Novel mechanism of cyclic AMP mediated extracellular signal regulated kinase activation in an intestinal cell line

The extracellular signal regulated kinase (ERK1/2) signaling cascade has been implicated as both a pro-apoptotic and anti-apoptotic pathway depending on cell type and context. In the T84 intestinal epithelial cell line, cAMP activates ERK1/2 resulting in the inhibition of apoptosis. Cyclic-AMP signaling relies on the binding and activation of a cAMP binding protein. In most cell types, the majority of this signaling occurs through an isoform of protein kinase A (PKAI or PKAII). Despite evidence to the contrary, we hypothesized that ERK1/2 activation is through a PKA isoform. Pharmacological activators and inhibitors of PKA as well as siRNA were used to further interrogate this potential signaling pathway. Our results demonstrate that at doses sufficient to increase PKA activity, PKAII specific cAMP analogs activate ERK1/2 while PKAI analogs do not. Pharmacological inhibition of the PKAII regulatory subunit and catalytic subunit as well as siRNA knockdown of the catalytic subunit blocks ERK1/2 activation. We conclude that in the T84 cell line, cAMP binding to the PKAII regulatory subunit leads to the subsequent phosphorylation of ERK1/2 and provides insight into the mechanism of cAMP mediated survival signaling in the intestinal epithelium. These results directly implicate PKAII as a mediator of cell survival in T84 cells and provide evidence for an additional means by which cAMP can influence intestinal cell turnover.     

Modulation of nitric oxide-induced apoptotic death of HL-60 cells by protein kinase C and protein kinase A through mitogen-activated protein kinases and CPP32-like protease pathways.

To define the signaling pathways during NO-induced apoptotic events and their possible modulation by two protein kinase systems, we explored the involvement of three structurally related mitogen-activated protein kinase subfamilies. Exposure of HL-60 cells to sodium nitroprusside (SNP) strongly activated p38 kinase, but did not activate c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In addition, SNP-induced apoptosis was markedly blocked by the selective p38 kinase inhibitor (SB203580) but not by MEK1 kinase inhibitor (PD098059), indicating that p38 kinase serves as a mediator of NO-induced apoptosis. In contrast, treatment of cells with phorbol 12-myristate 13-acetate (PMA) strongly activated not only JNK but also ERK, while not affecting p38 kinase. However, although SNP by itself weakly activated CPP32-like protease, SNP in combination with PMA markedly increased the extent of CPP32-like protease activation. Interestingly, N6,O2-dibutylyl cAMP (DB-cAMP) significantly blocked SNP- or SNP plus PMA-induced activation of CPP32-like protease and the resulting induction of apoptosis. DB-cAMP also blocked PMA-induced JNK activation. Collectively, these findings demonstrate the presence of specific up- or down-modulatory mechanisms of cell death pathway by NO in which (1) p38 kinase serves as a mediator of NO-induced apoptosis, (2) PKC acts at the point and/or upstream of JNK and provides signals to potentiate NO-induced CPP32-like protease activation, and (3) PKA lies upstream of either JNK or CPP32-like protease to protect NO- or NO plus PMA-induced apoptotic cell death in HL-60 cells.     

Epac activation converts cAMP from a proliferative into a differentiation signal in PC12 cells

Elevation of the intracellular cAMP concentration ([cAMP]i) regulates metabolism, cell proliferation, and differentiation and plays roles in memory formation and neoplastic growth. cAMP mediates its effects mainly through activation of protein kinase A (PKA) as well as Epac1 and Epac2, exchange factors activating the small GTPases Rap1 and Rap2. However, how cAMP utilizes these effectors to induce distinct biological responses is unknown. We here studied the specific roles of PKA and Epac in neuroendocrine PC12 cells. In these cells, elevation of [cAMP]i activates extracellular signal-regulated kinase (ERK) 1/2 and induces low-degree neurite outgrowth. The present study showed that specific stimulation of PKA triggered ERK1/2 activation that was considerably more transient than that observed upon simultaneous activation of both PKA and Epac. Unexpectedly, the PKA-specific cAMP analog induced cell proliferation rather than neurite outgrowth. The proliferative signaling pathway activated by the PKA-specific cAMP analog involved activation of the epidermal growth factor receptor and ERK1/2. Activation of Epac appeared to extend the duration of PKA-dependent ERK1/2 activation and converted cAMP from a proliferative into an anti-proliferative, neurite outgrowth-promoting signal. Thus, the present study showed that the outcome of cAMP signaling can depend heavily on the set of cAMP effectors activated.     

