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. 2013 Nov 7;32(45):5302-14.
doi: 10.1038/onc.2012.624. Epub 2013 Jan 21.

Role for Prdx1 as a Specific Sensor in Redox-Regulated Senescence in Breast Cancer

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Free PMC article

Role for Prdx1 as a Specific Sensor in Redox-Regulated Senescence in Breast Cancer

B Turner-Ivey et al. Oncogene. .
Free PMC article

Abstract

Recent studies suggest that Peroxiredoxin 1 (Prdx1), in addition to its known H₂O₂-scavenging function, mediates cell signaling through redox-specific protein-protein interactions. Our data illustrate how Prdx1 specifically coordinates p38MAPK-induced signaling through regulating p38MAPKα phosphatases in an H₂O₂ dose-dependent manner. MAPK phosphatases (MKP-1 and/or MKP-5), which are known to dephosphorylate and deactivate the senescence-inducing MAPK p38α, belong to a group of redox-sensitive phosphatases (protein tyrosine phosphatases) characterized by a low pKa cysteine in their active sites. We found that Prdx1 bound to both MKP-1 and MKP-5, but dissociated from MKP-1 when the Prdx1 peroxidatic cysteine Cys52 was over-oxidized to sulfonic acid, which in turn resulted in MKP-1 oxidation-induced oligomerization and inactivity toward p38MAPKα. Conversely, over-oxidation of Prdx1-Cys52 was enhancing in the Prdx1:MKP-5 complex with increasing amounts of H₂O₂ concentrations and correlated with a protection from oxidation-induced oligomerization and inactivation of MKP-5 so that activation toward p38MAPK was maintained. Further examination of this Prdx1-specific mechanism in a model of reactive oxygen species-induced senescence of human breast epithelial cells revealed the specific activation of MKP-5, resulting in decreased p38MAPKα activity. Taken together, our data suggest that Prdx1 orchestrates redox signaling in an H₂O₂ dose-dependent manner through the oxidation status of its peroxidatic cysteine Cys52.

