Potent inhibition of angiotensin AT1 receptor signaling by RGS8: importance of the C-terminal third exon part of its RGS domain.

R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in regulation of many GPCR-mediated responses. Multiple RGS proteins are usually expressed in a cell, and it is difficult to point out which RGS protein species are functionally important in the cell. To evaluate intrinsic potency of these RGS proteins, we compared inhibitory effects of RGS1, RGS2, RGS3, RGS4, RGS5, RGS8 and RGS16 on AT1 receptor signaling. Intracellular Ca(2+) responses to angiotensin II were markedly attenuated by transiently expressed RGS2, RGS3 and RGS8, compared to weak inhibition by RGS1, RGS4, RGS5 and RGS16. N-terminally deleted RGS2 (RGS2 domain) lost this potent inhibitory effect, whereas RGS domains of RGS3 and RGS8 showed strong inhibition similar to those of the full-length proteins. To investigate key determinants that specify the differences in potency, we constructed chimeric domains by replacing one or two of three exon parts of RGS8 domain with the corresponding part of RGS5. The chimeric RGS8 domains containing the first or the second exon part of RGS5 showed strong inhibitory effects similar to that of wild type RGS8, but the chimeric domain with the third exon part of RGS5 lost its activity. On the contrary, replacement of the third exon part of RGS5 with the corresponding residues of RGS8 increased the inhibitory effect. The role of the third exon part of RGS8 domain was further confirmed with the chimeric RGS8/RGS4 domains. These results indicate the potent inhibitory activity of RGS8 among R4/B subfamily proteins and importance of the third exon.

Bidirectional role of IL-6 signal in pathogenesis of lung fibrosis.

BACKGROUND: Various signals are known to participate in the pathogenesis of lung fibrosis. Our aim was to determine which signal is predominantly mobilized in the early inflammatory phase and thereafter modulates the development of lung fibrosis. METHODS: Mice received a single dose of 3 mg/kg body weight of bleomycin (BLM) and were sacrificed at designated days post-instillation (dpi). Lung homogenates and sections from mice in the early inflammatory phase were subjected to phospho-protein array analysis and immunofluorescence studies, respectively. Bronchoalveolar lavage fluid (BALF) from mice was subjected to an enzyme-linked immunosorbent assay (EIA) for interleukin (IL)-6 and evaluation of infiltrated cell populations. The effects of endogenous and exogenous IL-6 on the BLM-induced apoptotic signal in A549 cells and type 2 pneumocytes were elucidated. In addition, the effect of IL-6-neutralizing antibody on BLM-induced lung injury was evaluated. RESULTS: Phospho-protein array revealed that BLM induced phosphorylation of molecules downstream of the IL-6 receptor such as Stat3 and Akt in the lung at 3 dpi. At 3 dpi, immunofluorescence studies showed that signals of phospho-Stat3 and -Akt were localized in type 2 pneumocytes, and that BLM-induced IL-6-like immunoreactivity was predominantly observed in type 2 pneumocytes. Activation of caspases in BLM-treated A549 cells and type 2 pneumocytes was augmented by application of IL-6-neutralizing antibody, a PI3K inhibitor or a Stat3 inhibitor. EIA revealed that BLM-induced IL-6 in BALF was biphasic, with the first increase from 0.5 to 3 dpi followed by the second increase from 8 to 10 dpi. Blockade of the first increase of IL-6 by IL-6-neutralizing antibody enhanced apoptosis of type 2 pneumocytes and neutrophilic infiltration and markedly accelerated fibrosis in the lung. In contrast, blockade of the second increase of IL-6 by IL-6-neutralizing antibody ameliorated lung fibrosis. CONCLUSIONS: The present study demonstrated that IL-6 could play a bidirectional role in the pathogenesis of lung fibrosis. In particular, upregulation of IL-6 at the early inflammatory stage of BLM-injured lung has antifibrotic activity through regulating the cell fate of type 2 pneumocytes in an autocrine/paracrine manner.

