Basal Extracellular Signal-regulated Kinase Research Articles

Deletion of p66Shc dysregulates ERK and STAT3 activity in embryonic stem cells, enhancing their naïve-like self-renewal in the presence of LIF.

The ShcA adapter protein is necessary for early embryonic development. The role of ShcA in development is primarily attributed to its 52kDa and 46kDa isoforms, that transduce receptor tyrosine kinase (RTK) signaling via the extracellular signal regulated kinase (ERK). During embryogenesis, ERK acts as the primary signalling effector, driving fate acquisition and germ layer specification. P66Shc, the largest of the ShcA isoforms, has been observed to antagonize ERK in several contexts, however its role during embryonic development remains poorly understood. We hypothesized that p66Shc could act as a negative regulator of ERK activity during embryonic development, antagonizing early lineage commitment. To explore the role of p66Shc in stem cell self-renewal and differentiation, we created a p66Shc knockout (KO) murine embryonic stem cell (mESC) line. Deletion of p66Shc enhanced basal ERK activity, but surprisingly, instead of inducing mESC differentiation, loss of p66Shc enhanced the expression of core and naïve pluripotency markers. Using pharmacologic inhibitors to interrogate potential signalling mechanisms, we discovered that p66Shc deletion permits the self-renewal of naïve mESCs in the absence of conventional growth factors, by increasing their responsiveness to leukemia inhibitory factor (LIF). We discovered that loss of p66Shc enhanced not only increased ERK phosphorylation but also increased phosphorylation of Signal transducer and activator of transcription (STAT3(S727)) in mESCs, which may be acting to stabilize their naïve-like identity, desensitizing them to ERK-mediated differentiation cues. These findings identify p66Shc as a regulator of both LIF-mediated ESC pluripotency and of signaling cascades that initiate post-implantation embryonic development and ESC commitment.

Redundant roles of EGFR ligands in the ERK activation waves during collective cell migration.

Epidermal growth factor receptor (EGFR) plays a pivotal role in collective cell migration by mediating cell-to-cell propagation of extracellular signal-regulated kinase (ERK) activation. Here, we aimed to determine which EGFR ligands mediate the ERK activation waves. We found that epidermal growth factor (EGF)-deficient cells exhibited lower basal ERK activity than the cells deficient in heparin-binding EGF (HBEGF), transforming growth factor alpha (TGFα) or epiregulin (EREG), but all cell lines deficient in a single EGFR ligand retained the ERK activation waves. Surprisingly, ERK activation waves were markedly suppressed, albeit incompletely, only when all four EGFR ligands were knocked out. Re-expression of the EGFR ligands revealed that all but HBEGF could restore the ERK activation waves. Aiming at complete elimination of the ERK activation waves, we further attempted to knockout NRG1, a ligand for ErbB3 and ErbB4, and found that NRG1-deficiency induced growth arrest in the absence of all four EGFR ligand genes. Collectively, these results showed that EGFR ligands exhibit remarkable redundancy in the propagation of ERK activation waves during collective cell migration.

Molecular Mechanism of GPCR‐mediated ERK Pathway in Cancer

G protein-coupled receptors (GPCRs) are known to activate extracellular-signal-regulated kinase (ERK), a master regulator of cell proliferation and survival and a key oncogenic component. The co-occurrence of KRAS and GNAS (which encodes Gsa) mutations has been reported in several cancers including pancreatic ductal adenocarcinoma (PDAC), hinting at synergistic roles of these two pathways in activating pro-tumorigenic ERK. However, the precise mechanisms and the role underlying GPCR-mediated ERK regulation are not clearly understood. Here we investigated how the spatially organized β2-adrenergic receptor (b2AR) signaling controls ERK using FRET-based ERK activity biosensors targeted to various subcellular regions, which enable us to interrogate spatial and temporal regulation of ERK activity in living cells. In b2AR expressing HEK293 cells, epinephrine induces ERK activity at the endosome and in the nucleus, but not at the plasma membrane. This pool of ERK activity depends on endosome-localized active Gsa and requires ligand-stimulated b2AR endocytosis. The inhibition of endosomal b2AR and Gsa signaling using endosome-targeted nanobody and peptide inhibitor decreased basal nuclear ERK activity and proliferation in AsPC-1 cells, a PDAC cell line. The discovery of endosomal Gsa-mediated ERK activation could add the missing spatiotemporal information of GPCR regulation of ERK and also fill in the gap in our knowledge of GPCR signaling in cancer development, providing the basis for new therapeutic strategies for PDAC.

