Protocol of the RACB study: a multicenter, single-arm, prospective study to evaluate the efficacy of resection of initially unresectable hepatocellular carcinoma with atezolizumab combined with bevacizumab.

BACKGROUND: Although the standard therapy for advanced-stage hepatocellular carcinoma (HCC) is systemic chemotherapy, the combination of atezolizumab and bevacizumab (atezo + bev) with a high objective response rate may lead to conversion to resection in patients with initially unresectable HCC. This study aims to evaluate the efficacy of atezo + bev in achieving conversion surgery and prolonged progression-free survival (PFS) for initially unresectable HCC. METHODS: The RACB study is a prospective, single-arm, multicenter, phase II trial evaluating the efficacy of combination therapy with atezo + bev for conversion surgery in patients with technically and/or oncologically unresectable HCC. The main eligibility criteria are as follows: (1) unresectable HCC without a history of systemic chemotherapy, (2) at least one target lesion based on RECIST ver. 1.1, and (3) a Child‒Pugh score of 5-6. The definition of unresectable tumors in this study includes macroscopic vascular invasion and/or extrahepatic metastasis and massive distribution of intrahepatic tumors. Patients will be treated with atezolizumab (1200 mg/body weight) and bevacizumab (15 mg/kg) every 3 weeks. If the patient is considered resectable on radiological assessment 12 weeks after initial chemotherapy, the patient will be treated with atezolizumab monotherapy 3 weeks after combination chemotherapy followed by surgery 3 weeks after atezolizumab monotherapy. If the patient is considered unresectable, the patient will continue with atezo + bev and undergo a radiological assessment every 9 weeks until resectable or until disease progression. The primary endpoint is PFS, and the secondary endpoints are the overall response rate, overall survival, resection rate, curative resection rate, on-protocol resection rate, and ICG retention rate at 15 min after atezo + bev therapy. The assessments of safety and quality of life during the treatment course will also be evaluated. The number of patients has been set at 50 based on the threshold and the expected PFS rate at 6 months after enrollment of 40% and 60%, respectively, with a one-sided alpha error of 0.05 and power of 0.80. The enrollment and follow-up periods will be 2 and 1.5 years, respectively. DISCUSSION: This study will elucidate the efficacy of conversion surgery with atezo + bev for initially unresectable HCC. In addition, the conversion rate, safety and quality of life during the treatment course will also be demonstrated. TRIAL REGISTRATION: This study is registered in the Japan Registry of Clinical Trials (jRCTs051210148, January 7, 2022).

Microsomal prostaglandin E synthase-1 is a critical factor in dopaminergic neurodegeneration in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder of uncertain pathogenesis characterized by the loss of nigrostriatal dopaminergic neurons. Although increased production of prostaglandin E2 (PGE2) has been implicated in tissue damage in several pathological settings, the role of microsomal prostaglandin E synthase-1 (mPGES-1), an inducible terminal enzyme for PGE2 synthesis, in dopaminergic neurodegeneration remains unclear. Here we show that mPGES-1 is up-regulated in the dopaminergic neurons of the substantia nigra of postmortem brain tissue from PD patients and in neurotoxin 6-hydroxydopamine (6-OHDA)-induced PD mice. The expression of mPGES-1 was also up-regulated in cultured dopaminergic neurons stimulated with 6-OHDA. The genetic deletion of mPGES-1 not only abolished 6-OHDA-induced PGE2 production but also inhibited 6-OHDA-induced dopaminergic neurodegeneration both in vitro and in vivo. Nigrostriatal projections, striatal dopamine content, and neurological functions were significantly impaired by 6-OHDA administration in wild-type (WT) mice, but not in mPGES-1 knockout (KO) mice. Furthermore, in cultured primary mesencephalic neurons, addition of PGE2 to compensate for the deficiency of 6-OHDA-induced PGE2 production in mPGES-1 KO neurons recovered 6-OHDA toxicity to almost the same extent as that seen in WT neurons. These results suggest that induction of mPGES-1 enhances 6-OHDA-induced dopaminergic neuronal death through excessive PGE2 production. Thus, mPGES-1 may be a valuable therapeutic target for treatment of PD.

