Dysbindin-1, WAVE2 and Abi-1 form a complex that regulates dendritic spine formation.

Genetic variations in dysbindin-1 (dystrobrevin-binding protein-1) are one of the most commonly reported variations associated with schizophrenia. As schizophrenia could be regarded as a neurodevelopmental disorder resulting from abnormalities of synaptic connectivity, we attempted to clarify the function of dysbindin-1 in neuronal development. We examined the developmental change of dysbindin-1 in rat brain by western blotting and found that a 50 kDa isoform is highly expressed during the embryonic stage, whereas a 40 kDa one is detected at postnatal day 11 and increased thereafter. Immunofluorescent analyses revealed that dysbindin-1 is enriched at the spine-like structure of primary cultured rat hippocampal neurons. We identified WAVE2, but not N-WASP, as a binding partner for dysbindin-1. We also found that Abi-1, a binding molecule for WAVE2 involved in spine morphogenesis, interacts with dysbindin-1. Although dysbindin-1, WAVE2 and Abi-1 form a ternary complex, dysbindin-1 promoted the binding of WAVE2 to Abi-1. RNA interference-mediated knockdown of dysbindin-1 led to the generation of abnormally elongated immature dendritic protrusions. The present results indicate possible functions of dysbindin-1 at the postsynapse in the regulation of dendritic spine morphogenesis through the interaction with WAVE2 and Abi-1.

Essential roles of ERK-mediated phosphorylation of vinexin in cell spreading, migration and anchorage-independent growth.

Vinexin is an adaptor protein supposed to play pivotal roles in various cellular events such as cell adhesion, cytoskeletal organization, signaling and gene expression. Despite the possible importance, physiological functions and regulatory mechanisms of vinexin are largely unknown. In addition, although vinexin was reported to be phosphorylated by extracellular signal-regulated kinase (ERK), physiological significance of the phosphorylation remains to be elucidated. Here we carried out characterization of endogenous vinexin and found that it was enriched at the leading edge of migrating cells and focal adhesions of spread cells. In the analyses using ERK-phosphorylated vinexin-specific antibody, the phosphorylation signal was also detected at the leading edges of migrating cells and at cell periphery of spreading cells, whereas only faint signal was observed at focal adhesions of well-spread cells. We then established LNCaP cell lines stably expressing GFP-fused vinexinbeta or two mutants at Ser189 that mimic the ERK-phosphorylated or -unphosphorylated vinexin beta. Based on the analyses using the lines, the phosphorylation was likely to inhibit the cell spreading and migration. On the other hand, anchorage-independent cell growth was inhibited by unphosphorylated vinexinbeta. Taken together, ERK-mediated phosphorylation of vinexinbeta is strongly suggested to occur in a spatio-temporally regulated manner and play important roles in cell spreading, migration and anchorage-independent growth.

Menopausal status influences the relationship between serum ghrelin levels and fat mass in healthy women.

OBJECTIVE: To examine the relationships of fasting serum ghrelin levels to bone mass index (BMI) and body fat mass, focusing on the effects of menopausal status and changes in fat distribution in women after menopause. DESIGN: An observational study. PATIENTS: Fifty-nine pre-menopausal and 31 post-menopausal healthy Japanese women volunteers were enrolled in the present study. MEASUREMENTS: Total and regional body fat mass weight was measured by dual energy x-ray absorptiometry. Serum ghrelin was measured. Relationships of serum ghrelin levels to weight, BMI, total body and regional fat mass weight were separately examined in post-menopausal and pre-menopausal women. RESULTS: Serum ghrelin levels were significantly inversely correlated with weight (r = -0.377, p < 0.05, Pearson's correlation test), right arm fat mass (r = -0.408, p < 0.05), left arm fat mass (r = -0.386, p < 0.05), trunk fat mass (r = -0.361, p < 0.05) and total body fat mass (r = -0.383, p < 0.05) in the post-menopausal women but not in pre-menopausal women. CONCLUSIONS: Menopausal status may influence the relationship between serum ghrelin levels and fat mass in healthy women.

