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Biomedical subjects

Toshio Hirano

Publications and source records attributed to Toshio Hirano.

At least 19 recordsLinked to original sources
PubMed2003

Activation of gp130 transduces hypertrophic signal through interaction of scaffolding/docking protein Gab1 with tyrosine phosphatase SHP2 in cardiomyocytes.

Grb2-associated binder-1 (Gab1) is a scaffolding/docking protein and contains a Pleckstrin homology domain and potential binding sites for Src homology (SH) 2 and SH3 domains. Gab1 is tyrosine phosphorylated and associates with protein tyrosine phosphatase SHP2 and p85 phosphatidylinositol 3-kinase on stimulation with various cytokines and growth factors, including interleukin-6. We previously demonstrated that interleukin-6-related cytokine, leukemia inhibitory factor (LIF), induced cardiac hypertrophy through gp130. In this study, we report the role of Gab1 in gp130-mediated cardiac hypertrophy. Stimulation with LIF induced tyrosine phosphorylation of Gab1, and phosphorylated Gab1 interacted with SHP2 and p85 in cultured cardiomyocytes. We constructed three kinds of adenovirus vectors, those carrying wild-type Gab1 (AdGab1WT), mutated Gab1 lacking SHP2 binding site (AdGab1F627/659), and beta-galactosidase (Adbeta-gal). Compared with cardiomyocytes infected with Adbeta-gal, longitudinal elongation of cardiomyocytes induced by LIF was enhanced in cardiomyocytes infected with AdGab1WT but inhibited in cardiomyocytes infected with AdGab1F627/659. Upregulation of BNP mRNA expression by LIF was evoked in cardiomyocytes infected with Adbeta-gal and AdGab1WT but not in cardiomyocytes infected with AdGab1F627/659. In contrast, Gab1 repressed skeletal alpha-actin mRNA expression through interaction with SHP2. Furthermore, activation of extracellular signal-regulated kinase 5 (ERK5) was enhanced in cardiomyocytes infected with AdGab1WT compared with cardiomyocytes infected with Adbeta-gal but repressed in cardiomyocytes infected with AdGab1F627/659. Coinfection of AdGab1WT with adenovirus vector carrying dominant-negative ERK5 abrogated longitudinal elongation of cardiomyocytes induced by LIF. Taken together, these findings indicate that Gab1-SHP2 interaction plays a crucial role in gp130-dependent longitudinal elongation of cardiomyoctes through activation of ERK5.

Actins↗
PubMed2003

IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages.

Whereas interleukin-6 (IL-6) is a proinflammatory cytokine, IL-10 is an anti-inflammatory cytokine. Although signal transducer and activator of transcription 3 (STAT3) is essential for the function of both IL-6 and IL-10, it is unclear how these two cytokines have such opposing functions. Here we show that suppressor of cytokine signaling 3 (SOCS3) is a key regulator of the divergent action of these two cytokines. In macrophages lacking the Socs3 gene or carrying a mutation of the SOCS3-binding site in gp130, the lipopolysaccharide-induced production of tumor necrosis factor (TNF) and IL-12 is suppressed by both IL-10 and IL-6. SOCS3 specifically prevents activation of STAT3 by IL-6 but not IL-10. Taken together, these data indicate that SOCS3 selectively blocks signaling by IL-6, thereby preventing its ability to inhibit LPS signaling.

Animals↗
PubMed2003

Gab1 is required for EGF receptor signaling and the transformation by activated ErbB2.

