Inhibition of acetylcholine-induced activation of extracellular regulated protein kinase prevents the encoding of an inhibitory avoidance response in the rat

Phosphoinositide 3-kinase (PI3K) enzymes are critical in many cellular processes including cell survival. PI3Kγ, a member of the PI3K family, is activated in response to G-protein coupled receptor (GPCR) stimulation leading to extracellular regulated kinase (ERK) signal transduction cascade, a cell survival pathway. However, less is known about the underlying mechanisms of PI3Kγ-directed ERK activation. Knockdown of PI3Kγ showed that PI3Kγ not only regulates ERK phosphorylation in response to GPCR stimulation but also to receptor tyrosine kinase activation in HEK 293 cells. The key role of PI3Kγ in ERK activation was further validated by loss of insulin-stimulated ERK phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs). Surprisingly, ERK activation in KO MEFs post-insulin stimulation was completely rescued by expression of kinase-dead PI3Kγ mutant in KO MEFs demonstrating a kinase-independent role of PI3Kγ in regulating ERK function. Mechanistic studies showed that PI3Kγ regulates ERK activation by inhibiting ERK dephosphorylation following stimulation thereby, sustaining ERK phosphorylation and activation. Critically, PI3Kγ regulates ERK dephosphorylating phosphatase PP2A by interacting and sequestering PP2A from ERK maintaining ERK phosphorylation, which is evidenced by increased PP2A association with ERK in KO MEFs. Consistently, ERK activation was completely abolished in KO MEFs following carvedilol or insulin suggesting an essential role for PI3Kγ in ERK activation pathway. Correspondingly, primary cardiac fibroblasts isolated from KO mice showed complete loss of insulin-stimulated ERK phosphorylation compared to WT mice. This is intriguing given that GSK3 phosphorylation and not ERK phosphorylation is regulated by inhibition of PP2A through kinase-independent mechanism of PI3Kγ in the total cardiac lysates. Even though GSK3 and ERK are substrates for PP2A, our findings that ERK is regulated by kinase-independent function PI3Kγ suggest the existence of this unique regulation in fibroblasts and not in cardiomyocytes. Thus, kinase activity of PI3Kγ may contribute to cardiac-pathology while kinase-independent function could be beneficial and will be discussed in presentation.

Phosphoinositide 3-kinase (PI3K) enzymes are critical in many cellular processes including survival. PI3Kγ, a member of the PI3K family activated by G-protein coupled receptor (GPCR), is known to be a critical player in activation of extracellular regulated kinase (ERK) signal transduction cascade, a cell survival pathway. However, the exact mechanism by which PI3Kγ plays a role in ERK activation is not clearly understood. Our studies show that PI3Kγ plays a crucial role in enhancing the tone of ERK activation as use of PI3K inhibitors reduced GPCR stimulated ERK phosphorylation in HEK293 cells. siRNA knockdown of PI3Kγ resulted in loss of ERK phosphorylation through GPCRs (β-adrenergic) as well as receptor tyrosine kinases. The role of PI3Kγ in ERK activation was further corroborated by loss of insulin stimulated ERK phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs). Surprisingly, ERK activation in KO MEFs post-insulin stimulation was completely rescued by expression of kinase-dead PI3Kγ mutant in KO MEFs suggesting a kinase-independent role of PI3Kγ in regulating ERK function. Indepth mechanistic studies showed that PI3Kγ mediated activation of ERK by inhibiting ERK dephosphorylation following stimulation, thus stabilizing the ERK phosphorylation. PI3Kγ physically disrupts the interaction between ERK and ERK dephosphorylating phosphatase PP2A as evidenced by increase in phosphatase association with ERK in KO MEFs. Consistent with this observation, ERK activation was completely abolished in KO MEFs following carvedilol suggesting an essential role for PI3Kγ in cardio-protective ERK activation pathway. In this context, it is known that transverse aortic constriction (TAC) in mice leads to increase in ERK activation in the hearts and is also associated with concurrent up-regulation of PI3Kγ suggesting a key role for kinase-independent function of PI3Kγ in activating and maintaining the ERK signaling cascade. These indepth cellular studies and observation from our TAC studies led us to believe that kinase-dependent function of PI3Kγ may contribute to pathology while kinase-independent function may be cardio-protective through inhibition of PP2A by PI3Kγ. This novel signaling mechanism by PI3Kγ will be presented.