The leptin receptor in human osteoblasts and the direct effect of leptin on bone metabolism.

It is important to elucidate whether the leptin receptor, especially the long signal-transducing form of the leptin receptor (OB-Rb) is expressed in human osteoblasts. We detected the expression of human OB-Rb in cultured commercially available human osteoblasts (NHOst cells) using real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). After confirming the expression of OB-Rb, we investigated the effect of leptin on NHOst cells. Leptin enhanced cell proliferation of the cells shown by the MTT assay. Furthermore, leptin changed the copy numbers of Bax and Bcl-2 mRNAs in the cultured cells as shown by real-time quantitative RT-PCR, although the effect was not consistent. Leptin did not change the production of osteocalcin and osteopontin by the cells. Leptin did not change the expression of OB-Rb mRNA in the cells. In conclusion, OB-Rb mRNA is expressed in cultured commercially available human osteoblasts. Leptin may have some effects on bone metabolism by directly modulating cell proliferation and apoptosis of osteoblasts in humans.

IgE-dependent enhancement of Th2 cell-mediated allergic inflammation in the airways.

T helper 2 (Th2) cell-derived cytokines, including interleukin (IL)-4, IL-5 and IL-13, play important roles in causing allergic airway inflammation. In contrast to Th2 cells, however, the role of IgE and mast cells in inducing allergic airway inflammation is not understood fully. In the present study, we addressed this point using transgenic mice expressing trinitrophenyl (TNP)-specific IgE (TNP-IgE mice), which enable us to investigate the role of IgE without the influence of antigen-specific T cell activation and other immunoglobulins. When the corresponding antigen, TNP-BSA, was administered intranasally to TNP-IgE mice, a large number of CD4+ T cells were recruited into the airways. In contrast, TNP-BSA administration did not induce eosinophil recruitment into the airways or airway hyperreactivity. Furthermore, when ovalbumin (OVA)-specific Th2 cells were transferred to TNP-IgE mice and the mice were challenged with inhaled OVA, TNP-BSA administration increased OVA-specific T cell recruitment and then enhanced Th2 cell-mediated eosinophil recruitment into the airways. These results indicate that IgE-induced mast cell activation principally induces CD4+ T cell recruitment into the airways and thus plays an important role in enhancing Th2 cell-mediated eosinophilic airway inflammation by recruiting Th2 cells into the site of allergic inflammation.

Hsp27 suppresses the formation of inclusion bodies induced by expression of R120G alpha B-crystallin, a cause of desmin-related myopathy.

The R120G mutation in the small heat shock protein (sHSP) alpha B-crystallin has been identified in a family suffering from desmin-related myopathy. In this study, we characterized the features of transiently expressed R120G alpha B-crystallin in mammalian cells. In addition, we examined interactions of this mutant alpha B-crystallin with Hsp27, another representative sHSP. In HeLa cells, transiently expressed R120G alpha B-crystallin was mainly fractionated in the insoluble fraction, although wild-type alpha B-crystallin was predominantly found in the soluble fraction. In immunofluorescence studies, we found 15-25% of R120G alpha B-crystallin-expressing cells to contain multiple cytosolic inclusion bodies, in which Hsp27 was also localized. When R120G alpha B-crystallin and Hsp27 were transiently co-expressed in HeLa cells, the amount of R120G alpha B-crystallin in the soluble fraction was greater than with expression of R120G alpha B-crystallin alone. Moreover, co-expression resulted in reduced formation of inclusion bodies, suggesting that Hsp27 acts as a molecular chaperone for R120G alpha B-crystallin.

Activation of pulmonary T cells in corticosteroid-resistant and -sensitive interstitial pneumonitis in dermatomyositis/polymyositis.

