Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

Expression of Extracellular Signal-regulated Kinase and Angiotensin-converting Enzyme in Human Atria during Atrial Fibrillation

In order to investigate the changes in the expression of extracellular signal regulated kinase (ERK1/ERK2) and angiotensin converting enzyme (ACE) in the patients with atrial fibrillation (AF), 52 patients with rheumatic heart diseases were examined. Nineteen patients had chronic persistent AF (AF≥6 months, CAF), 12 patients had paroxymal AF (PAF) and 21 patients had no history of AF. The ERK expression was detected at the mRNA level by reverse transcription polymerase chain reaction, at the protein level by Western blotting and at atrial tissue level by immunohistochemistry. ERK activating kinases (MEK1/2) and ACE were determined by Western blotting techniques. The expression of ERK2 mRNA was increased in the patients with CAF (74±19 U vs sinus rhythm: 32±24 U, P <0.05). Activated ERK1/ERK2 and MEK1/2 were increased to more than 150 % in the patients with AF compared to those with sinus rhythm. No significant difference between CAF and PAF was found. The expression of ACE was three fold increased in the patients with CAF compared to those with sinus rhythm. Patients with AF showed an increased expression of ERK1/ERK2 in atrial interstitial cells and marked atrial fibrosis. An ACE dependent increase in the amounts of activated ERK1/ERK2 in atrial interstitial cells may be one of molecular mechanisms for the development of atrial fibrosis in the patients with AF. These findings may have important impact on the treatment of AF.

An experimental study of extracellular signal-regulated kinase and its interventional treatments in hepatic fibrosis

BACKGROUND: The pathogenesis of hepatic fibrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentiation. The aim of this study was to investigate the effects of PD98059, a specific inhibitor of ERK, on the cell cycle, cell proliferation, secretion of type I collagen and expression of cyclin D1 mRNA, CDK4 mRNA and transforming growth factor-beta 1 (TGF-beta 1) mRNA in rat hepatic stellate cells (HSCs) stimulated by acetaldehyde. METHODS: Rat HSCs stimulated by acetaldehyde were incubated with PD98059 at different concentrations. The cell cycle was analysed by flow cytometry. Cell proliferation was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of cyclin D1, CDK4 and TGF-beta 1 was examined using the reverse transcriptase-polymerase chain reaction. Type I collagen in the culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: 20, 50 and 100 mu mol/L PD98059 significantly inhibited the proliferation and provoked a G0/G1-phase arrest of acetaldehyde-induced HSCs in a dose-dependent manner. The secretion of type I collagen and the expression of cyclin D1, CDK4 and TGF-beta 1 mRNA in acetaldehyde-induced HSCs were markedly inhibited by 50 and 100 mu mol/L PD98059, respectively. CONCLUSIONS: The ERK pathway regulates the cell proliferation, secretion of type I collagen and the expression of TGF-beta 1 mRNA in rat HSCs stimulated by acetaldehyde, which is likely related to its regulative effect on the cell cycle.

p38 mitogen-activated protein kinase regulates type-Ⅰ vs type-Ⅱ phenotyping of human vascular endothelial cells

