Nobuyuki Takenaka

Crucial Role of Phospholipase Cε in Induction of Local Skin Inflammatory Reactions in the Elicitation Stage of Allergic Contact Hypersensitivity

Phospholipase Cε (PLCε) is an effector of Ras/Rap small GTPases. We previously demonstrated that PLCε plays a crucial role in development of phorbor ester-induced skin inflammation, which is intimately involved in the promotion of skin carcinogenesis. In this study, we have examined its role in local skin inflammatory reactions during development of contact hypersensitivity toward a hapten 2,4-dinitrofluorobenzene (DNFB). PLCε+/+ and PLCε−/− mice were sensitized with DNFB, followed by a DNFB challenge on the ears. PLCε−/− mice exhibited substantially attenuated inflammatory reactions compared with PLCε+/+ mice as shown by suppression of ear swelling, neutrophil infiltration, and proinflammatory cytokine production. In contrast, the extent and kinetics of CD4+ T cell infiltration showed no difference depending on the PLCε background. Adoptive transfer of CD4+ T cells from the sensitized mice to naive mice between PLCε+/+ and PLCε−/− backgrounds indicated that PLCε exerts its function in cells other than CD4+ T cells, presumably fibroblasts or keratinocytes of the skin, to augment inflammatory reactions during the elicitation stage of contact hypersensitivity. Moreover, dermal fibroblasts and epidermal keratinocytes cultured from the skin expressed proinflammatory cytokines in a PLCε-dependent manner on stimulation with T cell-derived cytokines such as IL-17, IFN-γ, TNF-α, and IL-4. These results indicate that PLCε plays a crucial role in induction of proinflammatory cytokine expression in fibroblasts and keratinocytes at the challenged sites, where infiltrated CD4+ T cells produce their intrinsic cytokines, thereby augmenting the local inflammatory reactions.

Akt2 regulates Rac1 activity in the insulin-dependent signaling pathway leading to GLUT4 translocation to the plasma membrane in skeletal muscle cells.

The small GTPase Rac1 plays a pivotal role in insulin-stimulated glucose uptake in skeletal muscle, which is mediated by GLUT4 translocation to the plasma membrane. However, regulatory mechanisms for Rac1 and its role in the signaling pathway composed of phosphoinositide 3-kinase and the serine/threonine kinase Akt remain obscure. Here, we investigate the role of Akt in the regulation of Rac1 in myocytes. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was suppressed by Akt inhibitor IV. Insulin-induced Rac1 activation, on the other hand, was completely inhibited by this inhibitor. Constitutively activated phosphoinositide 3-kinase induced Rac1 activation and GLUT4 translocation. This GLUT4 translocation was almost completely suppressed by Rac1 knockdown. Furthermore, constitutively activated phosphoinositide 3-kinase-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was suppressed by Akt2 knockdown. Finally, insulin-induced Rac1 activation was indeed inhibited by Akt2 knockdown. Together, these results reveal a novel regulatory mechanism involving Akt2 for insulin-dependent Rac1 activation.

Enhancement of ultraviolet B-induced skin tumor development in phospholipase Cε knockout mice is associated with decreased cell death

Phospholipase C (PLC) ε is a phosphoinositide-specific PLC regulated by small guanosine triphosphatases including Ras and Rap. Our previous studies revealed that PLCε gene-knockout (PLCε(-/-)) mice exhibit marked resistance to tumor formation in two-stage skin chemical carcinogenesis using 7,12-dimethylbenz(a)anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate as a promoter. In this model, PLCε functions in tumor promotion through augmentation of 12-O-tetradecanoylphorbol-13-acetate-induced inflammation. In this study, we have further assessed the role of PLCε in tumorigenesis using a mouse model of ultraviolet (UV) B-induced skin tumor development. We irradiated PLCε(+/+), PLCε(+/-) or PLCε(-/-) mice with doses of UVB increasing from 1 to 10 kJ/m(2) three times a week for a total of 25 weeks and observed tumor formation for up to 50 weeks. In sharp contrast to the results from the two-stage chemical carcinogenesis study, PLCε(-/-) mice developed a large number of neoplasms including malignant tumors, whereas PLCε(+/+) and PLCε(+/-) mice developed a relatively small number of benign tumors. However, UVB-induced skin inflammation was greatly suppressed in PLCε(-/-) mice, as observed with 12-O-tetradecanoylphorbol-13-acetate-induced inflammation, implying that PLCεs role in the suppression of UVB-induced tumorigenesis is not mediated by inflammation. Studies of the tumor initiation stage revealed that UVB-induced cell death in the skin was markedly suppressed in PLCε(-/-)mice. Our findings identify a novel function for PLCε as a critical molecule regulating UVB-induced cell death and suggest that resistance to UVB-induced cell death conferred by the absence of PLCε is closely related to the higher incidence of skin tumor formation.

