9 research outputs found

    The basal activity of PAC is Wnt/β-catenin pathway involved and dimer dependent.

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    <p>(A) The effects of the cell signaling inhibitors H-89, XAV939 and PAC1 dimerization inhibitor NAC on the remaining cell viabilities of PAC1-CHO, M-PAC1-CHO and pcDNA-CHO 24 h after serum withdrawal. All the data were plotted as the fold changes in the data of pcDNA-CHO because the changes in pcDNA-CHO had no correlation with PAC1. It was shown that the β-catenin signal inhibitor XAV939 (10 µM) and PAC1 dimerization inhibitor NAC (10 nM) significantly decreased the viabilities of PAC1-CHO cells (*, P<0.01, PAC1-CHO/XAV939 and PAC1-CHO/NAC vs. PAC1-CHO/no inhibitors), whereas the PKA inhibitor H-89 (100 µM) did not exert significant effect on the cells viability. The effects of H-89, XAV939 and NAC on the caspase3 activities (B) and Bcl-2 levels (C) in PAC1-CHO, M-PAC1-CHO and pcDNA-CHO 24 h after serum withdrawal showed that XAV939 (10 µM) and NAC (10 nM) exerted significant inhibitory effects on the anti-apoptotic activity of PAC1-CHO by increasing the caspase3 activity and decreasing the Bcl-2 level in the PAC1-CHO cells significantly (*, P<0.01, PAC1-CHO/XAV939 and PAC1-CHO/NAC vs. PAC1-CHO/no inhibitors), whereas H-89 (100 µM) did not have the similar inhibitory effects. (D)Top-flash assays. PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells were transfected with Top-flash + pRluc and Fop-flash + pRluc respectively. pRluc was used here as the internal control for transfection efficiency. After the transfection, cells were submitted to serum-withdraw induced apoptosis with or without the signal inhibitors H-89 (100 µM), XAV-939 (10 µM), and acetylcysteine (10 nM) for 24 h. And then cells were lysed and luciferase activities were measured. Relative luciferase activities were expressed as the ratio of TOP-flash/FOP-flash luciferase activity and the data were plotted as fold changes in the data from pcDNA-CHO cells. It was found that without inhibitors the relative luciferase activity in PAC1-CHO cells was significantly higher than that in M-PAC1-CHO and pcDNA-CHO (#, P<0.01, PAC1-CHO/no inhibitors vs. M-PAC1-CHO/no inhibitors and pcDNA-CHO/no inhibitors) after serum withdrawal. The addition of Wnt/β-catenin signal inhibitor XAV939 (10 µM) and PAC1 dimerization inhibitor NAC (10 nM) significantly decreased the relative luciferase activity in PAC1-CHO (*, P<0.01, PAC1-CHO/XAV939 and PAC1-CHO/NAC vs. PAC1-CHO/no inhibitors), indicating that the basal activity of PAC1 was Wnt/β-catenin involved and dimer dependent. The data were represented as the means ± S.E. of three independent experiments. (D) Western blotting showed XAV939 (10 µM) and NAC (10 nM) significantly decreased the β-catenin, cyclin D1 and c-myc levels in PAC1-CHO cells. (*, P<0.01, PAC1-CHO/XAV939 and PAC1-CHO/NAC vs. PAC1-CHO/no inhibitors), whereas H-89 (100 µM) did not have the similar inhibitory effects. The data were represented as the means ± S.E. of three independent experiments.</p

    The correlation of the PAC1 levels with its basal activity in Tet-on inducible expression system.

