The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon

Abstract

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn2+ are not well understood. Here, we determined a role of the colonocytic Zn2+ sensing receptor, ZnR/GPR39, in mediating Zn2+-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn2+-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn2+ by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases.

The zinc sensing receptor, ZnR/GPR39, triggers metabotropic calcium signalling in colonocytes and regulates occludin recovery in experimental colitis

“…Caco-2 colonocytic cells were grown in DMEM medium [20] containing 100 U ml -1 penicillin, 0.1 mg ml -1 streptomycin, 2 mM glutamine, 10% foetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), 1% (v/v) nonessential amino acids (Biological Industries) and 2 mM sodium pyruvate (Sigma-Aldrich, Rehovot, Israel) in a 5% CO 2 humidified atmosphere at 378C. Fluorescent imaging measurements were acquired on cells loaded with Fura-2 acetoxymethyl ester (AM; TEF-Labs, Austin, TX, USA) in Ringer's solution (30 min 2.5 mM).…”

“…Cells were harvested into lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 10 mM MgCl2, 25 mM NaF), in the presence of Protease Inhibitor Cocktail (1 : 50, Sigma-Aldrich). Protein concentrations were determined and whole-cell lysates (12 mg) were separated on 10% SDS-PAGE and blotted onto nitrocellulose membranes [20]. Antibodies raised against occludin (Invitrogen, Life Technologies) were used, and blots were quantified using IMAGEJ Gels Plugin.…”

“…A specific Zn 2þ sensing receptor (ZnR) was functionally described in this tissue [16,17] and was later associated with GPR39 as the molecular moiety mediating its activity [18,19]. The ZnR/GPR39 triggers cellular Ca 2þ signalling pathways and enhances cell proliferation, differentiation and survival [20]. The ZnR/GPR39 is prominently expressed in the gastrointestinal tract [21], and the analysis of ZnR/GPR39 knockout (KO) mice suggested a mild gastric phenotype of accelerated gastric emptying and higher volume of gastric fluid secretion [22].…”

Discovery and Characterization of Novel GPR39 Agonists Allosterically Modulated by Zinc

“…In a clinical study of LY2784544, diarrhea, nausea, anemia, and transient increases in serum creatinine, uric acid, and potassium have been reported and attributed to a "typical tumor lysis syndrome" (Tefferi, 2012); however, it seems conceivable that at least some of these effects might be due to activation of GPR39. Interestingly, GPR39 is highly expressed in human colorectal adenocarcinoma HT-29 cells, and Zn 21 and a GPR39 agonist stimulated Gq signaling and promoted survival in these cells (Cohen et al, 2012(Cohen et al, , 2014Boehm et al, 2013). By using the fluorescent imaging plate reader assay, we have shown that GPR39-C3, LY2784544, and GSK2636771 strongly activate the Gq pathway in HT-29 cells (Supplemental Fig.…”

“…GPR39 has been found to be expressed in mouse intestinal fibroblastlike cells (Zeng et al, 2012), human colon adenocarcinoma HT-29 cells (Yasuda et al, 2007;Cohen et al, 2012Cohen et al, , 2014, rat colon BRIN-BD11 cells (Moran et al, 2016), and mouse pancreatic epithelial NIT-1 cells (Fjellström et al, 2015). All of these cell types respond to Zn 21 via various signal transduction pathways.…”