To study the activation states and cytokine profiles of pulmonary T cells in corticosteroid-resistant and corticosteroid-sensitive interstitial pneumonitis (IP) in dermatomyositis (DM)/polymyositis (PM), we examined the activation markers and cytokine profiles of T cells in bronchoalveolar lavage fluids (BALF) from patients with IP in DM/PM before prednisolone therapy and then compared the activation states of T cells according to the therapeutic response of IP to prednisolone therapy. CD25+ CD4+ T cells in BALF were significantly increased in both corticosteroid-resistant and corticosteroid-sensitive IP in DM/PM as compared with those in controls without IP. Furthermore, CD25+ CD4+ T cells in BALF were significantly more increased in corticosteroid-resistant IP than those in cortico teroid- sensitive IP. Moreover, CD25+ CD8+ T cells in BALF were significantly increased only in corticosteroid-resistant IP, but not in corticosteroid-sensitive IP or controls without IP. IFN-gamma mRNA was detected in BALF T cells in corticosteroid-resistant and corticosteroid-sensitive IP but not in controls without IP, whereas IL-4 mRNA was virtually undetected in BALF T cells in both the IP groups. However, there were no significant differences in CD4/CD8 ratio of BALF T cells, HLA-DR+ BALF T cells or CD25+ and HLA-DR+ peripheral blood T cells between the two IP groups. These results indicate that activated Th1-type pulmonary T cells play an important role in the development of corticosteroid- resistant IP in DM/PM and that the increase in CD25+ CD8+ T cells in BALF is a useful indicator for corticosteroid-resistant IP in DM/PM and hence may be an indicator for early use of cyclosporin.

Increase of dendritic cells of type 2 (DC2) by altered response to IL-4 in atopic patients.

BACKGROUND: Recent studies have shown that there are 2 dendritic cell subpopulations, DC1 and DC2, which induce T(H)1 and T(H)2 cell differentiation in vitro, respectively. OBJECTIVE: The purpose of this study was to determine whether there exists a deviation of DC1 and DC2 subsets and to investigate their functional abnormalities in T(H)2 cell-mediated atopic diseases. METHODS: We analyzed the frequencies of DC1 (CD11c(+)CD123(-)) and DC2 (CD11c(-)CD123(+)) cells in peripheral blood of atopic patients; we also studied the responses of DC2 cells from atopic patients to IL-3 and IL-4 for their survival. RESULTS: DC2 cells but not DC1 cells were significantly increased in peripheral blood of atopic patients in comparison with that of healthy subjects. DC2 cell numbers were positively correlated with serum IgE levels and blood eosinophil counts, the increase of which reflects T(H)2-type immune response in atopic diseases. IL-4 inhibited IL-3-induced survival of DC2 cells from healthy controls, but IL-4 failed to suppress the IL-3-induced survival of DC2 cells from atopic patients. Furthermore, IL-4 alone enhanced the survival of DC2 cells from atopic patients but not from healthy controls. However, no significant differences were found in the expression levels of activation/maturation markers on DC2 cells between atopic patients and healthy controls. CONCLUSION: These results indicate that DC2 cells are preferentially increased in atopic patients in correlation with the state of atopic allergy and that DC2 cells in atopic patients, unlike those in healthy subjects, exhibit altered responses to IL-4 for survival, suggesting that DC2 cells in atopic patients might contribute to the enhanced T(H)2 cell differentiation in atopic diseases.

Essential role of Stat5 for IL-5-dependent IgH switch recombination in mouse B cells.

IL-5 stimulation of CD38-activated murine splenic B cells induces mu-gamma1 CSR at the DNA level leading to a high level of IgG1 production. Further addition of IL-4 in the system enhances IL-5-dependent mu-gamma1 CSR. Although some of the postreceptor signaling events initiated by IL-5 in activated B cells have been characterized, the involvement of Stat in IL-5 signaling has not been thoroughly evaluated. In this study, we examined the activation of Stat5 and activation-induced cytidine deaminase (AID) in CD38-activated murine splenic B cells by IL-5. The role of Stat5a and Stat5b in IL-5-induced mu-gamma1 CSR and also IgG1 and IgM production was documented, as IL-5 does not act on CD38-stimulated splenic B cells from Stat5a(-/-) and Stat5b(-/-) mice. Expression levels of CD38-induced germline gamma1 transcripts and AID in Stat5a(-/-) and Stat5b(-/-) B cells upon IL-5 stimulation were comparable to those of wild-type B cells. The impaired mu-gamma1 CSR by Stat5b(-/-) B cells, but not by Stat5a(-/-) B cells, was rescued in part by IL-4, as the addition of IL-4 to the culture of CD38- and IL-5-stimulated B cells induced mu-gamma1 CSR leading to IgG1 production. Analysis of cell division cycle number of wild-type B cells revealed that mu-gamma1 CSR was observed after five or six cell divisions. Stat5a(-/-) and Stat5b(-/-) B cells showed similar cell division cycles, but they did not undergo mu-gamma1 CSR. Our data support the notion that both Stat5a and Stat5b are essential for IL-5-dependent mu;-gamma1 CSR and Ig secretion; however, their major target may not be AID. Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function.