Resveratrol induces cervical cancer HeLa cell apoptosis through the activation and nuclear translocation promotion of FOXO3a.

Resveratrol (trans-3,4'V,5-trihydroxystilbene) presents antioxidant, anti-inflammatory, and cardioprotective functions in addition to its anticancer potential. In this study, we explored how resveratrol, as an anticancer agent, effectively influences cervical cancer HeLa cells. Our data showed that resveratrol could significantly inhibit HeLa cell proliferation and induce their apoptosis, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and flow cytometry. The immunofluorescence staining results in the present study suggested that resveratrol could facilitate FOXO3a nuclear translocation. We then focused on the mechanism of resveratrol in promoting HeLa cell apoptosis. The following experiments suggested that the possible initial mechanism involves the upregulation Forkhead box O (FOXO) 3a expression, which further increases the expression of Bcl-2 interacting mediator of cell death (BIM), the gene transcribed in apoptosis. Resveratrol could also inactivate the basal extracellular signal-regulated kinase (ERK) activity, causing FOXO3a activation and resulting in HeLa cell apoptosis. In summary, both mechanisms stimulated the accumulation of activated FOXO3a, promoted its nuclear translocation, and ultimately caused HeLa cell apoptosis. Thus, resveratrol may have a potential in the treatment of cervical cancer.

β-Arrestin-dependent signaling in GnRH control of hormone secretion from goldfish gonadotrophs and somatotrophs

In goldfish, two native isoforms of gonadotropin-releasing hormone (GnRH2 and GnRH3) stimulate luteinizing hormone (LH) and growth hormone (GH) release from pituitary cells through activation of cell-surface GnRH-receptors (GnRHRs) on gonadotrophs and somatotrophs. Interestingly, GnRH2 and GnRH3 induce LH and GH release via non-identical post-receptor signal transduction pathways in a ligand- and cell-type-selective manner. In this study, we examined the involvement of β-arrestins in the control of GnRH-induced LH and GH secretion from dispersed goldfish pituitary cells. Treatment with Barbadin, which interferes with β-arrestin and β2-adaptin subunit interaction, reduced LH responses to GnRH2 and GnRH3, as well as GH responses to GnRH2; but enhanced GnRH3-induced GH secretion. Barbadin also had positive influences on basal hormone release, and basal GH release in particular, as well as basal activity of extracellular signal-regulated kinase (ERK) and GnRH-induced ERK activation. These findings indicate that β-arrestins play permissive roles in the control of GnRH-stimulated LH release. However, in somatotrophs, β-arrestins, perhaps by mediating agonist-selective endosomal trafficking of engaged GnRHRs, participate in GnRH-isoform-specific GH release responses (stimulatory and inhibitory for GnRH2-GnRHR and GnRH3-GnRHR activation, respectively). The correlative stimulatory influences of Barbadin on basal hormone release and ERK activation suggest that β-arrestins may negatively regulate basal secretion through modulation of basal ERK activity. These results provide the first direct evidence of a role for β-arrestins in hormone secretion from an untransformed primary pituitary cell model, and establish these proteins as important receptor-proximal players in mediating functional selectivity downstream of goldfish GnRHRs.

Lapatinib-induced annexin A6 upregulation as an adaptive response of triple-negative breast cancer cells to EGFR tyrosine kinase inhibitors.

The epidermal growth factor receptor (EGFR) is a major oncogene in triple-negative breast cancer (TNBC), but the use of EGFR-targeted tyrosine kinase inhibitors (TKI) and therapeutic monoclonal antibodies is associated with poor response and acquired resistance. Understanding the basis for the acquired resistance to these drugs and identifying biomarkers to monitor the ensuing resistance remain a major challenge. We previously showed that reduced expression of annexin A6 (AnxA6), a calcium-dependent membrane-binding tumor suppressor, not only promoted the internalization and degradation of activated EGFR but also sensitized TNBC cells to EGFR-TKIs. Here, we demonstrate that prolong (>3 days) treatment of AnxA6-low TNBC cells with lapatinib led to AnxA6 upregulation and accumulation of cholesterol in late endosomes. Basal extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation was EGFR independent and significantly higher in lapatinib-resistant MDA-MB-468 (LAP-R) cells. These cells were more sensitive to cholesterol depletion than untreated control cells. Inhibition of lapatinib-induced upregulation of AnxA6 by RNA interference (A6sh) or withdrawal lapatinib from LAP-R cells not only reversed the accumulation of cholesterol in late endosomes but also led to enrichment of plasma membranes with cholesterol, restored EGFR-dependent activation of ERK1/2 and sensitized the cells to lapatinib. These data suggest that lapatinib-induced AnxA6 expression and accumulation of cholesterol in late endosomes constitute an adaptive mechanism for EGFR-expressing TNBC cells to overcome prolong treatment with EGFR-targeted TKIs and can be exploited as an option to inhibit and/or monitor the frequently observed acquired resistance to these drugs.

