Hirotoshi Utsunomiya

Calcium-dependent Epidermal Growth Factor Receptor Transactivation Mediates the Angiotensin II-induced Mitogen-activated Protein Kinase Activation in Vascular Smooth Muscle Cells

We have recently reported that angiotensin II (Ang II)-induced mitogen-activated protein kinase (MAPK) activation is mainly mediated by Ca2+-dependent activation of a protein tyrosine kinase through Gq-coupled Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMC). In the present study, we found Ang II rapidly induced the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with Shc and Grb2. These reactions were inhibited by the EGF receptor kinase inhibitor, AG1478. The Ang II-induced phosphorylation of the EGF receptor was mimicked by a Ca2+ionophore and completely inhibited by an intracellular Ca2+chelator. Thus, AG1478 abolished the MAPK activation induced by Ang II, a Ca2+ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. Moreover, Ang II induced association of EGF receptor with catalytically active c-Src. This reaction was not affected by AG1478. These data indicate that Ang II induces Ca2+-dependent transactivation of the EGF receptor which serves as a scaffold for pre-activated c-Src and for downstream adaptors, leading to MAPK activation in VSMC.

Murine double nullizygotes of the angiotensin type 1A and 1B receptor genes duplicate severe abnormal phenotypes of angiotensinogen nullizygotes.

Rodents are the unique species carrying duplicated angiotensin (Ang) type 1 (AT1) receptor genes, Agtr1a and Agtr1b. After separately generating Agtr1a and Agtr1b null mutant mice by gene targeting, we produced double mutant mice homozygous for both Agtr1a and Agtr1b null mutation (Agtr1a-/-; Agtr1b-/-) by mating the single gene mutants. Agtr1a-/-, Agtr1b-/- mice are characterized by normal in utero survival but decreased ex utero survival rate. After birth they are characterized by low body weight gain, marked hypotension, and abnormal kidney morphology including delayed maturity in glomerular growth, hypoplastic papilla, and renal arterial hypertrophy. These abnormal phenotypes are quantitatively similar to those found in mutant mice homozygous for the angiotensinogen gene (Agt-/-), indicating that major biological functions of endogenous Ang elucidated by the abnormal phenotypes of Agt-/- are mediated by the AT1 receptors. Infusion of Ang II, AT1 blockers, or an AT2 blocker was without effect on blood pressure in Agtr1a-/-; Agtr1b-/- mice, indicating that AT2 receptor does not exert acute depressor effects in these mice lacking AT1 receptors. Also, unlike Agt-/- mice, some Agtr1a-/-; Agtr1b-/- mice have a large ventricular septum defect, suggesting that another receptor such as AT2 is functionally activated in Agtr1a-/-, Agtr1b-/- mice.

Angiotensin II regulates vascular and endothelial dysfunction: recent topics of Angiotensin II type-1 receptor signaling in the vasculature.

Accumulating evidence strongly implicates angiotensin II (AngII) intracellular signaling in mediating cardiovascular diseases such as hypertension, atherosclerosis and restenosis after vascular injury. In vascular smooth muscle cells (VSMCs), through its G-protein-coupled AngII Type 1 receptor (AT(1)), AngII activates various intracellular protein kinases, such as receptor or non-receptor tyrosine kinases, which includes epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), c-Src, PYK2, FAK, JAK2. In addition, AngII activates serine/threonine kinases such as mitogen-activated protein kinase (MAPK) family, p70 S6 kinase, Akt/protein kinase B and various protein kinase C isoforms. In VSMCs, AngII also induces the generation of intracellular reactive oxygen species (ROS), which play critical roles in activation and modulation of above signal transduction. Less is known about endothelial cell (EC) AngII signaling than VSMCs, however, recent studies suggest that endothelial AngII signaling negatively regulates the nitric oxide (NO) signaling pathway and thereby induces endothelial dysfunction. Moreover, in both VSMCs and ECs, AngII signaling cross-talk with insulin signaling might be involved in insulin resistance, an important risk factor in the development of cardiovascular diseases. In fact, clinical and pharmacological studies showed that AngII infusion induces insulin resistance and AngII converting enzyme inhibitors and AT(1) receptor blockers improve insulin sensitivity. In this review, we focus on the recent findings that suggest the existence of novel signaling mechanisms whereby AngII mediates processes, such as activation of receptor or non-receptor tyrosine kinases and ROS, as well as cross-talk between insulin and NO signal transduction in VSMCs and ECs.

