Morphine induces ubiquitin-proteasome activity and glutamate transporter degradation

Abstract

Glutamate transporters play a crucial role in physiological glutamate homeostasis, neurotoxicity, and glutamatergic regulation of opioid tolerance. However, how the glutamate transporter turnover is regulated remains poorly understood. Here we show that chronic morphine exposure induced posttranscriptional down-regulation of the glutamate transporter EAAC1 in C6 glioma cells with a concurrent decrease in glutamate uptake and increase in proteasome activity, which were blocked by the selective proteasome inhibitor MG-132 or lactacystin but not the lysosomal inhibitor chloroquin. At the cellular level, chronic morphine induced the PTEN (phosphatase and tensin homolog deleted on chromosome Ten)-mediated up-regulation of the ubiquitin E3 ligase Nedd4 via cAMP/protein kinase A signaling, leading to EAAC1 ubiquitination and proteasomal degradation. Either Nedd4 or PTEN knockdown with small interfering RNA prevented the morphine-induced EAAC1 degradation and decreased glutamate uptake. These data indicate that cAMP/protein kinase A signaling serves as an intracellular regulator upstream to the activation of the PTEN/Nedd4-mediated ubiquitin-proteasome system activity that is critical for glutamate transporter turnover. Under an in vivo condition, chronic morphine exposure also induced posttranscriptional down-regulation of the glutamate transporter EAAC1, which was prevented by MG-132, and transcriptional up-regulation of PTEN and Nedd4 within the spinal cord dorsal horn. Thus, inhibition of the ubiquitin-proteasome-mediated glutamate transporter degradation may be an important mechanism for preventing glutamate overexcitation and may offer a new strategy for treating certain neurological disorders and improving opioid therapy in chronic pain management.

FIGURE 1.

Chronic morphine exposure down-regulates EAAC1 expression. C6 cells were exposed to morphine (MS, 10 μm) or morphine plus naloxone (Nal., 1 μm) for 0, 12, 24, 48, and 96 h. A, Western blotting of whole cell lysates. B, in vitro glutamate uptake activity. C, RT-PCR analysis. Results are representative of three independent experiments or the mean ± S.E. for at least three independent extract preparations. ^**, p < 0.01, compared with vehicle control using Student's t test. Tubulin, loading control.

FIGURE 2.

Chronic morphine induces ubiquitin-proteasome-mediated degradation of EAAC1. C6 cells were treated with morphine (MS, 48 h) or morphine plus the selective proteasome inhibitor MG-132 (20 μm for 12 h) or lactacystin (Lac., 10 μm for 12 h) or the lysosome inhibitor chloroquin (Chl., 50 μm for 12 h). A, Western blotting (WB) of whole cell lysates. B, in vitro glutamate uptake activity. C, 26 S proteasome activity assay. D, C6 cell lysates were co-immunoprecipitated with an anti-EAAC1 antibody (IP), followed by Western blotting with an anti-ubiquitin antibody and anti-EAAC1 antibody (WB). Ubn, the migration position of polyubiquitin conjugates; +, the EAAC1 position. Results are representative of three independent experiments or the mean ± S.E. for at least three independent extract preparations. ^*, p < 0.05 and ^**, p < 0.01, compared with vehicle control using Student's t test.

FIGURE 3.

The ubiquitin E3 ligase Nedd4 interacts with EAAC1. A, EAAC1 copurified with Nedd4. Extracts of C6 cells were subjected to immunoprecipitation using antibodies against several ubiquitin E3 ligases (lanes 2-6). EAAC1 co-purified with Nedd4 (lane 6) but not with a control IgG, Cbl, Parkin, or Mdm2. Morphine or morphine plus MG-132 did not disturb the interaction between EAAC1 and Nedd4 (lane 7 and 8). EAAC1 extract (lane 1) represents 4% of the amount used in the immunoprecipitation. B, Nedd4 co-purified with EAAC1. Extracts of C6 cells were immunoprecipitated with antibodies against EAAC1. Nedd4 was specifically copurified in EAAC1 precipitates (lane 3) but not in control IgG precipitates (lane 2). Extract (lane 1) represents 1% of the amount used in the immunoprecipitation. C, Nedd4 was markedly knocked down with siRNA. C6 cells were transfected with negative control siCONTROL non-targeting siRNA pool, Nedd4 siRNA pool, or vehicle, the Nedd4 expression level was examined at 48 h after each transfection. D, Nedd4 knockdown abolished the morphine-induced EAAC1 down-regulation. C6 cells transiently transfected with siCONTROL or siNedd4 were treated with vehicle, morphine (10 μm for 48 h), morphine plus MG-132, or MG-132 (MG-132 was added during the final 12 h of the 48-h morphine exposure), the EAAC1 expression level was examined in each group. E, C6 cells transiently transfected with siCONTROL or siNedd4 were treated with vehicle, morphine (10 μm for 48 h), morphine plus MG-132, or MG-132 (MG-132 was added during the final 12 h of the 48-h morphine exposure), cell lysates were co-immunoprecipitated with an anti-EAAC1 antibody (IP), followed by Western blotting with an anti-ubiquitin antibody (WB, Western blot). Ubn, the migration position of polyubiquitin conjugates; +, the EAAC1 position. Results are representative of three independent experiments. Actin, a loading control.