Protein kinase inhibitors can suppress stress-induced dissociation of Hsp27.

We previously showed that the aggregated form of Hsp27 in cultured cells becomes dissociated as a result of phosphorylation with various types of stress. In order to clarify the signal transduction cascade involved, the effects of various inhibitors of protein kinases and dithiothreitol on the dissociation of Hsp27 were here examined by means of an immunoassay after fractionation of cell extracts by sucrose density gradient centrifugation. The dissociation of Hsp27 induced by exposure of U251 MG human glioma cells to metals (NaAsO2 and CdCl2), hypertonic stress (sorbitol and NaCI), or anisomycin, an activator of p38 mitogen-activated protein (MAP) kinase, was completely suppressed by the presence of SB 203580 or PD 169316, inhibitors of p38 MAP kinase, but not by PD 98059 and Uo 126, inhibitors of MAP kinase kinase (MEK), nor by staurosporine, Go 6983, and bisindolylmaleimide I, inhibitors of protein kinase C. Phorbol ester (PMA)-induced dissociation of Hsp27 was completely suppressed by staurosporine, Go 6983, or bisindolylmaleimide I and partially suppressed by SB 203580, or PD 169316 but not by PD 98059 or Uo 126, indicating mediation by 2 cascades. The presence of 1 mM dithiothreitol in the culture medium during exposure to chemicals suppressed the dissociation of Hsp27 induced by arsenite and CdCl2 but not by other chemicals. These results suggest that the phosphorylation of Hsp27 is catalyzed by 2 protein kinases, p38 MAP kinase-activated protein (MAPKAP) kinase-2/3 and protein kinase C. In addition, metal-induced signals are sensitive to reducing power.

Role of CD4(+) CD25(+) regulatory T cells in T helper 2 cell-mediated allergic inflammation in the airways.

It has recently been shown that CD4(+) CD25(+) T cells are immunoregulatory T cells that prevent CD4(+) T cell-mediated organ-specific autoimmune diseases. To determine whether CD4(+) CD25(+) T cells downregulate Th2 cell-mediated allergic inflammation in the airways, we studied antigen-induced eosinophil recruitment in the airways in BALB/c Rag-2(-)(/-) mice transferred with CD4(+) CD25(+) T cell-depleted or unfractionated T cells from ovalbumin-specific TCR transgenic mice. Antigen-induced eosinophil recruitment into the airways was significantly decreased in the mice transferred with CD4(+) CD25(+) T cell-depleted splenocytes as compared with those transferred with unfractionated splenocytes. On the other hand, the depletion of CD4(+) CD25(+) T cells increased antigen-induced neutrophil and T cell recruitment in the airways of the mice. The depletion of CD4(+) CD25(+) T cells also decreased antigen-induced IL-4 and IL-5 production in the airways of the mice. Finally, the depletion of CD4(+) CD25(+) T cells prevented antigen-induced Th2 cell differentiation in vitro but increased the differentiation of Th1 cells. These results indicate that CD4(+) CD25(+) T cells modulate the Th1 and Th2 cell balance toward Th2 cells and thus upregulate Th2 cell-mediated allergic inflammation in the airways.

Regulation of the levels of small heat-shock proteins during differentiation of C2C12 cells.

