Kazuya Yamada

Zinc-fingers and homeoboxes (ZHX) 2, a novel member of the ZHX family, functions as a transcriptional repressor.

Zinc-fingers and homeoboxes (ZHX) 1 is a transcription factor that interacts with the activation domain of the A subunit of nuclear factor-Y (NF-YA). Using a yeast two-hybrid system, a novel ubiquitous transcription factor ZHX2 as a ZHX1-interacting protein was cloned. ZHX2 consists of 837 amino acid residues and contains two zinc-finger motifs and five homeodomains (HDs) as well as ZHX1. The mRNA is expressed among various tissues. ZHX2 not only forms a heterodimer with ZHX1, but also forms a homodimer. Moreover, ZHX2 interacts with the activation domain of NF-YA. Further analysis revealed that ZHX2 is a transcriptional repressor that is localized in the nuclei. Since ZHX2 shares a number of properties in common with ZHX1, we conclude that all these come under the ZHX family. The minimal functional domains of ZHX2 were then characterized. The dimerization domain with both ZHX1 and ZHX2 is the region containing HD1, the domain that interacts with NF-YA is the HD1 to HD2 region, the repressor domain is the HD1 to a proline-rich region. Lastly, using an immunoprecipitation assay, we showed that ZHX2 intrinsically interacts with NF-YA in HEK-293 cells and that ZHX2 represses the promoter activity of the cdc25C gene stimulated by NF-Y in Drosophila Schneider line 2 cells. Thus the ZHX family of proteins may participate in the expression of a number of NF-Y-regulated genes via a more organized transcription network.

Analysis of zinc-fingers and homeoboxes (ZHX)-1-interacting proteins: molecular cloning and characterization of a member of the ZHX family, ZHX3

Human zinc-fingers and homeoboxes (ZHX) 1, a transcriptional repressor, was originally cloned as an interacting protein with the activation domain of the A subunit of nuclear factor-Y (NF-YA). As the first step in investigating the mechanism by which ZHX1 acts as a transcriptional repressor, we conducted a search of ZHX1-interacting proteins using a yeast two-hybrid system. Nuclear proteins such as ZHX1, transcriptional co-factors and DNA-binding proteins, zyxin, androgen-induced aldose reductase and eleven-nineteen lysine-rich leukaemia gene, as well as some unknown proteins, were cloned. Molecular cloning and determination of the nucleotide sequence of the full-length cDNA encoding a novel protein revealed that it consists of 956 amino acid residues and contains two zinc-finger (Znf) motifs and five homeodomains (HDs) as well as ZHX1. We concluded that the protein forms the ZHX family with ZHX1 and denoted it ZHX3. ZHX3 not only dimerizes with both ZHX1 and ZHX3, but also interacts with the activation domain of the NF-YA. Further analysis revealed that ZHX3 is a ubiquitous transcriptional repressor that is localized in nuclei and functions as a dimer. Lastly, the dimerization domain, the interaction domain with NF-YA, and the repressor domain are mapped to a region including the HD1 region, and two nuclear localization signals are mapped to the N-terminal through Znf1 and the HD2 region, respectively.

Early Growth Response Gene-1 Regulates the Expression of the Rat Luteinizing Hormone Receptor Gene

Abstract LH receptor gene expression is primarily regulated via specific interactions of trans-acting proteins and cis-acting DNA sequences in the upstream region of the gene. In this study, we report, using luciferase assays, that the region between −171 and −137 base pairs (bp) is essential for basal expression of the rat LH receptor gene. To identify factors that interact with the region between −171 and −137 bp and regulate expression of the gene, a rat granulosa cell cDNA library was screened using a yeast one-hybrid system. A positive clone, isolated by the screening, encodes a transcription factor early growth response gene-1 (Egr-1). To determine the sequence to which Egr-1 protein binds, electrophoretic mobility shift assay (EMSA) was employed. The Egr-1 protein was produced by an in vitro transcription/translation system using a full-length rat Egr-1 cDNA. The upstream region between −171 and −137 bp contains 2 overlapping Egr-1 consensus sequences. The EMSA revealed that Egr-1 binds independently to both sites. The overexpression of Egr-1 in MA-10 cells caused an approximately 2-fold increase in reporter luciferase activity. However, no induction of the luciferase activity was observed when luciferase constructs that lacked or had mutations in either or both of the Egr-1 sites were used, indicating that Egr-1 positively regulates LH receptor gene expression. In differentiated granulosa cells that had been pretreated with FSH for 48 h, the levels of both mRNA and Egr-1 protein were induced by hCG or cAMP, reaching maximal levels approximately 1.5 h after treatment and then returning to basal levels 8 h thereafter. No Egr-1 mRNA or protein was detected in undifferentiated granulosa cells, even after stimulation with 8-bromoadenosine-cAMP. These results suggest that Egr-1 functions only in luteinized granulosa cells after stimulation with hCG or cAMP. In conclusion, the findings demonstrate that Egr-1 actually binds to the regulatory upstream region of the LH receptor gene and positively regulates receptor gene expression. In addition, Egr-1 expression was observed only in luteinized granulosa cells after stimulation with hCG or cAMP. The present study provides further support to the hypothesis that Egr-1 plays important roles in the pituitary-gonadal axis.

