Involvement of phosphatidylcholine-selective phospholipase C in activation of mitogen-activated protein kinase pathways in imidazoline receptor antisera-selected protein

Imidazoline receptor antisera-selected protein (IRAS) is considered as a candidate for the I1-imidazoline receptor (I1R), but the signaling pathway mediated by IRAS remains unknown. In our study, the signal transduction pathways of IRAS were investigated in CHO cells stably expressing IRAS (CHO-IRAS), and compared to the native I1R signaling pathways. Rilmenidine or moxonidine (10 nM-100 microM), I1R agonists, failed to stimulate [35S]-GTPgammaS binding in CHO-IRAS cell membrane preparations, suggesting that G protein may not be involved in IRAS signaling pathway. However, incubation of CHO-IRAS with rilmenidine or moxonidine for 5 min could induce an upregulation of phosphatidylcholine-selective phospholipase C (PC-PLC) activity, and an increase in the accumulation of diacylglycerol (DAG), the hydrolysate of PC-PLC, in a concentration-dependent manner. The elevated activation of PC-PLC by rilmenidine or moxonidine (100 nM) could be blocked by efaroxan, a selective I1R antagonist. Cells treated with rilmenidine or moxonidine showed an increased level of extracellular signal-regulated kinase (ERK) phosphorylation in a concentration-dependent manner, which could be reversed by efaroxan or D609, a selective PC-PLC inhibitor. These results suggest that the signaling pathway of IRAS in response to I1R agonists coupled with the activation of PC-PLC and its downstream signal transduction molecule, ERK. These findings are similar to those in the signaling pathways of native I1R, providing some new evidence for the relationship between I1R and IRAS.     

S1P-receptors in PC12 and transfected HEK293 cells: Molecular targets of hypotensive imidazoline I1 receptor ligands

The present study aimed at elucidating the molecular identity of the proposed “I₁-imidazoline receptors”, i.e. non-adrenoceptor recognition sites via which the centrally acting imidazolines clonidine and moxonidine mediate a major part of their effects. In radioligand binding experiments with [³H]clonidine and [³H]lysophosphatidic acid on intact, α₂-adrenoceptor-deficient PC12 cells, moxonidine, clonidine, lysophosphatidic acid and sphingosine-1-phosphate (S1P) competed for the specific binding sites of both radioligands with similar affinities. RNA interference with the rat S1P₁-, S1P₂- or S1P₃-receptor abolished specific [³H]lysophosphatidic acid binding. [³H]Clonidine binding was markedly decreased by siRNA targeting S1P₁- and S1P₃-receptors but not by siRNA against S1P₂-receptors. Finally, in HEK293 cells transiently expressing human S1P₃-receptors, sphingosine-1-phosphate, clonidine and moxonidine induced increases in intracellular calcium concentration, moxonidine being more potent than clonidine; this is in agreement with the known properties of the “I₁-imidazoline receptors”.The present results indicate that the “I₁-imidazoline receptors” mediating effects of clonidine and moxonidine in PC12 and the transfected HEK293 cells belong to the S1P-receptor family; in particular, the data obtained in PC12 cells suggest that the I₁ imidazoline receptors represent a mixture of S1P₁- and S1P₃-receptors and/or hetero-dimers of both.     

Augmentation of moxonidine-induced increase in ANP release by atrial hypertrophy

Imidazoline receptors are divided into I(1) and I(2) subtypes. I(1)-imidazoline receptors are distributed in the heart and are upregulated during hypertension or heart failure. The aim of this study was to define the possible role of I(1)-imidazoline receptors in the regulation of atrial natriuretic peptide (ANP) release in hypertrophied atria. Experiments were performed on isolated, perfused, hypertrophied atria from remnant-kidney hypertensive rats. The relatively selective I(1)-imidazoline receptor agonist moxonidine caused a decrease in pulse pressure. Moxonidine (3, 10, and 30 micromol/l) also caused dose-dependent increases in ANP secretion, but clonidine (an alpha(2)-adrenoceptor agonist) did not. Pretreatment with efaroxan (a selective I(1)-imidazoline receptor antagonist) or rauwolscine (a selective alpha(2)-adrenoceptor antagonist) inhibited the moxonidine-induced increases in ANP secretion and interstitial ANP concentration and decrease in pulse pressure. However, the antagonistic effect of efaroxan on moxonidine-induced ANP secretion was greater than that of rauwolscine. Neither efaroxan nor rauwolscine alone has any significant effects on ANP secretion and pulse pressure. In hypertrophied atria, the moxonidine-induced increase in ANP secretion and decrease in pulse pressure were markedly augmented compared with nonhypertrophied atria, and the relative change in ANP secretion by moxonidine was positively correlated to atrial hypertrophy. The accentuation by moxonidine of ANP secretion was attenuated by efaroxan but not by rauwolscine. These results show that moxonidine increases ANP release through (preferentially) the activation of atrial I(1)-imidazoline receptors and also via different mechanisms from clonidine, and this effect is augmented in hypertrophied atria. Therefore, we suggest that cardiac I(1)-imidazoline receptors play an important role in the regulation of blood pressure.     

