Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C2H2 zinc finger protein.

Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C2H2 motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.

The cDNA encoding early hematopoietic zinc finger protein

Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is upregulated during ischemic preconditioning by combining suppression subtractive hybridization and cDNA chip technology. It is currently designated as zinc finger protein 667 (ZNF667) by the Hugo Nomenclature committee and has GenBank accession number AY221750 (1,2). As a zinc finger nuclear transcriptional repressor, Mipu1 inhibits oxidative stress-induced cell injury, which is due to downregulation of expression of the apoptosis-related genes Fas and Bax (2-4). Electrophoretic mobility shift assay (EMSA) and luciferase reporter gene assays showed that hypoxia inducible factor 1α (HIF-1α) and cAMP-response element binding protein (CREB) bound to the Mipu1 promoter region and promoted its transcription during oxidative stress in cells (4,5).

The properties of Mipu1/ZNF667 are still only partially understood. However, its molecular features and expression profile as well as the biological functions so far identified suggest that it may play a role in the cardiovascular system. In this overview, we illustrate the data currently available on the structure, expression, interactions, and functional properties of this protein and discuss its possible significance in the cardiovascular field.

Biological characteristics of Mipu1

Mipu1, a typical N-terminal Krüppel-associated box (KRAB)/C2H2 zinc finger protein

A number of proteins with amino acid motifs capable of recognizing distinct DNA sequences via interaction with hydrogen donors and acceptors located in DNA major and minor grooves have been identified by bioinformatic analysis of DNA binding domains. The zinc finger domain can bind with DNA, the peptide, or histidine in the zinc finger protein and bind with divalent zinc ion to form a specific secondary structure. The zinc finger protein family has many subfamilies, among which C2H2 (or Kruppel) is the largest subfamily, in which the zinc finger sequence is CX_2CX_3FX_5LX_2HX_3H and the conserved sequence between the two zinc fingers is TGEKP(Y/F)X, where X represents any amino acid between conserved amino acids (6-9). The typical C2H2 zinc finger is a short protein motif with two histidine and two cysteine residues that hold a zinc ion with coordination bonds. It is obvious at present that they can also recognize various motifs in double-stranded DNA, single-stranded DNA, RNAs, and proteins (10-13). Depending on the domain at the N-terminal, C2H2 zinc finger proteins can be divided into four categories: FAX (finger-associated boxes), FAR (finger-associated repeats), POZ (pox virus and zinc fingers also known as Zin), and KRAB (Krüppel-associated box). The zinc finger proteins that contain KRAB, also called KRAB zinc finger proteins (KRAB-containing zinc finger proteins, KZNF), make up almost one-third (290 kinds) of all zinc finger proteins (799 kinds). They are the largest transcription repressor family in mammals and play an important role in embryonic development, cell differentiation, cell transformation, and cell cycle regulation (14-18) (Table 1; 19-27).

The full length of the Mipu1 open reading frame is 1827 base pairs (bp), encoding 608 amino acids; it is composed of five exons and four introns, and maps to chromosome 1q12.1 (2). The N-terminal region of the encoded peptide chain has a KRAB domain, whereas the C-terminal region has 14 C2H2 zinc fingers; therefore, it is a typical KRAB/C2H2 zinc finger protein. The six zinc fingers at the C-terminus of Mipu1 protein have been shown to combine with DNA, and Mipu1 has been identified as a transcription repressor that binds to the specific DNA binding site 5′-TGTCTTATCGAA-3′, with CTTA as the key sequence of the binding site (3,25,28,29) (Figure 1).

Figure 1

Structure of ZNF667. Arrows: zinc finger structure.

Promoter region of Mipu1 and its transcriptional regulation

Two different promoter prediction programs predicted two potential promoter regions for Mipu1: -104 to +146 bp, and -104 to +36 bp, with respect to the transcription start site. Both predicted Mipu1 promoters include the region between -104 and +36 bp, proposed to be the core promoter or the minimal promoter. Seven different deletion constructs were transiently transfected into an H9c2 cardiomyocyte cell line, and showed the luciferase activity of the seven constructs relative to the promoter-less construct. The results mapped the minimal promoter of Mipu1 to the region between -100 and +1 bp with respect to the transcription start site (30).

Lv et al. (30) showed that the GC box is essential for regulating the constitutive expression of Mipu1. However, the GC box has neither hypoxia-response nor stress-response elements, implying that other transcription factor binding sites within the Mipu1 promoter region might be responsible for its upregulation during pathological stress (ischemic or hypoxic stress). One CREB binding site and one hypoxia response element (HRE) site were identified using the MatInspector software (http://www.genomatix.de/cgi-bin/matinspector_prof/mat_fam.pl). Our previous studies showed that hypoxia-reoxygenation or H2O2-mediated inducible expression of Mipu1 is partially due to the activation of CREB (5,31). Recently using EMSA and luciferase reporter gene assays, Wang et al. (4) showed that HIF-1α bound to the HRE within the Mipu1 promoter region and promoted its transcription.

