Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor, FG4592, Induces Endogenous Metallothionein3 Expression in Human Neuronal Cell Line, ReNcell CX Cells

Abstract

Metallothionein (MT) is a small-molecule protein that functions in essential trace element homeostasis. Among MT isoforms, MT3 is involved in neuronal activity, and its expression is reported to be decreased in patients with neurodegenerative conditions such as Alzheimer’s disease; however, only a few effective drugs have been reported to induce MT3 expression. In this study, we evaluated existing drugs for the induction of MT3 expression in the neuronal cell line of ReNcell CX cells. Using recombinant proteins of MT isoforms with the 3× Flag tag, we performed Western blotting (WB) with the primary antibodies against MT3 or Flag tag, and this method of WB for MT3 was confirmed specifically to detect the MT3 protein. We treated ReNcell CX cells with several HIF-PH inhibitors and evaluated MT3 expression via real-time RT-PCR. We found that FG4592 significantly enhanced MT3 expression at both RNA and protein levels. FG4592 treatment increased the amount of hypoxia-inducible factor 1 alpha (HIF1α) binding to the MT3 promoter. These findings indicate that FG4592 induces MT3 expression via increased HIF1α. In conclusion, we found FG4592 to be an endogenous MT3 inducer in the cells of the nervous system in this study. The findings of this study are expected to lead to the development of new MT3-inducing drugs for neurodegenerative diseases based on FG4592.

INTRODUCTION

Metallothionein (MT) is a multifunctional low-molecular-weight protein with diverse functions, such as homeostasis of essential trace elements and antioxidant activity. MT is composed of approximately 68 amino acids and has 4 isoforms. MT1/2 is expressed in a variety of organs but less so in neurons.¹⁾ However, MT3 is localized in the central nervous system, including the cerebrum, striatum, and spinal cord, suggesting that MT3 has a function common to other MTs in neurons.²⁾ MT3 exhibits cytoprotective effects in neurons due to its potent reactive oxygen species (ROS)-trapping properties.³⁾

Regarding the association between MT3 and disease, it has been reported that MT3 is decreased in patients with various neurodegenerative diseases such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS).^4,5) MT3 also regulates actin polymerization, and because cytoskeletal disorders cause various neurodegenerative diseases, MT3 may be involved in these diseases.⁶⁾ There have been several reports on the therapeutic effects of MT3 in neurodegenerative diseases such as AD, including a report that MT3 directly administered into the brain of AD model mice improved the disease state.⁷⁾ Another report stated that MT3 directly regulates actin polymerization in astrocytes and is involved in the clearance of amyloid β (Aβ) by endocytosis.⁸⁾ In ALS, a decrease in MT3 has also been reported in the spinal cord of ALS patients,⁹⁾ and there are reports that MT3 overexpression in ALS model mice using an adenovirus vector has improved the disease phenotype.¹⁰⁾ MT1 and MT2 expression is readily induced by various stimuli, including many metals, oxidative stress, and cytokines,⁴⁾ while MT3 is rarely induced by these stimuli, and it has been reported that MT3 is induced by hypoxia.¹¹⁾ Therefore, although not many drugs induce MT3 expression, if endogenous MT3 could be induced, it could constitute one of the new treatment options for neurodegenerative diseases.

Hypoxia-inducible factor 1 alpha (HIF1α) is a transcription factor that plays a major role in the cellular response to hypoxic stress conditions.¹²⁾ HIF1α is an extremely short lived protein under oxygen conditions. HIF1α is hydroxylated by HIF-prolyl hydroxylase (HIF-PHD) and bound to the ubiquitin ligase complex. This leads to HIF1α ubiquitination, which is then degraded by the proteasome.^13,14) HIF-PHD inhibitors can promote HIF1α-mediated transcriptional induction via HIF-PHD inhibition. Regarding the association between HIF and MT3, it has been reported that the administration of a HIF-PHD inhibitor to human adipocytes significantly increased MT3 expression.¹⁵⁾

In this study, to explore MT3 expression induction agents in the brain, we used immortalized cells derived from the human fetal cerebrum (ReNcell CX cells) to evaluate MT3 induction by HIF-PHD inhibitors and to elucidate the mechanism involved.

