Temporal expression of connexins during neuronal differentiation of NT2/D1 cells

Abstract

Purpose: Gap junctions are known to be involved in growth and development, however the roles and regulation of specific connexin (Cx) subtypes in neuronal differentiation are not well understood. Changes in Cx expression may be involved in proper development of neurons, thus we have profiled the temporal expression of three neural Cxs during neuronal differentiation of NT2/D1 cells. Methods: Differentiation was induced via 4 weeks treatment with 10 μM retinoic acid (RA), followed by 2 weeks mitotic inhibitor treatment. Protein and mRNA were isolated from mixed cultures, in triplicate, at weekly timepoints during RA treatment, as well as from undifferentiated NT2/D1 cells and differentiated hNT neurons, and Cx expression assessed using immunoblotting and RT-PCR. Results: Cx43 protein was abundant in NT2/D1 cells. Cx43 protein was down-regulated five-fold by day 3 of RA treatment, relative to undifferentiated cells, remaining at this low level throughout RA treatment and was negligible in hNT neurons. Interestingly, Cx43 mRNA was highly expressed for the first 7 days of RA treatment before being downregulated by approximately half by day 14, indicating post-transcriptional regulation of Cxs was altered during differentiation. Cx30 and Cx36 mRNA were expressed at low levels in undifferentiated NT2/D1 cells. Cx30 mRNA was upregulated four-fold during RA treatment by day 14 before decreasing to two-fold of baseline expression by day 21. Cx36 mRNA expression increased two-fold and remained elevated at this level throughout the remaining RA treatment. Conclusion: We observed a tight regulation of Cx subtype expression during differentiation. The upregulation of Cx30 and Cx36 suggests these subtypes may be necessary for neuronal development, while Cx43 may be important for maintenance of an undifferentiated state.