Elsevier

Biochemical Pharmacology

Volume 80, Issue 3, 1 August 2010, Pages 316-324
Biochemical Pharmacology

Ferritin H induction by histone deacetylase inhibitors

https://doi.org/10.1016/j.bcp.2010.04.008Get rights and content

Abstract

Because both iron deficiency and iron excess are deleterious to normal cell function, the intracellular level of iron must be tightly controlled. Ferritin, an iron binding protein, regulates iron balance by storing iron in a bioavailable but nontoxic form. Ferritin protein comprises two subunits: ferritin H, which contains ferroxidase activity, and ferritin L. Here we demonstrate that ferritin H mRNA and protein are induced by histone deacetylase inhibitors (HDAC inhibitors), a promising class of anti-cancer drugs, in cultured human cancer cells. Deletion analysis and EMSA assays reveal that the induction of ferritin H occurs at a transcriptional level via Sp1 and NF-Y binding sites near the transcriptional start site of the human ferritin H promoter. Classically, HDAC inhibitors modulate gene expression by increasing histone acetylation. However, ChIP assays demonstrate that HDAC inhibitors induce ferritin H transcription by increasing NF-Y binding to the ferritin H promoter without changes in histone acetylation. These results identify ferritin H as a new target of HDAC inhibitors, and recruitment of NF-Y as a novel mechanism of action of HDAC inhibitors.

Introduction

Ferritin H plays an important role in iron metabolism. Iron is essential for normal cell growth, proliferation, energy metabolism and other critical functions of cells and tissues. However, excess iron is harmful, and can catalyze the formation of toxic reactive oxygen species (ROS) via Fenton chemistry and other mechanisms. Therefore, iron must be tightly controlled and compartmentalized [1]. Ferritin is the major iron storage protein of the cell. By sequestering excess iron in a nontoxic form, ferritin plays a critical role in the maintenance of intracellular iron balance. Ferritin consists of two subunit types, termed H and L, which are encoded by separate genes [2]. Twenty-four of these subunits assemble to form the apoferritin shell. Each apoferritin molecule can sequester up to 4500 iron atoms. The H subunit of ferritin, ferritin H, has inherent ferroxidase activity, and converts Fe(II) to Fe(III) as iron is internalized and sequestered in the ferritin mineral core [2]. Not surprisingly, homozygous murine knockouts of ferritin H are lethal [3]. Overexpression of ferritin H has been shown to cause an iron-deficient phenotype and reduce cell growth [4], [5].

Histone acetylation plays an important role in regulation of transcription [6]. Histone acetylation reduces the binding between histones and DNA, thus loosening chromatin structure and facilitating the access of RNA polymerase and other transcription factors to promoter regions. In contrast, histone deacetylation represses transcription by condensing chromatin structure. Histone acetylation and histone deacetylation are catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. HATs and HDACs do not directly bind to DNA, but are recruited to gene promoters by transcription factors, such as Sp1, Sp3, and NF-Y [7].

Histone deacetylase inhibitors (HDAC inhibitors) are a promising new class of anti-cancer drug. HDAC inhibitors inhibit cancer cell proliferation and lead to differentiation or apoptosis of cancer cells in vitro and in vivo[8]. Several HDAC inhibitors are currently in clinical trials and show significant anti-cancer activity [8], [9]. HDAC inhibitors not only induce cancer cells to undergo growth arrest and/or apoptosis, but also exhibit low toxicity against normal cells [10], [11]. HDAC inhibitors are of several chemical types, and range from simple chemicals (such as butyrate) to more complex agents such as hydroximates (such as trichostatin A [TSA], suberoylanilide hydroxamic acid [SAHA]), cyclic peptides (such as depsipeptide, apicidin), and benzamides (such as MS-275) [12], [13].

Regulation of gene expression is essential for the anti-tumor function of HDAC inhibitors, because inhibition of de novo protein synthesis suppresses HDAC inhibitor-induced apoptosis [14]. However, the detailed mechanism of HDAC inhibitor-induced cell death is not fully defined, and may also involve histone acetylation-independent mechanisms [15], [16]. Identification of target genes critical to the function of HDAC inhibitors will not only improve understanding of their fundamental mechanism of action, but may ultimately assist in their clinical application.

Here we reported that ferritin H is transcriptionally induced by HDAC inhibitors in human cancer cells. Unexpectedly, chromatin immunoprecipitation assays demonstrate that HDAC inhibitors do not act by increasing histone acetylation of the ferritin H promoter, but rather by recruiting NF-Y to the promoter. These results identify a novel mechanism of action of this important class of anti-cancer agent.

Section snippets

Chemicals and cell culture

Sodium butyrate and tricostatin A (TSA) were purchased from Sigma (St. Louis, MO). Human cervical carcinoma cells (HeLa) were obtained from American Type Culture Collection (ATCC) (Manassas, VA) and were maintained in DMEM (Invitrogen, Carlsbad, California) supplemented with 10% FBS (HyClone, Logan, UT), 100 units/ml penicillin, and 100 μg/ml streptomycin. PC3 cells were obtained from the ATCC and maintained in RPMI 1640 medium (Invitrogen) containing 10% FBS, 100 units/ml penicillin, and 100 μg/ml

HDAC inhibitors induce ferritin H mRNA and protein

We first tested whether HDAC inhibitors can induce ferritin H expression. HeLa cells were treated with various concentrations of the HDAC inhibitor tricostatin A (TSA) for 24 h, and effects on ferritin H expression were analyzed. As seen in Fig. 1A, Northern blot analysis demonstrated that TSA significantly upregulates ferritin H mRNA in a dose-dependent manner (Fig. 1A, upper panel); similar results were obtained using real-time RT-PCR (Fig. 1C). To assess the effect of TSA on ferritin H at the

Discussion

In this report, we demonstrate that the H subunit of ferritin, an iron storage protein, is induced by histone deacetylase inhibitors at transcriptional level via Sp1 and NF-Y sites in the ferritin H promoter.

HDAC inhibitors transcriptionally activate the ferritin H promoter by engaging elements of the human ferritin H promoter approximately 60 nucleotides 5′ of the transcriptional start site, a region known to be important in the transcriptional control of the human ferritin H gene. For

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    This work was supported in part by Public Health Service grant R37DK42412 from the NIDDK (FMT).

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