Yoshihiro Tsuchiya

Nuclear IKKβ Is an Adaptor Protein for IκBα Ubiquitination and Degradation in UV-Induced NF-κB Activation

Proinflammatory cytokines activate NF-kappaB using the IkappaB kinase (IKK) complex that phosphorylates inhibitory proteins (IkappaBs) at N-terminal sites resulting in their ubiquitination and degradation in the cytoplasm. Although ultraviolet (UV) irradiation does not lead to IKK activity, it activates NF-kappaB by an unknown mechanism through IkappaBalpha degradation without N-terminal phosphorylation. Here, we describe an adaptor function of nuclear IKKbeta in UV-induced IkappaBalpha degradation. UV irradiation induces the nuclear translocation of IkappaBalpha and association with IKKbeta, which constitutively interacts with beta-TrCP through heterogeneous ribonucleoprotein-U (hnRNP-U) leading to IkappaBalpha ubiquitination and degradation. Furthermore, casein kinase 2 (CK2) and p38 associate with IKKbeta and promote IkappaBalpha degradation by phosphorylation at C-terminal sites. Thus, nuclear IKKbeta acts as an adaptor protein for IkappaBalpha degradation in UV-induced NF-kappaB activation. NF-kappaB activated by the nuclear IKKbeta adaptor protein suppresses anti-apoptotic gene expression and promotes UV-induced cell death.

Peptidyl-prolyl Cis/Trans Isomerase NIMA-interacting 1 Associates with Insulin Receptor Substrate-1 and Enhances Insulin Actions and Adipogenesis

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.

Kupffer Cells May Autoregulate Interleukin 1 Production by Producing Interleukin 1 Inhibitor and Prostaglandin E2

Rat Kupffer cells stimulated with bacterial lipopolysaccharide (LPS) produced high levels of interleukin 1 (IL‐1), as determined by thymocyte proliferation assay. Indomethacin revealed a dose‐dependent augmentation in IL‐l production, in parallel with a dose‐dependent reduction in prostaglandin E2 production by Kupffer cells. The addition of exogenous prostaglandin E2, dibutyryl cAMP, or isoproterenol led to a dose‐dependent suppression of IL‐I production. The supernatant from UPS‐stimulated Kupffer cells also contained factors that Inhibited IL‐1‐induced thymocyte proliferation. Upon gel filtration, two inhibitory peaks, at apparent MW of 27,000 and 6000, were obtained. The latter but not the former fraction also affected Interleukin 2 (IL‐2)‐induced thymocyte proliferation. Increasing amounts of IL‐1 overcame the inhibitory activity derived from the 27,000 MW fraction. These results suggest to us that prostaglandin E2 and IL‐1 inhibitor released by Kupffer cells may be involved in negative self‐control in regulating IL‐1 production and its action.

Valsartan, independently of AT1 receptor or PPARγ, suppresses LPS-induced macrophage activation and improves insulin resistance in cocultured adipocytes

Macrophages are integrated into adipose tissues and interact with adipocytes in obese subjects, thereby exacerbating adipose insulin resistance. This study aimed to elucidate the molecular mechanism underlying the insulin-sensitizing effect of the angiotensin II receptor blocker (ARB) valsartan, as demonstrated in clinical studies. Insulin signaling, i.e., insulin receptor substrate-1 and Akt phosphorylations, in 3T3-L1 adipocytes was impaired markedly by treatment with tumor necrosis factor-α (TNFα) or in the culture medium of lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages, and valsartan had no effects on these impairments. However, in contrast, when cocultured with RAW 264.7 cells using a transwell system, the LPS-induced insulin signaling impairment in 3T3-L1 adipocytes showed almost complete normalization with coaddition of valsartan. Furthermore, valsartan strongly suppressed LPS-induced productions of cytokines such as interleukin (IL)-1β, IL-6, and TNFα with nuclear factor-κB activation and c-Jun NH(2)-terminal kinase phosphorylation in RAW 264.7 and primary murine macrophages. Very interestingly, this effect of valsartan was also observed in THP-1 cells treated with angiotensin II type 1 (AT1) siRNA or a peroxisome proliferator-activated receptor-γ (PPARγ) antagonist as well as macrophages from AT1a receptor-knockout mice. We conclude that valsartan suppresses the inflammatory response of macrophages, albeit not via PPARγ or the AT1a receptor. This suppression appears to secondarily improve adipose insulin resistance.