Regulation of NGFI-B/Nur77 gene expression in the rat ovary and in leydig tumor cells MA-10.

NR4A1, also called NGFI‐B in the rat, Nur77 in the mouse and TR3 in humans, belongs to the orphan nuclear steroid hormone receptor superfamily and is one of the immediate‐early genes. In the endocrine organs, including the gonads, NGFI‐B/Nur77 gene expression is rapidly induced by pituitary hormones. NGFI‐B/Nur77 expression was found to be rapidly reduced by an estrogenic endocrine disrupter, diethylstilbestrol (DES) in theca interna cells of immature rat ovaries. DES treatment also triggered a rapid decrease of serum luteinizing hormone (LH) levels, suggesting that DES acts on the hypothalamo–pituitary axis to suppress LH secretion from the pituitary. The transcriptional regulation of NGFI‐B/Nur77 by LH/human chorionic gonadotropin (hCG) or 8‐bromoadenosine 3′–5′‐cyclic monophosphate (8 Br‐cAMP) was examined in mouse Leydig tumor cells MA‐10. Luciferase assays using NGFI‐B/Nur77 promoter constructs and electric mobility shift assays (EMSA) showed that NGFI‐B/Nur77 gene expression was mediated through three of the four activator protein‐1 (AP‐1)‐like sites, namely the −233 AP‐1, −213 AP‐1 and −69 AP‐1 sites adjacent to the transcription start site of the NGFI‐B/Nur77 promoter. We also demonstrated here that both the Jun family and cAMP‐responsive element binding (CREB) proteins bind to the −233 AP‐1 site, whereas the main binding protein to the −213 AP‐1 site was CREB, and Jun family protein to the −69 AP‐1 site, respectively. The rapid induction of NGFI‐B/Nur77 gene expression by LH/hCG in MA‐10 cells appears to be mediated by both CREB and Jun family proteins through the cAMP‐protein kinase A (PKA) pathway. Mol. Reprod. Dev. 75: 931–939, 2008.

Remnant lipoprotein particles are taken up into myocardium through VLDL receptor--a possible mechanism for cardiac fatty acid metabolism.

The VLDL (very low-density lipoprotein) receptor is a peripheral lipoprotein receptor expressing in fatty acid active tissues abundantly. In the Balb/c fasting mice, VLDL receptor as well as LPL (lipoprotein lipase), FAT (fatty acid translocase)/CD36, H-FABP (heart-type fatty acid-binding protein), ACS (acyl-CoA synthetase) and LCAD (long-chain acyl-CoA dehydrogenase) expressions increased. An electron microscopic examination indicated the lipid droplets that accumulated in the hearts of fasting Balb/c mice. During the development of SD (Sprague-Dawley) rats, VLDL receptor, LPL, FAT/CD36, H-FABP, ACS, and LCAD mRNAs concomitantly increased with growth. However, PK (pyruvate kinase) mRNA expression was negligible. In cultured neonatal rat cardiomyocytes, VLDL receptor expression increased with days in culture. Oil red-O staining showed that cardiomyocytes after 7 days in culture (when the VLDL receptor protein is present) accumulated beta-migrating VLDL. Thereby, we showed that the cardiac VLDL receptor pathway for delivery of remnant lipoprotein particles might be part of a cardiac fatty acid metabolism.

SHARP‐2/Stra13/DEC1 as a potential repressor of phosphoenolpyruvate carboxykinase gene expression

The influence of the enhancer of split‐ and hairy‐related protein‐2 (SHARP‐2) transcriptional repressor on the expression of rat phosphoenolpyruvate carboxykinase (PEPCK) gene was examined. When H4IIE cells were treated with epigallocatechin gallate, a green tea constituent, an increase in SHARP‐2 mRNA levels and a decrease in PEPCK mRNA levels were observed. The adenovirus‐mediated overexpression of SHARP‐2 in H4IIE cells and primary cultured rat hepatocytes led to a decrease in the levels of PEPCK mRNA. Finally, when a SHARP‐2 expression plasmid was transiently transfected with various reporter plasmids into MH1C1 cells, the promoter activity of a PEPCK reporter plasmid was specifically decreased. Based on these findings, we conclude that SHARP‐2 is a potential repressor of PEPCK gene expression.

Activation of mouse phosphodiesterase 3B gene promoter by adipocyte differentiation in 3T3-L1 cells.

