TEMPORAL EXPRESSION OF CONNEXINS DURING
NEURONAL DIFFERENTIATION OF NT2/D1 CELLS
Wan C.K., O’Carroll S.J., Shaikh S.B., Green C.R. and Nicholson L.F.B.
The University of Auckland, Auckland, New Zealand.
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.