C-fos mediates cocaine inhibition of NGF-induced PC12 cell differentiation.
In utero cocaine exposure can affect CNS development. Previous studies showed that cocaine inhibits neuronal differentiation in a dose-dependent fashion, in nerve growth factor (NGF)-stimulated PC12 cells, without affecting cell viability. NGF activates intracellular signaling proteins, specific immediate-early genes (IEG) including a transient peak of c-fos expression, and induction of late genes expression, leading to the neuronal phenotype. We hypothesized that cocaine interferes with NGF signaling. Therefore, we examined the pattern of c-fos expression in our cellular model. Time course of c-fos expression up to 72 h was determined in cells treated with NGF 20 ng/ml and cocaine 10 microgram/ml (a moderately toxic level) by RT-PCR analysis. Total RNA was isolated from cells, and levels of c-fos mRNA were estimated using gene-specific primers. In both control and experimental conditions, c-fos level was maximal at 0.5 h. In the control cells, c-fos expression declined rapidly to less than 5% of the 0.5h value, while in the cocaine-treated cells, c-fos level persisted through the 72-h exposure. Adding c-fos antisense to cells treated with NGF and cocaine resulted in significant improvement of neurite out-growth, from 28% (NGF + cocaine) to 89% (NGF + cocaine + c-fos antisense) of control differentiation after 72 h of exposure (Dunnet's T < 3.24). Inhibitory effects of cocaine on NGF-induced PC12 differentiation may be attributed to alteration of c-fos expression. Further studies will be required to examine the role of D1 receptor activation in mediating c-fos expression and to explore the effects of cocaine on other IEGs.
Zachor
D A
DA
Department of Pediatrics, University of Alabama at Birmingham 35294, USA. dzachor@civmail.circ.uab.edu
Moore
J F
JF
Jin
J
J
Theibert
A B
AB
Percy
A K
AK
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D048429:Cell Size; D003042:Cocaine / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D016762:Genes, fos; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D016716:PC12 Cells; D016133:Polymerase Chain Reaction; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger; D051381:Rats; D013997:Time Factors; D015533:Transcriptional Activation
D009414:Nerve Growth Factors; D016376:Oligonucleotides, Antisense; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D003042:Cocaine