[A trial of simple and rapid carbapenemase big five gene analysis using LAMP method].

Reliable detection and typing of carbapenemase is important in the treatment of infectious diseases. In this study we newly designed LAMP primer based on the latest information, and established a detection method for Carbapenemase Big five gene. For DNA extraction from strains, alkaline boiling method and commercial kit were used. The reaction temperatures of the LAMP method was VIM: 65°C, NDM: 63°C, KPC: 65°C, OXA-48-like: 65°C, IMP: 61°C. And simultaneous LAMP method was at 63°C, for 60 min. It was possible to detect up to 103 copies/ml. The reactivity of LAMP using 36 strains verified by Multiplex-PCR was VIM (4/4: number of LAMP method positive strains/number of strains evaluated), NDM (2/2), KPC (4/4), OXA-48-like (4/4), IMP (17/17). The type of carbapenemase determined by the LAMP method were all consistent with multiplex PCR. All strains were detected within 30 min. In VIM, both VIM-1-like and VIM-2-like were able to detect. In this study, although the number and variation of the strains evaluated was limited, LAMP method was clinically useful as a simple and rapid carbapenemase detection method.

Optimal cutoff values of WHO-HPQ presenteeism scores by ROC analysis for preventing mental sickness absence in Japanese prospective cohort.

OBJECTIVES: Sickness absence due to mental disease in the workplace has become a global public health problem. Previous studies report that sickness presenteeism is associated with sickness absence. We aimed to determine optimal cutoff scores for presenteeism in the screening of the future absences due to mental disease. METHODS: A prospective study of 2195 Japanese employees from all areas of Japan was conducted. Presenteeism and depression were measured by the validated Japanese version of the World Health Organization Health and Work Performance Questionnaire (WHO-HPQ) and K6 scale, respectively. Absence due to mental disease across a 2-year follow-up was surveyed using medical certificates obtained for work absence. Socioeconomic status was measured via a self-administered questionnaire. Receiver operating curve (ROC) analysis was used to determine optimal cutoff scores for absolute and relative presenteeism in relation to the area under the curve (AUC), sensitivity, and specificity. RESULTS: The AUC values for absolute and relative presenteeism were 0.708 (95% CI, 0.618-0.797) and 0.646 (95% CI, 0.546-0.746), respectively. Optimal cutoff scores of absolute and relative presenteeism were 40 and 0.8, respectively. With multivariate adjustment, cohort participants with our proposal cutoff scores for absolute and relative presenteeism were significantly more likely to be absent due to mental disease (OR = 4.85, 95% CI: 2.20-10.73 and OR = 5.37, 95% CI: 2.42-11.93, respectively). The inclusion or exclusion of depressive symptoms (K6≥13) at baseline in the multivariate adjustment did not influence the results. CONCLUSIONS: Our proposed optimal cutoff scores of absolute and relative presenteeism are 40 and 0.8, respectively. Participants who scored worse than the cutoff scores for presenteeism were significantly more likely to be absent in future because of mental disease. Our findings suggest that the utility of presenteeism in the screening of sickness absence due to mental disease would help prevent such an absence.

Altered DNA methylation status of human brain derived neurotrophis factor gene could be useful as biomarker of depression.

Brain-derived neurotrophic factor (BDNF) is involved in the survival, development, and synaptic plasticity of neurons. BDNF is believed to be associated with the pathophysiology of psychiatric disorders. Several studies have suggested the relevance of DNA methylation in its promoter region with depression. Here, we report different methylation statuses in groups with different depressive scores or undergoing different levels of job-stress. DNA samples were extracted from the saliva of 774 Japanese workers, and the methylation status was determined using the Illumina HumanMethylation 450 K Microarray. Depressive symptoms were measured using the Kessler's K6 questionnaire. Job-stress scales were assessed via a self-administered questionnaire. Independent DNA pools were formed based on K6 and job-strain scores, and the methylation levels were compared among these pools. The average DNA methylation rate was significantly decreased in the highest K6 score group compared to the lowest group (methylated signals, 14.2% vs. 16.5%, P = 2 · 16 × 10(-198)). This difference remained for the CpG island in the promoter region (10.4% vs. 5.8%, P = 3 · 67 × 10(-133)). Regarding the job-strain score, there was a slight increase in the methylation level of the whole gene in the group with the highest score compared to that with the lowest score; however, these groups showed no difference in the promoter region. Our results revealed significant changes in the DNA methylation status of the complete human BDNF gene in persons with depression compared to normal individuals, especially in the promoter region of exon 1. This indicates that DNA methylation in this gene is a promising biomarker for diagnosing depression.

