Phosphorylation Of Extracellular Regulated Kinase Research Articles

Antagonism of Dopamine Receptor 2 Long Affects Cannabinoid Receptor 1 Signaling in a Cell Culture Model of Striatal Medium Spiny Projection Neurons.

Activation of dopamine receptor 2 long (D2L) switches the signaling of type 1 cannabinoid receptor (CB1) from Gαi to Gαs, a process thought to be mediated through CB1-D2L heteromerization. Given the clinical importance of D2 antagonists, the goal of this study was to determine if D2 antagonists could modulate CB1 signaling. Interactions between CB1 and D2L, Gαi, Gαs, and β-arrestin1 were studied using bioluminescence resonance energy transfer 2 (BRET(2)) in STHdh(Q7/Q7) cells. CB1-dependent extracellular regulated kinase (ERK)1/2, CREB phosphorylation, and CB1 internalization following cotreatment of CB1 agonist and D2 antagonist were quantified. Preassembled CB1-Gαi complexes were detected by BRET(2) Arachidonyl-2'-chloroethylamide (ACEA), a selective CB1 agonist, caused a rapid and transient increase in BRET efficiency (BRETEff) between Gαi-Rluc and CB1-green fluorescent protein 2 (GFP(2)), and a Gαi-dependent increase in ERK phosphorylation. Physical interactions between CB1 and D2L were observed using BRET(2) Cotreatment of STHdh(Q7/Q7) cells with ACEA and haloperidol, a D2 antagonist, inhibited BRETEff signals between Gαi-Rluc and CB1-GFP(2) and reduced the EMax and pEC50 of ACEA-mediated Gαi-dependent ERK phosphorylation. ACEA and haloperidol cotreatments produced a delayed and sustained increase in BRETEff between Gαs-Rluc and CB1-GFP(2) and increased the EMax and pEC50 of ACEA-induced Gαs-dependent cAMP response element-binding protein phosphorylation. In cells expressing CB1 and D2L treated with ACEA, binding of haloperidol to D2 receptors switched CB1 coupling from Gαi to Gαs In addition, haloperidol treatment reduced ACEA-induced β-arrestin1 recruitment to CB1 and CB1 internalization. D2 antagonists allosterically modulate cannabinoid-induced CB1 coupling, signaling, and β-arrestin1 recruitment through binding to CB1-D2L heteromers. These findings indicate that D2 antagonism, like D2 agonists, change agonist-mediated CB1 coupling and signaling.

Citalopram's mechanism for decreasing immobility time in the Tail Suspension Test

Different antidepressants often target common serotonergic and dopaminergic systems. However, antidepressants are not 100% effective suggesting that there are still multiple cellular mechanisms contributing to depression. Antidepressant responses by novel compounds are typically identified in preclinical models using the Tail Suspension Test (TST), a behavioral stress model that measures immobility time after exposure to an inescapable environment. The TST can predict chronic antidepressant efficacy after acute administration, where effective antidepressants decrease immobility time. Although the predictive validity of the TST is well established, the cellular mechanisms underlying the antidepressant‐driven immobility responses remain unknown. The purpose of this study is to identify the central nervous system cellular pathways that respond to the TST. Two antidepressants will be used in this study: olanzapine, and citalopram, which target the GABA and serotonin systems, respectively. Common pathways mediating the immobility effects of these antidepressants will be identified by immunohistological analysis using phosphorylated extracellular signal‐related kinases as markers of neuronal activity. What was found was that the phosphorylation of extracellular regulated kinases was a mediating step to citalopram's mechanism in decreasing immobility time. The implications of this study include the identification of cellular pathways activated by acute antidepressant treatment, the minimum cellular pathways that are necessary for efficacious therapeutic effects, and facilitating the synthesis of compounds that properly combat depression.

Saikosaponin-D Reduces H2O2-Induced PC12 Cell Apoptosis by Removing ROS and Blocking MAPK-Dependent Oxidative Damage.

