Transient ERK Activation Research Articles

Triiodothyronine (T3) suppresses hepatic tumorigenesis and development by inhibiting the phosphorylation of ERK.

The effect of triiodothyronine (T3) on the phosphorylation of ERK and the occurrence and development of hepatocellular carcinoma (HCC) is controversial and remains to be clarified. In the present study, both in vitro (hepatoma cell lines) and in vivo (wild-type mice [WT] and mouse models of HCC [HrasG12Vand KrasG12Dtransgenic mice (Hras-Tg and Kras-Tg)]) systems were used to investigate the effect of T3 on p-ERK and hepatocarcinogenesis. The results showed that, in vitro, T3 treatment elevated the levels of p-ERK in hepatoma cells within 30 min. However, p-ERK levels returned to normal after 1 h with no significant effects on cellular proliferation or apoptosis. Interestingly, in vivo, T3 induced early rapid and transient activation of ERK and later persistent downregulation of p-ERK in liver tissues of WT. In Hras-Tg, liver weight, liver/body weight ratio, hepatic tumor numbers and sizes were significantly reduced withT3treatment compared with the untreated group. Furthermore, the levels of albumin, HrasG12V, and p-ERK in hepatic precancerous and tumor tissues were all significantly downregulated with T3 treatment; however, the levels of endogenous Hras were not affected. In WT, T3 also induced downregulation of Albumin in liver tissues, but without influence on the expression of endogenous Hras and p-MEK. Especially, the inhibitory effect of T3 on p-ERK and hepatic tumorigenesis and development without influence on the levels of KrasG12D and p-MEK was further confirmed in Kras-Tg. In conclusion, T3 suppresses hepatic tumorigenesis and development by independently and substantially inhibiting the phosphorylation of ERK in vivo.

Cadmium promotes colorectal cancer metastasis through EGFR/Akt/mTOR signaling cascade and dynamics

Cadmium (Cd) is a hazardous environmental heavy metal with a prolonged biological half-life. Due to the main route of foodborne exposure, the intestinal tract is particularly vulnerable to Cd-induced toxicity. However, the chronic toxicity and underlying mechanisms of Cd in intestinal diseases, including colorectal cancer (CRC), still remain vague. Herein, we aim to investigate the long-term effects of Cd exposure on CRC development and the key signaling event. Our findings indicate that chronic and low-dose exposure to Cd promoted the invasion and metastasis capability of CRC cells in vitro and in mice, with a marginal increase in cell growth. The expression of cell junction-related genes was down-regulated while those molecules that facilitate cell mobility were significantly increased by Cd exposure. Epidermal growth factor receptor (EGFR) signaling was identified to play the dominant role in Cd-promoted CRC metastasis. Interestingly, Cd activated EGFR in a non-canonical manner that exhibited distinct signaling dynamics from the canonical ligand. In contrast to EGF, which induced transient EGFR signaling and ERK activation, Cd promoted sustained EGFR signaling to trigger Akt/mTOR cascade. The unique signaling dynamics of EGFR induced by Cd provoked responses that preferably enhanced the metastatic capacity rather than the growth. Furthermore, blockade of EGFR abrogated the promoting effects of Cd on the liver metastasis of CRC cells. In conclusion, this study provides a better understanding of the long-term influences of environmental Cd on CRC metastasis and reveals the unique EGFR signaling dynamics induced by Cd exposure.

A novel antiproliferative PKCα-Ras-ERK signaling axis in intestinal epithelial cells

