Abstract 483: The Signaling Pathways of Nicotine-induced ERK1/2 Phosphorylation in Rat Mesangial Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Ping Hua ◽  
Wenguang Feng ◽  
Gabriel Rezonzew ◽  
Phillip H Chumley ◽  
Edgar A Jaimes
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Kinase Inhibitors ◽  
Mesangial Cells ◽  
Protein Kinase Inhibitors ◽  
Biologically Active ◽  
Calcium Calmodulin Dependent ◽  
Nicotine Receptors ◽  
High Concentrations

Tobacco smoking is associated with accelerated progression of chronic kidney disease of different etiologies including diabetes and hypertension. However, the mechanisms involved are not well understood. We have previously reported that nicotine, a biologically active compound present in high concentrations in tobacco, induces cell proliferation and fibronectin production in mesangial cells which are prevented by ERK1/2 inhibition (AJP’05). In these studies we determined whether rat mesangial cells (MC) express nicotine receptors and characterized the signaling pathways that lead to ERK1/2 phosphorylation in response to nicotine. MC were grown in DMEM with 15% FBS in the presence of 0.4 mg/ml G418 and starved for 24 hours in DMEM without FBS before treatment. We first demonstrated that MC are endowed with several nicotinic Ach receptor (nAChR) subunits including α2-7 and β1-4 as assessed by western blot. Treatment of rat MC with nicotine at 10 -7 M caused a time-dependent ERK1/2 phosphorylation which peaked after 10 min of stimulation( N=3). Several protein kinase inhibitors were then used to identify the upstream kinases that mediate nicotine-induced ERK1/2 phosphorylation. The calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 (10 -7 M) decreased the ERK1/2 phosphorylation level by ∼57% (0.41 of 0.95) as compared to nicotine. The PKC inhibitor Go6983 at 10 -9 M, the PKA inhibitor H89 (10 -8 M) and the EGFR inhibitor AG 1478 (10 -7 M) also inhibited ERK1/2 phosphorylation by 60% (0.38 of 0.95), 48% (0.63 of 1.20) and 68% (0.40 of 1.23) respectively as compared to nicotine. Given the role of the nicotine receptors as agonist-regulated Ca 2+ channels, we determined the effects of Ca 2+ channel blockade on nicotine induced ERK1/2 phosphorylation. Treatment of MC with the calcium channel Verapamil (10 -9 M) resulted in 33% (0.49 of 0.73) inhibition of ERK1/2 phosphorylation as compared to nicotine. In summary, we have determined in these studies that rat MC are endowed with several nAChR subunits and that ERK1/2 phosphorylation in response to nicotine requires CaMK II, PKA, PKC and EGFR. In addition, we have demonstrated that these effects require Ca 2+ consistent with the role of the nAChR as agonist-regulated Ca 2+ channels in MC.

scholarly journals Polymerization of actin in RBL-2H3 cells can be triggered through either the IgE receptor or the adenosine receptor but different signaling pathways are used.

1994 ◽  
Vol 5 (3) ◽  
pp. 313-322 ◽  
Author(s):  
J R Apgar
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Adenosine Receptor ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Filamentous Actin ◽  
Ige Receptor ◽  
Ige Mediated ◽  
Rbl Cells ◽  
Stimulation Of