Ursodeoxycholic acid stimulates Nrf2-mediated hepatocellular transport, detoxification, and antioxidative stress systems in mice

The protective action of ursodeoxycholic acid (UDCA) in cholestatic liver diseases may be mediated by choleresis, detoxification, and cytoprotection against oxidative stress. Nrf2, one transcription factor, serves as a cellular stress sensor and is a key regulator for hepatic induction of detoxifying enzymes, antioxidative stress genes, and numerous Mrp family members. We aimed to investigate whether UDCA induces hepatic Mrp expression along with that of detoxifying enzymes and antioxidative stress genes via the Nrf2 transcriptional pathway. The protein level, subcellular localization, and mRNA level of Mrp family members were assessed in livers of Keap1 gene-knockdown (Keap1-kd) mice and those of UDCA-fed wild-type (WT) and Nrf2 gene-null (Nrf2-null) mice. Nuclear levels of Nrf2 in livers of Keap1-kd mice markedly increased, resulting in constitutive activation of Nrf2. Keap1-kd mice have high-level expression of hepatic Mrp2, Mrp3, and Mrp4 relative to WT mice. UDCA potently increased nuclear Nrf2 expression level in livers of WT mice, and the treatment showed maximal hepatic induction of Mrp2, Mrp3, and Mrp4 in association with enhanced membranous localizations in an Nrf2-dependent manner. UDCA similarly increased nuclear Nrf2 expression level in rat hepatocytes. Chromatin immunoprecipitation assays using mouse hepatocytes revealed the binding of Nrf2 to antioxidant response elements in the promoter regions of Mrp2, Mrp3, and Mrp4. These findings demonstrate an important role of Nrf2 in the induction of Mrp family members in livers and suggest that a therapeutic mechanism of UDCA action is, via Nrf2 activation, a stimulation of detoxification and antioxidative stress systems, along with Mrp-mediated efflux transport.

Clinical and histopathological analysis of proliferative potentials of recurrent and non-recurrent meningiomas

Abstract Proliferative potentials of meningiomas from 127 patients were examined immunohistochemically using the anti-Ki-67 monoclonal antibody, MIB-1, on paraffin sections, and the correlation among MIB-1 staining index (SI), histopathological finding, and clinial course of the disease was analyzed retrospectively. The mean MIB-1 SI of 50 male patients with meningioma was 5.5%, whereas that of 77 female patients was 2.7%. Higher MIB-1 SI were observed for younger patients. These age- and sex-related differences in MIB-1 SI were statistically significant. The patients were assigned to one of three groups: those with non-recurrent meningioma (n = 73); those with recurrent meningioma in whom the specimens obtained during the initial surgery were used to calculate the MIB-1 SI (n = 21); and those with recurrent meningioma for whom the specimens obtained during the surgery for recurrent tumors were used to calculate the MIB-1 SI (n = 33). The mean MIB-1 SI in these patients were 1.6%, 3.6%, and 8.8%, respectively, and there were statistically significant differences among these three groups. Statistical analyses reveal that meningiomas with a MIB-1 SI of 3% or more have a significantly high tendency for recurrence during the clinical courses, especially within the first 10-year follow-up periods. Moreover, there is statistically significant correlation between MIB-1 SI and recurrence in each Simpson’s grade. The time interval to the next recurrence for recurrent meningiomas is associated with the proliferative potential represented by the MIB-1 SI, and a correlation equation has been proposed to predict the date of the next recurrence. Analyses on cellularity of meningiomas revealed no statistically significant difference in cellularity between non-recurrent and recurrent meningiomas. There was no statistically significant relationship between cellularity and MIB-1 SI of meningiomas. In conclusion, examination on proliferative potentials of meningiomas using MIB-1 SI is very important for biological and histopathologicl analyses and the prediction of future recurrence.