Levels of the small heat-shock proteins (sHSPs) HSP27 and alphaB-crystallin during differentiation of mouse C2C12 cells were determined using specific immunoassays. Increases of these proteins were about 3-fold and 10-fold, respectively. Under the same conditions, however, the level of HSP70 in C2C12 cells barely increased, indicating selective accumulation of HSP27 and alphaB-crystallin with differentiation. While expression of mRNA for alphaB-crystallin was also markedly increased and that for HSP27 was but to a lesser extent, mRNA for HSP70 could barely be detected during differentiation. Activation of the heat-shock factor was not observed, in contrast to the case with heat-stressed undifferentiated cells. Various inhibitors of protein kinases affected the differentiation and the associated increase of sHSPs. Rapamycin, an inhibitor of p70 S6 kinase, completely inhibited the differentiation and suppressed the accumulation of HSP27 and alphaB-crystallin. SB203580, an inhibitor of p38 MAP kinase, also inhibited differentiation, but the accumulation of alphaB-crystallin was rather enhanced. PD98059, an inhibitor of MAP kinase kinase, significantly increased expression of a differentiation marker for muscle cells, creatine kinase M isozyme, as well as accumulation of alphaB-crystallin. These results suggest that accumulation of sHSPs during differentiation of C2C12 cells is regulated in a complex manner.

T cell vaccination eliminates antigen-specific T cells and prevents antigen-induced eosinophil recruitment into the tissue.

Asthma is characterized by airway inflammation with prominent eosinophil infiltrates. In a murine model of asthma, antigen-induced eosinophil recruitment into the airways of sensitized mice is mediated by CD4+ T cells and their cytokines, especially IL-5. In the present study, using ovalbumin-specific T cell receptor transgenic mice, we found that T cell vaccination, which was the administration of preactivated and attenuated antigen-specific T cells by the intraperitoneal route, prevented antigen-induced eosinophil recruitment into the airways. This effect was antigen-specific because ovalbumin-specific T cell vaccination did not affect BSA-induced eosinophil recruitment into the airways. We also found that antigen-specific IgE production as well as antigen-induced proliferation and cytokine production of splenocytes were diminished by T cell vaccination. Moreover, flow-cytometric analyses revealed that T cell vaccination eliminated antigen-specific T cells in the periphery. Together, these results indicate that T cell vaccination prevents antigen-induced eosinophil recruitment into the airways presumably by eliminating antigen-specific T cells.

Stat5a regulates T helper cell differentiation by several distinct mechanisms.

We have previously shown that CD4(+) T cell-mediated allergic inflammation is diminished in signal transducer and activator of transcription (Stat)5a-deficient (Stat5a(-/-)) mice. To determine whether Stat5a regulates T helper cell differentiation, we studied T helper (Th)1 and Th2 cell differentiation of Stat5a(-/-)CD4(+) T cells at single-cell levels. First, Th2 cell differentiation from antigen-stimulated splenocytes was significantly decreased in Stat5a(-/-) mice as compared with that in wild-type mice. Further, Th2 cell differentiation was also impaired in Stat5a(-/-) mice even when purified CD4(+) T cells were stimulated with anti-CD3 plus anti-CD28 antibodies in the presence of interleukin-4. Moreover, the retrovirus-mediated gene expression of Stat5a in Stat5a(-/-)CD4(+) T cells restored the Th2 cell differentiation at the similar levels to that in wild-type CD4(+) T cells. In addition, interleukin-4 normally phosphorylated Stat6 in CD4(+) T cells from Stat5a(-/-) mice. Second, the development of CD4(+)CD25(+) immunoregulatory T cells was impaired in Stat5a(-/-) mice, as indicated by a significant decrease in the number of CD4(+)CD25(+) T cells in Stat5a(-/-) mice. Furthermore, the depletion of CD4(+)CD25(+) T cells from wild-type splenocytes significantly decreased Th2 cell differentiation but increased Th1 cell differentiation, whereas the depletion of CD4(+)CD25(+) T cells from Stat5a(-/-) splenocytes had no significant effect on the Th1 and Th2 cell differentiation. Together, these results indicate that the intrinsic expression of Stat5a in CD4(+) T cells is required for Th2 cell differentiation and that Stat5a is involved in the development of CD4(+)CD25(+) immunoregulatory T cells that modulate T helper cell differentiation toward Th2 cells.