Therapeutic effect of lung mixed culture-derived epithelial cells on lung fibrosis.

Cell-based therapy is recognized as one of potential therapeutic options for lung fibrosis. However, preparing stem/progenitor cells is complicated and not always efficient. Here, we show easily prepared cell populations having therapeutic capacity for lung inflammatory disease that are named as 'lung mixed culture-derived epithelial cells' (LMDECs). LMDECs expressed surfactant protein (SP)-C and gave rise to type I alveolar epithelial cells (AECs) in vitro and in vivo that partly satisfied type II AEC-like characteristics. An intratracheal delivery of not HEK 293 cells but LMDECs to the lung ameliorated bleomycin (BLM)-induced lung injury. A comprehensive analysis of bronchoalveolar fluid by western blot array revealed that LMDEC engraftment could improve the microenvironment in the BLM-instilled lung in association with stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 signaling axis. SDF-1 enhanced both migration activity and differentiating efficiency of LMDECs. Further classification of LMDECs by flow cytometric study showed that a major population of LMDECs (LMDEC(Maj), 84% of total LMDECs) was simultaneously SP-C(+), CD44(+), CD45(+), and hematopoietic cell lineage(+) and that LMDECs included bronchioalveolar stem cells (BASCs) showing SP-C(+)Clara cell secretory protein(+)stem cell antigen (Sca)1(+) as a small population (1.8% of total LMDECs). CD44(+)-sorted LMDEC(Maj) and Sca1(+)-sorted LMDECs equally ameliorated fibrosis induced by BLM like LMDECs did. However, infiltrated neutrophils were observed in Sca1(+)-sorted LMDEC-treated alveoli that was not typical in LMDEC(Maj)- or LMDEC-treated alveoli. These findings suggest that the protective effect of LMDECs against BLM-induced lung injury depends greatly on that of LMDEC(Maj). Furthermore, the cells expressing both alveolar epithelial and hematopoietic cell lineage markers (SP-C(+)CD45(+)) that have characteristics corresponding to LMDEC(Maj) were observed in the alveoli of lung and increased approximately threefold in response to BLM instillation. Taken together, LMDECs newly classified in the present study are easily culture expanded and have a potential role in future regenerative cell therapy for pulmonary fibrosis.

p38 Mitogen-activated protein kinase accelerates emphysema in mouse model of chronic obstructive pulmonary disease.

CONTEXT: There are few short-term mouse models of chronic obstructive pulmonary disease (COPD) mimicking the human disease. In addition, p38 is recently recognized as a target for the treatment of COPD. However, the precise mechanism how p38 contributes to the pathogenesis of COPD is still unknown. OBJECTIVE: We attempted to create a new mouse model for COPD by intra-tracheal administration of a mixture of lipopolysaccharide (LPS) and cigarette smoke solution (CSS), and investigated the importance of the p38 mitogen-activated protein kinase (p38) pathway in the pathogenesis of COPD. METHODS: Mice were administered LPS + CSS once a day on days 0-4 and 7-11. Thereafter, CSS alone was administered to mice once a day on days 14-18. On day 28, histopathological changes of the lung were evaluated, and bronchoalveolar lavage fluid (BALF) was subjected to western blot array for cytokines. Transgenic (TG) mice expressing a constitutive-active form of MKK6, a p38-specific activator in the lung, were subjected to our experimental protocol of COPD model. RESULTS: LPS + CSS administration induced enlargement of alveolar air spaces and destruction of lung parenchyma. BALF analyses of the LPS + CSS group revealed an increase in expression levels of several cytokines involved in the pathogenesis of human COPD. These results suggest that our experimental protocol can induce COPD in mice. Likewise, histopathological findings of the lung and induction of cytokines in BALF from MKK6 c.a.-TG mice were more marked than those in WT mice. CONCLUSION: In a new experimental COPD mouse model, p38 accelerates the development of emphysema.

Mechanism for p38α-mediated experimental autoimmune encephalomyelitis.

