Biomedical subjects

Hitomi Matsuzaki

Publications and source records attributed to Hitomi Matsuzaki.

11 recordsLinked to original sources

PubMed2006

Mitogen-activated protein kinases, Erk and p38, phosphorylate and regulate Foxo1.

The members of the transcription factor Foxo family regulate the expression of genes concerned with the stress response, cell cycle and gluconeogenesis. Foxo1 (FKHR) contains 15 consensus phosphorylation sites for the mitogen-activated protein kinase (MAPK) family. Therefore, we hypothesized that MAPKs could directly regulate the transcriptional activity of Foxo1 via phosphorylation. In vitro kinase assay showed that Foxo1 was phosphorylated by extracellular signal-regulated kinase (Erk) and p38 MAPK (p38) but not by c-jun NH2-terminal kinase (JNK). In NIH3T3 cells, epidermal growth factor or anisomycin increased phosphorylation of exogenous Foxo1, which was significantly inhibited by pretreatment with an MEK 1 inhibitor, PD98059, or a p38 inhibitor, SB203580. Two-dimensional phosphopeptide mapping using mutation of phosphorylation sites for MAPK revealed that the nine serine residues in Foxo1 are specifically phosphorylated by Erk and that five of the nine residues are phosphorylated by p38 in vivo. Moreover, we also found that Foxo1 interacts with Ets-1 and functions as a coactivator for Ets-1 on the fetal liver kinase (Flk)-1 promoter in bovine carotid artery endothelial cells. Mutation of the nine phosphorylation sites for Erk in Foxo1 was shown to lead to less binding and synergistic activity for Ets-1 on the Flk-1 promoter when compared with wild-type Foxo1. These results suggest that Foxo1 is specifically phosphorylated by Erk and p38, and that this phosphorylation regulates the function of Foxo1 as a coactivator for Ets-1.

Animals↗

PubMed2005

Acetylation of Foxo1 alters its DNA-binding ability and sensitivity to phosphorylation.

The FOXO family of forkhead transcription factors plays a key role in a variety of biological processes, including metabolism, cell proliferation, and oxidative stress response. We previously reported that Foxo1, a member of the FOXO family, is regulated through reversible acetylation catalyzed by histone acetyltransferase cAMP-response element-binding protein (CREB)-binding protein (CBP) and NAD-dependent histone deacetylase silent information regulator 2, and that the acetylation at Lys-242, Lys-245, and Lys-262 of Foxo1 attenuates its transcriptional activity. However, the molecular mechanism by which acetylation modulates Foxo1 activity remains unknown. Here, we show that the positive charge of these lysines in Foxo1 contributes to its DNA-binding, and acetylation at these residues by CBP attenuates its ability to bind cognate DNA sequence. Remarkably, we also show that acetylation of Foxo1 increases the levels of its phosphorylation at Ser-253 through the phosphatidylinositol 3-kinase-protein kinase B signaling pathway, and this effect was overridden on the acetylation-deficient Foxo1 mutant. Furthermore, in in vitro kinase reactions, the association of wild-type Foxo1 and its target DNA sequence inhibits the protein kinase B-dependent phosphorylation of Foxo1, whereas mutated Foxo1 proteins, which mimic constitutively acetylated states, are efficiently phosphorylated even in the presence of the DNA. These results suggest that acetylation regulates the function of Foxo1 through altering the affinity with the target DNA and the sensitivity for phosphorylation.

Acetylation↗

PubMed2005

Genomic imprinting recapitulated in the human beta-globin locus.

A subset of genes in mammals are subject to genomic imprinting. The mouse H19 gene, for example, is active only when maternally inherited and the neighboring Igf2 gene is paternally expressed. This imprinted expression pattern is regulated by the imprinting control region (ICR) upstream of the H19 gene. A maternally inherited H19 ICR inhibits Igf2 gene activation by the downstream enhancer due to its insulator function while it suppresses H19 gene transcription by promoter DNA methylation when paternally inherited. These parent-of-origin specific functions depend on the allele-specific methylation of the ICR DNA, which is established during gametogenesis. Therefore, the ICR may also function as a landmark for epigenetic modifications. To examine whether the ICR confers these activities autonomously, we introduced a 2.9-kbp ICR-containing DNA fragment into a human beta-globin yeast artificial chromosome at the 3' end of the locus control region and established transgenic mouse lines. Expression of all of the beta-like globin genes was higher when the transgene was paternally inherited. In accord with this result, transgenic ICR DNA from nucleated erythrocytes was more heavily methylated when paternally transmitted. Chromatin immunoprecipitation assays confirmed that CCCTC binding factor is preferentially recruited to the maternal transgenic ICR in vivo. Surprisingly however, the parent-of-origin specific methylation pattern was not observed in germ cell DNA in testis, demonstrating that methylation was established after fertilization. Thus, the ICR autonomously recapitulated imprinting within the normally nonimprinted transgenic beta-globin gene locus, but the temporal establishment of imprinting methylation differs from that at the endogenous Igf2/H19 locus.