CB1 allosteric modulator Org27569 is an antagonist/inverse agonist of ERK1/2 signaling.

Introduction: Allosteric modulation of cannabinoid type-1 receptors (CB1) is a novel means through which signaling bias may be exerted. Org27569 remains the most-characterized CB1 allosteric modulator, yet there are conflicting reports regarding its effects on extracellular signal-regulated kinase (ERK) signaling. We conducted a systematic evaluation of Org27569's effects on cannabinoid agonists and ERK signaling.Materials and Methods: HEK293 cells transfected with the human cannabinoid type-1 receptor (hCB1) were treated with Org27569 alone or in combination with the endocannabinoid 2-arachidonoylglycerol (2-AG), the synthetic cannabinoid CP55,940, or the phytocannabinoid delta-9-tetrahydrocannabinol (THC) and ERK activation was measured by western blot. Overnight treatment with pertussis toxin (PTX) was used to determine the role of Gi/o in Org27569's inverse agonist effects. HEK293 cells transfected with green fluorescent protein tagged rat CB1 receptor were used to assess effects of Org27569 on CP55,940-induced receptor internalization. Subcellular fractionation was used to determine effects of Org27569 on ERK phosphorylation in both nuclear and cytosolic compartments.Results: We found that Org27569 is an antagonist of hCB1-mediated ERK signaling in HEK293 cells where it fully blocks CP55,940-but does not completely inhibit THC- and 2-AG-stimulated ERK1/2 activation following 5 min treatment. In rat CB1 HEK293 cells, CP55,940 (1 μM) treatment produced a significant increase in puncta at 20, 40, 60, and 120 min, consistent with receptor internalization. Org27569 (10 μM) co-treatment prevented internalization at each time point and alone had no effect. Org27569 reduced basal ERK phosphorylation in hCB1 HEK293 cells but not in untransfected cells following 20 min treatment. Overnight treatment with PTX abated this response. Following subcellular fractionation, Org27569 produced a significant decrease in ERK phosphorylation in the nuclear-enriched and cytosolic fractions.Conclusions: These data are consistent with previous studies demonstrating that CB1-mediated ERK1/2 activation is Gi/o-dependent and that Org27569 is an inverse agonist of CB1 receptors. Abrogation of Org27569's ability to reduce basal ERK phosphorylation following treatment with PTX and lack of inverse agonist effects in untransfected HEK293 cells demonstrates that Org27569 acts via CB1-Gi/o to produce this effect. To our knowledge, this is the first reported demonstration of inverse agonism of ERK signaling by Org27569.

Complement Factor I Promotes Progression of Cutaneous Squamous Cell Carcinoma

The incidence of cutaneous squamous cell carcinoma (cSCC) is rising worldwide. We have examined the role of complement components in the progression of cSCC. Analysis of cSCC cell lines (n=8) and normal human epidermal keratinocytes (n=11) with whole transcriptome profiling (SOLiD), quantitative real-time reverse transcriptase-PCR, and western blotting revealed marked overexpression of complement factor I (CFI) in cSCC cells. Immunohistochemical analysis for CFI in vivo showed stronger tumor cell-specific labeling intensity in invasive sporadic cSCCs (n=83) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n=7) than in cSCC in situ (n=65), premalignant epidermal lesions (actinic keratoses, n=64), benign epidermal papillomas (seborrheic keratoses, n=39), and normal skin (n=9). The expression of CFI was higher in the aggressive Ha-ras-transformed cell line (RT3) than in less tumorigenic HaCaT cell lines (HaCaT, A5, and II-4). The expression of CFI by cSCC cells was upregulated by IFN-γ and IL-1β. Knockdown of CFI expression inhibited proliferation and migration of cSCC cells and resulted in inhibition of basal extracellular signal-regulated kinase (ERK) 1/2 activation. Knockdown of CFI expression potently inhibited growth of human cSCC xenograft tumors in vivo. These results provide evidence for the role of CFI in the progression of cSCC and identify it as a potential therapeutic target in this nonmelanoma skin cancer.