Neuronal oxidative stress (OS) injury has been proven to be associated with many neurodegenerative diseases, and thus, antioxidation treatment is an effective method for treating these diseases. Saikosaponin-D (SSD) is a sapogenin extracted from Bupleurum falcatum and has been shown to have many pharmacological activities. The main purpose of this study was to investigate whether and how SSD protects PC12 cells from H2O2-induced apoptosis. The non-toxic level of SSD significantly mitigated the H2O2-induced decrease in cell viability, reduced the apoptosis rate, improved the nuclear morphology, and reduced caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage. Additionally, exogenous H2O2-induced apoptosis by damaging the intracellular antioxidation system. SSD significantly slowed the H2O2-induced release of malonic dialdehyde (MDA) and lactate dehydrogenase and increased the activity of superoxide dismutase (SOD) and the total antioxidant capacity, thereby reducing apoptosis. More importantly, SSD effectively blocked H2O2-induced phosphorylation of extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38MAPK), and specific inhibitors of ERK, JNK, and p38-reduced OS injury and apoptosis, suggesting that SSD reduces OS injury and apoptosis via MAPK signalling pathways. Finally, we confirmed that SSD significantly reduced H2O2-induced reactive oxygen species (ROS) accumulation, and the ROS inhibitor blocked the apoptosis caused by MAPK activation and cellular oxidative damage. In short, our study confirmed that SSD reduces H2O2-induced PC12 cell apoptosis by removing ROS and blocking MAPK-dependent oxidative damage.

Oridonin Inhibits Cell Proliferation and Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Oridonin, an active diterpenoid compound from Rabdosia rubescens, has anti-tumor effects. Rheumatoid arthritis fibroblast-like synoviocytes (RAFLS), a pathological hallmark of RA, exhibits "tumor-like" phenotype. Here, we investigated the effects of oridonin on the proliferation and apoptosis of RAFLS. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Apoptosis and mitochondrial membrane potential were detected by flow cytometry. Western blot analysis was performed to examine the phosphorylation of extra-cellular regulated kinases (ERK1/2), C-Jun N-Terminal Kinase (JNK), and p38 mitogen-activated protein kinases and the expression of apoptosis-related proteins. Oridonin inhibited cell proliferation and induced cell apoptosis in interleukin-1β (IL-1β)-treated FLS. z-VAD-fmk, a pan-caspase inhibitor, significantly (P < 0.05) attenuated oridonin-induced apoptosis of FLS. Oridonin suppressed IL-1β-mediated phosphorylation of ERK1/2 and JNK in a dose-dependent manner. Meanwhile, oridonin alone dose-dependently suppressed FLS proliferation, triggered cell apoptosis, and reduced mitochondrial membrane potential (ΔΨm) through activating caspase-3, caspase-9, and PARP, leading to translocation of cytochrome c into cytoplasm. z-VAD-fmk significantly (P < 0.05) inhibited oridonin-induced apoptosis. The accumulation of cellular reactive oxygen species (ROS) was about sevenfold increase in oridonin-treated cells. Pretreatment of N-acetylcysteine (NAC), an inhibitor of ROS, significantly attenuated oridonin-triggered apoptosis, indicating the involvement of ROS production in oridonin-induced mitochondrial apoptosis. Oridonin inhibits cell proliferation, induces cell apoptosis, and decreases the phosphorylation of ERK1/2 and JNK in IL-1β-exposed RAFLS. Oridonin induces mitochondrial apoptosis by enhancing the production of ROS in FLS.

Phosphatidylserine enhances IKBKAP transcription by activating the MAPK/ERK signaling pathway.

Familial dysautonomia (FD) is a genetic disorder manifested due to abnormal development and progressive degeneration of the sensory and autonomic nervous system. FD is caused by a point mutation in the IKBKAP gene encoding the IKAP protein, resulting in decreased protein levels. A promising potential treatment for FD is phosphatidylserine (PS); however, the manner by which PS elevates IKAP levels has yet to be identified. Analysis of ChIP-seq results of the IKBKAP promoter region revealed binding of the transcription factors CREB and ELK1, which are regulated by the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) signaling pathway. We show that PS treatment enhanced ERK phosphorylation in cells derived from FD patients. ERK activation resulted in elevated IKBKAP transcription and IKAP protein levels, whereas pretreatment with the MAPK inhibitor U0126 blocked elevation of the IKAP protein level. Overexpression of either ELK1 or CREB activated the IKBKAP promoter, whereas downregulation of these transcription factors resulted in a decrease of the IKAP protein. Additionally, we show that PS improves cell migration, known to be enhanced by MAPK/ERK activation and abrogated in FD cells. In conclusion, our results demonstrate that PS activates the MAPK/ERK signaling pathway, resulting in activation of transcription factors that bind the promoter region of IKBKAP and thus enhancing its transcription. Therefore, compounds that activate the MAPK/ERK signaling pathway could constitute potential treatments for FD.

