Induced Extracellular Signal-regulated Kinase Activation Research Articles

Extracellular signal-regulated kinase is activated in podocytes from patients with diabetic nephropathy.

In the past few decades, the global prevalence of diabetes has provided us with a warning about future chronic complications. Diabetic nephropathy (DN) is the main cause of end-stage kidney disease. Podocytes in the glomerulus play a critical role in regulating glomerular permeability, and podocyte injury is one of the main causes of DN. Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase family that plays critical roles in intracellular signal transduction. In human patients with DN, phosphorylated ERK (pERK), the active form of ERK, is increased in the glomeruli. However, information on the expression of pERK, specifically in podocytes in DN, is limited. Meanwhile, high glucose induces ERK activation in immortalized podocyte cell lines, suggesting the involvement of podocytic ERK in DN. We performed an immunohistochemical study using Wilms' tumor-1 (WT-1) as a podocyte-specific marker to investigate whether podocytic pERK levels are increased in patients with DN. In the glomeruli of the DN group, we observed remarkable co-staining for WT-1 and pERK. In contrast, the glomeruli of the control group contained only a few pERK-positive podocytes. Statistical analyses revealed that, relative to healthy controls, patients with DN showed significantly increased pERK expression levels in cells that were positive for WT-1 (DN: 51.3 ± 13.1% vs. control: 7.3 ± 1.6%, p = 0.0158, t-test, n = 4 for each group). This suggests that ERK activation in podocytes is involved in the pathogenesis of DN.

The ERK Signaling Cascade Inhibits Gonadotropin-Stimulated Steroidogenesis.

The response of granulosa cells to Luteinizing Hormone (LH) and Follicle- Stimulating Hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the Steroidogenic Acute Regulatory Protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.

RSU-1 interaction with prohibitin-2 links cell–extracellular matrix detachment to downregulation of ERK signaling

Cell–extracellular matrix (ECM) detachment is known to decrease extracellular signal–regulated kinase (ERK) signaling, an intracellular pathway that is central for control of cell behavior. How cell–ECM detachment is linked to downregulation of ERK signaling, however, is incompletely understood. We show here that focal adhesion protein Ras Suppressor 1 (RSU1) plays a critical role in cell–ECM detachment induced suppression of ERK signaling. We have identified prohibitin 2 (PHB2), a component of membrane lipid rafts, as a novel binding protein of RSU1, and mapped a major RSU1-binding site to PHB2 amino acids 150 to 206 in the C-terminal region of the PHB/SPFH (stomatin/prohibitin/flotillin/HflKC) domain. The PHB2 binding is mediated by multiple sites located in the N-terminal leucine-rich repeat region of RSU1. Depletion of PHB2 suppressed cell–ECM adhesion–induced ERK activation. Furthermore, cell–ECM detachment increased RSU1 association with membrane lipid rafts and interaction with PHB2. Finally, knockout of RSU1 or inhibition of RSU1 interaction with PHB2 by overexpression of the major RSU1-binding PHB2 fragment (amino acids 150–206) effectively suppressed the cell–ECM detachment induced downregulation of ERK signaling. Additionally, expression of venus-tagged wild-type RSU1 restored ERK signaling, while expression of venus-tagged PHB2-binding defective RSU1 mutant in which the N-terminal leucine-rich repeat region is deleted did not. Taken together, Our findings identify a novel RSU1-PHB2 signaling axis that senses cell–ECM detachment and links it to decreased ERK signaling.

Mechanisms of protein kinase C-induced sustained activation of extracellular signal-regulated kinase in the hippocampus

Protein Kinase C (PKC) and extracellular signal-regulated kinase (ERK) regulate synaptic plasticity and memory. PKC activation enhances long-term potentiation (LTP) in the hippocampal slices. In addition, activation of PKC by phorbol 12,13-diacetate (PDA) induces ERK activation. However, the mechanisms involved in PDA-induced activation of ERK are not well understood. Using hippocampal slices, we report that PDA induces a sustained activation of ERK. PDA-induced sustained ERK activation critically requires protein synthesis as well as transcription, the cellular processes that play crucial roles in long-lasting LTP and memory. In addition, the mammalian target of rapamycin activity is required for PDA-induced sustained ERK activation. Further, we show that growth factor signalling plays a critical role in PDA-induced sustained ERK activation. These results suggest that sustained ERK activation may have an important role in LTP.