In an in vitro model of human tuberculosis, monocyte-microglial networks regulate matrix metalloproteinase-1 and -3 gene expression and secretion via a p38 mitogen activated protein kinase-dependent pathway

BackgroundTuberculosis (TB) of the central nervous system (CNS) is characterized by extensive tissue inflammation, driven by molecules that cleave extracellular matrix such as matrix metalloproteinase (MMP)-1 and MMP-3. However, relatively little is known about the regulation of these MMPs in the CNS.MethodsUsing a cellular model of CNS TB, we stimulated a human microglial cell line (CHME3) with conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb). MMP-1 and MMP-3 secretion was detected using ELISAs confirmed with casein zymography or western blotting. Key results of a phospho-array profile that detects a wide range of kinase activity were confirmed with phospho-Western blotting. Chemical inhibition (SB203580) of microglial cells allowed investigation of expression and secretion of MMP-1 and MMP-3. Finally we used promoter reporter assays employing full length and MMP-3 promoter deletion constructs. Student’s t-test was used for comparison of continuous variables and multiple intervention experiments were compared by one-way ANOVA with Tukey’s correction for multiple pairwise comparisons.ResultsCoMTb up-regulated microglial MMP-1 and MMP-3 secretion in a dose- and time-dependent manner. The phospho-array profiling showed that the major increase in kinase activity due to CoMTb stimulation was in p38 mitogen activated protein kinase (MAPK), principally the α and γ subunits. p38 phosphorylation was detected at 15 minutes, with a second peak of activity at 120 minutes. High basal extracellular signal-regulated kinase activity was further increased by CoMTb. Secretion and expression of MMP-1 and MMP-3 were both p38 dependent. CoMTb stimulation of full length and MMP-3 promoter deletion constructs demonstrated up-regulation of activity in the wild type but a suppression site between -2183 and -1612 bp.ConclusionsMonocyte-microglial network-dependent MMP-1 and MMP-3 gene expression and secretion are dependent upon p38 MAPK in tuberculosis. p38 is therefore a potential target for adjuvant therapy in CNS TB.

Extracellular signal-regulated kinases (ERK1/2) signaling pathway plays a role in cortisol secretion in the long-term hypoxic ovine fetal adrenal near term

This study assessed the role of the extracellular signal-regulated kinase (ERK) signaling pathway on the previously observed enhanced cortisol secretion in response to adrenocorticotropic hormone (ACTH) treatment in fetal adrenocortical cells (FACs) from long-term hypoxic (LTH) ovine fetuses. Ewes were maintained at high altitude (3,820 m) from ~40 to 138-141 days gestation when FACs were collected and challenged with either ACTH (10 nM) or 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP, 10 mM) in the presence or absence of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MEK)/ERK inhibitor UO126 (10 μM). FACs from age-matched normoxic fetuses served as controls. Media and FACs were collected at selected time intervals after ACTH or 8-bromo-cAMP stimulation for cortisol measurement and Western analysis of ERK1/2 and phospho-ERK1 and -2 (pERK1/2). After ACTH or 8-bromo-cAMP treatment, cortisol production was greater in the LTH group compared with control (P < 0.05). UO126 reduced ACTH and 8-bromo-cAMP-mediated cortisol output in both groups (P < 0.01 vs. ACTH or 8-bromo-cAMP alone). Under basal conditions, ERK1/2 and pERK1/2 were not different between LTH and normoxic fetuses. In response to ACTH or 8-bromo-cAMP treatment, ERK1/2 were not different between groups; however, pERK1/2 were elevated in the LTH FACs compared with normoxic control FACs. ERK1/2 phosphorylation declined following ACTH treatment in the control group, but UO126 had no effect on ERK1/2 compared with untreated levels. Both ACTH and 8-bromo-cAMP treatment resulted in a decline of protein levels. UO126 pretreatment virtually eliminated pERK1/2 expression. We conclude that basal ERK signaling in FACs is necessary for normal cortisol production and sustained pERK in LTH adrenals enhances cortisol production.