Zn2+-dependent Activation of the Trk Signaling Pathway Induces Phosphorylation of the Brain-enriched Tyrosine Phosphatase STEP: MOLECULAR BASIS FOR ZN2+-INDUCED ERK MAPK ACTIVATION

Excessive release of Zn(2+) in the brain is implicated in the progression of acute brain injuries. Although several signaling cascades have been reported to be involved in Zn(2+)-induced neurotoxicity, a potential contribution of tyrosine phosphatases in this process has not been well explored. Here we show that exposure to high concentrations of Zn(2+) led to a progressive increase in phosphorylation of the striatal-enriched phosphatase (STEP), a component of the excitotoxic-signaling pathway that plays a role in neuroprotection. Zn(2+)-mediated phosphorylation of STEP61 at multiple sites (hyperphosphorylation) was induced by the up-regulation of brain-derived neurotropic factor (BDNF), tropomyosin receptor kinase (Trk) signaling, and activation of cAMP-dependent PKA (protein kinase A). Mutational studies further show that differential phosphorylation of STEP61 at the PKA sites, Ser-160 and Ser-221 regulates the affinity of STEP61 toward its substrates. Consistent with these findings we also show that BDNF/Trk/PKA mediated signaling is required for Zn(2+)-induced phosphorylation of extracellular regulated kinase 2 (ERK2), a substrate of STEP that is involved in Zn(2+)-dependent neurotoxicity. The strong correlation between the temporal profile of STEP61 hyperphosphorylation and ERK2 phosphorylation indicates that loss of function of STEP61 through phosphorylation is necessary for maintaining sustained ERK2 phosphorylation. This interpretation is further supported by the findings that deletion of the STEP gene led to a rapid and sustained increase in ERK2 phosphorylation within minutes of exposure to Zn(2+). The study provides further insight into the mechanisms of regulation of STEP61 and also offers a molecular basis for the Zn(2+)-induced sustained activation of ERK2.

Amomum tsao-ko fruit extract suppresses lipopolysaccharide-induced inducible nitric oxide synthase by inducing heme oxygenase-1 in macrophages and in septic mice.

Amomum tsao-ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti-inflammatory effects and the molecular mechanisms of the methanol extract of A.tsao-ko (AOM) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS-induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase-1 (HO-1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO-1) and silencing HO-1 using siRNA prevented the AOM-mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF-E2-related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N-acetyl-l-cysteine (NAC; a ROS scavenger) diminished the AOM-induced phosphorylation of ERK and JNK and AOM-induced HO-1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM-induced signalling of HO-1 expression. In LPS-induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO-1 expression and survival rates. These results indicate that AOM strongly inhibits LPS-induced NO production by activating the ROS/MAPKs/Nrf2-mediated HO-1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.

Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics.