Maximal beta3-adrenergic regulation of lipolysis involves Src and epidermal growth factor receptor-dependent ERK1/2 activation

Catecholamine-stimulated lipolysis is primarily a beta-adrenergic and cAMP-dependent event. In previous studies we established that the beta(3)-adrenergic receptor (beta(3)AR) in adipocytes utilizes a unique mechanism to stimulate extracellular signal-regulated kinases 1 and 2 (ERK) by direct recruitment and activation of Src kinase. Therefore, we investigated the role of the ERK pathway in adipocyte metabolism and found that the beta(3)AR agonist CL316,243 regulates lipolysis through both cAMP-dependent protein kinase (PKA) and ERK. Inhibition of PKA activity completely eliminated lipolysis at low (subnanomolar) CL316,243 concentrations and by 75-80% at higher nanomolar concentrations. The remaining 20-25% of PKA-independent lipolysis, as well as ERK activation, was abolished by inhibiting the activity of either Src (PP2 or small interfering RNA), epidermal growth factor receptor (EGFR with AG1478 or small interfering RNA), or mitogen-activated protein kinase kinase 1 or 2 (MKK1/2 with PD098059). PD098059 inhibited lipolysis by 53% in mice as well. Finally, the effect of estradiol, a reported acute activator of ERK and lipolysis, was also totally prevented by PP2, AG1478, and PD098059. These results suggest that ERK activation by beta(3)AR depends upon Src and epidermal growth factor receptor kinase activities and is responsible for the PKA-independent portion of the lipolytic response. Together these results illustrate the distinct and complementary roles for PKA and ERK in catecholamine-stimulated lipolysis.     

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.     

Modulation of CREB and NF-kappaB signal transduction by cannabinol in activated thymocytes

Cannabinoid compounds inhibit the cAMP signalling cascade in leukocytes. One of these compounds, cannabinol (CBN) has been shown to inhibit interleukin-2 (IL-2) expression and the activation of cAMP response element binding protein (CREB) and nuclear factor for immunoglobulin kappa chain in B cells (NF-kappaB) following phorbol-12-myristate-13 acetate (PMA) plus ionomycin (Io) treatment of thymocytes. Therefore, the objective of the present studies was to determine the role of cAMP and protein kinase A (PKA) in the CBN-mediated inhibition of IL-2, CREB, and NF-kappaB in PMA/Io-activated thymocytes. The inhibition of CREB/ATF-1 phosphorylation, or cAMP response element (CRE) or kappaB DNA binding activity produced by CBN in PMA/Io-activated thymocytes, could not be reversed by DBcAMP costimulation. Furthermore, DBcAMP failed to reverse the concentration-dependent inhibition of IL-2 protein secretion by CBN. Pretreatment of thymocytes with H89 produced a modest inhibition of PMA/Io-induced CREB/ATF-1 phosphorylation and CRE DNA binding activity but H89 had no effect on protein binding to a kappaB motif. Additionally, H89 modestly inhibited PMA/Io-induced IL-2 secretion. In light of the modest involvement of the cAMP pathway in CBN-mediated inhibition of CREB and IL-2 in PMA/Io-activated thymocytes, PD098059 (PD), the MEK inhibitor, was utilized to determine the role of ERK MAP kinases in thymocytes. ERKs play a critical role in IL-2 production but not for CREB phsophorylation. Collectively, these findings suggest that CBN may modulate several signalling pathways in activated T cells.     

PKA independent and cell type specific activation of the expression of caudal homeobox gene Cdx-2 by cyclic AMP

