Anisomycin induces COX-2 mRNA expression through p38(MAPK) and CREB independent of small GTPases in intestinal epithelial cells

Cyclooxygenase (COX)-2 expression in intestinal epithelial cells is associated with colorectal carcinogenesis. COX-2 expression is induced by numerous growth factors and gastrointestinal hormones through multiple protein kinase cascades. Here, the role of mitogen activated protein kinases (MAPKs) and small GTPases in COX-2 expression was investigated. Anisomycin and sorbitol induced COX-2 expression in non-transformed, intestinal epithelial IEC-18 cells. Both anisomycin and sorbitol activated p38(MAPK) followed by phosphorylation of CREB. SB202190 and PD169316 but neither PD98059 nor U0126 blocked COX-2 expression and CREB phosphorylation by anisomycin or sorbitol. Clostridium difficile toxin B inhibition of small GTPases did not affect anisomycin-induced COX-2 mRNA expression or phosphorylation of p38MAPK and CREB but did inhibit sorbitol-dependent COX-2 expression and phosphorylation of p38MAPK and CREB. Angiotensin (Ang) II-dependent induction of COX-2 mRNA and induced phosphorylation of p38MAPK and CREB were inhibited by toxin B. Reduction of CREB protein in cells transfected with CREB siRNAs inhibited anisomycin-induced COX-2 expression. These results indicate that activation of p38MAPK signaling is sufficient for COX-2 expression in IEC-18 cells. Ang II and sorbitol require small GTPase activity for COX-2 expression via p38MAPK while anisomycin-induced COX-2 expression by p38MAPK does not require small GTPases. This places small GTPase activity down-stream of the AT1 receptor and hyperosmotic stress and up-stream of p38MAPK and CREB.     

Inhibition of a signaling pathway in cardiac muscle cells by active mitogen-activated protein kinase kinase.

Signaling via the Ras pathway involves sequential activation of Ras, Raf-1, mitogen-activated protein kinase kinase (MKK), and the extracellular signal-regulated (ERK) group of mitogen-activated protein (MAP) kinases. Expression from the c-Fos, atrial natriuretic factor (ANF), and myosin light chain-2 (MLC-2) promoters during phenylephrine-induced cardiac muscle cell hypertrophy requires activation of this pathway. Furthermore, constitutively active Ras or Raf-1 can mimic the action of phenylephrine in inducing expression from these promoters. In this study, we tested whether constitutively active MKK, the molecule immediately downstream of Raf, was sufficient to induce expression. Expression of constitutively active MKK induce ERK2 kinase activity and caused expression from the c-Fos promoter, but did not significantly activate expression of reporter genes under the control of either the ANF or MLC-2 promoters. Expression of CL100, a phosphatase that inactivates ERKs, prevented expression from all of the promoters. Taken together, these data suggest that ERK activation is required for expression from the Fos, ANF, and MLC-2 promoters but MKK and ERK activation is sufficient for expression only from the Fos promoter. Constitutively active MKK synergized with phenylephrine to increase expression from a c-Fos- or an AP1-driven reporter. However, active MKK inhibited phenylephrine- and Raf-1-induced expression from the ANF and MLC-2 promoters. A DNA sequence in the MLC-2 promoter that is a target for inhibition by active MKK, but not CL100, was mapped to a previously characterized DNA element (HF1) that is responsible for cardiac specificity. Thus, activation of cardiac gene expression during phenylephrine-induced hypertrophy requires ERK activation but constitutive activation by MKK can inhibit expression by targeting a DNA element that controls the cardiac specificity of gene expression.     

Reciprocal signaling between heterotrimeric G proteins and the p21-stimulated protein kinase.

p21-activated protein kinase (PAK)-1 phosphorylated Galpha(z), a member of the Galpha(i) family that is found in the brain, platelets, and adrenal medulla. Phosphorylation approached 1 mol of phosphate/mol of Galpha(z) in vitro. In transfected cells, Galpha(z) was phosphorylated both by wild-type PAK1 when stimulated by the GTP-binding protein Rac1 and by constitutively active PAK1 mutants. In vitro, phosphorylation occurred only at Ser(16), one of two Ser residues that are the major substrate sites for protein kinase C (PKC). PAK1 did not phosphorylate other Galpha subunits (i1, i2, i3, o, s, or q). PAK1-phosphorylated Galpha(z) was resistant both to RGSZ1, a G(z)-selective GTPase-activating protein (GAP), and to RGS4, a relatively nonselective GAP for the G(i) and G(q) families of G proteins. Phosphorylation of Ser(27) by PKC did not alter sensitivity to either GAP. The previously described inhibition of G(z) GAPs by PKC is therefore mediated by phosphorylation of Ser(16). Phosphorylation of either Ser(16) by PAK1 or Ser(27) by PKC decreased the affinity of Galpha(z) for Gbetagamma; phosphorylation of both residues by PKC caused no further effect. PAK1 thus regulates Galpha(z) function by attenuating the inhibitory effects of both GAPs and Gbetagamma. In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by Gbetagamma, suggesting that PAK1 acts as a Gbetagamma-regulated effector protein. This inhibition of mammalian PAK1 by Gbetagamma contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the Gbetagamma homolog Ste4p/Ste18p.     