Activation of phosphodiesterase (PDE) 3B reduces free fatty acid output from adipocytes. Induction of PDE3B gene expression by adipocyte differentiation could improve insulin resistance. To examine whether the PDE3B promoter is activated by this differentiation, the 5′ flanking sequence of the mouse PDE3B gene was isolated. The transcription initiation site was determined to be located 195 bp upstream of the translation start site. No putative binding site for peroxisome proliferator‐activated receptor γ was found within 2 kb upstream of the transcription initiation site. This region had promoter activity, which was further activated on adipocyte differentiation in 3T3‐L1 cells.

Gene Expression of Basic Helix-Loop-Helix Transcription Factor, SHARP-2, Is Regulated by Gonadotropins in the Rat Ovary and MA-10 Cells

Abstract Basic helix-loop-helix (bHLH) proteins regulate transcription from the E box sequence (5′-CANNTG-3′) located in the regulatory region of most gene promoters. The rat enhancer of split- and hairy-related protein 2 (SHARP-2) is a member of the bHLH protein family. To analyze the possible role of SHARP-2 in the rat ovary, the regulation of the expression of the SHARP-2 gene was examined, and the SHARP-2 protein was characterized. Northern blot analysis revealed that the level of SHARP-2 mRNA abruptly and temporarily increases as the result of the action of LH, i.e., eCG or hCG treatment alone or hCG after eCG treatment, in the rat ovary, as indicated by the treatment of primary cultured rat granulosa cells with hCG after FSH treatment or of mouse Leydig MA-10 cells with hCG or 8-bromoadenosine 3′,5′-cyclic monophosphate. An in situ hybridization analysis showed that eCG treatment increases the level of the SHARP-2 transcript in theca interna cells and that hCG treatment, after the administration of eCG, increases the level of the SHARP-2 transcript in granulosa cells. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into primary cultured granulosa cells and MA-10 cells revealed that the entire coding sequence of SHARP-2 fused to the GFP is localized in the nucleus. The transcriptional activity of SHARP-2 also was examined using transient DNA transfection experiments. When an expression vector encoding the full length of SHARP-2 was cotransfected with thymidine kinase promoter-luciferase reporter plasmids, with or without E box sequences, into MA-10 cells, the luciferase activity was decreased in an E box-dependent manner. We conclude that the level of SHARP-2 mRNA is regulated by gonadotropins and that SHARP-2 functions as a transcriptional repressor localized in the nucleus.

Insulin Stimulates Expression of the Pyruvate Kinase M Gene in 3T3-L1 Adipocytes

M2-type pyruvate kinase (M2-PK) mRNA is produced from the PKM gene by an alternative RNA splicing in adipocytes. We found that insulin increased the level of M2-PK mRNA in 3T3-L1 adipocytes in both time- and dose-dependent manners. This induction did not require the presence of glucose or glucosamine in the medium. The insulin effect was blocked by pharmacological inhibitors of insulin signaling pathways such as wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), and PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase. A stable reporter expression assay showed that the promoter activity of an about 2.2-kb 5′-flanking region of the rat PKM gene was stimulated by insulin, but the extents of these stimulations were lower than those of the mRNA stimulation. Thus, we suggest that insulin increases the level of M2-PK mRNA in adipocytes by acting at transcriptional and post-transcriptional levels through signaling pathways involving both PI3K and MAPK kinase.

Nuclear Factor 1 Family Members Interact with Hepatocyte Nuclear Factor 1α to Synergistically Activate L-type Pyruvate Kinase Gene Transcription

Transcription of hepatic L-type pyruvate kinase (L-PK) gene is cell type-specific and is under the control of various nutritional conditions. The L-PK gene contains multiple cis-regulatory elements located within a 170-bp upstream region necessary for these regulations. These elements can synergistically stimulate L-PK gene transcription, although their mechanisms are largely unknown. Because nuclear factor (NF) 1 family members bind to specific cis-regulatory elements known as L-IIA and L-IIB and hepatocyte nuclear factor (HNF) 1α binds to the adjacent element L-I, we examined the functional and physical interactions between these two transcription factors. Reporter gene assay showed that these two factors synergistically activated the L-PK promoter containing the 5′-flanking region up to –189. Although two NF1-binding sites are required for the maximum synergistic effect of NF1 family members with HNF1α, significant functional interaction between the two factors was observed in the L-PK promoter containing two mutated NF1-binding sites and also in the promoter containing only the HNF1α-binding site, raising the possibility that NF1 proteins function as HNF1α co-activators. Chromatin immunoprecipitation assay revealed that both NF1 proteins and HNF1α bound to the promoter region of the L-PK gene in vivo. In vitro binding assay confirmed that NF1 proteins directly interacted mainly with the homeodomain of HNF1α via their DNA-binding domains. This interaction enhanced HNF1α binding to the L-I element and was also observed in rat liver by co-immunoprecipitation assay. Thus, we conclude that cooperative interaction between NF1 family members and HNF1α plays an important role in hepatic L-PK transcription.