AIM: To identify kinases involved in phenotype regulation of vascular endothelial cells(VECs): Proproliferative G-protein signaling 5(RGS5)^(high)(typeⅠ) vs anti-proliferative RGS5^(low)(typeⅡ) VECs.METHODS: Proteomic kinase assays were performed to identify the crucial kinase involved in the phenotype regulation of human VECs using typeⅠ VECs, which promotes the proliferation of human vascular smooth muscle cells(VSMCs), and typeⅡ VECs, which suppress the proliferation of human VSMCs. The assays were performed using multiple pairs of typeⅠ and typeⅡ VECs to obtain the least number of candidates. The involvement of the candidate kinases was verified by evaluating the effects of their specific inhibitors on the phenotype regulation of human VECs as well as the expression levels of regulator of RGS5, which is the causative gene for the "typeⅡ to typeⅠ" phenotype conversion of human VECs. RESULTS: p38α mitogen-activated protein kinase(p38α MAPK) was the only kinase that showed distinctive activities between typeⅠ and typeⅡ VECs: p38α MAPK activities were low and high in type-Ⅰand typeⅡ VECs, respectively. We found that an enforced expression of RGS5 indeed lowered p38α MAPK activitiesin typeⅡ VECs. Furthermore, treatments with a p38α MAPK inhibitor nullified the anti-proliferative potential in typeⅡ VECs. Interestingly, MAPK inhibitor treatments enhanced the induction of RGS5 gene. Thus, there is a vicious cycle between "RGS5 induction" and "p38α MAPK inhibition", which can explain the unidirectional process in the stress-induced "typeⅡ to typeⅠ" conversions of human VECs. To understand the upstream signaling of RGS5, which is known as an inhibitory molecule against the G protein-coupled receptor(GPCR)-mediated signaling, we examined the effects of RGS5 overexpression on the signaling events from sphingosine-1-phosphate(S1P) to N-cadherin, because S1 P receptors belong to the GPCR family gene and N-cadherin, one of their downstream effectors, is reportedly involved in the regulation of VEC-VSMC interactions. We found that RGS5 specifically bound with S1P1. Moreover, N-cadherin localization at intercellular junctions in typeⅡ VECs was abolished by "RGS5 overexpression" and "p38α MAPK inhibition".CONCLUSION: p38α MAPK plays crucial roles in "type-Ⅰ vs type-Ⅱ" phenotype regulations of human VECs at the downstream of RGS5.

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression

AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.

Pterygium epithelium abnormal differentiation related to activation of extracellular signal-regulated kinase signaling pathway in vitro

AIMTo investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase (ERK) signaling pathway in vitro.METHODSThe expression levels of phosphorylated ERK (P-ERK), keratin family members including K19 and K10 and the ocular master control gene Pax-6 were measured in 16 surgically excised pterygium tissues and 12 eye bank conjunctiva. In colony-forming cell assays, the differences in clone morphology and in K10, K19, P-ERK and Pax-6 expression between the head and body were investigated. When cocultured with the ERK signaling pathway inhibitor PD98059, the changes in clone morphology, colony-forming efficiency, differentiated marker K10, K19 and Pax-6 expression and P-ERK protein expression level were examined by immunoreactivity and Western blot analysis.RESULTSThe expression of K19 and Pax-6 decreased in the pterygium, especially in the head. No staining of K10 was found in the normal conjunctiva epithelium, but it was found to be expressed in the superficial cells in the head of the pterygium. Characteristic upregulation of P-ERK was observed by immunohistochemistry. The clone from the head with more differentiated cells in the center expressed more K10, and the clone from the body expressed more K19. The P-ERK protein level increased in the pterygium epithelium compared with conjunctiva and decreased when cocultured with PD98059. The same medium with the ERK inhibitor PD98059 was more effective in promoting clonal growth than conventional medium with 3T3 murine feeder layers. It was observed that the epithelium clone co-cultured with the inhibitor had decreased K10 expression and increased K19 and Pax-6 expression.CONCLUSIONWe suggest ERK signaling pathway activation might play a role in the pterygium epithelium abnormal differentiation.

XAF1 mediates apoptosis through an extracellular signal-regulated kinase pathway in colon cancer

Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-regulated kinase(ERK)pathway is thought to increase cell proliferation and to protect cells from apoptosis.The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.Methods:Four human colon cancer cell lines,HCT1116 and Lovo(wildtype p53),DLD1 and SW1116(mutant p53),were used.Lovo stable transfectants with XAF1 sense and antisense were established.The effects of dominant-negative MEK1(DN-MEK1)and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined.The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay.Cell proliferation was measured by MTT assay.Apoptosis was determined by Hoechst 33258 staining.Results:U0126 increased the expression of XAF1 in a time-and dose-dependent manner.A similar result was obtained in cells transfected with DN-MEK1 treatment.Conversely,the expression of XIAP was down-regulated.Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection.rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression.Overexpression of XAF1 was more sensitive to U0126-induced apoptosis,whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.Conclusions:XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation,which required de novo protein synthesis.The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.

