Morphine induces redox-based changes in global DNA methylation and retrotransposon transcription by inhibition of excitatory amino acid transporter type 3-mediated cysteine uptake

Abstract

Canonically, opioids influence cells by binding to a G protein-coupled opioid receptor, initiating intracellular signaling cascades, such as protein kinase, phosphatidylinositol 3-kinase, and extracellular receptor kinase pathways. This results in several downstream effects, including decreased levels of the reduced form of glutathione (GSH) and elevated oxidative stress, as well as epigenetic changes, especially in retrotransposons and heterochromatin, although the mechanism and consequences of these actions are unclear. We characterized the acute and long-term influence of morphine on redox and methylation status (including DNA methylation levels) in cultured neuronal SH-SY5Y cells. Acting via μ-opioid receptors, morphine inhibits excitatory amino acid transporter type 3-mediated cysteine uptake via multiple signaling pathways, involving different G proteins and protein kinases in a temporal manner. Decreased cysteine uptake was associated with decreases in both the redox and methylation status of neuronal cells, as defined by the ratios of GSH to oxidized forms of glutathione and S-adenosylmethionine to S-adenosylhomocysteine levels, respectively. Further, morphine induced global DNA methylation changes, including CpG sites in long interspersed nuclear elements (LINE-1) retrotransposons, resulting in increased LINE-1 mRNA. Together, these findings illuminate the mechanism by which morphine, and potentially other opioids, can influence neuronal-cell redox and methylation status including DNA methylation. Since epigenetic changes are implicated in drug addiction and tolerance phenomenon, this study could potentially extrapolate to elucidate a novel mechanism of action for other drugs of abuse.

Fig. 1.

Neuronal redox and methylation pathways. Neurons depend upon cysteine uptake to support GSH synthesis. Cysteine is provided by cleavage of GSH exported by astrocytes, and neurotrophic growth factors promote cysteine uptake via the EAAT3 transporter (Hodgson et al., 2013). Transsulfuration of HCY to cysteine is restricted in the human brain, increasing reliance upon EAAT3-mediated uptake. Methylation reactions, including DNA methylation, are regulated by methionine synthase, whose activity is dependent upon redox status and GSH levels. DNA-Me, DNA-methylation; PI3-kinase, phosphatidylinositol 3-kinase; PP+P_i, inorganic phosphate; THF, tetrahydrofolate.

Fig. 2.

Inhibition of cysteine uptake by morphine via µ-opioid receptors in SH-SY5Y cells. (A) Radiolabeled cysteine uptake by SH-SY5Y human neuroblastoma cells is reduced by increasing concentrations of morphine (0.1 nM to 10 µM) with an IC₅₀ of 2.4 nM. Pretreatment with the nonselective opioid antagonist naltrexone (1 µM) prevents the morphine-induced decrease of cysteine transport (n = 6). *Significant difference (P < 0.005) from control. (B) SH-SY5Y cells treated with 0.1 µM morphine showed a pattern of inhibition of cysteine uptake at different time points (0.5, 4, and 24 hours). Pretreatment with D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH₂ (CTAP; 0.1 μM), a specific μ-opioid receptor blocker, abrogated the effects of morphine. Pretreatment with naltrindole (NTI; 0.1 μM), a specific δ-opioid receptor blocker, did not inhibit the effects of morphine on cysteine uptake (n = 6). *Significant difference (P < 0.05) from no treatment control. Gray bar represents no treatment control (NTC). Nonspecific binding was subtracted from total cysteine uptake.

Fig. 3.

