Mechanisms of transcriptional modulation of the human anion exchanger SLC26A3 gene expression by IFN-γ

Two members of the SLC26 gene family, SLC26A3 or DRA (downregulated in adenoma) and SLC26A6 (putative anion transporter 1, PAT1), are known to play a major role in apical Cl−/OH− (HCO3−) exchange process in the human intestine. We have previously shown the inhibitory effects of IFN-γ (30 ng/ml, 24 h) on both SLC26A3 and A6 expression and promoter activity. We also demonstrated that the effects of IFN-γ on SLC26A6 gene expression were mediated via IRF-1 transcription factor. However, the molecular mechanisms underlying the transcriptional modulation of SLC26A3 gene expression by IFN-γ in the intestine are not known. The present studies were, therefore, designed to elucidate the signaling mechanisms and transcription factor(s) involved in mediating the inhibitory effects of IFN-γ on DRA promoter (p-−1183/+114) activity. Deletion analysis indicated that the IFN-γ response element is located within the −1183 to −790 region, and sequence analysis of this region revealed the presence of potential γ-activated site (GAS), a binding site (−933/−925 bp) for signal transducer and activator of transcription factor 1 (STAT1). Mutations in the potential GAS element abrogated the inhibitory effects of IFN-γ. These studies provide evidence for the involvement of STAT1 in the inhibition of SLC26A3 gene expression by IFN-γ in the human intestine

Stimulation of apical Cl−/HCO3−(OH−) exchanger, SLC26A3 by neuropeptide Y is lipid raft dependent

Neuropeptide Y (NPY), an important proabsorptive hormone of the gastrointestinal tract has been shown to inhibit chloride secretion and stimulate NaCl absorption. However, mechanisms underlying the proabsorptive effects of NPY are not fully understood. The present studies were designed to examine the direct effects of NPY on apical Cl−/HCO3−(OH−) exchange activity and the underlying mechanisms involved utilizing Caco2 cells. Our results showed that NPY (100 nM, 30 min) significantly increased Cl−/HCO3−(OH−) exchange activity (∼2-fold). Selective NPY/Y1 or Y2 receptor agonists mimicked the effects of NPY. NPY-mediated stimulation of Cl−/HCO3−(OH−) exchange activity involved the ERK1/2 MAP kinase-dependent pathway. Cell surface biotinylation studies showed that NPY does not alter DRA (apical Cl−/HCO3−(OH−) exchanger) surface expression, ruling out the involvement of membrane trafficking events. Interestingly, DRA was found to be predominantly expressed in the detergent-insoluble (DI) and low-density fractions (LDF) of human colonic apical membrane vesicles (AMVs) representing lipid rafts. Depletion of membrane cholesterol by methyl-β-cyclodextrin (MβCD, 10 mM, 1 h) remarkably decreased DRA expression in the DI fractions. Similar results were obtained in Triton-X 100-treated Caco2 plasma membranes. DRA association with lipid rafts in the DI and LDF fractions of Caco2 cells was significantly enhanced (∼45%) by NPY compared with control. MβCD significantly decreased Cl−/HCO3−(OH−) exchange activity in Caco2 cells as measured by DIDS- or niflumic acid-sensitive 36Cl− uptake (∼50%). Our results demonstrate that NPY modulates Cl−/HCO3−(OH−) exchange activity by enhancing the association of DRA with lipid rafts, thereby resulting in an increase in Cl−/HCO3−(OH−) exchange activity. Our findings suggest that the alteration in the association of DRA with lipid rafts may contribute to the proabsorptive effects of NPY in the human intestine