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In eukaryotic cells, heme production is tightly controlled by heme itself through negative feedback-mediated regulation of nonspecific 5-aminolevulinate synthase (ALAS1), which is a rate-limiting enzyme for heme biosynthesis. However, the mechanism driving the heme-dependent degradation of the ALAS1 protein in mitochondria is largely unknown. In the current study, we provide evidence that the mitochondrial ATP-dependent protease ClpXP, which is a heteromultimer of CLPX and CLPP, is involved in the heme-dependent degradation of ALAS1 in mitochondria. We found that ALAS1 forms a complex with ClpXP in a heme-dependent manner and that siRNA-mediated suppression of either CLPX or CLPP expression induced ALAS1 accumulation in the HepG2 human hepatic cell line. We also found that a specific heme-binding motif on ALAS1, located at the N-terminal end of the mature protein, is required for the heme-dependent formation of this protein complex. Moreover, hemin-mediated oxidative modification of ALAS1 resulted in the recruitment of LONP1, another ATP-dependent protease in the mitochondrial matrix, into the ALAS1 protein complex. Notably, the heme-binding site in the N-terminal region of the mature ALAS1 protein is also necessary for the heme-dependent oxidation of ALAS1. These results suggest that ALAS1 undergoes a conformational change following the association of heme to the heme-binding motif on this protein. This change in the structure of ALAS1 may enhance the formation of complexes between ALAS1 and ATP-dependent proteases in the mitochondria, thereby accelerating the degradation of ALAS1 protein to maintain appropriate intracellular heme levels.Heme is an essential molecule to almost all organisms. Heme functions as a prosthetic group on several types of proteins, including cytochromes, catalases, hemoglobin, and myoglobin. Moreover, it has been reported that heme is also involved in numerous regulatory systems in mammals (1), including those that govern transcription (2), translation (3), microRNA processing (4), and the circadian rhythm (5). Excess quantities of heme or heme precursors result in the generation of reactive oxygen species, causing oxidative stress in cells (6). Thus, the production of heme and heme precursors must be tightly regulated. This regulation occurs through the precise control of the intracellular expression of nonspecific 5-aminolevulinatye synthases (ALAS-N or ALAS1). ALAS1 is the first and the ratelimiting enzyme of the heme biosynthetic pathway in mammalian cells, except for in erythroid cells, in which erythroid-specific 5-aminolevulinate synthase (ALAS-E or ALAS2) regulates the first step of heme biosynthesis (7). Although ALAS2 expression increases during erythroid differentiation, ALAS1 expression is suppressed by heme at the transcriptional, translational, and post-translational levels (8). ALAS1 and ALAS2 are encoded by independent genes (9); however, they both contain a conserved amino acid sequence called the heme regulatory motif (HRM), 2 which is involved in heme-dependent inhib...

© F e r r a t a S t o r t i F o u n d a t i o nsequence of primers and probes used in this study are listed in the Online Supplementary Tables. Polymerase chain reaction-based quantitative chromatin immunoprecipitationReal-time PCR-based quantitative chromatin immunoprecipitation (ChIP-qPCR) analysis was conducted essentially as previously described. 22 Electrophoretic mobility shift assayElectrophoretic mobility shift assay (EMSA) was performed using "DIG Gel Shift Kit, 2 nd Generation" (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer's protocol. Sequences of oligonucleotides for probes are indicated by the horizontal bar in the relevant figures. Nuclear extracts were prepared, as described previously, 23 from K562 cells or HEK293 human embryonic kidney cells that were transfected with a GATA1-FLAG fusion protein expression vector or its backbone vector. Promoter/enhancer activity assaysEach target DNA fragment was prepared from genomic DNA from normal volunteers (WT) or patients with CSA (referred to as "GGTA" or "delGATA" in each reporter construct) and was cloned into pGL3basic plasmid (Promega Corporation, Madison, WI, USA). The human ALAS2 proximal promoter region (g.4820_5115, between -267 and +29 from the transcription start site) 16,24 was cloned into the multiple-cloning site of pGL3basic [referred to as pGL3-AEpro (-267)]. A single DNA fragment (5.2 kbp), carrying the ALAS2 proximal promoter, first exon, first intron and the untranslated region of the second exon, was subcloned into the multiple cloning site of pGL3basic [referred to as pGL3-AEpro(-267)+intron1]. A DNA fragment containing the GATA1-binding region in the first intron of the ALAS2 gene (corresponding to g.7488_7960), which was defined by ChIP-seq analysis, 22 is referred to as the ChIP-peak. The length of the WT ChIP-peak is 473 bp. In addition, a 130-bp fragment containing ALAS2int1GATA, the consensus sequence for the GATA1-binding site in the ChIP-peak, is referred to as ChIPmini. Several deletion mutants of ChIPmini were prepared using pGL3-AEpro(-267)+ChIPmini(WT) as a template. The pGL3-TKpro plasmid was constructed by cloning herpes simplex virus thymidine kinase promoter into the multiple cloning site of pGL3basic plasmid. Each reporter vector and pEF-RL25 were introduced into K562 cells or HEK293 cells. Luciferase activity was determined using a dualluciferase reporter system (Promega).Disruption of a GATA binding element causes CSA haematologica | 2014; 99 (2) 253 Figure 1. Identification of a functional GATA1 element in the first intron of the ALAS2 gene. (A) Chromatin immunoprecipitation assay. Fragmented genomic DNA segments were immunoprecipitated with anti-GATA1 antibody or control IgG, and then precipitated fragments were quantified using real-time PCR as described in the Online Supplementary Methods. PC or NC indicates positive control or negative control, respectively, for the ChIP assay using anti-GATA1 in K562 cells. 22 One GATA element is present in the proximal promoter region and ...

