Neuroprotective Effects of the DPP4 Inhibitor Vildagliptin in In Vivo and In Vitro Models of Parkinson's Disease.

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the midbrain. Restoration of nigrostriatal dopamine neurons has been proposed as a potential therapeutic strategy for PD. Because currently used PD therapeutics only help relieve motor symptoms and do not treat the cause of the disease, highly effective drugs are needed. Vildagliptin, a dipeptidyl peptidase 4 (DPP4) inhibitor, is an anti-diabetic drug with various pharmacological properties including neuroprotective effects. However, the detailed effects of vildagliptin against PD are not fully understood. We investigated the effects of vildagliptin on PD and its underlying molecular mechanisms using a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model and a 1-methyl-4-phenylpyridium (MPP+)-induced cytotoxicity model. Vildagliptin (50 mg/kg) administration significantly attenuated MPTP-induced motor deficits as evidenced by rotarod, pole, and nest building tests. Immunohistochemistry and Western blot analysis revealed that vildagliptin increased tyrosine hydroxylase-positive cells in the SNpc and striatum, which was reduced by MPTP treatment. Furthermore, vildagliptin activated MPTP-decreased PI3k/Akt and mitigated MPTP-increased ERK and JNK signaling pathways in the striatum. Consistent with signaling transduction in the mouse striatum, vildagliptin reversed MPP+-induced dephosphorylation of PI3K/Akt and phosphorylation of ERK and JNK in SH-SY5Y cells. Moreover, vildagliptin attenuated MPP+-induced conversion of LC3B-II in SH-SY5Y cells, suggesting its role in autophagy inhibition. Taken together, these findings indicate that vildagliptin has protective effects against MPTP-induced motor dysfunction by inhibiting dopaminergic neuronal apoptosis, which is associated with regulation of PI3k/Akt, ERK, and JNK signaling transduction. Our findings suggest vildagliptin as a promising repurposing drug to treat PD.

Limonene has anti-anxiety activity via adenosine A2A receptor-mediated regulation of dopaminergic and GABAergic neuronal function in the striatum.

BACKGROUND: Limonene, a common terpene found in citrus fruits, is assumed to reduce stress and mood disorders. Dopamine and γ-aminobutyric acid (GABA) have been reported to play an important role in modulating anxiety in different parts of the brain. HYPOTHESIS/PURPOSE: Herein, we report the anxiolytic activity of limonene. In addition, we identified a possible mechanism underlying the effect of limonene on DAergic and GABAergic neurotransmission. STUDY DESIGN: In this study, mice were injected with saline in the control group and limonene in the test group before behavioral analysis. We performed immunoblotting and high-performance liquid chromatography (HPLC) analysis after the behavioral study. RESULTS: The limonene treated group showed increased locomotor activity and open-arm preference in the elevated plus maze experiment. Limonene treatment increased the expression of both tyrosine hydroxylase and GAD-67 proteins and significantly upregulated dopamine levels in the striatum. Furthermore, tissue dopamine levels were increased in the striatum of mice following limonene treatment, and depolarization-induced GABA release was enhanced by limonene pre-treatment in PC-12 cells. Interestingly, limonene-induced anxiolytic activity and GABA release augmentation were blocked by an adenosine A2A receptor (A2AR) antagonist. CONCLUSION: Our results suggest that limonene inhibits anxiety-related behavior through A2A receptor-mediated regulation of DAergic and GABAergic neuronal activity.

Sulfuretin Attenuates MPP⁺-Induced Neurotoxicity through Akt/GSK3ß and ERK Signaling Pathways.

