Mariko Inoue

Impaired Insulin Signaling in Endothelial Cells Reduces Insulin-Induced Glucose Uptake by Skeletal Muscle

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.

The Spatial Patterning of Mouse Cone Opsin Expression Is Regulated by Bone Morphogenetic Protein Signaling through Downstream Effector COUP-TF Nuclear Receptors

Cone photopigments, known as opsins, are pivotal elements and the first detection module used in color vision. In mice, cone photoreceptors are distributed throughout the retina, and short-wavelength (S) and medium-wavelength (M) opsins have unique expression patterns in the retina with a gradient along the dorsoventral axis; however, the mechanisms regulating the spatial patterning of cone opsin expression have not been well documented. The purpose of this study was to define the mechanisms regulating the spatial patterning of cone opsin expression. By analyzing knock-outs for bone morphogenetic protein (BMP) signaling, we found an essential role for BMP in forming cone opsin expression patterns in the retina; however, BMP signaling is activated only transiently in the dorsal half of the retina during early retinal development. Thus, BMP is not likely to play a direct role in opsin gene expression, which starts at a later stage of retinal development. We identified the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) nuclear receptor as a link between BMP and opsin expression. BMP signaling is essential for the correct dorsoventral spatial expression of COUP-TFI and COUP-TFII. Through gain- and loss-of-function analyses, we found that both COUP-TFI and COUP-TFII are required to suppress S-opsin expression in the dorsal retina but that only COUP-TFI plays an essential role in suppressing M-opsin expression in the ventral retina. Based on these findings, we propose a new molecular cascade involving BMP and COUP-TFs that conveys dorsoventral information to direct the expression of cone opsins during retinal development.

TCF7L2 in mouse pancreatic beta cells plays a crucial role in glucose homeostasis by regulating beta cell mass

Aims/hypothesisCommon genetic variations of the transcription factor 7-like 2 gene (encoded by TCF7L2), one of the T cell factor/lymphoid enhancer-binding factor transcription factors for the converging wingless-type MMTV integration site family (Wnt)/β-catenin signalling pathway, are known to be associated with type 2 diabetes. Individuals with at-risk alleles of TCF7L2 exhibit impaired insulin secretion. Although previous studies using animal models have revealed the existence of a relationship between the Wnt/β-catenin signalling pathway and glucose homeostasis, it remains unclear whether TCF7L2 in the pancreatic beta cells might be causally involved in insulin secretion in vivo. In this study, we investigated the role of TCF7L2 expressed in the pancreatic beta cells in glucose homeostasis.MethodsThree independent groups of genetically engineered mice (DN mice) were generated, in which expression of the dominant-negative form of Tcf7l2 was driven under a rat insulin promoter. Phenotypes of both adult and newborn mice were evaluated. The levels of genes and proteins expressed in isolated islets were determined by reverse transcription-quantitative PCR and western blot analysis, respectively.ResultsAdult DN mice showed impaired glucose tolerance and decreased insulin secretion in both oral and intraperitoneal glucose tolerance tests. Marked reduction of the beta cell area and whole-pancreas insulin content was observed in both the adult and newborn DN mice. Islets from the DN mice showed decreased gene expressions of Ccnd1, Ccnd2, Irs1, Irs2, Ins1, Ins2 and Mafa, consistent with the deleterious effects of the dominant-negative form of Tcf7l2 on beta cell proliferation and insulin production.Conclusions/interpretationTCF7L2 expressed in the pancreatic beta cells plays a crucial role in glucose metabolism through regulation of the beta cell mass.

TGF-β3-expressing CD4+CD25−LAG3+ regulatory T cells control humoral immune responses

Autoantibodies induce various autoimmune diseases, including systemic lupus erythematosus (SLE). We previously described that CD4+CD25−LAG3+ regulatory T cells (LAG3+ Treg) are regulated by Egr2, a zinc-finger transcription factor required for the induction of T-cell anergy. We herein demonstrate that LAG3+ Treg produce high amounts of TGF-β3 in an Egr2- and Fas-dependent manner. LAG3+ Treg require TGF-β3 to suppress B-cell responses in a murine model of lupus. Moreover, TGF-β3- and LAG3+ Treg-mediated suppression requires PD-1 expression on B cells. We also show that TGF-β3-expressing human LAG3+ Treg suppress antibody production and that SLE patients exhibit decreased frequencies of LAG3+ Treg. These results clarify the mechanism of B-cell regulation and suggest therapeutic strategies.

