by We thank C. and mechanical hypersensitivity, and spared nerve injury-induced mechanical hypersensitivity. However, ERK1 deletion did delay formalin-induced long-term heat hypersensitivity, without affecting formalin-induced mechanical hypersensitivity, suggesting that ERK1 partially shapes long-term responses to formalin. Interestingly, ERK1 deletion resulted in elevated basal ERK2 phosphorylation. However, this did not appear to influence nociceptive processing, since inflammation-induced ERK2 phosphorylation and pERK1/2 immunoreactivity in spinal cord were not elevated in ERK1 KO mice. Additionally, systemic MEK inhibition with SL327 (-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile) attenuated formalin-induced spontaneous behaviors similarly in wild-type and ERK1 KO mice, indicating that unrelated signaling pathways do not functionally compensate for the loss of ERK1. Together, these results suggest that ERK1 plays a limited role in nociceptive sensitization and support a predominant role for ERK2 in these processes. == Introduction == Extracellular signal-regulated kinases (ERKs), ERK1 and ERK2, are JAK2-IN-4 mitogen-activated protein kinases (MAPKs) (Pearson et al., 2001) that have been identified as critical players in sensitization to noxious stimuli after peripheral inflammation and nerve damage (Ji et al., 1999,2002;Karim et al., 2001;Ciruela et al., 2003;Obata et al., 2003;Song et al., 2005). A variety of acute noxious stimuli and chronic pain models result in ERK1/2 activation (phosphorylation) at many levels of the nociceptive sensory system including dorsal root ganglion (DRG), spinal cord, and amygdala (Ji et al., 2009). The use of inhibitors that block activation of both ERK1 and ERK2 by inhibiting their shared upstream MAP kinase kinases (MEK1/2) and transgenic expression in neurons of a dominant-negative form of MEK1, which suppresses MEK1/2ERK1/2 signaling, have demonstrated that ERK1/2 is necessary for nociceptive sensitization (Ji et al., 1999,2002,2009;Karim et al., 2001,2006;Song et al., 2005;Hu et al., 2006;Seino et al., 2006). Although much is known about MEK1/2ERK1/2 signaling, little is known about the specific functions of JAK2-IN-4 each ERK isoform. Functional redundancy has been a working model because the isoforms are 90% homologous (Boulton et al., 1991) and no isoform-specific inhibitors currently exist. However, there is emerging evidence that ERK1 and ERK2 may be functionally distinct. ERK1 knock-outs (ERK1 KOs) are viable but exhibit behavioral abnormalities correlated with altered synaptic plasticity in striatum (Pags et al., 1999;Selcher et al., 2001;Mazzucchelli et al., 2002), whereas ERK2 knock-outs are embryonic lethal at embryonic day 8.5 (Krens et al., 2006). Alternative methods for Mouse monoclonal antibody to TAB1. The protein encoded by this gene was identified as a regulator of the MAP kinase kinase kinaseMAP3K7/TAK1, which is known to mediate various intracellular signaling pathways, such asthose induced by TGF beta, interleukin 1, and WNT-1. This protein interacts and thus activatesTAK1 kinase. It has been shown that the C-terminal portion of this protein is sufficient for bindingand activation of TAK1, while a portion of the N-terminus acts as a dominant-negative inhibitor ofTGF beta, suggesting that this protein may function as a mediator between TGF beta receptorsand TAK1. This protein can also interact with and activate the mitogen-activated protein kinase14 (MAPK14/p38alpha), and thus represents an alternative activation pathway, in addition to theMAPKK pathways, which contributes to the biological responses of MAPK14 to various stimuli.Alternatively spliced transcript variants encoding distinct isoforms have been reported200587 TAB1(N-terminus) Mouse mAbTel+86- targeting ERK2, including reduced expression from a hypomorphic mutant allele and conditional deletion in telencephalon, have revealed a requirement for ERK2 in several learning and memory paradigms (Satoh et al., 2007;Samuels et al., 2008). In cell culture, genetic targeting or RNA interference (RNAi) experiments suggest specific roles for ERK1 and ERK2 (Mazzucchelli et al., 2002;Vantaggiato et al., 2006). Evidence to support this hypothesis includes the observations that ERK1 exclusively interacts with the MEKERK signaling scaffold MP1 (Schaeffer et al., 1998) and the fact that differences in amino acid sequence between ERK1 and ERK2 occur in domains that may affect MEK1/2 binding, ERK dimerization, and subcellular localization (Boulton et al., 1991;Zhang et al., 1994;Cobb and Goldsmith, 2000). Indeed, ERK1 and ERK2 have different rates of shuttling between the cytoplasm and nucleus because of sequence differences in the N terminus (Marchi et al., 2008). Currently, the importance of ERK1 in nociceptive sensitization remains unknown. Therefore, we tested the necessity of ERK1 in acute noxious sensitization and in models of chronic inflammatory and neuropathic pain using ERK1 KO mice. Although ERK1 is activated in these models, genetic deletion of ERK1 had JAK2-IN-4 a minimal impact on these ERK-dependent behaviors. Interestingly, ERK1 deletion increased basal ERK2 phosphorylation without affecting inflammation-induced changes in ERK2 phosphorylation. Our observations indicate that ERK1 is not required for nociceptive sensitization and suggest that ERK2 plays a predominant role. == Materials and Methods == == == == == == Animals. == All experiments were performed according to the guidelines of the National Institutes of Health and were approved by the Animal Care and Use Committee of Washington University School of Medicine. Mice were housed with a 12 h light/dark cycle andad libitumaccess to food and water. Targeted deletion of ERK1 was achieved by homologous recombination in a process described in detail previously (Nekrasova et al., 2005). Briefly, the targeting vector deleted exons 16 ofmapk3, which includes the kinase active site and phosphorylation loop, and replaced them with a Neo cassette flanked with loxP sites. Embryonic stem cells from 129 Sv mice were transfected and selected for homologous recombination. Successfully targeted clones were injected into CD1 blastocysts, which were implanted in pseudopregnant CD1 females. Chimeras with.