Steroid resistance is a significant problem in management of chronic inflammatory diseases, including asthma. Accessible biomarkers are needed to identify steroid resistant patients to optimize their treatment. This study examined corticosteroid resistance in severe asthma. 24 asthmatics with forced expiratory volume in one second of less then 80% predicted were classified as steroid resistant or steroid sensitive based on changes in their lung function following a week of treatment with oral prednisone. Heparinised blood was collected from patients prior to oral prednisone administration. Phosphorylated mitogen activated kinases (MAPK) (extracellular regulated kinase (ERK), p38 and jun kinase (JNK)) were analyzed in whole blood samples using flow cytometry. Activation of phospho-p38 MAPK and phospho-mitogen- and stress-activated protein kinase 1 (MSK1) in asthmatics’ peripheral blood mononuclear cells (PBMC) were confirmed by Western blot. Dexamethasone suppression of the LPS-induced IL-8 mRNA production by steroid resistant asthmatics PBMC in the presence of p38 and ERK inhibitors was evaluated by real time PCR. Flow cytometry analysis identified significantly stronger p38 phosphorylation in CD14+ monocytes from steroid resistant than steroid sensitive asthmatics (p = 0.014), whereas no difference was found in phosphorylation of ERK or JNK in CD14+ cells from these two groups of asthmatics. No difference in phosphorylated p38, ERK, JNK was detected in CD4+, CD8+ T cells, B cells and NK cells from steroid resistant vs. steroid sensitive asthmatics. P38 MAPK pathway activation was confirmed by Western blot, as significantly higher phospho-p38 and phospho-MSK1 levels were detected in the PBMC lysates from steroid resistant asthmatics. P38 inhibitor significantly enhanced DEX suppression of LPS-induced IL-8 mRNA by PBMC of steroid resistant asthmatics. This is the first report demonstrating selective p38 MAPK pathway activation in blood monocytes of steroid resistant asthmatics, suggesting that p38 and MSK1 phosphorylation can serve as blood biomarkers of steroid resistance.

Activation of extracellular regulated kinase and mechanistic target of rapamycin pathway in focal cortical dysplasia

Neuropathology of resected brain tissue has revealed an association of focal cortical dysplasia (FCD) with drug-resistant epilepsy (DRE). Recent studies have shown that the mechanistic target of rapamycin (mTOR) pathway is hyperactivated in FCD as evidenced by increased phosphorylation of the ribosomal protein S6 (S6) at serine 240/244 (S(240/244) ), a downstream target of mTOR. Moreover, extracellular regulated kinase (ERK) has been shown to phosphorylate S6 at serine 235/236 (S(235/236) ) and tuberous sclerosis complex 2 (TSC2) at serine 664 (S(664) ) leading to hyperactive mTOR signaling. We evaluated ERK phosphorylation of S6 and TSC2 in two types of FCD (FCD I and FCD II) as a candidate mechanism contributing to mTOR pathway dysregulation. Tissue samples from patients with tuberous sclerosis (TS) served as a positive control. Immunostaining for phospho-S6 (pS6(240/244) and pS6(235/236) ), phospho-ERK (pERK), and phospho-TSC2 (pTSC2) was performed on resected brain tissue with FCD and TS. We found increased pS6(240/244) and pS6(235/236) staining in FCD I, FCD II and TS compared to normal-appearing tissue, while pERK and pTSC2 staining was increased only in FCD IIb and TS tissue. Our results suggest that both the ERK and mTOR pathways are dysregulated in FCD and TS; however, the signaling alterations are different for FCD I as compared to FCD II and TS.

ZDHHC17 promotes axon outgrowth by regulating TrkA-tubulin complex formation.

Correct axonal growth during nervous system development is critical for synaptic transduction and nervous system function. Proper axon outgrowth relies on a suitable growing environment and the expression of a series of endogenous neuronal factors. However, the mechanisms of these neuronal proteins involved in neuronal development remain unknown. ZDHHC17 is a member of the DHHC (Asp-His-His-Cys)-containing family, a family of highly homologous proteins. Here, we show that loss of function of ZDHHC17 in zebrafish leads to motor dysfunction in 3-day post-fertilization (dpf) larvae. We performed immunolabeling analysis to reveal that mobility dysfunction was due to a significant defect in the axonal outgrowth of spinal motor neurons (SMNs) without affecting neuron generation. In addition, we found a similar phenotype in zdhhc17 siRNA-treated neural stem cells (NSCs) and PC12 cells. Inhibition of zdhhc17 limited neurite outgrowth and branching in both NSCs and PC12. Furthermore, we discovered that the level of phosphorylation of extracellular-regulated kinase (ERK) 1/2, a major downstream effector of tyrosine kinase (TrkA), was largely upregulated in ZDHHC17 overexpressing PC12 cells by a mechanism independent on its palmitoyltransferase (PAT) activity. Specifically, ZDHHC17 is necessary for proper TrkA-tubulin module formation in PC12 cells. These results strongly indicate that ZDHHC17 is essential for correct axon outgrowth in vivo and in vitro. Our findings identify ZDHHC17 as an important upstream factor of ERK1/2 to regulate the interaction between TrkA and tubulin during neuronal development.