CD73, a Novel Cell Surface Antigen That Characterizes Retinal Photoreceptor Precursor Cells

PURPOSE The authors sought to identify cell surface markers of photoreceptor and its precursor cells. METHODS The expression of surface CD antigens that label both temporally and spatially distinct populations of mouse retinal cells were examined. Of the antibodies that showed positive signals in retinal cells, CD73 was focused on for more detailed analyses. RESULTS Mouse retinal subpopulations that expressed CD73 first appeared around birth and subsequently increased dramatically in number, eventually representing more than 90% of the retinal cells in the adult. CD73(+) cells were postmitotic and mostly rhodopsin-negative at postnatal day 1. However, in the adult retina, most of these cells expressed rhodopsin but not s-opsin. In reaggregation cultures, CD73(+) cells differentiated into rhodopsin-positive cells more rapidly than CD73(-) cells, which supports the idea that CD73 is an early photoreceptor lineage marker. The effects of ectopic expression in retinal cells of Nrl and Crx, both of which are transcription factors known to be expressed in photoreceptor lineage, suggest that CD73 is genetically downstream of Crx in the rod cell differentiation lineage. Adult retina of the common marmoset monkey also showed correlation of the expression pattern of rhodopsin and CD73. CONCLUSIONS CD73 is a cell surface marker of cone/rod common precursors and mature rod cells in mice and is genetically localized between Nrl and Crx. The expression of CD73 was conserved in primate rod cells, and CD73 provides an useful tool to purify photoreceptor cells for transplantation aimed at the regeneration of photoreceptors.

Revisiting the regulatory roles of the TGF-β family of cytokines

TGF-β family members are multipotent cytokines that are involved in many cellular processes, including cell differentiation, organ development, wound healing and immune regulation. TGF-β has pleiotropic effects on adaptive immunity, especially in the regulation of CD4(+) T cell and B cell responses. Furthermore, identification of CD4(+) T cell subsets that produce TGF-β3 revealed unexpected roles of TGF-β3 in the control of adaptive immunity. In contrast to TGF-β1, which induces extensive fibrosis, TGF-β3 induces non-scarring wound healing and counteracts tissue fibrosis. Recent progress in the understanding of the activation mechanism of TGF-β may enable us to develop novel biologic therapies based on advanced protein engineering.

Degree of agreement between weight perception and body mass index of Japanese workers: MY Health Up Study.

Degree of Agreement between Weight Perception and Body Mass Index of Japanese Workers: MY Health Up Study: Mariko Inoue, et al. Department of Public Health, Graduate School of Medicine, University of Tokyo—The objective of this study was to assess the degree of agreement between body mass index (BMI) and weight perception of Japanese office workers between the ages of 20 and 65. We sent 43,064 self‐administered health‐related questionnaires to all employees of a financial firm in Japan in October 2004. The questionnaire asked the respondents for their height, weight, and their weight perception. The kappa coefficient was calculated to investigate the degree of agreement between the BMI calculated using the self‐reported data and weight perception for three categories. Of the questionnaires, 34,921 (81.1%) were returned and 33,514 responses (77.8%) were used for the analysis. Based on Japanese obesity criteria, 2,202 men (31.2%) and 5,145 women (19.5%) were obese; and 179 men (2.5%) and 2,769 women (10.5%) were underweight. The kappa coefficient was 0.374 for men and 0.297 for women. The kappa coefficients for different age groups—twenties, thirties, forties, fifties, and sixties (up to 65)—were respectively as follows: 0.315, 0.355, 0.374, 0.406, and 0.425 for men; and 0.194, 0.275, 0.285, 0.334, and 0.355 for women. In conclusion, the degree of agreement between BMI and weight perception differed by age and sex. The degree of agreement was smaller among women than among men and the degree of agreement among younger women was weaker than among older women.

