Cd. Mobarak et al., The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells, MOL BIOL CE, 11(9), 2000, pp. 3191-3203
The RNA-binding protein HuD binds to a regulatory element in the 3' untrans
lated region (3' UTR) of the GAP-43 mRNA. To investigate the functional sig
nificance of this interaction, we generated PC12 cell lines in which HuD le
vels were controlled by transfection with either antisense (pDuH) or sense
(pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP
-43 protein and mRNA, and these levels remained low even after nerve growth
factor (NGF) stimulation, a treatment that is normally associated with pro
tein kinase C (PKC)dependent stabilization of the GAP-43 mRNA and neuronal
differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mR
NA had a shorter half-life in these cells. In agreement with their deficien
t GAP-43 expression, pDuH cells failed to grow neurites in the presence of
NGF or phorbol esters. These cells, however, exhibited normal neurite outgr
owth when exposed to dibutyryl-cAMP, an agent that induces outgrowth indepe
ndently from GAP-43. We observed opposite effects in pcHuD-transfected cell
s. The GAP-43 mRNA was stabilized in these cells, leading to an increase in
the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to
grow short spontaneous neurites, a process that required the presence of GA
P-43. In conclusion, our results suggest that HuD plays a critical role in
PKC-mediated neurite outgrowth in PC12 cells and that this protein does so
primarily by promoting the stabilization of the GAP-43 mRNA.