Galphaq signaling is required for Rho-dependent transcriptional activation of the cyclooxygenase-2 promoter in fibroblasts

Previously, we demonstrated that the gastrin releasing peptide (GRP) induces cyclooxygenase-2 (COX-2) expression through a Rho-dependent, protein kinase C (PKC)-independent signaling pathway in fibroblasts (Slice et al., 1999, J Biol Chem 274:27562-27566). However, the specific role of heterotrimeric guanine nucleotide binding regulatory proteins (G-proteins) that are coupled to the GRP receptor in Rho-dependent COX-2 expression has not been elucidated. In this report, we utilize embryonic fibroblasts from transgenic mice containing double gene knock-outs (DKO) for Galpha(q/11) and Galpha(12/13) to demonstrate that COX-2 promoter activation by GRP requires Galpha(q). Furthermore, we show that GRP-dependent COX-2 gene expression, as assessed by a COX-2 reporter luciferase assay, was induced in cells lacking Galpha(12/13) but was blocked in cells that did not express Galpha(q/11). GRP-dependent COX-2 promoter induction in Galpha(q/11) deficient cells was rescued by expression of wild type Galpha(q) but blocked by inhibition of calcium signaling in calcium-free media or in cells treated with 2-aminoethoxydiphenylborate (2-APB). Co-stimulation of transfected Galpha(q/11) deficient cells with GRP and thapsigargin (TG) induced the COX-2 promoter. Activation of endogenous Rho by expression of Onco-lbc or expression of Rho A Q63L resulted in COX-2 promoter activation in Galpha(q/11) deficient cells. Inhibition of Rho by Clostridium botulinum C3 toxin blocked COX-2 promoter induction. Expression of Galpha(q) Q209L in the well-characterized fibroblast cell line, NIH3T3, induced the COX-2 promoter which was blocked by expression of C3 toxin. These results demonstrate that calcium signaling mediated by Galpha(q) and Rho play critical roles in GRP-dependent COX-2 expression in fibroblasts.     

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.     

G(i)-dependent activation of c-Jun N-terminal kinase in human embryonal kidney 293 cells

Heterotrimeric G proteins stimulate the activities of two stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase in mammalian cells. In this study, we examined whether alpha subunits of G(i) family activate JNK using transient expression system in human embryonal kidney 293 cells. Constitutively activated mutants of Galpha(i1), Galpha(i2), and Galpha(i3) increased JNK activity. In contrast, constitutively activated Galpha(o) and Galpha(z) mutants did not stimulate JNK activity. To examine the mechanism of JNK activation by Galpha(i), kinase-deficient mutants of mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7), which are known to be JNK activators, were transfected into the cells. However, Galpha(i)-induced JNK activation was not blocked effectively by kinase-deficient MKK4 and MKK7. In addition, activated Galpha(i) mutant failed to stimulate MKK4 and MKK7 activities. Furthermore, JNK activation by Galpha(i) was inhibited by dominant-negative Rho and Cdc42 and tyrosine kinase inhibitors, but not dominant-negative Rac and phosphatidylinositol 3-kinase inhibitors. These results indicate that Galpha(i) regulates JNK activity dependent on small GTPases Rho and Cdc42 and on tyrosine kinase but not on MKK4 and MKK7.     

Activation of p21-activated kinase 6 by MAP kinase kinase 6 and p38 MAP kinase

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.     

RGS17/RGSZ2, a novel regulator of Gi/o, Gz, and Gq signaling

To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.     

Differential regulation of cyclooxygenase 2 expression by small GTPases Ras, Rac1, and RhoA

Cyclooxygenase 2 (COX-2) is an immediate early gene induced by a variety of stimuli and its expression is stimulated by individual activation of Ras or Rho GTPases. Here we investigate the role of coordinate activation of Ras and Rho GTPases in the induction of COX-2. Individual expression of constitutively active Ras, RhoA, or Rac1 was capable of stimulating COX-2 expression in NIH3T3 cells, but co-expression of constitutively active RhoA with either constitutively active Ras or Rac1 was required for full stimulation of COX-2 expression. Serum growth factors differentially activated Ras, RhoA, and Rac1, which correlated with the activation of Raf-1, ERK, and c-Jun as well as with induction of COX-2. Inhibition of Ras significantly blocked the activation of Raf-1, ERK, and c-Jun and the stimulation of COX-2 expression in response to serum. In contrast, inhibition of Rho family GTPases partially blocked serum induction of ERK activation but had little effects on COX-2 expression. Both inhibitors of MEK (PD098059) and JNK (SP600125) inhibited serum induction of COX-2. PD98059 only inhibited constitutively active Ras-induced COX-2 expression, while SP600125 significantly inhibited both constitutively active Ras- and RhoA-induced COX-2 expression. Together, our data suggest that constitutively active oncogenic Ras and Rho coordinately stimulate COX-2 expression whereas transient activation of Ras but not RhoA or Rac1 mediates the induction of COX-2 in response to serum. Furthermore, ERK and JNK activation are both required for serum- and oncogenic Ras-mediated COX-2 expression whereas only JNK activation is required for oncogenic RhoA-mediated stimulation of COX-2 expression.     