We have previously shown that the serine/threonine kinase PKCα triggers MAPK/ERK kinase (MEK)-dependent G1→S cell cycle arrest in intestinal epithelial cells, characterized by downregulation of cyclin D1 and inhibitor of DNA-binding protein 1 (Id1) and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Here, we use pharmacological inhibitors, genetic approaches, siRNA-mediated knockdown, and immunoprecipitation to further characterize antiproliferative ERK signaling in intestinal cells. We show that PKCα signaling intersects the Ras-Raf-MEK-ERK kinase cascade at the level of Ras small GTPases and that antiproliferative effects of PKCα require active Ras, Raf, MEK, and ERK, core ERK pathway components that are also essential for pro-proliferative ERK signaling induced by epidermal growth factor (EGF). However, PKCα-induced antiproliferative signaling differs from EGF signaling in that it is independent of the Ras guanine nucleotide exchange factors (Ras-GEFs), SOS1/2, and involves prolonged rather than transient ERK activation. PKCα forms complexes with A-Raf, B-Raf, and C-Raf that dissociate upon pathway activation, and all three Raf isoforms can mediate PKCα-induced antiproliferative effects. At least two PKCα–ERK pathways that collaborate to promote growth arrest were identified: one pathway requiring the Ras-GEF, RasGRP3, and H-Ras, leads to p21Cip1 upregulation, while additional pathway(s) mediate PKCα-induced cyclin D1 and Id1 downregulation. PKCα also induces ERK-dependent SOS1 phosphorylation, indicating possible negative crosstalk between antiproliferative and growth-promoting ERK signaling. Importantly, the spatiotemporal activation of PKCα and ERK in the intestinal epithelium in vivo supports the physiological relevance of these pathways and highlights the importance of antiproliferative ERK signaling to tissue homeostasis in the intestine.

Robustness of epithelial sealing is an emerging property of local ERK feedback driven by cell elimination.

SummaryWhat regulates the spatiotemporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination where simultaneous death of neighboring cells could impair epithelial sealing. Here, using the Drosophila pupal notum (a single-layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first showed that death of clusters of at least three cells impaired epithelial sealing; yet, such clusters were almost never observed in vivo. Accordingly, statistical analysis and simulations of cell death distribution highlighted a transient and local protective phase occurring near every cell death. This protection is driven by a transient activation of ERK in cells neighboring extruding cells, which inhibits caspase activation and prevents elimination of cells in clusters. This suggests that the robustness of epithelia with high rates of cell elimination is an emerging property of local ERK feedback.

Biphasic spatiotemporal regulation of GRB2 dynamics by p52SHC for transient RAS activation.

RTK-RAS-MAPK systems are major signaling pathways for cell fate decisions. Among the several RTK species, it is known that the transient activation of ERK (MAPK) stimulates cell proliferation, whereas its sustained activation induces cell differentiation. In both instances however, RAS activation is transient, suggesting that the strict temporal regulation of its activity is critical in normal cells. RAS on the cytoplasmic side of the plasma membrane is activated by SOS through the recruitment of GRB2/SOS complex to the RTKs that are phosphorylated after stimulation with growth factors. The adaptor protein GRB2 recognizes phospho-RTKs both directly and indirectly via another adaptor protein, SHC. We here studied the regulation of GRB2 recruitment under the SHC pathway using single-molecule imaging and fluorescence correlation spectroscopy in living cells. We stimulated MCF7 cells with a differentiation factor, heregulin, and observed the translocation, complex formation, and phosphorylation of cell signaling molecules including GRB2 and SHC. Our results suggest a biphasic regulation of the GRB2/SOS-RAS pathway by SHC: At the early stage (<10 min) of stimulation, SHC increased the amplitude of RAS activity by increasing the association sites for the GRB2/SOS complex on the plasma membrane. At the later stage however, SHC suppressed RAS activation and sequestered GRB2 molecules from the membrane through the complex formation in the cytoplasm. The latter mechanism functions additively to other mechanisms of negative feedback regulation of RAS from MEK and/or ERK to complete the transient activation dynamics of RAS.

S-allyl cysteine inhibits TNF-α-induced inflammation in HaCaT keratinocytes by inhibition of NF- κB-dependent gene expression via sustained ERK activation.

Tumor necrosis factor-α (TNF-α)-induced keratinocyte inflammation plays a key role in the pathogenesis of multiple inflammatory skin diseases. Here we investigated the anti-inflammatory effect of S-allyl cysteine (SAC) on TNF-α-induced HaCaT keratinocyte cells and the mechanism behind its anti-inflammatory potential. SAC was found to inhibit TNF-α-stimulated cytokine expression. Further, SAC was found to inhibit TNF-α-induced activation of p38, JNK and NF-κB pathways. Interestingly, SAC was found to differentially regulate ERK MAP kinase in cells. TNF-α-induced transient ERK activation and SAC treatment resulted in sustained ERK activation both in the presence and absence of TNF-α. Additionally, SAC failed to inhibit the TNF-α-induced expression of the pro-inflammatory cytokines TNF-α and IL-1β when cells were treated with the MEK inhibitor PD98059, suggesting that the anti-inflammatory effect of SAC is via sustained activation of the ERK pathway. Since ERK activation has been reported to negatively regulate NF-κB-driven gene expression and we find that SAC activates ERK and negatively regulates NF-κB, we investigated whether there existed any crosstalk between the ERK and the NF-κB pathways. NF-κB-dependent reporter assay, visualization of the nuclear translocation of NF-κB-p65 subunit and determination of the cellular levels of I-κB, the inhibitor of NF-κB, revealed that SAC inhibited TNF-α-induced NF-κB activation, and PD98059 treatment reversed this effect. These results collectively suggest that SAC inhibits TNF-α-induced inflammation in HaCaT cells via a combined effect entailing the inhibition of the p38 and the JNK pathways and NF-κB pathway via the sustained activation of ERK.