Protein kinase C alpha, betaI, and betaII isozymes regulate cytokine production in mast cells through MEKK2/ERK5-dependent and -independent pathways

Regulation of MAPK pathways by PKC isoforms was examined in murine bone marrow-derived mast cells (BMMCs). The PKCalpha, betaI, and betaII isoforms showed the most robust activation after FcepsilonR1-mediated stimulation by anti-ovalbumin specific IgE and ovalbumin (IgE-ova). PKCalpha, betaI, and betaII were all involved in activation of JNK, MEKK2, and ERK5, with differential relative contributions of each isoform to specific MAPK pathway components. BMMCs from mice lacking MEKK2 showed reduced production (50-60%) of IL-6, IL-13, and TNF-alpha after stimulation, demonstrating MEKK2-dependent and -independent pathways for cytokine production. Cytokine production was stimulated by over-expression of PKC in cells from MEKK2-deficient and wild-type mice. Activation of ERK5 did not occur in BMMCs lacking MEKK2, indicating that MEKK2-independent cytokine production was also ERK5-independent. Since MAPK modules differentially regulate mast cell functions, including degranulation and cytokine production, it is suggested that specific functions could be targeted by inhibiting specific PKC isoforms.     

Activation of protein kinase C beta II by the stereo-specific phosphatidylserine receptor is required for phagocytosis of apoptotic thymocytes by resident murine tissue macrophages

We showed previously that protein kinase C (PKC) is required for phagocytosis of apoptotic leukocytes by murine alveolar (AM?) and peritoneal macrophages (PM?) and that such phagocytosis is markedly lower in AM? compared with PM?. In this study, we examined the roles of individual PKC isoforms in phagocytosis of apoptotic thymocytes by these two M? populations. By immunoblotting, AM? expressed equivalent PKC eta but lower amounts of other isoforms (alpha, betaI, betaII, delta, epsilon, mu, and zeta), with the greatest difference in betaII expression. A requirement for PKC betaII for phagocytosis was demonstrated collectively by phorbol 12-myristate 13-acetate-induced depletion of PKC betaII, by dose-response to PKC inhibitor Ro-32-0432, and by use of PKC betaII myristoylated peptide as a blocker. Exposure of PM? to phosphatidylserine (PS) liposomes specifically induced translocation of PKC betaII and other isoforms to membranes and cytoskeleton. Both AM? and PM? expressed functional PS receptor, blockade of which inhibited PKC betaII translocation. Our results indicate that murine tissue M? require PKC betaII for phagocytosis of apoptotic cells, which differs from the PKC isoform requirement previously described in M? phagocytosis of other particles, and imply that a crucial action of the PS receptor in this process is PKC betaII activation.     

Overexpression of protein kinase C isoforms protects RAW 264.7 macrophages from nitric oxide-induced apoptosis: involvement of c-Jun N-terminal kinase/stress-activated protein kinase, p38 kinase, and CPP-32 protease pathways

Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macrophages. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-induced apoptosis, we generated clones of RAW 264.7 cells that overexpress one of the PKC isoforms and explored the possible interactions between PKC and three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO-generating agent, activated both c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellular signal-regulated kinase (ERK)-1 and ERK-2. In addition, SNP-induced apoptosis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC-beta II, -delta, and -eta) showed substantial protection from cell death induced by the exposure to NO donors such as SNP and S-nitrosoglutathione (GSNO). In contrast, in RAW 264.7 parent or in empty vector-transformed cells, these NO donors induced internucleosomal DNA cleavage. Moreover, overexpression of PKC isoforms significantly suppressed SNP-induced JNK/SAPK and p38 kinase activation, but did not affect ERK-1 and -2. We also explored the involvement of CPP32-like protease in the NO-induced apoptosis. Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7 parent cells. In addition, SNP dramatically activated CPP32 in the parent or in empty vector-transformed cells, while slightly activated CPP32 in PKC transfectants. Therefore, we conclude that PKC protects NO-induced apoptotic cell death, presumably nullifying the NO-mediated activation of JNK/SAPK, p38 kinase, and CPP32-like protease in RAW 264.7 macrophages.     

