Abstract
Background: Zinc is a mineral that is essential for biological molecules, such as transcription factors, and is involved in the maintenance of intestinal homeostasis. Vitamin D signaling is mediated by vitamin D receptor (VDR) activated by 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] and is also important in intestinal functions, such as calcium absorption and epithelial barrier maintenance. However, the crosstalk between vitamin D signaling and zinc signaling in intestinal cells remains poorly understood. Materials and Methods: Colon cancer SW480 and HCT116 cells were treated with zinc chloride (ZnCl2) with/without 1,25(OH)2D3. Expression of zinc-inducible genes [metallothionein 1A (MT1A) and MT2A] and VDR target genes [cytochrome P450 family 24 subfamily A member 1 (CYP24A1), transient receptor potential cation channel subfamily V member 6 (TRPV6) and cadherin 1 (CDH1)] was examined. Results: Treatment of cells with ZnCl2 effectively induced MT1A and MT2A mRNA expression, and interestingly suppressed mRNA expression of CDH1, which was induced by 1,25(OH)2D3 in both cell lines. ZnCl2 also reduced the CDH1 protein level in HCT116 cells. Conclusion: Zinc signaling suppresses VDR-induced expression of CDH1.
Zinc is a trace element that plays essential physiological roles in catalytic, structural and regulatory function of biological molecules, such as enzymes and transcription factors, and is necessary for development, differentiation, neurological functions, and protein synthesis [reviewed in (1)]. Bioinformatics analysis has revealed that over 10% of human proteins bind to zinc (2). Zinc deficiency causes immune, endocrine and neuronal dysfunctions, skin and intestinal disorders, and growth retardation (1, 3, 4). Although zinc excess induces toxicity, such as nausea, vomiting and lethargy (1, 5), the pharmacological properties of zinc are utilized in the treatment of gastric ulcers (6). Cellular zinc levels are tightly regulated by transporters, including the zinc transporter/solute carrier 30A (SLC30A) family and the Zrt/Irt-like protein (ZIP)/SLC39A family (1, 4). Growing evidence from characterization of zinc transporters by knockout studies in mice and human genetic studies highlights the importance of ‘zinc signaling’ in cellular mechanisms (1).
The active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], regulates numerous physiological processes, including calcium and bone metabolism, cellular growth and differentiation, and immunity (7, 8). The biological actions of 1,25(OH)2D3 are mainly mediated by the vitamin D receptor (VDR), a zinc-finger transcription factor of the nuclear receptor superfamily (9, 10). Ligand binding induces a conformational change of VDR, and the structural rearrangement results in the dynamic interaction with retinoid X receptor and exchange of cofactor complexes, leading to transcriptional induction of specific target genes that usually have a vitamin D response element, a two-hexanucleotide (AGGTCA or a related sequence) direct repeat motif separated by three nucleotide (direct repeat 3), such as the gene encoding cytochrome P450 family 24 subfamily A member 1 (CYP24A1) and that encoding transient receptor potential cation channel subfamily V member 6 (TRPV6) (10). Genome-wide analyses show the existence of more than 1,000 VDR-binding sites, some of which do not contain a putative vitamin D response element (11-13).
The intestine is a principal target organ of VDR signaling, and the major physiological effect of vitamin D is to enhance calcium absorption (14). Vitamin D deficiency results in rickets and osteomalacia, which are caused by impaired calcium absorption in the intestine. Intestinal epithelial VDR is also involved in the maintenance of mucosal barrier function and epithelial differentiation by inhibiting β-catenin activity and inducing the gene encoding cadherin 1 (E-cadherin; CDH1) (15-17), and in protection of epithelial cells from inflammatory stress by inducing autophagy (18). In addition to vitamin D signaling, zinc plays important roles in intestinal epithelial functions. SLC39A7 (also called ZIP7), a zinc influx transporter of the ZIP family, is highly expressed in intestinal crypts, and mice with an intestinal epithelium-specific Slc39a7 deletion are vulnerable to endoplasmic stress in intestinal proliferative progenitor cells, indicating that zinc signaling is important in the homeostasis of the intestinal epithelium (19). However, the crosstalk between vitamin D signaling and zinc signaling remains poorly understood. In this study, we examined the effect of zinc on the expression of VDR target genes in colon cancer cells.