Alteration in MARCKS phosphorylation and expression by methylmercury in SH-SY5Y cells and rat brain.

The molecular mechanisms mediating methylmercury (MeHg)-induced neurotoxicity are not completely understood. Because myristoylated alanine-rich C kinase substrate (MARCKS) plays an essential role in the differentiation and development of neuronal cells, we studied the alteration of MARCKS expression and phosphorylation in MeHg-induced neurotoxicity of neuroblastoma SH-SY5Y cells and in the rat brain. Exposure to MeHg induced a decrease in cell viability of SH-SY5Y cells, which was accompanied by a significant increase in phosphorylation and a reduction in MARCKS expression. Pretreatment of cells with a protein kinase C inhibitor or an extracellular Ca(2+) chelator suppressed MeHg-induced MARCKS phosphorylation. In MARCKS knock-down cells, MeHg-induced cell death was significantly augmented in comparison to control siRNA. In brain tissue from MeHg-treated rats, MARCKS phosphorylation was enhanced in the olfactory bulb in comparison to control rats. The present study may indicate that alteration in MARCKS expression or phosphorylation has consequences for MeHg-induced neurotoxicity.

Rates of serious intracellular infections in autoimmune disease patients receiving initial glucocorticoid therapy.

BACKGROUND/AIMS: The Japanese National Hospital Organization evidence-based medicine (EBM) Study group for Adverse effects of Corticosteroid therapy (J-NHOSAC) is a Japanese hospital-based cohort study investigating the safety of the initial use of glucocorticoids (GCs) in patients with newly diagnosed autoimmune diseases. Using the J-NHOSAC registry, the purpose of this observational study is to analyse the rates, characteristics and associated risk factors of intracellular infections in patients with newly diagnosed autoimmune diseases who were initially treated with GCs. METHODOLOGY/PRINCIPAL FINDINGS: A total 604 patients with newly diagnosed autoimmune diseases treated with GCs were enrolled in this registry between April 2007 and March 2009. Cox proportional-hazards regression was used to determine independent risk factors for serious intracellular infections with covariates including sex, age, co-morbidity, laboratory data, use of immunosuppressants and dose of GCs. Survival was analysed according to the Kaplan-Meier method and was assessed by the log-rank test. There were 127 serious infections, including 43 intracellular infections, during 1105.8 patient-years of follow-up. The 43 serious intracellular infections resulted in 8 deaths. After adjustment for covariates, diabetes (Odds ratio [OR]: 2.5, 95% confidence interval [95% CI] 1.1-5.9), lymphocytopenia (≦1000/µl, OR: 2.5, 95% CI 1.2-5.2) and use of high-dose (≧30 mg/day) GCs (OR: 2.4, 95% CI 1.1-5.3) increased the risk of intracellular infections. Survival curves showed lower intracellular infection-free survival rate in patients with diabetes, lymphocytopaenia and high-dose GCs treatments. CONCLUSIONS/SIGNIFICANCE: Patients with newly diagnosed autoimmune diseases were at high risk of developing intracellular infection during initial treatment with GCs. Our findings provide background data on the risk of intracellular infections of patients with autoimmune diseases. Clinicians showed remain vigilant for intracellular infections in patients with autoimmune diseases who are treated with GCs.

Glucocorticoid Therapy and the Risk of Infection in Patients With Newly Diagnosed Autoimmune Disease.