Protein kinase CbetaI interacts with the beta1-adrenergic signaling pathway to attenuate lipolysis in rat adipocytes

We have shown previously that insulin attenuates beta1-adrenergic receptor (beta1-AR)-mediated lipolysis via activation of protein kinase C (PKC) in rat adipocytes. This antilipolysis persists after removal of insulin and is independent of the phosphodiesterase 3B activity, and phorbol 12-myristate 13-acetate (PMA) could substitute for insulin to produce the same effect. Here, we attempted to identify the PKC isoform responsible for antilipolysis. Isolated adipocytes were treated with high and low concentrations of PMA for up to 6 h to degrade specific PKC isoforms. In the PMA-treated cells, the downregulation profiles of PKC isoforms alpha and betaI, but not betaII, delta, epsilon, or zeta, correlated well with a decrease of lipolysis-attenuating effect of PMA. After rats fasted for 24 h, adipocyte expression of PKC isoform alpha increased, while expression of PKCdelta decreased. Fasting did not change the potency of PMA to attenuate lipolysis, however. The lipolysis-attenuating effect of PMA was blocked by the PKCbetaI/betaII inhibitor LY 333531, but not by the PKCbetaII inhibitor CGP 53353 or the PKCdelta inhibitor rottlerin. These data suggest that PKCbetaI interacts with beta1-AR signaling and attenuates lipolysis in rat adipocytes.     

Ethanol-induced mitogen activated protein kinase activity mediated through protein kinase C.

The aim of this study was to determine the pathway(s) by which ethanol activates mitogen-activated protein kinase (MAPK) signaling and to determine the role of Ca2+ in the signaling process. MAPK signaling was determined by assessing MAPK activity, measuring phosphorylated extracellular signaling-regulated kinase (pp 44 ERK-1 and pp 42 ERK-2) expression and ERK activity by measuring ERK-2-dependent phosphorylation of a synthetic peptide as a MAPK substrate in rat vascular smooth muscle cells. Ethanol activated extracellular signal-regulated kinase expression (ERK 1 and 2) could be observed when vascular smooth muscle cells (VSMCs) were stimulated for 5 min or less, but was inhibited when cells are treated for 10 min or more with 1-16 mM of ethanol. Maximum ethanol-induced MAPK activity was observed within 5 min with 4 or 8 mM. Ethanol stimulated MAPK activity was blocked by the protein kinase C (PKC) inhibitor (GF109203X) and epidermal growth factor (EGF) receptor antagonist (PD153035) by 41 +/- 24 and 34 +/- 12.3%, respectively. The calcium channel blocker, diltiazem and the chelating agent, BAPTA, reduced the activation of MAPK activity by ethanol, significantly. The data demonstrate that ethanol-stimulated MAPK expression is mediated partially through both the EGF-receptor and PKC intermediates and that activation through the PKC intermediate is calcium-dependent.     

Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled receptor-EGF receptor transactivation pathway.

In GN4 rat liver epithelial cells, angiotensin II (Ang II) produces intracellular calcium and protein kinase C (PKC) signals and stimulates ERK and JNK activity. JNK activation appears to be mediated by a calcium-dependent tyrosine kinase (CADTK). To define the ERK pathway, we established GN4 cells expressing an inhibitory Ras(N17). Induction of Ras(N17) blocked EGF- but not Ang II- or phorbol ester (TPA)-dependent ERK activation. In control cells, Ang II and TPA produced minimal increases in Ras-GTP level and Raf kinase activity. PKC depletion by chronic TPA exposure abolished TPA-dependent ERK activation but failed to diminish the effect of Ang II. In PKC-depleted cells, Ang II increased Ras-GTP level and activated Raf and ERK in a Ras-dependent manner. In PKC depleted cells, Ang II stimulated Shc and Cbl tyrosine phosphorylation, suggesting that without PKC, Ang II activates another tyrosine kinase. PKC-depletion did not alter Ang II-dependent tyrosine phosphorylation or activity of p125(FAK), CADTK, Fyn or Src, but PKC depletion or incubation with GF109203X resulted in Ang II-dependent EGF receptor tyrosine phosphorylation. In PKC-depleted cells, EGF receptor-specific tyrosine kinase inhibitors blocked Ang II-dependent EGF receptor and Cbl tyrosine phosphorylation, and ERK activation. In summary, Ang II can activate ERK via two pathways; the latent EGF receptor, Ras-dependent pathway is equipotent to the Ras-independent pathway, but is masked by PKC action. The prominence of this G-protein coupled receptor to EGF receptor pathway may vary between cell types depending upon modifiers such as PKC.     