Abstract 273: Phosphoinositide 3-Kinase γ Regulates Cardio-protective ERK by Kinase Independent Mechanism

Phosphoinositide 3-kinase (PI3K) enzymes are critical in many cellular processes including survival. PI3Kγ, a member of the PI3K family activated by G-protein coupled receptor (GPCR), is known to be a critical player in activation of extracellular regulated kinase (ERK) signal transduction cascade, a cell survival pathway. However, the exact mechanism by which PI3Kγ plays a role in ERK activation is not clearly understood. Our studies show that PI3Kγ plays a crucial role in enhancing the tone of ERK activation as use of PI3K inhibitors reduced GPCR stimulated ERK phosphorylation in HEK293 cells. siRNA knockdown of PI3Kγ resulted in loss of ERK phosphorylation through GPCRs (β-adrenergic) as well as receptor tyrosine kinases. The role of PI3Kγ in ERK activation was further corroborated by loss of insulin stimulated ERK phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs). Surprisingly, ERK activation in KO MEFs post-insulin stimulation was completely rescued by expression of kinase-dead PI3Kγ mutant in KO MEFs suggesting a kinase-independent role of PI3Kγ in regulating ERK function. Indepth mechanistic studies showed that PI3Kγ mediated activation of ERK by inhibiting ERK dephosphorylation following stimulation, thus stabilizing the ERK phosphorylation. PI3Kγ physically disrupts the interaction between ERK and ERK dephosphorylating phosphatase PP2A as evidenced by increase in phosphatase association with ERK in KO MEFs. Consistent with this observation, ERK activation was completely abolished in KO MEFs following carvedilol suggesting an essential role for PI3Kγ in cardio-protective ERK activation pathway. In this context, it is known that transverse aortic constriction (TAC) in mice leads to increase in ERK activation in the hearts and is also associated with concurrent up-regulation of PI3Kγ suggesting a key role for kinase-independent function of PI3Kγ in activating and maintaining the ERK signaling cascade. These indepth cellular studies and observation from our TAC studies led us to believe that kinase-dependent function of PI3Kγ may contribute to pathology while kinase-independent function may be cardio-protective through inhibition of PP2A by PI3Kγ. This novel signaling mechanism by PI3Kγ will be presented.

Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy.

AimsActivation of Ca2+/Calmodulin protein kinase II (CaMKII) is an important step in signaling of cardiac hypertrophy. The molecular mechanisms by which CaMKII integrates with other pathways in the heart are incompletely understood. We hypothesize that CaMKII association with extracellular regulated kinase (ERK), promotes cardiac hypertrophy through ERK nuclear localization.Methods and ResultsIn H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction. Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses. To determine the role of CaMKII in cardiac hypertrophy in vivo, spontaneously hypertensive rats were subjected to intramyocardial injections of AntCaNtide or tat-CN17β. Left ventricular hypertrophy was evaluated weekly for 3 weeks by cardiac ultrasounds. We observed that the treatment with CaMKII inhibitors induced similar but significant reduction of cardiac size, left ventricular mass, and thickness of cardiac wall. The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart.ConclusionThese results indicate that CaMKII and ERK interact to promote activation in hypertrophy; the inhibition of CaMKII-ERK interaction offers a novel therapeutic approach to limit cardiac hypertrophy.

Cavin-2 in oral cancer: A potential predictor for tumor progression.

