Activation of ERK1/2 by Store-Operated Calcium Entry in Rat Parotid Acinar Cells

The regulation of intracellular Ca2+ concentration ([Ca2+]i) plays a critical role in a variety of cellular processes, including transcription, protein activation, vesicle trafficking, and ion movement across epithelial cells. In many cells, the activation of phospholipase C-coupled receptors hydrolyzes membrane phosphoinositides and produces the depletion of endoplasmic reticulum Ca2+ stores, followed by the sustained elevation of [Ca2+]i from Ca2+ entry across the plasma membrane via store-operated Ca2+ entry (SOCE). Ca2+ entry is also increased in a store-independent manner by arachidonate-regulated Ca2+ (ARC) channels. Using rat parotid salivary gland cells, we examined multiple pathways of Ca2+ entry/elevation to determine if they activated cell signaling proteins and whether this occurred in a pathway-dependent manner. We observed that SOCE activates extracellular signal-related kinases 1 and 2 (ERK1/2) to ∼3-times basal levels via a receptor-independent mechanism when SOCE was initiated by depleting Ca2+ stores using the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (TG). TG-initiated ERK1/2 phosphorylation increased as rapidly as that initiated by the muscarinic receptor agonist carbachol, which promoted an increase to ∼5-times basal levels. Notably, ERK1/2 phosphorylation was not increased by the global elevation of [Ca2+]i by Ca2+ ionophore or by Ca2+ entry via ARC channels in native cells, although ERK1/2 phosphorylation was increased by Ca2+ ionophore in Par-C10 and HSY salivary cell lines. Agents and conditions that blocked SOCE in native cells, including 2-aminoethyldiphenyl borate (2-APB), SKF96363, and removal of extracellular Ca2+, also reduced TG- and carbachol-stimulated ERK1/2 phosphorylation. TG-promoted ERK1/2 phosphorylation was blocked when SRC and Protein Kinases C (PKC) were inhibited, and it was blocked in cells pretreated with β-adrenergic agonist isoproterenol. These observations demonstrate that ERK1/2 is activated by a selective mechanism of Ca2+ entry (SOCE) in these cells, and suggest that ERK1/2 may contribute to events downstream of SOCE

UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation

Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′-triphosphate (UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the most potent agonist among several nucleotides tested. Adenosine 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also potent agonists, characteristic of the P2Y2 or P2Y4 receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2 phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is mediated by a P2Y2 or P2Y4 receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR

Proline-rich tyrosine kinase 2 mediates gonadotropin-releasing hormone signaling to a specific extracellularly regulated kinase-sensitive transcriptional locus in the luteinizing hormone beta-subunit gene

G protein-coupled receptor regulation of gene transcription primarily occurs through the phosphorylation of transcription factors by MAPKs. This requires transduction of an activating signal via scaffold proteins that can ultimately determine the outcome by binding signaling kinases and adapter proteins with effects on the target transcription factor and locus of activation. By investigating these mechanisms, we have elucidated how pituitary gonadotrope cells decode an input GnRH signal into coherent transcriptional output from the LH β-subunit gene promoter. We show that GnRH activates c-Src and multiple members of the MAPK family, c-Jun NH(2)-terminal kinase 1/2, p38MAPK, and ERK1/2. Using dominant-negative point mutations and chemical inhibitors, we identified that calcium-dependent proline-rich tyrosine kinase 2 specifically acts as a scaffold for a focal adhesion/cytoskeleton-dependent complex comprised of c-Src, Grb2, and mSos that translocates an ERK-activating signal to the nucleus. The locus of action of ERK was specifically mapped to early growth response-1 (Egr-1) DNA binding sites within the LH β-subunit gene proximal promoter, which was also activated by p38MAPK, but not c-Jun NH(2)-terminal kinase 1/2. Egr-1 was confirmed as the transcription factor target of ERK and p38MAPK by blockade of protein expression, transcriptional activity, and DNA binding. We have identified a novel GnRH-activated proline-rich tyrosine kinase 2-dependent ERK-mediated signal transduction pathway that specifically regulates Egr-1 activation of the LH β-subunit proximal gene promoter, and thus provide insight into the molecular mechanisms required for differential regulation of gonadotropin gene expression

Tyrphostin AG126 exerts neuroprotection in CNS inflammation by a dual mechanism

© 2015 Wiley Periodicals, Inc. Acknowledgement Grant sponsor: State of Lower Saxony-Israel Research Cooperation; Grant number: ZN2035; Grant sponsor:German Research Council; Grant number: SFB/TRR43 and FOR1336; Grant sponsor: Parkinson UK; Grant number: K-1001; Grant sponsor: ProFutura Program (University of Gottingen); Grant sponsor: Else Kroner Fresenius Stiftung;Grant number: A69/2010; Grant sponsor: DFG; Grant number: WE 3547/4–1; Grant sponsor: US National Multiple Sclerosis Society; Grant numbers: NMSS; PP 1660. The authors thank Elke Pralle, Susanne Kiecke and Caroline Jaß (University of Gottingen) for excellent technical assistance.Peer reviewedPostprin