The synergistic induction of cyclooxygenase-2 in lung fibroblasts by angiotensin II and pro-inflammatory cytokines

Although we have demonstrated that Angiotensin II (Ang II) signaling plays a role in colon and lung tumorigenesis, the precise mechanisms by which Ang II stimulates tumorigenesis remain unclear. The aim of this study was to investigate the synergistic induction of COX-2 by Ang II and pro-inflammatory cytokines in lung fibroblasts. We also compared the efficiencies of Ang II-dependent COX-2 induction in lung epithelial cells and stromal cells. Ang II induced COX-2 expression in lung fibroblasts in a dose-dependent manner (10(-9) to 10(-7) M) through the Ang II subtype 1 receptor (AT(1)). In addition, Ang II synergistically stimulated the induction of COX-2 by pro-inflammatory cytokines, IL-1beta, or TNF-alpha. Our results indicate that the pro-tumorigenic function of Ang II is attributable, in part, to its strong stimulatory effect of COX-2 expression in lung fibroblasts in which synergistic stimulation with pro-inflammatory cytokines was evident. It is also suggested that the AT(1) receptor in lung fibroblasts may be a rational target for chemoprevention of lung cancer.     

Mechanisms of regulation of phospholipase D1 and D2 by the heterotrimeric G proteins G13 and Gq.

Our earlier studies of rat brain phospholipase D1 (rPLD1) showed that the enzyme could be activated in cells by alpha subunits of the heterotrimeric G proteins G(13) and G(q). Recently, we showed that rPLD1 is modified by Ser/Thr phosphorylation and palmitoylation. In this study, we first investigated the roles of these post-translational modifications on the activation of rPLD1 by constitutively active Galpha(13)Q226L and Galpha(q)Q209L. Mutations of Cys(240) and Cys(241) of rPLD1, which abolish both post-translational modifications, did not affect the ability of either Galpha(13)Q226L or Galpha(q)Q209L to activate rPLD1. However, the RhoA-insensitive mutants, rPLD1(K946A,K962A) and rPLD1(K962Q), were not activated by Galpha(13)Q226L, although these mutant enzymes responded to phorbol ester and Galpha(q)Q209L. On the contrary, the PKC-insensitive mutant rPLD1(DeltaN168), which lacks the first 168 amino acids of rPLD1, responded to Galpha(13)Q226L but not to Galpha(q)Q209L. In addition, we found that rPLD2 was strongly activated by Galpha(q)Q209L and phorbol ester. However, surprisingly, the enzymatic activity of rPLD2 was suppressed by Galpha(13)Q226L and constitutively active V14RhoA in COS-7 cells. Abolition of the post-translational modifications of rPLD2 did not alter the effects of Galpha(q)Q209L or Galpha(13)Q226L. The suppressive effect of Galpha(13)Q226L on rPLD2 was reversed by dominant negative N19RhoA and the C3 exoenzyme of Clostridium botulinum, further supporting a role for RhoA. In summary, Galpha(13) activation of rPLD1 in COS-7 cells is mediated by Rho, while Galpha(q) activation requires PKC. rPLD2 is activated by Galpha(q), but is inhibited by Galpha(13). Neither Ser/Thr phosphorylation nor palmitoylation is required for these effects.     

Regulator of G protein signaling 8 (RGS8) requires its NH2 terminus for subcellular localization and acute desensitization of G protein-gated K+ channels

Functional roles of the NH(2)-terminal region of RGS (regulators of G protein signaling) 8 in G protein signaling were studied. The deletion of the NH(2)-terminal region of RGS8 (DeltaNRGS8) resulted in a partial loss of the inhibitory function in pheromone response of yeasts, although Galpha binding was not affected. To examine roles in subcellular distribution, we coexpressed two fusion proteins of RGS8-RFP and DeltaNRGS8-GFP in DDT1MF2 cells. RGS8-RFP was highly concentrated in nuclei of unstimulated cells. Coexpression of constitutively active Galpha(o) resulted in translocation of RGS8 protein to the plasma membrane. In contrast, DeltaNRGS8-GFP was distributed diffusely through the cytoplasm in the presence or absence of active Galpha(o). When coexpressed with G protein-gated inwardly rectifying K(+) channels, DeltaNRGS8 accelerated both turning on and off similar to RGS8. Acute desensitization of G protein-gated inwardly rectifying K(+) current observed in the presence of RGS8, however, was not induced by DeltaNRGS8. Thus, we, for the first time, showed that the NH(2) terminus of RGS8 contributes to the subcellular localization and to the desensitization of the G protein-coupled response.