Mycophenolate mofetil therapy for rapidly progressive interstitial lung disease in a patient with clinically amyopathic dermatomyositis

Rapidly progressive interstitial lung disease (ILD) associated with clinically amyopathic dermatomyositis (CADM) is often lethal despite corticosteroid therapy combined with cyclophosphamide and cyclosporine or tacrolimus. Several recent studies have described the treatment of ILD in patients with systemic sclerosis (SSc) or other connective tissue diseases with mycophenolate mofetil (MMF), a noncytotoxic immunosuppressant [1–9]. To our knowledge, this is the first report of MMF therapy as an alternative to intravenous pulses of cyclophosphamide (IVCY) for rapidly progressive ILD associated with CADM in combination with high-dose corticosteroids and oral tacrolimus. A 55-year-old female was admitted with a 1-week history of low-grade fever, muscle pain in the thigh, Gottron’s papules, and heliotrope rash. The white blood cell count was 3,230/ll with 75.9 % neutrophils, and C-reactive protein was 0.27 mg/dl (\ 0.3 mg/dl). The serum antinuclear antibody titre was not elevated and no anti-Jo-1 antibodies were detected. Anti-CADM 140 antibody was positive. She had no muscle weakness and her serum creatine kinase (156 IU/l; normal 45–163 IU/l) and aldolase (6.8 IU/l; normal 2.7–7.5 IU/l) levels were normal. Quadriceps magnetic resonance imaging and electromyography detected muscle inflammation. Slight fine crackles were audible bilaterally. Arterial blood gas analysis showed pH 7.472, PaO2 76.4 Torr, and PaCO2 34.5 Torr. The serum KL-6 was elevated at 695 U/ml (\ 499 U/ml). A chest X-ray revealed faint reticular shadows in both lower lung fields. Chest high-resolution computed tomography (HRCT) showed bilateral subpleural opacity with groundglass attenuation (GGA) (Fig. 1a). She was diagnosed with CADM and progressive ILD, and given intravenous pulse methylprednisolone therapy at 1 g/day for 3 days followed by oral prednisolone 1 mg/kg/day (50 mg/day). After 3 weeks, the muscle pain and skin manifestations had improved, while the chest images had deteriorated gradually with lung shrinkage on chest X-ray and increasing GGA with patchy consolidation on HRCT (Fig. 1b). Biweekly IVCY at 500 mg/body dose and oral tacrolimus were added immediately. The trough serum tacrolimus level was maintained at 8–10 ng/ml. Over the next 13 weeks, she received six sessions of IVCY therapy, the pulmonary lesion extended (Fig. 1c) and hypoxemia requiring oxygen therapy manifested. Intravenous pulse methylprednisolone therapy at 250 mg/day for 3 days was administrated followed by prednisolone 30 mg/day. Nevertheless, the chest images and her respiratory condition had not improved 4 weeks after these additional therapies. Furthermore, IVCY could not be continued because of sustained leukocytopaenia. We switched from IVCY to oral MMF, and gradual improvement of the hypoxemia and pulmonary lesions was obtained. Thirty-one weeks after starting MMF, when the prednisolone had been tapered to 10 mg/day, the lung GGA and exertional dyspnoea resolved (Fig. 1d) and the serum KL-6 level decreased from 4,837 to 563 U/ml (Fig. 2). During the combination therapy with MMF, she suffered from cytomegalovirus infection, which was successfully treated with ganciclovir. Her ILD and CADM have been well controlled without exacerbation for 2 years. H. Tsuchiya (&) H. Tsuno M. Inoue Y. Takahashi H. Yamashita H. Kaneko T. Kano A. Mimori Division of Rheumatic Diseases, National Center for Global Health and Medicine, 1-21-1 Toyama, shinjuku-ku, Tokyo 162-8655, Japan e-mail: elephantscale@yahoo.co.jp

Egr2 and Egr3 in regulatory T cells cooperatively control systemic autoimmunity through Ltbp3-mediated TGF-β3 production