One of the mitogen-activated protein kinases, p38, has been found to play a crucial role in various inflammatory responses. In this study, we analyzed the roles of p38α in multiple sclerosis, using an animal model, experimental autoimmune encephalomyelitis (EAE). p38α(+/-) mice (p38α(-/-) showed embryonic lethality) showed less severe neurological signs than WT mice. Adoptive transfer of lymph node cells (LNC) from sensitized WT mice with MOG(35-55) to naive WT-induced EAE was much more severe compared with the case using LNC from sensitized p38α(+/-) mice. Comprehensive analysis of cytokines from MOG(35-55)-challenged LNC by Western blot array revealed that production of IL-17 was significantly reduced by a single copy disruption of the p38α gene or a p38 inhibitor. Likewise, by a luciferase reporter assay, an electrophoresis mobility shift assay, and characterization of the relationship between p38 activity and IL-17 mRNA expression, we confirmed that p38 positively regulates transcription of the Il17 gene. Furthermore, oral administration of a highly specific p38α inhibitor (UR-5269) to WT mice at the onset of EAE markedly suppressed the progression of EAE compared with a vehicle group. These results suggest that p38α participates in the pathogenesis of EAE through IL-17 induction.

N-type calcium channel in the pathogenesis of experimental autoimmune encephalomyelitis.

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca(2+) channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca(2+) channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca(2+) channel α(1B)-deficient (α(1B)(-/-)) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35-55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α(1B)(-/-) mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α(1B)(-/-) mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α(1B)(-/-) mice and was significantly inhibited by a selective N-type Ca(2+) channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca(2+). These results suggest that the N-type Ca(2+) channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.

Functional analysis of guinea pig ß1-adrenoceptor.

Although similarity of pharmacological responses to certain stimuli between guinea pigs and humans has been reported, this has been poorly defined by a molecular biological approach. In this study, we cloned the gene of guinea pig ?1-adrenoceptor (ADRB1). The deduced amino acid sequence of guinea pig ADRB1 (467-aa) showed 91% and 92% identity with the human and rat ADRB1 sequences, respectively. Using HEK293T cells expressing guinea pig, human and rat ADRB1s independently, we elucidated the functional characteristics of each ADRB1. The ligand-binding profiles and the concentration-response relationships for isoprenaline-induced cyclic adenosine monophosphate (cAMP) production were similar among the three ADRB1s. Isoprenaline also induced phosphorylation of extracellular-signal related kinases (ERK) through ADRB1s in a concentration-dependent manner. The minimum effective concentration of isoprenaline for phosphorylation of ERK, through guinea pig ADRB1 was the same as through human ADRB1, but markedly lower than that of through rat ADRB1. ERK phosphorylation through guinea pig ADRB1 was sensitive to pertussis toxin, a dominant-negative ras and PD98059, indicating that a G(i)-mediated pathway is involved in the ADRB1/ERK signaling loop. These results suggest that the G(i)-coupling efficacy of guinea pig and human ADRB1s may be higher than that of rat ADRB1.

Cooperative action of APJ and α1A-adrenergic receptor in vascular smooth muscle cells induces vasoconstriction.

The apelin receptor (APJ), a receptor for apelin and elabela/apela, induces vasodilation and vasoconstriction in blood vessels. However, the prolonged effects of increased APJ-mediated signalling, involving vasoconstriction, in smooth muscle cells have not been fully characterized. Here, we investigated the vasoactive effects of APJ gain of function under the control of the smooth muscle actin (SMA) gene promoter in mice. Transgenic overexpression of APJ (SMA-APJ) conferred sensitivity to blood pressure and vascular contraction induced by apelin administration in vivo. Interestingly, ex vivo experiments showed that apelin markedly increased the vasoconstriction of isolated aorta induced by noradrenaline (NA), an agonist for α- and ß-adrenergic receptors, or phenylephrine, a specific agonist for α1-adrenergic receptor (α1-AR). In addition, intracellular calcium influx was augmented by apelin with NA in HEK293T cells expressing APJ and α1A-AR. To examine the cooperative action of APJ and α1A-AR in the regulation of vasoconstriction, we developed α1A-AR deficient mice using a genome-editing technique, and then established SMA-APJ/α1A-AR-KO mice. In the latter mouse line, aortic vasoconstriction induced by a specific agonist for α1A-AR, A-61603, were significantly less than in SMA-APJ mice. These results suggest that the APJ-enhanced response requires α1A-AR to contract vessels coordinately.