Cavin-2 (CVN2) affects formation of large caveolae, which are membrane-rich cholesterol domains associated with several functions in signal transduction. Accumulating evidence suggests that CVN2 is present in many cellular types; however, the molecular mechanisms of CVN2 in cancers and its clinical relevance are unknown. We proposed a mechanism by which CVN2 regulates caveolin-1 expression leading to slow cellular proliferation by inactivation of the extracellular regulated kinase (ERK) pathway. Quantitative reverse transcriptase-polymerase chain reaction and immunoblot analyses were used to assess the CVN2 regulation mechanism in oral squamous cell carcinoma (OSCC). Immunohistochemistry (IHC) was performed to analyze the correlation between CVN2 expression and clinical behavior in 115 patients with OSCC. A CVN2 overexpressed model of OSCC cells (oeCVN2 cells) was used for functional experiments. CVN2 expression was down-regulated significantly (P < 0.05) in OSCCs compared with normal counterparts in vitro and in vivo. In addition to the findings that a serum deprivation culture induced up-regulation of CVN2 and slowed cellular proliferation, oeCVN2 cell growth decreased because of cell-cycle arrest at the G1 phase resulting from up-regulated cyclin-dependent kinase inhibitors (p21(Cip1) and p27(Kip1) ) and down-regulated cyclins (cyclin D1, cyclin E) and cyclin-dependent kinases (CDK2, CDK4, and CDK6). Interestingly, CVN2 overexpression facilitated caveolin-1 recruitment and colocalization with each other. We also found decreased ERK phosphorylation levels, an upstream event in cell-cycle arrest. Clinically, IHC data from primary OSCCs showed high tumoral progression in CVN2-negative patients with OSCC. CVN2 may be a possible key regulator of OSCC progression via the CVN2/caveolin-1/ERK pathway and a potential therapeutic target for developing new treatments for OSCCs. © 2015 Wiley Periodicals, Inc.

An 8-O-4′ norlignan exerts oestrogen-like actions in osteoblastic cells via rapid nongenomic ER signaling pathway

Ethnopharmacological relevanceSambucus williamsii Hance (SWH), which belongs to the Caprifoliaceae family distributed in various regions of China, Korea and Japan, has been used as a folk medicine for treatment of bone and joint diseases in China for thousands of years. In previous studies, SWH was shown to possess anti-osteoporosis, healing fracture, anti-inflammatory and analgesic activities. Our previous studies showed that SWH extract effectively suppressed ovariectomy-induced increase in bone turnover and improved bone mineral density and bone biomechanical strength in rats as well as in mice. An 8-O-4′ norlignan, (7R,8S)-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-propanediol (PPD) was previously isolated and identified as the bioactive ingredient in SWH. The present study aimed to characterize the bone protective effects as well as its mechanism of actions in osteoblasts. Materials and methodsBone protective actions of PPD on different cells were determined by proliferation assay, alkaline phosphatase (ALP) activity assay, calcium deposition as well as real-time reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, estrogen receptor (ER) antagonist ICI182,780 and mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocking assays, competitive ER radioligand binding assay, ERE-dependent luciferase reporter assay and immunoblotting were used to determine if PPD activated ER and if the effects of PPD on osteoblastic functions were ER dependent. ResultsPPD exerted anabolic effects in osteoblasts and its effects were abolished by co-incubation with ICI182,780 or U0126. PPD induced mRNA expressions of Runx2, ALP, osteocalcin, and increased the ratio of osteoprotegerin/receptor activator of nuclear factor κB (OPG/RANKL). PPD failed to bind to either ERα or ERβ and did not activate ERE-luciferase activity via ER. PPD induced the phosphorylation of extracellular regulated kinases (ERK) and its effect was completely abolished by U0126. It also induced the phosphorylation of ERα at serine 118. ConclusionThese data show that PPD is a bioactive compound in SWH that exerts oestrogen-like actions in osteoblast-like cells via ligand-independent, estrogen response element (ERE)-independent and mitogen-activated protein (MAP) Kinase-mediated rapid nongenomic ER signaling pathway.

PJ-34 inhibits PARP-1 expression and ERK phosphorylation in glioma-conditioned brain microvascular endothelial cells