Src family kinase activity drives cytomegalovirus reactivation by recruiting MOZ histone acetyltransferase activity to the viral promoter

Human cytomegalovirus (HCMV) latency and reactivation rely on a complex interplay between cellular differentiation, cell signaling pathways, and viral gene functions. HCMV reactivation in dendritic cells (DCs) is triggered by IL-6 and extracellular signal-regulated kinase (ERK)–mitogen-activated protein kinase signaling. However, activation of the same pathway fails to reactivate HCMV in other myeloid cell types, despite this signaling axis being active in those cells. We hypothesized that IL-6–induced ERK activation initiates the changes in chromatin structure required for viral reactivation but that a concomitant signal is necessary to complete the changes in chromatin structure required for gene expression to occur. Using a differential phosphoproteomics approach in cells that do or do not support IL-6–induced viral reactivation, we identified the concomitant activation of an Src family kinase (SFK), hematopoietic cell kinase (HCK), specifically in DCs in response to IL-6. Pharmacological and genetic inhibition of HCK activity indicated that HCK is required for HCMV reactivation. Furthermore, the HCK/SFK activity was linked to recruitment of the monocytic leukemia zinc finger protein (MOZ) histone acetyltransferase to the viral promoter, which promoted histone acetylation after ERK-mediated histone phosphorylation. Importantly, pharmacological and genetic inhibition of MOZ activity prevented reactivation. These results provide an explanation for the selective activation of viral gene expression in DCs by IL-6, dependent on concomitant SFK and ERK signaling. They also reveal a previously unreported role for SFK activity in the regulation of chromatin structure at promoters in eukaryotic cells via MOZ histone acetyltransferase activity.

Abstract B33: Persistent activation of ERK and SMAD4 nuclear retention by high free fatty acids promote breast cancer metastasis

Abstract Obesity increases the risk of developing distant metastatic recurrence, and diminishes overall and breast cancer-specific survival; however, the mechanisms behind this correlation and relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are usually elevated in obese individuals. Here, using human breast cancer cell lines, we found that TGF-β transiently activates the extracellular signal-regulated kinase (ERK) and subsequently marks SMAD4 for phosphorylation, which facilitates SMAD4-ubiquitin specific peptidase 9, X-linked (USP9x) interaction, SMAD4 nuclear retention, SMAD3-SMAD4 complex nuclear accumulation, and stimulated TGF-β/SMAD3–mediated transcriptional activity of Twist and Snail. USP9x competitively inhibited the E3 ubiquitin-protein ligase transcriptional intermediary factor 1 γ (TIF1γ) from binding and monoubiquitinating SMAD4. This process maintained SMAD4 nuclear retention and stabilized the SMAD3/SMAD4 complex in the nucleus. Inhibition of ERK resulted in SMAD4 de-phosphorylation, USP9x-SMAD4 disassociation and SMAD4-TIF1γ interaction, which ultimately marks SMAD4 for mono-ubiquitination, leading to SMAD4 nuclear export and termination of the transcription of Twist and Snail. In addition, loss of USP9x abolished TGF-β–induced SMAD4 nuclear retention and SMAD3/SMAD4 formation, leading to inhibition of Twist and Snail expression. We also found that ERK-induced SMAD4 T277 phosphorylation and SMAD4-USP9x interaction was required for TGF-β–induced breast cancer invasion, which was attributed to SMAD3/SMAD4-dependent up-regulation of the transcription factors, Twist and Snail. Importantly, FFA further facilitates TGF-β–induced ERK activation, SMAD4 phosphorylation, SMAD4 nuclear retention and SMAD3/SMAD4 formation, thus promoting TGF-β–dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. Additionally, phospho-ERK and -SMAD4 levels correlated with activated TGF-β signaling and metastasis in obese patient breast cancer specimens. Altogether, we demonstrated that USP9x promotes breast cancer invasion by promoting nuclear retention of SMAD4 and identified crosstalk between USP9x and TGF-β/ERK/SMAD4/SMAD3 pathways. Our study highlights the vital interaction of USP9x and SMAD4 for governing TGF-β signaling and dyslipidemia-induced aberrant TGF-β activation during breast cancer metastasis. Citation Format: Yong Wu, Xiaoting Yu, Kruttika Bhat, Sami Dwabe, Mohammad Atefi, Jay Vadgama. Persistent activation of ERK and SMAD4 nuclear retention by high free fatty acids promote breast cancer metastasis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B33.