Effects of tick saliva on the migratory and invasive activity of Saos-2 osteosarcoma and MDA-MB-231 breast cancer cells

In previous studies we showed that tick saliva modulates the migratory activity of cells involved in the wound healing response. Since cell migration is a prerequisite for tumor invasion and metastasis, we examined the effects of tick saliva on the migratory and invasive activity of Saos-2 osteosarcoma and MDA-MB-231 (MB-231) breast cancer cells and the potential signaling pathways that may be affected. Saliva inhibited basal and agonist-induced Saos-2 and MB-231 migration and invasion through a matrigel-coated filter. In the Saos-2 cells, saliva suppressed epidermal growth factor (EGF)-activation of Akt/Protein Kinase B, however, only basal extracellular signal-regulated kinase (ERK) activity was affected in MB-231 cells. EGF receptor (EGFR) overexpression masked the effect of saliva on MB-231 cells, but its ability to inhibit MB-231 migration was enhanced by the EGFR inhibitor PD 168393 and MEK inhibitor U0126. Our data indicate that the mechanisms ticks have evolved to regulate the wound healing response have generalized effects on the migratory and invasive activities of metastatic cancer cells.

Combinatorial topography and cell-type specific regulation of the ERK pathway by dopaminergic agonists in the mouse striatum

Therapeutic agents and drugs of abuse regulate the extracellular signal-regulated kinase (ERK) cascade signaling in the medium-sized spiny neurons (MSNs) of the striatum. However, whether this regulation is associated with specific cortical and thalamic inputs has never been studied. We used Drd2-EGFP BAC-transgenic mice to undertake a topographical and cell-type specific analysis of ERK phosphorylation and two of its downstream targets histone H3 and ribosomal protein S6 (rS6) in the dorsal striatum following injection of SKF81297 (D1R-like agonist), quinpirole (D2R-like agonist) or apomorphine (non selective DA receptor agonist). In striatal areas receiving inputs from the cingulate/prelimbic, visual and auditory cortex, SKF81297 treatment increased phosphorylation of ERK, histone H3 and rS6 selectively in EGFP-negative MSNs of Drd2-EGFP mice. In contrast, no regulation was found in striatal region predominantly targeted by the sensorimotor and motor cortex. Apomorphine slightly enhanced ERK and rS6, but not histone H3 phosphorylation. This regulation occurred exclusively in EGFP-negative neurons mostly in striatal sectors receiving connections from the insular, visual and auditory cortex. Quinpirole administration inhibited basal ERK activation but did not change histone H3 and rS6 phosphorylation throughout the rostrocaudal axis of the dorsal striatum. This anatomo-functional study indicates that D1R and D2R agonists produce a unique topography and cell-type specific regulation of the ERK cascade signaling in the mouse striatum, and that those patterns are closely associated with particular cortical and thalamic inputs. This work evidences the need of a precise identification of the striatal areas under study to further understand striatal plasticity.

Normal epidermal growth factor receptor signaling is dispensable for bone anabolic effects of parathyroid hormone

Although the bone anabolic properties of intermittent parathyroid hormone (PTH) have long been employed in the treatment of osteoporosis, the molecular mechanisms behind this action remain largely unknown. Previous studies showed that PTH increases the expression and the activity of epidermal growth factor receptor (EGFR) in osteoblasts, and activation of ERK1/2 by PTH in osteoblasts was demonstrated to induce the proteolytical release of EGFR ligands and EGFR transactivation. However, conclusive evidence for an important role of the EGFR system in mediating the anabolic actions of intermittent PTH on bone in vivo is lacking. Here, we evaluated the effects of intermittent PTH on bone in Waved-5 (Wa5) mice which carry an antimorphic Egfr allele whose product acts as a dominant negative receptor. Heterozygous Wa5 females and control littermates received a subcutaneous injection of PTH (80 μg/kg) or buffer on 5 days per week for 4 weeks. Wa5 mice had slightly lower total bone mineral density (BMD), but normal cancellous bone volume and turnover in the distal femoral metaphysis. The presence of the antimorphic Egfr allele neither influenced the PTH-induced increase in serum osteocalcin nor the increases in distal femoral BMD, cortical thickness, cancellous bone volume, and cancellous bone formation rate. Similarly, the PTH-induced rise in lumbar vertebral BMD was unchanged in Wa5 relative to wild-type mice. Wa5-derived osteoblasts showed considerably lower basal extracellular signal-regulated kinase 1/2 (ERK1/2) activation as compared to control osteoblasts. Whereas activation of ERK1/2 by the EGFR ligand amphiregulin was largely blocked in Wa5 osteoblasts, treatment with PTH induced ERK1/2 activation comparable to that observed in control osteoblasts, relative to baseline levels. Our data indicate that impairment of EGFR signaling does not affect the anabolic action of intermittent PTH on cancellous and cortical bone.