Parkinson's disease (PD) is the second most common neurodegenerative disease. It is caused by the death of dopaminergic neurons in the substantia nigra pars compacta. Oxidative stress and mitochondrial dysfunction contribute to the loss of dopaminergic neurons in PD. Sulfuretin is a potent antioxidant that is reported to be beneficial in the treatment of neurodegenerative diseases. In this study, we examined the protective effect of sulfuretin against 1-methyl-4-phenyl pyridinium (MPP⁺)-induced cell model of PD in SH-SY5Y cells and the underlying molecular mechanisms. Sulfuretin significantly decreased MPP⁺-induced apoptotic cell death, accompanied by a reduction in caspase 3 activity and polyADP-ribose polymerase (PARP) cleavage. Furthermore, it attenuated MPP⁺-induced production of intracellular reactive oxygen species (ROS) and disruption of mitochondrial membrane potential (MMP). Consistently, sulfuretin decreased p53 expression and the Bax/Bcl-2 ratio. Moreover, sulfuretin significantly increased the phosphorylation of Akt, GSK3ß, and ERK. Pharmacological inhibitors of PI3K/Akt and ERK abolished the cytoprotective effects of sulfuretin against MPP⁺. An inhibitor of GSK3ß mimicked sulfuretin-induced protection against MPP⁺. Taken together, these results suggest that sulfuretin significantly attenuates MPP⁺-induced neurotoxicity through Akt/GSK3ß and ERK signaling pathways in SH-SY5Y cells. Our findings suggest that sulfuretin might be one of the potential candidates for the treatment of PD.

Vitis labruscana leaf extract ameliorates scopolamine-induced impairments with activation of Akt, ERK and CREB in mice.

BACKGROUND: Grapes are among the most widely consumed plants and are used as a folk medicine. Vitis species have been traditionally used as anti-inflammatory, analgesic, and memory-enhancing agents, but, their biological activities of discarded grape leaves are not completely understood. PURPOSE: We investigated the effects of alcoholic aqueous leaf extract of Vitis labruscana (LEVL) in a mouse model of memory impairment and tried to ascertain its mechanism. We also evaluated its effects in SH-SY5Y cells. METHODS: LEVL (50, 100, and 150 mg/kg) was administered to ICR mice once daily for 7 days. Memory impairment was induced with intraperitoneal scopolamine injections (1 mg/kg) and measured with the Y-maze test and a passive avoidance task. LEVL-induced signaling was evaluated in SH-SY5Y cells and mouse hippocampi. RESULTS: We first identified quercetin-3-O-glucuronide as LEVL's major component. We then showed that LEVL promoted phosphorylation of Akt, extracellular regulated kinase (ERK), and cyclic AMP response element binding protein (CREB) and proliferation of SH-SY5Y cells. Oral LEVL administration (100 mg/kg) for 7 days significantly reversed scopolamine-induced reductions of spontaneous alternation in the Y-maze test and scopolamine-induced shortening of latency times in the passive avoidance task's retention trial. Consistent with the cell experiment results, LEVL restored scopolamine-decreased phosphorylation of Akt, ERK, and CREB and scopolamine-reduced expression of brain-derived neuroprotective factor expression in mouse hippocampi. CONCLUSION: Our results suggest that LEVL promotes phosphorylation of Akt, ERK, and CREB in the hippocampus and ameliorates scopolamine-induced memory impairment in mice.

ROS Production and ERK Activity Are Involved in the Effects of d-ß-Hydroxybutyrate and Metformin in a Glucose Deficient Condition.