Involvement of medial septal glutamate and GABA A receptors in behaviour-induced acetylcholine release in the hippocampus: A dual probe microdialysis study

MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrated differential MEKK subcellular localization: MEKK1 was nuclear and in post-Golgi vesicular-like structures; MEKK2 and 4 were localized to distinct Golgi-associated vesicles that were dispersed by brefeldin A. MEKK1 and 2 were activated by EGF, and kinase-inactive mutants of each MEKK partially inhibited EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibited EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associated with Rac and Cdc42 and kinase-inactive mutants blocked Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1-4 did not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42 independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli.

Sphingosine-1-Phosphate Inhibition of Apoptosis Requires Mitogen-Activated Protein Kinase Phosphatase-1 in Mouse Fibroblast C3H10T½ Cells

The anti-cancer agent [Arg(6), D-Trp(7,9), N(me)Phe(8)]-substance P (6-11) (SP-G) modulates gastrin releasing peptide (GRP) and arginine vasopressin signalling in small cell lung cancer cells leading to growth arrest and apoptosis. We have shown that SP-G acts as a biased agonist at GRP receptors. This work examines the hypothesis that SP-G acts as a biased agonist at the V(1A) vasopressin receptor. The human V(1A) receptor was expressed in CHO-K1 cells. Extracellular regulated kinase (ERK) activation and intracellular Ca(2+) were measured using activation state-specific antibodies and Fura-2-AM respectively. The effect of SP-G on tumourigenicity was assessed by colony assay. In V(1A) receptor expressing cells, SP-G caused a sustained activation of ERK via a stimulation of V(1A) receptor coupling to G(i). Inhibition of G(i) with Pertussis toxin attenuated the inhibition by SP-G of the growth of CHO-K1 cells stably expressing the V(1A) receptor. Chimeric V(1A) receptors containing the second or third intracellular loop of the V(2) receptor were capable of binding vasopressin and SP-G but had altered ability to activate phospholipase C (PLC) and ERK. The second intracellular loop of the V(1A) receptor was essential for vasopressin-stimulated PLC and ERK activation but not for SP-G-induced ERK activation. This work provides mechanistic insight, for biased agonists at V(1A) receptors and highlights a potential role for such agents as anti-cancer agents.

A consistent pattern of response has been observed when FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have been used as monotherapy to treat patients with relapsed or refractory FLT3- internal tandem duplication (ITD) acute myeloid leukaemia (AML). Circulating blasts are cleared from the peripheral blood, while bone marrow blasts are either unaffected or are cleared from the marrow at a much slower rate. We used an in vitro model of FLT3-ITD AML blasts co-cultured with normal human bone marrow stromal cells to investigate the basis for this dichotomous response pattern to FLT3 inhibitors. We have found that in blasts on stroma, potent FLT3 inhibition predominantly results in cell cycle arrest rather than apoptosis. The anti-apoptotic effect is mediated through a combination of direct cell-cell contact and soluble factors. The addition of exogenous FLT3 ligand (FL) augments the protection, primarily by shifting the 50% inhibitory concentration for FLT3 inhibition upwards. Cytokine-activated extracellular regulated kinase (ERK), rather than STAT5, appears to be the most important downstream signalling protein mediating the protective effect, and inhibition of MEK significantly abrogates stromal-mediated resistance. These findings explain the phenomenon of peripheral blood versus bone marrow blast responses and suggest that the combination of potent FLT3 inhibition and MEK inhibition is a promising strategy for the treatment of FLT3-ITD AML.

The mitogen activated protein (MAP) kinase cascade, leading to extracellular-regulated kinase (ERK) activation, is a key regulator of cell growth and proliferation. The effects of ERK are mediated by differences in ERK signalling dynamics, including magnitude and duration. In vivo, ERK signalling is stimulated by both growth factors and adhesion signals. A model for adhesion-mediated ERK activation is presented. Outputs of the model such as ERK and FAK activation, as well as responses to different ligand densities, are compared with published experimental data. The model then serves as a basis for understanding how adhesion may contribute to ERK signalling through changes in the dynamics of focal adhesion kinase activation. The main parameters influencing ERK are determined through screening analyses and parameter variation. With these parameters, key points in the pathway that give rise to changes in downstream signalling dynamics are identified. In particular, oncogenic Raf and Ras promote cell growth by increasing the magnitude and duration, respectively, of ERK activity.

