Krüppel-like factor 4 regulates adaptive expression of the zinc transporter Zip4 in mouse small intestine

Abstract

Epithelial cells of the small intestine are the site of zinc absorption. Intestinal uptake of zinc is inversely proportional to the dietary supply of this essential micronutrient. The mechanism responsible for this adaptive differential in apical zinc transport is not known. The zinc transporter Zip4 (Slc39a4) is essential for adequate enteric zinc uptake. In mice, Zip4 expression is upregulated at low zinc intakes with a concomitant ZIP4 localization to the apical enterocyte plasma membrane. With the present experiments, we show that the zinc finger transcription factor Krüppel-like factor 4 (KLF4), produced in high abundance in the intestine, is expressed at elevated levels in mice fed a low-zinc diet. In the murine intestinal epithelial cell (IEC) line MODE-K, zinc depletion of culture medium with cell-permeant and cell-impermeant chelators increased Zip4 and Klf4 mRNA and Zip4 heterogeneous nuclear RNA expression. Zinc depletion led to increased KLF4 in nuclear extracts. Knockdown of KLF4 using small interfering RNA transfection drastically limited ZIP4 induction upon zinc depletion and reduced 65Zn uptake by depleted IECs. EMSAs with nuclear extracts of IECs showed KLF4 binding to cis elements of the mouse Zip4 promoter, with increased binding under zinc-limited conditions. Reporter constructs with the Zip4 promoter and mutation studies further demonstrated that Zip4 is regulated through a KLF4 response element. These data from experiments with mice and murine IECs demonstrate that KLF4 is induced during zinc restriction and is a transcription factor involved in adaptive regulation of the zinc transporter ZIP4.

Fig. 1.

Characterization of Zip4 and Klf4 expression in intestines of control (ZnA) and zinc-depleted (ZnD) mice. mRNA levels in total RNA from intestines of the mice were determined by quantitative PCR (qPCR) and normalized to 18S rRNA levels. A: Mt mRNA, a positive control, demonstrates zinc depletion in ZnD mice. B–D: Zip4, Klf4, and alkaline phosphatase transcripts. Values are means ± SD (n = 3–4 mice per group). *P < 0.05; **P < 0.02 vs. ZnA.

Fig. 2.

Immunofluoresence localization of Krüppel-like factor 4 (KLF4) and ZIP4 (both detected separately with Texas Red) in mouse intestine. A and C: KLF4 and ZIP4, respectively, in ZnA mice. B and D: KLF4 and ZIP4, respectively, in ZnD mice. Sections were stained with 4′,6-diamidino-2-phenylindole nuclear stain. KLF4 images were captured by confocal microscopy and ZIP4 images by epifluoresence microscopy. Images represent findings from numerous experiments. Magnification: ×63 + ×4 (A and B) ×630 (C and D).

Fig. 3.

Western blot (top) and densitometric (bottom) analysis of KLF4 in nuclear extracts from intestines of ZnA and ZnD mice. Data represent results from replicated experiments.

Fig. 4.

Expression of Zip4 and Klf4 in small intestine (SI) and lung of ZnA (solid bars) and ZnD (gray bars) mice. Transcript abundance is normalized to level of 18S rRNA. Values are means ± SD (n = 3 mice per group). *P < 0.05; **P < 0.02 vs. ZnA.

Fig. 5.

Response of Zip4 and Klf4 mRNA and Zip4 heterogeneous nuclear RNA (hnRNA) expression to zinc depletion in the intestinal epithelial cell line MODE-K. Cells were incubated for 24 h in medium alone (control), medium containing 4 μM N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN; with or without 4 μM zinc), or medium containing 50 μM diethylenetriamine pentaacetic acid (DTPA; with or without 50 μM zinc). Abundance of Zip4 and/or Klf4 mRNAs was measured by qPCR and normalized to level of 18S rRNA. A and B: TPEN comparison groups. C and D: DTPA comparison groups. E: relative abundance of Zip4 hnRNA after incubation for 24 h with or without 50 μM DTPA. Values are means ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 vs. other groups.

Fig. 6.

Knockdown of Klf4 with small interfering RNA (siRNA) in intestinal epithelial cells limits Zip4 induction by zinc depletion. Cells were transfected with control (nontargeting) siRNA or mouse Klf4 siRNA and then incubated for 24 h in medium alone or medium containing 50 μM DTPA. Abundance of Zip4 mRNA or Klf4 mRNA was measured by qPCR and normalized to level of 18S rRNA. A: Klf4 mRNA knockdown by Klf4 siRNA transfection. *P < 0.05 vs. control. B: knockdown of Zip4 mRNA by Klf4 siRNA transfection in response to DTPA. Values are means ± SD (n = 3). *P < 0.05; **P < 0.01 vs. non-DTPA-treated cells using nontargeting and Klf4 siRNA, respectively. C: densitometry of Zip4 expression. Western blot analysis demonstrates that upregulation of ZIP4 produced by DTPA is inhibited by Klf4 siRNA. Data represent results from numerous experiments.

Fig. 7.

Knockdown of KLF4 with siRNA limits ⁶⁵Zn transport activity of intestinal epithelial cells in response to zinc depletion. Nearly confluent cells were transfected with control (nontargeting) siRNA or mouse Klf4 siRNA and cultured in medium containing 50 μM DTPA for 24 h. Cells were then placed in fresh medium (2.5 μM Zn) containing ⁶⁵Zn (57,000 cpm/ml) without DTPA and incubated for up to 60 min at 37°C. Cells were also incubated at 0°C as a control. Values are means ± SD (n = 3). *P < 0.05 vs. control.

Fig. 8.

Representative chemiluminescence-based EMSA demonstrates KLF4 binding to cis elements in mouse Zip4 promoter. Cells were incubated for 48 h in medium alone or medium containing 50 μM DTPA, and nuclear extracts were prepared. In one reaction, unlabeled (cold) probe was added at an excess (100×). Another reaction included anti-KLF4 antibody to obtain evidence for a supershift of the complex.

Fig. 9.

Effect of zinc depletion on activity of the Zip4 promoter. Confluent cells were cultured in medium containing 50 μM DTPA for 48 h and transiently transfected with native or mutated Zip4 promoter luciferase constructs. M1 indicates the mutated KLF4 binding element of the Zip4 promoter. Firefly luciferase activity of lysed cells was normalized to Renilla luciferase activity. Values are means ± SD (n = 4). ***P < 0.05 vs. control.