Hypoglycemia, a complication of insulin or sulfonylurea therapy in diabetic patients, leads to brain damage. Furthermore, glucose replenishment following hypoglycemic coma induces neuronal cell death. In this study, we investigated the molecular mechanism underlying glucose deficiency-induced cytotoxicity and the protective effect of d-ß-hydroxybutyrate (D-BHB) using SH-SY5Y cells. The cytotoxic mechanism of metformin under glucose deficiency was also examined. Cell viability under 1 mM glucose (glucose deficiency) was significantly decreased which was accompanied by increased production of reactive oxygen species (ROS) and decreased phosphorylation of extracellular signal-regulated kinase (ERK) and glycogen synthase 3 (GSK3ß). ROS inhibitor reversed the glucose deficiency-induced cytotoxicity and restored the reduced phosphorylation of ERK and GSK3ß. While metformin did not alter cell viability in normal glucose media, it further increased cell death and ROS production under glucose deficiency. However, D-BHB reversed cytotoxicity, ROS production, and the decrease in phosphorylation of ERK and GSK3ß induced by the glucose deficiency. ERK inhibitor reversed the D-BHB-induced increase in cell viability under glucose deficiency, whereas GSK3ß inhibitor did not restore glucose deficiency-induced cytotoxicity. Finally, the protective effect of D-BHB against glucose deficiency was confirmed in primary neuronal cells. We demonstrate that glucose deficiency-induced cytotoxicity is mediated by ERK inhibition through ROS production, which is attenuated by D-BHB and intensified by metformin.

Cearoin Induces Autophagy, ERK Activation and Apoptosis via ROS Generation in SH-SY5Y Neuroblastoma Cells.

Neuroblastomas are the most common solid extracranial tumors in childhood. We investigated the anticancer effect of cearoin isolated from Dalbergia odorifera in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were treated with various doses of cearoin. The viability was measured by MTT assay. DCFDA fluorescence assay and Griess assay were used for the measurement of intracellular reactive oxygen species (ROS) and nitric oxide (NO), respectively. Western blot analysis was performed to clarify the molecular pathway involved. Cearoin induced cell death in a dose-dependent manner. Cearoin increased the phosporylation of ERK, the conversion of LC3B-I to LC3B-II, decrease in Bcl2 expression, the activation of caspase-3, and the cleavage of PARP, indicating the induction of autophagy and apoptosis. Furthermore, cearoin treatment increased the production of ROS and NO. Co-treatment with the antioxidant N-acetylcysteine completely abolished cearoin-mediated autophagy, ERK activation and apoptosis, suggesting the critical role of ROS in cearoin-induced anticancer effects. Moreover, co-treatment with ERK inhibitor PD98059 partially reversed cearoin-induced cell death, indicating the involvement of ERK in cearoin anticancer effects. These data reveal that cearoin induces autophagy, ERK activation and apoptosis in neuroblastoma SH-SY5Y cells, which is mediated primarily by ROS generation, suggesting its therapeutic application for the treatment of neuroblastomas.

Quercetin-3-O-glucuronide promotes the proliferation and migration of neural stem cells.

Quercetin is a bioactive compound exerting therapeutic effects on in vivo animal models of neurodegeneration or neurotoxicity. However, the narrow therapeutic dose-range of quercetin has been a point of concern since previous studies have demonstrated that quercetin induces cytotoxicity in vitro. Quercetin is metabolized to quercetin glucuronates such as quercetin-3-O-glucuronide (Q3GA), primarily detected in the plasma and the brain. Here, we examined whether and how quercetin or Q3GA regulates neural stem cells (NSCs) in vivo and in vitro. Immunohistochemistry showed that oral administration of quercetin increased nestin-, DCX-, BrdU/DCX-, and BrdU/NeuN-positive cells in the dentate gyrus of mice. However, quercetin decreased the viability of human embryonic NSCs in culture, accompanied by decreased Akt phosphorylation and increased cleavage of caspase-3 and PARP. In contrast, Q3GA increased BrdU-positive cell proliferation, Akt phosphorylation, and cyclin D1 expression. PI3K/Akt inhibitor LY294002 reversed Q3GA-induced Akt phosphorylation and cyclin D1 expression, thereby reducing Q3GA-induced proliferation. Furthermore, Q3GA increased the protein secretion of BDNF and its blockade using anti-BDNF antibody reversed Q3GA-induced proliferation. Under differentiation state, Q3GA promotes NSC migration, along with increased mRNA expression of CXCR4. Moreover, Q3GA significantly reversed scopolamine-induced reduction of Akt phosphorylation in the mouse hippocampus and ameliorated scopolamine-induced memory impairments. Our results demonstrate that quercetin and its metabolite Q3GA control NSC viability in a converse manner through contrary regulation of Akt, accounting for the conflicting effects of quercetin in vivo and in vitro. This study provides a novel mechanism for the positive effects of Q3GA on neurogenesis and suggests its therapeutic potential in neurodegenerative diseases.