Impaired placental neovascularization in mice with pregnancy-associated hypertension.

Preeclampsia is a serious disorder that may result in severe morbidity and mortality for mother and fetus, and it is thought that the placental dysfunction is important in the pathogenesis of preeclampsia. As the model of preeclampsia, we previously generated a transgenic mouse model that developed pregnancy-associated hypertension (PAH) by mating females expressing human angiotensinogen with males expressing human renin. In PAH mice, maternal blood pressure started to rise from days 12 to 13 of gestation (E12-13) to term (E19-20), which is accompanied by the fetal intrauterine growth retardation and systemic maternal disorders including proteinuria and convulsion. To understand the pathology of the complications in PAH mice that overlap with those in human preeclampsia, we analyzed the PAH placenta sequentially from the onset of hypertension to the term of delivery. In PAH placenta, histological analysis revealed that the microvessel densities of fetal vasculature at term were significantly lower than those of normal placenta, and the majority of terminal vessels of PAH placenta were lacking for pericytes and basement membrane. The interaction between fetal vasculature and maternal blood canal at labyrinth of PAH placenta was morphologically distorted, and the expression patterns of key molecules in neovascularization of PAH placenta were distinct from those of normal placenta during pregnancy. In addition, maternal plasma level of soluble form of vascular endothelial growth factor receptor-1 (sVEGFR-1) was significantly increased in PAH at E19. Furthermore, in uteroplacental site, in situ proteolytic activity of PAH mice was suppressed from E16 to term compared to that of normal pregnancy, and the expression of matrix metalloproteinase-2 mRNA was strikingly downregulated at E16 in PAH mice. Collective data suggest that the impairments of fetoplacental neovascularization and uteroplacental remodeling contribute to the development of complications in PAH.

Up-regulation of CXC chemokines and their receptors: implications for proinflammatory microenvironments of ovarian carcinomas and endometriosis.

Molecular abnormalities in the epithelial cells of endometriosis and their relevance to carcinogenesis of the ovary have been well studied. On the other hand, the differences of proinflammatory microenvironments between endometriosis and ovarian carcinomas have not been well documented yet. In this study, the expression patterns of CXC chemokines (IL-8, ENA-78, GRO-alpha, I-TAC, Mig, and SDF-1) and their receptors (CXCR2, CXCR3, and CXCR4) were compared among 12 ovarian carcinomas, 8 endometriosis, and 6 normal ovaries using quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. The CXCR3-mediated signaling in ovarian carcinoma cells in vitro was also investigated. In quantitative reverse transcriptase polymerase chain reaction, ENA-78 was up-regulated both in endometriosis and carcinomas, whereas I-TAC was detected exclusively in carcinomas. CXCR3 was up-regulated both in carcinomas and endometriosis. However, immunohistochemical studies revealed that the localization of CXCR3 in carcinomas was distinctively different from that in endometriosis. In carcinoma-endometriosis coexisting cases, CXCR3-positive lymphocytes in benign lesions decreased in proportion as CXCR3-positive tumor cells replaced the tissues. CXCR3 was also detected in ovarian carcinoma cell lines in vitro. Administration of interferon gamma (IFN-gamma)-inducible chemokines induced extracellular signal-regulated kinase phosphorylation in these carcinoma cells. The results indicated that CXC chemokines might contribute to the progression of ovarian carcinomas and endometriosis in different manners. Aberrant expression of IFN-gamma-inducible chemokines and CXCR3 in carcinoma cells in association with reduced CXCR3-positive immune cells raised the possibility that IFN-gamma-inducible chemokines might not exert effective antitumor immune responses but that they might work in favor of tumor progression.