Materials and Methods
Compounds. 1,25(OH)2D3 and zinc chloride (ZnCl2) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).
Cell culture. Human intestinal carcinoma SW480 cells and HCT116 cells (American Type Culture Collection, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium containing 10% inactivated fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2 (20).
Viable cell evaluation. Viable cells were evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (21). Cells were seeded at 1×104 cells/well in a 96-well plate 1 day before the addition of ZnCl2 and treated with a range of ZnCl2 concentration (0-200 μM) for 24 h. For viable cell evaluation, cells were incubated with 5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Nacalai Tesque, Inc., Kyoto, Japan) for 4 h, and the cells were dissolved in 100 μl of dimethyl sulfoxide. Absorbance at 560 nm was measured with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Reverse transcription and quantitative real-time polymerase chain reaction. Cells were seeded at 2×105 cells/well in a 6-well plate 1 day before treating with 100 or 200 μM ZnCl2 alone for 3 or with/without 100 nM 1,25(OH)2D3 for 24 h. Total RNA from cells was prepared by the acid guanidine thiocyanate-phenol/chloroform method (22). cDNAs were synthesized using the ImProm-II Reverse Transcription system (Promega Corporation, Madison, WI, USA). Real-time polymerase chain reaction was performed on the ABI PRISM 7000 Sequence Detection System (Thermo Fisher Scientific, Waltham, MA, USA) with Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). Primer sequences for genes encoding metallothionein 1A (MT1A) and MT2A were as follows: MT1A, 5’-GCC TCT CAA CTT CTT GCT TG-3’ and 5’-GCA CAC TTG GCA CAG CTC AT-3’; MT2A, 5’-TGC AAC CTG TCC CGA CTC TA-3’ and 5’-TTT GCA GAT GCA GCC CTG GG-3’. For relative mRNA expression, the mRNA values were normalized to those of glyceraldehyde 3-phosphate dehydrogenase and primer sequences for CYP24A1, TRPV6, CDH1 and glyceraldehyde 3-phosphate dehydrogenase were as reported previously (20, 23).
Western blot analysis. Cells (3.6×105 cells) were seeded in a 60 mm dish 1 day before the treating with 200 μM ZnCl2 with/without 100 nM 1,25(OH)2D3 for 24 h. Whole cell lysates were homogenized in RIPA buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 4 mM EDTA, 0.1% sodium dodecyl sulfate, 1% deoxycholate, 1% Triton X-100, 10% glycerol) containing 10 mM NaF, 1 mM Na2VO4 and protease inhibitor cocktail (Merck KGaA, Darmstadt, Germany), and centrifuged at 14,000 × g at 4°C for 10 minutes to remove debris as reported previously with minor modification (16, 24). The proteins (5 μg for each sample) were electrophoresed on a 7.5% sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane for immunoblotting. Western blot analysis was performed using antibody to CDH1 (BD Biosciences, San Jose, CA, USA), visualized with an ECL plus western blotting detection system (GE Healthcare, Chalfont St. Giles, UK), and using an anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA), visualized with an alkaline phosphatase conjugate substrate system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), as reported previously (23, 25). CDH1 protein levels were quantified with Image J 1.45 (National Institutes of Health, Bethesda, MD, USA), and normalized with those of β-actin protein.
Statistics. Data are presented as means±S.D of triplicate assays. All quantitative data were analyzed by one-way factorial analysis of variance (ANOVA) followed by Tukey’s post hoc test using Prism 8 (GraphPad Software, La Jolla, CA, USA).