MATERIALS AND METHODS

Cell Culture

Immortalized cells derived from the human fetal cerebral cortex (ReNcell CX cells; Cat. No. SCC007; Millipore-Sigma, St. Louis, MO, U.S.A.) were used as the cell line for this study. Cells were grown in a dish coated with laminin (20 μg/mL; Millipore-Sigma) in a growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Thermo Fisher Scientific, Waltham, MA, U.S.A.) containing heparin (10 U/mL; Millipore-Sigma), B27 supplement (1×; Thermo Fisher Scientific), basic fibroblast growth factor (bFGF) (20 ng/mL; REPROCELL, Yokohama, Japan), and rhEGF (20 ng/mL; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) under 5% CO₂ at 37 °C.

Cell Treatment

The cells were seeded at 3.5 × 10⁵ cells/mL and cultured for 24 h. To determine MT3 induction by several HIF-PH inhibitors, cells were treated with 50 μM dimethyloxalyglycine (DMGO), 50 μM vadadustat, 10 μM daprodustat, 10 μM enarodustat, and 50 μM roxadustat (FG4592) for 24 h. For cell toxicity and cell viability experiments, FG4592 5, 10, 50, 100, and 200 μM were used to treat cells for 24 h.

RNA Isolation and Real-Time RT-PCR

Total RNA was isolated from cells using the Maxwell RSC simplyRNA Cells Kit (Promega, Madison, WI, U.S.A.) or the PureLink RNA Mini Kit (Thermo Fisher Scientific). Total RNA (1 μg) was reverse-transcribed using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). The real-time RT-PCR analysis was performed using THUNDERBIRD® SYBR qPCR Mix (Toyobo) or Thermo Scientific SYBR Green qPCR Master Mix (Thermo Fisher Scientific), with amplification achieved using a StepOne Real-Time PCR System or QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific). The primers used for real-time RT-PCR are outlined in Supplementary Table 1. The sequence information of the primers for the human MT isoforms was used from the previous study.¹⁶⁾

Cell Toxicity and Cell Viability

Cell toxicity was determined using the LDH assay kit (Dojindo, Kumamoto, Japan), and cell viability was determined using the Cell Counting Kit-8 (Dojindo). Measurements were performed as per the manufacturer’s protocols. Briefly, in the LDH assay, a cell culture medium was added to the working solution, and absorbance at 490 nm was measured. While in Cell Counting Kit-8, CCK-8 solution was added to cells and absorbance at 450 nm was measured.

Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE) and Western Blotting (WB)

The cells were lysed with the SDS sample buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol). For MT detection, we performed protein sample modification based on the previous study.¹⁷⁾ Briefly, the protein samples were treated with 37.5 mM iodoacetamide, 0.75% tri-butylphosphine, and 0.75 mM ethylenediaminetetraacetic acid (EDTA) at 60 °C for 50 min. After centrifugation, the supernatants were used for SDS-PAGE. SDS-PAGE and WB were performed based on previous studies.^18,19) The primary antibodies used in this study were as follows: rabbit anti-flag antibody (Abcam, Cambridge, U.K.; 1 : 1,000 dilution; RRID: AB_439687), rabbit anti-MT3 antibody (in house), rabbit anti-HIF1α antibody (Abcam; ab228649), and mouse anti-β-actin antibody (Millipore-Sigma; 1:1000 dilution; RRID: AB_476692). The second set of antibodies used in this study was as follows: goat anti-mouse immunoglobulin G (IgG) antibody, peroxidase-conjugated, H + L (1:5000 dilution; Merck KGaA, Darmstadt, Germany; RRID: AB_90456) and goat anti-rabbit IgG antibody, peroxidase-conjugated, H + L (1:5000 dilution; Merck KGaA; RRID: AB_90264). The band intensity was determined by the ImageJ software program (National Institutes of Health (NIH)).

