared with heat shock protein 27 (Hsp-27), 5-LO was a weak substrate for MK2. However, the addition of unsaturated fatty acids (i.e. arachidonate 1-50 microm) up-regulated phosphorylation of 5-LO, but not of Hsp-27, by active MK2 in vitro, resulting in a similar phosphorylation as for Hsp-27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca(2+)/calmodulin-dependent kinase II), but these activities were not increased by fatty acids. HeLa cells expressing wild type 5-LO or S271A-5-LO, showed prominent 5-LO activity when incubated with Ca(2+)-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower as compared with wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus that does not prominently increase intracellular Ca(2+) and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation.     

pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine

An hepatic protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) on Ser/Thr residues is markedly activated after intraperitoneal injection of cycloheximide in the rat. The enzyme has been purified greater than 10,000-fold to near homogeneity and corresponds to a 54-kDa polypeptide, based on auto-phosphorylation, renaturation of activity from sodium dodecyl sulfate gels, and gel filtration. The protein kinase activity is unaffected by prior autophosphorylation, Ca2+, diacylglycerol and phospholipids, cyclic nucleotides, staurosporine, and protein kinase inhibitor, but can be totally and specifically deactivated by the Ser/Thr protein phosphatase 2A. The enzyme is inhibited completely but reversible by transition metals and p-chloromercuribenzoate, and is strongly stimulated by poly-L-lysine toward most, but not all protein substrates. The activity of the cycloheximide-stimulated MAP-2 kinase (pp54 MAP-2 kinase) toward potential polypeptide substrates was compared to that of an insulin-stimulated MAP-2 kinase (pp42 MAP-2 kinase). Although both MAP-2 kinases exhibited little or no ability to phosphorylate histones and casein, the two kinases had a distinguishable substrate specificity. At comparable MAP-2 phosphorylating activities, pp42 MAP-2 kinase, but not pp54 MAP-2 kinase, phosphorylated and activated the Xenopus S6 protein kinase II. Moreover, pp42 MAP-2 kinase phosphorylated myelin basic protein at 10-12-fold higher rates than did pp54 MAP-2 kinase. Cycloheximide-activated pp54 MAP-2 protein kinase appears to be a previously uncharacterized protein kinase that is itself regulated through Ser/Thr phosphorylation and, perhaps, polypeptide regulators with basic domains. The identity of the upstream regulatory elements and the native substrates remain to be established.     

Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway.

Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L. cv. Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores. We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea. Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors. Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions. However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC). The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC). On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein. A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein. These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DeltapH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC.     

Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: Role of calcium and protein kinase A and C

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E₂ (PGE₂) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE₂ provoked a 3.9 ± 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca⁺⁺ channel blockers, verapamil and nifedipine, and the Ca⁺⁺/calmodulin inhibitor, W-7. The Ca⁺⁺ ionophore, ionomycin, mimicked the effects of PGE₂ as did the phorbol ester PMA, which activates Ca⁺⁺-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE₂ did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE₂ secretagogue, IL-1β, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 ± 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE₂ does not mediate the effect of its secretagogue and that IL-1β signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE₂ receptor signalling pathways. Taken together, our results suggest that PGE₂ modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism. © 1996 Wiley-Liss, Inc.