Expression of Mipu1

Mipu1 mRNA is expressed in the heart, liver, spleen, lung, kidney, intestine, brain, and skeletal muscle of normal mice, with the highest level of expression in spleen and lung, a very high level of expression in heart and skeletal muscle, a very low level of expression in liver and brain, and the lowest level of expression in intestine. Mipu1 protein has a very high level of expression in the heart and liver of normal rats and is mainly located in the nuclei of H9c2 myogenic cells, but it has a very low level of expression in liver, testis, kidney, and skeletal muscle and shows no signs of expression in spleen and lung (25,32,33). In studies of rat myocardial ischemia-reperfusion, Mipu1 expression increased at 3 h of reperfusion, following 30 min of myocardial ischemia, reached its peak level 6 h later, and maintained that level until a further 12 h later. In addition, Mipu1 expression in H9c2 cells could be induced by hydrogen peroxide (26), and it had an obviously higher expression in cerebral cortex and hippocampus after 12 and 24 h of reperfusion, after 3 min of ischemic preconditioning, than that of the sham surgery groups (32,34). Our results indicated that Mipu1 mRNA expression was significantly increased during hypoxia-reoxygenation or H2O2 stimulation in H9c2 cells (5,31).

Cytoprotection effects of Mipu1

It has been demonstrated that Mipu1 has a high expression in rat heart and is mainly located in the nuclei of H9c2 myogenic cells (25). The expression pattern and nuclear localization suggest that Mipu1 plays a role in the regulation of gene transcription in the cardiovascular system. Upregulation of Mipu1 is induced after myocardial infarction mainly in the infarcted area, and to some extent in the remote noninfarcted myocardium, suggesting that it may play an important role in myocardial infarction; however, further studies are needed to identify the mechanism (26). Overexpression of Mipu1 can reduce H9c2 cell injury caused by CoCl2-serum-free culture (1). At the same time, promoter activity and expression of Mipu1 increased significantly during the hypoxia-reoxygenation process, which suggests that it may be involved in the injury of H9c2 cells (1). Being a zinc finger nuclear transcriptional repressor, its DNA binding sequence is 5-TGTCTTATCGAA-3, within which CTTA is the core sequence binding site (25). Recent studies have also shown that Mipu1 can reduce apoptosis of H9c2 induced by H2O2 and tumor necrosis factor alpha (TNF-α), and can repress the expression of the apoptosis-related genes Fas and Bax (25-27). Overexpression of Mipu1 represses transcriptional activity of serum response element (SRE) and activator protein-l (AP-1), and inhibition of Mipu1 expression by RNAi can increase the transcriptional activity of SRE and AP-1; that is, Mipu1 may be involved in the function of SRE and AP-1 during the transcriptional regulation process and plays an important role in the pathological process of heart and vascular diseases through regulating the mitogen-activated protein kinase (MAPK) signaling pathway (35).

HIF-1 serves as an important endogenous cytoprotective gene that maintains oxygen homeostasis by inducing the expression of cluster genes, such as EPO, HO-1, and iNOS, at the transcriptional level (36-40). Recently, Wang et al. (4) reported that HIF-1α bound to the HRE within the Mipu1 promoter region and promoted its transcription, leading to cytoprotection of HIF-1 against H2O2-mediated injury in H9c2 cells partly through regulation of Mipu1 expression. Our previous studies also found that hypoxia-reoxygenation or H2O2-induced upregulation of Mipu1 in H9c2 cardiomyocytes was mediated by cAMP/protein kinase A (PKA)-dependent CREB activation, and that the cytoprotection of CREB against hypoxia-reoxygenation or H2O2-mediated injury in H9c2 cells occurs partly through regulation of Mipu1 expression (5).

Expression of Mipu1 is markedly increased in endotoxemia, which may have an important role in the inflammatory reaction process induced by lipopolysaccharide (LPS) (41). Further analysis of the role of Mipu1 and its mechanism in the inflammatory process caused by LPS may provide new ideas and experimental clues for the prevention and cure of sepsis and other related diseases.

Perspectives

In summary, Mipu1 is a nuclear factor with a variety of biological functions, such as participation in the process of myocardial ischemic preconditioning, protection of the myocardium from ischemic disease, and inflammation. Analysis of the function of Mipu1 in ischemic heart disease is beneficial because it may provide new ideas for clinical treatment and prevention of ischemic heart disease. However, further development of related technologies is needed to obtain a comprehensive and detailed understanding of the function of Mipu1 and its role in ischemic-related diseases.