Figure 1.
HuD protein specifically binds to NEP mRNA in human SK-N-SH cells.
A, RIP assay showing HuD-bound NEP mRNA in cultured SK-N-SH cells. Cells were lysed and immunoprecipitated with normal rabbit immunoglobulin G (NRIgG), HuD antibody, or antibody solution with HuD peptide and then total RNA was isolated from immunoprecipitates and used for RT-qPCR with specific human NEP primers. As a control, human GAPDH mRNA was amplified from RT-qPCR of total RNA (Mean ± SEM of three independent experiments, ***P<0.001, compared with NRIgG). B, RNA-EMSA assay using recombinant HuD protein and a biotin-labeled oligoriboprobe corresponding to the candidate ARE sequence in 3′-UTR region of NEP mRNA and the 3′-UTR of GAPDH mRNA as a negative control, respectively. Unlabeled oligoriboprobe was used as a competitor for the inhibition reaction.
Figure 2.
Figure 2. Activated PKCε stabilizes NEP mRNA.
A, Cells were untreated or incubated with HuD siRNA or control siRNA (Con siRNA) for 4 days and then treated with actinomycin D (ActD, 10 µg/ml) for 2, 4, 6, 8, and 10 hrs. Total RNA was isolated and NEP mRNA was quantified by real time RT-qPCR. NEP mRNA at each time point was compared with the initial mRNA level (100%). A nonlinear regression analysis was conducted to calculate the first-order decay constant (k). Average mRNA half-life (t1/2) was calculated as 0.693/k and reported in the table (Mean ± SEM of the three independent experiments, *P<0.05, HuD siRNA+ActD compared with untreated; #P<0.05, HuD siRNA+ActD compared with Con siRNA-treated). B, mRNA stability assay from cells treated with ActD or ActD+bryostatin (Bryo, 0.5 nM), or pre-treated with Ro 32-0432 (Ro, 2 µM) for 30 min and then treated with ActD+Bryo for 2, 4, 6, 8, and 10 hrs. Average mRNA half-life (t1/2) was calculated as 0.693/k and reported in the table (Mean ± SEM of the three independent experiments, **P<0.01, ActD+Bryo compared with ActD to assess the bryostatin effect; #P<0.01, ActD+Bryo compared with ActD+Ro+Bryo to assess the Ro 32-0432 effect). C, Quantitative RT-qPCR analysis showing remaining NEP mRNA from untreated cells or cells incubated with HuD siRNA or Con siRNA for 4 days (Mean ± SEM for three independent experiments, **P<0.01, compared with untreated). D, RIP analysis to detect NEP mRNA associated with HuD protein in untreated cells or cells treated with 0.5 nM Bryo or pre-treated with 2 µM Ro for 30 min and then treated with Bryo for 1 hr. Relative amounts of NEP mRNA bound to HuD were analyzed by real-time RT-qPCR (Mean ± SEM for three independent experiments, ***P<0.001, compared with untreated; #P<0.001, compared with Bryo-treated). E, RIP analysis to detect NEP mRNA associated with HuD protein in untreated cells or cells incubated with PKCε siRNA or Con siRNA, and then treated without/with Bryo for 1 hr (Mean ± SEM for three independent experiments, **P<0.01, compared with untreated; n.s. = not specific).
Figure 3.
Figure 3. Activated PKCε enhances HuD binding to NEP mRNA and increases NEP protein expression.
A, Immunoblot analyses to detect HuD and NEP protein levels in untreated cells or cells incubated with control siRNA (Con siRNA) or HuD siRNA (Mean ± SEM, **P<0.01, compared with untreated). B, Relative expression of NEP protein was measured by immunoblot analysis after treatment with bryostatin (Bryo, 0.5 nM) or actinomycin D (ActD, 10 µg/ml), or pre-treatment with ActD for 1 hr prior to bryostatin (ActD+Bryo) for 24 hr (Mean ± SEM of three independent experiments, **P<0.01, ***P<0.001, compared with untreated control). C, Immunoblot analyses to detect PKCε and NEP protein levels in untreated cells or cells incubated with Con siRNA or PKCε siRNA (Mean ± SEM, *P<0.05, **P<0.01, compared with untreated).