Construction of p3 × FLAG-CMV-C-Terminal-MT Isoform Vectors

The p3 × FLAG-CMV-C-terminal vector (TaKaRa) was digested with HindIII (Thermo Fisher) and BamHI (Thermo Fisher) restriction enzymes. The cDNAs of the human MT isoforms (MT1A, 1B, IE, 1F, 1G, 1H, 1M, 1X, 2A, and 3) were amplified by PCR using Prime Star Max (TaKaRa) from pcDNA3.1 MTs hygro vectors. The primers used for MT isoforms cDNA are shown in Supplementary Table 2. Restriction enzyme-treated samples and purified PCR products were cloned using the In-Fusion HD Cloning Kit (TaKaRa Bio, Shiga, Japan) per the manufacturer’s protocol.

Construction of pET28a MT Isoform 3 × FLAG Vectors

The pET28a vector (Sigma-Millipore) was digested with HindIII and NotI (Thermo Fisher) restriction enzymes. The cDNAs of the MT isoforms with 3 × Flag were amplified by PCR using Prime Star Max from p3 × FLAG-CMV-C-terminal-hMT isoform vectors. The primers used for the MT isoforms 3xFLAG cDNA are shown in Supplementary Table 3. Restriction enzyme-treated samples and purified PCR products were cloned using the In-Fusion HD Cloning Kit as per the manufacturer’s protocol.

Preparation of MT Isoform Proteins

The pET28a-MT isoform-3× FLAG plasmid vectors were transformed into BL21 (Nippon gene, Tokyo, Japan) and cultured in 3 mL of LB liquid medium containing kanamycin at 37 °C for 5 h at 250 rpm with shaking and MT3 isoform-3× FLAG proteins were induced by IPTG treatment. The Escherichia coli pellet was suspended with 100 μL of buffer A (50 mM Tris pH 9.5, 0.3 M NaCl) and then crushed using a probe sonicator. Then 36 μL of 4 × Dye (10% SDS, 20% sucrose, 0.25 M Tris–HCl, 50 μM dithiothreitol) was added, centrifuged at 9000 × g for 5 min, and the supernatant was removed as the soluble fraction. Fifty microliters of 1× Dye was added to the pellet and suspended to make the insoluble fraction. The insoluble fractions were used for the WB of the MT isoforms.

Chromatin Immunoprecipitation (ChIP) Assay

The ChIP assay was performed based on a previous study.²⁰⁾ The antibodies used were as follows: rabbit anti-HIF1α antibody (Abcam; ab228649) and rabbit normal IgG (MBL International, Tokyo, Japan; RRID: AB_10805234). The primers used for amplifying the MT3 promoter region are shown as follows (forward: 5′-ACAGATCTGGCGTCCTGGAG-3′; reverse: 5′-TTTCCGTTTGTGCGCTTGGG-3′). ChIP-DNA levels are presented as a percentage of the input DNA. Owing to the differences in the enrichment rates between the input DNA and template, ΔCt was calculated as follows:

ΔCt = Ct^{ChIP DNA} – [Ct ^{diluted input DNA} – log₂(dilution factor of diluted input DNA to total input DNA)].

Additionally, 2^–ΔCt × 100 was determined as the percentage of inputs.

Statistical Analysis

Data were presented as the mean ± standard error (S.E.) of the mean. All statistical analyses were performed using StatView (Abacus, Baltimore, MD, U.S.A.) and IBM SPSS Statistics ver.19.0 (IBM, Armonk, NY, U.S.A.). The threshold for statistical analysis was set at p < 0.05.

RESULTS

The Levels of MT3 mRNA Were Increased by HIF-PH Inhibitors

The levels of MT3 mRNA were significantly increased by treatment with HIF-PH inhibitors, except DMOG and vadadustat (Fig. 1A). We focused on FG4592 as a candidate MT3 inducer, and then cellular viability and cytotoxicity were determined under FG4592 treatment. Cytotoxicity was significantly increased by 200 μM FG4592 treatment (Fig. 1B). Cellular viability was significantly decreased by 200 μM FG4592 treatment (Fig. 1C). Also, we determined the time course experiment of MT3 mRNA changes after 50 μM FG4592 treatment. The levels of MT3 mRNA were significantly increased after 24 h of FG4592 treatment (Fig. 1D). From these results HIF-PH inhibitors would be candidate drugs for endogenous MT3 induction.

