The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells

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.