Animals↗

PubMed2004

Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity.

Longevity regulatory genes include the Forkhead transcription factor FOXO and the NAD-dependent histone deacetylase silent information regulator 2 (Sir2). Genetic studies demonstrate that Sir2 acts to extend lifespan in Caenorhabditis elegans upstream of DAF-16, a member of the FOXO family, in the insulin-like signaling pathway. However, the molecular mechanisms underlying the requirement of DAF-16 activity in Sir2-mediated longevity remain unknown. Here we show that reversible acetylation of Foxo1 (also known as FKHR), the mouse DAF-16 ortholog, modulates its transactivation function. cAMP-response element-binding protein (CREB)-binding protein binds and acetylates Foxo1 at the K242, K245, and K262 residues, the modification of which is involved in the attenuation of Foxo1 as a transcription factor. Conversely, Sir2 binds and deacetylates Foxo1 at residues acetylated by cAMP-response element-binding protein-binding protein. Sir2 is recruited to insulin response sequence-containing promoter and increases the expression of manganese superoxide dismutase and p27(kip1) in a deacetylase-activity-dependent manner. Our findings establish Foxo1 as a direct and functional target for Sir2 in mammalian systems.

Acetylation↗

PubMed2004

Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo.

APJ is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin, was identified recently. They are highly expressed in the cardiovascular system, suggesting that APJ is important in the regulation of blood pressure. To investigate the physiological functions of APJ, we have generated mice lacking the gene encoding APJ. The base-line blood pressure of APJ-deficient mice is equivalent to that of wild-type mice in the steady state. The administration of apelin transiently decreased the blood pressure of wild-type mice and a hypertensive model animal, a spontaneously hypertensive rat. On the other hand, this hypotensive response to apelin was abolished in APJ-deficient mice. This apelin-induced response was inhibited by pretreatment with a nitric-oxide synthase inhibitor, and apelin-induced phosphorylation of endothelial nitric-oxide synthase in lung endothelial cells from APJ-deficient mice disappeared. In addition, APJ-deficient mice showed an increased vasopressor response to the most potent vasoconstrictor angiotensin II, and the base-line blood pressure of double mutant mice homozygous for both APJ and angiotensin-type 1a receptor was significantly elevated compared with that of angiotensin-type 1a receptor-deficient mice. These results demonstrate that APJ exerts the hypotensive effect in vivo and plays a counterregulatory role against the pressor action of angiotensin II.

Alleles↗

PubMed2004

Bile acids regulate gluconeogenic gene expression via small heterodimer partner-mediated repression of hepatocyte nuclear factor 4 and Foxo1.

Bile acid homeostasis is tightly controlled by the feedback mechanism in which an atypical orphan nuclear receptor (NR) small heterodimer partner (SHP) inactivates several NRs such as liver receptor homologue-1 and hepatocyte nuclear factor 4. Although NRs have been implicated in the transcriptional regulation of gluconeogenic genes, the effect of bile acids on gluconeogenic gene expression remained unknown. Here, we report that bile acids inhibit the expression of gluconeogenic genes, including glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase, and fructose 1,6-bis phosphatase in an SHP-dependent fashion. Cholic acid diet decreased the mRNA levels of these gluconeogenic enzymes, whereas those of SHP were increased. Reporter assays demonstrated that the promoter activity of phosphoenolpyruvate carboxykinase and fructose 1,6-bis phosphatase via hepatocyte nuclear factor 4, or that of G6Pase via the forkhead transcription factor Foxo1, was down-regulated by treatment with chenodeoxicholic acid and with transfected SHP. Remarkably, Foxo1 interacted with SHP in vivo and in vitro, which led to the repression of Foxo1-mediated G6Pase transcription by competition with a coactivator cAMP response element-binding protein-binding protein. These findings reveal a novel mechanism by which bile acids regulate gluconeogenic gene expression via an SHP-dependent regulatory pathway.

Animals↗

PubMed2003

Insulin-induced phosphorylation of FKHR (Foxo1) targets to proteasomal degradation.

Forkhead transcription factor FKHR (Foxo1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. It has been shown that FKHR is phosphorylated via insulin or growth factor signaling cascades, resulting in its cytoplasmic retention and the repression of target gene expression. Here, we investigate the fate of FKHR after cells are stimulated by insulin. We show that insulin treatment decreases endogenous FKHR proteins in HepG2 cells, which is inhibited by proteasome inhibitors. FKHR is ubiquitinated in vivo and in vitro, and insulin enhances the ubiquitination in the cells. In addition, the signal to FKHR degradation from insulin is mediated by the phosphatidylinositol 3-kinase pathway, and the mutation of FKHR at the serine or threonine residues phosphorylated by protein kinase B, a downstream target of phosphatidylinositol 3-kinase, inhibits the ubiquitination in vivo and in vitro. Finally, efficient ubiquitination of FKHR requires both phosphorylation and cytoplasmic retention in the cells. These results demonstrate that the insulin-induced phosphorylation of FKHR leads to the multistep negative regulation, not only by the nuclear exclusion but also the ubiquitination-mediated degradation.