Glucocorticoid (GC) therapy is associated with the risk of life-threatening adverse events in patients with autoimmune disease. To determine accurately the incidence and predictors of GC-related adverse events during initial GC treatment, we conducted a cohort study. Patients with autoimmune disease who were initially treated with GCs in Japan National Hospital Organization (NHO) hospitals were enrolled. Cox proportional hazard regression was used to determine the independent risks for GC-related serious adverse events and mortality. Survival was analyzed according to the Kaplan-Meier method and was assessed with the log-rank test.The 604 patients had a total follow-up of 1105.8 person-years (mean, 1.9 year per patient). One hundred thirty-six patients had at least 1 infection with objective confirmation, and 71 patients had serious infections. Twenty-two cardiovascular events, 55 cases of diabetes, 30 fractures, 23 steroid psychosis events, and 4 avascular bone necrosis events occurred during the follow-up period. The incidence of serious infections was 114.8 (95% confidence interval, 95.7-136.6) per 1000 person-years. After adjustment for covariates, the following independent risk factors for serious infection were found: elderly age (hazard ratio [HR], 1.25/10-yr age increment; p = 0.016), presence of interstitial lung disease (HR, 2.01; p = 0.011), high-dose GC use (≥29.9 mg/d) (HR, 1.71; p = 0.047), and low performance status (Karnofsky score, HR, 0.98/1-score increment; p = 0.002). During the follow-up period, 73 patients died, 35 of whom died of infection. Similarly, elderly age, the presence of interstitial lung disease, and high-dose GC use were found to be significant independent risk factors for mortality. The incidence of serious and life-threatening infection was higher in patients with autoimmune disease who were initially treated with GCs. Although the primary diseases are important confounding factors, elderly age, male sex, the presence of interstitial lung diseases, high-dose GCs, and low performance status were shown to be risk factors for serious infection and mortality.

Japanese dietary pattern consistently relates to low depressive symptoms and it is modified by job strain and worksite supports.

BACKGROUND: This study examined the association between traditional Japanese dietary pattern and depressive symptoms in Japanese workers, employing large-scale samples, considering socioeconomic status (SES) and job stress factors. METHODS: A cross-sectional study of 2266 Japanese employees aged 21-65 years from all areas of Japan was conducted as part of the Japanese Study of Health, Occupation and Psychosocial factors related Equity (J-HOPE). Habitual diet was assessed by FFQ (BDHQ). The depression degree and job stress factors (job demand, job control, and worksite support) were measured by K6 and Job Content Questionnaire. RESULTS: Participants with high scores for the balanced Japanese dietary pattern were significantly less likely to show probable mood/anxiety disorders (K6≥9) with multivariate adjustment including SES and job stress factors (odds ratio=0.66 [0.51-0.86], trend P=0.002). Other dietary patterns were not associated with depressive symptoms. Even after stratification by job stress factors, the Japanese dietary pattern was consistently protective against depressive symptoms. Furthermore, a highly significant difference between the first and third tertiles of the dietary pattern was observed in participants with active strain (high demand and high control) with low worksite supports (8.5 vs. 5.2, P=0.011). LIMITATIONS: Female participant sample was relatively small. CONCLUSIONS: Japanese dietary pattern consistently related to low depressive symptoms in this large-scale cohort of Japanese workers, even after adjusting for SES and job stress factors. The protective impact is especially strong for workers with active strain and low support. Making better use of traditional dietary patterns may facilitate reducing social disparities in mental health.

MARCKS dephosphorylation is involved in bradykinin-induced neurite outgrowth in neuroblastoma SH-SY5Y cells.