Crosslinking of the IgE receptor on rat basophilic leukemia (RBL) cells using the multivalent antigen DNP-BSA leads to a rapid and sustained increase in the filamentous actin content of the cells. Stimulation of RBL cells through the adenosine receptor also induces a very rapid polymerization of actin, which peaks in 45-60 s and is equivalent in magnitude to the F-actin response elicited through stimulation of the IgE receptor. However, in contrast to the IgE mediated response, which remains elevated for over 30 min, the F-actin increase induced by the adenosine analogue 5'-(N-ethylcarboxamido)-adenosine (NECA) is relatively transient and returns to baseline values within 5-10 min. While previous work has shown that the polymerization of actin in RBL cells stimulated through the IgE receptor is mediated by protein kinase C (PKC), protein kinase inhibitors have no effect on the F-actin response activated through the adenosine receptor. In contrast, pretreatment of the cells with pertussis toxin completely inhibits the F-actin response to NECA but has relatively little effect on the response induced through the IgE receptor. Stimulation of RBL cells through either receptor causes increased production of phosphatidylinositol mono-phosphate (PIP) and phosphatidylinositol bis-phosphate (PIP2), which correlates with the F-actin response. Production of PIP and PIP2 may be important downstream signals since these polyphosphoinositides are able to regulate the interaction of gelsolin and profilin with actin. Thus the polymerization of actin can be triggered through either the adenosine receptor or the IgE receptor, but different upstream signaling pathways are being used. The IgE mediated response requires the activation of PKC while stimulation through the adenosine receptor is PKC independent but involves a G protein.

scholarly journals Protein kinase inhibitors--potential chemotherapeutic agents.

1995 ◽  
Vol 42 (4) ◽  
pp. 405-418 ◽  
Author(s):  
D Shugar
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Chemotherapeutic Agents ◽  
Protein Kinase Inhibitors ◽  
Donor And Acceptor ◽  
Catalytic Sites ◽  
Competitive Inhibitors ◽  
Preclinical Trials ◽  
Antiviral Chemotherapy

Protein kinase inhibitors, widely exploited for elucidation of the biological functions of kinases, have more recently come under active consideration as potential chemotherapeutic agents for tumour and other diseases. A brief overview is presented of diverse approaches to the design and development of selective protein kinase inhibitors, and related problems such as donor and acceptor specificities, stereochemical aspects, emerging relationships between protein, sugar and nucleoside kinases. In particular, and contrary to popular belief that ATP-competitive inhibitors cannot be selective because of the close homology of the ATP catalytic sites, numerous examples are presented of such inhibitors which are both potent and selective for a given kinase or class of kinases. Some of these are undergoing preclinical trials. Attention is also directed to the role of cellular and viral protein kinases in the life cycle of viruses, and the potential of these enzymes, especially those encoded by, and essential for replication of, a given virus as targets for antiviral chemotherapy.

scholarly journals Signaling pathways regulating FSH- and amphiregulin-induced meiotic resumption and cumulus cell expansion in the pig

Reproduction ◽  
2012 ◽  
Vol 144 (5) ◽  
pp. 535-546 ◽  
Author(s):  
R Prochazka ◽  
M Blaha ◽  
L Nemcova
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Kinase Inhibitors ◽  
Egf Receptor ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Protein Kinase Inhibitors ◽  
Akt Inhibitor ◽  
Cumulus Expansion

To define signaling pathways that drive FSH- and epidermal growth factor (EGF)-like peptide-induced cumulus expansion and oocyte meiotic resumption, in vitro cultured pig cumulus–oocyte complexes were treated with specific protein kinase inhibitors. We found that FSH-induced maturation of oocytes was blocked in germinal vesicle (GV) stage by protein kinase A (PKA), MAPK14, MAPK3/1, and EGF receptor (EGFR) tyrosine kinase inhibitors (H89, SB203580, U0126, and AG1478 respectively) whereas phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog (PI3K/AKT) inhibitor (LY294002) blocked maturation of oocytes in metaphase I (MI). Amphiregulin (AREG)-induced maturation of oocytes was efficiently blocked in GV by U0126, AG1478, and low concentrations of LY294002; H89, SB203580, and high concentrations of LY294002 allowed the oocytes to undergo breakdown of GV and blocked maturation in MI. Both FSH- and AREG-induced cumulus expansion was incompletely inhibited by H89 and completely inhibited by SB203580, U0126, AG1478, and LY294002. The inhibitors partially or completely inhibited expression of expansion-related genes (HAS2, PTGS2, and TNFAIP6) with two exceptions: H89 inhibited only TNFAIP6 expression and LY294002 increased expression of PTGS2. The results of this study are consistent with the idea that PKA and MAPK14 pathways are essential for FSH-induced transactivation of the EGFR, and synthesis of EGF-like peptides in cumulus cells and MAPK3/1 is involved in regulation of transcriptional and posttranscriptional events in cumulus cells required for meiotic resumption and cumulus expansion. PI3K/AKT signaling is important for regulation of cumulus expansion, AREG-induced meiotic resumption, and oocyte MI/MII transition. The present data also indicate the existence of an FSH-activated and PKA-independent pathway involved in regulation of HAS2 and PTGS2 expression in cumulus cells.