Negative regulation of the LKB1/AMPK pathway by ERK in human acute myeloid leukemia cells

Adenosine monophosphate-activated protein kinase (AMPK) is a sensor for cellular energy status. When the cellular energy level is decreased, AMPK is activated and functions to suppress energy-consuming processes, including protein synthesis. Recently, AMPK has received attention as an attractive molecular target for cancer therapy. Several studies have revealed that the activation of AMPK by chemical stimulators, such as metformin, induces apoptosis in a variety of hematologic malignant cells. From another perspective, these results suggest that the function of AMPK is impaired in hematologic tumor cells. However, the precise mechanisms by which this impairment occurs are not well understood. In melanoma cells, oncogenic BRAF constitutively activates the extracellular signal-regulated kinase (ERK) pathway and phosphorylates liver kinase B1, an upstream activator of 5' adenosine monophosphate-activated protein kinase (AMPK), resulting in the inactivation of liver kinase B1 and AMPK. In this study, we analyzed whether ERK is involved in the suppression of AMPK activity using established and primary human leukemia cells. We found an inverse correlation between the intensity of ERK activity and the degree of AMPK activation after stimulation with either glucose deprivation or metformin. We also found that the inhibition of ERK activity by U0126 restored AMPK activation after metformin treatment. Furthermore, a combined treatment with metformin and U0126 enhanced the antileukemic activity of metformin. Importantly, metformin induced ERK activation by suppressing the protein levels of dual specificity phosphatase 6, a negative regulator of ERK. This crosstalk between AMPK and ERK could diminish the antileukemic activity of metformin. Taken together, our present observations suggest a novel therapeutic strategy for improving the efficacy of metformin in treating leukemia.

Spry2 is a novel therapeutic target for periodontal tissue regeneration through fibroblast growth factor receptor signaling and epidermal growth factor signaling

Sprouty2 (Spry2) inhibits the activation of the extracellular signal-regulated kinase (ERK) pathway via receptor tyrosine kinase signaling. In a recent paper published in Journal of Cellular Biochemistry, we demonstrated that transfection of a dominant-negative mutant of Spry2 enhanced fibroblast growth factor (FGF)- and epidermal growth factor (EGF)-induced ERK activation in osteoblasts. In contrast, it decreased their activation in gingival epithelial cells. Consistent with these observations, the sequestration of Spry2 increased osteoblast proliferation by FGFR and EGFR stimulation, whereas it decreased gingival epithelial cell proliferation via the ubiquitination and degradation of EGF receptors (EGFR). In addition, reduction of Spry2 activity upregulated Runx2 expression and downregulated Twist, a negative regulator of Runx2 through FGFR and EGFR signaling, resulting in enhanced osteoblastogenesis in osteoblasts. Furthermore, we also found that suppression of Spry2 upregulated cell proliferation and migration in human periodontal ligament cell lines when they were stimulated by both FGF and EGF, and led to a shift in macrophage polarization, exerted immunosuppressive and tissue-repairing effects in macrophages. These results suggest that the application of a Spry2 inhibitor may effectively resolve inflammation by periodontitis and allow periodontal ligament and alveolar bone to grow and block the ingrowth of gingival epithelial cells in bony defects, biologically mimicking the barrier effect seen in conventional GTR. This approach has potential for developing a new regeneration strategy.

Insufficient radiofrequency ablation promotes human hepatoma SMMC7721 cell proliferation by stimulating vascular endothelial growth factor overexpression.

The aims of the current study were to investigate the influence of insufficient radiofrequency ablation (RFA) on the cell proliferation of the human hepatocellular carcinoma (HCC) cells, SMMC7721, and to elucidate the underlying mechanism. SMMC7721 cells were subjected to a 47°C treatment regimen to simulate insufficient RFA, in the presence or absence of KN93 [a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII)], PD98059 [a specific inhibitor of extracellular signal-regulated kinase (ERK)], or axitinib (a specific inhibitor of vascular endothelial growth factor (VEGF) receptor]. Cell proliferation was determined using a thiazolyl terazolium assay (MTT). The levels of CaMKII, phospho-CaMKII, ERK, phospho-ERK and VEGF were observed by western blot analysis. The results demonstrated that the 47°C treatment regimen: i) Triggered upregulation of VEGF expression in the SMMC7721 cells, which was reduced by CaMKII or ERK inhibition; ii) induced ERK activation was prevented by KN93; and iii) promoted SMMC7721 cell proliferation, which was greatly inhibited by axitinib, KN93 and PD98059. In conclusion, the results indicated that insufficient RFA promotes SMMC7721 cell proliferation by activating CaMKII/ERK-dependent VEGF overexpression.