Phosphorylation of Ser166 in RGS5 by protein kinase C causes loss of RGS function.

RGS5 is a member of regulators of G protein signaling (RGS) proteins that attenuate heterotrimeric G protein signaling by functioning as GTPase-activating proteins (GAPs). We investigated phosphorylation of RGS5 and the resulting change of its function. In 293T cells, transiently expressed RGS5 was phosphorylated by endogenous protein kinases in the basal state. The phosphorylation was enhanced by phorbol 12-myristate 13-acetate (PMA) and endothelin-1 (ET-1), and suppressed by protein kinase C (PKC) inhibitors, H7, calphostin C and staurosporine. These results suggest involvement of PKC in phosphorylation of RGS5. In in vitro experiments, PKC phosphorylated recombinant RGS5 protein at serine residues. RGS5 protein phosphorylated by PKC showed much lower binding capacity for and GAP activity toward Galpha subunits than did the unphosphorylated RGS5. In cells expressing RGS5, the inhibitory effect of RGS5 on ET-1-induced Ca(2+) responses was enhanced by staurosporine. Mass spectrometric analysis of the phosphorylated RGS5 revealed that Ser166 was one of the predominant phosphorylation sites. Substitution of Ser166 by aspartic acid abolished the binding capacity to Galpha subunits and the GAP activity, and markedly reduced the inhibitory effect on ET-1-induced Ca(2+) responses. These results indicate that phosphorylation at Ser166 of RGS5 by PKC causes loss of the function of RGS5 in G protein signaling. Since this serine residue is conserved in RGS domains of many RGS proteins, the phosphorylation at Ser166 by PKC might act as a molecular switch and have functional significance.

Pathophysiology of tumor neovascularization.

Neovascularization is essential to the process of development and differentiation of tissues in the vertebrate embryo, and is also involved in a wide variety of physiological and pathological conditions in adults, including wound repair, metabolic diseases, inflammation, cardiovascular disorders, and tumor progression. Thanks to cumulative studies on vasculature, new therapeutic approaches have been opened for us to some life-threatening diseases by controlling angiogenesis in the affected organs. In cancer therapy, for example, modulation of factors responsible for tumor angiogenesis may be beneficial in inhibiting of tumor progression. Several antiangiogenic approaches are currently under preclinical trial. However, the mechanisms of neovascularization in tumors are complicated and each tumor shows unique features in its vasculature, depending on tissue specificity, angiogenic micromilieu, grades and stages, host immunity, and so on. For better understanding and effective therapeutic approaches, it is important to clarify both the general mechanism of angiogenic events and the disease-specific mechanism of neovascularization. This review discusses the general features of angiogenesis under physiological and pathological conditions, mainly in tumor progression. In addition, recent topics such as contribution of the endothelial progenitor cells, tumor vasculogenic mimicry, markers for tumor-derived endothelial cells and pericytes, and angiogenic/angiostatic chemokines are summarized.

Cytodifferentiation enhances Erk activation induced by endothelin-1 in primary cultured astrocytes.

We have previously demonstrated that endothelin-1 (ET-1)-induced extracellular signal-regulated kinase (Erk) activity via the ETB receptor (EDNRB) is mediated through two independent pathways, a protein kinase C-dependent pathway and a pertussis toxin (PTX)-sensitive pathway, in astrocytes. In this study, we showed that the molar potency of ET-1 to induce Erk activation was two orders of magnitude higher in dibutyryl cAMP (DBcAMP)-treated astrocytes than in quiescent astrocytes. This DBcAMP-enhanced molar potency of ET-1 in Erk activation was selectively inhibited by pretreatment of astrocytes with PTX. The expression level of EDNRB in astrocytes was markedly upregulated by DBcAMP-induced cytodifferentiation. However, this up-regulation was simply attributed to the high expression of low-affinity sites. The molar potency of ET-1 to induce both stimulation of inositol trisphosphate production and activation of protein kinase C in DBcAMP-treated astrocytes was similar to that in quiescent astrocytes. On the contrary, the molar potency of ET-1 to induce accumulation of Ras-GTP was two orders of magnitude higher in DBcAMP-treated astrocytes than in quiescent astrocytes, which was consistent with the case of ET-1-induced Erk activation. Moreover, the ET-1-induced Ras activation was PTX sensitive. These results suggest that cytodifferentiation selectively enhances the PTXsensitive Ras/Erk pathway induced by ET-1 in astrocytes, and that cytodifferentiation-induced EDNRB up-regulation might not contribute to this selective potentiation of ET-1 signaling.