Significance Transcription factors early growth response gene 2 (Egr2) and Egr3 have long been regarded as negative regulators of T-cell activation. Egr2 is also known as a susceptibility gene for systemic lupus erythematosus characterized by dysregulated humoral immune responses to autoantigens. Previously, we reported that Egr2-expressing CD4+CD25-LAG3+ regulatory T cells regulate lupus pathogenesis via production of TGF-β3. However, the role of Egr2 and Egr3 in the regulation of humoral immunity is unclear. Here we report that Egr2 and Egr3 regulate germinal center reactions by promoting TGF-β3 production from regulatory T cells. Egr2 and Egr3 induce the expression of latent TGF-β binding protein 3 (Ltbp3), which is required for TGF-β3 secretion. These findings suggest that Egr2 and Egr3 in T cells may be potential novel therapeutic targets for autoantibody-mediated autoimmune diseases. Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by multiorgan inflammation induced by autoantibodies. Early growth response gene 2 (Egr2), a transcription factor essential for T-cell anergy induction, controls systemic autoimmunity in mice and humans. We have previously identified a subpopulation of CD4+ regulatory T cells, CD4+CD25−LAG3+ cells, that characteristically express both Egr2 and LAG3 and control mice model of lupus via TGF-β3 production. However, due to the mild phenotype of lymphocyte-specific Egr2-deficient mice, the presence of an additional regulator has been speculated. Here, we show that Egr2 and Egr3 expressed in T cells cooperatively prevent humoral immune responses by supporting TGF-β3 secretion. T cell-specific Egr2/Egr3 double-deficient (Egr2/3DKO) mice spontaneously developed an early onset lupus-like disease that was more severe than in T cell-specific Egr2-deficient mice. In accordance with the observation that CD4+CD25−LAG3+ cells from Egr2/3DKO mice completely lost the capacity to produce TGF-β3, the excessive germinal center reaction in Egr2/3DKO mice was suppressed by the adoptive transfer of WT CD4+CD25−LAG3+ cells or treatment with a TGF-β3–expressing vector. Intriguingly, latent TGF-β binding protein (Ltbp)3 expression maintained by Egr2 and Egr3 was required for TGF-β3 production from CD4+CD25−LAG3+ cells. Because Egr2 and Egr3 did not demonstrate cell intrinsic suppression of the development of follicular helper T cells, Egr2- and Egr3-dependent TGF-β3 production by CD4+CD25−LAG3+ cells is critical for controlling excessive B-cell responses. The unique attributes of Egr2/Egr3 in T cells may provide an opportunity for developing novel therapeutics for autoantibody-mediated diseases including SLE.

COUP-TFI and -TFII nuclear receptors are expressed in amacrine cells and play roles in regulating the differentiation of retinal progenitor cells

Chicken ovalbumin upstream promoter transcription factors (COUP-TFs) are members of the steroid/thyroid hormone receptor superfamily. We have shown that two homologous COUP-TF genes, COUP-TFI and COUP-TFII, are expressed in developing mouse retina with a unique gradient along the dorsal-ventral axis. In this work, we aimed to characterize the detailed expression patterns of COUP-TFs in mature retina. Their functions in retinal progenitor cell differentiation into subtypes of mature retinal cells were also examined. Immunostaining of frozen mouse retinal sections with antibodies against COUP-TFs and markers for retinal subtypes revealed that COUP-TFI and -TFII are expressed in amacrine cells, especially in a glycinergic subtype in mature mouse retina. Forced expression of COUP-TFI and -TFII in mouse retinal explant culture by retrovirus-mediated gene transfer promoted amacrine and cone photoreceptor cell differentiation, whereas that of rod photoreceptors decreased. Cell proliferation and apoptosis were not affected by the perturbation of COUP-TFI and -TFII expression levels. Using the Y79 retinoblastoma cell line, we observed that COUP-TFI and -TFII suppressed the transcriptional activation of the Nrl gene. We then analyzed one another member of COUP-TF transcription factors, COUP-TFgamma, whose structure is relatively distant from those of COUP-TFI and -TFII. It is expressed mainly in horizontal cells and has weak activity in inducing amacrine cells when COUP-TFgamma was ectopically expressed in retinal explants. In summary, we found that COUP-TFI and -TFII play roles in amacrine cell differentiation, and COUP-TFgamma has distinct expression pattern and roles during retinal development.