Dexamethasone suppresses DU145 cell proliferation and cell cycle through inhibition of the extracellular signal-regulated kinase 1 /2 pathway and cyclin D1 expression

Aim： To determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth. Methods： The cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were determined by proliferation assay and fluorescence-activated cell sorter. Western blot analysis was carried out to evaluate the effects of dexamethasone on phosphorylation of extracellular signal-regulated kinase （ERK）1/2 and expression of cyclin D1 in DU145 cells with or without glucocorticoid receptor （GR） antagonist RU486. Reverse transcription- polymerase chain reaction verified the expression of GR mRNA in DU145 cells. Results： Dexamethasone significantly inhibited DU 145 cell proliferation at the G0/G1 phase. Westem blot analysis showed a dramatic reduction of ERK1/2 activity and cyclin D1 expression in dexamethasone-treated cells. The decreased phosphorylation of ERK1/2 in dexamethasone-treated cells was attenuated by GR blockade. Additionally, the effects of dexamethasone in inhibiting cyclin D1 expression were altered by GR blockade. Conclusion： Dexamethasone suppresses DU145 cell proliferation and cell cycle, and the underlying mechanisms are through the inhibition of phosphorylation of ERK1/2 and cyclin D1 expression. The inhibition of ERK1/2 phosphorylation and cyclin D1 expression is attenuated by GR blockade, suggesting that GR regulates ERK1/2 and cyclin D1 pathways. These observations suggest that dexamethasone has a potential clinical application in prostate cancer therapy.

Effect of extracellular signal-regulated kinase and nitric oxide on compressive neuralgia formation and maintenance

BACKGROUND： Previous studies have shown that extracellular signal-regulated kinase 1/2 （ERK1/2） and nitric oxide activation play a pivotal role in central sensitization and long-term neuronal plasticity induced by noxious stimulation. However, their effects on compressive neuralgia formation and maintenance remain poorly understood.OBJECTIVE： To investigate effects of the specific inhibitor of ERK1/2 signal pathway U0126 on neuronal nitric oxide synthase （nNOS） expression in the dorsal horn of the spinal cord in a compressive neuralgia rat model.DESIGN, TIME AND SETTING： A randomized, controlled experiment was performed at the Institute of Otolaryngology, Head and Neck Surgery, First Hospital of Jilin University from July 2008 to March 2009.MATERIALS： U0126 （Bio-Mol, USA） was used in this study.METHODS： A total of 84 rats were randomly assigned to two groups. In the first part of the experiment, 24 rats were used for behavioral testing, and they were randomly assigned to three sub-groups （n =8）： U0126, dimethyl sulfoxide （DMSO） and model control. In the second part of the experiment, 60 rats were used for immunofluorescence and Western blot analysis, and they were randomly assigned to six sub-groups （n = 10）： sham surgery, model control, U0126 post-injection at 0.5, 2, 12 and 24 hours. Neuropathic pain was produced by chronic compression to the dorsal root ganglion in rats from each sub-group. Rats in the U0126 group were administered a 5-ug U0126 intrathecal injection, and rats in the DMSO group were administered a 10-μL 5% DMSO intrathecal injection.MAIN OUTCOME MEASURES： Changes in mechanical and thermal hyperalgesia were observed using von Frey filaments and thermalqia stimular. Thermal and mechanical hyperalgesia were stimulated at different time points following intrathecal injection of U0126. nNOS activation and expression in the spinal cord dorsal horn were determined by immunofluorescence and Western blot analysis.RESULTS： Intrathecal injection of U0126 significantly attenuated chronic compression of dorsal root ganglion-induced mechanical and thermal hyperalgesia. Immunofluorescence staining results demonstrated that, compared to the sham surgery group, the number of nNOS-positive neurons was significantly increased in the injured spinal dorsal horn in the model control group （P〈0.01）. However, compared to the model control group, there were significantly decreasing numbers of nNOS-positive neurons in the U0126 post-injection groups at 0.5-hour, 2-hour, and 12-hour （P〈0.05）. Western blot analysis revealed similar results. CONCLUSION： Decreased activity in the ERK signal pathway resulted in down regulated nNOS expression in the dorsal horn of the spinal cord. These results suggested that ERK is involved in nitric oxide reaction to neuropathic pain.