Altered ADAR 2 equilibrium and 5HT2CR editing in the prefrontal cortex of ADAR 2 transgenic mice

Modulation of serotonin signaling by RNA editing of the serotonin 2C receptor (5HT(2C) R) may be relevant to affective disorder as serotonin functions regulate mood and behavior. Previously, we observed enhanced endogenous behavioral despair in ADAR2 transgenic mice. As the transcript of the 5HT(2C) R is a substrate of ADAR2, we hypothesized that perturbed ADAR2 equilibrium in the prefrontal cortex of ADAR2 transgenic mice alters the normal distribution of edited amino acid isoforms of the 5HT(2C) R and modifies the receptor function in downstream basal extracellular signal-regulated kinase (ERK) signaling. We examined groups of naive control and ADAR2 transgenic mice and found significantly increased ADAR2 expression, increased RNA editing at A, C, D and E sites and significantly altered normal distribution of edited amino acid isoforms of the 5HT(2C) R with increased proportions of valine asparagine valine, valine serine valine, valine asparagine isoleucine, isoleucine asparagine valine and decreased isoleucine asparagine isoleucine amino acid isoforms of the 5HT(2C) R in ADAR2 transgenic mice. Localized serotonin levels (5-HT) were unchanged and perturbed ADAR2 equilibrium coincides with dysregulated edited amino acid isoforms of the 5HT(2C) R and reduced basal ERK signaling. These results altogether suggest that altered 5HT(2C) R function could be contributing to enhanced depression-like behavior of ADAR2 transgenic mice and further implicate ADAR2 as a contributing factor in cases of affective disorder.

Dysregulation of p38 and MKP-1 in Response to NOD1/TLR4 Stimulation in Sarcoid Bronchoalveolar Cells

Sarcoidosis is a systemic inflammatory disorder characterized by distinct up-regulation of Th1 cytokines, such as tumor necrosis factor (TNF)-α and IL-12. The mechanism underlying this up-regulation remains unclear. Recognition of microbial moieties through Toll-like or Nod-like receptors evokes sequential activation of mitogen-activated protein kinases (MAPKs), which plays a role in Th1-immune response. To test the hypothesis that dysregulation in MAPK signaling in response to microbial stimulation is important in mediating Th1 response in sarcoidosis. Ex vivo cultured bronchoalveolar lavage (BAL) cells isolated from patients with sarcoidosis and control subjects were stimulated with low-dose Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 1 (NOD1) ligands as a model of microbial stimulation, and MAPK signaling and inflammatory response were analyzed. BAL cells from patients with sarcoidosis exhibited higher basal p38 activity, greater p38 phosphorylation, and more robust production of TNF-α and IL-12/IL-23p40 on stimulation with NOD1 and TLR4 agonists than cells isolated from control subjects. In contrast, control BAL cells had greater basal extracellular signal-regulated kinase (ERK) activity and NOD1 and TLR4 agonists preferentially activated the ERK pathway. Inhibition of p38, but not ERK, attenuated production of both IL12/IL23p40 and TNF-α. Interestingly, stimulation of cells from patients with sarcoidosis with either NOD1 or TLR4 ligand failed to induce MAPK phosphatase 1 (MKP-1). Adenovirus-mediated overexpression of MKP-1 attenuated p38 activation and decreased the production of IL12/IL23p40 and TNF-α in sarcoid BAL cells. Our results suggest that enhanced p38 signaling in response to microbial products is caused by abnormal regulation of MKP-1 and contributes to heightened inflammation in sarcoidosis.

