STAT5-glucocorticoid receptor interaction and MTF-1 regulate the expression of ZnT2 (Slc30a2) in pancreatic acinar cells

Abstract

The exocrine pancreas plays an important role in endogenous zinc loss by regulating excretion into the intestinal tract and hence influences the dietary zinc requirement. The present experiments show that the zinc transporter ZnT2 (Slc30a2) is localized to the zymogen granules and that dietary zinc restriction in mice decreased the zinc concentration of zymogen granules and ZnT2 expression. Excess zinc given orally increased ZnT2 expression and was associated with increased pancreatic zinc accumulation. Rat AR42J acinar cells when induced into a secretory phenotype, using the glucocorticoid analog dexamethasone (DEX), exhibited increased ZnT2 expression and labile zinc as measured with a fluorophore. DEX administrated to mice also induced ZnT2 expression that accompanied a reduction of the pancreatic zinc content. ZnT2 promoter analyses identified elements required for responsiveness to zinc and DEX. Zinc regulation was traced to a MRE located downstream from the ZnT2 transcription start site. Responsiveness to DEX is produced by two upstream STAT5 binding sites that require the glucocorticoid receptor for activation. ZnT2 knockdown in the AR42J cells using siRNA resulted in increased cytoplasmic zinc and decreased zymogen granule zinc that further demonstrated that ZnT2 may mediate the sequestration of zinc into zymogen granules. We conclude, based upon experiments with intact mice and pancreatic acinar cells in culture, that ZnT2 participates in zinc transport into pancreatic zymogen granules through a glucocorticoid pathway requiring glucocorticoid receptor and STAT5, and zinc-regulated signaling pathways requiring MTF-1. The ZnT2 transporter appears to function in a physiologically responsive manner involving entero-pancreatic zinc trafficking.

Fig. 1.

Tissue and serum zinc concentrations and the expression of ZnT1 and ZnT2 in mouse pancreas. (A–C) Mice were fed either a zinc-adequate (ZnA) or a zinc-deficient diet (ZnD) for 21 days. (A) Cytoplasmic, nuclear, and zymogen granule compartments were isolated along with serum. Zinc is expressed as mg zinc/g protein or μg zinc/ml. (B,D) qPCR analysis of MT, ZnT1 and ZnT2 mRNA levels in pancreas. Values are relative mRNA normalized to 18S rRNA. (C) Abundance of ZnT1 and ZnT2 proteins in isolated zymogen granules (ZG) and plasma membranes (PM) of ZnA and ZnD mice were analyzed by Western blotting. Na⁺/K⁺ ATPase and amylase are loading controls for PM and ZG fractions, respectively. (D) Mice were given a dose of 35 μg zinc/g body weight or saline orally. MT, ZnT1, and ZnT2 mRNAs were measured 3 h and 8 h after gavage. n = 3–4.

Fig. 2.

ZnT2 promoter and transport activity in transfected cells in response to zinc. (A) HeLa cells were transfected with murine ZnT2 promoter constructs over the range (-815 to +93) to (-95 to +93) ligated into pGL3-Basic vector. One construct (-95 to +93) was also mutated at the MRE consensus sequence (+53 to +59). Luciferase activity was measured 48 h after transfection (values are relative luminescence units; firefly/renilla). Zinc (100 μM) was added for the last 24 h. (B) Activity of -95 to +93 promoter construct in response to 0, 20, 40, 80, or 120 μM Zn added for the last 24 h. (C) HEK293 cells were cotransfected with an hMTF-1 expression vector and the -95 to +93 ZnT2 promoter construct for 48 h with 100 μM Zn added for the last 24 h. (D,E) AR42J cells were transfected with a control or pCMV-ZnT2-flag vector for 48 h. (D) Western blot of total cell lysate showing ZnT2-flag overexpression. (E) Cells were preincubated with ⁶⁵Zn for 24 h. Efflux of the ⁶⁵Zn is expressed on a cpm/cell protein basis. Bars with different superscripts indicate the means are statistically different at P < 0.05.

Fig. 3.

Dexamethasone increases ZnT2 expression in rat AR42J cells and mouse pancreas. (A, B) AR42J cells were incubated with DEX for up to 48 h. ZnT1, ZnT2, and amylase mRNA levels were measured by qPCR. (C) Mice were injected i.p. with either DEX or saline and killed 3, 8, or 16 h thereafter. mRNA levels were measured by qPCR. (D, E) Confocal immunofluorescence microscopy of purified zymogen granules from pancreas of (D) saline or (E) DEX-treated mice. ZnT2 was detected with affinity-purified antibody. n = 3–4.

Fig. 4.

Dexamethasone-induced ZnT2 expression in AR42J pancreatic acinar cells is mediated by STAT5 through a pathway that requires the glucocorticoid receptor. (A) Cells were cultured in the medium supplemented with or without DEX, CpdA, and/or RU486 for 12 h. (B) Cells were cultured with either the JAK2 inhibitor, AG490 or the STAT5 inhibitor (CNH) for 24 h, prior to DEX for 12 h. qPCR was used to measure MT and ZnT2 mRNA levels. (C,D) AR42J cells were treated with to DEX for 5 h, two-step cross-linking ChIP assays were performed with GR (C) or STAT5 (D) antibodies, and DNA enrichment was analyzed by qPCR. n = 3.

Fig. 5.

Zinc trafficking in rat AR42J pancreatic acinar cells is influenced by ZnT2 expression. (A) Cells were transfected with either ZnT2 siRNA or control siRNA and cultured with or without DEX for 48 h. ZnT2 mRNA was measured by qPCR. (B) Western blots showing effect of ZnT2 siRNA inhibition on ZnT2 protein in AR42J cells. Control vs. ZnT2 siRNA treated cells (Upper Panels). Comparative effects of DEX and ZnT2 siRNA on ZnT1 vs. ZnT2 (Lower Panels). (C) Cells were transfected with ZnT2 siRNA and harvested at various times. MT mRNA was measured by qPCR. (D, E) ⁶⁵Zn retention was used as a direct measure of zinc accumulation. Some cells were transfected with ZnT2 siRNA (D) and some were treated with DEX (E). After 48 h in the presence of ⁶⁵Zn the cells cytoplasmic (CP), crude nuclear (Nuc), and zymogen granule (ZG) fractions were isolated. ⁶⁵Zn content was measured and the specific activity was used to calculate zinc retention. (F) Cellular labile zinc was measured using a FluoZin 3-AM fluorescence assay. Cells were treated with zinc (40 μM) or DEX or both for 48 h. Values with a different superscript are significantly different at P < 0.05 or greater. n = 4.

Fig. 6.

Proposed model of zinc transport and secretion in pancreatic acinar cells. Zinc influx is influenced by ZIP5 located at the basolateral plasma membrane. Sources of zinc for export are derived previously from the Golgi as metalloproteins (MP), metallothionein (MT), and the labile intracellular pool [Zn]. Zinc secretion at the apical plasma membrane is regulated through two different pathways. Zinc is transported into the ductal lumen of the pancreatic acinar cells through ZnT1 localized on the apical plasma membrane. Cytosolic zinc is sequestrated into zymogen granules by ZnT2 and is released during regulated exocytosis. Expression of ZnT1 and ZnT2 is mediated by MTF-1, depending on the intracellular zinc level. MT and ZnT2 expression is regulated by GC through the glucocorticoid receptor (GR). ZnT2 expression requires interaction of the GR with STAT5. ZnT2 expression is more likely to be associated with secretory stimulation of digestive enzymes.