Figure 4.
Activated PKCε increases phosphorylation and membrane localization of NEP.
A, Cells were untreated or treated with bryostatin (Bryo, 0.5 nM) or pre-treated with Ro 32-0432 (Ro, 2 µM) for 30 min and then treated with Bryo for 1 hr, lysed, and then used for immunoprecipitation using NEP antibody. Immunocomplexes were further analyzed to detect phosphorylated and biotinylated NEP protein in immunoblot analyses after normalization for NEP protein level (Mean ± SEM, *P<0.05, compared with untreated). B, Cells were untreated or incubated with control siRNA (Con siRNA) or PKCε siRNA for 4 days without/with bryostatin (1 nM) treatment for 1 hr, lysed, and then used for immunoprecipitation using NEP antibody. Immunocomplexes were further analyzed to detect phosphorylated and biotinylated NEP protein in immunoblot analyses after normalization for NEP protein level (Mean ± SEM, **P<0.01, compared with Con siRNA). C, Cells were untreated or incubated with Con siRNA or HuD siRNA for 4 days, lysed, and then used for immunoprecipitation using NEP antibody. Immunocomplexes were further analyzed to detect phosphorylated and biotinylated NEP protein in immunoblot analyses after normalization for NEP protein level.
Figure 5.
PKCε activation enhances NEP activity.
A, Fluorometric measurement of NEP activity from cells in the absence (untreated) or presence of bryostatin (Bryo) at 0.27, 0.5, 1, or 2 nM for 1 hr (Mean ± SEM of three independent experiments, *P<0.05, **P<0.01, Bryo compared with untreated). B, NEP activity in cells in the absence (untreated) or presence of 1 nM Bryo for 15 min, 30 min, 1 hr, or 3 hr (Mean ± SEM of three independent experiments, *P<0.05, **P<0.01, Bryo compared with untreated). C, NEP activity in cells in the absence (untreated) or presence of 1 nM Bryo, pre-incubated with 2 µM Ro for 30 min, pre-incubated with phosphoramidon (PA, 10 µM, a specific NEP inhibitor) for 5 min, or pre-incubated with Ro+PA before Bryo treatment for 1 hr (Mean ± SEM of three independent experiments, **P<0.01, compared with untreated; #P<0.01, compared with Bryo). D, NEP activity measurement in untreated cells or cells incubated with control siRNA (Con siRNA) or HuD siRNA without or with treatment of 1 nM Bryo or 2 µM Ro+Bryo for 1 hr (Mean ± SEM for three independent experiments, **P<0.01, compared with Con siRNA; #P<0.05, compared with HuD siRNA; $P<0.05, compared with HuD siRNA+Bryo). E, NEP activity measurement in untreated cells or cells incubated with Con siRNA or PKCε siRNA without or with treatment of 1 nM Bryo or 2 µM Ro+Bryo for 1 hr (Mean ± SEM for three independent experiments, ***P<0.001, compared with Con siRNA).
Figure 6.
PKCε activation rescues oligomeric Aβ-mediated inhibition of HuD-NEP mRNA interaction and NEP protein expression.