Pin1 Associates with and Induces Translocation of CRTC2 to the Cytosol, Thereby Suppressing cAMP-responsive Element Transcriptional Activity

Pin1 is a unique regulator, which catalyzes the conversion of a specific phospho-Ser/Thr-Pro-containing motif in target proteins. Herein, we identified CRTC2 as a Pin1-binding protein by overexpressing Pin1 with Myc and FLAG tags in mouse livers and subsequent purification of the complex containing Pin1. The association between Pin1 and CRTC2 was observed not only in overexpression experiments but also endogenously in the mouse liver. Interestingly, Ser136 in the nuclear localization signal of CRTC2 was shown to be involved in the association with Pin1. Pin1 overexpression in HepG2 cells attenuated forskolin-induced nuclear localization of CRTC2 and cAMP-responsive element (CRE) transcriptional activity, whereas gene knockdown of Pin1 by siRNA enhanced both. Pin1 also associated with CRTC1, leading to their cytosol localization, essentially similar to the action of CRTC2. Furthermore, it was shown that CRTC2 associated with Pin1 did not bind to CREB. Taken together, these observations indicate the association of Pin1 with CRTC2 to decrease the nuclear CBP·CRTC·CREB complex. Indeed, adenoviral gene transfer of Pin1 into diabetic mice improved hyperglycemia in conjunction with normalizing phosphoenolpyruvate carboxykinase mRNA expression levels, which is regulated by CRE transcriptional activity. In conclusion, Pin1 regulates CRE transcriptional activity, by associating with CRTC1 or CRTC2.

DPP-IV inhibitor anagliptin exerts anti-inflammatory effects on macrophages, adipocytes, and mouse livers by suppressing NF-κB activation

Dipeptidyl peptidase IV (DPP-IV) expression in visceral adipose tissue is reportedly increased in obese patients, suggesting an association of DPP-IV with inflammation. In this study, first, lipopolysaccharide (LPS)- or palmitate-induced elevations of inflammatory cytokine mRNA expressions in RAW264.7 macrophages were shown to be significantly suppressed by coincubation with a DPP-IV inhibitor, anagliptin (10 μM), despite low DPP-IV expression in the RAW264.7 cells. Regarding the molecular mechanism, LPS-induced degradation of IκBα and phosphorylations of p65, JNK, and p38, as well as NF-κB and AP-1 promoter activities, were revealed to be suppressed by incubation with anagliptin, indicating suppressive effects of anagliptin on both NF-κB and AP-1 signaling pathways. Anagliptin also acted on 3T3-L1 adipocytes, weakly suppressing the inflammatory cytokine expressions induced by LPS and TNFα. When 3T3-L1 and RAW cells were cocultured and stimulated with LPS, the effects of anagliptin on the suppression of cytokine expressions in 3T3-L1 adipocytes were more marked and became evident at the 10 μM concentration. Anti-inflammatory effects of anagliptin were also observed in vivo on the elevated hepatic and adipose expressions and serum concentrations of inflammatory cytokines in association with the suppression of hepatic NF-κB transcriptional activity in LPS-infused mice. Taking these observations together, the anti-inflammatory properties of anagliptin may be beneficial in terms of preventing exacerbation of diabetes and cardiovascular events.

4F2hc stabilizes GLUT1 protein and increases glucose transport activity

Glucose transporter 1 (GLUT1) is widely distributed throughout various tissues and contributes to insulin-independent basal glucose uptake. Using a split-ubiquitin membrane yeast two-hybrid system, we newly identified 4F2 heavy chain (4F2hc) as a membrane protein interacting with GLUT1. Though 4F2hc reportedly forms heterodimeric complexes between amino acid transporters, such as LAT1 and LAT2, and regulates amino acid uptake, we investigated the effects of 4F2hc on GLUT1 expression and the associated glucose uptake. First, FLAG-tagged 4F2hc and hemagglutinin-tagged GLUT1 were overexpressed in human embryonic kidney 293 cells and their association was confirmed by coimmunoprecipitation. The green fluorescent protein-tagged 4F2hc and DsRed-tagged GLUT1 showed significant, but incomplete, colocalization at the plasma membrane. In addition, an endogenous association between GLUT1 and 4F2hc was demonstrated using mouse brain tissue and HeLa cells. Interestingly, overexpression of 4F2hc increased the amount of GLUT1 protein in HeLa and HepG2 cells with increased glucose uptake. In contrast, small interfering RNA (siRNA)-mediated 4F2hc gene suppression markedly reduced GLUT1 protein in both cell types, with reduced glucose uptake. While GLUT1 mRNA levels were not affected by overexpression or gene silencing of 4F2hc, GLUT1 degradation after the addition of cycloheximide was significantly suppressed by 4F2hc overexpression and increased by 4F2hc siRNA treatment. Taken together, these observations indicate that 4F2hc is likely to be involved in GLUT1 stabilization and to contribute to the regulation of not only amino acid but also glucose metabolism.