Constitutive Activity of Serotonin2C Receptors at G Protein-Independent Signaling: Modulation by RNA Editing and Antidepressants

Serotonin (5-HT)(2C) receptor is a G(q)-coupled receptor exhibiting a high degree of constitutive activity toward phospholipase C effector pathway, a process regulated by receptor mRNA editing. In addition to G protein-dependent signaling, 5-HT(2C) receptors also activate the extracellular signal-regulated kinase (ERK) 1/2 pathway independently of receptor coupling to G proteins. Constitutive activity at ERK signaling has not yet been explored. Transient expression of unedited 5-HT(2C-INI) receptors in human embryonic kidney (HEK) 293 cells resulted in a marked increase in ERK1/2 phosphorylation compared with nontransfected cells. No increase in ERK1/2 phosphorylation was measured in cells expressing fully edited (5-HT(2C-VGV)) receptors. Basal ERK1/2 phosphorylation in 5-HT(2C-INI) receptor-expressing cells was abolished by 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole (SB206,553), a 5-HT(2C) inverse agonist toward phospholipase C. This effect was prevented by the neutral antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-ylcarbamoyl]indoline (SB242,084), which alone did not alter basal activity. Similar observations were made in vivo in mouse choroid plexus, a structure rich in constitutively active 5-HT(2C) receptors. Reminiscent of agonist-induced ERK1/2 phosphorylation, basal activity in HEK 293 cells was unaffected by cellular depletion of Gα(q/11) and Gα(13) proteins but strongly reduced in cells expressing a dominant-negative β-arrestin or depleted of β-arrestin by RNA interference and in cells expressing a dominant-negative calmodulin or a 5-HT(2C-INI) receptor mutant not capable of interacting with calmodulin. The tetracyclic antidepressants mirtazapine and mianserin likewise reduced basal ERK activation. On the other hand, the m-chlorophenylpiperazine derivative trazodone and the selective serotonin reuptake inhibitor fluoxetine were inactive alone but blocked 5-HT-induced ERK1/2 phosphorylation. Together, these data provide the first evidence of constitutive activity of a G protein-coupled receptor toward G-independent, β-arrestin-dependent, receptor signaling.

Oncogenic KRAS Desensitizes Colorectal Tumor Cells to Epidermal Growth Factor Receptor Inhibition and Activation

Epidermal growth factor receptor (EGFR)-targeting therapeutics have shown efficacy in the treatment of colorectal cancer patients. Clinical studies have revealed that activating mutations in the KRAS protooncogene predict resistance to EGFR-targeted therapy. However, the causality between mutant KRAS and resistance to EGFR inhibition has so far not been demonstrated. Here, we show that deletion of the oncogenic KRAS allele from colorectal tumor cells resensitizes those cells to EGFR inhibitors. Resensitization was accompanied by an acquired dependency on the EGFR for maintaining basal extracellular signal-regulated kinase (ERK) activity. Deletion of oncogenic KRAS not only resensitized tumor cells to EGFR inhibition but also promoted EGF-induced NRAS activation, ERK and AKT phosphorylation, and c-FOS transcription. The poor responsiveness of mutant KRAS tumor cells to EGFR inhibition and activation was accompanied by a reduced capacity of these cells to bind and internalize EGF and by a failure to retain EGFR at the plasma membrane. Of 16 human colorectal tumors with activating mutations in KRAS, 15 displayed loss of basolateral EGFR localization. Plasma membrane localization of the EGFR could be restored in vitro by suppressing receptor endocytosis through Rho kinase inhibition. This caused an EGFR-dependent increase in basal and EGF-stimulated ERK phosphorylation but failed to restore tumor cell sensitivity to EGFR inhibition. Our results demonstrate a causal role for oncogenic KRAS in desensitizing tumor cells not only to EGFR inhibitors but also to EGF itself.