Primary malignant melanoma of cervix and vagina.

Primary malignant melanoma (MM) accounts for 1% of all cancers, and only 3% to 7% of these tumors occur in the female genital tract. Data are limited with respect to the basis for treatment recommendations because of the rarity of MM. The overall prognosis of melanomas of the female genital tract is very poor. Two cases of MM of the female genital tract are presented. The first case is of a 70-year-old female patient who complained of left thigh pain and underwent magnetic resonance imaging that showed cervical cancer with involvement of the vagina, bladder, and parametrium, in addition to multiple bony metastases of the proximal femur, acetabulum, and both iliac bones. The second case is of a 35-year-old female patient who suffered from vaginal bleeding for 5 months, and she was diagnosed as having primary vaginal melanoma. The patient underwent radical surgery and two additional surgeries because of recurrence of cancer in both inguinal areas. After surgery, the patient received adjuvant immunotherapy, radiation therapy, and chemotherapy. In both the aforementioned cases, the pathologic diagnosis was made after immunohistochemical analysis, i.e., the tumor cells were stained with HMB-45 and S100, and were found to be positive for both immunostains.

A Prenylated Xanthone, Cudratricusxanthone A, Isolated from Cudrania tricuspidata Inhibits Lipopolysaccharide-Induced Neuroinflammation through Inhibition of NF-κB and p38 MAPK Pathways in BV2 Microglia.

Cudrania tricuspidata Bureau (Moraceae) is an important source of traditional Korean and Chinese medicines used to treat neuritis and inflammation. Cudratricusxanthone A (1), a prenylated xanthone, isolated from C. tricuspidata, has a variety of biological and therapeutic activities. The goal of this study was to examine the effects of compound 1 on neuroinflammation and characterize its mechanism of action in lipopolysaccharide (LPS)-stimulated BV2 microglia. Cudratricusxanthone A (1) suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 enzymes and decreased the production of iNOS-derived nitric oxide and COX-2-derived prostaglandin E2 in LPS-stimulated mouse BV2 microglia. The compound also decreased tumor necrosis factor-α, interleukin (IL)-1ß, and IL-12 production; inhibited the phosphorylation and degradation of IκB-α; and blocked the nuclear translocation of p50 and p65 in mouse BV2 microglia induced by LPS. Cudratricusxanthone A (1) had inhibitory effects on nuclear factor kappa B DNA-binding activity. Additionally, it inhibited the p38 mitogen-activated protein kinase signaling pathway. Our data suggests that cudratricusxanthone A (1) may be a useful therapeutic agent in the treatment of neurodegenerative diseases caused by neuroinflammation.

Citron Rho-interacting kinase mediates arsenite-induced decrease in endothelial nitric oxide synthase activity by increasing phosphorylation at threonine 497: Mechanism underlying arsenite-induced vascular dysfunction.