Influence of Ren and Du meridian electro-acupuncture on neural stem cell proliferation and extracellular signal-regulated kinase pathway in a rat model of focal cerebral ischemia injury

BACKGROUND： Studies have shown that electro-acupuncture at the Ren meridian could improve proliferation of subventricular zone neural stem cells in cerebral-ischemic rats. However, there are few reports on the influence of electro-acupuncture at the Du meridian on neural stem cell proliferation. OBJECTIVE： To observe the influence of electro-acupuncture at Ren and Du meridians on neural stem cell proliferation in the subventricular zone and altered signal transduction in cerebral ischemia rats. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Human Anatomy, Medical College of Sun Yat-sen University from May 2006 to February 2008. MATERIALS： Mouse anti-rat bromodeoxyuridine （BrdU） monoclonal antibody was provided by Sigma, USA; mouse anti-rat nestin monoclonal antibody and extracellular signal-regulated protein kinase （ERK） specific inhibitor PD98059 were provided by Calbiochem, Germany; acupuncture needle was provided by Suzhou Acupuncture Supplies, China. METHODS： A total of 126 rats were randomly assigned to four groups： model （n = 36）, Du meridian （n = 36）, Ren/Du meridian （n = 36）, and Ren/Du meridian ＋ PD98059 （n = 18）. Rats in the Ren/Du meridian ＋ PD98059 group were observed on days 7 （n = 6） and 14 （n = 12） after cerebral ischemia injury. Rats in the model, Du meridian, and Ren/Du meridian groups were observed on days 7, 14, and 28 after cerebral ischemia injury, with 12 rats per group at each time point. Thread occlusion was used to establish middle cerebral artery occlusion models. Electro-acupuncture was performed at Renzhong （DU 26） and Baihui （DU 20） acupoints in the Du meridian group, as well as Chengjiang （RN 24）, Guanyuan （RN 4）, Renzhong, and Baihuiacupoints in the Ren/Du meridian and Ren/Du meridian ＋ PD98059 groups 2 days after model establishment. In addition, electro-acupuncture stimulation with disperse-dense waves was performed, with 30 Hz disperse wave, 100 Hz dense wave, and 5 V intensity for 20 minutes. Rats in the Ren/Du meridian ＋ PD98059 group were treated with 0.2 pg PD98059 injection into the subventricular zone, 2 pL per rat. Rats in the model group were not treated with electro-acupuncture. MAIN OUTCOME MEASURES： BrdU/nestin immunofluorescent staining was used to detect proliferating neural stem cells in the subventricular zone of cerebral ischemia rats; Western blot was used to determine phosphorylated ERK1 and 2 （pERK1/2） expression in the subventricular zone. RESULTS： On days 14 and 28 after cerebral ischemia, there were significantly more BrdU-positive and BrdU/nestin-positive cells in the Ren/Du meridian group compared with the Du meridian group （P 〈 0.05）. PD98059 decreased the number of BrdU-positive and BrdU/nestin-positive cells induced by electro-acupuncture at the/：ten and Du meridians （P 〈 0.05）. On days 7, 14, and 28 after treatment, pERK1/2 expression was significantly greater in the Du meridian and Ren/Du meridian groups compared with the model group （P 〈 0.05）. The promoting effect of electro-acupuncture at Ren and Du meridians on ERK1/2 phosphorylation was superior to electro-acupuncture at the Du meridian alone on day 14 after model induction （P 〈 0.05）. However, PD98059 completely abolished the promoting effect of electro-acupuncture at Ren/Du meridians on pERK1/2 expression （P 〈 0.05）. CONCLUSION： Electro-acupuncture at Ren and Du meridians increased proliferation of subventricular zone neural stem cells, which was related to activation of the ERK pathway in a rat model of cerebral ischemia injury.