Inhibitors of PARP-1(Poly(ADP-ribose) polymerase-1) act by competing with NAD+, the enzyme physiological substrate, which play a protective role in many pathological conditions characterized by PARP-1 overactivation. It has been shown that PARP-1 also promotes tumor growth and progression through its DNA repair activity. Since angiogenesis is an essential requirement for these activities, we sought to determine whether PARP inhibition might affect rat brain microvascular endothelial cells (GP8.3) migration, stimulated by C6-glioma conditioned medium (CM). Through wound-healing experiments and MTT analysis, we demonstrated that PARP-1 inhibitor PJ-34 [N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide] abolishes the migratory response of GP8.3 cells and reduces their viability. PARP-1 also acts in a DNA independent way within the Extracellular-Regulated-Kinase (ERK) signaling cascade, which regulates cell proliferation and differentiation. By western analysis and confocal laser scanning microscopy (LSM), we analyzed the effects of PJ-34 on PARP-1 expression, phospho-ERK and phospho-Elk-1 activation. The effect of MEK (mitogen-activated-protein-kinase-kinase) inhibitor PD98059 (2-(2-Amino-3-methoxyphenyl)-4 H-1-benzopyran-4-one) on PARP-1 expression in unstimulated and in CM-stimulated GP8.3 cells was analyzed by RT-PCR. PARP-1 expression and phospho-ERK activation were significantly reduced by treatment of GP8.3 cells with PJ-34 or PD98059. By LSM, we further demonstrated that PARP-1 and phospho-ERK are coexpressed and share the same subcellular localization in GP8.3 cells, in the cytoplasm as well as in nucleoplasm. Based on these data, we propose that PARP-1 and phospho-ERK interact in the cytosol and then translocate to the nucleus, where they trigger a proliferative response. We also propose that PARP-1 inhibition blocks CM-induced endothelial migration by interfering with ERK signal-transduction pathway.

MTUS1/ATIP3a down-regulation is associated with enhanced migration, invasion and poor prognosis in salivary adenoid cystic carcinoma

BackgroundMicrotubule-associated tumor suppressor gene (MTUS1) has been identified as tumor suppressor gene in many malignant tumors. In this study, we investigated the role of MTUS1 in the development of salivary adenoid cystic carcinoma (SACC) and its functional effect on the migration and invasion of SACC.MethodsArchival clinical samples including 49 primary SACC were examined for MTUS1 expression by immunohistochemistry. Statistical analyses were performed to evaluate the correlation between MTUS1 with histopathological features and survival. The expression of MTUS1/ATIP (AT2 receptor-interacting protein) isoforms was determined in SACC tissue samples and cell lines using quantitative RT-PCR assays. Then we investigated whether the migration and invasion of SACC were mediated by MTUS1/ATIP3a using in vitro cell migration and invasion assay.ResultsWe confirmed that the down-regulation of MTUS1 was a frequent event in SACC, and was correlated with distant metastasis and associated with reduced overall survival and disease free survival. Isoform specific quantitative RT-PCR assays revealed that ATIP1, ATIP3a and ATIP3b were the major isoforms of the MTUS1 gene products in SACC, and were significant down-regulation in SACC as compared to matching normal tissues. For functional analyses, we found that SACC-LM cells (SACC cell line with higher migration and invasion ability) possessed a lower expression level of ATIP3a compared to SACC-83 cells (lower migration and invasion ability). Restoration of ATIP3a expression in SACC-LM cells induced anti-proliferative activity and inhibited the migration and invasion ability. Knockdown of ATIP3a promoted the proliferation, migration and invasion ability of SACC-83 cells. Restoration of ATIP3a inhibited the phosphorylation of ERK (extracellular-regulated kinase) 1/2, the expression of Slug and Vimentin in SACC-LM cells, while knockdown of ATIP3a increased the phosphorylation of ERK1/2, the expression of Slug and Vimentin in SACC-83 cells.ConclusionsOur studies confirm that MTUS1 plays an important role in the progression of SACC, and may serve as a biomarker or therapeutic target for patients with SACC. MTUS1/ATIP3a down-regulation contributes to the proliferation, migration and the invasion abilities of SACC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1209-x) contains supplementary material, which is available to authorized users.

복분자 온수추출물의 VEGF-유도성 혈관신생 억제효과

The interruption of angiogenesis using herbal extracts is now recognized as a useful approach for treating many solid tumors. To date, the best-validated antitumor approach is to target the vascular endothelial growth factor (VEGF)-induced angiogenic pathway. In the present study, we first identified the antiangiogenic activity of a hot-water extract of Rubus coreanus Miquel (RCMHE) in vitro and ex vivo. This extract suppressed VEGF-induced angiogenesis, the phosphorylation of extracellular regulated kinase (ERK), p38 and the activation of matrix metalloproteinases (MMPs). RCMHE also inhibited the VEGF-responsive phosphorylation of VEGFR2. These results clearly show that RCMHE may have potential therapeutic value for angiogenesis-associated human diseases through the suppression of angiogenesis and the interruption of the phosphorylation of VEGFR2.