scholarly journals Development of a Fluorescence Polarization Bead-Based Coupled Assay to Target Different Activity/Conformation States of a Protein Kinase

2004 ◽  
Vol 9 (4) ◽  
pp. 309-321 ◽  
Author(s):  
Zhuomei Lu ◽  
Zhizhang Yin ◽  
Linda James ◽  
Rosalinda Syto ◽  
Jill M. Stafford ◽  
...  
Keyword(s):  
Protein Kinase ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Catalytic Domain ◽  
Protein Kinase Inhibitors ◽  
Biologically Active ◽  
Specific Substrate ◽  
Inactive Form ◽  
Substrate Peptide ◽  
Coupled Assay

Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP™)-based couple™d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z′ of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC™ and validated with the protein kinase C inhibitor staurosporine. The IC50 value generated was comparable to the value obtained by the radioactive 33P-γ-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.

scholarly journals Activated Coagulation Factor X: A Novel Mitogenic Stimulus for Human Mesangial Cells

2001 ◽  
Vol 12 (5) ◽  
pp. 891-899 ◽  
Author(s):  
RAFFAELLA MONNO ◽  
GIUSEPPE GRANDALIANO ◽  
ROBERTA FACCIO ◽  
ELENA RANIERI ◽  
CARMELA MARTINO ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Signaling Pathways ◽  
Phospholipase C ◽  
Serine Protease ◽  
Mesangial Cells ◽  
Factor X ◽  
Mitogenic Effect ◽  
Human Mesangial Cells

Abstract. Intraglomerular activation of the coagulation cascade is a common feature of mesangioproliferative glomerulonephritis. Besides thrombin, very little is known about the cellular effects of other components of the coagulation system. This study investigated the effect of activated factor X (FXa) on cultured human mesangial cells. This serine protease induced a significant and dose-dependent increase in DNA synthesis. In addition to its mitogenic effect, FXa caused a striking upregulation of platelet-derived growth factor (PDGF) A and B chain gene expression. Next, the intracellular mitogenic signaling pathways activated by FXa were investigated. FXa induced a rapid spike in cytosolic calcium concentration followed by a sustained plateau. This response was not influenced by the downregulation of thrombin receptors. In addition, FXa stimulated a significant upregulation of different tyrosine-phosphorylated proteins. One of these phosphorylated cellular proteins was represented by the c-jun N-terminal kinase, a member of the mitogen-activated protein kinase family. To evaluate the role of FXa enzymatic activity and of PDGF autocrine secretion, FXa-induced DNA synthesis was studied in the presence of leupeptin, a specific serine protease inhibitor, and neutralizing anti-PDGF antibody. To investigate the role of tyrosine kinase (TK) activation on FXa mitogenic effect, FXa-stimulated thymidine uptake was evaluated in the presence of genistein and herbimycin A, two powerful and specific TK inhibitors. FXa-elicited DNA synthesis was also examined after protein kinase C (PKC) downregulation by prolonged incubation with phorbol-12-myristate-13-acetate to study the influence of the phospholipase C-PKC axis. The proliferative effect of FXa required its proteolytic activity, and the activation of TK was only partially dependent on PKC activation while it was PDGF independent. Finally, it was shown by reverse transcription-PCR that mesangial cells do not express the signaling splicing variant of the putative FXa receptor, effector protease receptor-1. In conclusion, the present study demonstrated that FXa is a powerful mitogenic factor for human mesangial cells, and it induces its cellular effect not through effector protease receptor-1, but most likely by binding a protease-activated receptor and activating phospholipase C—PKC and TK signaling pathways.

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