Mechanisms of cAMP-induced sustained activation of extracellular signal-regulated kinase in the hippocampus

Protein phosphorylation is known to regulate synaptic plasticity and memory. Protein kinases including protein kinase A and extracellular signal-regulated kinase (ERK) play important roles in these processes. Forskolin, a protein kinase A activator, induces long-term potentiation (LTP) in the hippocampus. Forskolin also induces ERK activation, which plays important roles in LTP. However, the mechanisms of forskolin-induced ERK activation are not clearly understood. Here we show that forskolin induces sustained ERK activation in the hippocampal slices. Further, blockade of protein synthesis or transcription inhibits forskolin-induced sustained ERK activation. In contrast, forskolin-induced immediate ERK activation is unaffected by inhibition of protein synthesis or transcription. Sustained ERK activation may contribute to forskolin-induced LTP in the hippocampus.

Suppressive Effects of SuHeXiang Wan on Amyloid-β42-Induced Extracellular Signal-Regulated Kinase Hyperactivation and Glial Cell Proliferation in a Transgenic <i>Drosophila</i> Model of Alzheimer’s Disease

SuHeXiang Wan (SHXW), a Chinese traditional medicine, has been used to treat infantile convulsions, seizures and strokes. Previously, we reported that modified SHXW, called KSOP1009, suppressed the hyper-activation of c-Jun N-terminal kinase (JNK) and Alzheimer's disease (AD)-like phenotypes in amyloid-β42 (Aβ42)-expressing Drosophila AD models. In the present study, we, further, investigated the detailed mechanism by which KSOP1009 suppresses the AD-like phenotypes of the model flies. As seen in the brains of AD patients, pan-neuronal expression of Aβ42 in Drosophila increased activation of extracellular signal-regulated kinase (ERK), which was monitored by its phosphorylation level, and the number of glial cells in the brain. Suppression of caspase activity did not affect these phenomena, suggesting that Aβ42 induces ERK activation and glial cell proliferation independently of apoptotic processes. KSOP1009 intake significantly reduced the level of ERK activation and the number of glial cells. Moreover, KSOP1009 intake also effectively decreased the defects in the wing vein formation induced by Epidermal growth factor receptor (Egfr) overexpression in fly wings, suggesting that it may contain an inhibitory substance that inhibits the EGFR/ERK signaling pathway. In addition, the Aβ42-induced locomotive defect was partially rescued by inhibition of the elevated ERK activity through its antagonistic drug treatment. Taken together, these results suggest that KSOP1009 exerts its therapeutic effect by inhibiting the EGFR/ERK pathway and glial cell proliferation and by suppressing the JNK pathway and apoptosis.

P3‐034: Rapamycin downregulates tau phosphorylation in SH‐SY5Y cells

P3‐034: Rapamycin downregulates tau phosphorylation in SH‐SY5Y cells

The PP242 Mammalian Target of Rapamycin (mTOR) Inhibitor Activates Extracellular Signal-regulated Kinase (ERK) in Multiple Myeloma Cells via a Target of Rapamycin Complex 1 (TORC1)/ Eukaryotic Translation Initiation Factor 4E (eIF-4E)/RAF Pathway and Activation Is a Mechanism of Resistance

Activation of PI3-K-AKT and ERK pathways is a complication of mTOR inhibitor therapy. Newer mTOR inhibitors (like pp242) can overcome feedback activation of AKT in multiple myeloma (MM) cells. We, thus, studied if feedback activation of ERK is still a complication of therapy with such drugs in this tumor model. PP242 induced ERK activation in MM cell lines as well as primary cells. Surprisingly, equimolar concentrations of rapamycin were relatively ineffective at ERK activation. Activation was not correlated with P70S6kinase inhibition nor was it prevented by PI3-kinase inhibition. ERK activation was prevented by MEK inhibitors and was associated with concurrent stimulation of RAF kinase activity but not RAS activation. RAF activation correlated with decreased phosphorylation of RAF at Ser-289, Ser-296, and Ser-301 inhibitory residues. Knockdown studies confirmed TORC1 inhibition was the key proximal event that resulted in ERK activation. Furthermore, ectopic expression of eIF-4E blunted pp242-induced ERK phosphorylation. Since pp242 was more potent than rapamycin in causing sequestering of eIF-4E, a TORC1/4E-BP1/eIF-4E-mediated mechanism of ERK activation could explain the greater effectiveness of pp242. Use of MEK inhibitors confirmed ERK activation served as a mechanism of resistance to the lethal effects of pp242. Thus, although active site mTOR inhibitors overcome AKT activation often seen with rapalog therapy, feedback ERK activation is still a problem of resistance, is more severe than that seen with use of first generation rapalogs and is mediated by a TORC1- and eIF-4E-dependent mechanism ultimately signaling to RAF.