Bradykinin (BK) plays a major role in producing peripheral sensitization in response to peripheral inflammation and in pain transmission in the central nerve system (CNS). Because BK activates protein kinase C (PKC) through phospholipase C (PLC)-ß and myristoylated alanine-rich C kinase substrate (MARCKS) has been found to be a substrate of PKC, we explored the possibility that BK could induce MARCKS phosphorylation and regulate its function. BK stimulation induced transient MARCKS phosphorylation on Ser159 with a peak at 1 min in human neuroblastoma SH-SY5Y cells. By contrast, PKC activation by the phorbol ester phorbol 12,13-dibutyrate (PDBu) elicited MARCKS phosphorylation which lasted more than 10 min. Western blotting analyses and glutathione S-transferase (GST) pull-down analyses showed that the phosphorylation by BK was the result of activation of the PKC-dependent RhoA/Rho-associated coiled-coil kinase (ROCK) pathway. Protein phosphatase (PP) 2A inhibitors calyculin A and fostriecin inhibited the dephosphorylation of MARCKS after BK-induced phosphorylation. Moreover, immunoprecipitation analyses showed that PP2A interacts with MARCKS. These results indicated that PP2A is the dominant PP of MARCKS after BK stimulation. We established SH-SY5Y cell lines expressing wild-type MARCKS and unphosphorylatable MARCKS, and cell morphology changes after cell stimulation were studied. PDBu induced lamellipodia formation on the neuroblastoma cell line SH-SY5Y and the morphology was sustained, whereas BK induced neurite outgrowth of the cells via lamellipodia-like actin accumulation that depended on transient MARCKS phosphorylation. Thus these findings show a novel BK signal cascade-that is, BK promotes neurite outgrowth through transient MARCKS phosphorylation involving the PKC-dependent RhoA/ROCK pathway and PP2A in a neuroblastoma cell line.

Inhibition of prostaglandin E2 EP3 receptors improves stroke injury via anti-inflammatory and anti-apoptotic mechanisms.

Although deletion of EP3 receptors is known to ameliorate stroke injury in experimental stroke models, the underlying mechanisms and the effects of EP3-specific antagonists remain poorly understood. Here we demonstrate the protective effect of postischemic treatment with an EP3 antagonist, ONO-AE3-240, through anti-inflammatory and anti-apoptotic effects. In transient focal ischemia models, peritoneal injection of an EP3 antagonist after occlusion-reperfusion reduced infarction, edema and neurological dysfunctions to almost the same levels of those in EP3 knockout (KO) mice. Furthermore, neuronal apoptosis in the ischemic cortex investigated by terminal dUTP nick-end labeling (TUNEL) and caspase-3 immunostaining were ameliorated in EP3 antagonist-treated mice or EP3 KO mice as compared with vehicle-treated mice or wild-type (WT) mice, respectively. There were no significant differences between ONO-AE3-240-injected or EP3 KO mice and vehicle-injected or WT mice, respectively, in mean arterial blood pressure, cerebral blood flow or body temperature. The double-immunostaining showed that EP3 receptor-positive cells were also positive for CD-11b and partially for Neu-N, the marker for microglia and neurons. Deletion of EP3 receptors also reduced damage of the blood-brain barrier, activation of microglia and infiltration of neutrophils into the ischemic cortex. These results suggest that EP3 receptors are involved in stroke injury through the enhancement of inflammatory and apoptotic reactions in the ischemic cortex. Thus, EP3 antagonists may be valuable for the treatment of human stroke.

MARCKS regulates lamellipodia formation induced by IGF-I via association with PIP2 and beta-actin at membrane microdomains.

Myristoylated alanine-rich C kinase substrate (MARCKS) is considered to participate in formation of F-actin-based lamellipodia, which represents the first stage of neurite formation. However, the mechanism of how MARCKS is involved in lamellipodia formation is not precisely unknown. Using SH-SY5Y cells, we demonstrated here that MARCKS was translocated from cytosol to detergent-resistant membrane microdomains, known as lipid rafts, within 30 min after insulin-like growth factor-I (IGF-I) stimulation, which was accompanied by MARCKS dephosphorylation, beta-actin accumulation in lipid rafts, and lamellipodia formation. The protein kinase C inhibitor, Ro-31-8220, and Rho-kinase inhibitors, HA1077 and Y27632, themselves decreased basal phosphorylation levels of MARCKS and coincidently elicited translocation of MARCKS to lipid rafts. On the other hand, the phosphoinositide 3-kinase inhibitor, LY294002, abolished IGF-I-induced dephosphorylation, translocation of MARCKS to lipid rafts, and lamellipodia formation. Treatment of cells with neomycin, a PIP2-masking reagent, attenuated the translocation of MARCKS to lipid rafts and the lamellipodia formation induced by IGF-I, although dephosphorylation of MARCKS was not affected. Immunocytochemical and immunoprecipitation analysis indicated that IGF-I stimulation induced the translocation of MARCKS to lipid rafts in the edge of lamellipodia and formation of the complex with PIP2. Moreover, we demonstrated that knockdown of endogenous MARCKS resulted in significant attenuation of IGF-I-induced beta-actin accumulation in the lipid rafts and lamellipodia formation. These results suggest a novel role for MARCKS in lamellipodia formation induced by IGF-I via the translocation of MARCKS, association with PIP2, and accumulation of beta-actin in the membrane microdomains.