We reported that arsenite causes an acute decrease in nitric oxide (NO) production by increasing phosphorylation of endothelial NO synthase at threonine 497 (eNOS-Thr(497)); however, the detailed mechanism has not yet been clarified. Here, we investigated the kinase involving in arsenite-stimulated eNOS-Thr(497) phosphorylation. Although treatment with H-89, a known protein kinase A (PKA) inhibitor, inhibited arsenite-stimulated eNOS-Thr(497) phosphorylation, no inhibition was found in cells treated with other PKA inhibitors, including Rp-8-Br-cAMPS or PKI. Based on previous reports, we also tested whether RhoA mediates arsenite-stimulated eNOS-Thr(497) phosphorylation and found that arsenite causes an acute increase in RhoA activity. Ectopic expression of dominant negative (DN)-RhoA significantly reversed arsenite-stimulated eNOS-Thr(497) phosphorylation. An in vitro phosphorylation assay also revealed that the well-known Rho effectors, Rho-associated protein kinase 1/2 (ROCK1/2), directly phosphorylate eNOS-Thr(497). Y27632, a selective ROCK inhibitor, reversed arsenite-stimulated eNOS-Thr(497) phosphorylation. However, overexpression of a small interfering RNA (siRNA) against ROCK1/2 or DN-ROCK did not reverse arsenite-stimulated eNOS-Thr(497) phosphorylation, thereby providing no conclusive evidence of a role for ROCK1/2. Knockdown of PKC-related protein kinase 1/2, another Rho effector, also did not reverse arsenite-stimulated eNOS-Thr(497) phosphorylation. In contrast, we found that transfection with an siRNA against citron Rho-interacting kinase (CRIK), the other downstream effector of Rho, significantly reversed the arsenite-induced eNOS-Thr(497) phosphorylation that was accompanied by restoration of eNOS enzymatic activity repressed by arsenite. Moreover, CRIK directly phosphorylated eNOS-Thr(497)in vitro. Finally, we also found that arsenite increased eNOS-Thr(497) phosphorylation and decreased acetylcholine-induced vessel relaxation in rat aortas. In conclusion, we demonstrate that arsenite acutely inhibits eNOS enzymatic activity and vessel relaxation in part by increasing the RhoA/CRIK/eNOS-Thr(497) phosphorylation signaling axis, which provides a molecular mechanism underlying arsenite-induced impaired vascular diseases.

Telmisartan attenuates hyperglycemia-exacerbated VCAM-1 expression and monocytes adhesion in TNFα-stimulated endothelial cells by inhibiting IKKß expression.

Uncontrolled hyperglycemia accelerates endothelial damage and vascular inflammation caused by proinflammatory cytokines including tumor necrosis factor α (TNFα), which leads to arteriosclerotic cardiovascular diseases such as myocardial infarction. Telmisartan, an angiotensin II type 1 receptor blocker (ARB), is prescribed for treatment of hypertensive patients with concurrent diabetes mellitus (DM). Although a few clinical trials have suggested that telmisartan decreases cardiovascular complications in diabetic patients, the molecular mechanism for the beneficial effects remains elusive. Here, we investigated a molecular mechanism and effects of telmisartan on the expression of vascular cell adhesion molecule-1 (VCAM-1) and attachment of monocytes onto endothelial cells induced by TNFα in hyperglycemia-treated bovine aortic endothelial cells (BAEC). Telmisartan dose-dependently decreased hyperglycemia-aggravated IκB kinase ß (IKKß) expression and nuclear factor-κB (NF-κB) p65-Ser(536) phosphorylation, which accompanied a decrease in VCAM-1 expression and THP-1 monocytes adhesion. Among ARBs, including losartan and fimasartan, only telmisartan showed the inhibitory effects on expression of VCAM-1 and IKKß, and phosphorylation of NF-κB p65-Ser(536). The telmisartan's beneficial effects were not changed by pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist, although GW9662 clearly inhibited rosiglitazone-induced CD36 expression. Finally, ectopic expression of wild type (WT)-IKKß significantly restored telmisartan-attenuated VCAM-1 expression, NF-κB p65-Ser(536) phosphorylation, and THP-1 monocytes adhesion. Taken together, our findings demonstrate that telmisartan ameliorates hyperglycemia-exacerbated vascular inflammation, at least in part, by decreasing expression of IKKß and VCAM-1 independently of PPARγ. Telmisartan may be useful for the treatment of DM-associated vascular inflammation and cardiovascular diseases.

Arsenite Acutely Decreases Nitric Oxide Production via the ROS-Protein Phosphatase 1-Endothelial Nitric Oxide Synthase-Thr(497) Signaling Cascade.