Cell Line↗

PubMed2003

Hepatocyte nuclear factor-4 is a novel downstream target of insulin via FKHR as a signal-regulated transcriptional inhibitor.

Previous studies have shown that FKHR, a member of the forkhead family of transcription factors, acts as a DNA binding-independent cofactor of nuclear receptors, including estrogen, retinoid, and thyroid hormone receptors, in addition to the original function as a DNA binding transcription factor that redistributes from the nucleus to the cytoplasm by insulin-induced phosphorylation. Here, we demonstrated the physical interaction of FKHR with hepatocyte nuclear factor (HNF)-4, a member of steroid/thyroid nuclear receptor superfamily, and the repression of HNF-4 transactivation by FKHR. FKHR interacted with the DNA binding domain of HNF-4 and inhibited HNF-4 binding to the cognate DNA. Furthermore, the binding affinity of HNF-4 with phosphorylated FKHR significantly decreased in comparison to that with unphosphorylated FKHR. Therefore, a phosphorylation of FKHR by insulin followed by its dissociation from HNF-4 and the redistribution of FKHR from the nucleus to the cytoplasm would expect to induce the transcriptional activation of HNF-4 by facilitating to the access of HNF-4 to its DNA element. Indeed, most intriguingly, insulin stimulation reversed the repression of HNF-4 transcriptional activity by phosphorylation-sensitive (wild-type) FKHR, but not by phosphorylation-deficient FKHR. These results suggest that insulin regulates the transcriptional activity of HNF-4 via FKHR as a signal-regulated transcriptional inhibitor.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2003

Regulation of PGC-1 promoter activity by protein kinase B and the forkhead transcription factor FKHR.

Peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) plays a major role in mediating hepatic gluconeogenesis in response to starvation, during which PGC-1 is induced by the cyclic AMP response element binding protein. Although it is observed that insulin counteracts PGC-1 transcription, the mechanism by which insulin suppresses the transcription of PGC-1 is still unclear. Here, we show that forkhead transcription factor FKHR contributes to mediating the effects of insulin on PGC-1 promoter activity. Reporter assays demonstrate that insulin suppresses the basal PGC-1 promoter activity and that coexpression of protein kinase (PK)-B mimics the effect of insulin in HepG2 cells. Insulin response sequences (IRSs) are addressed in the PGC-1 promoter as the direct target for FKHR in vivo. Coexpression of FKHR stimulates the PGC-1 promoter activity via interaction with the IRSs, while coexpression of FKHR (3A), in which the three putative PKB sites in FKHR are mutated, mainly abolishes the suppressive effect of PKB. Whereas deletion of the IRSs prevents the promoter stimulation by FKHR, that activity is still partially inhibited by insulin. These results indicate that signaling via PKB to FKHR can partly account for the effect of insulin to regulate the PGC-1 promoter activity via the IRSs.

Androstadienes↗

PubMed2003

Negative regulation of forkhead transcription factor AFX (Foxo4) by CBP-induced acetylation.

AFX (also called Foxo4), a member of mammalian FOXO forkhead transcription factors, is the homolog of DAF-16, which contributes longevity and oxidative stress in Caenorhabditis elegans. Here, we show that CREB-binding protein (CBP) interacts with AFX via CH1 region and acetylates it at the three lysine residues (K186, K189, and K408). Trichostatin A (TSA) repressed the AFX-stimulated transcription, and substitution of the lysine residues with arginine (K186, 189, 408R) enhanced the transcriptional activity of AFX. Furthermore, TSA suppressed the expression of p27kip1 gene induced by AFX in HEK293T cells. Compared to the coexpression of CBP wild-type, a HAT activity-deficient mutant (DeltaHAT) efficiently leads the cooperative activation of AFX transcription in HepG2 cells. Taken together, these results demonstrate that CBP-induced acetylation of AFX is a novel modification mechanism by which AFX keeps the transcriptional activity mitigating in the nucleus.

Animals↗

PubMed2002

Regulation of alkaline phosphatase promoter activity by forkhead transcription factor FKHR.

Alkaline phosphatase (ALP) is a basic marker of osteoblast maturation and osteogenesis. However, the mechanisms of the ALP gene regulation in osteoblasts remain elusive. In this study, we examined the expression of forkhead transcription factor FKHR, a regulator of hepatic glucose metabolic and proapoptotic genes, in osteogenic cells and the effect of FKHR on transcription of the ALP gene. RT-PCR and immunoblot analyses revealed the expression of FKHR in osteogenic MC3T3-E1, SaOS2, and UMR 106 cells. Reporter assays demonstrated that the overexpression of FKHR stimulated ALP promoter activity through the forkhead response element in its promoter. These results suggest that ALP is a target gene regulated by FKHR and that FKHR contributes to osteoblast maturation and osteogenesis.

3-Phosphoinositide-Dependent Protein Kinases↗