Ethyl-3,4-dephostatin Inhibits PTPN2 and Induces ERK Activation

The mitogen-activated protein kinases (MAPKs) are key signaling enzymes that play critical roles in cellular signal transduction pathways such as cell survival, proliferation, differentiation, and apoptosis. The three major groups of MAPK that are expressed in mammals are p38, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK). Signaling by MAPKs is processed following their phosphorylation at conserved threonine and tyrosine residues within the activation loop of MAPKs mediated by their upstream kinases in activation. Protein tyrosine phosphatases (PTPs) are responsible for regulation of MAPK activity through dephosphorylation of phospho-threonine and phospho-tyrosine residues on MAPKs. The human genome encodes 107 PTPs. PTPs can be classified into four groups based on amino-acid sequences of catalytic domains. They are class I Cys-based PTPs, class II Cys-based PTPs, class III Cys-based PTPs, and Asp-based PTPs. Especially, protein tyrosine phosphatase non-receptor type 2 (PTPN2) that is also known as T cell protein tyrosine phosphatase (TC-PTP) belongs to class I Cys-based PTPs as the intracellular, non-receptor PTPs (NRPTPs) with a high degree of sequence and structural homology within the catalytic domain. PTPN2 has been reported to modulate cytokine receptor signaling, including IFN-γ signaling. PTPN2 dephosphorylates and inactivates Src tyrosine kinases to suppress downstream signaling through ERK and production of interleukin 6. In a way to study biological functions of PTPN2, we performed the regulatory mechanism of PTPN2 activity using a specific chemical compound, ethyl-3,4-dephostatin. Ethyl-3,4-dephostatin (Fig. 1) was originally identified as a potent PTP-1B and src homology-2-containing protein tyrosine phosphatase-1 (SHP-1) inhibitor and showed inhibitory effect on several PTPs in vitro. Recently, dual-specificity phosphatase 22 (DUSP22) was identified as an additional target of ethyl-3,4-dephostatin. To investigate whether ethyl-3,4-dephostatin was able to inhibit PTPN2 activity, we treated various concentrations of ethyl-3,4-dephostatin on purified active PTPN2. PTPN2 activity was decreased by ethyl-3,4-dephostatin in a dosedependent manner. The half maximal inhibitory concentration (IC50) of PTPN2 was found to be about 6.5 ± 0.54 μM by using the curve fitting program PRISM 3.0 (Fig. 2(a)). In addition, the Lineweaver-Burk plot shows that the Ki was 8.6 μM (Fig. 2(b)). This result indicates that ethyl-3,4dephostatin acts as a competitive inhibitor of PTPN2, suggesting that ethyl-3,4-dephostatin suppresses the activity of PTPN2 through binding to the catalytic site. We further examined whether ethyl-3,4-dephostatin influences PTPN2-mediated ERK inhibition in vivo. Since PTPN2 inactivates Src tyrosine kinases that are located upstream of ERK signaling, we overexpressed PTPN2 in HEK 293 cells and detected phosphorylation levels of endogenous ERK after treatment with ethyl-3,4-dephostatin. HEK 293 cells were transfected with FLAG-PTPN2 expression plasmid and

Tricyclic Antidepressant Amitriptyline Activates Fibroblast Growth Factor Receptor Signaling in Glial Cells: INVOLVEMENT IN GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR PRODUCTION

