Lee W. Slice

Activation of Protein Kinase D by Signaling through the α Subunit of the Heterotrimeric G Protein Gq

Protein kinase D (PKD/PKCμ) immunoprecipitated from COS-7 cells transiently transfected with either a constitutively active mutant of Rho (RhoQ63L) or the Rho-specific guanine nucleotide exchange factor pOnco-Lbc (Lbc) exhibited a marked increase in basal activity. Addition of aluminum fluoride to cells co-transfected with PKD and wild type Gα13 also induced PKD activation. Co-transfection of Clostridium botulinum C3 toxin blocked activation of PKD by RhoQ63L, Lbc, or aluminum fluoride-stimulated Gα13. Treatment with the protein kinase C inhibitors GF I or Ro 31-8220 prevented the increase in PKD activity induced by RhoQ63L, Lbc, or aluminum fluoride-stimulated Gα13. PKD activation in response to Gα13 signaling was also completely prevented by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Co-expression of C. botulinum C3 toxin and a COOH-terminal fragment of Gαq that acts in a dominant-negative fashion blocked PKD activation in response to agonist stimulation of bombesin receptor. Expression of the COOH-terminal region of Gα13 also attenuated PKD activation in response to bombesin receptor stimulation. Our results show that Gα13 contributes to PKD activation through a Rho- and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both Gαq and Gα13 in response to bombesin receptor stimulation.

Amino Acid-stimulated Ca2+ Oscillations Produced by the Ca2+-sensing Receptor Are Mediated by a Phospholipase C/Inositol 1,4,5-Trisphosphate-independent Pathway That Requires G12, Rho, Filamin-A, and the Actin Cytoskeleton

The G protein-coupled Ca2+-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca2+ and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca2+ concentration ([Ca2+]i). Here, we examined whether these differing patterns of [Ca2+]i oscillations produced by the CaR are mediated by separate signal transduction pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in single cells, we found that stimulation of CaR by an increase in the extracellular Ca2+ concentration ([Ca2+]o) leads to periodic synthesis of inositol 1,4,5-trisphosphate, whereas l-phenylalanine stimulation of the CaR does not induce any detectable change in the level this second messenger. Furthermore, we identified a novel pathway that mediates transient [Ca2+]i oscillations produced by the CaR in response to l-phenylalanine, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the G12 subfamily, the C-terminal region of the CaR, and the scaffolding protein filamin-A. Our model envisages that Ca2+ or amino acids stabilize unique CaR conformations that favor coupling to different G proteins and subsequent activation of distinct downstream signaling pathways.

Cooperation of Gq, Gi, and G12/13 in Protein Kinase D Activation and Phosphorylation Induced by Lysophosphatidic Acid

To examine the contribution of different G-protein pathways to lysophosphatidic acid (LPA)-induced protein kinase D (PKD) activation, we tested the effect of LPA on PKD activity in murine embryonic cell lines deficient in Gαq/11(Gαq/11 KO cells) or Gα12/13(Gα12/13 KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control. In RK and Gα12/13 KO cells, LPA induced PKD activation through a phospholipase C/protein kinase C pathway in a concentration-dependent fashion with maximal stimulation (6-fold for RK cells and 4-fold for Gα12/13 KO cells in autophosphorylation activity) achieved at 3 μm. In contrast, LPA did not induce any significant increase in PKD activity in Gαq/11 KO cells. However, LPA induced a significantly increased PKD activity when Gαq/11 KO cells were transfected with Gαq. LPA-induced PKD activation was modestly attenuated by prior exposure of RK cells to pertussis toxin (PTx) but abolished by the combination treatments of PTx and Clostridium difficile toxin B. Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Gα12/13 KO cells. Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an antibody that detects the phosphorylated state of this residue. Our results indicate that Gq is necessary but not sufficient to mediate LPA-induced PKD activation. In addition to Gq, LPA requires additional G-protein pathways to elicit a maximal response with Gi playing a critical role in Gα12/13 KO cells. We conclude that LPA induces PKD activation through Gq, Gi, and G12 and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways.

Angiotensin II and Epidermal Growth Factor Induce Cyclooxygenase-2 Expression in Intestinal Epithelial Cells through Small GTPases Using Distinct Signaling Pathways