Grb2-associated binder-1 (Gab1) is a pleckstrin homology (PH) domain-containing adapter molecule that is believed to function downstream of receptors for growth factors and cytokines. We previously found that deficiency in the mouse Gab1 gene led to embryonic lethality and defects in ERK activation in response to growth factors and cytokines. Here, we established immortalized Gab1-/- cell lines and analysed roles of Gab1 in growth factor-mediated signaling and oncogenesis. EGF-dependent activation of c-Raf and Mek1/2, which function upstream of ERKs, was perturbed in Gab1-/- cells. EGF-mediated upregulation of GTP-bound form of Ras was also reduced in these cells. EGF-dependent GTP/GDP exchange activity for Ras was suppressed in the Gab1-/- cells and expression of a constitutively active Sos restored ERK activation in these cells, indicating that Gab1 functions upstream of Ras. Furthermore, activated form of ErbB2 (active ErbB2)-mediated transformation, such as colony formation in soft agar and tumor formation in nude mice, was strongly suppressed when the Gab1-/- cells were transfected with active ErbB2, whereas the active Sos efficiently induced transformation of Gab1-/- cells. The data show that Gab1 plays an essential role in EGF-receptor/ErbB-mediated cell proliferation and oncogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Gads/Grb2-mediated association with LAT is critical for the inhibitory function of Gab2 in T cells.

A docking protein, Gab2, is recruited to the vicinity of the TCR complex and inhibits downstream signaling by interaction with negative regulators. However, the molecular mechanisms of this recruitment remain unclear. We have found that Gab2 associates with LAT upon TCR stimulation and that LAT is essential for Gab2 phosphorylation. By analysis of several Gab2 mutants, the c-Met binding domain (MBD) of Gab2 was found to be both necessary and sufficient for stimulation-induced LAT binding. Within the MBD domain, a novel Grb2 SH3 binding motif, PXXXR, is critical for constitutive association with Gads/Grb2. Through this association, Gab2 is recruited to the lipid raft after TCR ligation and exerts inhibitory function. The in vivo significance of this association is illustrated by the fact that T-cell responses are impaired in transgenic mice expressing wild-type Gab2 but not in mice expressing mutant Gab2 lacking the motif. Furthermore, T cells from Gab2-deficient mice showed enhanced proliferative responses upon TCR stimulation. These results indicate that Gads/Grb2-mediated LAT association is critical for the inhibitory function of Gab2, implying that Gab2 induced in stimulated T cells may exert an efficient negative feedback loop by recruiting inhibitory molecules to the lipid raft and competing with SLP-76 through Gads binding.

Adaptor Proteins, Signal Transducing↗
PubMed2003

A homeobox gene, pnx, is involved in the formation of posterior neurons in zebrafish.

A homeobox gene, pnx, is expressed in prospective posterior neurogenic regions and later in primary neurons. pnx expression was regulated by a signal from the non-axial mesendoderm and by Notch signaling. Pnx contains an Eh1 repressor domain, which interacted with Groucho and acted as a transcriptional repressor. Misexpression of pnx increased neural precursor cells and postmitotic neurons, which express neurogenin1 and elavl3/HuC, respectively. Expression of an antimorphic Pnx (VP16Pnx) or inhibition of Pnx by antisense morpholino oligonucleotide led to the reduction in the number of a subset of primary neurons. Misexpression of pnx promoted neurogenesis independent of Notch signaling. Epistatic analyses showed that Pnx also functions downstream of the Notch signal. These data indicate that pnx is a novel repressor-type homeobox gene that regulates posterior neurogenesis.

Amino Acid Sequence↗
PubMed2003

Ogon/Secreted Frizzled functions as a negative feedback regulator of Bmp signaling.

The zebrafish mutant ogon (also called mercedes and short tail) displays ventralized phenotypes similar to the chordino (dino) mutant, in which the gene for the Bmp antagonist Chordin is mutated. We isolated the gene responsible for ogon by a positional cloning strategy and found that the ogon locus encodes a zebrafish homolog of Secreted Frizzled (Sizzled), which has sequence similarity to a Wnt receptor, Frizzled. Unlike other secreted Frizzled-related proteins (sFrps) and the Wnt inhibitor Dickkopf1, the misexpression of Ogon/Sizzled dorsalized, but did not anteriorize, the embryos, suggesting a role for Ogon/Sizzled in Bmp inhibition. Ogon/Sizzled did not inhibit a Wnt8-dependent transcription in the zebrafish embryo. ogon/sizzled was expressed on the ventral side from the late blastula through the gastrula stages. The ventral ogon/sizzled expression in the gastrula stage was reduced or absent in the swirl/bmp2b mutants but expanded in the chordino mutants. Misexpression of ogon/sizzled did not dorsalize the chordino mutants, suggesting that Ogon/Sizzled required Chordin protein for dorsalization and Bmp inhibition. These data indicate that Ogon/Sizzled functions as a negative regulator of Bmp signaling and reveal a novel role for a sFrp in dorsoventral patterning.