Neuropilin 1 Mediates Keratinocyte Growth Factor Signaling in Adipose-Derived Stem Cells: Potential Involvement in Adipogenesis

Adipogenesis is regulated by a complex network of molecules, including fibroblast growth factors. Keratinocyte growth factor (KGF) has been previously reported to promote proliferation on rat preadipocytes, although the expression of its specific receptor, FGFR2-IIIb/KGFR, is not actually detected in mesenchymal cells. Here, we demonstrate that human adipose-derived stem cells (ASCs) show increased expression of KGF during adipogenic differentiation, especially in the early steps. Moreover, KGF is able to induce transient activation of ERK and p38 MAPK pathways in these cells. Furthermore, KGF promotes ASC differentiation and supports the activation of differentiation pathways, such as those of PI3K/Akt and the retinoblastoma protein (Rb). Notably, we observed only a low amount of FGFR2-IIIb in ASCs, which seems not to be responsible for KGF activity. Here, we demonstrate for the first time that Neuropilin 1 (NRP1), a transmembrane glycoprotein expressed in ASCs acting as a coreceptor for some growth factors, is responsible for KGF-dependent pathway activation in these cells. Our study contributes to clarify the molecular bases of human adipogenesis, demonstrating a role of KGF in the early steps of this process, and points out a role of NRP1 as a previously unknown mediator of KGF action in ASCs.

Cot kinase plays a critical role in Helicobacter pylori-induced IL-8 expression.

Helicobacter pylori is a major pathogen causing various gastric diseases including gastric cancer. Infection of H. pylori induces pro-inflammatory cytokine IL-8 expression in gastric epithelial cells in the initial inflammatory process. It has been known that H. pylori can modulate Ras-Raf-Mek-Erk signal pathway for IL-8 induction. Recently, it has been shown that another signal molecule, cancer Osaka thyroid oncogene/tumor progression locus 2 (Cot/Tpl2) kinase, activates Mek and Erk and plays a role in the Erk pathway, similar to MAP3K signal molecule Raf kinase. Therefore, the objective of this study was to determine whether Cot kinase might be involved in IL-8 induction caused by H. pylori infection. AGS gastric epithelial cells were infected by H. pylori strain G27 or its isogenic mutants lacking cagA or type IV secretion system followed by treatment with Cot kinase inhibitor (KI) or siRNA specific for Cot kinase. Activation of Erk was assessed by Western blot analysis and expression of IL-8 was measured by ELISA. Treatment with Cot KI reduced both transient and sustained Erk activation. It also reduced early and late IL-8 secretion in the gastric epithelial cell line. Furthermore, siRNA knockdown of Cot inhibited early and late IL-8 secretion induced by H. pylori infection. Taken together, these results suggest that Cot kinase might play a critical role in H. pylori type IV secretion apparatus-dependent early IL-8 secretion and CagA-dependent late IL-8 secretion as an alternative signaling molecule in the Erk pathway.

Abstract 1453: CXCR4 and CXCR7 homodimers and heterodimers play differential roles in breast cancer