Eradication of Helicobacter pylori prevents cancer development in subjects with mild gastric atrophy identified by serum pepsinogen levels

A longitudinal cohort study was conducted in Helicobactor pylori‐infected middle‐aged Japanese males to evaluate the preventive effects of H. pylori eradication on the development of gastric cancer according to the extent of chronic atrophic gastritis (CAG). The extent of CAG was monitored by baseline serum pepsinogen (PG) levels. We followed 3,656 subjects with persistent H. pylori infection and 473 subjects with successful H. pylori eradication for cancer development for a mean (SD) of 9.3 (0.7) years. Groups with and without extensive CAG were categorized based on PG test‐positive criteria to detect extensive CAG of PG I ≤ 70 ng/ml and PG I/II ratio ≤ 3.0. During the study period, 5 and 55 gastric cancers developed in H. pylori‐eradicated and the noneradicated subjects, respectively, indicating no significant reduction in cancer incidence after H. pylori eradication. Among the noneradicated subjects, 1,329 were PG test‐positive and 2,327 were PG test‐negative. Gastric cancer was confirmed in 30 and 25 subjects, respectively. Among subjects whose infection was eradicated, 155 were PG test‐positive and 318 were PG test‐negative. Of these subjects, gastric cancer was confirmed in 3 and 2 subjects, respectively. Significant reduction in cancer incidence after eradication was observed only in PG test‐negative subjects (p < 0.05; log‐rank test). The results of this study strongly indicate that cancer development after eradication depends on the presence of extensive CAG before eradication and that H. pylori eradication is beneficial to most PG test‐negative subjects with mild CAG as defined by the aforementioned criteria.

Cancer High-Risk Subjects Identified by Serum Pepsinogen Tests: Outcomes after 10-Year Follow-up in Asymptomatic Middle-Aged Males

Background: Gastric cancer screening using the pepsinogen filter test is receiving wide recognition in Japan owing to convenience, freedom from discomfort or risk, efficiency, and economy. Because the long-term outcomes of cancer development in extensive atrophic gastritis detected by pepsinogen test are unclear, test-positive and test-negative subjects were investigated in a longitudinal cohort study. Methods: Subjects comprised 5,209 middle-aged men with measured serum pepsinogen levels who were followed for 10 years. Cancer development based on “atrophy-positive” and “atrophy-negative” criteria used for cancer screening was investigated. Results: During the study, 63 cases of cancer developed in the cohort, representing an incidence rate of 125 per 100,000 person-years. Pepsinogen test screening using the most widely used atrophy-positive criterion (pepsinogen I, ≤70 ng/mL; pepsinogen I/II ratio, ≤3.0) displayed 58.7% sensitivity, 73.4% specificity, and 2.6% positive predictive value. Cancer incidence rate was 276 per 100,000 person-years for the atrophy-positive group and 70 per 100,000 person-years for the atrophy-negative group. Incidence rate was higher in groups fulfilling stricter positive criteria detecting more extensive atrophy, reaching 424 per 100,000 person-years. In addition, 9.2% of atrophy-negative subjects with pepsinogen I of >70 ng/mL and pepsinogen I/II ratio of ≤3.0 (reflecting putative inflammation-based high pepsinogen II level) are at high risk for cancer, particularly diffuse-type cancer, with a cancer incidence rate comparable with atrophy-positive subjects (216 per 100,000 person-years). Conclusion: Atrophy-positive subjects by pepsinogen filter test, particularly those fulfilling stricter criteria, and atrophy-negative subjects with low pepsinogen I/II ratio reflecting putative extensive active inflammation constitute populations at high risk for gastric cancer, requiring thorough endoscopic examination. (Cancer Epidemiol Biomarkers Prev 2008;17(4):838–45)

Genipin enhances Mrp2 (Abcc2)-mediated bile formation and organic anion transport in rat liver