Role of RalA downstream of Rac1 in insulin-dependent glucose uptake in muscle cells.

The small GTPase RalA has been implicated in glucose uptake in insulin-stimulated adipocytes, although it remains unclear whether RalA has a similar role in insulin signaling in other types of cells. Recently, we have demonstrated that the Rho family GTPase Rac1 has a critical role in insulin-dependent glucose uptake in myoblast culture and mouse skeletal muscle. However, the mechanisms underlying Rac1-dependent glucose uptake, mostly mediated by the plasma membrane translocation of the glucose transporter GLUT4, remain largely unknown. The purpose of this study is to examine the involvement of RalA in Rac1 regulation of the translocation of GLUT4 to the plasma membrane in muscle cells. Ectopic expression of a constitutively activated RalA mutant indeed stimulated GLUT4 translocation, suggesting an important role of RalA also in muscle cells. GLUT4 translocation induced by constitutively activated mutation of Rac1 or more physiologically by upstream Rac1 regulators, such as phosphoinositide 3 kinase and the guanine nucleotide exchange factor FLJ00068, was abrogated when the expression of RalA was downregulated by RNA interference. The expression of constitutively activated Rac1, on the other hand, caused GTP loading and subcellular redistribution of RalA. Collectively, we propose a novel mechanism involving RalA for Rac1-mediated GLUT4 translocation in skeletal muscle cells.

Application of fast neutron radiography to three-dimensional visualization of steady two-phase flow in a rod bundle

Abstract Three-dimensional void fraction distribution of air–water two-phase flow in a 4×4 rod-bundle near a spacer was visualized by fast neutron radiography using a CT method. One-dimensional cross sectional averaged void fraction distribution was also calculated. The behaviors of low void fraction (thick water) two-phase flow in the rod bundle around the spacer were clearly visualized. It was shown that the void fraction distributions were visualized with a quality similar to those by thermal neutron radiography for low void fraction two-phase flow which is difficult to visualize by thermal neutron radiography. It is concluded that the fast neutron radiography is efficiently applicable to two-phase flow studies.

Three-dimensional visualization of void fraction distribution in steady two-phase flow by thermal neutron radiography

Abstract Three-dimensional void fraction distributions of a steady air–water two-phase flow in a 4×4 rod-bundle with circular ferrule type spacers were measured by neutron radiography using a CT method. The high flux thermal neutron radiography system at JRR-3M in JAERI was used. Two-phase flow was visualized with a SIT tube camera and time-averaged one-dimensional cross sectional averaged void fraction distributions were calculated. Visualization with high spatial resolution up to 0.18 mm was carried out by using a cooled CCD camera. Projections in 250 directions were obtained and were reconstructed by a filtered back projection method after using some image processing techniques. Animations were made to show the three-dimensional distributions. One-dimensional and three-dimensional void fraction distributions of the steady state two-phase flow in the rod bundle near the spacer were clearly visualized.

A critical role of the small GTPase Rac1 in Akt2‐mediated GLUT4 translocation in mouse skeletal muscle

Insulin promotes glucose uptake in skeletal muscle by inducing the translocation of the glucose transporter GLUT4 to the plasma membrane. The serine/threonine kinase Akt2 has been implicated as a key regulator of this insulin action. However, the mechanisms whereby Akt2 regulates multiple steps of GLUT4 translocation remain incompletely understood. Recently, the small GTPase Rac1 has been identified as a skeletal muscle‐specific regulator of insulin‐stimulated glucose uptake. Here, we show that Rac1 is a critical downstream component of the Akt2 pathway in mouse skeletal muscle as well as cultured myocytes. GLUT4 translocation induced by constitutively activated Akt2 was totally dependent on the expression of Rac1 in L6 myocytes. Moreover, we observed the activation of Rac1 when constitutively activated Akt2 was ectopically expressed. Constitutively activated Akt2‐triggered Rac1 activation was diminished by knockdown of FLJ00068, a guanine nucleotide exchange factor for Rac1. Knockdown of Akt2, on the other hand, markedly reduced Rac1 activation by a constitutively activated mutant of phosphoinositide 3–kinase. In mouse skeletal muscle, constitutively activated mutants of Akt2 and phosphoinositide 3‐kinase, when ectopically expressed, induced GLUT4 translocation. Muscle‐specific rac1 knockout markedly diminished Akt2‐ or phosphoinositide 3‐kinase‐induced GLUT4 translocation, highlighting a crucial role of Rac1 downstream of Akt2. Taken together, these results strongly suggest a novel regulatory link between Akt2 and Rac1 in insulin‐dependent signal transduction leading to glucose uptake in skeletal muscle.