Long-term Forskolin Stimulation Induces AMPK Activation and Thereby Enhances Tight Junction Formation in Human Placental Trophoblast BeWo Cells

BeWo cells, derived from human choriocarcinoma, have been known to respond to forskolin or cAMP analogues by differentiating into multinucleated cells- like syncytiotrophoblasts on the surfaces of chorionic villi of the human placenta. In this study, we demonstrated that long-term treatment with forskolin enhances the tight junction (TJ) formation in human placental BeWo cells. Interestingly, AMPK activation and phosphorylation of acetyl-CoA carboxylase (ACC), a molecule downstream from AMPK, were induced by long-term incubation (>12h) with forskolin, despite not being induced by acute stimulation with forskolin. In addition, co-incubation with an AMPK inhibitor, compound C, as well as overexpression of an AMPK dominant negative mutant inhibited forskolin-induced TJ formation. Thus, although the molecular mechanism underlying AMPK activation via the forskolin stimulation is unclear, the TJ formation induced by forskolin is likely to be mediated by the AMPK pathway. Taking into consideration that TJs are present in the normal human placenta, this mechanism may be important for forming the placental barrier system between the fetal and maternal circulations.

Role of Pin1 Protein in the Pathogenesis of Nonalcoholic Steatohepatitis in a Rodent Model

Background: NASH is a disease characterized by fat accumulation and chronic inflammation in the liver. Results: Pin1 expression was increased in NASH model mouse livers. Pin1 KO mice were resistant to NASH development. Conclusion: Pin1 plays critical roles in NASH development. Significance: A Pin1 inhibitor might be a novel agent for treating NASH. Nonalcoholic steatohepatitis (NASH) is a disorder characterized by simultaneous fat accumulation and chronic inflammation in the liver. In this study, Pin1 expression was revealed to be markedly increased in the livers of mice with methionine choline-deficient (MCD) diet-induced NASH, a rodent model of NASH. In addition, Pin1 KO mice were highly resistant to MCD-induced NASH, based on a series of data showing simultaneous fat accumulation, chronic inflammation, and fibrosis in the liver. In terms of Pin1-induced fat accumulation, it was revealed that the expression levels of peroxisome proliferator-activated receptor α and its target genes were higher in the livers of Pin1 KO mice than in controls. Thus, resistance of Pin1 KO mice to hepatic steatosis is partially attributable to the lack of Pin1-induced down-regulation of peroxisome proliferator-activated receptor α, although multiple other mechanisms are apparently involved. Another mechanism involves the enhancing effect of hematopoietic Pin1 on the expressions of inflammatory cytokines such as tumor necrosis factor and monocyte chemoattractant protein 1 through NF-κB activation, eventually leading to hepatic fibrosis. Finally, to distinguish the roles of hematopoietic or nonhematopoietic Pin1 in NASH development, mice lacking Pin1 in either nonhematopoietic or hematopoietic cells were produced by bone marrow transplantation between wild-type and Pin1 KO mice. The mice having nonhematopoietic Pin1 exhibited fat accumulation without liver fibrosis on the MCD diet. Thus, hepatic Pin1 appears to be directly involved in the fat accumulation in hepatocytes, whereas Pin1 in hematopoietic cells contributes to inflammation and fibrosis. In summary, this is the first study to demonstrate that Pin1 plays critical roles in NASH development. This report also raises the possibility that hepatic Pin1 inhibition to the appropriate level might provide a novel therapeutic strategy for NASH.

The effects of a high dose, short course of interferon on hepatitis C

To shorten the period of interferon (IFN) treatment for chronic hepatitis C, we investigated the clinical efficacy of a regimen using a higher dose and a shorter treatment period. Fifty chronic hepatitis C patients who were hepatitis C virus (HCV)‐RNA positive and who were histologically diagnosed as having chronic hepatitis, took part in the study. Virus levels were measured before and 2 weeks after starting the treatment. We administered natural IFNα, 10 MU, i.m. daily for 2 consecutive weeks and then three times per week for the subsequent 14 weeks (total dose 560 MU). Patients who were HCV‐RNA negative at the completion of the therapy and 6 months later, were evaluated as sustained responders (SR; 32%). Those who were not HCV‐RNA negative at the two time points were evaluated as non‐responders. Nucleotide and clone differences in the hypervariable region (HVR) and predictive factors for prognosis were also analysed. Low virus level and HCV‐RNA genotype 2a/2b were the predictors for good prognosis, whereas the numbers of nucleotide differences and clone differences in HVR were not. Sustained responder patients became HCV‐RNA negative 2 weeks after starting the treatment at a significantly higher rate, whereas no non‐responder patients were HCV‐RNA negative at that time. The SR rate (32%) was equivalent to those reported in previous 24 week treatment studies. This IFN therapy using a higher dose and a shorter period was useful.