Differential involvement of G proteins and downstream signaling kinases in morphine-induced inhibition of cysteine uptake by SH-SY5Y cells. (A) Short-term (0.5 and 4 hours) morphine treatment (0.1 μM) inhibited cysteine uptake by SH-SY5Y human neuroblastoma cells, and this effect was blocked by pretreatment with PTX, a G_i/o inhibitor (0.5 μg/ml, 24 hours), but not by CTX, a G_s inhibitor (0.5 μg/ml, 24 hours) (n = 6). Long-term (24 hours) morphine treatment (0.1 μM) induced inhibition of cysteine uptake that was blocked by pretreatment with cholera toxin, but not pertussis toxin. (B) The short-term (4 hours) inhibitory effect of morphine on cysteine uptake was blocked by pretreatment with the PKA inhibitor H-89 (0.1 μM) (n = 6). The long-term (24 hours) effect of morphine (0.1 μM) on cysteine uptake was blocked by pretreatment with the mitogen-activated protein kinase kinase inhibitor PD98059 (10 μM) (n = 6). *Significant difference (P < 0.005) from no treatment control (NTC; gray bar).

Fig. 4.

Effect of morphine on cellular cysteine concentration and GSH/GSSG and SAM/SAH ratios in SH-SY5Y cells. Intracellular cysteine, GSH/GSSG, and SAM/SAH ratios were decreased by morphine treatment at 4 hours (A) and 24 hours (B). Pretreatment with the PKA inhibitor H-89 (0.1 µM) blocked the effects of morphine (0.1 μM) at 4 hours, and the mitogen-activated protein kinase kinase inhibitor PD98059 (10 µM) blocked the effects of morphine at 24 hours (n = 4). *Significant difference (P < 0.05) from untreated control values.

Fig. 5.

Effect of morphine on global DNA methylation in SH-SY5Y cells. Cells were treated with morphine (0.1 μM) for 4 and 24 hours in the presence or absence of H-89 (0.1 μM) and PD98059 (10 μM), respectively (n = 6). Global methylation was quantified using an anti–5-methylcytosine antibody, measured by enzyme-linked immunosorbent assay. No significant changes were observed after 4 hours of morphine treatment, but 24 hours of morphine treatment induced significant hypomethylation. *Significant difference (P < 0.05) from untreated controls.

Fig. 6.

Morphine induces hypomethylation in LINE-1 repetitive elements and increases LINE-1 mRNA levels. (A) Morphine (0.1 μM) induced changes in site-specific methylation content for individual CpG sites on promoter regions of the LINE-1Hs family, as determined by bisulfite conversion and pyrosequencing. Pos1–4 indicates individual CpG sites, normalized against control values. *Significant difference (P < 0.05) compared with control; **P < 0.01 compared with control. (B) Promoter methylation (TSS ± 500 base pairs) of LINE-1. Morphine increased promoter methylation after 4 hours, which returned to normal levels after 24 hours of morphine treatment (N = 5). (C) qRT-PCR analysis of LINE-1 mRNA levels. Four hours of morphine treatment decreased LINE-1 mRNA levels, whereas 24 hours of morphine treatment induced a large increase. Pretreatment with H-89 or PD98059 blocked the changes in LINE-1 mRNA levels after 4 or 24 hours of morphine treatment, respectively. *Significant difference (P < 0.05) from untreated controls.

Fig. 7.

Proposed mechanism. In SH-SY5Y cells, morphine inhibition of EAAT3 via the μ-opioid receptor is mediated by MAPK and PKA signaling via Gα_i/o and Gα_s. Activation of the μ-opioid receptor can also lead to glutamate transporter regulator 3-18 activation (Lin et al., 2001; Ikemoto et al., 2002), which reduces the level of EAAT3 surface expression. PKA pathways can activate transcriptional factors, such as CREB, but can also activate the PTEN/ubiquitin/Nedd4 pathway, which promotes degradation of EAAT3. Further, the PKA and ERK pathway can also mediate phosphorylation of EAAT3, which can affect surface expression and/or EAAT3 activity directly. Thus, morphine treatment reduces GSH/GSSG and SAM/SAH ratios, leading to epigenetic changes subsequently affecting transcription. CREB, cAMP response element-binding; DNMT, DNA methyl transferase enzyme; Nedd4, neural precursor cell expressed developmentally downregulated protein 4; PTEN, phosphatase and tensin homolog; TFs, transcription factors.