Long‐Term Ethanol Exposure Impairs Neuronal Differentiation of Human Neuroblastoma Cells Involving Neurotrophin‐Mediated Intracellular Signaling and in Particular Protein Kinase C

Revealing the molecular changes in chronic ethanol-impaired neuronal differentiation may be of great importance for understanding ethanol-related pathology in embryonic development but also in the adult brain. In this study, both acute and long-term effects of ethanol on neuronal differentiation of human neuroblastoma cells were investigated. We focused on several aspects of brain-derived neurotrophic factor (BDNF) signaling because BDNF activates the extracellular signal-regulated kinase (ERK) cascade, promoting neuronal differentiation including neurite outgrowth. The effects of ethanol exposure on morphological differentiation, cellular density, neuronal marker proteins, basal ERK activity, and ERK responsiveness to BDNF were measured over 2 to 4 weeks. qRT-PCR and Western blotting were performed to investigate the expression of neurotrophin receptor tyrosin kinase B (TrkB), members of the ERK-cascade, protein kinase C (PKC) isoforms and Raf-Kinase-Inhibitor-Protein (RKIP). Chronic ethanol interfered with the development of a neuronal network consisting of cell clusters and neuritic bundles. Furthermore, neuronal and synaptic markers were reduced, indicating impaired neuronal differentiation. BDNF-mediated activation of the ERK cascade was found to be continuously impaired by ethanol. This could not be explained by expressional changes monitored for TrkB, Raf-1, MEK, and ERK. However, BDNF also activates PKC signaling which involves RKIP, which finally leads to ERK activation as well. Therefore, we hypothesized that ethanol impairs this branch of BDNF signaling. Indeed, both PKC and RKIP were significantly down-regulated. Chronic ethanol exposure impaired neuronal differentiation of neuroblastoma cells and BDNF signaling, particularly the PKC-dependent branch. RKIP, acting as a signaling switch at the merge of the PKC cascade and the Raf/MEK/ERK cascade, was associated with neuronal differentiation and significantly reduced in ethanol treatment. Moreover, PKC expression itself was even more strongly reduced. In contrast, members of the Raf-1/MEK/ERK cascade were less affected and the observed changes were not associated with impaired differentiation. Thus, reduced RKIP and PKC levels and subsequently reduced positive feedback on ERK activation provide an explanation for the striking effects of long-term ethanol exposure on BDNF signal transduction and neuronal differentiation, respectively.

Genetic Predictors of MEK Dependence in Non–Small Cell Lung Cancer

Hyperactivated extracellular signal-regulated kinase (ERK) signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS, and upstream receptor tyrosine kinases. To characterize the mitogen-activated protein kinase/ERK kinase (MEK)/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n = 87) and tumors (n = 916) for BRAF mutations. We found that non-small cell lung cancers (NSCLC) cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition compared with those harboring mutations in epidermal growth factor receptor (EGFR), KRAS, or ALK and ROS kinase fusions. Supporting its classification as a "driver" mutation in the cells in which it is expressed, MEK inhibition in (V600E)BRAF NSCLC cells led to substantial induction of apoptosis, comparable with that seen with EGFR kinase inhibition in EGFR mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF mutant cell lines were resistant to EGFR inhibition. These data, together with the nonoverlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hotspot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared with those whose tumors harbor kinase domain mutations in EGFR.

Inhibitory crosstalk between ERK and AMPK in the growth and proliferation of cardiac fibroblasts

Extracellular signal-regulated kinase (ERK) is one of the key protein kinases that regulate the growth and proliferation in cardiac fibroblasts (CFs). As an energy sensor of cellular metabolism, AMP-activated protein kinase (AMPK) is found recently to be involved in myocardial remodeling. In this study, we investigated the crosstalk between ERK and AMPK in the growth and proliferation of CFs. In neonatal rat cardiac fibroblasts (NRCFs), we found that serum significantly inhibited basal AMPK phosphorylation between 10 min and 24 h and also partially inhibited AMPK phosphorylation by AMPK activator, 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR). Furthermore, ERK inhibitor could greatly reverse the inhibition of AMPK by serum. Conversely, activation of AMPK by AICAR also showed a significant inhibition of basal and serum-induced ERK phosphorylation but it showed a delayed and steadfast inhibition which appeared after 60 min and lasted until 12 h. Moreover, inhibition of ERK could repress the activation of p70S6K, an important kinase in cardiac proliferation, and AICAR could also inhibit p70S6K phosphorylation. In addition, under both serum and serum-free medium, AICAR significantly inhibited the DNA synthesis and cell numbers, and reduced cells at S phase. In conclusion, AMPK activation with AICAR inhibited growth and proliferation in cardiac fibroblasts, which involved inhibitory interactions between ERK and AMPK. This is the first report that AMPK could be a target of ERK in growth factors-induced proliferation, which may give a new mechanism that growth factors utilize in their promotion of proliferation in cardiac fibroblasts.