Role of Gap Junctional Intercellular Communication (GJIC) through p38 and ERK1/2 Pathway in the Differentiation of Rat Neuronal Stem Cells

Abstract

ABSTRACT. Gap junctional intercellular communications (GJIC) contributes to neural function in development and differentiation of CNS. In this study, we have investigated the expression of GJIC during the differentiation of neuronal stem cells and 12-Ø-tetradecanoylphorbol-13-acetate (TPA)-induced neuronal stem cell-derived cells from rat brain. During neuronal stem cell differentiation, expressions of Cx43 and 32 were increased for the duration of 72 hr, however the effect were decreased on the 7d. In the neuronal stem cell-derived cells, pretreatments with p38 MAP kinase inhibitor, SB203580, and MEK inhibitor, PD98059, could protect GJIC against TPA-induced inhibition of GJIC. Our data suggest that GJIC plays an important role during neuronal stem cell differentiation, and ERK1/2 and p38 MAP kinase signaling pathway may be closely related functionally to regulate gap junction in rat neuronal stem cell-derived cells. Neuronal stem cells are able to differentiate into astrocytes, neurons, and oligodendrocytes [3,10,23], when given the appropriate signals [2,7]. During the stage where the brain develops, gap junctional intercellular communication (GJIC) allows the diffusional exchange of ions and metabolites for communication between neighboring cells [8,14,20]. Connexin 32 and 43 were expressed in the neurons of adult rat or mouse [15,17], and Cx43 was the most abundant gap junction protein in the CNS [4,5]. However, no study has yet been performed to the role of gap junctions in rat neuronal stem cell differentiation. In the present study, we have examined protein expression of Cx43 and Cx32 during the neuronal stem cell differentiation and induction of Cx43 and Cx32. This can be elicited in neuronal stem cell-derived cells by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) and this induction showed that it can be prevented by the p38 MAP-Kinase inhibitor SB203580 and the MEK inhibitor PD98059. MATERIALS AND METHODSCell culture: Neuronal stem cells were prepared by culturing cells from cerebrum of embryonic day 17 (E17) Sprague-Dawley rat (Bio Genomics, Korea) fetus. Briefly, fresh cerebrums were dissociated in the presence of trypsin and DNase I and planted on a Petri dish (Nunc, IL). Cells were seeded at a density of 1~5 × 10 6 cells/ml in a mixture (1:1) of Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12; Gibco) replenished with 2% B27 (Gibco), gentamycin (50 µg/ml), anti-PPLO (pleuro-pneumonia-like organism) reagent (100 µg/ml), recombinant human basic fibroblast growth factor (bFGF 20 ng/ml, Sigma), and epidermal growth factor (10 ng/ml).Scrape loading/ dye transfer (SL/DT) assay: Cells were treated with TPA for 1 hr. The GJIC assay was conducted at non-cytotoxic dose levels of the samples, as determined by the MTT assay. Following incubation, the cells were washed twice with 2 ml of PBS, Lucifer yellow was added to the washed cells and three scrapes were made with a surgical steel-bladed scalpel at low light intensities. These three scrapes were performed to ensure that the scrape travers...

Tetramethylpyrazine Suppresses Transient Oxygen-Glucose Deprivation-Induced Connexin32 Expression and Cell Apoptosis via the ERK1/2 and p38 MAPK Pathway in Cultured Hippocampal Neurons

“…Introducing gap junction proteins (Cxs) into cell lines has been shown to enhance bystander cell death [41]. In addition, gap junction blocker carbenoxolone could reduce OGD-induced neuronal death in hippocampal slice cultures [42]. Consistent with these reports, in this study, we found the quantitative inverse relationship between Cx32 expression and hippocampal neuron viability.…”

Non-classical Signalling Mechanisms in Stem Cells

“…Lastly, rat brain derived NS/PC express Cx43 and Cx45 and communicate through GJIC, which is essential for their survival and proliferation . In these cells, Cx32 and Cx43 are upregulated during differentiation (Yang et al, 2005). Similar data were observed in mouse fetal NS/PC, where the closure of gap junctions decreases cell proliferation and reduces cell survival Duval et al, 2002).…”

Role of Gap Junctions in Embryonic and Somatic Stem Cells

“…Neural stem cells isolated from the rat developing brain express Cx43 and Cx45 and are capable of forming GJIC, where GJIC is essential for survival and proliferation in these cells [14]. Moreover, Cx43 and Cx32 levels were observed to increase temporarily during differentiation in these cells [131]. Future studies on human neural stem cells will help address whether this role of gap junctions is conserved among species.…”