Recently, both clinical and animal studies demonstrated neuronal and glial plasticity to be important for the therapeutic action of antidepressants. Antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production through monoamine-independent protein-tyrosine kinase, extracellular signal-regulated kinase (ERK), and cAMP responsive element-binding protein (CREB) activation in glial cells (Hisaoka, K., Takebayashi, M., Tsuchioka, M., Maeda, N., Nakata, Y., and Yamawaki, S. (2007) J. Pharmacol. Exp. Ther. 321, 148-157; Hisaoka, K., Maeda, N., Tsuchioka, M., and Takebayashi, M. (2008) Brain Res. 1196, 53-58). This study clarifies the type of tyrosine kinase and mechanism of antidepressant-induced GDNF production in C6 glioma cells and normal human astrocytes. The amitriptyline (a tricyclic antidepressant)-induced ERK activation was specifically and completely inhibited by fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors and siRNA for FGFR1 and -2. Treatment with amitriptyline or several different classes of antidepressants, but not non-antidepressants, acutely increased the phosphorylation of FGFRs and FGFR substrate 2α (FRS2α). Amitriptyline-induced CREB phosphorylation and GDNF production were blocked by FGFR-tyrosine kinase inhibitors. Therefore, antidepressants activate the FGFR/FRS2α/ERK/CREB signaling cascade, thus resulting in GDNF production. Furthermore, we attempted to elucidate how antidepressants activate FGFR signaling. The effect of amitriptyline was inhibited by heparin, non-permeant FGF-2 neutralizing antibodies, and matrix metalloproteinase (MMP) inhibitors. Serotonin (5-HT) also increased GDNF production through FGFR2 (Tsuchioka, M., Takebayashi, M., Hisaoka, K., Maeda, N., and Nakata, Y. (2008) J. Neurochem. 106, 244-257); however, the effect of 5-HT was not inhibited by heparin and MMP inhibitors. These results suggest that amitriptyline-induced FGFR activation might occur through an extracellular pathway, in contrast to that of 5-HT. The current data show that amitriptyline-induced FGFR activation might occur by the MMP-dependent shedding of FGFR ligands, such as FGF-2, thus resulting in GDNF production.

Differential involvement of medial prefrontal cortex and basolateral amygdala extracellular signal-regulated kinase in extinction of conditioned taste aversion is dependent on different intervals of extinction following conditioning

Extinction reflects a decrease in the conditioned response (CR) following non-reinforcement of a conditioned stimulus. Behavioral evidence indicates that extinction involves an inhibitory learning mechanism in which the extinguished CR reappears with presentation of an unconditioned stimulus. However, recent studies on fear conditioning suggest that extinction erases the original conditioning if the time interval between fear acquisition and extinction is short. The present study examined the effects of different intervals between acquisition and extinction of the original memory in conditioned taste aversion (CTA). Male Long-Evans rats acquired CTA by associating a 0.2% sucrose solution with malaise induced by i.p. injection of 4 ml/kg 0.15 M LiCl. Two different time intervals, 5 and 24 h, between CTA acquisition and extinction were used. Five or 24 h after CTA acquisition, extinction trials were performed, in which a bottle containing 20 ml of a 0.2% sucrose solution was provided for 10 min without subsequent LiCl injection. If sucrose consumption during the extinction trials was greater than the average water consumption, then rats were considered to have reached CTA extinction. Rats subjected to extinction trials lasting 24 h, but not 5 h, after acquisition re-exhibited the extinguished CR following injection of 0.15 M LiCl alone 7 days after acquisition. Extracellular signal-regulated kinase (ERK) in the medial prefrontal cortex (mPFC) and basolateral nucleus of the amygdala (BLA) was examined by Western blot after the first extinction trial. ERK activation in the mPFC was induced after the extinction trial beginning 5 h after acquisition, whereas the extinction trial performed 24 h after acquisition induced ERK activation in the BLA. These data suggest that the original conditioning can be inhibited or retained by CTA extinction depending on the time interval between acquisition and extinction and that the ERK transduction pathway in the mPFC and BLA is differentially involved in these processes.