Cdx-2 is a transactivator for the proglucagon gene in pancreatic and intestinal endocrine cells. Cdx-2 is also expressed in differentiated intestinal epithelia of nonendocrine origin. Cdx-2-/- mice are embryonic lethal, while Cdx-2+/- mutants show multiple malfunctions including the formation of intestinal polyps. Within the polyps, the remaining wild type Cdx-2 allele ceases its expression, while the expression of both Cdx-2 and proglucagon in the endocrine cells remains unaltered, indicating that Cdx-2 could be haplo-insufficient for nonendocrine cells, but not for proglucagon producing endocrine cells. We propose that mechanisms underlying Cdx-2 expression and auto-regulation [Xu F, Li H & Jin T (1999), J Biol Chem274, 34310-34316] differ in these two types of cells. We show here that forskolin and cAMP upregulate Cdx-2 expression in proglucagon producing cells, but not in colon cancer cells and primary intestinal cell cultures. It is unlikely that the activation is mainly mediated by PKA, because the activation was observed in a PKA deficient cell line. Co-transfecting a dominant negative Ras expression plasmid substantially repressed the Cdx-2 promoter, in contrast to a previous finding that Ras is a negative factor for Cdx-2 expression in colon cancer cells. Furthermore, forskolin activated ERK1/2 phosphorylation in the endocrine cells, and attenuation of ERK1/2 phosphorylation by its inhibitor is associated with attenuated Cdx-2 expression. Finally, an Epac pathway specific cAMP analogue stimulated both ERK1/2 phosphorylation and Cdx-2 expression. Taken together, our observations suggest that Cdx-2 expression is regulated by the second messenger cAMP, cell-type specifically, via the Epac pathway.     

Erk-dependent cytosolic phospholipase A2 activity is induced by CD95 ligand cross-linking in the mouse derived Sertoli cell line TM4 and is required to trigger apoptosis in CD95 bearing cells

In the present study we demonstrated that CD95L cross-linking generated reverse signalling in the mouse derived Sertoli cell line TM4. Treatment of TM4 cells with mAb anti-CD95L induced activation of the cytosolic phospholipase A2 (cPLA2). Cytosolic PLA2 activation was controlled by the MAPK pathway as indicated by the ability of the specific MEK inhibitor, PD098059, to abolish cPLA2 activation. In addition, Western blot experiments showed a rapid increase in phosphorylated Erk1/2 following CD95L cross-linking, while no effect on the phosphorylation of other MAPK, p38 or JNK, was observed. CD95L cross-linking by mAb increased the levels of soluble CD95L and apoptotic activity of TM4 cell supernatants, which was blocked by co-incubation with the PLA2 inhibitor, AACOCF3 or PD098059. Finally, pre-treatment of TM4 cells with AACOCF3 or PD098059 completely abolished TM4-induced apoptosis of Jurkat T cells, thus indicating that the Erk/cPLA2 pathway is required for CD95L-induced apoptosis.     

Mitogen-activated protein kinase activation by stimulation with thyrotropin-releasing hormone in rat pituitary GH3 cells.

We examined whether mitogen-activated protein (MAP) kinase is activated by thyrotropin-releasing hormone (TRH) in GH3 cells, and whether MAP kinase activation is involved in secretion of prolactin from these cells. Protein kinase inhibitors--such as PD098059, calphostin C, and genistein--and removal of extracellular Ca2+ inhibited MAP kinase activation by TRH. A cAMP analogue activated MAP kinase in these cells. Effects of cAMP on MAP kinase activation were inhibited by PD098059. TRH-induced prolactin secretion was not inhibited by levels of PD098059 sufficient to i activation but was inhibited by wortmannin (1 microM) and KN93. Treatment of GH3 cells with either TRH or cAMP significantly inhibited DNA synthesis and induced morphological changes. The effects stimulated by TRH were reversed by PD098059 treatment, but the same effects stimulated by cAMP were not. Treatment of GH3 cells with TRH for 48 h significantly increased the prolactin content in GH3 cells and decreased growth hormone content. The increase in prolactin was completely abolished by PD098059, but the decrease in growth hormone was not. These results suggest that TRH-induced MAP kinase activation is involved in prolactin synthesis and differentiation of GH3 cells, but not in prolactin secretion.     

Initiation of cell locomotility is a morphogenetic checkpoint in thyroid epithelial cells regulated by ERK and PI3-kinase signals.