Chronic (>24 h) exposure of arsenite, an environmental toxicant, has shown the decreased nitric oxide (NO) production in endothelial cells (EC) by decreasing endothelial NO synthase (eNOS) expression and/or its phosphorylation at serine 1179 (eNOS-Ser(1179) in bovine sequence), which is associated with increased risk of vascular diseases. Here, we investigated the acute (<24 h) effect of arsenite on NO production using bovine aortic EC (BAEC). Arsenite acutely increased the phosphorylation of eNOS-Thr(497), but not of eNOS-Ser(116) or eNOS-Ser(1179), which was accompanied by decreased NO production. The level of eNOS expression was unaltered under this condition. Treatment with arsenite also induced reactive oxygen species (ROS) production, and pretreatment with a ROS scavenger N-acetyl-L-cysteine (NAC) completely reversed the observed effect of arsenite on eNOS-Thr(497) phosphorylation. Although protein kinase C (PKC) and protein phosphatase 1 (PP1) were reported to be involved in eNOS-Thr(497) phosphorylation, treatment with PKC inhibitor, Ro318425, and overexpression of various PKC isoforms did not affect the arsenite-stimulated eNOS-Thr(497) phosphorylation. In contrast, treatment with PP1 inhibitor, calyculin A, mimicked the observed effect of arsenite on eNOS-Thr(497) phosphorylation. Lastly, we found decreased cellular PP1 activity in arsenite-treated cells, which was reversed by NAC. Overall, our study demonstrates firstly that arsenite acutely decreases NO production at least in part by increasing eNOS-Thr(497) phosphorylation via ROS-PP1 signaling pathway, which provide the molecular mechanism underlying arsenite-induced increase in vascular disease.

Aggressive angiomyxoma of the vulva: A case report.

Aggressive angiomyxoma (AA) is an unusual mesenchymal tumor. AA occurs most commonly in women of reproductive age and is located in the perineal or pelvic region. This is a distinct soft tissue tumor that has a prominent myxoid matrix and numerous thin-walled blood vessels and may have an aggressive local recurrence. The tumors have the characteristics of large size (usually greater than 10 cm) and slow growth, and are not painful. The standard treatment for AA is total excision and close follow-up. We announce a case of a 35 year-old female presenting with a pedunculated AA on the right labium majora that has not relapsed for seven years.

Angiomyofibroblastoma of the vulva.

Angiomyofibroblastoma (AMFB) is an uncommon benign mesenchymal tumor. AMFB occurs almost in the vulvo-vaginal area of women. The gross features of AMFB are well-circumscribed so it clinically is often thought as Bartholin gland cyst or aggressive angiomyxoma. Usually, most tumors grow slowly, and patients do not feel pain. It also has low tendency for local recurrence. The histologic findings of the tumors are abundant thin-walled blood vessels with hypocellular and hypercellular areas. Almost all tumor cells have immunoreactivity for both desmin and vimentin. It also has estrogen and/or progesterone receptors, but staining for cytokeratin is negative. Here is a case of AMFB of the vulva occurring in a 40-year-old woman, involving the right labia majora. The patient described that her vulva mass grew in about few months. The maximum dimension of the tumor was measured as 2 cm, and we resected the tumor one month after as her second visit.

Trichostatin A epigenetically increases calpastatin expression and inhibits calpain activity and calcium-induced SH-SY5Y neuronal cell toxicity.

