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.

Fig. 1.

Phorbol 12-myristate 13-acetate (PMA)-induced Na⁺/H⁺ exchanger 2 (NHE2) promoter activity is triggered by activation of novel PKC. A: schematic illustration of the NHE2 5′-flanking region harbored in p-1051/+150, NHE2 promoter-reporter construct. The relevant cis-elements are shown. C2BBe1 cells were transiently transfected with p-1051/+150 and treated with or without PMA (100 nM, 16 h). To examine the effects of signaling pathways, before PMA treatment some samples were treated with pathway-specific inhibitors (5 μM each): Gö6976 (B), Gö6983 (C), and Gö6850 (D) for 1 h. Luciferase activities were determined 48 h posttransfection as described in materials and methods. Data are presented as means ± SE and are representative of 3 independent experiments performed in triplicate. Asterisks denote differences between PMA vs. inhibitor-plus PMA treatments and are statistically significant at P < 0.05. PRE, PMA response element. Egr-1, early growth response-1; Sp1, specificity protein-1.

Fig. 2.

siRNA-mediated PKCδ knockdown blocks PMA-induced NHE2 promoter activity. C2BBe1 cells were transiently transfected with PKCδ siRNA before transfection with p-415/+150. Cells were incubated in serum-reduced media for 16 h before PMA treatment. Cells were either treated with 100 nM PMA or vehicle. Cells were collected 48 h after transfection, and luciferase activities were determined in 10 μg of total cell proteins. The results are presented as average of 3 independent experiments performed in triplicate (n = 3). Asterisk indicates differences between luciferase activities of the PMA-treated control siRNA transfected cells vs. similarly treated cells transfected with PKCδ siRNA, P < 0.05.

Fig. 3.

PMA stimulates PKCδ phosphorylation. C2BBe1 cells were serum deprived overnight and treated with PMA (100 nM) (A) for various time points or with Gö6850 (B) (1, 2.5, and 5 μM) for 1 h before addition of PMA for 5 min. Total cell lysates (TCL) were prepared, and 20 μg/lane were resolved on a 10% SDS-PAGE. Western blot analysis performed as described in materials and methods using phospho-PKCδ (Thr-507) antibody. The blot in A was stripped and reprobed with anti-Pan-PKCδ as loading control. A similarly loaded gel was used as a loading control and probed with actin in B.

Fig. 4.

Early events after PMA exposure mediate transcriptional activation of the NHE2 promoter. C2BBe1 cells were transiently transfected with NHE2 promoter-reporter construct, p-415/+150. Cells were incubated in serum-reduced media for 16 h before PMA treatment for the indicated times. After each time point PMA was washed off, and cells were incubated in serum-reduced media (1% FBS) up to 16 h. Cells were harvested 48 h posttransfection, and luciferase activities were determined in 10 μg of total cell proteins. The results are presented as average of 3 independent experiments performed in triplicate (n = 3). *P < 0.05.

Fig. 5.

Effects of ERK1/2 and phosphatidylinositol 3-kinase (PI3-K) pathway inhibitors on the NHE2 promoter activity. C2BBe1 cells were serum starved and treated with ERK1/2 inhibitor U0126 (50 μM) (A) or LY294002 (50 μM) (B), a PI3-K inhibitor, for 1 h and subsequently stimulated with PMA (100 nM) for 16 h. Luciferase actives were determined as described in materials and methods. *Activity significantly different from PMA treatment (P < 0.05).

Fig. 6.

ERK1/2 activation in response to PMA in C2BBe1 cells. A: C2BBe1 cells were serum starved and treated with PMA for 15 min to 16 h. TCL were prepared and resolved in SDS-PAGE and probed with anti-phospho-ERK1/2 antibody. A similarly loaded gel was processed and probed with Pan-ERK1/2 antibody as loading control. B and C: after serum starvation and pretreatment (1 h) with different doses of U0126 or Gö6850, respectively, cells were incubated with or without PMA (100 nM) for 1 h. Western blot analyses were performed on TCL using anti-phospho-ERK antibody. The membrane in B was stripped and reprobed with Pan-ERK antibody as loading control.

Fig. 7.

Egr-1 expression in response to PMA and pathway-specific inhibitors. A: C2BBe1 cells were serum starved and treated with PMA for 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 6 h, and 16 h. Nuclear proteins were prepared subjected to Western blot analysis using antibody against Egr-1 transcription factor. The membrane was then stripped and reprobed with actin antibody for loading control. In other experiments serum-starved cells were pretreated with PKC inhibitor Gö6850 (1–5 μM) (B), PKCδ-specific inhibitor rottlerin (10 μM) (C), or ERK1/2 inhibitor U0126 (10–50 μM) (D) for 1 h. Cells were then stimulated with or without PMA (100 nM) for 1 h. Western blot analyses were performed using anti-Egr-1 antibody as described in materials and methods. Actin was used as loading control.

Fig. 8.

Effect of ERK1/2 inhibition on the PMA-induced Egr-1 nuclear expression. C2BBe1 cells were grown on glass coverslips to ∼90% confluence, then treated with or without PMA. To assess the effect of U0126, slides were pretreated with U0126 (50 μM, 1 h) before PMA treatments (100 nM, 2 h). The cells were fixed, stained, and visualized under fluorescence microscope as described in materials and methods. In each panel, a horizontal XY-optical section and the corresponding Z stacks (reconstructed serial vertical ZY optical sections of 0.5 μM each) are shown. Egr-1 was visualized with Alexa Fluor 488 (green), and nuclei were stained with DAPI (blue). The positions of vertical cuts are indicated with dashed white lines. In untreated (UT) cells (top) Egr-1 is predominantly located in the cell cytoplasm, whereas confocal images of cells treated with PMA distinctly show that Egr-1 is localized to the nuclei (middle). In contrast, treatment with U0126 before PMA exposure exhibits inhibition of Egr-1 accumulation in the nuclei (bottom). ZY-sections confirm the presence of Egr-1 in the nuclei of PMA-treated cells.

Fig. 9.

PMA-induced Egr-1 interacts with NHE2-PRE, and ERK1/2 inhibitor U0126 prevents this interaction. ³²P-end-labeled double-stranded oligonucleotide containing the NHE2 PRE at nucleotides −339 to −324 was incubated with nuclear proteins (5 μg) from untreated (lanes 1–7) or PMA-treated (100 nM, 2 h) (lanes 8–14) cells for 20 min at room temperature. The reactions were carried out in the absence (lanes 1 and 6) or presence of unlabeled Sp1 and NHE2-PRE (100-fold excess) oligonucleotides (lanes 2 and 11, respectively) or in the presence of specific antibodies (lanes 2–4, 8, and 12–14) as shown on top of the image. ss, supershifted band. DNA-protein complexes were resolved on 5% native gels, dried, and visualized by autoradiography.