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.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Active Transport, Cell Nucleus / drug effects
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Cell Line, Tumor
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Cell Nucleus / drug effects
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Cell Nucleus / metabolism
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Early Growth Response Protein 1 / metabolism
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Epithelial Cells / drug effects
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Epithelial Cells / metabolism*
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Gene Expression / drug effects
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Gene Expression / genetics
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Humans
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Intestinal Mucosa / cytology*
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MAP Kinase Kinase 1 / antagonists & inhibitors
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MAP Kinase Kinase 1 / metabolism
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MAP Kinase Kinase 2 / antagonists & inhibitors
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MAP Kinase Kinase 2 / metabolism
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Mitogen-Activated Protein Kinase 1 / antagonists & inhibitors
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Mitogen-Activated Protein Kinase 1 / metabolism*
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Mitogen-Activated Protein Kinase 3 / antagonists & inhibitors
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Mitogen-Activated Protein Kinase 3 / metabolism*
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphoinositide-3 Kinase Inhibitors
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Phosphorylation / drug effects
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Protein Binding / drug effects
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Protein Binding / genetics
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Protein Kinase C / antagonists & inhibitors
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Protein Kinase C / metabolism
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Protein Kinase C-delta / antagonists & inhibitors
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Protein Kinase C-delta / metabolism*
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Protein Kinase Inhibitors / pharmacology
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RNA, Small Interfering / administration & dosage
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RNA, Small Interfering / genetics
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Response Elements / genetics
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Signal Transduction / drug effects
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Signal Transduction / physiology
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Sodium-Hydrogen Exchangers / genetics*
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Sp1 Transcription Factor / metabolism
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Sp3 Transcription Factor / metabolism
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Tetradecanoylphorbol Acetate / administration & dosage
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Tetradecanoylphorbol Acetate / pharmacology
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Transfection
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Up-Regulation / genetics*
Substances
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EGR1 protein, human
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Early Growth Response Protein 1
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Phosphoinositide-3 Kinase Inhibitors
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Protein Kinase Inhibitors
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RNA, Small Interfering
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SLC9A2 protein, human
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SP3 protein, human
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Sodium-Hydrogen Exchangers
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Sp1 Transcription Factor
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Sp3 Transcription Factor
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MAP2K2 protein, human
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Protein Kinase C
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Protein Kinase C-delta
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MAPK1 protein, human
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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MAP Kinase Kinase 1
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MAP Kinase Kinase 2
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MAP2K1 protein, human
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Tetradecanoylphorbol Acetate