Dermatophagoides farinae extract induces severe atopic dermatitis in NC/Nga mice, which is effectively suppressed by the administration of tacrolimus ointment.

Atopic dermatitis (AD) is a chronic inflammatory skin disease, which is accompanied by marked increases in the levels of inflammatory cells, including mast cells and eosinophils as well as T cells and macrophages. To investigate the expression pattern of chemokines in AD, a house dust mite, Dermatophagoides farinae extracts (DfE)-induced NC/Nga AD model was developed in mice, and this model was used to determine the expression levels of chemokines in atopic lesions using DNA microarrays and RT-PCR. When NC/Nga mice were repeatedly treated with DfE for 4 to 7 weeks on the back skin, the mRNA expression levels of CCL20/LARC, CCL24/eotaxin-2, CCL17/TARC, and CCL11/eotaxin-1 were markedly induced and lesser of CCL2/MCP-1, within the inflammatory lesion of the back skin. Immunohistochemical staining revealed the expression of these chemokines in the epidermis and dermis of DfE-treated NC/Nga mice. Interestingly, repeated application of tacrolimus ointment potently inhibited DfE-induced atopic dermatitis in NC/Nga mice concomitant with the inhibition of these changes in chemokine gene and protein expression levels particularly of CCL20/LARC, CCL17/TARC, and CCL11/eotaxin-1. These data indicate that severe atopic dermatitis induced by DfE accompanies elevated chemokine levels, and it was proposed that tacrolimus ointment is beneficial for the treatment of severe AD.

Unphosphorylated MARCKS is involved in neurite initiation induced by insulin-like growth factor-I in SH-SY5Y cells.

Myristoylated alanine-rich C kinase substrate (MARCKS) has been suggested to be involved in various aspects of neuronal cell differentiation, including neurite outgrowth. However, the precise mechanisms by which MARCKS phosphorylation is regulated, and how MARCKS contributes to neurite outgrowth, are poorly understood. Here, we found that treatment of SH-SY5Y cells with insulin-like growth factor-I (IGF-I) induced a rapid and transient decrease in the level of phosphorylated MARCKS (P-MARCKS) to below the basal level. The decrease in P-MARCKS induced by IGF-I was blocked by pretreatment of cells with phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 and wortmannin. A decrease in P-MARCKS was also observed in cells treated with a Rho-dependent kinase (ROCK) inhibitor, Y27632. Furthermore, IGF-I induced transient inactivation of RhoA, an upstream effector of ROCK. We showed that MARCKS was translocated to the membrane and colocalized with F-actin at the lamellipodia and the tips of neurites in the cells stimulated with IGF-I. Finally, overexpression of wild-type MARCKS or an unphosphorylatable mutant of MARCKS enhanced the number of neurite-bearing cells relative to vector-transfected cells. Taken together, these findings suggest that unphosphorylated MARCKS is involved in neurite initiation, and highlight the important role played by MARCKS in organization of the actin cytoskeleton.

Linalyl acetate as a major ingredient of lavender essential oil relaxes the rabbit vascular smooth muscle through dephosphorylation of myosin light chain.

