Mitogenic signaling via endogenous kappa-opioid receptors in C6 glioma cells: evidence for the involvement of protein kinase C and the mitogen-activated protein kinase signaling cascade

Abstract

As reports on G protein-coupled receptor signal transduction mechanisms continue to emphasize potential differences in signaling due to relative receptor levels and cell type specificities, the need to study endogenously expressed receptors in appropriate model systems becomes increasingly important. Here we examine signal transduction mechanisms mediated by endogenous kappa-opioid receptors in C6 glioma cells, an astrocytic model system. We find that the kappa-opioid receptor-selective agonist U69,593 stimulates phospholipase C activity, extracellular signal-regulated kinase 1/2 phosphorylation, PYK2 phosphorylation, and DNA synthesis. U69,593-stimulated extracellular signal-regulated kinase 1/2 phosphorylation is shown to be upstream of DNA synthesis as inhibition of signaling components such as pertussis toxin-sensitive G proteins, L-type Ca2+ channels, phospholipase C, intracellular Ca2+ release, protein kinase C, and mitogen-activated protein or extracellular signal-regulated kinase kinase blocks both of these downstream events. In addition, by overexpressing dominant-negative or sequestering mutants, we provide evidence that extracellular signal-regulated kinase 1/2 phosphorylation is Ras-dependent and transduced by Gbetagamma subunits. In summary, we have delineated major features of the mechanism of the mitogenic action of an agonist of the endogenous kappa-opioid receptor in C6 glioma cells.

FIG. 1

κ-Opioid stimulation of DNA synthesis in C6 cells. Cells were maintained in “low MEM” for 48 h before agonist and inhibitor treatment. Inhibitors were added to the same medium before opioid. [³H]Thymidine (0.5 μCi/ml) was included 30 min after U69,593 (U69) for the final 23 h in the presence of the other reagents. A: U69 stimulates [³H]thymidine incorporation in a dose-dependent manner. Nonlinear regression analysis of the average ± SEM (bars) values of three independent experiments performed in triplicate indicates an EC₅₀ of 1.03 nM. B: U69 (1 μM) stimulation of DNA synthesis is blocked by the following agents with the indicated concentrations and times before U69 treatment—the κ-OR antagonist nor-BNI (1 μM, 1 h), PTX (100 ng/ml, overnight), nifedipine (1 μM, 30 min), U73,122 (1 μM, 30 min), dantrolene (1 μM, 30 min), or BAPTA (50 μM, 30 min). *Significantly greater than all points, p < 0.01 by ANOVA. Data are mean ± SEM (bars) values from three to eight experiments performed in triplicate. Basal [³H]thymidine incorporation was measured as 13,560 ± 1,233 dpm per well.

FIG. 2

κ-Opioid stimulation of PI turnover. C6 cells were maintained in “low MEM” for 48 h and then labeled with 2 μCi/ml myo-[³H]inositol per well in the same medium for 24 h before activation. Medium was changed, and cells were incubated in “low MEM” plus 5 mM LiCl for 1 h before drug treatment. U69,593 (U69; 1 μM) stimulation of PLC activity ensued for 1 h in the presence of the indicated inhibitors. The following reagents were applied at the indicated concentrations and times before U69 treatment: nor-BNI (1 μM, 1 h), PTX (100 ng/ml, overnight), nifedipine (1 μM, 30 min), and U73,122 (1 μM, 30 min). *Significantly greater than control, ^#significantly less than U69 alone, p < 0.01 by ANOVA. Data are mean ± SEM (bars) values from three to seven experiments performed in triplicate. Basal ³H-IP_x levels were measured as 31,850 ± 4,239 dpm per well.

FIG. 3

Inhibitors of PKC and MEK block κ-opioid stimulation of [³H]thymidine incorporation. C6 cells were maintained in “low MEM” for 48 h before treatment. Opioids, inhibitors, and [³H]thymidine were added to the same medium as described in Fig. 1. The following inhibitors were used at the concentrations and times before U69,593 (U69) treatment indicated: PMA (1 μM, overnight), GFX (100 nM, 30 min), and PD98059 (1 μM, 30 min). *Significantly greater than control, p < 0.01 by ANOVA. Data are mean ± SEM (bars) values from three to eight experiments performed in triplicate.