Adenosine A(2A) receptor mRNA regulation by nerve growth factor is TrkA-, Src-, and Ras-dependent via extracellular regulated kinase and stress-activated protein kinase/c-Jun NH(2)-terminal kinase

We have shown previously that nerve growth factor (NGF) down-regulates adenosine A(2A) receptor (A(2A)AR) mRNA in PC12 cells. To define cellular mechanisms that modulate A(2A)AR expression, A(2A)AR mRNA and protein levels were examined in three PC12 sublines: i) PC12nnr5 cells, which lack the high affinity NGF receptor TrkA, ii) srcDN2 cells, which overexpress kinase-defective Src, and iii) 17.26 cells, which overexpress a dominant-inhibitory Ras. In the absence of functional TrkA, Src, or Ras, NGF-induced down-regulation of A(2A)AR mRNA and protein was significantly impaired. However, regulation of A(2A)AR expression was reconstituted in PC12nnr5 cells stably transfected with TrkA. Whereas NGF stimulated the mitogen-activated protein kinases p38, extracellular regulated kinase 1 and 2 (ERK1/ERK2), and stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) in PC12 cells, these kinases were activated only partially or not at all in srcDN2 and 17.26 cells. Inhibiting ERK1/ERK2 with PD98059 or inhibiting SAPK/JNK by transfecting cells with a dominant-negative SAPKbeta/JNK3 mutant partially blocked NGF-induced down-regulation of A(2A)AR expression in PC12 cells. In contrast, inhibiting p38 with SB203580 had no effect on the regulation of A(2A)AR mRNA and protein levels. Treating SAPKbeta/JNK3 mutant-transfected PC12 cells with PD98059 completely abolished the NGF-induced decrease in A(2A)AR mRNA and protein levels. These results reveal a role for ERK1/ERK2 and SAPK/JNK in regulating A(2A)AR expression.     

Hepatocyte resistance to oxidative stress is dependent on protein kinase C-mediated down-regulation of c-Jun/AP-1

The prevention of injury from reactive oxygen species is critical for cellular resistance to many death stimuli. Resistance to death from the superoxide generator menadione in the hepatocyte cell line RALA255-10G is dependent on down-regulation of the c-Jun N-terminal kinase (JNK)/AP-1 signaling pathway by extracellular signal-regulated kinase 1/2 (ERK1/2). Because protein kinase C (PKC) regulates both oxidant stress and JNK signaling, the ability of PKC to modulate hepatocyte death from menadione through effects on AP-1 was examined. PKC inhibition with Ro-31-8425 or bisindolylmaleimide I sensitized this cell line to death from menadione. Menadione treatment led to activation of PKCmicro, or protein kinase D (PKD), but not PKCalpha/beta, PKCzeta/lambda, or PKCdelta/. Menadione induced phosphorylation of PKD at Ser-744/748, but not Ser-916, and translocation of PKD to the nucleus. PKC inhibition blocked menadione-induced phosphorylation of PKD, and expression of a constitutively active PKD prevented death from Ro-31-8425/menadione. PKC inhibition led to a sustained overactivation of JNK and c-Jun in response to menadione as determined by in vitro kinase assay and immunoblotting for the phosphorylated forms of both proteins. Cell death from PKC inhibition and menadione treatment resulted from c-Jun activation, since death was blocked by adenoviral expression of the c-Jun dominant negative TAM67. PKC and ERK1/2 independently down-regulated JNK/c-Jun, since inhibition of either kinase failed to affect activation of the other kinase, and simultaneous inhibition of both pathways caused additive JNK/c-Jun activation and cell death. Resistance to death from superoxide therefore requires both PKC/PKD and ERK1/2 activation in order to down-regulate proapoptotic JNK/c-Jun signaling.     