Epithelial locomotility is a fundamental determinant of tissue patterning that is subject to strict physiological regulation. The current study sought to identify cellular signals that initiate cell migration in cultured thyroid epithelial cells. Porcine thyroid cells cultured as 3-dimensional follicles convert to 2-dimensional monolayers when deprived of agents that stimulate cAMP/PKA signaling. This morphogenetic event is driven by the activation of cell-on-substrate locomotility, providing a convenient assay for events that regulate the initiation of locomotion. In this system, the extracellular signal regulated kinase (ERK) pathway became activated as follicles converted to monolayer, as demonstrated by immunoblotting for activation-specific phosphorylation and nuclear accumulation of ERK. Inhibition of ERK activation using the drug PD98059 effectively prevented cells from beginning to migrate. PD98059 inhibited cell spreading, actin filament reorganization and the assembly of focal adhesions, cellular events that mediate the initiation of thyroid cell locomotility. Akt (PKB) signaling was also activated during follicle-to-monolayer conversion and the phosphoinositide 3-kinase (PI3-kinase) inhibitor, wortmannin, also blocked the initiation of cell movement. Wortmannin did not, however, block activation of ERK signaling. These findings, therefore, identify the ERK and PI3-kinase signaling pathways as important stimulators of thyroid cell locomotility. These findings are incorporated into a model where the initiation of thyroid cell motility constitutes a morphogenetic checkpoint regulated by coordinated changes in stimulatory (ERK, PI3-kinase) and tonic inhibitory (cAMP/PKA) signaling pathways.     

Angiotensin-(1-7)-induced activation of ERK1/2 is cAMP/protein kinase A-dependent in glomerular mesangial cells

The renin-angiotensin system (RAS) plays an important role in renal physiology and kidney injury. Although the cellular effects of the RAS activation are generally attributed to angiotensin II (ANG II), the recent identification of angiotensin-converting enzyme 2 has shifted the focus to other peptides including Ang-(1-7). The G protein-coupled receptor for Ang-(1-7), mas, is expressed by mesangial cells (MC) but the signal transduction pathways activated by Ang-(1-7) in MC have not been fully elucidated. Accordingly, we studied the effect of Ang-(1-7) on extracellular signal-related kinase (ERK)1/2 activation in rat MC. Ang-(1-7)-induced ERK1/2 phosphorylation in MC is time- and concentration-dependent. Pretreatment of MC with the mas receptor antagonist A-779 but not the AT(1) antagonist losartan or the AT(2) antagonist PD123319 abrogated ERK1/2 activation. Neither pretreatment with the NADPH oxidase inhibitors diphenyleneiodonium and apocynin nor pretreatment with the epidermal growth factor (EGF) receptor antagonists AG1478 and PD158780 attenuated Ang-(1-7)-induced activation of ERK1/2. Even though each of these compounds abolished ANG II-induced activation of ERK1/2. Ang-(1-7) increased intracellular cAMP levels and activated protein kinase A (PKA) and inhibition of either adenylyl cyclase or PKA activity attenuated Ang-(1-7)-induced ERK1/2 activation. In conclusion, Ang-(1-7)-induced activation of ERK1/2 is cAMP/PKA-dependent in MC, but independent of NADPH oxidase and the EGF receptor.     

Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation

Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.     

Mitogen-activated protein kinase activation and regulation of cyclooxygenase 2 expression by platelet-activating factor and hCG in human endometrial adenocarcinoma cell line HEC-1B

The activation of mitogen-activated protein kinase (MAP kinase) and the regulation of cyclooxygenase 2 (COX-2) were investigated in the human endometrial adenocarcinoma cell line HEC-1B by treatment with platelet-activating factor (PAF) and hCG. Pre-treatment of the cells with both oestradiol and medroxyprogesterone acetate was required for MAP kinase activation and COX-2 expression to respond to PAF and hCG. PAF-induced MAP kinase activation was sensitive to MAP kinase kinase (MEK) inhibitor, PD098059, and phosphatidylinositol-3-OH kinase (PI3K) inhibitor, wortmannin. In contrast, hCG-induced MAP kinase activation was sensitive to PD098059 and protein kinase A inhibitor, H-89, but not to wortmannin. PAF-induced COX-2 expression was insensitive to PD098059 but sensitive to wortmannin, whereas hCG-induced COX-2 expression was sensitive to PD098059 and H-89 but insensitive to wortmannin. 8-(4-chlorophenylthio)-cAMP, a potent cAMP analogue, induced activation of MAP kinase and expression of COX-2. These results indicate that MAP kinase is activated with PAF and hCG in HEC-1B cells. In addition, COX-2 expression is stimulated through the MAP kinase activation pathway with hCG and the wortmannin sensitive pathway with PAF in HEC-1B cells. These results also imply that protein kinase A remains upstream of hCG-induced activation of MAP kinase in HEC-1B cells.     