Cocaine induces ubiquitination of Egr-1 in the rat dorsal striatum.

Early growth response (Egr) is a member of the zinc finger family of transcription factors that reflects neuronal activity induced by various stimuli. Acute cocaine administration elicits rapid and transient induction of several immediate early genes in brain neurons. However, the mechanism regulating the degradation of the Egr-1 protein is not clearly understood. In this study, rats were injected with cocaine and the relationships among locomotor activity, Egr-1 protein level, phosphorylation of upstream kinase extracellular regulated kinase (ERK)1/2, Egr-1 mRNA expression, and ubiquitination of the Egr-1 protein were measured in the dorsal striatum and the frontal cortex. Locomotor activity reached a peak at about 15 min, and phosphorylation of ERK1/2 and Egr-1 mRNA level also increased at that time. However, the Egr-1 protein level decreased initially in the dorsal striatum, probably due to ubiquitination-mediated degradation. When locomotor activity decreased substantially at 30 min, the phosphorylation of ERKs and expression levels of Egr-1 mRNA and protein reached their peak levels and the protein level subsequently increased. These findings indicate that immediate early gene protein levels would not be a reliable indicator of increased regional activity in the brain. Thus, observations spanning multiple time periods or the examination of mRNA rather than protein would be recommended in these situations.

Reduced phosphorylation of synapsin I in the hippocampus of Engrailed-2 knockout mice, a model for autism spectrum disorders

Mice lacking the homeodomain transcription factor Engrailed-2 (En2−/− mice) are a well-characterized model for autism spectrum disorders (ASD). En2−/− mice present molecular, neuropathological and behavioral deficits related to ASD, including down-regulation of ASD-associated genes, cerebellar hypoplasia, interneuron loss, enhanced seizure susceptibility, decreased sociability and impaired cognition. Specifically, impaired spatial learning in the Morris water maze (MWM) is associated with reduced expression of neurofibromin and increased phosphorylation of extracellular-regulated kinase (ERK) in the hippocampus of En2−/− adult mice. In the attempt to better understand the molecular cascades underlying neurofibromin-dependent cognitive deficits in En2 mutant mice, we investigated the expression and phosphorylation of synapsin I (SynI; a major target of neurofibromin-dependent signaling) in the hippocampus of wild-type (WT) and En2−/− mice before and after MWM. Here we show that SynI mRNA and protein levels are down-regulated in the hippocampus of naïve and MWM-treated En2−/− mice, as compared to WT controls. This down-regulation is paralleled by reduced levels of SynI phosphorylation at Ser549 and Ser553 residues in the hilus of mutant mice, before and after MWM. These data indicate that in En2−/− hippocampus, neurofibromin-dependent pathways converging on SynI phosphorylation might underlie hippocampal-dependent learning deficits observed in En2−/− mice.

Effect of lavage with artificial cerebrospinal fluid on neural cell apoptosis and the ERK pathway after rat traumatic brain injury

To explore the eff ect of lavage with artificial cerebrospinal fluid on neural cell apoptosis and the extracellular regulated kinase (ERK) pathway aft er traumatic brain injury. A total of 192 SD rats were randomly divided into a sham group, a traumatic brain injury model group, a local artificial cerebrospinal fluid group, and a local saline group. Each group was divided into 4 sub-groups by the sacrificed time at 6 h, 12 h, 1 d and 3 d aft er the operation. Th e phosphorylation of extracellular regulated kinase 2 (P-ERK2), TNF-α and cellular apoptosis were examined. Th e levels of P-ERK2 protein and TNF-α protein, as well as the number of apoptotic cells at each time point in the local artificial cerebrospinal fluid group were lower than those in the model group or in the saline group (P<0.05). Lavage with artificial cerebrospinal fluid can reduce apoptosis of neural cells after brain injury through the ERK pathway.