Phospholipase Cɛ has a crucial role in ultraviolet B-induced neutrophil-associated skin inflammation by regulating the expression of CXCL1/KC.

Phospholipase C (PLC) ɛ is a phosphoinositide-specific PLC regulated by small GTPases including Ras and Rap. We previously demonstrated that PLCɛ has an important role in the development of phorbol ester-induced skin inflammation. In this study, we investigated the role of PLCɛ in ultraviolet (UV) B-induced acute inflammatory reactions in the skin. Wild-type (PLCɛ+/+) and PLCɛ gene knockout (PLCɛ−/−) mice were irradiated with a single dose of UVB at 1, 2.5, and 10 kJ/m2 on the dorsal area of the skin, and inflammatory reactions in the skin were histologically evaluated up to 168 h after irradiation. In PLCɛ+/+ mice, irradiation with 1 and 2.5 kJ/m2 UVB resulted in dose-dependent neutrophil infiltration in the epidermis at 24 and 48 h after irradiation. When mice were irradiated with 10 kJ/m2 of UVB, most mice developed skin ulcers by 48 h and these ulcers became more severe at 168 h. In PLCɛ−/− mice, UVB (1 or 2.5 kJ/m2)-induced neutrophil infiltration was markedly suppressed compared with PLCɛ+/+ mice. The suppression of neutrophil infiltration in PLCɛ−/− mice was accompanied by attenuation of UVB-induced production of CXCL1/keratinocyte-derived chemokine (KC), a potent chemokine for neutrophils, in the whole skin. Cultured epidermal keratinocytes and dermal fibroblasts produced CXCL1/KC in a PLCɛ-dependent manner after UVB irradiation, and the UVB-induced upregulation of CXCL1/KC in these cells was significantly abolished by a PLC inhibitor. Furthermore, UVB-induced epidermal thickening was noticeably reduced in the skin of PLCɛ−/− mice. These results indicate that PLCɛ has a crucial role in UVB-induced acute inflammatory reactions such as neutrophil infiltration and epidermal thickening by at least in part regulating the expression of CXCL1/KC in skin cells such as keratinocytes and fibroblasts.

A METHOD FOR QUANTITATIVE MEASUREMENT BY THERMAL NEUTRON RADIOGRAPHY

Abstract A quantitative measurement method by thermal neutron radiography was proposed for two-dimensional void fraction measurement in two-phase flow. The umbra method was modified for two-dimensional measurement by using a neutron absorber grid. Image processing methods to compensate for the effects of neutrons scattered in the object and optical rays scattered in camera were developed. A step made from acrylic resin was tested with a B4C grid 3mm in width and 3mm in interval. It was shown that two dimensional quantitative measurement was possible with this method in sacrifice of the spatial resolution.

Overexpression of phospholipase Cε in keratinocytes upregulates cytokine expression and causes dermatitis with acanthosis and T‐cell infiltration

Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases. We showed previously using PLCε‐deficient mice that PLCε plays a critical role in activation of cytokine production in non‐immune skin cells in a variety of inflammatory reactions. For further investigation of its role in inflammation, we created transgenic mice overexpressing PLCε in epidermal keratinocytes. The resulting transgenic mice spontaneously developed skin inflammation as characterized by formation of adherent silvery scales, excessive growth of keratinocytes, and aberrant infiltration of immune cells such as T cells and DC. Development of the skin symptoms correlated well with increased expression of factors implicated in human inflammatory skin diseases, such as IL‐23, in keratinocytes, and with the accumulation of CD4+ T cells producing IL‐22, a potent inducer of keratinocyte proliferation. Intradermal injection of a blocking antibody against IL‐23 as well as treatment with the immunosuppressant FK506 reversed these skin phenotypes, which was accompanied by suppression of the IL‐22‐producing T‐cell infiltration. These results reveal a crucial role of PLCε in the development of skin inflammation and suggest a mechanism in which PLCε induces the production of cytokines including IL‐23 from keratinocytes, leading to the activation of IL‐22‐producing T cells.