Characterization of a novel C. elegans RGS protein with a C2 domain: evidence for direct association between C2 domain and Galphaq subunit.

RGS (regulator of G protein signaling) proteins are GTPase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits and negatively regulate G protein-mediated signal transduction. In this study, we determined the cDNA sequence of a novel Caenorhabditis elegans (C. elegans) RGS protein. The predicted protein, termed C2-RGS, consists of 782 amino acids, and contains a C2 domain and an RGS domain. C2 domains are typically known to be Ca(2+) and phospholipid binding sites, found in many proteins involved in membrane traffic or signal transduction, and most of their biological roles are not identified. To study the function of C2-RGS protein, a series of six truncated versions of C2-RGS were constructed. When the full-length protein of C2-RGS was expressed transiently in AT1a-293T cells, ET-1-induced Ca(2+) responses were strongly suppressed. When each of the mutants with either RGS domain or C2 domain was expressed, the Ca(2+) responses were suppressed moderately. Furthermore, we found that C2 domain of PLC-beta1 also had a similar moderate inhibitory effect. RGS domain of C2-RGS bound to mammalian and C. elegans Galphai/o and Galphaq subunits only in the presence of GDP/AlF(4)(-), and had GAP activity to Galphai3. On the other hand, C2 domains of C2-RGS and PLC-beta1 also bound strongly to Galphaq subunit, in the presence of GDP, GDP/AlF(4)(-), and GTPgammaS, suggesting the stable persistent association between these C2 domains and Galphaq subunit at any stage during GTPase cycle. These results indicate that both the RGS domain and the C2 domain are responsible for the inhibitory effect of the full-length C2-RGS protein on Galphaq-mediated signaling, and suggest that C2 domains of C2-RGS and PLC-beta1 may act as a scaffold module to organize Galphaq and the respective whole protein molecule in a stable signaling complex, both in the absence and presence of stimulus.

Endothelin regulates function of IL-17-producing T cell subset.

AIMS: Although endothelin (ET) is known to play pleiotropic roles in various pathological conditions, its relation to autoimmune disease has not been elucidated. Here, we focused on interleukin (IL)-17, which is closely related to the pathogenesis of multiple sclerosis, and investigated the effect of ET receptor blockers on the production of IL-17 by T lymphocytes. MAIN METHODS: Lymph node cells from mice at 8 days post-immunization with MOG35-55 were stimulated in vitro with MOG35-55 in the presence or absence of an ET receptor blocker (BQ123 for ETA or BQ788 for ETB). Naïve T cells from mice were subjected to an in vitro model of Th17 differentiation, and ET-mediated IL-17 production was investigated under the states of Th17 differentiation and activation. KEY FINDINGS: ELISA revealed that MOG35-55-induced IL-17 production was significantly inhibited by BQ123 but not BQ788. Consistent with the ELISA results for IL-17, the frequency of CD4(+) T cells producing IL-17 but not IFN-γ was reduced by BQ123. Under the differentiating state from naïve T cells to Th17 cells, the spontaneous release of IL-17 from CD4(+) T cells was increased, which was insensitive to BQ123, indicating that ET/ETA signaling did not affect Th17 differentiation. After the time period of Th17 differentiation, however, the increase in IL-17 production by restimulation of the cells with anti-CD3 plus anti-CD28 antibodies was significantly inhibited by BQ123. SIGNIFICANCE: We demonstrated that ET/ETA signaling plays a crucial role in IL-17 production by Th17. BQ123 might be expected to be a future therapeutic drug for multiple sclerosis.