Heme oxygenase consists of two structurally related isozymes, heme oxygenase‐1 and and heme oxygenase‐2, each of which cleaves heme to form biliverdin, iron and carbon monoxide. Expression of heme oxygenase‐1 is increased or decreased depending on cellular microenvironments, whereas little is known about the regulation of heme oxygenase‐2 expression. Here we show that hypoxia (1% oxygen) reduces the expression levels of heme oxygenase‐2 mRNA and protein after 48 h of incubation in human cell lines, including Jurkat T‐lymphocytes, YN‐1 and K562 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma, as judged by northern blot and western blot analyses. In contrast, the expression level of heme oxygenase‐1 mRNA varies under hypoxia, depending on the cell line; it was increased in YN‐1 cells, decreased in HeLa and HepG2 cells, and remained undetectable in Jurkat and K562 cells. Moreover, heme oxygenase‐1 protein was decreased in YN‐1 cells under the conditions used, despite the induction of heme oxygenase‐1 mRNA under hypoxia. The heme oxygenase activity was significantly decreased in YN‐1, K562 and HepG2 cells after 48 h of hypoxia. To explore the mechanism for the hypoxia‐mediated reduction of heme oxygenase‐2 expression, we showed that hypoxia shortened the half‐life of heme oxygenase‐2 mRNA (from 12 h to 6 h) in YN‐1 cells, without affecting the half‐life of heme oxygenase‐1 mRNA (9.5 h). Importantly, the heme contents were increased in YN‐1, HepG2 and HeLa cells after 48 h of incubation under hypoxia. Thus, the reduced expression of heme oxygenase‐2 may represent an important adaptation to hypoxia in certain cell types, which may contribute to the maintenance of the intracellular heme level.

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ABSTRACT(Pro)renin receptor ((P)RR) is a specific receptor for renin and prorenin. The aim of the present study is to clarify expression of (P)RR and pathophysiological roles of (P)RR in human breast carcinomas. (P)RR expression was studied in 69 clinical cases of breast carcinoma by immunohistochemistry. Effects of (P)RR on cell proliferation were examined in cultured human breast carcinoma cells using (P)RR specific small interference RNA. Immunohistochemistry showed that (P)RR immunoreactivity was detected in the breast carcinoma cells in 50 of 69 cases of breast carcinoma (72%). The analysis on association between (P)RR immunoreactivity and clinicopathological parameters showed that the number of (P)RR positive cases was significantly greater in Ki-67 (a cell proliferation marker) ≥ 10% group than in Ki-67 < 10% group (P = 0.02). (P)RR was expressed in 4 types of human breast carcinoma cell lines. (P)RR specific small interference RNA inhibited proliferation of both MCF-7 (ERα positive) and SK-BR-3 (ERα negative) cells. The present study has shown, for the first time, the expression of (P)RR in human breast carcinoma tissues and cultured breast carcinoma cell lines. These findings have raised the possibility that the blockade of the (P)RR signaling may be a novel therapeutic strategy against breast carcinomas.The renin-angiotensin system (RAS) plays important roles, not only in homeostatic regulation of blood pressure and water-electrolyte metabolism, but also in tumor biology including breast cancers. All the components of the RAS, including prorenin, renin, angiotensinogen (AGT), angiotensin I converting enzyme (ACE) and angiotensin receptors were localized in normal and cancerous breast tissues (28). Angiotensin II (Ang II) acts on the angiotensin II type 1 receptor (AT1R) to promote cell proliferation in breast cancer cells, via the protein kinase C/Ca 2+ / phosphoinositide 3-kinase pathways, and extracellular signal-related kinase (ERK) activation (32). Ang II promoted cell adhesion, invasion and metastasis of breast carcinoma by regulating the expression of integrins, vascular endothelial growth factor and matrix metalloproteinase-9 (22, 23). Clinicopathological study showed that AT1R expression in breast carcinoma tissues correlated with axillary lymph node metastasis (2). (Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, consists of 350 amino acids

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(Pro)renin receptor [(P)RR], a specific receptor for renin and prorenin, is a 350 amino acid protein with a single transmembrane domain. In the present study, the expression of (P)RR in the human brain and pituitary, and its co-localisation with arginine vasopressin and oxytocin in the human hypothalamus were studied by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. Human brain and pituitary tissues were obtained at autopsy from the subjects without neurological or endocrinological disorders. The antiserum against (P)RR was raised in a rabbit by injecting the peptide fragment of human (P)RR corresponding to 224-237 amino acids conjugated with bovine serum albumin. Quantitative RT-PCR showed that (P)RR mRNA was widely expressed in every region of brain examined and pituitary, with the highest expression levels found in the pituitary and frontal lobe. Immunocytochemistry showed that (P)RR was expressed in the paraventricular and supraoptic nuclei of human hypothalami, and in anterior pituitary cells. Immunostaining of serial sections showed that (P)RR was co-localised with arginine vasopressin and oxytocin in the magnocellular neurones of the paraventricular and supraoptic nuclei. The preabsorption of the antibody by the antigen peptide abolished the immunostaining of (P)RR in the human hypothalamus. The present study has shown that (P)RR mRNA is widely expressed in the human brain and pituitary, consistent with the hypothesis that (P)RR is related to the various brain functions, such as cognitive function and brain development. Co-localisation of (P)RR with vasopressin in the hypothalamus raised the possibility that (P)RR may be related to the central control of water-electrolyte metabolism and blood pressure.