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    <p>The fluorescence microscopic observation (A) and the fluorescence density assays (B) of the PAC1-YFP expression induced by Dox (0–100 ng/mL) in Tet-on inducible system. The fluorescence microscopic images showed that the numbers of cells with YFP fluorescence increased following increases in the concentration of Dox (1–100 ng/mL), whereas there was no fluorescence observed without induction by Dox (0 ng/mL). Bar, 20 µm. The YFP fluorescence densities, assayed using the Victor3 1420 multi-label counter, increased with the concentration of Dox (1–100 ng/mL), indicating that the expression levels of PAC1 were controlled by Dox in a concentration-dependent manner. (C) Western blotting of the inducible expression of PAC1-YFP. The western blotting with goat polyclonal IgG against the C-terminus of PAC1 using reductive SDS-PAGE showed the bands corresponding to PAC1-YFP deepened with the increase of Dox (1–100 ng/mL), while no band corresponding to PAC1-YFP was found in the treatment without Dox (0 ng/mL). The remaining cell viabilities (D), the caspase3 activity (E) and the Bcl-2 levels (F) after serum withdrawal in the double-stable Tet-on advanced inducible cells treated with Dox (1–100 ng/mL) were plotted as the fold changes in the data from the cells treated without Dox (0 ng/mL). It was shown that the higher concentrations of Dox induced higher expression levels of PAC1-YFP, which in turn led to the higher anti-apoptotic activity of the cells, including higher remaining cell viability, lower caspase3 activity and higher Bcl-2 level. (G) Top-flash assays. In double-stable Tet-on advanced inducible cells, after the transfection with Top-flash + pRluc or Fop-flash + pRluc, cells were submitted to serum-withdraw induced apoptosis with Dox (1–100 ng/mL) or without Dox for another 24 h. And then cells were lysed and luciferase activities were measured. Relative luciferase activities were expressed as the ratio of TOP-flash/FOP-flash luciferase activity and the data were plotted as fold changes in the data from the cells treated without Dox (0 ng/mL). It was shown that the relative luciferase activities increased following the increase of Dox (1–100 ng/mL), indicating that the higher expression levels of PAC1-YFP induced by higher concentration of Dox resulted into stronger Wnt/β-catenin signals. (H) Western blotting of β-catenin, cyclin D1 and c-myc corresponding to Wnt/β-catenin pathway. After the protein expression levels were normalized by the corresponding levels of the control nucleoporin-p62 and plotted as the fold changes of the cells treated without Dox, it was shown that in the cells expressing a range of PAC1-YFP induced by Dox (0–100 ng/mL), β-catenin, cyclin D1 and c-myc levels increased following the increases of the PAC1 levels. All these data suggested the significant positive correlation of the PAC1 levels with the anti-apoptotic activities involved with Wnt/β-catenin signals. The data were represented as the means ± S.E. of three independent experiments.</p

    The mechanism for the ligan-independent basal activity of PAC1 dimers (A) and the ligand-dependent activation of PAC1.

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    <p>(A) In ligand-free situation, the disturbance of the plasma induced entocytosis of PAC1 dimers, which triggered the activation of the basal activity of PAC1 dimers involved with Wnt/β-catenin signal pathway to protect the cells against apoptosis. (B) In ligand-dependent manner, the binding of the ligands for PAC1 disrupted the dimerization of PAC1 and induced internalization of PAC1 monomer, which inhibited the basal activity of PAC1 dimers.</p

    Effects of the NAC on the dimerization of PAC1.

