ZINC INHIBITS CYCLIC AMP AGONISTS-INDUCED CHLORIDE SECRETION IN HUMAN INTESTINAL EPITHELIAL CELLS AND MOUSE INTESTINE VIA CALCIUM-SENSING RECEPTOR ACTIVATION

Paneth cells reside at the bottom of the crypts in the small intestine and are crucial for maintaining homeostasis of the epithelium by engendering host protection from enteric pathogens. Defects in Paneth cells compromise the intestinal epithelial host defense and lead to a range of mucosal pathologies. MicroRNA-195 (miR-195) is evolutionally conserved among different species and it regulates the stability and translation of target mRNAs and is involved in many aspects of cell processes. MiR-195 can interact with RNA-binding proteins including HuR to jointly regulate target gene expression synergistically or antagonistically. We recently reported that intestinal epithelial tissue-specific transgenic expression of miR-195 resulted in Paneth cell dysfunction in mice, but the exact mechanism underlying miR-195 in the regulation of Paneth cell function remains unknown. Sox9 is a specific transcription factor essential for Paneth cell differentiation and its deletion stops Paneth cell development. Here we tested the hypothesis that miR-195 regulates Paneth cells by altering Sox9 expression via interaction with HuR. Methods: Studies were conducted in our newly generated miR-195 transgenic (miR195-Tg) mice, HuR knockout (IE-HuR-/-) mice, and cultured HEK cells. Paneth cells were examined by lysozyme-immunostaining assays. The functions of miR-195 and HuR were determined by their gene silencing or overexpression in vitro. Levels of Sox9 mRNA and protein were measured by qPCR and Western blotting analyses. The interactions of Sox9 mRNA with HuR or miR-195 were examined by RNP or biotin immunoprecipitation (IP)/qPCR analysis. Results: miR195-Tg mice exhibited reduced levels of Sox9 protein (by ~70%) in the small intestinal mucosa, which was associated with a significant decrease in the number of Paneth cells. Ectopically overexpressed miR-195 in HEK cells also decreased cellular abundance of Sox9 primarily by inhibiting Sox9 translation since miR-195 overexpression decreased the levels of newly synthesized Sox9 protein without effect on its mRNA content. In contrast, HuR overexpression increased Sox9 expression in HEK cells. IP/qPCR analysis revealed that miR-195 did not directly bind to the Sox9 mRNA but prevented HuR association with Sox9 transcript. Ectopically expressed HuR rescued Sox9 expression in cells overexpressing miR-195. Double mutant mice bearing a miR-195 transgene and HuR ablation were generated by crossbreeding miR195-Tg mice and IE-HuR-/- mice and displayed further decreases in the levels of Sox9 and Paneth cells in the small intestine. Conclusions: These results indicate that 1) miR-195 inhibits Sox9 translation through interaction with HuR; and 2) control of Sox9 expression by miR-195 and HuR plays an important role in Paneth cell development and function.

Diarrhea is an important global health problem and secretory diarrhea outbreaks, such as cholera, remain a major cause of mortality in certain regions. Current treatment for secretory diarrhea is primarily supportive, and fluid replacement with oral rehydration solution (ORS) is the mainstay of cholera treatment. In addition to ORS, the WHO currently recommends oral zinc use for 10-14 days in children with diarrhea. Zinc has antidiarrheal efficacy in patients with and without zinc deficiency suggesting that it has direct antidiarrheal effects; however its exact mechanism of antidiarrheal action is not well understood. Extracellular calcium-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for secretory diarrhea. We recently showed that CaSR activator cinacalcet has marked antidiarrheal effects in human intestinal epithelial cells and mouse intestine. Cinacalcet effect was via stimulating cAMP hydrolysis that leads to inhibition of cAMP-gated ion channels (CFTR and basolateral membrane K⁺ channels). CaSR is physiologically activated by divalent and trivalent cations, and zinc (Zn²⁺) is a divalent heavy metal. We hypothesized that CaSR activation is a key mechanism for antidiarrheal effects of zinc. In human intestinal T84 cells natively expressing CaSR, zinc concentration-dependently inhibited forskolin-induced secretory I_sc by up to 60%, which was comparable to CaSR activator cinacalcet effect. Similarly, 100 μM zinc inhibited I_sc induced by clinically relevant cAMP agonists cholera toxin and vasoactive intestinal peptide (VIP) by 65%. Interestingly, 100 μM zinc had no effect on cGMP-agonist heat-stable E. coli enterotoxin-induced secretory I_sc in T84 cells, suggesting its antisecretory effects are specific to cAMP agonists. Using selective membrane permeabilization and ion gradients in T84 cells, we found that zinc inhibits apical membrane CFTR Cl^- conductance and basolateral membrane K⁺ channel conductance by ~65%. Zinc had no effect on forskolin-induced secretory I_sc in CFTR-transfected Fischer Rat Thyroid cells that do not express CaSR. Zinc treatment resulted in intracellular Ca²⁺ elevation (secondary messenger for CaSR) in CaSR-transfected HEK293 cells, but not in wild type HEK293 cells, suggesting that zinc is a CaSR agonist. In mouse intestine, zinc (100 μM) inhibited forskolin-induced secretory I_sc by 40% in wildtype mice, with no effect in intestinal epithelia-specific CaSR knockout mice (Vil1-Cre; Casr-flox). Our findings suggest that CaSR activation is a major mechanism for the antidiarrheal effects of zinc. In addition to its current clinical use in cholera, zinc can also be effective in other cAMP-mediated diarrheas such as VIP-secreting tumors and bile acid diarrhea.