In a preliminary experiment, we found that lavender essential oil relaxes vascular smooth muscle. Thus, the present experiments were designed to investigate the relaxation mechanism of linalyl acetate as the major ingredient of lavender essential oil in rabbit carotid artery specimens. Linalyl acetate produced sustained and progressive relaxation during the contraction caused by phenylephrine. The relaxation effect of linalyl acetate at a concentration near the EC50 was partially but significantly attenuated by nitroarginine as an inhibitor of nitric oxide synthase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one as an inhibitor of guanylyl cyclase, or by the denudation of endothelial cells. In specimens without endothelium, the phenylephrine-induced contraction and phosphorylation of myosin light chain (MLC) were significantly attenuated after the pretreatment with linalyl acetate. The relaxation caused by linalyl acetate in the endothelium-denuded specimens was clearly inhibited by calyculin A as an inhibitor of MLC phosphatase, although not by ML-9 as an inhibitor of MLC kinase. Furthermore, suppression of the phenylephrine-induced contraction and MLC phosphorylation with linalyl acetate was canceled by the pretreatment with calyculin A. These results suggest that linalyl acetate relaxes the vascular smooth muscle through partially activation of nitric oxide/cyclic guanosine monophosphate pathway, and partially MLC dephosphorylation via activating MLC phosphatase.

Microsomal prostaglandin E synthase-1 is a critical factor of stroke-reperfusion injury.

Although augmented prostaglandin E(2) (PGE(2)) synthesis and accumulation have been demonstrated in the lesion sites of rodent transient focal ischemia models, the role of PGE(2) in neuronal survival has been controversial, showing both protective and toxic effects. Here we demonstrate the induction of microsomal PGE synthase 1 (mPGES-1), an inducible terminal enzyme for PGE(2) synthesis, in neurons, microglia, and endothelial cells in the cerebral cortex after transient focal ischemia. In mPGES-1 knockout (KO) mice, in which the postischemic PGE(2) production in the cortex was completely absent, the infarction, edema, apoptotic cell death, and caspase-3 activation in the cortex after ischemia were all reduced compared with those in wild-type (WT) mice. Furthermore, the behavioral neurological dysfunctions observed after ischemia in WT mice were significantly ameliorated in KO mice. The ameliorated symptoms observed in KO mice after ischemia were reversed to almost the same severity as WT mice by intracerebroventricular injection of PGE(2) into KO mice. Our observations suggest that mPGES-1 may be a critical determinant of postischemic neurological dysfunctions and a valuable therapeutic target for treatment of human stroke.

Investigation of the pronounced synergism between prostaglandin E2 and other constrictor agents on rat femoral artery.

This study investigates the pronounced synergism between the weak contractile action of prostaglandin E(2) (PGE(2)) and strong actions of phenylephrine, U-46619 and K(+) on rat isolated femoral artery. The potency ranking for synergism was SC-46275 (prostanoid receptor agonist selectivity: EP(3)>>EP(1))=sulprostone (EP(3)>EP(1))>17-phenyl PGE(2) (EP(1)>EP(3)). The novel EP(3) antagonist L-798106 (0.2-1microM) blocked the enhanced action of sulprostone (pA(2)=7.35-8.10), while the EP(1) antagonist SC-51322 (1microM) did not (pA(2)<6.0). Matching responses to priming agent and priming agent/sulprostone were similarly suppressed by nifedipine (300nM) and the selective Rho-kinase inhibitors H-1152 (0.1-1microM) and Y-27632 (1-10microM). Our findings implicate an EP(3) receptor in the prostanoid component of contractile synergism. While the synergism predominantly operates through a Ca(2+) influx-Rho-kinase pathway, the EP(3) receptor does not necessarily transduce via Rho-kinase.

Rho-kinase mediates spinal nitric oxide formation by prostaglandin E2 via EP3 subtype.

