Shousaku Itoh

Dissection of Signaling Cascades through gp130 In Vivo: Reciprocal Roles for STAT3- and SHP2-Mediated Signals in Immune Responses

We generated a series of knockin mouse lines, in which the cytokine receptor gp130-dependent STAT3 and/or SHP2 signals were disrupted, by replacing the mouse gp130 gene with human gp130 mutant cDNAs. The SHP2 signal-deficient mice (gp130F759/F759 were born normal but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXQ/FXXQ) died perinatally, like the gp130-deficient mice (gp130D/D). The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation, indicating a negative regulatory role for SHP2. Th1-type cytokine production and IgG2a and IgG2b production were increased in the gp130F759/F759 mice, while they were decreased in the gp130FXXQ/FXXQ immune system. These results indicate that the balance of positive and negative signals generated through gp130 regulates the immune responses.

Interleukin-6 Directly Inhibits Osteoclast Differentiation by Suppressing Receptor Activator of NF-κB Signaling Pathways

Interleukin-6 (IL-6) is a multifunctional cytokine produced by various cells to regulate hematopoiesis, inflammation, immune responses, and bone homeostasis. IL-6 is also known to modulate the differentiation of osteoblasts and osteoclasts. IL-6 is believed to play a positive regulatory role in osteoclast differentiation by inducing the expression of receptor activator of NF-κB ligand (RANKL) on the surface of osteoblasts: RANKL then interacts with RANK expressed on osteoclast progenitors, inducing osteoclast differentiation via the RANK signaling pathway, which involves NF-κB, JNK, and p38. In this report, we demonstrate that IL-6 can also directly act on osteoclast progenitors to suppress their differentiation via an inhibition of RANK signaling pathways. IL-6 specifically suppressed RANK-mediated IκB degradation and JNK activation. Microarray analysis revealed that costimulation with IL-6 and RANKL up-regulates the transcription of MKP1 and MKP7, which encode enzymes that dephosphorylate JNK, and down-regulates the transcription of Senp2 and Cul4A, which are related to the ubiquitin pathway. Thus, IL-6 directly acts on osteoclast progenitors and suppresses their differentiation by regulating the transcription of specific genes related to MAPK phosphatases and the ubiquitin pathway.

Tissue-Specific Autoregulation of the stat3 Gene and Its Role in Interleukin-6-Induced Survival Signals in T Cells

ABSTRACT Signal transducer and activator of transcription 3 (STAT3) mediates signals of various growth factors and cytokines, including interleukin-6 (IL-6). In certain IL-6-responsive cell lines, thestat3 gene is autoregulated by STAT3 through a composite IL-6 response element in its promoter that contains a STAT3-binding element (SBE) and a cyclic AMP-responsive element. To reveal the nature and roles of the stat3 autoregulation in vivo, we generated mice that harbor a mutation in the SBE (stat3 mSBE ). The intact SBE was crucial for IL-6-induced stat3 gene activation in the spleen, especially in the red pulp region, the kidney, and both mature and immature T lymphocytes. The SBE was not required, however, for IL-6-induced stat3 gene activation in hepatocytes. T lymphocytes from the stat3 mSBE/mSBE mice were more susceptible to apoptosis despite the presence of IL-6 than those from wild-type mice. Consistent with this, IL-6-dependent activation of the Pim-1 and junB genes, direct target genes for STAT3, was attenuated in T lymphocytes of thestat3 mSBE/mSBE mice. Thus, the tissue-specific autoregulation of the stat3 gene operates in vivo and plays a role in IL-6-induced antiapoptotic signaling in T cells.

An explanation of the mineralization mechanism in osteoblasts induced by calcium hydroxide.

Calcium hydroxide (Ca(OH)(2)) has been broadly used in endodontics, including apexification to obtain apical closure by mineralization. However, the detailed mechanism of mineralization induced by Ca(OH)(2) is still unclear. This study focuses on the function of calcium and hydroxyl ions which dissociate from Ca(OH)(2) during the mineralization process. Though primary osteoblasts cultured in the medium without or with 0.025mgml(-1) Ca(OH)(2) did not show mineralization, they did exhibit mineralization when they were cultured with a higher concentration of Ca(OH)(2) (0.25mgml(-1)). Mineralization induced in the presence of 0.25mgml(-1) Ca(OH)(2) was greater at pH 7.4 than at pH 8.5. The high mineralization activity observed under neutral conditions was caused by the prolonged activation of p38 and JNK. Hydroxyl ions did not have any effect on the mineralization. The results demonstrate that calcium ions dissociated from Ca(OH)(2) are critical for inducing the mineralization of osteoblasts.

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-δ-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.

Essential Role for the gtfA Gene Encoding a Putative Glycosyltransferase in the Adherence of Porphyromonas gingivalis

ABSTRACT Porphyromonas gingivalis, an oral bacterium, might play a role in the pathogenesis or progression of adult periodontitis. In this study, we isolated from P. gingivalis a putative glycosyltransferase gene, designated gtfA, which had a consensus domain for glycosyltransferase in its N terminus. GtfA consisted of 248 amino acids and its predicted molecular mass was 28 kDa; however, as the molecular mass of endogenous GtfA protein was around 40 kDa, this suggested that GtfA had undergone some posttranslational modifications. To reveal the role of the gtfA gene in P. gingivalis, we established gtfA-deficient strains by allelic replacement. Morphologically, gtfA-deficient P. gingivalis lacked mature fimbriae. gtfA-deficient P. gingivalis also showed a very low ability for autoaggregation, and its ability to attach to epithelial cells was severely impaired. Thus, the results indicate that the gtfA gene is required for P. gingivalis autoaggregation as well as attachment to epithelial cells. These results suggest that GtfA might have an important role in the pathogenicity of P. gingivalis by regulating adhesion.