Regulation of extracellular signal-regulated kinase 1/2 influences hippocampal neuronal survival in a rat model of diabetic cerebral ischemia

Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from injury in a diabetic model after cerebral ischemia/reperfusion. In this study, transient whole-brain ischemia was induced by four-vessel occlusion in normal and diabetic rats, and extracellular signal-regulated kinase 1/2 inhibitor （U0126） was administered into diabetic rats 30 minutes before ischemia as a pretreatment. Results showed that the number of surviving neurons in the hippocampal CA1 region was reduced, extracellular signal-regulated kinase 1/2 phosphorylation and KuT0 activity were decreased, and pro-apoptotic Bax expression was upregulated after intervention using U0126. These findings demonstrate that inhibition of extracellular signal-regulated kinase 1/2 activity aggravated neuronal loss in the hippocampus in a diabetic rat after cerebral ischemia/reperfusion, further decreased DNA repairing ability and ac- celerated apoptosis in hippocampal neurons. Extracellular signal-regulated kinase 1/2 activation plays a neuroprotective role in hippocampal neurons in a diabetic rat after cerebral ischemia/ reperfusion.

Neurotransmitter regulation of extracellular signal-regulated kinase expression following subarachnoid hemorrhage

BACKGROUND： Very few studies have addressed neuronal injury in cerebral vasospasm and subarachnoid hemorrhage （SAH）, and the role of neurotransmitters in the regulation of extracellular signal-regulated kinase 1/2 （ERK1/2） expression following SAH. OBJECTIVE： To analyze neurotransmitter regulation of ERK1/2 expression through the use of signal transduction, and to investigate cerebral injury mechanisms following SAH. DESIGN, TIME AND SETTING： A completely randomized grouping and controlled animal experiment was performed at the Experimental Center of Medical College of Xi＇an Jiaotong University from March to December 2008. MATERIALS： Extraceliular signal-regulated ERK1/2 polyclonal antibody and streptavidin-peroxidase method kits were purchased from Beijing Biosynthesis Biotechnology, China; DAB kit was purchased from Zhongshan Golden Bridge Biotechnology, China; TUNEL kit was purchased from Promega, USA. METHODS： A total of 114 male, Sprague Dawley rats, aged 55-63 days old, were randomly assigned to five groups： SAH （n = 30）, saline control （n = 30）, puncture control （n = 30）, normal control （n = 6）, and neurotransmitter-treated （n = 18）. The SAH model was established by twice injecting blood through the cisterna magna. The neurotransmitters-treated group was subdivided into three groups according to drugs injected into the lateral cerebral ventricle： acetylcholine chloride, norepinephrine, and saline, with six animals in each group. MAIN OUTCOME MEASURES： Rats from the SAH, saline control, and puncture control groups were respectively sacrificed at 6, 12, and 24 hours, as well as 3 and 5 days, with six rats at each time point. The normal control group rats were sacrificed at 6 hours, and the neurotransmitter group rats were sacrificed 3 days following neurotransmitter injection. Morphological cellular changes were observed by hematoxylin and eosin staining. Immunohistochemical SP method was used to detect expression of ERK1/2 in the cortex, and cortical apoptosis was detected using the TUNEL method. RESULTS： Neural tissue edema, apoptosis, and necrosis occurred in the cortex of the SAH group. ERK1/2-positive cells were first observed at 6 hours, peaked at 12 hours following SAH in the cortex, and gradually decreased thereafter. Cellular apoptosis was observed in the cortex at 6 hours and peaked at 24 hours following SAH. ERK1/2 distribution in the brain overlapped apoptotic cells to a great degree. The number of ERK1/2-positive and apoptotic cells was significantly greater in the SAH group compared with the three control groups （P 〈 0.05）. Compared to the number of ERK1/2-positive cells in the saline-treated group, acetylcholine chloride treatment resulted in decreased ERK1/2 expression and apoptosis （P 〈 0.05）. Norepinephrine resulted in increased ERK1/2 expression, but there was no significance in apoptosis compared to the saline-treated group （P 〉 0.05）. CONCLUSION： Apoptosis was observed early in the rat cortex following SAH. In addition, ERK1/2 was expressed earlier than apoptosis. Acetylcholine chloride treatment resulted in decreased numbers of apoptotic cells following SAH, possibly by down-regulating ERK1/2 expression.

Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway in depressive disorder

The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.

Role of Extracelluar Regulated Protein Kinases in FTY720-induced Apoptosis of Leukemia Cell Lines HL-60 and U937

The effects of a novel immunosuppressive agent FTY720 on proliferation inhibition and apoptosis of acute leukemia cell lines HL 60 and U937, and the role of extracelluar regulated protein kinase (ERK) in the course of proliferation inhibition and apoptosis induced by FTY720 were studied. The proliferation inhibition rate of HL 60 and U937 cells by various concentrations of FTY720 was detected by MTT assay. Cell apoptosis was detected by DNA fragment analysis and flow cytometry. The phosphorylated ERK1/2 protein expression was observed by Western blotting. The change of intracellular distribution of ERK1/2 protein was identified by SP immunohistochemical staining. The results showed that FTY720 could inhibit the growth of HL 60 and U937 cells effectively in a dose dependent manner. After incubation with FTY720 for 24 h, apoptosis was observed in HL 60 and U937 cells. The intracellular expression of phosphorylated ERK1/2 protein was also down regulated and the distribution of ERK1/2 protein in cell nuclear was reduced during FTY720 induced apoptosis. So, that FTY720 inhibited ERK1/2 phosphorylation might mediate the role of FTY720 induced apoptosis and proliferation inhibition of leukemia cells.

Regulative Function of Telomerase and Extracelluar Regulated Protein Kinases to Leukemic Cell Apoptosis

In order to investigate the regulative function of telom erase and phosphorylated (acti- vated) extracelluar regulated protein kinase (ERK) 1and 2 in the leukemic cell lines HL - 6 0 and K5 6 2 proliferation inhibition and apoptosis,three chemotherapeutic drugs Harringtonine(HRT) , Vincristine(VCR) and Etoposide(Vp16 ) were selected as inducers.The proliferation inhibition rate was detected by MTT m ethod,the cell cycle and cell apoptosis was analyzed by flow cytometry and the telom erase activity was detected by the telom eric repeat am plification protocol(TRAP) assay and bioluminescence analysis method.The phosphorylated ERK 1/ 2 protein expression was detected by western blot method.The results showed that HRT,VCR and Vp16 could inhibit cell proliferation,induce apoptosis,inhibit telomerase activity and down- regulate the protein expres- sion of phosphorylated ERK.Itwas suggested that ERK signal transduction pathway was involved in the down- regulation of telomerase activity and the onset of apoptosis in the leukem ic cells treat- ed by HRT,VCR and Vp16 .

Extracellular signal-regulated kinase, substance P and neurokinin-1 are involved in the analgesic mechanism of herb-partitioned moxibustion

Herb-partitioned moxibustion can effectively mitigate visceral pain, a major symptom in inflammatory bowel disease, but the analgesic lnechanism is still unclear. Moreover, extracellular signal-regulated kinase, substance P, and neurokinin-1 are involved in formation of central hyperalgesia. Thus, we postulated that the analgesic effect of herb-partitioned moxibustion may be associated with these factors. Accordingly, in this study, we established an inflammatory bowel disease visceral pain model in rat by enema with a mixed solution of 5% trinitrobenzenesulfonic acid and 50% ethanol. Bilateral Tianshu （ST25） and Qihai （CV6） points were selected for herb-partitioned moxi- bustion. Our results showed that herb-partitioned moxibustion improved visceral pain and down-regulated extracellular signal-regulated kinase, substance P, and neurokinin-1 protein and mRNA expression in dorsal root ganglia. These results indicate that down-regulation of extracellular signal-regulated kinase, substance E and neurokinin-1 protein and mRNA may be a central mechanism for the analgesic effect of herb-partitioned moxibustion.