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    <p>(A) BiFC assays. Shown were YFP fluorescence intensity re-produced by the transfection of the receptor constructs as indicated. The cells without transfection were used as negative control and the cells transfected with PAC-YFP as positive control. Exogenous NAC (10 nM) decreased the YFP fluorescence intensity produced by PAC-Y/N+PAC-Y/C significantly (*, P<0.01 PAC-Y/N+PAC-Y/C+NAC vs. PAC-Y/N+PAC-Y/C), while the transfection of M+PAC-Y/N+M+PAC-Y/C produced no YFP fluorescence signals. Data were presented as means ± S.E. of three independent experiments. (B) Saturation BRET. Shown were the BRET saturation curves plotted as a ratio of YFP fluorescence to Rlu luminescence that were observed for tagged receptor constructs studied with a fixed amount of donor and increasing amounts of acceptor. PAC-Rluc/PAC-YFP receptor constructs yielded exponential curves that reached asymptotes indicating significant homo-dimerization of PAC1, while M-PAC-Rluc/M-PAC-YFP yielded curves not different from a straight line, indicating that D-PAC1 lost the ability to form dimers. The addition with NAC (10 nM) at 2 h before the BRET signal assay lowered the curves significantly (*, P<0.01 PAC-Rluc/PAC-YFP+NAC vs. PAC-Rluc/PAC-YFP). The data were represented as the means ± S.E. of three independent experiments. (C) Static BRET. BRET ratios for CHO cells expressing receptor constructs as indicated. For static BRET, a total of 1.0 µg of DNA per well divided equally among the noted constructs in each condition was utilized. The shaded area represents the nonspecific BRET signal generated between PAC-Rlu and soluble YFP protein, with BRET signals above this area considered to be significant. As shown the BRET ratio in PAC-Rluc/PAC-YFP CHO cells incubated with NAC (10 nM) was significantly lower than that in cells without treatment with NAC (*, P<0.01 PAC-Rluc/PAC-YFP+NAC vs. PAC-Rluc/PAC-YFP). The data were presented as the means ± S.E. of three independent experiments. (D) Western blotting analysis with a goat polyclonal IgG against the C-terminus of PAC1 using non-reductive SDS-PAGE. When PAC-YFP expressing cells incubated with exogenous NAC (10 nM), as shown, the band with the molecular weight (about 160 kD) consistent with the molecular weight of the PAC1 dimer was weakened by the presence of NAC (10 nM). All these results showed that NAC was an inhibitor of the dimerization of PAC1, which offered us a tool to analysis the relation of the dimerization of PAC1 with its basal activity.</p

    The endocytosis of PAC1 dimers using BiFC during serum withdrawal.

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    <p>(A) The fluorescence confocal microscopic images of BiFC signals 2 h after serum withdrawal. It was shown that after the nuclear staining with DAPI in CHO cells transfected with PAC-Y/N+PAC-Y/C, the BiFC signals (YFP fluorescence reproduced by the dimerization), representing the PAC1 dimers, were mostly located into the cells and close to the nucleus 2 h after serum withdrawal, while CHO cells transfected with M-PAC-Y/N+M-PAC-Y/C displayed no BiFC signals. Bar, 10 µm. (B) The fluorescence confocal microscopic images of live CHO cells transfected with PAC-Y/N+PAC-Y/C submitted to serum withdrawal. At the beginning of the serum withdrawal, the BiFC signals, representing the PAC1 dimers, were mostly located on or near plasma membranes. Then, at 0.5 h after serum withdrawal, the BiFC signals trafficked into the cells in some type of vehicles, and at 2 h after serum withdrawal, most of the BiFC signals were inside the cells and aggregated around the nucleus. Bar, 5 µm.</p

    The ligand-dependent activation of PAC1 and M-PAC1 by PACAP.

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    <p>(A) Western blotting of endogenous PACAP in CHO-K1 and neuro2a cells. As shown CHO-K1 had no detectable endogenous PACAP, while neuro2a cells produced endogenous PACAP. (B) The expression of PAC1-YFP and M-PAC1-YFP detected by immunofluorescence. Shown were immunofluorescence results of PAC1-CHO and M-PAC1-CHO cells cultured in DMEM with 0.5% CS-FBS at 37°C overnight, which indicated that both PAC1 and M-PAC1 trafficked normally to the plasma membrane and 0.5% CS-FBS induced no significant receptors endocytosis. (C) Fluorescence densities assays. Shown were the YFP fluorescence densities in the whole cell lysate detected using the Victor3 1420 multi-label counter with excitation (460±30 nm) and emission (535±30 nm), indicating that the expression levels of PAC1-YFP in CHO cells were equal to those of M-PAC1-YFP. (D) Western blotting assays using reductive SDS-PAGE. Western blotting with a goat polyclonal IgG against the C-terminus of PAC1 in the reductive condition showed that there were similar bands with the molecular weight about 160 kD in PAC1-CHO and M-PAC1-CHO, but not in CHO. (E) The cell viabilities of PAC1-CHO and M-PAC1-CHO cells promoted by PACAP. The data were plotted as the fold changes of the treatment without PACAP (0 nM). After the cells were submitted the addition of PACAP (1–100 nM) in the absence of CS-FBS for 24 h, MTT assays showed that PACAP exerted more significant proliferative effects on M-PAC1-CHO than on PAC1-CHO (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO), indicating that the activation level of PAC1 by PACAP was lower than that of M-PAC1. (F) The intracellular cAMP levels induced by PACAP (1–100 nM) in PAC1-CHO and M-PAC1-CHO cells. After the data were plotted as the fold changes of the treatment with 0 nM PACAP, it was shown that the intracellular cAMP levels in M-PAC1-CHO cells induced by PACAP were significantly higher than the intracellular cAMP levels in PAC1-CHO cells induced by PACAP (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments.</p

    The ligand independent activity of PAC1 and M-PAC1 against serum withdrawal induced apoptosis.