Abstract The majority of breast cancer deaths are due to the metastasis of tumor cells to secondary organs. One of the most studied metastatic processes is the migration of chemokine receptor-expressing cancer cells towards their ligands in other organs, a mechanism where the receptor CXCR4 and the chemokine CXCL12 play key roles. This process, however, may be affected by the expression of the atypical receptor CXCR7, which has recently been linked to breast cancer progression. Indeed, staining of CXCR7 in patient samples is strongly positive in both lobular and ductal breast cancers. Given CXCR7's ability to form heterodimers with CXCR4, we investigated how dual expression of both receptors differs from their lone expression in terms of their signaling and internalization pathways. In this study we created stable single CXCR4 or CXCR7 CHO transfectants or double CXCR4-CXCR7 transient transfectants to examine ligand-dependent receptor internalization. The presence of heterodimers was evaluated using fluorescence resonance energy transfer (FRET). We found that in CHO-CXCR4 the receptor was degraded after a 30 minute stimulation with 10 nM CXCL12, whilst in CHO-CXCR7 the receptor returned to the surface in under 2 hours, reaching higher CXCR7 levels than before treatment. RT-PCR data suggested that this increase was partially due to induction of further CXCR7 expression. When the receptors were coexpressed, a similar pattern could be seen, although the CXCR7 recycling levels were not as high. CXCR4 degradation could be partially inhibited by pretreating cells with 1 μM lactacystin, a proteasome inhibitor. When cells were stimulated for 1 hour with 1 nM VUF11207, a CXCR7 agonist, no significant effect was seen on CHO-CXCR4 whilst CHO-CXCR7 cells showed internalization and recycling. Supporting RT-PCR data showed that the agonist did not trigger CXCR7 expression like seen with CXCL12. Interestingly, when coexpressed, both CXCR4 and CXCR7 showed degradation after internalization. Similar results were observed with MDA-MB-231 cells transfected with CXCR4 or CXCR7. The effects of the binding were also assessed by measuring ERK phosphorylation by Western blot and cell-based ELISA. An early but transient (5-15 minutes) ERK activation was seen in CHO-CXCR4 cells, whilst CHO-CXCR7 cells showed a sustained activation of up to 2 hours. On the other hand, CHO-CXCR4-CXCR7 cells showed a very strong phosphorylation at 5 minutes that quickly diminished. The role of the receptors in invasion and migration in the presence of CXCL12 was also assessed. In summary, this study highlights how CXCR4/CXCR7 heterodimerization can create a distinct signaling entity with unique properties and alter receptor pharmacology. This could be used to create therapeutic compounds to reduce breast cancer cell migration. Citation Format: Irene del Molino del Barrio, Simi Ali, John Kirby, Annette Meeson. CXCR4 and CXCR7 homodimers and heterodimers play differential roles in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1453.

Defined spatiotemporal features of RAS-ERK signals dictate cell fate in MCF-7 mammary epithelial cells.

Signals conveyed through the RAS-ERK pathway are essential for the determination of cell fate. It is well established that signal variability is achieved in the different microenvironments in which signals unfold. It is also known that signal duration is critical for decisions concerning cell commitment. However, it is unclear how RAS-ERK signals integrate time and space in order to elicit a given biological response. To investigate this, we used MCF-7 cells, in which EGF-induced transient ERK activation triggers proliferation, whereas sustained ERK activation in response to heregulin leads to adipocytic differentiation. We found that both proliferative and differentiating signals emanate exclusively from plasma membrane-disordered microdomains. Of interest, the EGF signal can be transformed into a differentiating stimulus by HRAS overexpression, which prolongs ERK activation, but only if HRAS localizes at disordered membrane. On the other hand, HRAS signals emanating from the Golgi complex induce apoptosis and can prevent heregulin-induced differentiation. Our results indicate that within the same cellular context, RAS can exert different, even antagonistic, effects, depending on its sublocalization. Thus cell destiny is defined by the ability of a stimulus to activate RAS at the appropriate sublocalization for an adequate period while avoiding switching on opposing RAS signals.

Frequency modulation of ERK activation dynamics rewires cell fate.

Transient versus sustained ERK MAP kinase (MAPK) activation dynamics induce proliferation versus differentiation in response to epidermal (EGF) or nerve (NGF) growth factors in PC-12 cells. Duration of ERK activation has therefore been proposed to specify cell fate decisions. Using a biosensor to measure ERK activation dynamics in single living cells reveals that sustained EGF/NGF application leads to a heterogeneous mix of transient and sustained ERK activation dynamics in distinct cells of the population, different than the population average. EGF biases toward transient, while NGF biases toward sustained ERK activation responses. In contrast, pulsed growth factor application can repeatedly and homogeneously trigger ERK activity transients across the cell population. These datasets enable mathematical modeling to reveal salient features inherent to the MAPK network. Ultimately, this predicts pulsed growth factor stimulation regimes that can bypass the typical feedback activation to rewire the system toward cell differentiation irrespective of growth factor identity.