Role of cAMP-dependent pathway in eosinophil apoptosis and survival

The survival and apoptosis of eosinophils is of pivotal importance for controlling allergic diseases such as asthma and rhinitis. In this study we have investigated the role for cAMP in regulating eosinophil survival and apoptosis in the absence of eosinophil-active cytokines. The treatment with dibutyryl cyclic AMP (dbcAMP) increased eosinophil survival with a concomitant decrease of apoptosis in a dose-dependent manner. The pretreatment with a protein kinase A (PKA) inhibitor blocked the effects of dbcAMP on survival and apoptosis of eosinophils. The catalytic subunit of PKA was translocated to nucleus in parallel with a robust increase of intracellular cAMP levels upon exposure to dbcAMP but not IL-5, suggesting the separation of PKA activation from the IL-5-induced suppression of eosinophil apoptosis. When eosinophils were treated with pharmacological inhibitors of protein kinases prior to exposure to dbcAMP or IL-5, only the mitogen-activating protein kinase (MAPK) inhibitor, PD098059, was partly able to block dbcAMP-induced augmentation of eosinophil viability, whereas both Janus kinase 2 and MAPK inhibitors effectively interrupted the IL-5-induced prolongation of eosinophil survival. The effects of dbcAMP and these protein kinase inhibitors on eosinophil apoptosis were confirmed by morphologic analysis. We propose that a cAMP-dependent pathway may constitute an important component for regulating eosinophil survival/apoptosisand that cAMP may inhibit eosinophil apoptosis through the activation of PKA and of subsequent MAPK in part.     

cAMP activates TRPC6 channels via the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB)-mitogen-activated protein kinase kinase (MEK)-ERK1/2 signaling pathway

cAMP is an important second messenger that executes diverse physiological function in living cells. In this study, we investigated the effect of cAMP on canonical TRPC6 (transient receptor potential channel 6) channels in TRPC6-expressing HEK293 cells and glomerular mesangial cells. The results showed that 500 μm 8-Br-cAMP, a cell-permeable analog of cAMP, elicited [Ca(2+)](i) increases and stimulated a cation current at the whole-cell level in TRPC6-expressing HEK293 cells. The effect of cAMP diminished in the presence of the PI3K inhibitors wortmannin and LY294002 or the MEK inhibitors PD98059, U0126, and MEK inhibitor I. 8-Br-cAMP also induced phosphorylation of MEK and ERK1/2. Conversion of serine to glycine at an ERK1/2 phosphorylation site (S281G) abolished the cAMP activation of TRPC6 as determined by whole-cell and cell-attached single-channel patch recordings. Experiments based on a panel of pharmacological inhibitors or activators suggested that the cAMP action on TRPC6 was not mediated by PKA, PKG, or EPAC (exchange protein activated by cAMP). Total internal fluorescence reflection microscopy showed that 8-Br-cAMP did not alter the trafficking of TRPC6 to the plasma membrane. We also found that, in glomerular mesangial cells, glucagon-induced [Ca(2+)](i) increases were mediated through the cAMP-PI3K-PKB-MEK-ERK1/2-TRPC6 signaling pathway. In summary, this study uncovered a novel TRPC6 activation mechanism in which cAMP activates TRPC6 via the PI3K-PKB-MEK-ERK1/2 signaling pathway.     

A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation

Classically, the FSH receptor (FSH-R) mediates its effects through coupling to guanine nucleotide-binding protein alpha S subunit (Galpha(s)) and activation of the cAMP/protein kinase A (PKA) signaling pathway. beta-Arrestins are rapidly recruited to the FSH-activated receptor and play key roles in its desensitization and internalization. Here, we show that the FSH-R expressed in HEK 293 cells activated ERK by two temporally distinct pathways dependent, respectively, on Galpha(s)/PKA and beta-arrestins. Galpha(s)/PKA-dependent ERK activation was rapid, transient, and blocked by H89 (a PKA inhibitor), but it was insensitive to small interfering RNA-mediated depletion of beta-arrestins. beta-Arrestin-dependent ERK activation was slower but more sustained and was insensitive to H89. We identified five Ser/Thr residues in the C terminus of the receptor (638-644) as a major phosphorylation site. Mutation of these residues into Ala (5A FSH-R) significantly reduced the stability of FSH-induced beta-arrestin 1 and 2 interaction when compared with the wild-type receptor. As expected, the 5A FSH-R-mediated cAMP accumulation was enhanced, and its internalization was reduced. In striking contrast, the ability of the 5A FSH-R to activate ERK via the beta-arrestin-dependent pathway was increased. G protein-coupled receptor kinase 5 (GRK5) and GRK6 were required for beta-arrestin-dependent ERK activation by both the wild-type and 5A FSH-R. By contrast, GRK2 depletion enhanced ERK activation by the wild-type FSH-R but not by the 5A FSH-R. In conclusion, we demonstrate the existence of a beta-arrestin-dependent, GRK-regulated mechanism for ERK activation by the FSH-R. A phosphorylation cluster in the C terminus of the FSH-R, identified as a site of beta-arrestin recruitment, positively regulated both desensitization and internalization but negatively regulated beta-arrestin-dependent ERK activation.     