Light touch induces ERK activation in superficial dorsal horn neurons after inflammation: involvement of spinal astrocytes and JNK signaling in touch‐evoked central sensitization and mechanical allodynia

Activation of extracellular signal-regulated kinase (ERK) in spinal cord neurons could serve as a marker for sensitization of dorsal horn neurons in persistent pain. ERK is normally activated by high-threshold noxious stimuli. We investigated how low-threshold mechanical stimuli could activate ERK after complete Freund's adjuvant (CFA)-induced inflammation. Unilateral injection of CFA induced ipsilateral heat hyperalgesia and bilateral mechanical allodynia. CFA-induced ERK activation in ipsilateral dorsal horn neurons declined after 2 days. Interestingly, low-threshold mechanical stimulation given by light touch either on the inflamed paw or the contralateral non-inflamed paw dramatically increased ERK phosphorylation in the dorsal horn ipsilateral to touch stimulation. Notably, light touch induced ERK phosphorylation mainly in superficial neurons in laminae I-IIo. Intrathecal administration of the astroglial toxin L-α-aminoadipate on post-CFA day 2 reversed CFA-induced bilateral mechanical allodynia but not heat hyperalgesia. Furthermore, L-α-aminoadipate, the glial inhibitor fluorocitrate, and a peptide inhibitor of c-Jun N-terminal Kinase all reduced light touch-evoked ERK activation ipsilateral to touch. Collectively, these data suggest that (i) ERK can be activated in superficial dorsal horn neurons by low-threshold mechanical stimulation under pathological condition and (ii) ERK activation by light touch is associated with mechanical allodynia and requires an astrocyte network.

MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show here that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21 WAF1 and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin α vβ 3 were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin.

AVS-1357 inhibits melanogenesis via prolonged ERK activation.

In this study, we demonstrated that a derivative of imidazole, AVS-1357, is a novel skin-whitening compound. AVS-1357 was found to significantly inhibit melanin production in a dose-dependent manner; however, it did not directly inhibit tyrosinase. Furthermore, we found that AVS-1357 induced prolonged activation of extracellular signal-regulated kinase (ERK) and Akt, while it downregulated microphthalmia-associated transcription factor (MITF) and tyrosinase. It has been reported that the activation of ERK and/or Akt is involved in melanogenesis. Therefore, we examined the effects of AVS-1357 on melanogenesis in the absence or presence of PD98059 (a specific inhibitor of the ERK pathway) and/or LY294002 (a specific inhibitor of the Akt pathway). PD98059 dramatically increased melanogenesis, whereas LY294002 had no effect. Furthermore, PD98059 attenuated AVS-1357 induced ERK activation, as well as the downregulation of MITF and tyrosinase. These findings suggest that the effects of AVS-1357 occur via downregulation of MITF and tyrosinase, which is caused by AVS-1357-induced prolonged ERK activation. Taken together, our results indicate that AVS-1357 has the potential as a new skin whitening agent.

Cyclooxygenase-2 Up-Regulates Ataxia Telangiectasia and Rad3 Related through Extracellular Signal-Regulated Kinase Activation

Cyclooxygenase-2 (COX-2) overexpression caused prolonged G2 arrest after exposure to ionizing radiation (IR) in our previous study. We were therefore interested in investigating the function of COX-2 in the G2 checkpoint pathway. Interestingly, we found that cells in which COX-2 is overexpressed showed up-regulated ataxia telangiectasia and Rad3 related (ATR) expression compared with control cells. In this study, we investigated the mechanism of ATR up-regulation by COX-2 and tested our hypothesis that COX-2-induced extracellular signal-regulated kinase (ERK) activation mediates up-regulation of ATR by COX-2. To investigate the relationship between COX-2 and ATR, we used two stable COX-2-overexpressing cancer cell lines (HCT116-COX-2 and H460-COX-2), a COX-2 knockdown A549 lung cancer cell line (AS), and an ATR knockdown HCT116 cell line. Cells were treated with various drugs [celecoxib, prostaglandin E2 (PGE2), PD98059, U0126, and hydroxyurea] and were then analyzed using reverse transcription-PCR, confocal microscopy, Western blotting, and clonogenic assay. COX-2-overexpressing cells were shown to have increased ERK phosphorylation and ATR expression compared with control cells, whereas AS cells were shown to have decreased levels of phospho-ERK and ATR. In addition, exogenously administered PGE2 increased ERK phosphorylation. Inhibition of ERK phosphorylation decreased ATR expression in both HCT116-COX-2 and A549 cells. HCT116-COX-2 cells were resistant to IR or hydroxyurea compared with HCT116-Mock cells, whereas administration of ATR shRNA showed the opposite effect. COX-2 stimulates ERK phosphorylation via PGE2. This COX-2-induced ERK activation seems to increase ATR expression and activity in endogenous COX-2-overexpressing cancer cells as well as in COX-2-overexpressing stable cell lines.