A novel purification method for multipotential skeletal stem cells

At least some cells within bone marrow stromal populations are multipotential (i.e., differentiate in vitro into osteoblasts, chondrocytes, and adipocytes) and thus designated skeletal stem cells (SSCs) or mesenchymal stem cells (MSCs) amongst other names. Recently, a subpopulation of stromal cells, notably osteoblasts or their progenitors, has been identified as a definitive regulatory component of the hematopoietic stem cell (HSC) niche. Thus, the development of methods for purifying not only SSCs but cells comprising the HSC niche is of interest. Here, we report a method for purifying a novel bone marrow‐derived population with a high frequency of osteoprogenitors and high expression levels of osteoblast differentiation markers (highly purified osteoprogenitors (HipOPs)) as well as markers of the bone niche for HSCs. In vivo transplantation experiments demonstrated that donor HipOPs differentiated into not only osteoblasts, osteocytes and cells around sinusoids but also hematopoietic cells. Thus, HipOPs represent a novel population for simultaneous reconstruction of bone and bone marrow microenvironments. J. Cell. Biochem. 108: 368–377, 2009.

First Mouse Model for Combined Osteogenesis Imperfecta and Ehlers-Danlos Syndrome

By using a genome‐wide N‐ethyl‐N‐nitrosourea (ENU)‐induced dominant mutagenesis screen in mice, a founder with low bone mineral density (BMD) was identified. Mapping and sequencing revealed a T to C transition in a splice donor of the collagen alpha1 type I (Col1a1) gene, resulting in the skipping of exon 9 and a predicted 18‐amino acid deletion within the N‐terminal region of the triple helical domain of Col1a1. Col1a1Jrt/+ mice were smaller in size, had lower BMD associated with decreased bone volume/tissue volume (BV/TV) and reduced trabecular number, and furthermore exhibited mechanically weak, brittle, fracture‐prone bones, a hallmark of osteogenesis imperfecta (OI). Several markers of osteoblast differentiation were upregulated in mutant bone, and histomorphometry showed that the proportion of trabecular bone surfaces covered by activated osteoblasts (Ob.S/BS and N.Ob/BS) was elevated, but bone surfaces undergoing resorption (Oc.S/BS and N.Oc/BS) were not. The number of bone marrow stromal osteoprogenitors (CFU‐ALP) was unaffected, but mineralization was decreased in cultures from young Col1a1Jrt/+ versus +/+ mice. Total collagen and type I collagen content of matrices deposited by Col1a1Jrt/+ dermal fibroblasts in culture was ∼40% and 30%, respectively, that of +/+ cells, suggesting that mutant collagen chains exerted a dominant negative effect on type I collagen biosynthesis. Mutant collagen fibrils were also markedly smaller in diameter than +/+ fibrils in bone, tendon, and extracellular matrices deposited by dermal fibroblasts in vitro. Col1a1Jrt/+ mice also exhibited traits associated with Ehlers‐Danlos syndrome (EDS): Their skin had reduced tensile properties, tail tendon appeared more frayed, and a third of the young adult mice had noticeable curvature of the spine. Col1a1Jrt/+ is the first reported model of combined OI/EDS and will be useful for exploring aspects of OI and EDS pathophysiology and treatment.

LIF/STAT3/SOCS3 Signaling Pathway in Murine Bone Marrow Stromal Cells Suppresses Osteoblast Differentiation

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that belongs to the interleukin‐6 family and is expressed by multiple tissue types. This study analyzed the effect of LIF on osteoblast differentiation using primary murine bone marrow stromal cells (BMSCs). Colony‐forming unit‐osteoblast formation by BMSCs was significantly suppressed by LIF treatment. To clarify the mechanism underlying the LIF suppressive effect on osteoblast differentiation, we analyzed the downstream signaling pathway of LIF. LIF/signal transducer and activator of transcription 3 (STAT3) signaling induces the expression of suppressor of cytokine signaling 3 (SOCS3). SOCS3 knockdown experiments have previously demonstrated that short‐hairpin SOCS3‐BMSCs reversed the LIF suppressive effect. Our results demonstrated that LIF suppresses osteoblast differentiation through the LIF/STAT3/SOCS3 signaling pathway. J. Cell. Biochem. 115: 1262–1268, 2014.

Gab2 plays distinct roles in bone homeostasis at different time points

Grb2-associated binder 2 (Gab2) is an adaptor molecule that can be tyrosine phosphorylated by various growth factors and cytokines. Gab2 is known to play a role in signaling pathways downstream of cytokines that regulate bone homeostasis, including M-CSF, RANKL, and IL-6. To clarify the role of Gab2 in bone homeostasis during distinct phases of skeletal development, we compared phenotypic changes in bone homeostasis in Gab2−/− mice at two different ages. Although Gab2−/− mice showed increased bone volume at both time points, the reasons underlying the increased bone volume differed. At 6 weeks, the increased bone volume was due to enhanced bone resorption and bone formation, indicating that Gab2 plays a negative regulatory role for both osteoclastogenesis and osteoblast differentiation. At 12 weeks, the increased bone volume resulted from reduced osteoclast differentiation, indicating that Gab2 plays a positive regulatory role for osteoclastogenesis. Thus, Gab2 plays opposite roles in osteoclastogenesis during the phases of skeletal development and maintenance.