TNF‐α requires cAMP/calcium response elements (CRE) to inhibit slc38a2 transcription for SNAT2 mediated Met uptake in enterocytes

Abstract

Background Methionine, an essential amino acid, serves as a methyl donor for biosynthesis of many biomolecules. Previously we observed that immunoinflammatory cytokine, Tumor Necrosis Factor (TNF-α) inhibits Na+-methionine cotransporter (SNAT2, solute carrier, slc38a2) numbers and mRNA abundance in enterocytes. Aim How TNF-α regulates the transcriptionof SNAT2 in enterocytes to inhibit Met cotransport is unknown. Methods [3H]-Met uptake was measured in 10 days postconfluent rat intestinalepithelial cells (IEC-6) grown on transwell plates. IEC-6 cells were treated with different inhibitors intercepting different checkpoints of pathways for TNF-α mediated inhibition of SNAT2 at 9 days postconfluence. Results TNF-α treatment decreased SNAT2 activity and increased more than 2.5-fold cytosolic Ca2+, and cAMP. PKC inhibitor did not affect theTNF-α mediated inhibition of SNAT2 activity. However, cAMP and PKA inhibitors independently antagonized TNF-α effect on SNAT2 activity. It has been shown that cAMP response elements (CRE) in the promoter that controls the expression of the rat SNAT2 gene regulated by cAMP. ChIP and Luciferase assays show that TNF-α inhibits CRE to inhibit SNAT2 mRNA transcription.When taken together along with Kinetics, Western blotting, immunocytochemistry, and qRT-PCR demonstrated that TNF-α transcriptionally inhibits SNAT2 via CRE in IEC-6 cells. Conclusion Thus, this study provides new insights into how the TNF-α inhibits Met uptake through the transcriptional regulation of the SNAT2 transporter via CRE in enterocytes. Supported by NSF Grant #HRD-1332459