Colorectal carcinogenesis is a multistep process involving genetic mutations and alterations in rigorously controlled signaling pathways and gene expression that control intestinal epithelial cell proliferation, differentiation, and apoptosis. Cyclooxygenase-2 (COX-2) is aberrantly expressed in premalignant adenomatous polyps and colorectal carcinomas and is associated with increased epithelial cell proliferation, decreased apoptosis, and increased cell invasiveness. Currently, knowledge of the regulation of expression of COX-2 by endogenous cell-surface receptors is inadequate. Recently, in a non-transformed rat intestinal epithelial cell line (IEC-18), we showed induction of cell proliferation and DNA synthesis by angiotensin II (Ang II) via the endogenous Ang II type 1 receptor (Chiu, T., Santiskulvong, C., and Rozengurt, E. (2003) Am. J. Physiol. 285, G1–G11). We report that Ang II potently stimulated expression of COX-2 mRNA and protein as an immediate-early gene response through the Ang II type 1 receptor, correlating with an increase in prostaglandin I2 production. Ang II induced Cdc42 activation and filopodial formation. COX-2 expression was induced by epidermal growth factor (EGF), which activated Rac with lamellipodial formation. Inhibition of small GTPases by Clostridium difficile toxin B blocked COX-2 expression by Ang II and EGF. Inhibition of ERK activation by U0126 or PD98059 significantly decreased EGF-dependent COX-2 expression, but did not affect Ang II-dependent COX-2 expression. Conversely, inhibition of p38MAPK by SB202190 or PD169316 inhibited COX-2 expression by Ang II, but did not block COX-2 induction by EGF. Ang II caused Ca2+ mobilization. Inhibition of Ca2+ signaling by 2-aminobiphenyl borate blocked Ang II-dependent COX-2 expression. EGF did not induce Ca2+ mobilization, and 2-aminobiphenyl borate did not inhibit EGF-dependent COX-2 expression. Inhibition of COX-2 expression correlated with inhibition of prostaglandin I2 production. Luciferase promoter assays showed that Ang II-dependent transcriptional activation of the COX-2 promoter was dependent on activation of small GTPases and p38MAPK and on Ca2+ signaling via the cAMP-responsive element/activating transcription factor cis-acting element.

Galpha(13) stimulates Rho-dependent activation of the cyclooxygenase-2 promoter.

Cyclooxygenase-2 (COX-2) gene expression is rapidly increased by cytokines, tumor promoters, and growth factors and is markedly enhanced in various cancer cells. Here, we examine the regulation of COX-2 promoter activity by α subunits of heterotrimeric G proteins in NIH 3T3 cells. Using a transient transfection assay with a reporter vector in which the murine COX-2 promoter drives the production of luciferase and expression vectors encoding for α subunits of G-proteins, we show that overexpression of wild type and constitutively active Gα13 and Gαqinduced transcription from the COX-2 promoter. The highest level of induced luciferase activity (5.8-fold) occurred in cells expressing the constitutively active Gα13(Q226L). We also show that expression of a constitutively active mutant of Rho (RhoQ63L) also induced transcription from the COX-2 promoter. Co-expression ofClostridium botulinum C3 toxin specifically blocked induction of the COX-2 promoter by either Gα13Q226L or RhoQ63L but did not prevent the activation of this promoter by Ras, Rac, v-src, or forskolin. We conclude that Gα13 signals through a Rho-dependent pathway leading to activation of the COX-2 promoter. This pathway is not inhibited by either cytochalasin D, which disrupts actin filament organization, or genistein, a broad spectrum tyrosine kinase inhibitor, indicating a bifurcation of the signaling pathway used by Gα13/Rho to induce COX-2 expression from that used to induce stress fiber formation and tyrosine phosphorylation of focal adhesion proteins.

Ang II and EGF Synergistically Induce COX-2 Expression Via CREB in Intestinal Epithelial Cells

Cyclooxygenase (COX)‐2 derived prostaglandins (PGs) play a major role in intestinal inflammation and colorectal carcinogenesis. Because COX‐2 is the rate‐limiting step in the production of PGs, mechanisms that regulate COX‐2 expression control PG production in the cell. Using the non‐tumorigenic, rat intestinal epithelial cell, IEC‐18, we demonstrate that co‐activation of endogenously expressed AT1 receptor and EGFR resulted in synergistic expression of COX‐2 mRNA and protein involving transcriptional and post‐transcriptional mechanisms. Ang II and EGF induced transient phosphorylation of ERK, p38MAPK and CREB. Co‐stimulation with Ang II and EGF prolonged phosphorylation of ERK, p38MAPK, and CREB. The p38MAPK selective inhibitor, SB202190, but not the MEK selective inhibitor, PD98059, or the EGFR kinase inhibitor, AG1478, inhibited Ang II‐dependent COX‐2 expression and CREB phosphorylation. EGF‐dependent COX‐2 expression and CREB phosphorylation were inhibited by SB202190, PD98059, and AG1478. Inhibition of CREB expression using two separate RNAi methods blocked COX‐2 expression by Ang II and EGF. Expression of a dominant negative CREB mutant inhibited Ang II‐ and EGF‐dependent induction of the COX‐2 promoter. Ang II induced luciferase expression in cells transfected with the CRE‐luc reporter vector and cells co‐transfected with Gal4‐luc reporter vector and a Gal4‐CREB expression vector. Chromatin immunoprecipitation assays demonstrated CREB binding to the proximal rat COX‐2 promoter region containing a CRE cis‐acting element. These results indicate that co‐stimulation with Ang II and EGF synergistically induced COX‐2 expression in these intestinal epithelial cells through p38MAPK mediated signaling cascades that converge onto CREB. J. Cell. Physiol. 214:96–109, 2008.

