Diarrhea associated with inflammatory bowel disease has been attributed to stimulated secretion of proinflammatory cytokines like IFN-gamma and TNF-alpha, which have been shown to downregulate the expression of the sodium-hydrogen exchanger-3 (NHE3) gene. In this study, we have investigated the mechanism of NHE3 gene regulation by IFN-gamma and TNF-alpha in C2BBe1 cells. In response to both IFN-gamma (30 ng/ml) and TNF-alpha (20 ng/ml), the construct containing the bp -95 to +5 region of the human NHE3 promoter, which harbors a number of cis-elements including four potential Sp1 binding sites, showed a maximum repression of 60%. Knockdown of Sp1 and Sp3 expression using small interfering RNA resulted in a significant inhibition of the NHE3 promoter activity and resistance to cytokines effects. These cytokines showed no effects on the expression of Sp1 and Sp3 mRNA and protein levels as assessed by RT-PCR and Western blot analyses, respectively. After treatment with cytokines, the binding of Sp1 and Sp3 proteins to NHE3 promoter decreased significantly, as seen by gel mobility shift assays and chromatin immunoprecipitation assays. The inhibitory effects of both cytokines on the NHE3 promoter were completely blocked by the broad-range kinase inhibitor staurosporine and the selective protein kinase A (PKA) inhibitor 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer. The binding affinity of Sp1 and Sp3 proteins for NHE3 Sp1 probe was significantly decreased after in vitro phosphorylation of nuclear proteins by the alpha-catalytic subunit of PKA. Our data indicate that IFN-gamma and TNF-alpha may repress the NHE3 promoter activity in C2BBe1 cells by PKA-mediated phosphorylation of Sp1 and Sp3 transcription factors.

NHE3 (Na+/H+ exchanger 3) is essential for Na+ absorption in the ileum and is expressed in a cell-specific manner in the apical membrane of the intestinal epithelial cells. In the present study, we report the stimulatory effect of PMA on the hNHE3 (human NHE3) transcription. Pretreatment with actinomycin D or cycloheximide blocked the up-regulation of the NHE3 mRNA by PMA, indicating that the increased level of NHE3 mRNA expression is regulated by transcriptional activation and is dependent on de novo protein synthesis. 5'-Deletion of the promoter region and transfection analysis in C2BBe1 cells revealed that the PMA effect is mediated through a GC-rich DNA region between nt -88 and -69. Gel mobility-shift assays demonstrated that in nuclear extracts from C2BBe1 cells grown under the basal growth conditions, Sp1 (stimulating protein-1) and Sp3 interact with this GC-rich DNA region, while, in PMA-treated nuclear extracts, PMA-induced EGR-1 (early growth response gene product 1) transcription factor binds to the same site. Binding of EGR-1 diminished the Sp1 and Sp3 interactions with this promoter region significantly. Co-transfection of Sp1 or Sp3 into SL2 cells activated the NHE3-reporter constructs, suggesting that Sp1 and Sp3 act as positive regulators of the NHE3 expression. In addition, overexpression of EGR-1 was sufficient to transactivate the NHE3-reporter gene activity, and knockdown of EGR-1 with gene-specific small interfering RNA resulted in inhibition of the PMA-induced up-regulation of the endogenous NHE3 mRNA expression. Furthermore, the PKC (protein kinase C) inhibitor chelerythrine chloride did not affect PMA-induced NHE3 promoter activity, suggesting that PMA stimulation of the hNHE3 gene expression may be PKC-independent.

Transcription of human immunodeficiency virus type 1 (HIV-1) is regulated by cis-acting DNA elements located in the viral long terminal repeats, by viral transregu-latory proteins, and by cellular transcription factors acting in concert to modulate the degree of viral expression. We demonstrate that a DNA fragment corresponding to the central portion of the HIV-1 genome exhibits enhancer activity when cloned upstream of the thymidine kinase promoter of herpes simplex virus. This enhancer is inducible by phorbol 12-myristate 13-acetate in HeLa cells and is independent of its position and orientation with respect to the promoter. We have mapped the activity of the enhancer to two independent domains encompassing nucleotides 4079-4342 (end of the pol gene) and nucleotides 4781-6026 (vifgene and frst coding exon of tat). This intragenic enhancer and its subdomains demonstrate cellular specificity because they are only active in specific cell lines. The presence of similar intragenic enhancer elements in other retroviruses suggests that they might be a conserved feature of this family of viruses.

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