Fig. 1. MT3 mRNA Levels by HIF-PH Inhibitors and Cell Viability and Cytotoxicity by FG4592 Treatment

(A) The level of MT3 mRNA was determined at 24 h after treatment with HIF-PH inhibitors (biological replicates n = 4/group). (B) Cytotoxicity was determined 24 h after FG4592 treatment (biological replicates n = 6/group). (C) Cell viability was determined at 24 h after FG4592 treatment (biological replicates n = 4/group). (D) The level of MT3 mRNA was determined at 24–72 h after 50 μM FG4592 treatment (biological replicates n = 3/group). Data are presented as mean ± S.E. Comparisons were performed using the 1-way ANOVA, followed by the post hoc Bonferroni’s test (* p < 0.05).

Only MT3 mRNA Was Induced in FG4592 Compared with Other MT Isoforms

We also determined other MT isoform mRNA levels by FG4592 treatment to compare with MT3 mRNA changes. The levels of MT3 mRNA were significantly increased by FG4592 treatment, whereas the levels of other MT isoforms were significantly decreased by FG4592 treatment (Fig. 2). The level of MT1B mRNA could not be detected due to the low endogenous expression level in ReNcell CX cells (data not shown). We found that FG4592 induced MT3, while the expression of other MT isoforms was reduced.

Fig. 2. The mRNA Level of MT Isoforms by FG4592 Treatment

The mRNA levels of the MT isoform were determined 24 h after 5–100 μM FG4592 treatment (biological replicates n = 4/group). Data are presented as mean ± S.E. Comparisons were performed using the one-way ANOVA, followed by the post hoc Bonferroni’s test (* p < 0.05).

MT3 Protein Levels Were Significantly Increased by FG4592 Treatment

Recombinant human MT isoforms tagged with 3 × FLAG were prepared to confirm the Western blotting experimental conditions for the detection of the MT3 protein. Only the MT3 recombinant protein was detected by our anti-MT3 antibody (Fig. 3A). In addition, we confirmed that all MT isoform proteins were expressed in this experiment using an antibody against Flag tag (Fig. 3B). We determined the endogenous MT3 protein levels and MT3 protein levels were significantly increased by FG4592 treatment accompanied by the increase in the HIF1α protein (Fig. 3C). We confirmed the method for detecting MT3 protein and FG4592 increased MT3 at protein level.

Fig. 3. Confirmation of the WB System for Detecting Human MT3 and the Level of MT3 Protein by FG4592 Treatment

(A) The result of WB using the anti-MT3 antibody applied to the recombinant protein of human MT isoforms was presented. (B) The result of WB using the anti-Flag antibody applied to the recombinant protein of human MT isoforms was presented. (C) The levels of MT3, HIF1α, and β-actin protein were determined at 72 h after 50 μM FG4592 treatment (biological replicates n = 4/group). Lysate from HEK293 cells transfected with the pcDNA3.1-MT3 vector was used as a positive control. Data are presented as mean ± S.E. Statistical significance was determined using the Student’s t-test (* p < 0.05).

The Amount of HIF1α Binding to the MT3 Promoter Was Increased by FG4592 Treatment

We found some hypoxia-responsive element (HRE) in the human MT3 promoter region (Fig. 4A, Supplementary Fig. 1); therefore, the amount of HIF1α binding to the MT3 promoter was determined using the ChIP assay. The amount of HIF1α binding to the MT3 promoter was significantly increased by FG4592 treatment (Fig. 4B). These results showed that HIF1α could bind to the promoter of the MT3 gene to induce MT3 transcription.

Fig. 4. The Amount of HIF1α Binding to the MT3 Promoter Region under FG4592 Treatment

(A) The location of HREs and primers in the MT3 gene promoter for ChIP assay is shown. (B) The level of HIF1α binding to the MT3 promoter region was determined at 12 h after 50 μM FG4592 treatment. IgG was used as a negative control for the ChIP assay. Data are presented as mean ± S.E. Comparisons were performed using Student’s t-test (* p < 0.05).