Role of the AMP kinase in cytokine-induced human EndoC-βH1 cell death

The aim of the present investigation was to delineate cytokine-induced signaling and death using the EndoC-βH1 cells as a model for primary human beta-cells. The cytokines IL-1β and IFN-γ induced a rapid and transient activation of NF-κB, STAT-1, ERK, JNK and eIF-2α signaling. The EndoC-βH1 cells died rapidly when exposed to IL-1β + IFN-γ, and this occurred also in the presence of the actinomycin D. Inhibition of NF-κB and STAT-1 did not protect against cell death, nor did the cytokines activate iNOS expression. Instead, cytokines promoted a rapid decrease in EndoC-βH1 cell respiration and ATP levels, and we observed protection by the AMPK activator AICAR against cytokine-induced cell death. It is concluded that EndoC-βH1 cell death can be prevented by AMPK activation, which suggests a role for ATP depletion in cytokine-induced human beta-cell death.

MCP‐1‐Induced Protein Promotes Human Pulmonary Fibroblast Migration Induced by SiO2 via MAPKs and PI3K Signaling

Silicosis is a systemic disease caused by inhalation of silicon dioxide (SiO2). Phagocytosis of SiO2 in the lung causes inflammatory cascade resulting in fibroblast proliferation and migration with subsequent fibrosis. Clinical evidence indicated that activation of alveolar macrophage induced by SiO2 produces a rapid and sustained inflammation characterized by generation of monocyte chemotactic protein 1 (MCP-1), which, in turn, induces fibrosis. Our previous studies suggested that MCP-1-induced protein 1 (MCPIP1, ZC3H12A), downstream of MCP-1, played a critical role in fibroblast proliferation and migration. Here we investigated the mechanism of fibroblast migration induced by SiO2 with emphasis on MCPIP1 Experiments in primary cultured human pulmonary fibroblast (adult) showed: 1). SiO2 induced release of MCPIP1 in a concentration and time dependent manner. 2). Cellular migration was up-regulated in both scratch assay and 3D collagen matrix model in the presence of SiO2 (50µg/cm2). 3). Rapid and transient activation of ERK, p38, JNK and Akt also occurred after SiO2; pharmacologic inhibition of each kinase reduced MCPIP1 expression and blunted the migration induced by SiO2. 4). RNA interference of MCPIP1 prevented the increased migration which occurred after SiO2. These data demonstrated that up-regulation of MCPIP1 was involved in cell migration of pulmonary fibroblast induced by SiO2. Signaling events upstream of MCPIP1 induction included activation of MAP kinases and PI3K. Our study deciphered the link between MCPIP1 and cellar migration induced by SiO2, providing a novel insight into the potential of MCPIP1 in terms of opening up novel therapeutic avenues for silicosis.

Insulin is involved in transcriptional regulation of NKCC and the CFTR Cl(-) channel through PI3K activation and ERK inactivation in renal epithelial cells.

It is is well known that insulin stimulates glucose transport and epithelial Na(+) channel (ENaC)-mediated Na(+) reabsorption; however, the action of insulin on Cl(-) secretion is not fully understood. In this study, we investigated the action of insulin on Na(+)-K(+)-2Cl(-) cotransporter (NKCC)-mediated Cl(-) secretion in epithelial A6 cells. Interestingly, insulin treatment remarkably enhanced the forskolin-stimulated Cl(-) secretion associated with an increase in apical Cl(-) conductance by upregulating mRNA expression of both CFTR and NKCC, although insulin treatment alone had no effect on the basal Cl(-) secretion or apical Cl(-) conductance without forskolin application. We next elucidated a role of phosphoinositide 3-kinase (PI3K) in the insulin-induced enhancement of the Cl(-) secretion, since insulin actually activated PI3K, resulting in activation of Akt, a downstream molecule of PI3K. LY294002 (a PI3K inhibitor) reduced the Cl(-) secretion by suppressing mRNA expression of NKCC, whereas insulin still had a stimulatory action on mRNA expression of CFTR even in the presence of LY294002. On the other hand, we found that a MEK inhibitor (PD98059) further enhanced the insulin-stimulated CFTR mRNA expression and the Cl(-) secretion in forskolin-stimulated A6 cells and that insulin induced slight, transient activation of ERK followed by significant inactivation of ERK. These observations suggest that: (1) insulin respectively upregulates mRNA expression of NKCC and CFTR through activation of PI3K and inactivation of ERK; (2) insulin signals on mRNA expression of NKCC and CFTR are not enough to stimulate transepithelial Cl(-) secretion, but enhance the stimulatory action of cAMP on transepithelial Cl(-) secretion.