FIGURE 4.

PTEN regulates the Nedd4 expression. A, C6 cells were treated with morphine (MS, 10 μm) or morphine plus naloxone (Nal, 1 μm) for 0, 8, 12, 24, and 48 h. Western blotting of PTEN or Nedd4 was performed using whole cell lysates. B, time course effect of morphine on the level of Nedd4 protein in PTEN-null U87 cells. C, PTEN copurified with Nedd4. Lysates from the PTEN-expressing 293T and C6 cell lines and the PTEN-null U87 cell line were IP with Nedd4 (lane 1) or normal rabbit IgG (lane 2) and were Western blotted (WB) with PTEN, μ-opioid receptor (MOR), or Nedd4. PTEN, but not μ-opioid receptor, copurified with Nedd4 (lane 1), but not control IgG (lane 2) in 293T and C6 cells. D, Nedd4 copurified with PTEN. Extracts of 293T, C6, and U87 cells were immunoprecipitated with antibodies against PTEN (lane 1) or normal rabbit IgG (lane 2) and Western blotted (WB) with Nedd4, μ-opioid receptor, and PTEN. Nedd4 but not μ-opioid receptor specifically copurified in PTEN precipitates (lane 1) but not in control IgG precipitates (lane 2) in 293T and C6 cells. PTEN-null U87 cells were used as control. E, time course PTEN and Need4 mRNA level (RT-PCR) after each treatment in C6 cells. F, intensity (-fold) quantification and statistical analysis of the RT-PCR results in E. Results are the mean ± S.E. for at least three independent extract preparations. ^*, p < 0.05, as compared with the vehicle group using Student's t test.

FIGURE 5.

Morphine recruited PTEN through the cAMP/PKA signaling. A, 293T, C6, and U87 cells were pretreated with the adenylyl cyclase inhibitor ddA (30 μm), PKA inhibitor H89 (30 μm), phosphatidylinositol 3-kinase inhibitor LY294002 (10 μm), cGMP-dependent protein kinase inhibitor RP-8-PCPT (5 μm), or PKC inhibitor Go6976 (1 μm) for 30 min and then co-treated with morphine (10 μm) for 48 h. Western blotting of whole lysate was made. B, 26 S proteasome activity assay of these same samples from panel A. Results are representative of three independent experiments or the mean ± S.E. for at least three independent extract preparations. ^*, p < 0.05, as compared with the vehicle group using Student's t test.

FIGURE 6.

PTEN is a negative regulator in EAAC1 expression in C6 cells. C6 cells were stably transfected with vector-based short hairpin RNA constructs: PTEN/pRNAT-H1.1/Neo/GFP or scramble pRNAT-H1.1/Neo/GFP. A, fluorescence microscopy (top row) and Western blotting of the wild type (Con), scramble (Scr), and 3 siPTEN clones (C1-3) with anti-PTEN (middle row) or tubulin (bottom row). B, immunocytochemistry of scramble siPTEN (ssiPTEN) and siPTEN (C1) with anti-EAAC1 (red) and Hoechst (blue) nuclear staining. There was a marked increase in intracellular labeling of EAAC1 with large vesicle-like structures and clusters accumulated in the perinuclear area (bottom row). Ninety percent of the PTEN knocked down C6 cells represented enhanced EAAC1 immunoreactivities. Among them, 80% exhibits this pattern. In contrast, the scramble siPTEN cell exhibited dispersed, punctuating, and weak intracellular labeling of EAAC1 (top row). These images are representative of multiple fields examined for each treatment from two independent immunofluorescence experiments. C, glutamate uptake activity assay in wild type, SsiPTEN, and siPTEN-transfected cells. ^*, p < 0.05 as compared with the wide type (WT) control using Student's t test.

FIGURE 7.

The proteasome inhibitor MG-132 prevented spinal cord EAAC1 down-regulation. Rats (n = 6) were administrated intrathecally (twice daily) with vehicle, morphine (MS, 15 nmol), morphine plus MG-132 (5 nmol), or MG132 (5 nmol) alone for seven consecutive days. A, Western blotting of glutamate transporter EAAC1, PTEN, and Need4 within the spinal cord dorsal horn. B, the mRNA level of EAAC1, PTEN, and Nedd4 examined using RT-PCR after each treatment. C, densitometric quantification of RT-PCR results are shown in B. Analysis of variance, ^*, p < 0.05, as compared with the vehicle control. Lane 1, vehicle; lane 2, morphine (15 nmol); lane 3, morphine (15 nmol) plus MG-132 (5 nmol); lane 4, MG-132 (5 nmol) alone. Results are representative of three independent experiments or the mean ± S.E. from at least three independent extract preparations. Tubulin, loading control.