The RNA-binding Protein HuD Is Required for GAP-43 mRNA Stability, GAP-43 Gene Expression, and PKC-dependent Neurite Outgrowth in PC12 Cells

Abstract

The RNA-binding protein HuD binds to a regulatory element in the 3Ј untranslated region (3Ј UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels 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 protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. 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 GAP-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. INTRODUCTIONIn addition to transcriptional factors, RNA-binding proteins play a critical role in the developmental control of gene expression. Among these is ELAV (embryonic lethal abnormal vision), an RNA-binding protein identified in Drosophila, where the gene is required for normal development and maintenance of the nervous system (Campos et al., 1985;Robinow et al., 1988). In higher vertebrates and mammals, four members of the ELAV-like family have been identified. These are also referred to as Hu proteins, namely HuR (also known as HuA), HuB (Hel-N1), HuC, and HuD, because these are targets of anti-Hu antibodies present in the sera of patients with paraneoplastic encephalomyelitis (Dalmau et al., 1992). HuR is ubiquitously expressed (Ma et al., 1996), while HuB, HuC, and HuD are expressed uniquely in the nervous system. Recent studies indicate that overexpression of neural ELAV-like proteins is sufficient to induce neuronal differentiation in vitro and in vivo (Wakamatsu and Weston, 1997; Akamatsu et al., 1999;Antic et al., 1999;Kasashima et al., 1999). While the exact function and targets of ELAV/Hu proteins remain to be fully elucidated, it seems likely that this family of RNA-binding proteins controls neuronal differentiation by selectively modulating the expression of neural-specific, growth-associated genes.The growth-associated protein GAP-43 is expressed in neurons primarily during the initial establishment and regeneration of neural connections (Skene, 1989;Benowitz and Routtenberg, 1997 Eggen et al., 1995;Chiaramello et al., 1996;Kinney et ...

Posttranscriptional regulation of gene expression in learning by the neuronal ELAV-like mRNA-stabilizing proteins

“…4d shows that there was indeed such a decrease in GAP-43 expression levels; a significant down-regulation of GAP-43 mRNA, revealed by one-way ANOVA and post hoc analysis, was observed for the anti-HuC antisense treatment by quantitative RT-PCR and confirmed by in situ hybridization in both the CA1 and CA3 hippocampal subfields (in all cases after pairwise comparisons AS ODN-and DN ODN-treated mice were different at P Ͻ 0.001). Similar to this finding, in PC12 cells antisense-mediated down-regulation of another ELAV-like gene, HuD, resulted in a correlated decrease in GAP-43 mRNA steady-state levels and stability and in defective neurite outgrowth (30).…”

p53 is required for nerve growth factor-mediated differentiation of PC12 cells via regulation of TrkA levels

“…Unlike the induction of p21, p53 alone is not sufficient, but required for, NGF to induce GAP-43. Since Figure 7 A model of p53 for neurite outgrowth in PC12 cells p53 is required for neuronal differentiation J Zhang et al the increased expression of GAP-43 by NGF is due to stabilization of GAP-43 mRNA, 27 it is likely that p53 may modulate the expression of GAP-43 indirectly by regulating other neuronal differentiation-related genes, including TrkA.…”

Molecular Mechanisms, Biological Actions, and Neuropharmacology of the Growth-Associated Protein GAP-43

“…The RNA binding protein HuD is required for GAP-43 mRNA stability and GAP-43 gene expression [65]. HuD is an ELAV (embryonic lethal abnormal vision) protein, and is in a group that includes the proteins HuB and HuC [70].…”