Differential activation of MAPKs by individual and combined ochratoxin A and citrinin treatments in porcine kidney PK15 cells

The aim of this study was to investigate the underlying mechanisms of OTA and CTN individual and combined toxicity in porcine kidney PK15 cells of proximal tubule origin. Activation and expression of mitogen-activated protein kinases (MAPKs) ERK, JNK and p38 were determined by Western blot analysis. MAPKs were differentially activated by single or dual OTA and CTN treatments. Single OTA and CTN stimulated transient ERK and prolonged JNK activation, while phospho-p38 signal was more persistent after OTA treatment. Mycotoxin mixture provoked significant down-regulation of ERK activation, more prolonged phospho-p38 signal, and two-stage JNK phosphorylation pattern. In order to define the role of particular MAPKs in mycotoxin(s) cytotoxicity, we performed MTT assay with specific MAPKs inhibitors. In both individual and combined treatments JNK and p38 inhibition significantly induced cell survival. When cells were exposed to toxin mixture, inhibition of ERK also promoted cell survival, although to a lesser extent that JNK and p38 inhibition. Next we investigated the association between calcium (Ca2+) and MAPKs after OTA and/or CTN treatments, and we employed Ca2+ chelator BAPTA-AM. We demonstrated that p38 activation was significantly down-regulated in cells treated with CTN alone or OTA + CTN suggesting the role of Ca2+ in mycotoxin-induced cell death.

Sos1 regulates sustained TCR‐mediated Erk activation

The duration and/or the magnitude of Ras-Erk activation are known to be crucial for cell-fate decisions. In T cells, sustained Erk activation correlates with differentiation/proliferation, whereas transient Erk activation parallels with unresponsiveness/apoptosis. The mechanism by which Son of sevenless (Sos) proteins and Ras guanyl-releasing protein 1 (RasGRP1) contribute to dynamics of Erk activation in mature T cells is not yet known. Here, we have assessed this issue using stimuli inducing either transient or sustained TCR signaling and RNA interference mediated suppression of Sos1, Sos2, and RasGRP1 expression in primary human T cells. We found that transient Erk activation depends on RasGRP1 but not on Sos. Conversely, sustained Erk signaling and T-cell activation depend on both Sos1 and RasGRP1. In summary, our data show for the first time that the two guanine nucleotide exchange factors expressed in T cells are differentially involved in the regulation of the duration of Erk phosphorylation and T-cell activation.

Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells

The activation kinetics of MAPK Erk are critical for T cell development and activation. In particular, sustained Erk signaling is required for T cell activation and effector functions, such as IL-2 production. Although Raf-1 triggers transient Erk activation, B-Raf is implicated in sustained Erk signaling after TCR stimulation. In this study, we show that B-Raf is dephosphorylated on its inhibitory serine 365 upon TCR triggering. However, it is unknown how B-Raf activation is coupled to the TCR. Using mass spectrometry, we identified protein kinase D-interacting substrate of 220 kDa (Kidins220)/ankyrin repeat-rich membrane spanning protein, mammalian target of rapamycin, Rictor, Dock2, and GM130 as novel B-Raf interaction partners. We focused on Kidins220, a protein that has been studied in neuronal cells and found that it associated with the pre-TCR, αβTCR, and γδTCR. Upon prolonged TCR stimulation, the Kidins220-TCR interaction was reduced, as demonstrated by immunoprecipitation and proximity ligation assays. We show that Kidins220 is required for TCR-induced sustained, but not transient, Erk activation. Consequently, induction of the immediate early gene products and transcription factors c-Fos and Erg-1 was blocked, and upregulation of the activation markers CD69, IL-2, and IFN-γ was reduced. Further, Kidins220 was required for optimal calcium signaling. In conclusion, we describe Kidins220 as a novel TCR-interacting protein that couples B-Raf to the TCR. Kidins220 is mandatory for sustained Erk signaling; thus, it is crucial for TCR-mediated T cell activation.

