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Mitogen-activated protein kinase modulates ethanol inhibition of cell adhesion mediated by the L1 neural cell
Xiaowei Dou1, Michael F Wilkemeyer, Carrie E Menkari
1Veterans Affairs Boston Healthcare System, Department of Neurology, Harvard Medical School, West Roxbury, MA 02132, USA.
Abstract:
There is a genetic contribution to fetal alcohol spectrum disorders (FASD), but the identification of candidate genes has been elusive. Ethanol may cause FASD in part by decreasing the adhesion of the developmentally critical L1 cell adhesion molecule through interactions with an alcohol binding pocket on the extracellular domain. Pharmacologic inhibition or genetic knockdown of ERK2 did not alter L1 adhesion, but markedly decreased ethanol inhibition of L1 adhesion in NIH/3T3 cells and NG108-15 cells. Likewise, leucine replacement of S1248, an ERK2 substrate on the L1 cytoplasmic domain, did not decrease L1 adhesion, but abolished ethanol inhibition of L1 adhesion. Stable transfection of NIH/3T3 cells with human L1 resulted in clonal cell lines in which L1 adhesion was consistently sensitive or insensitive to ethanol for more than a decade. ERK2 activity and S1248 phosphorylation were greater in ethanol-sensitive NIH/3T3 clonal cell lines than in their ethanol-insensitive counterparts. Ethanol-insensitive cells became ethanol sensitive after increasing ERK2 activity by transfection with a constitutively active MAP kinase kinase 1. Finally, embryos from two substrains of C57BL mice that differ in susceptibility to ethanol teratogenesis showed corresponding differences in MAPK activity. Our data suggest that ERK2 phosphorylation of S1248 modulates ethanol inhibition of L1 adhesion by inside-out signaling and that differential regulation of ERK2 signaling might contribute to genetic susceptibility to FASD. Moreover, identification of a specific locus that regulates ethanol sensitivity, but not L1 function, might facilitate the rational design of drugs that block ethanol neurotoxicity.
Insights
Genetic factors influence fetal alcohol spectrum disorders (FASD). This study reveals ERK2 signaling and L1 cell adhesion molecule phosphorylation as key modulators of ethanol sensitivity, potentially explaining genetic susceptibility to FASD.
Area of Science:
- Neuroscience
- Genetics
- Cell Biology
Background:
- Fetal alcohol spectrum disorders (FASD) have a genetic component, but causative genes remain unidentified.
- Ethanol exposure can impair L1 cell adhesion molecule (L1CAM) function, crucial for neural development, potentially contributing to FASD.
- The precise molecular mechanisms linking ethanol exposure, L1CAM, and genetic susceptibility to FASD are not fully understood.
Purpose of the Study:
- To investigate the role of ERK2 signaling in modulating ethanol's effects on L1CAM adhesion.
- To identify specific molecular players involved in ethanol sensitivity related to FASD.
- To explore potential genetic variations contributing to differential susceptibility to ethanol teratogenesis.
Main Methods:
- Utilized NIH/3T3 and NG108-15 cell lines with genetic manipulation (knockdown, transfection) of ERK2 and L1CAM.
- Employed site-directed mutagenesis to alter the S1248 phosphorylation site on L1CAM.
- Analyzed ERK2 activity, L1CAM adhesion, and MAPK activity in mouse models with varying ethanol susceptibility.
Main Results:
- ERK2 inhibition or knockdown did not affect basal L1CAM adhesion but significantly reduced ethanol's inhibitory effect.
- Mutating the S1248 site on L1CAM abolished ethanol's inhibition of L1CAM adhesion.
- Ethanol-sensitive cell lines exhibited higher ERK2 activity and S1248 phosphorylation compared to insensitive lines.
- Increased ERK2 activity rendered ethanol-insensitive cells sensitive to ethanol's effects on L1CAM adhesion.
- Mouse substrains differing in ethanol teratogenesis susceptibility showed corresponding differences in MAPK activity.
Conclusions:
- ERK2-mediated phosphorylation of L1CAM at S1248 is a critical mechanism in regulating ethanol's inhibitory effects on L1CAM adhesion via inside-out signaling.
- Differential regulation of ERK2 signaling pathways may underlie genetic susceptibility to FASD.
- Identifying genetic loci controlling ethanol sensitivity, independent of L1CAM function, could enable targeted therapeutic strategies against ethanol neurotoxicity.
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