Endothelin B receptor-mediated encephalopathic events in mouse sepsis model.

AIMS: We evaluated whether pathophysiological events in the brain in sepsis are mediated by ET-1/ETB receptor axis. MAIN METHODS: We prepared raw fecal fluid from soft stool of mice. Mice were randomly divided into three groups: pre-PBS+raw fecal fluid group (Sepsis, easy stool method (ESM) group); pre-BQ788+raw fecal fluid group (BQ group); and pre-BQ788+PBS group (PBS group). According to each experimental condition, PBS or BQ788 was intravenously injected into mice prior to intraperitoneal administration of fecal fluid or PBS. All groups of mice were sacrificed at 8h after administration, and then brain samples were prepared. KEY FINDINGS: In the ESM group, an increase of apoptotic neuroblasts was demonstrated in the subgranular zone of the hippocampal dentate gyrus, enhanced expression of c-FOS was observed in arginine-vasopressin-containing neurons in the hypothalamic paraventricular nucleus, and various cytokines involving TNF-α were upregulated in the brain, compared with those in the PBS group. In the region corresponding to their findings, the number of reactive microglia and vascular leakage was markedly increased. BQ788 inhibited the induction of c-FOS expression, neuroblast apoptosis, cytokine upregulation and reactive microglia without affecting vascular leakage. SIGNIFICANCE: We demonstrated that BQ788 could protect the brain from the following sepsis-associated pathophysiological output: neural cell death, inflammatory response and the Hans Selye's environmental stress reaction.

Brefeldin A is an estrogenic, Erk1/2-activating component in the extract of Agaricus blazei mycelia.

We purified an Erk1/2-activating component in Agaricus blazei and identified it as brefeldin A (BFA). The extract of A. blazei mycelia (ABE) previously showed an estrogenic gene-expression profile and positive effects in patients with cardiovascular symptoms. Here, we demonstrate that BFA has estrogenic activity in reporter gene assays and stimulates an estrogen-receptor pathway revealed by activation of Erk1/2, although BFA had no growth-stimulating activity in breast cancer MCF-7 cells. The presence of estrogenic activity without any explicit growth-stimulating effect is unique to BFA, and such components are termed here "silent estrogens". To test this hypothesis, we examined the target-gene transcription and signaling pathways induced by BFA. Furthermore, BFA was found in the mycelium but not fruiting body of A. blazei, suggesting the potential use of ABE for therapeutics and its supplementary use in traditional medicines and functional foods.

Glucagon-like peptide-1 secretion by direct stimulation of L cells with luminal sugar vs non-nutritive sweetener.

UNLABELLED: Aims/Introduction: Oral ingestion of carbohydrate triggers secretion of glucagon-like peptide (GLP)-1, which inhibits the postprandial rise in blood glucose levels. However, the mechanism of carbohydrate-induced GLP-1 secretion from enteroendocrine L cells remains unclear. In the present study, GLP-1 secretion was examined by meal tolerance tests of healthy Japanese volunteers. MATERIALS AND METHODS: Twenty-one healthy Japanese men participated in the study. The meal tolerance test was performed with modified nutrient compositions, with or without pretreatment with the α-glucosidase inhibitor acarbose, or with substitution of sucrose with an equivalent dose of sweeteners in the meal. Blood concentrations of glucose, insulin, GLP-1, and apolipoprotein (Apo) B-48 were measured. RESULTS: GLP-1 secretion started concomitant with the increase in blood glucose levels 10 min after meal ingestion. Insulin secretion started at 5 min, before the increase in blood glucose levels, reflecting the contribution of direct nutrient stimulation on the former parameter and neural regulation in the latter. Carbohydrate retention in the gut lumen induced by acarbose pretreatment extended postprandial GLP-1 secretion and negated the increase in serum ApoB-48 levels. GLP-1 secretion was markedly decreased by a reduction in the amount of sucrose in the meal and was not restored by an equivalent dose of sweeteners used to compensate for the sweet taste. CONCLUSIONS: The results indicate that direct stimulation of L cells with sugar, but not sweetener, is required for carbohydrate-induced GLP-1 secretion. In addition, inhibition of digestion of dietary carbohydrate by α-glucosidase inhibitors may prevent postprandial hyperglycemia by increasing GLP-1 secretion and by inhibiting glucose absorption. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00163.x, 2011).