Calpains are involved in calcium-induced neuronal cell toxicity, which is associated with the pathophysiology of Alzheimer's disease (AD). The activity of calpains is regulated by the inhibitor calpastatin, and increased activity of calpains and decreased calpastastin are often found in AD. Histone deacetylase (HDAC) inhibitors are implicated in AD treatment through the improvement of learning and memory but the underlying mechanism is yet to be understood. Here, using SH-SY5Y neuroblastoma cells and a calcium ionophore ionomycin, we examined whether and how HDAC inhibitor trichostatin A (TSA) inhibits calcium-induced neuronal cell death. TSA increased both the mRNA and protein levels of calpastatin, with no alterations in those of calpain 1 and calpain 2. Furthermore, TSA-stimulated increase of calpastatin was accompanied by a significant attenuation of ionomycin-induced autolysis of calpain 1, but not of calpain 2, and calpain-dependent 150 kDa αII spectrin cleavage. Under these conditions, however, caspase activity was unaltered. Moreover, ectopic expression of small interfering RNA of calpastatin reversed the inhibitory effect of TSA on ionomycin-induced calpain 1 autolysis and αII spectrin cleavage. Chromatin immunoprecipitation assay revealed the increased levels of acetylation at lysine 5 of histone H4 (H4K5-Ac), H3K9-Ac and H3K14-Ac within the calpastatin promoter region in TSA-treated cells relative to control cells. Finally, TSA significantly decreased ionomycin-induced cell toxicity. This study demonstrates that TSA attenuates calcium-induced neuronal cell death by the inhibition of calpain activity which is mediated in part by increased calpastatin expression via histone hyperacetylation within the calpastatin promoter region. Our study provides a novel mechanism for the neuroprotective effect of HDAC inhibitors on AD.

Coordinated regulation of angiopoietin-1 and vascular endothelial growth factor by arsenite in human brain microvascular pericytes: implications of arsenite-induced vascular dysfunction.

Arsenite is an environmental toxicant that is associated with vascular disease; however, the underlying mechanism of its toxicity has yet to be elucidated. Vascular stability appears to be tightly regulated by several vasoactive proteins produced by two adjacent vascular cells, endothelial cells (EC) and pericytes. The disruption of vascular stability may be involved in arsenite toxicity. The roles of angipoietins (Ang) and vascular endothelial growth factor (VEGF) in this process have been evaluated, but these studies have mostly been limited to EC. In this study, we used human brain microvascular pericytes (HBMP) to evaluate the effects of arsenite on Ang-1 and VEGF regulation. Ang-2 was reported to be not detected in HBMP. Arsenite decreased Ang-1 secretion in a time and dose-dependent manner, while it increased VEGF secretion. Although arsenite did not alter Ang-1 mRNA expression, it increased intracellular Ang-1 protein levels in a dose-dependent manner, suggesting a role for arsenite in the intracellular trapping of Ang-1. Contrary to Ang-1, the expression of VEGF mRNA was dose-dependently up-regulated by arsenite. Treatment with N-actyl-l:-cysteine (NAC) alone decreased the release of Ang-1, but failed to attenuate the arsenite-induced decrease in Ang-1 secretion, while NAC completely blocked the arsenite-stimulated VEGF secretion. These results indicate that reactive oxygen species are involved in the regulation of VEGF, but not of Ang-1, secretion in response to arsenite treatment in pericytes. Furthermore, immunocytochemical analysis using confocal microscopy revealed a colocalization of Ang-1 with actin filaments that occurred independently of tubulin. In conclusion, arsenite decreases Ang-1 secretion and increases VEGF secretion, which may offer new insight into understanding the arsenite toxicity associated with vascular instability and subsequent development of vascular disease.

Blockage by SP600125 of Fcepsilon receptor-induced degranulation and cytokine gene expression in mast cells is mediated through inhibition of phosphatidylinositol 3-kinase signalling pathway.

