Ethanol-induced small heat shock protein genes in the differentiation of mouse embryonic neural stem cells

Abstract

Neural stem cells (NSCs) of the neuroepithelium differentiate into one of three central nervous system (CNS) cell lineages: neurons, astrocytes, or oligodendrocytes. In this study, the differentiation potential of NSCs from the forebrain of embryonic day 15 (E15) mouse embryos was analyzed using immunocytochemistry. NSCs were differentiated early in the presence or absence of ethanol (50 mM), and gene expression patterns among NSCs, differentiated cells and ethanol-treated differentiated cells were assessed by microarray and real-time PCR analysis. Genes that were up-regulated in differentiated cells both in the presence and in the absence of ethanol when compared to NSCs were related to the Wnt signaling pathway, including Ctnna1, Wnt5a, Wnt5b, Wnt7a, Fzd3, and Fzd2; genes related to cell adhesion, including Cadm1, Ncam1, and Ncam2; and genes encoding small heat shock proteins, including HspB2, HspB7, and HspB8. In particular, the expression levels of HspB2 and HspB7 were elevated in ethanol-treated differentiated cells compared to non-treated differentiated cells. The gene expression patterns of various heat shock transcription factors (HSFs), proteins that regulate the transcription of heat shock genes, were also analyzed. The expression levels of HSF2 and HSF5 increased in differentiated cells in the presence and absence of ethanol when compared to NSCs. Of these two genes, HSF5 demonstrated an enhanced up-regulation, particularly in ethanol-treated differentiated cells compared to cells that were differentiated in the absence of ethanol. These results imply that HspB2 and HspB7, which are small heat shock proteins with tissue-restricted expression profiles, might be up-regulated by ethanol during the short-term differentiation of NSCs.

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“…The rat embryos were decapitated on embryonic day 17 (E17), and the hippocampus was quickly removed and placed on a dish with ice cold Hank’s balanced salt solution containing penicillin (100 U/ml)/streptomycin (100 ug/ml) [8,9]. Next, the hippocampus was minced, incubated in 0.25% trypsin–ethylenediaminetetraacetic acid (trypsin/EDTA) for 5 min at 37°C and then incubated with 0.05% DNase for 5 min at 37°C.…”

Alcohol stress, membranes, and chaperones

“…When adult mice were injected with a single dose of ethanol, the nuclear translocation of HSF1 was enhanced and various Hsp genes were activated in the cerebral cortex (Pignataro et al 2007). The levels of sHsps were increased in neural stem cells derived from the forebrain of mouse embryos when the cells were differentiated in the presence of 50 mM ethanol, and the expression of the HSF genes was also upregulated (Choi et al 2011).…”

Ethanol-related alterations in gene expression patterns in the developing murine hippocampus

“…We pooled all fetuses and total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. The resulting cDNA was transcribed to cRNA, and the resulting biotinylated cRNA was hybridized to microarrays according to the manufacturer's specifications [16]. Image acquisition was conducted using an Affymetrix GeneChip ® Scanner 3000 7G.…”

“…qRT-PCR was performed as described by Choi et al [16]. In brief, qRT-PCR was performed with synthesized cDNA, SYBR Premix Ex TaqTM II (TaKaRa, Shiga, Japan), and appropriate primers using ABI 7500 real-time thermal cycler (Applied Biosystems, Carlsbad, USA).…”