Inchin‐ko‐to (ICKT), an herbal medicine, and its ingredients exert potent choleretic effects by a “bile acid‐independent” mechanism. The current study was designed to determine whether ICKT or its ingredients potentiate multidrug resistance‐associated protein 2 (Mrp2; Abcc2)‐mediated choleresis in vivo. Biliary secretion of Mrp2 substrates and the protein mass, subcellular localization, and messenger RNA (mRNA) level of Mrp2 were assessed in rat liver after infusion of genipin, an intestinal bacterial metabolite of geniposide, a major ingredient of ICKT. The function of Mrp2 was also assessed by the adenosine triphosphate (ATP)‐dependent uptake of Mrp2‐specific substrates using canalicular membrane vesicles (CMVs) from the liver. Infusion of genipin increased bile flow by 230%. It also increased biliary secretion of bilirubin conjugates and reduced glutathione (GSH) by 513% and 336%, respectively, but did not increase bile acid secretion. The ATP‐dependent uptake of estradiol 17‐β‐D‐glucuronide (E217βG; by 265%), leukotriene C4 (LTC4; by 161%), taurolithocholate‐3‐sulfate (TLC‐3S; by 266%), and methotrexate (MTX; by 234%) was significantly stimulated in the CMVs from the liver. These effects were not observed in Mrp2‐deficient rats. Under these conditions, genipin treatment increased the protein mass of Mrp2 in the CMVs but not the mRNA level. In immunoelectron microscopic studies, a marked increase in Mrp2 density in the canalicular membrane (CM) and microvilli was observed in the genipin‐treated liver tissue sections when compared with the vehicle‐treated liver tissue sections. In conclusion, genipin may enhance the bile acid‐independent secretory capacity of hepatocytes, mainly by stimulation of exocytosis and insertion of Mrp2 in the bile canaliculi. ICKT may be a potent therapeutic agent for a number of cholestatic liver diseases. (HEPATOLOGY 2004;39:167–178.)

Prognostic significance of neuroendocrine differentiation, proliferation activity and androgen receptor expression in prostate cancer

Androgen, acting via the androgen receptor (AR), is associated with the development and progression of prostate cancer. Anti‐androgen therapy is widely used to manage prostate cancer. However, the conversion of the tumor from a hormone‐sensitive to a hormone‐insensitive status causes such therapy to fail. Several mechanisms have now been put forward for this conversion, including neuroendocrine (NE) differentiation of the tumor cells. In this study, we evaluated the prognostic significance of tumor‐cell proliferation activity, NE differentiation and AR expression. Formalin‐fixed, paraffin‐embedded sections were prepared from 42 patients with adenocarcinoma of the prostate. Using antibodies to AR, the Ki‐67 antigen (MIB‐1), chromogranin A and synaptophysin, immunohistochemical expression of AR, tumor proliferation activity and NE differentiation were analyzed. Our study revealed that AR expression was significantly lower in adenocarcinoma (52.2 ± 27.1%) than in non‐tumorous prostate tissue (68.3 ± 18.3%; P < 0.001). NE differentiation was found in 50% of the tumors, which was correlated with the Gleason score (P < 0.05). An univariate analysis revealed a significant correlation between progression‐free survival with both AR expression (P < 0.01) and proliferation activity (P < 0.001). NE differentiation was not a prognostic factor in this study.

Cancer development based on chronic active gastritis and resulting gastric atrophy as assessed by serum levels of pepsinogen and Helicobacter pylori antibody titer

Our study investigated the relationship between gastric cancer development and activity of Helicobacter pylori‐associated chronic gastritis or the resulting chronic atrophic gastritis (CAG). A cohort of 4,655 healthy asymptomatic subjects, in whom serum pepsinogen (PG) and H. pylori antibody titer had been measured to assess the activity and stage of H. pylori‐associated chronic gastritis, was followed for up to 16 years, and cancer development was investigated. In subjects with a serologically diagnosed healthy stomach (H. pylori‐negative/CAG‐negative), cancer incidence rate was low, at 16/100,000 person‐years. With the establishment of H. pylori infection and progression of chronic gastritis, significant stepwise cancer risk elevations were seen from CAG‐free subjects (H. pylori‐positive/CAG‐negative) [hazard ratio (HR) = 8.9, 95% confidence interval (CI) = 2.7–54.7] to subjects with CAG (H. pylori‐positive/CAG‐positive) (HR = 17.7, 95% CI = 5.4–108.6) and finally to subjects with metaplastic gastritis (H. pylori‐negative/CAG‐positive) (HR = 69.7, 95% CI = 13.6–502.9). In H. pylori‐infected CAG‐free subjects, significantly elevated cancer risk was observed in the subgroup with active inflammation‐based high PG II level or potent immune response‐based high H. pylori antibody titer; the former was associated with a particularly high risk of diffuse‐type cancer, and both subgroups showed high cancer incidence rates of around 250/100,000 person‐years, comparable to that in subjects with CAG. No such risk elevation was observed in H. pylori‐infected subjects with CAG. These results clearly indicate that gastric cancer develops mainly from the gastritis‐atrophy‐metaplasia‐cancer sequence and partly from active inflammation‐based direct carcinogenesis, and that serum levels of PG and H. pylori antibody titer provide indices of cancer development in H. pylori‐infected subjects.