Amino Acid Sequence↗
PubMed2002

Molecular basis of the cell specificity of cytokine action.

The molecular cloning and biological analyses of cytokines have led us to a general understanding of their pleiotropism and redundancy. These features have been ascribed to the composition of cytokine receptor complexes, which include a signal-transducing receptor subunit that is used by all members of a cytokine family and a binding subunit that is specific for each cytokine. Even though a given cytokine uses the same receptor complex when binding to various cell types, the cytokine elicits quite specific and distinct biological responses in different types of cells. Even in the same type of cell, the responses to a given cytokine could vary depending on the location of the cell and the condition of its microenvironment. Important mediators for the main cytokine signal-transduction pathway are the Janus kinases (Jaks) and signal transducer and activator of transcription (STATs). Selective usage of members of the Jak and STAT families by a given cytokine receptor is partly responsible for the specificity of cytokine action. In addition to the Jak-STAT pathway, a cytokine receptor complex can simultaneously operate multiple signal-transduction pathways, which usually express contradictory properties. These contradictory signals from a single cytokine are orchestrated to evoke a unified biological response in the cell. Here we discuss the molecular mechanisms that regulate how the cell specificity of cytokine signals is regulated, especially focusing on the IL-6/gp130 system.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Gab1 and SHP-2 promote Ras/MAPK regulation of epidermal growth and differentiation.

In epidermis, Ras can influence proliferation and differentiation; however, regulators of epidermal Ras function are not fully characterized, and Ras effects on growth and differentiation are controversial. EGF induced Ras activation in epidermal cells along with phosphorylation of the multisubstrate docking protein Gab1 and its binding to SHP-2. Expression of mutant Gab1Y627F deficient in SHP-2 binding or dominant-negative SHP-2C459S reduced basal levels of active Ras and downstream MAPK proteins and initiated differentiation. Differentiation triggered by both Gab1Y627F and SHP-2C459S could be blocked by coexpression of active Ras, consistent with Gab1 and SHP-2 action upstream of Ras in this process. To study the role of Gab1 and SHP-2 in tissue, we generated human epidermis overexpressing active Gab1 and SHP-2. Both proteins stimulated proliferation. In contrast, Gab1Y627F and SHP-2C459S inhibited epidermal proliferation and enhanced differentiation. Consistent with a role for Gab1 and SHP-2 in sustaining epidermal Ras/MAPK activity, Gab1-/- murine epidermis displayed lower levels of active Ras and MAPK with postnatal Gab1-/- epidermis, demonstrating the hypoplasia and enhanced differentiation seen previously with transgenic epidermal Ras blockade. These data provide support for a Ras role in promoting epidermal proliferation and opposing differentiation and indicate that Gab1 and SHP-2 promote the undifferentiated epidermal cell state by facilitating Ras/MAPK signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2002

A point mutation of Tyr-759 in interleukin 6 family cytokine receptor subunit gp130 causes autoimmune arthritis.

We generated a mouse line in which the src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-2 binding site of gp130, tyrosine 759, was mutated to phenylalanine (gp130(F759/F759)). The gp130(F759/F759) mice developed rheumatoid arthritis (RA)-like joint disease. The disease was accompanied by autoantibody production and accumulated memory/activated T cells and myeloid cells. Before the disease onset, the T cells were hyperresponsive and thymic selection and peripheral clonal deletion were impaired. The inhibitory effect of IL-6 on Fas ligand expression during activation-induced cell death (AICD) was augmented in gp130(F759/F759) T cells in a manner dependent on the tyrosine residues of gp130 required for signal transducer and activator of transcription 3 activation. Finally, we showed that disease development was dependent on lymphocytes. These results provide evidence that a point mutation of a cytokine receptor has the potential to induce autoimmune disease.