Prostaglandin E2 (PGE2), the principal pro-inflammatory prostanoid, is known to play versatile roles in pain transmission via four PGE receptor subtypes, EP1-EP4. We recently demonstrated that continuous production of nitric oxide (NO) by neuronal NO synthase (nNOS) following phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) and NMDA receptor NR2B subunits is essential for neuropathic pain. These phosphorylation and nNOS activity visualized by NADPH-diaphorase histochemistry were blocked by indomethacin, a PG synthesis inhibitor. To clarify the interaction between cyclooxygenase and nNOS pathways in the spinal cord, we examined the effect of EP subtype-selective agonists on NO production. NO formation was stimulated in the spinal superficial layer by EP1, EP3, and EP4 agonists. While the EP1- and the EP4-stimulated NO formation was markedly blocked by MK-801, an NMDA receptor antagonist, the EP3-stimulated one was completely inhibited by H-1152, a Rho-kinase inhibitor. Phosphorylation of MARCKS and NADPH-diaphorase activity stimulated by the EP3 agonist were also blocked by H-1152. These results suggest that PGE2 stimulates NO formation by Rho-kinase via EP3, a mechanism(s) different from EP1 and EP4.

MARCKS is a major PKC-dependent regulator of calmodulin targeting in smooth muscle.

Calmodulin (CaM) is a ubiquitous transducer of intracellular Ca(2+) signals and plays a key role in the regulation of the function of all cells. The interaction of CaM with a specific target is determined not only by the Ca(2+)-dependent affinity of calmodulin but also by the proximity to that target in the cellular environment. Although a few reports of stimulus-dependent nuclear targeting of CaM have appeared, the mechanisms by which CaM is targeted to non-nuclear sites are less clear. Here, we investigate the hypothesis that MARCKS is a regulator of the spatial distribution of CaM within the cytoplasm of differentiated smooth-muscle cells. In overlay assays with portal-vein homogenates, CaM binds predominantly to the MARCKS-containing band. MARCKS is abundant in portal-vein smooth muscle ( approximately 16 microM) in comparison to total CaM ( approximately 40 microM). Confocal images indicate that calmodulin and MARCKS co-distribute in unstimulated freshly dissociated smooth-muscle cells and are co-targeted simultaneously to the cell interior upon depolarization. Protein-kinase-C (PKC) activation triggers a translocation of CaM that precedes that of MARCKS and causes multisite, sequential MARCKS phosphorylation. MARCKS immunoprecipitates with CaM in a stimulus-dependent manner. A synthetic MARCKS effector domain (ED) peptide labelled with a photoaffinity probe cross-links CaM in smooth-muscle tissue in a stimulus-dependent manner. Both cross-linking and immunoprecipitation increase with increased Ca(2+) concentration, but decrease with PKC activation. Introduction of a nonphosphorylatable MARCKS decoy peptide blocks the PKC-mediated targeting of CaM. These results indicate that MARCKS is a significant, PKC-releasable reservoir of CaM in differentiated smooth muscle and that it contributes to CaM signalling by modulating the intracellular distribution of CaM.

Microglia-specific expression of microsomal prostaglandin E2 synthase-1 contributes to lipopolysaccharide-induced prostaglandin E2 production.

Microsomal prostaglandin E2 synthase (mPGES)-1 is an inducible protein recently shown to be an important enzyme in inflammatory prostaglandin E2 (PGE2) production in some peripheral inflammatory lesions. However, in inflammatory sites in the brain, the induction of mPGES-1 is poorly understood. In this study, we demonstrated the expression of mPGES-1 in the brain parenchyma in a lipopolysaccharide (LPS)-induced inflammation model. A local injection of LPS into the rat substantia nigra led to the induction of mPGES-1 in activated microglia. In neuron-glial mixed cultures, mPGES-1 was co-induced with cyclooxygenase-2 (COX-2) specifically in microglia, but not in astrocytes, oligodendrocytes or neurons. In microglia-enriched cultures, the induction of mPGES-1, the activity of PGES and the production of PGE2 were preceded by the induction of mPGES-1 mRNA and almost completely inhibited by the synthetic glucocorticoid dexamethasone. The induction of mPGES-1 and production of PGE2 were also either attenuated or absent in microglia treated with mPGES-1 antisense oligonucleotide or microglia from mPGES-1 knockout (KO) mice, respectively, suggesting the necessity of mPGES-1 for microglial PGE2 production. These results suggest that the activation of microglia contributes to PGE2 production through the concerted de novo synthesis of mPGES-1 and COX-2 at sites of inflammation of the brain parenchyma.