Zinc finger transcription factor Egr-1 is involved in stimulation of NHE2 gene expression by phorbol 12-myristate 13-acetate

Malakooti, Jaleh; Sandoval, Ricardo; Memark, Vanchad C.; Dudeja, Pradeep K.; Ramaswamy, Krishnamurthy

doi:10.1152/ajpgi.00010.2005

Cited by 15 publications

(35 citation statements)

References 57 publications

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“…3). These findings agree with our previous reports, where PMA exposure led to a time-dependent increase (1-6 h) in the endogenous NHE2 mRNA levels (36). These data strongly suggest that the initial phase of PKC␦ activation by PMA may be responsible for PMA effect on NHE2 expression.…”

Section: Discussionsupporting

confidence: 93%

“…PMA stimulates the expression of NHE2 through overexpression of transcription factor Egr-1 and its interaction with the NHE2 promoter (36). In accordance with our previous data, PMA-induced Egr-1 protein expression was transient, peaked between 1-2 h and returned to the basal level by 6 h (Fig.…”

Section: Inhibition Of Pkc␦ and Mek-erk Activation Prevent Egr-1 Exprsupporting

confidence: 91%

“…By functional and mutational assays we mapped the PMA-responsive region to the promoter region between bp Ϫ415 to Ϫ227. PMA responsiveness was further localized to a PMA response element (PRE) at nucleotides Ϫ339 to Ϫ324, and these findings were corroborated by mutational analyses of PRE in transient transfection assays (36). The NHE2-PRE motif, which is composed of overlapping binding sites for specificity protein-1 (Sp1) and early growth response-1 (Egr-1), interacts with transcription factors Sp1 and Sp3 in the basal growth conditions and Egr-1 in response to PMA (36,43).…”

Section: Namentioning

confidence: 76%

“…DNAprotein binding reactions were carried out as described previously (36). Briefly, 5 g of the nuclear extracts was incubated with reaction buffer at 50,000 counts per min (cpm) of the probe at room temperature for 20 min.…”

Section: Methodsmentioning

confidence: 99%

“…Previously we reported the presence of a cis-element in the NHE2 promoter that mediates the positive regulation of NHE2 transcription in response to PMA (36). By functional and mutational assays we mapped the PMA-responsive region to the promoter region between bp Ϫ415 to Ϫ227.…”

Section: Namentioning

confidence: 96%

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PKCδ-dependent activation of ERK1/2 leads to upregulation of the human NHE2 transcriptional activity in intestinal epithelial cell line C2BBe1

Muthusamy

Shukla

Amin

et al. 2012

American Journal of Physiology-Gastrointestinal and Liver Physi

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Muthusamy S, Shukla S, Amin Md. R, Cheng M, Orenuga T, Dudeja PK, Malakooti J. PKC␦-dependent activation of ERK1/2 leads to upregulation of the human NHE2 transcriptional activity in intestinal epithelial cell line C2BBe1. Am J Physiol Gastrointest Liver Physiol 302: G317-G325, 2012. First published November 3, 2011; doi:10.1152/ajpgi.00363.2011.-The apical Na ϩ /H ϩ exchanger (NHE) isoform NHE2 is involved in transepithelial Na ϩ 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, PKC␦, 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 PKC␦. Small inhibitory RNA-mediated knockdown of PKC␦ blocked the stimulatory effect of PMA on the NHE2 promoter activity. In addition, blockade of PKC␦ by rottlerin, a PKC␦-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 PKC␦, subsequent PKC␦-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. Naϩ /H ϩ exchanger; signal transduction pathway; phorbol 12-myristate 13-acetate; early growth response-1

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Section: Discussionsupporting

confidence: 93%

Section: Inhibition Of Pkc␦ and Mek-erk Activation Prevent Egr-1 Exprsupporting

confidence: 91%

Section: Namentioning

confidence: 76%

Section: Methodsmentioning

confidence: 99%

Section: Namentioning

confidence: 96%

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PKCδ-dependent activation of ERK1/2 leads to upregulation of the human NHE2 transcriptional activity in intestinal epithelial cell line C2BBe1