Polyglutamylase activity of tubulin tyrosine ligase-like 4 is negatively regulated by the never in mitosis gene A family kinase never in mitosis gene A-related kinase 5

BACKGROUND Tubulins,building blocks of microtubules,are modified substrates of diverse post-translational modifications including phosphorylation,polyglycylation and polyglutamylation.Polyglutamylation of microtubules,catalyzed by enzymes from the tubulin tyrosine ligase-like(TTLL)family,can regulate interactions with molecular motors and other proteins.Due to the diversity and functional importance of microtubule modifications,strict control of the TTLL enzymes has been suggested.AIM To characterize the interaction between never in mitosis gene A-related kinase 5(NEK5)and TTLL4 proteins and the effects of TTLL4 phosphorylation.METHODS The interaction between NEK5 and TTLL4 was identified by yeast two-hybrid screening using the C-terminus of NEK5(a.a.260–708)as bait and confirmed by immunoprecipitation.The phosphorylation sites of TTLL4 were identified by mass spectrometry and point mutations were introduced.RESULTS Here,we show that NEK5 interacts with TTLL4 and regulates its polyglutamylation activity.We further show that NEK5 can also interact with TTLL5 and TTLL7.The silencing of NEK5 increases the levels of polyglutamylation of proteins by increasing the activity of TTLL4.The same effects were observed after the expression of the catalytically inactive form of NEK5.This regulation of TTLL4 activity involves its phosphorylation at Y815 and S1136 amino acid residues.CONCLUSION Our results demonstrate,for the first time,the regulation of TTLL activity through phosphorylation,pointing to NEK5 as a potential effector kinase.We also suggest a general control of tubulin polyglutamylation through NEK family members in human cells.

OVER-EXPRESSION OF EXTRACELLULAR SIGNAL-REGULATED KINASE IN VASCULAR SMOOTH MUSCLE CELL OF HYPERTENSIVE RATS

Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery.The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively.The control group were sham operated age-matched Wistar rats.Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats.Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00±33.00 mm Hg at the end of experiment, significantly higher than that in the control rats (P<0.01).Blood pressure in SHR4w (108.00±11.25 mm Hg) was similar to that in the controls.However, it rose to 122.25±21.75 mm Hg in SHR8w, and even up to 201.75±18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls (P<0.01).The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P<0.05).Hyaline degeneration of the afferent arterioles was found in WHR.In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w, and SHR16w.Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2.The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7.09%±1.75%, 14.57%±4.58%, 29.44%±7.35%, and 13.63%±3.85%, respectively) than that of the controls(P<0.01).The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR16w were significantly higher (12.09%±1.40%, 24.17%±6.92%, 32.44%±4.05%, and 18.61%±3.35%, respectively) than that of the controls (P<0.01), too.The expression of ERK1/2 protein of kidney in WHR and SHR16w was significantly higher than that in the controls by Western blotting assay (P<0.01).Conclusion Extracellular signal transduction system are highly expressed in kidney VSMC of two-kidney one clip WHR and SHR.Phospho-ERK1/2 may play an important role in VSMC hypertrophy and hyperplasia under hypertension.