Pizotifen Activates ERK and Provides Neuroprotection in vitro and in vivo in Models of Huntington's Disease

Huntington's disease (HD) is a dominantly inherited neurodegenerative condition characterized by dysfunction in striatal and cortical neurons. There are currently no approved drugs known to slow the progression of HD. To facilitate the development of therapies for HD, we identified approved drugs that can ameliorate mutant huntingtin-induced toxicity in experimental models of HD. A chemical screen was performed in a mouse Hdh(Q111/Q111) striatal cell model of HD. This screen identified a set of structurally related approved drugs (pizotifen, cyproheptadine, and loxapine) that rescued cell death in this model. Pizotifen was subsequently evaluated in the R6/2 HD mouse model. We found that in striatal Hdh(Q111/Q111) cells, pizotifen treatment caused transient ERK activation and inhibition of ERK activation prevented rescue of cell death in this model. In the R6/2 HD mouse model, treatment with pizotifen activated ERK in the striatum, reduced neurodegeneration and significantly enhanced motor performance. These results suggest that pizotifen and related approved drugs may provide a basis for developing disease modifying therapeutic interventions for HD.

Modeling anhydrobiosis: activation of the mitogen‐activated protein kinase ERK by dehydration in both human cells and nematodes

Anhydrobiosis ("life without water") is the state of suspended animation that certain organisms, including some nematodes, tardigrades, and bdelloid rotifers, enter during desiccation. Extreme water loss imposes considerable stress on biomolecules, cells, and tissues, and must require specific sensing and response mechanisms for survival. However, these mechanisms are poorly understood, in part owing to the lack of amenable model systems. We have, therefore, begun to develop mammalian cell lines as tools for investigating the eukaryotic response to desiccation, and have an additional long-term goal of generating a desiccation-tolerant mammalian cell. Here, we investigate the role of the mitogen-activated protein kinases (MAPKs) in controlling gene expression in response to evaporative water loss. We report that the ERK MAPK pathway inhibitor U0126 can almost completely block induction of desiccation early response genes in a human cell line, suggesting a role for the ERK signal transduction pathway in the stress response. Accordingly, ERK is activated by phosphorylation during desiccation of human cells. Importantly, nematodes also activate ERK on drying, showing that the mammalian cell model behaves similarly to invertebrates experiencing similar stress conditions. We further reveal that, in response to desiccation, human cells can rapidly initiate complex stress signaling networks involving all three MAPK pathways, with transient activation of ERK and sustained activation of JNK and p38. These results are consistent with a role for MAPK pathways in anhydrobiotic adaptation and suggest that non-anhydrobiotes are able to sense and, at least to some extent, respond appropriately to evaporative water loss.

Modulation of Gonadotropin-Releasing Hormone-Induced Extracellular Signal-Regulated Kinase Activation by Dual-Specificity Protein Phosphatase 1 in LβT2 Gonadotropes

As the regulator of pituitary reproductive hormone synthesis, the hypothalamic neuropeptide GnRH is the central regulator of reproduction. A hallmark of GnRH action is the differential control of gene expression in pituitary gonadotropes through varied pulsatile stimulation. Among other signaling events, GnRH activation of the ERK family of MAPKs plays a significant role in the transcriptional regulation of the luteinizing hormone β-subunit gene and regulation of cap-dependent translation. We evaluated the ERK response to different GnRH pulse amplitudes in the gonadotrope cell line LβT2. We found that low-amplitude stimulation with GnRH invokes a rapid and transient ERK activation, whereas high-amplitude stimulation invokes a prolonged activation specifically in the cytoplasm fraction of LβT2 cells. Nuclear and cytoplasmic targets of ERK, Ets-like gene 1, and eukaryotic initiation factor 4E, respectively, are similarly activated. Feedback control of ERK activation occurs mainly through the dual-specificity protein phosphatases (DUSPs). DUSP1 is localized to the nucleus in LβT2 cells but DUSP4, another member implicated in GnRH feedback, exists in both the nucleus and cytoplasm. Manipulation of nuclear DUSP activity through overexpression or knockdown of Dusp1 modulates the ERK response to low and high GnRH pulse amplitudes and activation of the Lhb promoter. Dusp1 overexpression abolishes sustained ERK activation and inhibits Lhb promoter activity induced by high amplitude pulses. Conversely, Dusp1 knockdown enhances ERK activation by low-amplitude stimulation and increases stimulation of Lhb promoter activity. We conclude that DUSP1 feedback activity modulates ERK activation and the transcriptional response to GnRH.