Results
As ZnCl2 has been reported to induce gastrin gene promoter activity in human intestinal SW480 cells (26), we examined the effect of ZnCl2 on VDR target gene expression in this cell line. Firstly, we determined non-toxic concentrations of ZnCl2 in SW480 cells by evaluating cell viability. Treatment of SW480 cells with ZnCl2 at 1 μM and 10 μM slightly increased the proportion of viable cells, while ZnCl2 at 200 μM markedly reduced it (Figure 1A). As zinc exposure had been shown to increase mRNA expression of MT1A and MT2A from 2 to 6 h by activating metal regulatory transcription factor 1 (MTF1) in human embryonic kidney 293 cells (27), we next treated SW480 cells with ZnCl2 at the highest non-toxic concentration (100 μM) for 3 h and examined mRNA expression of MT1A and MT2A to assess whether zing signaling is active in SW480 cells. We observed significant induction of MT1A and MT2A genes (Figure 1B), indicating that ZnCl2 treatment activates cellular zinc signaling.
Effect of ZnCl2 on cell viability and expression of metallothionein 1A (MT1A) and MT2A in SW480 cells. A: Cell viability. Cells were treated with a range of concentrations of ZnCl2 for 24 h, and cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Absorbance values of treated cells were compared to those of control cells. Significantly different at *p<0.05 and ***p<0.001 (one-way analysis of variance followed by Tukey’s multiple comparisons). B: mRNA expression of MT1A and MT2A. Cells were treated with 0 (vehicle control) or 100 μM ZnCl2 for 3 h, and expression of the zinc-inducible genes was evaluated with reverse transcription and quantitative real-time polymerase chain reaction. *Significantly different at p<0.05 (Student’s t-test).
As reported previously (20, 25), 1,25(OH)2D3 treatment effectively increased the mRNA expression of VDR target genes, CYP24A1, TRPV6 and CDH1 (Figure 2). While treatment with ZnCl2 alone did not affect gene expression, ZnCl2 reduced 1,25(OH)2D3-induced expression of CDH1 but did not significantly affect that of CYP24A1 or TRPV6 in SW480 cells treated with 1,25(OH)2D3 (Figure 2).
Effect of ZnCl2 on mRNA expression of vitamin D receptor target genes in SW480 cells. Cells were treated with vehicle control, or 100 μM ZnCl2 with/without 100 nM 1,25(OH)2D3 for 24 h, and mRNA expression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1), transient receptor potential cation channel subfamily V member 6 (TRPV6) and cadherin 1 (CDH1) was evaluated. ***Significantly different at p<0.001 (one-way analysis of variance followed by Tukey’s multiple comparisons).
We examined the effect of ZnCl2 in another intestinal colon cancer cell line, HCT116. ZnCl2 at 200 μM was not toxic to HCT116 cells (data not shown), and effectively induced mRNA expression of MT1A and MT2A in HCT116 cells (Figure 3A). Treatment of HCT116 cells with 200 μM ZnCl2 reduced expression of CYP24A1 and CDH1 induced by 1,25(OH)2D3, but the effect of the combination on TRPV6 expression was not significant (Figure 3B). We observed a concentration-dependent effect of ZnCl2 on CDH1 expression induced by 1,25(OH)2D3 in HCT116 cells (Figure 3C). Thus, ZnCl2 was found to reduce 1,25(OH)2D3-induced CDH1 expression in both SW480 and HCT116 cells.
Effect of ZnCl2 on expression of zinc-inducible genes and vitamin D receptor (VDR) target genes in HCT116 cells. A: Cells were untreated or treated with 200 μM ZnCl2 for 3 h. mRNA expression of zinc-inducible genes, metallothionein 1A (MT1A) and MT2A. was then determined by polymerase chain reaction (PCR). B: Cells were treated with vehicle control, or 200 μM ZnCl2 with/without 100 nM 1,25(OH)2D3 for 24 h. mRNA expression of VDR target genes, cytochrome P450 family 24 subfamily A member 1 (CYP24A1), transient receptor potential cation channel subfamily V member 6 (TRPV6) and cadherin 1 (CDH1) was then determined by PCR. C: Cells were treated with 0-200 μM ZnCl2 in the absence or presence of 100 nM 1,25(OH)2D3 for 24 h. CDH1 mRNA expression was determined by PCR and found to be ZnCl2 concentration-dependent. Significantly different at *p<0.05, **p<0.01 and ***p<0.001 (one-way analysis of variance followed by Tukey’s multiple comparisons).