Background/Aims: To verify whether human IgG induces proinflammatory activation of human proximal tubular epithelial cells (PTEC) independent of the metabolic overload of protein reabsorption. Methods: Cultured PTEC were incubated with normal IgG, IgG from systemic lupus erythematosus (SLE) patients, albumin or transferrin. IL-6 secretion and extracellular regulated kinase (ERK) activation (dual-phosphorylated ERK) were measured by ELISA and by Western blotting of PTEC extracts, respectively; renal biopsy specimens from patients with IgG and non-IgG proteinuria were analyzed by immunohistochemistry and in situ hybridization to detect ERK-P and IL-6. Results: Normal and SLE IgG, but not albumin or transferrin, induced an early significant increase in IL-6 secretion by PTECs. Also ERK activation was found after 1-hour incubation of PTEC with IgG, but not with control medium and albumin-treated PTEC. Activated ERK and IL-6 were found to colocalize in tubular cells in the kidney specimens of patients with IgG proteinuria only. Conclusion: IgG-dependent early activation of ERK and increased IL-6 secretion in PTEC suggest that IgG filtered during nonselective proteinuria may play a specific role in tubulointerstitial disease. Such a role could be particularly relevant in diseases associated with abnormal IgG pool compositions, such as SLE. Preliminary results on human renal biopsy specimens suggest that our in vitro observations may also be relevant in vivo.

Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion and acts upon pancreatic β-cells potentiating glucose-stimulated insulin secretion and stimulating β-cell proliferation, differentiation, survival and gene transcription. These effects are mediated through the activation of multiple signal transduction pathways including the extracellular regulated kinase (ERK) pathway. We have previously reported that GLP-1 activates ERK through a mechanism dependent upon the influx of extracellular Ca2+ through L-type voltage gated Ca2+ channels (VGCC). However, the mechanism by which L-type VGCCs couple to the ERK signalling pathway in pancreatic β-cells is poorly understood. In this report, we characterise the relationship between L-type VGCC mediated changes in intracellular Ca2+ concentration ([Ca2+]i) and the activation of ERK, and demonstrate that the sustained activation of ERK (up to 30 min) in response to GLP-1 requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [Ca2+]i or Ca2+ efflux from the endoplasmic reticulum. Moreover, sustained elevation of [Ca2+]i induced by ionomycin is insufficient to stimulate the prolonged activation of ERK. Using the cell permeant Ca2+ chelators, EGTA-AM and BAPTA-AM, to determine the spatial dynamics of L-type VGCC-dependent Ca2+ signalling to ERK, we provide evidence that a sustained increase in Ca2+ within the microdomain of the L-type VGCC is sufficient for signalling to ERK and that this plays an important role in GLP-1- stimulated ERK activation.

Activation of ERK (Extra-cellular Regulated Kinase) signal plays a major role in metabolic diseases such as cancer and Alzheimer's. Ingenuity Pathway Analysis (IPA) of RNA-Seq data from long-term O-GlcNAcase (OGA) enzyme inhibitor- Thiamet-G (TMG) treated SH-SY5Y (neuroblastoma) cells, which elevates O-GlcNAc levels, indicated ERK Signaling as a top upregulated pathway. O-GlcNAcylation consists of the addition of a single N-acetyl-glucosamine residue (GlcNAc) to specific serine/threonine residues of proteins by the enzyme OGT (O-GlcNAc Transferase) and the removal of O-GlcNAc is catalyzed by the enzyme OGA. O-GlcNAc is highly abundant and is known to modulate kinase activity. Thus, to further investigate the mechanism by which O-GlcNAcylation activates ERK, we did a serum reactivation time-course and found that there is an amplification in ERK phosphorylation after long-term TMG treatment in HeLa and SH-SY5Y cells. Next, TGF-β stimulation that specifically activates ERK signaling also indicated an increase in ERK phosphorylation with long-term TMG treatment, confirming a robust ERK activation with TMG treatment. ERK phosphorylation oscillates with time after mitogen activation; therefore, we wanted to see if TMG treatment influenced ERK oscillation for a period of 24 hours. Serum reactivation revealed an oscillation in phosphorylated-ERK expression within a 24-hour period, where it reaches a maximum at 4 hours and slowly decreases at 8,12 and 24 hours upon long-term TMG treatment. Next, we wanted to further evaluate the mechanism by which TMG treatment increases ERK phosphorylation. Of note, we did not measure any O-GlcNAcylation on ERK; hence, we probed the expression of activated mitogen activated protein kinase-kinase (phosphorylated MEK) which phosphorylates ERK and Dual specificity phosphatases (DUSPs), which dephosphorylate ERK. Interestingly, serum reactivation time course showed an increase in both phosphorylated-MEK and total DUSP4 with TMG treatment. In addition to the use of pharmacological OGA inhibitor, we measured the effect of OGT knock-down (KD) and OGA KD on ERK signaling in SH-SY5Y and HeLa cells. There was amplification in ERK phosphorylation even after OGT knock-down (OGT KD) which lowers the level of O-GlcNAcylation, in contrast to long-term TMG treatment which increases O-GlcNAcylation. These data suggest that the cycling of the modification on and off substrates is key to ERK pathway regulation. Recent research also shows APOE4 stimulates the transcription of amyloid precursor protein (APP) via a non-canonical ERK signaling pathway, leading to increased amyloid-beta secretion in Alzheimer's disease pathogenesis. Therefore, we wanted to see if there is an increase in the expression of APP in the long-term TMG treated SH-SY5Y cells after serum reactivation, and we saw an increase in APP expression correlating with amplification of ERK signaling. Further evaluation of these molecular mechanisms to elucidate how O-GlcNAcylation amplifies ERK signaling and understand if O-GlcNAcylation and ERK activation work together to increase the transcription of APP are necessary.