Time-dependent effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in focal cerebral ischemia rats

BACKGROUND： The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and function of brain cells. OBJECTIVE： To observe the effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in the lateral cerebral ventricle wall of rats with focal cerebral ischemia. The effects were analyzed at different time points after intervention. DESIGN： Randomized controlled study. SETTING： Department of Anatomy, Sun Yat-Sen University. MATERIALS： A total of 60 healthy adult male Wistar rats weighing （250±10） g were provided by the Experimental Animal Center, Medical College of Sun Yat-Sen University. The animal experiment was conducted with confirmed consent by the local ethics committee. The GB6805-Ⅱ electric acupuncture apparatus was provided by Shanghai Medical Equipment High-techno Company. METHODS： The experiment was performed at the Laboratory of Anatomy, Sun Yat-Sen University, from February to July 2007. All experimental animals were randomly divided into the following groups： normal group （n = 6）, sham operation group （n = 18）, model group （n = 18）, and electroacupuncture group （n = 18）. Middle cerebral artery occlusion （MCAO） was performed in the model group and electroacupuncture group. Zea Longa＇s grading standard was used to assess neurological impairment after reperfusion; animals whose grades were between l and 4 were included in this study. The normal control group was not exposed to MCAO. In sham operation animals, the right common carotid artery （CCA） was isolated, and the external carotid artery （ECA） was damaged, but no embolism was induced. The electroacupuncture group was given acupuncture on the second day after surgery. The acupoint locations were chosen according to Experimental Acupuncture （People＇s Publishing House; 1997; First Edition）. The Chengjiang, Qihai, and Guanyuan acupoints were labeled and connected to a G6805 electroacupuncture apparatus with sparse-dense waves （sparse waves were 30 Hz, dense waves were 100 Hz）, with a frequency of 6-15 V. The duration was 20 minutes. Two days after surgery, the model and sham operation groups were placed with their backs on the operating table, but they received no acupuncture. However, the normal group received acupuncture. The experimental animals under anesthesia were sacrificed on days 7, 14, and 28 post-surgery. Western blot analysis was used to measure expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall. Expression was measured in the normal group at time points corresponding to the sham operation group. MAIN OUTCOME MEASURES： Expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall at different time points after intervention. RESULTS： All 60 rats were included in the final analysis, without any loss. Seven days after MCAO, there was no significant difference in extracellular signal-regulated kinases 1/2 expression in the electroacupuncture group compared to the model group （P 〉 0.05）. However, extracellular signal-regulated kinases 1/2 expression significantly increased in the model group at 14 and 28 days after treatment （P 〈 0.05）. CONCLUSION： Electroacupuncture at the Ren channel can enhance extracellular signal-regulated kinasesl/2 expression in the inferior region of the lateral cerebral ventricle wall of rats with focal cerebral ischemia. However, this effect is not apparent until 14 days after electroacupuncture intervention.

Transcriptional regulation of human polo-like kinases and early mitotic inhibitors

Human polo-like kinases （PLK1-PLK4） have been implicated in mitotic regulation and carcinogenesis. PLK1 phosphorylates early mitotic inhibitor 1 （Emil） to ensure mitosis entry, whereas Emi2 plays a key role during the meiotic cell cycle. Transcription factor E2F is primarily considered to regulate the G1/S transition of the cell cycle but its involvement in the regulation of mitosis has also been recently suggested. A gap still exists between the molecular basis of E2F and mitotic regulation. The present study was designed to characterize the transcriptional regulation of human PLK and Emi genes. Adenoviral overexpression of E2F1 increased PLK1 and PLK3 mRNA levels in A549 cells. A reporter gene assay revealed that the putative promoter regions of PLK1, PLK3, and PLK4 genes were responsive to activators E2F, E2F1-E2F3. We further characterized the putative promoter regions of Emil and Emi2 genes, and these could be regulated by activators E2F and E2F1-E2F4, respectively. Finally, PLK1-PLK4, Emil, and Emi2 mRNA expression levels in human adult, fetal tissues, and several cell lines indicated that each gene has a unique expression pattern but is uniquely expressed in common tissues and cells such as the testes and thymus. Collectively, these results indicate that E2F can integrate G1/S and G2/M to oscillate the cell cycle by regulating mitotic genes PLK and Emi, leading to determination of the cell fate.