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Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667--a multifunctional KRAB/[C.sub.2][H.sub.2] zinc finger protein.

The cDNA encoding early hematopoietic zinc finger protein

Myocardial ischemic preconditioning upregulated protein 1 (M/pu1) 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, M/pu1 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 1[alpha] (HIF-1[alpha]) and cAMP-response element binding protein (CREB) bound to the M/pu1 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 Mipul

Mipul, a typical N-terminal Kruppel-associated box (KRAB)/[C.sub.2][H.sub.2] 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 [C.sub.2][H.sub.2] (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 [C.sub.2][H.sub.2] 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, [C.sub.2][H.sub.2] 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 (Kruppel-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 Mipul 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 [C.sub.2][H.sub.2] zinc fingers; therefore, it is a typical KRAB/[C.sub.2][H.sub.2] zinc finger protein. The six zinc fingers at the C-terminus of Mipul protein have been shown to combine with DNA, and Mipul 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).

Promoter region of Mipul and its transcriptional regulation

Two different promoter prediction programs predicted two potential promoter regions for Mipul: -104 to + 146 bp, and -104 to +36 bp, with respect to the transcription start site. Both predicted Mipul 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 Mipul 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 Mipul. However, the GC box has neither hypoxia-response nor stress-response elements, implying that other transcription factor binding sites within the Mipul 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 [H.sub.2][O.sub.2]-mediated inducible expression of Mipul 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-1[alpha] bound to the HRE within the Mipul promoter region and promoted its transcription.

Expression of Mipul

Mipul 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, Mipul 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 Mipul mRNA expression was significantly increased during hypoxia-reoxygenation or [H.sub.2][O.sub.2] stimulation in H9c2 cells (5,31).

Cytoprotection effects of Mipul

It has been demonstrated that Mipul 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 Mipul 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 Mipul can reduce H9c2 cell injury caused by Co[Cl.sub.2]-serum-free culture (1). At the same time, promoter activity and expression of Mipul 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 Mipul can reduce apoptosis of H9c2 induced by [H.sub.2][O.sub.2] and tumor necrosis factor alpha (TNF-[alpha]), and can repress the expression of the apoptosis-related genes Fas and Bax (25-27). Overexpression of Mipul represses transcriptional activity of serum response element (SRE) and activator protein-l (AP-1), and inhibition of Mipul expression by RNAi can increase the transcriptional activity of SRE and AP-1; that is, Mipul 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-1[alpha] bound to the HRE within the Mipul promoter region and promoted its transcription, leading to cytoprotection of HIF-1 against [H.sub.2][O.sub.2]-mediated injury in H9c2 cells partly through regulation of Mipul expression. Our previous studies also found that hypoxia-reoxygenation or [H.sub.2][O.sub.2]-induced upregulation of Mipul in H9c2 cardiomyocytes was mediated by cAMP/ protein kinase A (PKA)-dependent CREB activation, and that the cytoprotection of CREB against hypoxia-reoxygenation or [H.sub.2][O.sub.2]-mediated injury in H9c2 cells occurs partly through regulation of Mipul expression (5).

Expression of Mipul 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 Mipul 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, Mipul 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 Mipul 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 Mipul and its role in ischemic-related diseases.

http://dx.doi.org/10.1590/1414-431X20144029

Received April 2, 2014. Accepted July 23, 2014. First published online October 31, 2014.

Acknowledgments

Research supported by the National Natural Science Foundation of China (#81100212, #81170277, and #8110 0106), the PhD Programs Foundation of the Ministry of Education of the People's Republic of China (#20114324 120004 and #20124324110003), the China Postdoctoral Science Foundation (#2012 M511383), the Scientific Research Fund of Hunan Provincial Education Department (#11C1094 and #11C1095), the Science and Technology Project of Hunan Province (#2014FJ3014), the Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province (#2008-244), and the Construct Program of the Key Discipline in Human Hunan Province (#2011-76).

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D. Han [1] *, C. Zhang [1]*, W.J. Fan [1,2]*, W.J. Pan [1], D.M. Feng [1], S.L. Qu [1] and Z.S. Jiang [1]

[1] Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province, PR China

[2] The Second Affiliated Hospital, University of South China, Hengyang City, Hunan Province, PR China

Correspondence: Shunlin Qu: <qushunlin78@126.com>; Zhisheng Jiang: <zsjiang2005@163.com>.

* These authors are co-first authors.

Caption: Figure 1. Structure of ZNF667. Arrows: zinc finger structure.
Table 1. Role of C2H2 ZNF.

C2H2 ZNF                 Role               Evidence for the role

ZNF139         Increased multi-drug        Promoting the expression
                 resistance                  of bcl-2 and
                                            inhibiting the
                                             expression of Bax
ZNF268         Contributes to cervical     Enhancing NF-Kb
                 carcinogenesis              signaling
ZNFD           Transcriptional activator   Activates the
                 in PKC signal pathway       transcriptional
                                             activities of AP1
               Development of mouse        Transcriptional
                 testis                      regulation of HSE
HNF-4[alpha]   Regulation of fatty         Regulating human
                 acid metabolism             intestinal fatty acid
                                             binding protein
                                             (hFABP2) expression
               Regulation of bile acid     Regulation of expression
                 synthesis in human          of cholesterol
                 liver                       7[alpha]-hydroxylase
                                             (Cyp7a1)
ZNF667         Regulation of cell          Repress expression of
                 apoptosis                   Fas and Bax

C2H2 ZNF                 Role              Reference

ZNF139         Increased multi-drug           19
                 resistance
ZNF268         Contributes to cervical        20
                 carcinogenesis
ZNFD           Transcriptional activator      21
                 in PKC signal pathway
               Development of mouse           22
                 testis
HNF-4[alpha]   Regulation of fatty            23
                 acid metabolism
               Regulation of bile acid        24
                 synthesis in human
                 liver
ZNF667         Regulation of cell            25-27
                 apoptosis

ZNF: zinc finger; HNF-4[alpha]: hepatocyte nuclear factor 4a.
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Author:Han, D.; Zhang, C.; Fan, W.J.; Pan, W.J.; Feng, D.M.; Qu, S.L.; Jiang, Z.S.
Publication:Brazilian Journal of Medical and Biological Research
Article Type:Report
Date:Jan 1, 2015
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