Potent and selective inhibition of angiotensin AT1 receptor signaling by RGS2: roles of its N-terminal domain.

Emerging evidence indicates that R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in functional regulation in the cardiovascular system. In this study, we compared effects of three R4/B subfamily proteins, RGS2, RGS4 and RGS5 on angiotensin AT1 receptor signaling, and investigated roles of the N-terminus of RGS2. In HEK293T cells expressing AT1 receptor stably, intracellular Ca(2+) responses induced by angiotensin II were much more strongly attenuated by RGS2 than by RGS4 and RGS5. N-terminally deleted RGS2 proteins lost this potent inhibitory effect. Replacement of the N-terminal residues 1-71 of RGS2 with the corresponding residues (1-51) of RGS5 decreased significantly the inhibitory effect. On the other hand, replacement of the residues 1-51 of RGS5 with the residues 1-71 of RGS2 increased the inhibitory effect dramatically. Furthermore, we investigated functional contribution of N-terminal subdomains of RGS2, namely, an N-terminal region (residues 16-55) with an amphipathic α helix domain (the subdomain N1), a probable non-specific membrane-targeting subdomain, and another region (residues 56-71) between the α helix and the RGS box (the subdomain N2), a probable GPCR-recognizing subdomain. RGS2 chimera proteins with the residues 1-33 or 34-52 of RGS5 showed weak inhibitory activity, and either of RGS5 chimera proteins with residues 1-55 or 56-71 of RGS2 showed strong inhibitory effects on AT1 receptor signaling. The present study indicates the essential roles of both N-terminal subdomains for the potent inhibitory activity of RGS2 on AT1 receptor signaling.

CD151 dynamics in carcinoma-stroma interaction: integrin expression, adhesion strength and proteolytic activity.

A member of tetraspanin CD151 is a scaffold protein of laminin-binding integrins and it plays an important role in stable interaction between cells and basement membrane. Although the upregulation of CD151 in tumor cells is thought to accelerate tumor invasion and metastasis, detailed pathological investigation on CD151 and its association with integrins has not been well documented, yet. In the present study, we showed that the expression levels of CD151 and its associated integrin subunits in epidermal carcinoma cell HSC5 were higher than those in immortalized epidermal cell HaCaT. By the stimulation of epidermal growth factor, CD151 was dissociated from cell surface and dispersed in the cytoplasm, and alpha3beta1 integrin was concomitantly internalized. To understand the significance of CD151 in tumor cell dynamics, CD151 in HSC5 was knocked down (HSC5(CD151-)), and the expression of integrin subunits and matrix metalloproteinases (MMPs) were investigated. In HSC5(CD151-), striking morphological alteration on Matrigel and laminin, and cytoskeletal rearrangements were demonstrated. alpha3beta1 integrin was internalized in part, and alpha6beta4 integrin was re-distributed from basal site to cell periphery. Quantitative RT-PCR, Western blot and zymography revealed that the expression levels of MMP2, MMP7 and MMP9 were markedly downregulated in HSC5(CD151-). Immunoprecipitation assay demonstrated that MMP7 was co-immunoprecipitated with CD151. In double stainings, MMP7 was colocalized with CD151 at the leading edge of lamellipodia under migratory status. These results elucidated the importance of CD151 as one of the key molecules for integrin-dependent carcinoma-stroma interaction. It is indicated that CD151 might contribute not only to cell stabilization by associating with adhesion complexes but also to cell migration by inducing integrins re-localization and MMPs production.