A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase c pathways

Mitogen-activated protein kinase (MAPK) cascades underlie long-term mitogenic, morphogenic, and secretory activities of purinergic receptors. In HEK-293 cells, N-ethylcarboxamidoadenosine (NECA) activates endogenous A2BARs that signal through Gs and Gq/11. UTP activates P2Y2 receptors and signals only through Gq/11. The MAPK isoforms, extracellular-signal regulated kinase 1/2 (ERK), are activated by NECA and UTP. H-89 blocks ERK activation by forskolin, but weakly affects the response to NECA or UTP. ERK activation by NECA or UTP is unaffected by a tyrosine kinase inhibitor (genistein), attenuated by a phospholipase C inhibitor (U73122), and is abolished by a MEK inhibitor (PD098059) or dominant negative Ras. Inhibition of protein kinase C (PKC) by GF 109203X failed to block ERK activation by NECA or UTP, however, another PKC inhibitor, Ro 31-8220, which unlike GF 109203X, can block the zeta-isoform, and prevents UTP- but not NECA-induced ERK activation. In the presence of forskolin, Ro 31-8220 loses its ability to block UTP-stimulated ERK activation. PKA has opposing effects on B-Raf and c-Raf-1, both of which are found in HEK-293 cells. The data are explained by a model in which ERK activity is modulated by differential effects of PKC zeta and PKA on Raf isoforms.     

Galactose specific adhesin of enteroaggregative E. coli induces IL-8 secretion via activation of MAPK and STAT-3 in INT-407 cells

Background

Enteroaggregative Escherichia coli (EAEC) is one of the most common bacterial pathogens associated with the etiology of persistent diarrhea. A characteristic feature of EAEC-pathogenesis is the induction of profound inflammatory response in the intestinal epithelium. The present study was designed to investigate the underlying mechanism of inflammatory responses induced by a novel galactose specific adhesin of T7 strain of EAEC (EAEC-T7) in human intestinal epithelial cell line (INT-407).

Methods

INT-407 cells were stimulated with the adhesin in the absence and presence of anti-adhesin (IgG_AD)/d-galactose/H7/staurosporin (inhibitor of PKC)/PD098059 (inhibitor of MEK)/SB203580 (inhibitor of p38-MAPkinase)/AG490 (inhibitor of JAK (-2,-3)/STAT-3 pathway). The expression of activated Raf-1, MEK-1, ERK1/2, JNK, p38-MAPK and STAT-3 was analyzed by Western immunoblot. Release of interleukin-8 (IL-8) was measured by ELISA.

Results

The adhesin was found to induce activation of Raf-1, MEK-1, ERK1/2, p38-MAPK and STAT-3, which was reduced in the presence of IgG_AD/d-galactose. The activation of Raf-1 was found to be attenuated in the presence of H7/staurosporin. The expression of phosphorylated STAT-3 was downregulated in the presence of AG490 and PD098059. Further, the adhesin induced IL-8 secretion was reduced in the presence of the inhibitors of MEK (PD098059), p38-MAPK (SB203580) and JAK (-2,-3)/STAT-3 pathway (AG490).

Conclusions

We propose that STAT-3 activation is quintessential for the galactose specific adhesin induced IL-8 secretion by INT-407 cells and must occur in concert with the activation of ERK1/2.

General significance

Our contribution regarding the galactose specific adhesin mediated signaling leads to an improved understanding of the EAEC-pathogenesis and may provide novel therapeutic approaches to combat EAEC infection.     

Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38 MAPK, pathways

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-kappaB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IkappaBalpha phosphorylation, IkappaBalpha degradation, and the formation of a NF-kappaB-specific DNA-protein complex. The PMA-mediated increase in kappaB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.