A, RIP analysis to detect NEP mRNA associated with HuD protein in untreated cells or cells treated with Aβ (Abeta, 1 µM), Aβ+bryostatin (Bryo, 0.5 nM) or pre-treated with Ro 32-0432 (Ro, 2 µM) for 30 min and then treated with Abeta+Bryo for 6 hr. Relative amounts of NEP mRNA bound to HuD were analyzed by real-time RT-qPCR (Mean ± SEM for three independent experiments, **P<0.01, compared with untreated; #P<0.01, compared with Abeta; $P<0.01, compared with Abeta+Bryo). B, Relative change in NEP protein expression was determined based on immunoblot analyses of untreated cells or cells treated with 1 µM Abeta or Abeta +0.5 nM Bryo, or pre-treated with 2 µM Ro for 1 hr prior to Abeta+Bryo for 24 hr (Mean ± SEM of the three independent experiments, *P<0.05, compared with untreated; #P<0.05, compared with Abeta; $P<0.05, compared with Abeta+Bryo).
Figure 7.
PKCε activation recovers NEP membrane localization inhibited by oligomeric Aβ peptides.
A, Cells were untreated or treated with oligomeric Aβ (Abeta; 1 µM) or Abeta+bryostatin (Bryo, 1 nM), or pre-incubated with Ro 32-0432 (Ro, 2 µM) for 30 min before Abeta+Bryo treatment for 1 hr, and then used for biotin-labeling. Phosphorylated and biotinylated NEP proteins were detected by immunoblot from immunoprecipitated NEP protein and were compared (Mean ± SEM of the three independent experiments, *P<0.05, **P<0.01, compared with untreated; #P<0.05, compared with Abeta; $P<0.05, compared with Abeta+Bryo). B, NEP activity assay from untreated cells or cells treated with 0.5 or 1 µM Abeta, or Abeta +1 nM Bryo, or pre-incubated with 2 µM Ro for 30 min before Abeta+Bryo treatment for 1 hr (Mean ± SEM for three independent experiments, *P<0.05, **P<0.01, compared with untreated; #P<0.01, compared with Abeta; $P<0.01, compared with Abeta+Bryo).
Figure 8.
Activated PKCε stimulates NEP activity to protect SK-N-SH cells against Aβ neurotoxicity.
A, Cells were untreated or treated with bryostatin (Bryo, 1 nM), phosphoramidon (PA, 10 µM)+Bryo, Ro 32-0432 (Ro, 2 µM)+Bryo, or PA+Ro+Bryo for 1 hr and incubated with monomeric Aβ 1–42 peptide (Abeta, 2.5 µg) for additional 4 hr. Aβ 1–42 peptide was precipitated from the reactions by 20% TCA and immunoblotted with anti-Aβ peptide antibody 6E10 (Mean ± SEM for three independent experiments, **P<0.01, compared with untreated; #P<0.05, compared with Bryo). B, Aβ degradation assay in cells incubated with control siRNA (Con siRNA) or HuD siRNA without or with treatment of bryostatin (Bryo, 1 nM) or Ro 32-0432 (Ro, 2 µM)+Bryo for 1 hr (Mean ± SEM for three independent experiments, ***P<0.001, compared with Con siRNA; #P<0.001, compared with HuD siRNA; $P<0.001, compared with HuD siRNA+Bryo). C, Aβ degradation assay in cells incubated with Con siRNA or NEP siRNA without or with treatment of 1 nM Bryo or 2 µM Ro+Bryo for 1 hr (Mean ± SEM for three independent experiments, ***P<0.001, compared with Con siRNA; #P<0.05, compared with NEP siRNA; $P<0.05, compared with NEP siRNA+Bryo). D, Cells were incubated in absence (untreated) or presence of DMSO (vehicle) or oligomeric Aβ1–42 peptide (1 µM) for 24 hr. Cells were co-treated with Abeta +0.5 or 1 nM Bryo or pre-treated with 2 µM Ro for 1 hr or phosphoramidon (PA, 10 µM) for 10 min prior to co-treatment with Abeta+Bryo (1 nM). The viability of the cells after treatment was determined by cell viability assay and results are expressed as a percentage of viable cells compared with untreated control cells (Mean ± SEM of three independent experiments, **P<0.01, compared with untreated; #P<0.05, compared with Abeta; $P<0.01, compared with Abeta+Bryo).