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    <p>(A) The remaining cell viabilities of PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells 48 h after serum withdrawal. When the data were plotted as the percentage of the initial cell viability without serum withdrawal, it was shown that PAC1-CHO had remaining cell viability (57.34±5.91%) that was significantly higher than that of M-PAC1-CHO (36.96±6.85%) or pcDNA-CHO (37.89±7.11%) (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). (B) The intracellular caspase3 activities after serum withdrawal. The reactions of pcDNA-CHO were considered not result from PAC1 because pcDNA-CHO did not express PAC1 or PACAP; therefore, all the data were plotted as fold changes in pcDNA-CHO. As shown, PAC1-CHO had significantly lower caspase3 activity than M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO), whereas there was no significant difference between M-PAC1-CHO and pcDNA-CHO. (C) The intracellular Bcl-2 levels after serum withdrawal. After the data were plotted as the fold changes of pcDNA-CHO, it was shown that PAC1-CHO had significantly higher Bcl-2 level about 2 folds of that in M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments. (D) The detection of β-catenin, cyclin D1 and c-myc levels in PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells by western blotting. The western blotting results and the statistical analysis showed that the levels of β-catenin, cyclin D1 and c-myc (tow targets of β-catenin) in PAC1-CHO cells were significantly higher than those in M-PAC1-CHO or pcDNA-CHO cells (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). These findings indicated that overexpression of wild type PAC1 endowed CHO with anti-apoptotic activities against serum withdrawal, suggesting that PAC1 had ligand independent basal activity, while M-PAC1 did not. And Wnt/β-catenin signals were involved in the anti-apoptotic activity of PAC1-CHO. The data were represented as the means ± S.E. of three independent experiments.</p

    The correlation of PAC1 knockdown with its basal activity in Neuro2a.

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    <p>(A) Knockdown of endogenous PACAP and PAC1 with shRNA in Neuro2a. Western blotting assays showed that shRNA against PACAP significantly diminished the expression of endogenous PACAP in neuro2a/PACAP<sup>-</sup>, and further transfection with shRNA plasmids against PAC1 (+) to neuro2a/PACAP<sup>-</sup> cells decreased the PAC1 levels significantly, while control plasmids (-) did not interfere with expression of PAC1. The knockdown of PACAP and PAC1 in neuro2a produced a chance for the detection of the correlation of PAC1 down-regulation with its ligand independent basal activity. (B) The remaining cell viabilities of nero2a/PACAP<sup>-</sup> transfected with PAC1 shRNA plasmids (+) or control plasmid (-). After the data were plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that down-regulation of PAC1 with PAC1 shRNA plasmids (+) decreased the remaining cell viabilities to almost a half of the remaining cell viabilities transfected with control plasmids (-) 48 h after serum withdrawal (*, P<0.01, shRNA + vs. shRNA-). (C) Western blotting of β-catenin, cyclin D1 and c-myc in the nero2a/PACAP<sup>-</sup> cells transfected with PAC1 shRNA plasmids (+) or control plasmids (-). After the relative protein levels were normalized by the corresponding levels of the control nucleoporin-p62 and plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that PAC1 shRNA plasmids (+) significantly decreased the levels of β-catenin, cyclin D1 and c-myc compared with control plasmids (+)(*, P<0.01, shRNA+ vs. shRNA-). These data suggested that down-regulation of PAC1 in the natural cells such neuro2a with high expression of PAC1 inhibited the anti-apoptotic activities in the ligand free condition. The data were represented as the means ± S.E. of three independent experiments.</p
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