Muthusamy

Shukla

Amin

et al. 2012

American Journal of Physiology-Gastrointestinal and Liver Physi

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Tumor necrosis factor-α represses the expression of NHE2 through NF-κB activation in intestinal epithelial cell model, C2BBe1

Amin¹,

Orenuga²,

Tyagi³

et al. 2011

Inflammatory Bowel Diseases

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Background High levels of proinflammatory cytokines are linked to pathogenesis of diarrhea in inflammatory bowel diseases (IBD). Na+ absorption is compromised in IBD. The studies were designed to determine the effect of tumor necrosis factor-α (TNF-α) on the expression and activity of NHE2, a Na+/H+ exchanger (NHE) that is involved in trans-epithelial Na+ absorption in intestinal epithelial cells. Methods NHE2 regulation was examined in TNF-α treated C2BBe1 cells by RT-PCR, reporter gene assays and Western blot analysis. NHE isoform activities were measured as ethyl-isopropyl-amiloride- and HOE694-sensitive 22Na-uptake. In vitro and in vivo protein-DNA interactions were assessed by gel mobility shift assays and chromatin immunoprecipitation studies. Results TNF-α treatment of C2BBe1 cells led to repression of NHE2 promoter activity, mRNA and protein levels; and inhibited both NHE2 and NHE3 mediated 22Na-uptake. 5′-deletion analysis of the NHE2 promoter-reporter constructs identified bp −621 to −471 as TNF-α responsive region (TNF-RE). TNF-α activated NF-κB subunits, p50 and p65, and their DNA-binding to a putative NF-κB motif within TNF-RE. Mutations in the NF-κB motif abolished NF-κB-DNA interactions and abrogated TNF-α-induced repression. Ectopic over-expression of NF-κB resulted in repression of NHE2 expression. Two functionally distinct inhibitors of NF-κB blocked the inhibitory effect of TNF-α. Conclusions The human NHE2 isoform is a direct target of transcription factor NF-κB. TNF-α-mediated activation of NF-κB decreases the expression and activity of NHE2 in intestinal epithelial cell line, C2BBe1. These findings implicate NF-κB in the modulation of Na+ absorption during intestinal inflammatory conditions such as IBD where high level of TNF-α is detected.

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Factor binding and chromatin modification in the promoter of murine Egr1 gene upon induction

Tur

Georgieva

Gagete

et al. 2010

Cell. Mol. Life Sci.

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The influence of chromatin on immediate-early gene expression has been studied in a model of Egr1 induction in intact mouse cells. ChIP analysis of factor and RNA polymerase binding reveals that the gene is constitutively poised for transcription in nonstimulated cells, but a repressing chromatin structure hampers productive transcription. Stimulation with phorbol esters results in a transient activation, which starts at 5 min and peaks at 30 min. Quantitative mapping of promoter occupancy by the different factors shows for the first time that no direct competition between SP1 and EGR1 occurs. The phosphorylation of ELK1 and CREB, which involves both the cascades of MEK1/2 and p38 kinases, is required for gene expression, which ceases following the binding of NAB1 and NAB2 to the promoter. The changes in histone acetylation and the differential recruitment of histonemodifying complexes further show the role of chromatin in the activation of this immediate-early gene.

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Zinc finger transcription factor Egr-1 is involved in stimulation of NHE2 gene expression by phorbol 12-myristate 13-acetate

Cited by 15 publications

References 57 publications

PKCδ-dependent activation of ERK1/2 leads to upregulation of the human NHE2 transcriptional activity in intestinal epithelial cell line C2BBe1

PKCδ-dependent activation of ERK1/2 leads to upregulation of the human NHE2 transcriptional activity in intestinal epithelial cell line C2BBe1

Tumor necrosis factor-α represses the expression of NHE2 through NF-κB activation in intestinal epithelial cell model, C2BBe1

Factor binding and chromatin modification in the promoter of murine Egr1 gene upon induction

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