Amino Acid Substitution↗
PubMed2002

STAT3 down-regulates the expression of cyclin D during liver development.

As the expression of cyclin D1 is induced during liver regeneration and also in hepatic tumor cells, cyclin D1 is likely to play an important role in the proliferation and transformation of hepatocytes. However, the role of cyclin D1 in liver development remains unknown. Here we show that the expression of D-type cyclins including cyclin D1, D2, and D3 is down-regulated along with liver development. In addition, oncostatin M (OSM), an interleukin-6 family cytokine, down-regulated the expression of cyclin D1 and D2 in a primary culture of fetal hepatocytes in which OSM induces hepatic differentiation. Ectopic expression of receptor mutants defective in the activation of either STAT3 or SHP-2/Ras indicated that the down-regulation of D1 and D2 cyclins by OSM was mediated by STAT3 but not by SHP-2/Ras. Consistently, expression of dominant negative STAT3 but not Ras relieved OSM-induced suppression of cyclin D expression. Activation of STAT3 in fetal hepatocytes of transgenic mice expressing the STAT3-estrogen receptor fusion protein by 4-hydroxytamoxifen resulted in the suppression of cyclin D1 and D2 expression. These results indicate that STAT3 activation is necessary and sufficient for down-regulation of D1 and D2 cyclins in fetal hepatocytes. Furthermore, STAT3-C, a constitutively active form of STAT3, suppressed transcription of the cyclin D1 promoter in fetal hepatocytes, whereas it activated the transcription in hepatic tumor cells, huH7 and HepG2. Thus, STAT3-mediated down-regulation of cyclin D expression is rather specific to fetal hepatocytes that are undergoing maturation processes including a reduction of their proliferation potential.

Animals↗
PubMed2002

Adapter molecule Grb2-associated binder 1 is specifically expressed in marginal zone B cells and negatively regulates thymus-independent antigen-2 responses.

Grb2-associated binder 1 (Gab1) is a member of the Gab/daughter of sevenless family of adapter molecules involved in the signal transduction pathways of a variety of growth factors, cytokines, and Ag receptors. To know the role for Gab1 in hematopoiesis and immune responses in vivo, we analyzed radiation chimeras reconstituted with fetal liver (FL) cells of Gab1(-/-) mice, because Gab1(-/-) mice are lethal to embryos. Transfer of Gab1(-/-) FL cells of 14.5 days post-coitum rescued lethally irradiated mice, indicating that Gab1 is not essential for hematopoiesis. Although mature T and B cell subsets developed normally in the peripheral lymphoid organs, reduction of pre-B cells and increase of myeloid cells in the Gab1(-/-) FL chimeras suggested the regulatory roles for Gab1 in hematopoiesis. The chimera showed augmented IgM and IgG1 production to thymus-independent (TI)-2 Ag, although they showed normal responses for thymus-dependent and TI-1 Ags, indicating its negative role specific to TI-2 response. Gab1(-/-) splenic B cells stimulated with anti-delta-dextran plus IL-4 plus IL-5 showed augmented IgM and IgG1 production in vitro that was corrected by the retrovirus-mediated transfection of the wild-type Gab1 gene, clearly demonstrating the cell-autonomous, negative role of Gab1. Furthermore, we showed that the negative role of Gab1 required its Src homology 2-containing tyrosine phosphatase-2 binding sites. Cell fractionation analysis revealed that nonfollicular B cells were responsible for the augmented Ab production in vitro. Consistent with these results, the Gab1 gene was expressed in marginal zone B cells but not follicular B cells. These results indicated that Gab1 is a unique negative regulator specific for TI-2 responses.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Requirement of Gab2 for mast cell development and KitL/c-Kit signaling.