Vaccinia-induced epidermal growth factor receptor-MEK signalling and the anti-apoptotic protein F1L synergize to suppress cell death during infection

F1L is a functional Bcl-2 homologue that inhibits apoptosis at the mitochondria during vaccinia infection. However, the extent and timing of cell death during ΔF1L virus infection suggest that additional viral effectors cooperate with F1L to limit apoptosis. Here we report that vaccinia growth factor (VGF), a secreted virulence factor, promotes cell survival independently of its role in virus multiplication. Analysis of single and double knockout viruses reveals that VGF acts synergistically with F1L to protect against cell death during infection. Cell survival in the absence of F1L is dependent on VGF activation of the epidermal growth factor receptor. Furthermore, signalling through MEK kinases is necessary and sufficient for VGF-dependent survival. We conclude that VGF stimulates an epidermal growth factor receptor-MEK-dependent pro-survival pathway that synergizes with F1L to counteract an infection-induced apoptotic pathway that predominantly involves the BH3-only protein Bad

Rottlerin stimulates apoptosis in pancreatic cancer cells through interactions with proteins of the Bcl-2 family

Rottlerin is a polyphenolic compound derived from Mallotus philipinensis. In the present study, we show that rottlerin decreased tumor size and stimulated apoptosis in an orthotopic model of pancreatic cancer with no effect on normal tissues in vivo. Rottlerin also induced apoptosis in pancreatic cancer (PaCa) cell lines by interacting with mitochondria and stimulating cytochrome c release. Immunoprecipitation results indicated that rottlerin disrupts complexes of prosurvival Bcl-xL with Bim and Puma. Furthermore, siRNA knockdown showed that Bim and Puma are necessary for rottlerin to stimulate apoptosis. We also showed that rottlerin and Bcl-2 and Bcl-xL inhibitor BH3I-2' stimulate apoptosis through a common mechanism. They both directly interact with mitochondria, causing increased cytochrome c release and mitochondrial depolarization, and both decrease sequestration of BH3-only proteins by Bcl-xL. However, the effects of rottlerin and BH3I-2' on the complex formation between Bcl-xL and BH3-only proteins are different. BH3I-2' disrupts complexes of Bcl-xL with Bad but not with Bim or Puma, whereas rottlerin had no effect on the Bcl-xL interaction with Bad. Also BH3I-2', but not rottlerin, required Bad to stimulate apoptosis. In conclusion, our results demonstrate that rottlerin has a potent proapoptotic and antitumor activity in pancreatic cancer, which is mediated by disrupting the interaction between prosurvival Bcl-2 proteins and proapoptotic BH3-only proteins. Thus rottlerin represents a promising novel agent for pancreatic cancer treatment

PKCδ Sensitizes Neuroblastoma Cells to L-Buthionine-Sulfoximine and Etoposide Inducing Reactive Oxygen Species Overproduction and DNA Damage

Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB) cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH), which is crucial in counteracting the endogenous production of reactive oxygen species (ROS). We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC) δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO), a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches

Agonist-induced internalisation of the glucagon-like peptide-1 receptor is mediated by the Gαq pathway

The glucagon like peptide-1 receptor (GLP-1R) is a G-protein coupled receptor (GPCR) and an important target in the treatment of type 2 diabetes mellitus (T2DM). Upon stimulation with agonist, the GLP-1R signals through both Gαs and Gαq coupled pathways to stimulate insulin secretion. The agonist induced GLP-1R internalisation has recently been shown to be important for insulin secretion. However, the molecular mechanisms underlying GLP-1R internalisation remain unknown. The aim of this study was to determine the role of GLP-1R downstream signalling pathways in its internalisation. Agonist induced human GLP-1R (hGLP-1R) internalisation and activity were examined using a number of techniques including immunoblotting, ELISA, immunofluorescence, and luciferase assays to determine cAMP production, intracellular Ca2+ accumulation and ERK phosphorylation. Agonist induced hGLP-1R internalisation is dependent on caveolin-1 and dynamin. Inhibition of the Gαq pathway but not the Gαs pathway affected hGLP-1R internalisation. Consistent with this, hGLP-1R mutant T149 M and small molecule agonists (compound 2 and compound B), which activate only the Gαs pathway, failed to induce internalisation of the receptor. Chemical inhibitors of the Gαq pathway, PKC and ERK phosphorylation significantly reduced agonist induced hGLP-1R internalisation. These inhibitors also suppressed agonist induced ERK1/2 phosphorylation demonstrating that the phosphorylated ERK acts downstream of the Gαq pathway in the hGLP-1R internalisation. In summary, agonist induced hGLP-1R internalisation is mediated by the Gαq pathway. The internalised hGLP-1R stimulates insulin secretion from pancreatic β-cells, indicating the importance of GLP-1 internalisation for insulin secretion