SP600125 is used as a specific inhibitor of c-Jun N-terminal kinase (JNK). We initially aimed to examine physiological roles of JNK in mast cells that play a central role in inflammatory and immediate allergic responses. We found that Fc receptor for IgE (FcepsilonRI)-induced degranulation (serotonin release) and cytokine gene expression [interleukin (IL)-6, tumour necrosis factor-alpha and IL-13] in bone marrow-derived mast cells, were almost completely inhibited by SP600125. However, the time course of FcepsilonRI-induced JNK activation did not correlate with that of serotonin release. Furthermore, FcepsilonRI-induced degranulation and cytokine gene expression were not impaired in a JNK activator, MKK7-deficient mast cells, in which JNK activation was lost. These results indicate that the inhibitory effects by SP600125 are not due to impaired JNK activation. Instead, we found that SP600125 markedly inhibited the FcepsilonRI-induced activation of phosphatidylinositol 3-kinase (PI3K) and Akt, the same as a PI3K inhibitor, wortmannin. Finally, we found that SP600125 specifically inhibits delta form of p110 catalytic subunit (p110delta) of PI3K. Thus, SP600125 exerts its influence on mast cell functions by inhibiting the kinase activity of PI3K, but not JNK.

SEK1/MKK4-mediated SAPK/JNK signaling participates in embryonic hepatoblast proliferation via a pathway different from NF-kappaB-induced anti-apoptosis.

Mice lacking the stress-signaling kinase SEK1 die from embryonic day 10.5 (E10.5) to E12.5. Although a defect in liver formation is accompanied with the embryonic lethality of sek1(-/-) mice, the mechanism of the liver defect has remained unknown. In the present study, we first produced a monoclonal antibody specifically recognizing murine hepatoblasts for the analysis of liver development and further investigated genetic interaction ofsek1 with tumor necrosis factor-alpha receptor 1 gene (tnfr1) and protooncogene c-jun, which are also responsible for liver formation and cell apoptosis. The defective liver formation in sek1(-/-) embryos was not protected by additionaltnfr1 mutation, which rescues the embryonic lethality of mice lacking NF-kappaB signaling components. There was a progressive increase in the hepatoblast cell numbers of wild-type embryos from E10.5 to E12.5. Instead, impaired hepatoblast proliferation was observed in sek1(-/-) livers from E10.5, though fetal liver-specific gene expression was normal. The impaired phenotype in sek1(-/-) livers was more severe than in c-jun(-/-) embryos, and sek1(-/-) c-jun(-/-) embryos died more rapidly before E8.5. The hepatoblast proliferation required no hematopoiesis, since liver development was not impaired in AML1(-/-) mice that lack hematopoietic functions. Stimulation of stress-activated protein kinase/c-Jun N-terminal kinase by hepatocyte growth factor was attenuated in sek1(-/-) livers. Thus, SEK1 appears to play a crucial role in hepatoblast proliferation and survival in a manner apparently different from NF-kappaB or c-Jun.

Activation of extracellular signal-regulated kinase by ultraviolet is mediated through Src-dependent epidermal growth factor receptor phosphorylation. Its implication in an anti-apoptotic function.

Ultraviolet (UV) irradiation stimulates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) superfamily and implicated in stress-induced apoptosis. UV also induces the activation of another MAPK member, extracellular signal-regulated kinase (ERK), which is typically involved in a growth-signaling cascade. However, the UV-induced signaling pathway leading to ERK activation, together with the physiological role, has remained unknown. Here we examined the molecular mechanism and physiological function of UV-induced ERK activation in human epidermoid carcinoma A431 cells that retain a high number of epidermal growth factor (EGF) receptors. UV-induced ERK activation was accompanied with the Tyr phosphorylation of EGF receptors, and both responses were completely abolished in the presence of a selective EGF receptor inhibitor (AG1478) or the Src inhibitor PP2 and by the expression of a kinase-dead Src mutant. On the other hand, SAPK/JNK activation by UV was partially inhibited by these inhibitors. UV stimulated Src activity in a manner similar to the ERK activation, but the Src activation was insensitive to AG1478. UV-induced cell apoptosis measured by DNA fragmentation and caspase 3 activation was enhanced by AG1478 and an ERK kinase inhibitor (U0126) but inhibited by EGF receptor stimulation by the agonist. These results indicate that UV-induced ERK activation, which provides a survival signal against stress-induced apoptosis, is mediated through Src-dependent Tyr phosphorylation of EGF receptors.