NGF/PI3K signaling-mediated epigenetic regulation of delta opioid receptor gene expression.

The G protein-coupled delta opioid receptor (DOR) not only plays a critical role in opioid-mediated pain control, drug tolerance and addiction [1], but it is also associated with neuronal differentiation, cell survival, neuroprotection, and neurogenesis [2-6]. Nerve growth factor (NGF), which is the prototype of neurotrophins, plays a critical role in the development and maintenance of the central and peripheral nervous systems [7]. The rat adrenal pheochromocytoma PC12 cell line is a well-established NGF-responsive model for the study of gene expression during neuronal differentiation and development [8]. NGF temporally induces the expression of the dor gene in PC12 cells[9-11]. Neurotrophins regulate the expression of many neuronal genes during neuronal development. Moreover, the dor gene is expressed both spatially and temporally in specific developmental stages [12,13]. Token together, elucidating the molecular mechanisms of NGF-induced dor gene expression might shed light on the general mechanism of neurotrophin-mediated gene expression during neuronal differentiation and development. The signal transduction pathways from the cell surface to the nucleus in NGF-responsive PC12 cells have been investigated extensively [14], but the temporal mechanism of NGF-induced gene expression is not well understood at the chromatin level. Phosphorylation and acetylation of histone H3 at the N-terminal position are associated with mitogen-mediated induction of the immediate-early (IE) genes, c-fos and c-jun [15-17]. NGF induces many IE genes and late phase genes in PC12 cells [18]. NGF signaling apparently induces immediate-early genes by the recruitment of transcriptional coactivators/acetyltransferases to chromatin-remodeling complexes on the promoters of induced genes [19]. However, little is known of how NGF induces late phase genes such as dor. We previously observed that sustained activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling is important for the expression of the late-phase dor gene [11]. It is still unknown how NGF induces the expression of the dor gene after PI3K survival signaling reaches the nucleus. One possible mechanism for NGF-induced gene expression in the nucleus is that the NGF signal leads to the regulation of locus-specific modifications of histone tails, which subsequently bind to the promoter of an NGF-induced gene to initiate transcription and elongation. Such epigenetic modifications of histone tails can be a critical step for NGF-induced late phase gene expression. To test this idea, we examined the global and locus-specific effects of NGF signaling on modifications of histone H3. We also analyzed the status of H3 trimethylation in the rat dor promoter region in living cells using chromatin immunoprecipitation (ChIP) assays. We provide several lines of evidence showing that NGF treatment results in modifications of H3 Lys9 (H3K9), which correlate temporally with NGF-induced dor gene expression in PC12 cells. NGF/PI3K-mediated gene expression has been associated with a plethora of important biological processes, including cell survival, development, differentiation, and cell cycles [7,20-22]. Hence, NGF/PI3K-induced H3K9 modifications might have broad implications for cell survival, neuronal differentiation and development.