The involvement of transcription factors in endothelial cell differentiation is largely unknown. We demonstrated that arachidonic acid (AA) mediates growth factor-induced gene expression. Herein, we hypothesized that AA mediated transcriptional regulation of endothelial cell differentiation. Challenge of endothelial cells with AA activated the c-jun amino terminal kinase (JNK) and the extracellular regulated kinases (ERKs). Inhibition of cyclooxygenase 1 and 2 had no effect on AA-induced JNK or ERK activation, suggesting that AA acts independently of its transformation into prostaglandins. AA stimulated AP-1 activity, and this effect was blocked by JNK inhibition, but had no effect on Egr-1. Basic fibroblast growth factor (bFGF), also activated JNK and ERK, and stimulated AA release. Inhibition of cPLA2 decreased bFGF-induced endothelial cell differentiation, AA release and AP-1 activation, while had no effect on ERK and Egr-1 activation. AA and bFGF induced neovessel formation in vivo in a JNK-dependent manner. These results suggest that AA mediates bFGF-induced endothelial cell differentiation via the JNK/cjun/AP-1 pathway. Given the role of AA in vascular pathophysiology, targeting the signaling pathways activated by AA could represent a novel approach for the prevention or treatment of diseases associated with endothelial cell dysfunction. (Showalter Foundation).

Lipid peroxidation triggers both c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase (ERK) activation and neointimal hyperplasia induced by cessation of blood flow in the mouse carotid artery

Neuroprotective effects of Polygonum multiflorum extract against glutamate-induced oxidative toxicity in HT22 hippocampal cells

5-HT(1A) receptors have been hypothesized to mediate some of the neuronal plasticity and behavioral responses stimulated by serotonin selective reuptake inhibitors. Although the cellular signaling pathways required for inducing these actions have not yet been determined, roles for the neuroprotective extracellular-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and Akt pathways have been suggested. In the current studies we have utilized primary cultures to directly determine whether hippocampal 5-HT(1A) receptors couple to activation of Akt and ERK. We found that E18 hippocampal neurons exhibit a twofold activation of Akt when exposed to nanomolar concentrations of 5-HT. The 5-HT(1/7) receptor-selective agonist 5-carboxamidotryptamine maleate (5-CT) and the 5-HT(1A/7) receptor-selective agonist 8-hydroxy-N,N-dipropyl-aminotetralin (8-OH-DPAT) maleate were found to activate Akt with equal efficacy, and similar potency, to 5-HT. p-MPPI and WAY-100635, antagonists selective for 5-HT(1A) receptors, completely inhibited 5-CT- stimulated Akt activation. Activation of Akt was also inhibited by pretreatment with pertussis toxin as well as the phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002. In contrast, the 5-HT selective antagonist, SB269970, caused no inhibition. Although the density of 5-HT(1A) receptors expressed by cultured neurons was sufficient to activate Akt, no activation of ERK was observed. These findings suggest that Akt, and not ERK, may be relevant to previous reports of hippocampal 5-HT(1A) receptors mediating neurotrophic responses.