Selective reduction and recovery of invariant Valpha24JalphaQ T cell receptor T cells in correlation with disease activity in patients with systemic lupus erythematosus.

OBJECTIVE: To study the regulatory role of CD4-CD8- double-negative (DN) invariant T cell receptor (TCR) Valpha24JalphaQ T cells, a human counterpart of murine NK 1 + T cells, in the autoimmune process of systemic lupus erythematosus (SLE). METHODS: We carried out a 2 step frequency analysis of DN Valpha24JalphaQ T cells in patients with SLE before and after prednisolone therapy; the frequency of DN Valpha24+ T cells was determined by 3 color FACS analysis and subsequently the frequency of Valpha24JalphaQ rearrangement among DN Valpha24+ T cells was determined by sequencing. RESULTS: DN Valpha24+ T cells were significantly increased in patients with active SLE compared to healthy subjects. In healthy subjects, invariant Valpha24JalphaQ TCR dominated in DN Valpha24+ T cells at a high frequency (93-100%). However, the invariant Valpha24JalphaQ TCR was not detected in DN Valpha24+ T cells from patients with active SLE, and instead 2 to 9 Jalpha genes other than the invariant JalphaQ were oligoclonally expanded in the patients. In inactive SLE induced by prednisolone therapy, the invariant Valpha24JalphaQ TCR could be detected in DN Valpha24+ T cells from all the patients and dominated in most of the patients. Further, oligoclonally expanded Valpha24+ clones other than the invariant JalphaQ gene in active disease states were significantly decreased by prednisolone therapy. CONCLUSION: The selective reduction of DN invariant Valpha24JalphaQ T cells is related to the disease progression of SLE, while DN TCR Valpha24 T cells other than Valpha24JalphaQ T cells constitute autoaggressive T cells in SLE.

Interaction between Smad anchor for receptor activation and Smad3 is not essential for TGF-beta/Smad3-mediated signaling.

Regulation of subcellular localization of Smad proteins is supposed to be critical for the effective initiation and maintenance of TGF-beta signaling. Recently, Smad anchor for receptor activation (SARA) has been identified as a Smad2 binding protein. SARA regulates the subcellular localization of Smad2 and is required for TGF-beta/Smad2-mediated signaling. In this study, we determined whether the interaction between SARA and Smad3 is essential for TGF-beta/Smad3-mediated signaling. We found that a mutant Smad3 (Smad3NS) that lacked the binding to SARA was phosphorylated by TGF-beta type I receptor at the similar level to that in wild-type Smad3 (Smad3WT). Smad3NS also formed complexes with Smad4 and translocalized into the nucleus. Moreover, Smad3NS and Smad3WT equally enhanced TGF-beta-induced transcription. Therefore, these findings indicate that, in contrast to SARA/Smad2 interaction, SARA/Smad3 interaction is not essential for TGF-beta/Smad3-mediated signaling.

Ser-59 is the major phosphorylation site in alphaB-crystallin accumulated in the brains of patients with Alexander's disease.

The phosphorylation state of alphaB-crystallin accumulated in the brains of two patients with Alexander's disease (one infantile and one juvenile type) was determined by means of SDS-PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against alphaB-crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25-28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of alphaB-crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser-59 in both cases. A significant level of phosphorylation at Ser-45 but not Ser-19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. alphaB-crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. AlphaB-crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser-59 in all cases. Immunohistochemically, alphaB-crystallin was stained in Rosenthal fibers in brains of patients with Alexander's disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser-59. These results indicate that the major phosphorylation site in alphaB-crystallin in brains of patients with Alexander's disease or Alzheimer's disease as well as in aged controls is Ser-59.