DISCUSSION

This study aimed to explore prototype drugs that induce endogenous human MT3. Few stimuli or compounds have been reported to induce MT3, and studies using human adipocytes have shown that MT3 is induced under hypoxic conditions.¹⁵⁾ The development of drugs that induce the endogenous expression of MT3, which is localized in the nerves and is thought to be associated with neural function, could lead to the development of therapeutic agents for neurodegenerative diseases. Therefore, we used an inhibitor of HIF-PH, a degradative enzyme of the hypoxia-responsive transcription factor HIF1α, to confirm MT3 induction in the cells of the nervous system. Among the various HIF-PH inhibitors, endogenous MT3 mRNA in ReNcell CX cells was significantly increased by HIF-PH inhibitor treatments other than DMOG and vadadustat (Fig. 1A). Although DMOG has been reported to increase MT3 mRNA in human adipocytes,¹⁵⁾ it did not induce MT3 in the nervous system cells in this study. Although different HIF-PH inhibitors may differ in their ability to induce MT3 and the level of MT3 induction should be determined by all HIF-PH inhibitor treatments used in this study under the same experimental condition, we focused on FG4592 among HIF-PH inhibitors for further analysis. FG4592 treatment resulted in a significant increase in MT3 mRNA expression in a time-dependent and concentration-dependent manner (Figs. 1D, 2). In contrast, the mRNA expression levels of other MT isoforms were significantly reduced by FG4592 treatment (Fig. 2). There have not been many reports on the molecular mechanisms that may suppress MT expression levels. The suppression of MT expression by transcriptional repression through microRNA or DNA methylation has been reported^21,22); however, it has not been studied in detail. A previous report showed that the binding of MTF1 and HIF1α to MREs is involved in the induction of mouse MT1 expression by hypoxia²³⁾; however, the detailed mechanism is unclear. In the present study, HIF1α is thought to be involved in the reduction of MT isoforms by FG4592 treatment; however, it is unclear whether HIF1α acts directly on the gene promoters of MT isoforms to repress their transcription. The decrease in other MTs is considered a disadvantage for MT3 inducers intended for therapeutic use; however, it may be necessary in the future to reduce the decrease in other MT isoforms by chemical modification of the compound or other modifications.

In this study, the increase in MT3 by FG4592 was confirmed at the protein level (Fig. 3C). Our WB system with the MT3 antibody was able to specifically detect MT3 at the protein level among human MT isoforms (Figs. 3A, 3B). Furthermore, the binding of HIF1α to the MT3 promoter was confirmed by the presence of HRE sequences that bind HIF1α to the MT3 gene promoter. FG4592 treatment increased the amount of HIF1α binding to the MT3 promoter (Fig. 4). These results indicate that FG4592 induces MT3 expression via increased HIF1α. It should be better to determine the relationship between HIF1α and MT3 induction, and further study would be required using stable HIF1α knockout cells.

In conclusion, we found FG4592 to be an endogenous MT3 inducer in cells of the nervous system in this study. FG4592 has already been used as a treatment for renal anemia,²⁴⁾ but it is also transferable to the brain, and there are reports of studies on its use as a treatment for Parkinson’s disease.²⁵⁾ It is expected that the results of this study will lead to the development of new MT3-inducing drugs for neurodegenerative diseases based on FG4592.

Acknowledgments

A Grant of the Suzuken Memorial Foundation to H.K. and a Grant-in-Aid for Scientific Research on Innovative Areas JSPS KAKENHI (JP19H05767A02) to I.H. were partly used to support this study.

Author Contributions

H.K., M.T., T.I., K.K., F.K., Y.N., T.M., and K.O. designed experiments, performed experiments, and analyzed data. H.K., M.T., T.I., K.K., F.K., Y.N., T.M., K.O., I.H., and M.I. interpreted and discussed results. M.T., T.I. wrote the initial draft of the manuscript. Y.S., T.K., N.M., and Y.K. contributed to reagents, materials, and methods. H.K. supervised and conceived project. Y.S., T.K., N.M., Y.K., I.H., and M.I. validated and revised manuscript. All authors approved the final version.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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