Expression of the high molecular weight fibroblast growth factor-2 isoform of 210 amino acids is associated with modulation of protein kinases C delta and epsilon and ERK activation

The high molecular weight (HMW) fibroblast growth factor (FGF)-2 isoform of 210 amino acids initiated at a CUG start codon possesses a nuclear localization sequence and is not secreted. In contrast, the low molecular weight (LMW) isoform of 155 amino acids initiated at the AUG start codon can be secreted and activates the cell surface FGF receptors. The two isoforms possess different biological properties; however, little is known about the intracrine regulatory mechanisms involved in the biological effects of the HMW FGF-2 isoform. Using pancreatic cells stably transfected with cDNAs leading to the expression of either the HMW FGF-2 (A3 cells) or the LMW form (A5 cells), we provide evidence that the two FGF-2 isoforms differentially modulate PKC levels. The LMW FGF-2 up-regulated the PKC epsilon levels by 1.6-fold; by contrast the HMW isoform down-regulated the level of this PKC isotype by about 3-fold and increased the amount of PKC delta by 1.7-fold. PKC mRNAs were also modified, suggesting that PKC expression was regulated at a pretranslational level. Additionally, expression of different levels of the HMW FGF-2 with an inducible expression system confirmed the role of this isoform on PKC delta and epsilon expressions. Increased activation of ERK-1 and -2 was also observed in cells expressing the HMW FGF-2. By using different PKC inhibitors and a dominant negative PKC delta, it was found that ERK activation was PKC delta-dependent. These data indicate that expression of HMW FGF-2 can modify PKC levels by acting at the intracellular level and that the overexpression of PKC delta induces ERK-1/2 activation. The expression of a dominant negative FGFR1 did not reduce ERK-1/2 activation by the HMW FGF-2, suggesting that ERK activation does not require FGFR activity. The signaling cascade downstream of ERK might be involved in the known mitogenic effect exerted by this FGF-2 isoform.     

Centrally Acting Imidazolines Stimulate Vascular Alpha 1A-Adrenergic Receptors in Rat-Tail Artery

  1. Centrally acting imidazoline antihypertensive agents clonidine and moxonidine also act peripherally to contract blood vessels. While these agents act at both I₁-imidazoline and alpha 2 adrenergic receptors centrally, the receptor types by which they mediate contraction require further definition. We therefore characterized the receptor subtype by which these agents mediate contraction of proximal rat-tail artery.2. Dose–response curves were determined for phenylephrine and for several imidazoline ligands, using endothelium denuded, isolated ring segments, of tail arteries from adult male Sprague-Dawley rats. Ring segments were mounted on a force transducer with platinum wires and immersed in a tissue bath containing Krebs solution, to which drugs could be added. Signals were digitized and recorded by a computer.3. Tail artery contractions expressed as a percent of contraction to 106 mM potassium were phenylephrine (96%), moxonidine (88%), clonidine (52%), and UK14304 (30%). Neither rilmenidine nor harmane caused contraction. Contraction of tail artery to moxonidine or clonidine could be blocked by alpha 1 antagonist urapidil or prazosin, and also by alpha 1A subtype selective antagonist WB4101. Schild plots were generated and a calculated pA2 value of 9.2 for prazosin in the presence of clonidine confirms clonidine as an agonist at alpha 1A receptors in proximal segments of rat-tail artery.4. Our work suggests that clonidine and moxonidine are promiscuous compounds at micromolar concentrations and that harmane and rilmenidine are more selective compounds for in vivo imidazoline research.*This work represents a portion of a dissertation to be submitted to the School of Graduate Studies, Loma Linda University, for the degree of Doctor of Philosophy.     

