title: PKCδ-dependent activation of ERK1/2 leads to upregulation of the human NHE2 transcriptional activity in intestinal epithelial cell line C2BBe1.
abstract: The apical Na+/H+ exchanger (NHE) isoform NHE2 is involved in transepithelial Na+ absorption in the intestine. Our earlier studies have shown that mitogenic agent phorbol 12-myristate 13-acetate (PMA) induces the expression of NHE2 through activation of transcription factor early growth response-1 (Egr-1) and its interactions with the NHE2 promoter. However, the signaling pathways involved in transcriptional stimulation of NHE2 in response to PMA in the intestinal epithelial cells are not known. Chemical inhibitors and genetic approaches were used to investigate the signaling pathways responsible for the stimulation of NHE2 expression by PMA via Egr-1 induction. We show that, in response to PMA, PKCδ, a member of novel PKC isozymes, and MEK-ERK1/2 pathway of mitogen-activated protein kinases stimulate the NHE2 expression in C2BBe1 intestinal epithelial cells. PMA rapidly and transiently induced activation of PKCδ. Small inhibitory RNA-mediated knockdown of PKCδ blocked the stimulatory effect of PMA on the NHE2 promoter activity. In addition, blockade of PKCδ by rottlerin, a PKCδ-specific inhibitor, and ERK1/2 by U0126, a MEK-ERK inhibitor, abrogated PMA-induced Egr-1 expression. Immunofluorescence studies revealed that inhibition of ERK1/2 activation prevents translocation of PMA-induced Egr-1 into the nucleus. Consistent with these data, PMA-induced Egr-1 interaction with the NHE2 promoter region was prevented in nuclear extracts from U0126-pretreated cells. In conclusion, our data provide the first evidence that the stimulatory effect of PMA on NHE2 expression is mediated through the initial activation of PKCδ, subsequent PKCδ-dependent activation of MEK-ERK1/2 signaling pathway, and stimulation of Egr-1 expression. Furthermore, we show that transcription factor Egr-1 acts as an intermediate effector molecule that links the upstream signaling cues to the long-term stimulation of NHE2 expression by PMA in C2BBe1 cells.
authors: Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA.
authors: Saminathan
authors: S
authors: Muthusamy
authors: Sagar
authors: S
authors: Shukla
authors: Md Ruhul
authors: MR
authors: Amin
authors: Ming
authors: M
authors: Cheng
authors: Temitope
authors: T
authors: Orenuga
authors: Pradeep K
authors: PK
authors: Dudeja
authors: Jaleh
authors: J
authors: Malakooti
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000008:administration & dosage / Q000494:pharmacology; D014162:Transfection; D015854:Up-Regulation / Q000235:genetics*
chemical_list: D051766:Early Growth Response Protein 1; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D017923:Sodium-Hydrogen Exchangers; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D013755:Tetradecanoylphorbol Acetate; C497325:EGR1 protein, human; C081517:SLC9A2 protein, human; C497121:SP3 protein, human; C482128:MAP2K2 protein, human; C535150:MAPK1 protein, human; C482125:MAP2K1 protein, human