Mast cells are thought to participate in a variety of immune responses, such as parasite resistance and the allergic reaction. Mast cell development depends on stem cell factor (Kit ligand) and its receptor, c-Kit. Gab2 is an adaptor molecule containing a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab2 is phosphorylated on tyrosine after stimulation with cytokines and growth factors, including KitL. Gab2-deficient mice were created to define the physiological requirement for Gab2 in KitL/c-Kit signaling and mast cell development. In Gab2-deficient mice, the number of mast cells was reduced markedly in the stomach and less severely in the skin. Bone marrow-derived mast cells (BMMCs) from the Gab2-deficient mice grew poorly in response to KitL. KitL-induced ERK MAP kinase and Akt activation were impaired in Gab2-deficient BMMCs. These data indicate that Gab2 is required for mast cell development and KitL/c-Kit signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Crystallographic studies on human BST-1/CD157 with ADP-ribosyl cyclase and NAD glycohydrolase activities.

cADPR is the novel second messenger that elicits calcium release from intracellular calcium stores and works independently of IP(3). In mammals, the ADP-ribosyl cyclase function is found in two membrane proteins, CD38 and BST-1/CD157. These enzymes, exposed extracellularly, bear cADPR hydrolase and NAD glycohydrolase activities. In spite of its functional importance, the structural basis of these enzymatic reactions remains elusive. We determined the crystal structures of the extracellular region of human BST-1 at atomic resolution in the free form and in complexes with five substrate analogues: nicotinamide, NMN, ATPgammaS, ethenoNADP, and ethenoNAD. The three-dimensional structural views of the reaction centre with these ligands revealed the mode of substrate binding and the catalytic mechanism of the multifunctional enzymatic reactions. In each catalytic cleft of the dimeric enzyme, substrates are recognized predominantly through van der Waals interactions with two tryptophan residues, and thereby the N-glycosidic bond of NAD is correctly exposed near a catalytic glutamate residue. Its carboxyl side-chain stabilizes the catalytic intermediate of the S(N)-1 type reaction. This conformation of the catalytic cleft also implies the mechanism of cyclization between the adenine base and the ribose. The three key residues are invariant among the sequences of BST-1, CD38, and Aplysia cyclase, and hence this substrate recognition mode and catalytic scheme appear to be common in the cyclase family.

ADP-ribosyl Cyclase↗
PubMed2002

A novel repressor-type homeobox gene, ved, is involved in dharma/bozozok-mediated dorsal organizer formation in zebrafish.

Dharma/Bozozok (Dha/Boz) is a homeodomain protein containing an Engrailed homology (Eh) 1 repressor motif. It is important in zebrafish dorsal organizer formation. Dha/Boz interacted with a co-repressor Groucho through the Eh1 motif. Expression of a Dha/Boz fused to the transcriptional activator VP16 repressed dorsal axis formation and the expression of organizer genes but led to the dorsal expansion of expression of the homeobox gene vox/vega1, indicating that Dha/Boz functions as a transcriptional repressor for dorsal axis formation. We also isolated a novel homeobox gene, ved, whose expression was negatively regulated by dha/boz. ved's sequence and expression profile were similar to those of vox/vega1 and vent/vega2. Like Vox/Vega1 and Vent/Vega2, Ved acted as a transcriptional repressor. The combined inhibition of ved, vox/vega1, and vent/vega2, by antisense morpholino injection, strongly dorsalized the embryos and elicited ventral expansion of organizer gene expression, compared with the effect of inhibiting each of these genes alone. These results suggest that ved is a target for the repressor Dha/Boz. Ved functions redundantly with vox/vega1 and vent/vega2 to restrict the organizer domain.

Amino Acid Motifs↗
PubMed2002

Stat3 Controls Cell Movements during Zebrafish Gastrulation.

Vertebrate axis formation requires both the correct specification of cell fates and the coordination of gastrulation movements. We report that the zebrafish signal transducer and activator of transcription 3 (Stat3) is activated on the dorsal side by the maternal Wnt/beta-catenin pathway. Zebrafish embryos lacking Stat3 activity display abnormal cell movements during gastrulation, resulting in a mispositioned head and a shortened anterior-posterior axis, but show no defects in early cell fate specification. Time course analysis, cell tracing, and transplantation experiments revealed that Stat3 activity is required cell autonomously for the anterior migration of dorsal mesendodermal cells and non-cell autonomously for the convergence of neighboring paraxial cells. These results reveal a role for Stat3 in controlling cell movements during gastrulation.

Animals↗