p53 directly regulates the transcription of the human frataxin gene and its lack of regulation in tumor cells decreases the utilization of mitochondrial iron
Introduction
Friedreich ataxia, caused by reduced expression of mitochondrial frataxin by a homozygous GAA repeat expansion, involves neurodegradation of large sensory neurons, cardiomyopathy, impaired glucose tolerance, and diabetes mellitus, and is designated as a neurodegradative inherited disease (Calabrese et al., 2005, Patel and Isaya, 2001). Knockout of the frataxin homolog Yfh1p in yeast impaired respiration and caused the deletion of mitochondrial DNA and iron accumulation (Babcock et al., 1997). Moreover, hepatic-specific knockdown of the frataxin gene in mouse reduced the life span and caused tumor formation, which resembles the conditions of patients with Friedreich ataxia who suffer various types of cancer and reduced life span (Thierbach et al., 2005). Conversely, overexpression of frataxin in tumor cells enhanced respiratory metabolism, decreased the generation of reactive oxygen species and cancer growth (Schulz et al., 2006), and also prevented the malignant transformation of fibroblasts by oxidative stress-inducing drug (Schulz et al., 2006), implying its tumor-suppressor function.
Frataxin is an iron-binding protein, contributing to the biogenesis of iron–sulfur clusters (Ye and Rouault, 2010). The iron–sulfur cluster-containing proteins in mitochondria include the complexes of the electron transport chain and ferrochelatase, the enzyme for the last step of heme biosynthesis (Campuzano et al., 1997, Taketani, 2005). Thus, low expression of frataxin impairs the utilization of iron in mitochondria and their functions. We have reported that protoporphyrin in cancerous cells accumulates owing to the limited capacity for ferrochelatase reaction (Ohgari et al., 2005, Ohgari et al., 2011). The reduced ferrochelatase reaction can be ascribed to frataxin defects, which cause not only down-regulation of the expression of the enzyme but also decreased supply of iron for enzymatic reactions in tumor cells (Ohgari et al., 2011, Schoenfeld et al., 2005). We have shown the possibility that tumor-suppressor factor p53 controls the expression of mouse frataxin and the defects in tumor cells may cause the accumulation of ALA-induced protoporphyrin (Sawamoto et al., 2013). However, the molecular mechanisms underlying the abnormal utilization of iron in mitochondria of human cancer and the function of frataxin are still unknown.
To clarify the decreased expression of frataxin in cancerous cells by the dysfunction of p53, we examined the p53-dependent regulation of frataxin in non-tumor cells. Here, we report that down-regulation of p53 decreased the level of human frataxin and identified the p53-responsive element in the promoter of the human frataxin gene. Improvement of the utilization of iron for heme biosynthesis by the expression of frataxin is also shown.
Section snippets
Materials
Restriction endonucleases and DNA-modifying enzymes were from Takara Co. (Tokyo, Japan) and Toyobo Co. (Tokyo, Japan). Pifithrin-α was a product of Sigma-Aldrich (St. Louis, MI). Iron citrate was prepared as described previously (Taketani and Tokunaga, 1981). Antibodies for p53 and frataxin were as previously described (Sawamoto et al., 2013). Polyclonal antibody for actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were of analytical grade.
Cell Cultures and Analysis of Protoporphyrin
Human epithelial
Expression of Frataxin Is Regulated by p53
To examine whether the expression of human frataxin is regulated by p53, non-tumor HEK293T cells were treated with an inhibitor of the p53 function, 5 μM pifithrin-α, for 16 h and the RT-PCR was carried out after the isolation of RNA. As shown in Fig. 1A, the level of frataxin mRNA in pifithrin-α-treated cells was markedly decreased, compared with that in untreated cells. Immunoblot analysis revealed that frataxin in HEK293T cells was decreased by the treatment with pifithrin-α for 24 h (Fig. 1B).
Discussion
The present study first gave direct evidence that the expression of human frataxin is dependent on p53. Evidently, the inhibition of p53 function by pifithrin-α and the knockdown of p53 decreased the levels of frataxin mRNA and protein. The reporter activity of the promoter in the human frataxin gene carrying p53RE located at − 209 to − 200 bp was high in non-tumor HEK293T cells, but did not change in cancerous HeLa cells (Fig. 2B), compared with that without p53RE. The expression of the wild-type
Acknowledgments
This study was supported in part by grants from the Ministry of Health, Labor and Welfare of Japan (H24-Nanchi-S-006), and from the Ministry of Education, Science, Sports, and Culture of Japan (23510256).
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Present address: Department of Biochemistry, Hanoi Medical University, Hanoi, Vietnam.