Intestinal sugar absorption is regulated by phosphorylation and turnover of protein kinase C betaII mediated by phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent pathways

Stimulation of intestinal fructose absorption by phorbol 12-myristate 13-acetate (PMA) results from rapid insertion of GLUT2 into the brush-border membrane and correlates with protein kinase C (PKC) betaII activation. We have therefore investigated the role of phosphatidylinositol 3 (PI3)-kinase and mammalian target of rapamycin in the regulation of fructose absorption by PKC betaII phosphorylation. In isolated jejunal loops, stimulation of fructose absorption by PMA was inhibited by preperfusion with wortmannin or rapamycin, which blocked GLUT2 activation and insertion into the brush-border membrane. Antibodies to the last 18 and last 10 residues of the C-terminal region of PKC betaII recognized several species differentially in Western blots. Extensive cleavage of native enzyme (80/78 kDa) to a catalytic domain product of 49 kDa occurred. PMA and sugars provoked turnover and degradation of PKC betaII by dephosphorylation to a 42-kDa species, which was converted to polyubiquitylated species detected at 180 and 250+ kDa. PMA increased the level of the PKC betaII 49-kDa species, which correlates with the GLUT2 level; wortmannin and rapamycin blocked these effects of PMA. Rapamycin and wortmannin inhibited PKC betaII turnover. PI3-kinase, PDK-1, and protein kinase B were present in the brush-border membrane, where their levels were increased by PMA and blocked by the inhibitors. We conclude that GLUT2-mediated fructose absorption is regulated through PI3-kinase and mammalian target of rapamycin-dependent pathways, which control phosphorylation of PKC betaII and its substrate-induced turnover and ubiquitin-dependent degradation. These findings suggest possible mechanisms for short term control of intestinal sugar absorption by insulin and amino acids.     

Macrophage colony-stimulating factor induces the expression of mitogen-activated protein kinase phosphatase-1 through a protein kinase C-dependent pathway.

M-CSF triggers the activation of extracellular signal-regulated protein kinases (ERK)-1/2. We show that inhibition of this pathway leads to the arrest of bone marrow macrophages at the G0/G1 phase of the cell cycle without inducing apoptosis. M-CSF induces the transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), which correlates with the inactivation of ERK-1/2. Because the time course of ERK activation must be finely controlled to induce cell proliferation, we studied the mechanisms involved in the induction of MKP-1 by M-CSF. Activation of ERK-1/2 is not required for this event. Therefore, M-CSF activates ERK-1/2 and induces MKP-1 expression through different pathways. The use of two protein kinase C (PKC) inhibitors (GF109203X and calphostin C) revealed that M-CSF induces MKP-1 expression through a PKC-dependent pathway. We analyzed the expression of different PKC isoforms in bone marrow macrophages, and we only detected PKCbetaI, PKCepsilon, and PKCzeta. PKCzeta is not inhibited by GF109203X/calphostin C. Of the other two isoforms, PKCepsilon is the best candidate to mediate MKP-1 induction. Prolonged exposure to PMA slightly inhibits MKP-1 expression in response to M-CSF. In bone marrow macrophages, this treatment leads to a complete depletion of PKCbetaI, but only a partial down-regulation of PKCepsilon. Moreover, no translocation of PKCbetaI or PKCzeta from the cytosol to particulate fractions was detected in response to M-CSF, whereas PKCepsilon was constitutively present at the membrane and underwent significant activation in M-CSF-stimulated macrophages. In conclusion, we remark the role of PKC, probably isoform epsilon, in the negative control of ERK-1/2 through the induction of their specific phosphatase.     

Protein kinase C zeta plays a central role in activation of the p42/44 mitogen-activated protein kinase by endotoxin in alveolar macrophages

Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC zeta, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC zeta, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK. LPS did not activate Raf-1, the classic activator of MEK. Pseudosubstrate-specific peptides with attached myristic acid are cell permeable and can be used to block the activity of specific PKC isoforms in vivo. We found that a peptide specific for PKC zeta partially blocked activation of both MEK and ERK by LPS. We also found that this peptide blocked in vivo phosphorylation of MEK after LPS treatment. In addition, we found that LPS caused PKC zeta to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC zeta. PKC zeta has been shown in other systems to be phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase. We found that LPS activates PI 3-kinase and causes the formation of a PKC zeta/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC zeta activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC zeta.     

Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras,protein kinase C,and protein kinase A in neuronal cells

The small GTPases Ras or Rap1 were suggested to mediate the stimulatory effect of some G protein-coupled receptors on ERK activity in neuronal cells. Accordingly, we reported here that pituitary adenylate cyclase-activating polypeptide (PACAP), whose G protein-coupled receptor triggers neuronal differentiation of the PC12 cell line via ERK1/2 activation, transiently activated Ras and induced the sustained GTP loading of Rap1. Ras mediated peak stimulation of ERK by PACAP, whereas Rap1 was necessary for the sustained activation phase. However, PACAP-induced GTP-loading of Rap1 was not sufficient to account for ERK activation by PACAP because 1) PACAP-elicited Rap1 GTP-loading depended only on phospholipase C, whereas maximal stimulation of ERK by PACAP also required the activity of protein kinase A (PKA), protein kinase C (PKC), and calcium-dependent signaling; and 2) constitutively active mutants of Rap1, Rap1A-V12, and Rap1B-V12 only minimally stimulated the ERK pathway compared with Ras-V12. The effect of Rap1A-V12 was dramatically potentiated by the concurrent activation of PKC, the cAMP pathway, and Ras, and this potentiation was blocked by dominant-negative mutants of Ras and Raf. Thus, this set of data indicated that GPCR-elicited GTP loading of Rap1 was not sufficient to stimulate efficiently ERK in PC12 cells and required the permissive co-stimulation of PKA, PKC, or Ras.     

CREB-dependent cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression is mediated by protein kinase C and calcium

Cellular production of prostaglandins (PGs) is controlled by the concerted actions of cyclooxygenases (COX) and terminal PG synthases on arachidonic acid in response to agonist stimulation. Recently, we showed in an ileal epithelial cell line (IEC-18), angiotensin II-induced COX-2-dependent PGI2 production through p38MAPK, and calcium mobilization (J. Biol. Chem. 280: 1582-1593, 2005). Agonist binding to the AT1 receptor results in activation of PKC activity and Ca2+ signaling but it is unclear how each pathway contributes to PG production. IEC-18 cells were stimulated with either phorbol-12,13-dibutyrate (PDB), thapsigargin (TG), or in combination. The PG production and COX-2 and PG synthase expression were measured. Surprisingly, PDB and TG produced PGE2 but not PGI2. This corresponded to induction of COX-2 and mPGES-1 mRNA and protein. PGIS mRNA and protein levels did not change. Activation of PKC by PDB resulted in the activation of ERK1/2, JNK, and CREB whereas activation of Ca2+ signaling by TG resulted in the delayed activation of ERK1/2. The combined effect of PKC and Ca2+ signaling were prolonged COX-2 and mPGES-1 mRNA and protein expression. Inhibition of PKC activity, MEK activity, or Ca2+ signaling blocked agonist induction of COX-2 and mPGES-1. Expression of a dominant negative CREB (S133A) blocked PDB/TG-dependent induction of both COX-2 and mPGES-1 promoters. Decreased CREB expression by siRNA blocked PDB/TG-dependent expression of COX-2 and mPGES-1 mRNA. These findings demonstrate a coordinated induction of COX-2 and mPGES-1 by PDB/TG that proceeds through PKC/ERK and Ca2+ signaling cascades, resulting in increased PGE2 production.     

Androgens activate mitogen-activated protein kinase signaling: role in neuroprotection

Recent evidence indicates that testosterone is neuroprotective, however, the underlying mechanism(s) remains to be elucidated. In this study, we investigated the hypothesis that androgens induce mitogen-activated protein kinase (MAPK) signaling in neurons, which subsequently drives neuroprotection. We observed that testosterone and its non-aromatizable metabolite dihydrotestosterone (DHT) rapidly and transiently activate MAPK in cultured hippocampal neurons, as evidenced by phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2. Importantly, pharmacological suppression of MAPK/ERK signaling blocked androgen-mediated neuroprotection against beta-amyloid toxicity. Androgen activation of MAPK/ERK and neuroprotection also was observed in PC12 cells stably transfected with androgen receptor (AR), but in neither wild-type nor empty vector-transfected PC12 cells. Downstream of ERK phosphorylation, we observed that DHT sequentially increases p90 kDa ribosomal S6 kinase (Rsk) phosphorylation and phosphorylation-dependent inactivation of Bcl-2-associated death protein (Bad). Prevention of androgen-induced phosphorylation of Rsk and Bad blocked androgen neuroprotection. These findings demonstrate AR-dependent androgen activation of MAPK/ERK signaling in neurons, and specifically identify a neuroprotective pathway involving downstream activation of Rsk and inactivation of Bad. Elucidation of androgen-mediated neural signaling cascades will provide important insights into the mechanisms of androgen action in brain, and may present a framework for therapeutic intervention of age-related neurodegenerative disorders.