FIG. 4

Time- and concentration-dependent κ-opioid phosphorylation of ERK1/2. C6 cells were maintained in “low MEM” for 48 h before treatment. Opioids were added to the same medium. Cell lysates were prepared, and 15 μg of protein was loaded per lane on 10% sodium dodecyl sulfate-polyacrylamide mini-gels for western blot analysis. A: Time course. ERK1/2 phosphorylation was measured after exposure for 0.5, 1, 5, 10, 30, and 60 min to 5 nM U69,593. Data are mean ± SEM (bars) values of three to five separate experiments. B: Concentration dependence. ERK1/2 phosphorylation was assayed after a 5-min exposure to 0.5, 1, 5, 10, or 100 nM concentrations of U69,593. Data are mean ± SEM (bars) values of three to five separate experiments. Data were compiled by densitometric analysis (NIH Image software) and represented as the fold increased intensity as normalized by untreated controls in each experiment. Also shown are representative membranes blotted first with anti-phospho(P)ERK1/2 (top) and then stripped and reblotted with anti-ERK1 (bottom).

FIG. 5

Inhibition of κ-opioid phosphorylation of ERK1/2. C6 cells were kept in “low MEM” for 48 h before treatment. Opioids and inhibitors were added to the same medium. Cell lysates were prepared, and 15 μg of protein was loaded per lane on 10% sodium dodecyl sulfate-polyacrylamide mini-gels. U69,593 (U69; 10 nM, 10 min) was used to stimulate ERK1/2 phosphorylation. The following inhibitors were applied at the following concentrations and times before U69 treatment: (A) Nor-BNI (1 μM, 1 h), PTX (100 ng/ml, overnight), nifedipine (1 μM, 30 min), U73,122 (1 μM, 30 min), dantrolene (1 μM, 30 min), and BAPTA (50 μM, 30 min) and (B) PMA (1 μM, overnight), GFX (100 nM, 30 min), and PD98059 (1 μM, 30 min). *Significantly greater than all points, p < 0.01 by ANOVA. Data are mean ± SEM (bars) values from three to nine experiments. Data were compiled by densitometric analysis (NIH Image software) and represented as the fold increased intensity as normalized by untreated controls in each experiment. Also shown are representative membranes blotted first with anti-phospho(P)ERK1/2 (top) and then stripped and reblotted with anti-ERK1 (bottom).

FIG. 6

κ-OR mediates phosphorylation of ERK1/2 via Gβγ proteins. C6 cells were grown as described and then were transfected with 1 μg of CD8 or CD8-βARK-C cDNA and 2 μl of LIPOFECTAMINE per well. Before agonist stimulation, cells were maintained in “low MEM” for 48 h. U69,593 (U69; 10 nM, 10 min) phosphorylation was expressed as fold activation over the respective transfected control (control = 1.0). CD8 and CD8-βARK transfection was confirmed in parallel samples by immunoblotting with anti-CD8 Ab. *Significantly greater than un-stimulated CD8 vector controls, p < 0.01 by Student’s t test; ^#significantly less than U69 plus CD8 vector, no different than CD8-βARK control, p < 0.001 by Student’s t test. Data are mean ± SEM (bars) values from three to five experiments. Data were compiled by densitometric analysis (NIH Image software). Also shown is a representative membrane blotted first with anti-phospho(P)ERK1/2 (top) and then stripped and reblotted with anti-ERK1 (bottom).

FIG. 7

N17Ras blocks κ-opioid phosphorylation of ERK1/2. Cells were grown as described and then were transfected with 1 μg of N17Ras-pcDNAIII or the vector alone and 2 μl of LIPOFECTAMINE per well. Before agonist stimulation, cells were maintained in “low MEM” for 48 h. U69,593 (U69; 10 nM, 10 min) phosphorylation was expressed as fold activation over the respective transfected control (control = 1.0). N17Ras transfection was confirmed (compared with vector-transfected cells) in parallel samples by immunoblotting with anti-Ras. *Significantly greater than un-stimulated vector controls, p < 0.01 by Student’s t test; ^#significantly less than U69 plus vector, no different than N17Ras control, p < 0.001 by Student’s t test. Data are mean ± SEM (bars) values from three to seven experiments. Data were compiled by densitometric analysis (NIH Image software). Also shown are representative membranes blotted first with anti-phospho(P)ERK1/2 (top) and then stripped and reblotted with anti-ERK1 (bottom).

FIG. 8

κ-Opioid-stimulated PYK2 phosphorylation. Cells were plated equally in 100-mm-diameter dishes as described. After 48 h in “low MEM” cells were either treated with 10 nM U69,593 (U69) for 3 min or lysed immediately (untreated controls). After incubation with anti-phosphotyrosine Abs plus protein A-Sepharose for 4 h at 4°C, immunoprecipitates were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels. Immunoblotting with anti-PYK2 Ab reveals increased phosphorylated PYK2 in treated cells. Shown are a representative blot and the average densitometry gained from three separate experiments. Data are average ± SEM (bars) densitometry from three experiments as normalized by the average of the control band densities. *Significantly greater than control, p < 0.01 by Student’s t test.