Y2 receptors decrease human pancreatic cancer growth and intracellular cyclic adenosine monophosphate levels

BACKGROUNDnPeptide YY (PYY), a 36 amino acid enteric hormone, is known to decrease pancreatic exocrine and endocrine function. Previous studies with BIM-43004-1, a modified PYY(22-36) Y2 receptor agonist, have revealed diminished mitochondrial activity in pretreated pancreatic cancer cells in vitro. We investigated the effects of both PYY and BIM-43004-1 on pancreatic cancer growth in vivo.nnnMETHODSnThe 100,000 to 150,000 human pancreatic cancer cells, Mia PaCa-2, were orthotopically transplanted into 48 male athymic mice. After 1 week animals were treated with either PYY or BIM-43004-1 at 200 pmol/kg/hr via miniosmotic pumps for 2, 3, or 4 weeks. Paired controls received saline solution. At death tumor size and mass were measured. Receptor binding studies and intracellular cyclic adenosine monophosphate (cAMP) levels were measured in vitro.nnnRESULTSnAll mice had significant human cancer growth within the pancreas by histologic sections at 2, 3, and 4 weeks. Tumor mass was decreased by 60.5% in BIM-43004-1 treated mice and 27.1% in PYY treated mice. Receptor binding studies revealed binding of [125I]-BIM-43004-1 and displacement of ligand on competitive addition of nonradioactive BIM-43004-1. K dissociation constant of 4.5 nmol and 27,000 receptors per cell were quantitated by receptor binding studies. In BIM-43004-1 treated pancreatic cells a 52.5% decrease in intracellular cAMP levels was noted, whereas a 15.3% decrease was seen in PYY treated cells.nnnCONCLUSIONSnBIM-43004-1, a novel Y2 synthetic agonist, specifically binds to human pancreatic cancer cells, decreases intracellular cAMP levels, and suppresses tumor growth in vivo. Adjuvant hormonal treatment with this Y2 receptor analog may be beneficial in the treatment of patients with pancreatic adenocarcinoma.

Cytotoxic necrotizing factor from Escherichia coli induces RhoA-dependent expression of the cyclooxygenase-2 gene

ABSTRACT Cytotoxic necrotizing factor 1 (CNF) is a toxin produced by some isolates of Escherichia coli that cause extraintestinal infections. CNF can initiate signaling pathways that are mediated by the Rho family of small GTPases through a covalent modification that results in constitutive activation. In addition to regulating the assembly of actin stress fibers and focal adhesion complexes, RhoA can also regulate gene expression at the level of transcription. Here we demonstrate for the first time, by using a luciferase-based reporter system, that the transcription of cyclooxygenase-2 (COX-2) is strongly upregulated in NIH 3T3 fibroblasts treated with CNF and that this effect is dependent upon the activation of RhoA by the toxin. Subsequent protein tyrosine phosphorylation events modulate the induction, but the transcription signal is not mediated by Rho-associated kinase (p160/ROCK) and so must rely upon another effector that is activated by RhoA. CNF therefore induces COX-2 expression via a RhoA-dependent signaling pathway that diverges from the pathway that regulates cytoskeletal rearrangements in response to RhoA activation.

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. J. Cell. Biochem.

Protein kinase D mediates synergistic expression of COX-2 induced by TNF-α and bradykinin in human colonic myofibroblasts

Myofibroblasts have recently been identified as major mediators of tumor necrosis factor-alpha (TNF-alpha)-associated colitis, but the precise mechanism(s) involved remains incompletely understood. In particular, the possibility that TNF-alpha signaling cross talks with other proinflammatory mediators, including bradykinin (BK), has not been examined in these cells. Here we show that treatment of 18Co cells, a model of human colonic myofibroblasts, with BK and TNF-alpha induced striking synergistic COX-2 protein expression that was paralleled by increases in the levels of transcripts encoding COX-2 and microsomal prostaglandin E synthase 1 (mPGES-1) and by the production of PGE(2). COX-2 expression in 18Co cells treated with BK and TNF-alpha was prevented by the B(2) BK receptor antagonist HOE-140, the preferential protein kinase C (PKC) inhibitors Ro31-8220 and GF-109203X, and Gö-6976, an inhibitor of conventional PKCs and protein kinase D (PKD). In a parallel fashion, TNF-alpha, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation induced by BK, as measured by PKD phosphorylation at its activation loop (Ser(744)) and autophosphorylation site (Ser(916)). BK-induced PKD activation was also inhibited by HOE-140, Ro31-8220, and Gö-6976. Transfection of 18Co cells with small interfering RNA targeting PKD completely inhibited the synergistic increase in COX-2 protein in response to BK and TNF-alpha, demonstrating, for the first time, a critical role of PKD in the pathways leading to synergistic expression of COX-2. Our results imply that cross talk between TNF-alpha and BK amplifies a PKD phosphorylation cascade that mediates synergistic COX-2 expression in colonic myofibroblasts. It is plausible that PKD increases COX-2 expression in colonic myofibroblasts to promote an inflammatory microenvironment that supports tumor growth.