Finally, we examined CDH1 protein levels in HCT116 cells treated with ZnCl2 and/or 1,25(OH)2D3. Consistent with mRNA expression of CDH1 (Figure 3), 1,25(OH)2D3 increased the CDH1 protein level whilst combined treatment with ZnCl2 reduced 1,25(OH)2D3-induced CDH1 protein expression (Figure 4).
ZnCl2 reduces the cadherin 1 (CDH1) protein level in HCT116 cells. Cells were untreated (vehicle control), or treated with 200 μM ZnCl2 with/without 100 nM 1,25(OH)2D3 for 24 h. Western blotting was then performed for CDH1 and β-actin. The CDH1 protein level was quantified with Image J 1.45 and normalized with that of β-actin. Significantly different at *p<0.05 and **p<0.01 (one-way analysis of variance followed by Tukey’s multiple comparisons).
Discussion
In this study, we found that zinc signaling affects expression of VDR target genes, specifically CDH1, the gene encoding cadherin 1, in colon cancer cells. Firstly, we observed that treatment with non-toxic concentrations of ZnCl2 effectively increased zinc-inducible expression of MT1A and MT2A indicating active zinc signaling through MTF1 (27). Treatment with ZnCl2 alone did not increase expression of VDR target genes. Although VDR is a zinc finger transcription factor (9, 10), zinc signaling cannot induce VDR activation in the absence of its ligand. The active VDR ligand 1,25(OH)2D3 effectively induced expression of CDH1, CYP24A1 and TRPV6 in SW480 and HCT116 cells, and ZnCl2 combination suppressed expression of CDH1 in SW480 cells and of CDH1 and CYP24A1 in HCT116 cells. A direct effect of zinc signaling on VDR transactivation activity is unlikely to account for this gene-selective effect. Zinc signaling may regulate CDH1 transcription through VDR-independent mechanism(s). Knockdown of a zinc import transporter of the ZIP/SLC39A family, namely SLC39A6 (formerly LIV-1), in hepatocellular carcinoma cells was shown to increase mRNA and protein expression of CDH1 (28, 29), suggesting that cellular zinc signaling represses CDH1 expression.
VDR plays an important role in the maintenance of mucosal barrier function and epithelial differentiation by regulating β-catenin signaling and inducing CDH1 expression (15-17). VDR activation also inhibits epithelial-to-mesenchymal transition (EMT) of colon cancer cells (30, 31). Elevated expression of SLC39A6 was associated with an aggressive phenotype of pancreatic cancer by inducing EMT (32). SLC39A6 also induced EMT in cervical cancer (33) and prostate cancer (34) cells. These findings suggest that intracellular zinc accumulation and consequent activation of zinc signaling are involved in EMT of cancer cells. Although zinc signaling plays a role in the homeostasis of the intestinal epithelium (19), zinc accumulation may be involved in colon carcinogenesis by suppressing VDR-dependent expression of CDH1. Our results provide insights into the crosstalk between zing signaling and vitamin D signaling in intestinal cancer cells.
Acknowledgements
The Authors thank members of the Makishima laboratory for technical assistance, the late Professor Kazumasa Ikeda of Nihon University College of Bioresource Sciences for helpful support, and Dr. Andrew I. Shulman for editorial assistance.
Footnotes
Authors’ Contributions
M.I. performed experiments, analyzed the data and wrote the article. A.H. performed experiments and analyzed the data. M.M. supervised experiments, and wrote and edited the article.
Conflicts of Interest
The Authors declare no conflicts of interest in regard to this study.
- Received September 6, 2021.
- Revision received September 22, 2021.
- Accepted September 24, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