Figures

Figure 1
Figure 1. Lack of Prdx1 promotes senescence in murine fibroblasts and mammary epithelial cells
A. Primary Prdx1−/− and +/+MEFs were immortalized following the 3T3 protocol. Cells were counted every three days and cell numbers were plotted on the y-Axis; passages were plotted on the x-axis. Arrows indicate senescence: cell numbers did not exceed the number of cells plated. B. Left panel: Primary Prdx1−/− and +/+MEFs of different passages were stained for SA-βgal activity. Right panel: Quantification of B, (up to 4000 cells per passage and genotype were analyzed). C. Primary Prdx1−/− and +/+MEFs were treated with increasing doses of H2O2 for 8 hours and analyzed by Western blotting for phosphorylation of p53 on Ser15 and p53 protein levels. D. Primary Prdx1−/− and +/+MEFs were passaged in the absence and presence of 3μM and 6 μM of the p38MAPKα inhibitor SB203580, and stained for SA-βgal activity at passages 3, 4, 5, and 6. Representative pictures showing senescent morphology and confluency were also taken. Quantification of cells positive for SA-βgal staining was performed; “*” denotes statistical significance. P-values for Prdx1−/− MEFs include: 0.027 (passage 3, 0μM SB203580 vs. 3μM); 0.007, 0.0036 (passage 4, 0μM SB203580 vs. 3μM, and 0μM SB203580 vs. 6μM, respectively); 0.000, 0.000 (passage 5, 0μM SB203580 vs. 3μM, and 0μM SB203580 vs. 6μM, respectively); and 0.022, 0.003 (passage 6, 0μM SB203580 vs. 3μM, and 0μM SB203580 vs. 6μM, respectively). For Prdx1+/+MEFs: 0.007, 0.0036 (passage 4, 0μM SB203580 vs. 3μM, and 0μM SB203580 vs. 6μM, respectively), and 0.000, 0.0029 (passage 6, 0μM SB203580 vs. 3μM, and 0μM SB203580 vs. 6μM, respectively). E. Prdx1-/− and +/+MEFs were plated on 10cm plates at 1.8 × 105 and serum-starved for 48 h in 0.25% FBS. Cells were treated with increasing amounts of H2O2 (25-200 μM) for 10min in serum-free medium. Following treatment, plates were washed with cold 1X PBS (pH 7.2-7.4) two times. Cell lysates were analyzed by SDS-PAGE and Western blotting for phosphorylation of p38MAPKα on Thr180 and Tyr 182, phosphorylation of the p38MAPKα substrate ATF2 on Thr 69 and 71, p38MAPKα protein levels, and actin as loading control. F. Quantification of three independent experiments analyzing phosphorylation of p38MAPKα and its substrate ATF2 as described under E. Density of Western blot bands were analyzed using Image J software (http://rsbweb.nih.gov/ij/). Densities of phosphorylated p38MAPKα and ATF-2 proteins bands were normalized to density of p38MAPKα protein bands. Values were normalized to density of untreated WT cells as 1. Three different sets of Prdx1 WT and KO clones were tested. “*” indicates statistical significance (Students T-test) of KO and matching WT clones treated with H2O2. P-values for phosphorylation of p38MAPKα were 0.038 (25μM H2O2), 0.039 (50 μM H2O2), 0.008 (100 μM H2O2) and 0.044 (200 μM H2O2). P-values for phosphorylation of ATF-2 were 0.022 (25μM H2O2), 0.037 (100 μM H2O2) and 0.044 (200 μM H2O2). G. Prdx1−/− and +/+MEFs were plated as above and treated with increasing amounts of PDGF for 1-10 mins in phenol-free DMEM supplemented with 0.1% BSA. Following treatment, plates were washed with cold 1X PBS two times before lysing. Protein lysates were analyzed as above. H. Quantification of three independent experiments as described under F for protein lysates used in G. Four different sets of Prdx1 WT and KO clones were tested. “*” indicates statistical significance (students T-test) of KO and matching WT clone treated with PDGF. P-values for phosphorylation of p38MAPKα were 0.003 (0.5min), 0.004 (1min), 0.04 (5min), <0.0001 (7min) and 0.021 (10min). P-values for phosphorylation of ATF-2 were 0.015 (5min), 0.018 (7min) and 0.009 (10min).
Figure 2
Figure 2. Prdx1 prevents ROS-induced senescence in breast epithelial cells
A. Mammary glands from 12-14 month old MMTV-H-Ras-Prdx1+/+ and MMTV-H-Ras-Prdx1−/− were stained for SA-βgal activity. B. Prdx1 expression was decreased in various human breast epithelial cells using lentiviral shRNA. MCF-10A, MCF-7 and MDA-MB-231 cells expressing vector control (empty vector = EV) or shPrdx1 were plated at 35,000 cells/well in 6-well plates overnight. Cells were treated with H2O2 in DMEM containing 10% FBS for 4 days. Following treatment, cells were washed with sterile 1X PBS and incubated in fresh medium for 24 h, and sub-cultured at low confluency for 10 days. Plates were stained for SA-βgal activity as previously described (53). Up to 6000 cells per treatment and genotype were quantified. C. MCF-7 cells were plated overnight at 35,000 cells/well in 6-well plates, and treated as in B with the addition of the p38MAPKα inhibitor SB203580 every two days before staining for SA-βgal activity. D-F. MCF-10A, MCF-7 and MDA-MB-231 cells expressing vector control (empty vector = EV) or shPrdx1 were plated in 6 cm dishes overnight at 8.0 X 104. The following day, medium was removed and the plates washed in 1X sterile PBS two times, and incubated in serum free medium for 120 minutes. Following equilibration in serum-free medium, cells were treated with 100 μM H2O2 for up to 2 h for MCF-7 cells, and 30 min for MCF-10A and MDAMB-231 cells. Cells were lysed with 150 μl of lysis buffer, and protein lysates were analyzed by Western blotting for p38MAPKα phosphorylation, p38MAPKα, Prdx1 knock down, and actin protein levels. Quantification of three to four independent experiments for each human breast epithelial cell line analyzing phosphorylation of p38MAPKα is placed below each cell line. Density of phosphorylated p38MAPKα Western blot bands were analyzed using Image J software (http://rsbweb.nih.gov/ij/) and normalized to densities of p38MAPKα protein bands. Lastly, values were normalized to density of untreated EV cells as 1. “*” indicates statistical significance (Students T-test) of shPrdx1 and matching EV clone treated with H2O2. P-values of p38MAPKα phosphorylation for MCF-10A: 0.049 (5min), 0.031 (10min) and 0.019 (20min); MBA-MD-231: 0.018 (5min) and 0.013 (10min); MCF-7: 0.011 (90min) and 0.028 (120min).
Figure 3
Figure 3. Non-covalent binding of Prdx1 to MKP-1 and MKP-5
A. Pull down of recombinant Catalase, MKP-1 and 5 by Prdx1 conjugated protein G agarose. The far left lane represents protein agarose G only to serve as a negative control for non-specific binding. B. Purified Prdx1 protein (2.0 nM) labeled with Alexa Fluor® 546 was titrated with indicated amount of the purified phosphatase proteins labeled with QSY® 35. Alexa546 fluorescence decrease (normalized to initial Alexa546 emission) was recorded and processed using a “One Site Saturation model” (Pharmacology application, SigmaPlot 10.0, SyStat, MA) with best hyperbolic fit (R2≥ 0.99) according to equation: Y= Bmax*X / (KD + X) where: X – is a concentration of added PTEN (MKP-1 or MKP-5) protein; Y – is a normalized decrease of Alexa®546 fluorescence corresponding to specific binding of phosphatases; Bmax – is a saturated number of binding sites with apparent equilibrium dissociation constant KD. Data represent mean±SD for 3 independent experiments. The lines represent hyperbolic fit of experimental data for the titration of 2.0 nM of Alexa546-labeled Prx1 with QSY35-labeled: PTEN – solid line (R2=0.99); MKP-1 - dashed line (R2=0.99); and MKP-5 - doted and dashed line (R2=0.99). C and D. 293T HEK cells were transfected with 2.0μg of Flag-MKP-1 and Flag-MKP-5, and treated with increasing amounts of H2O2 for 30 min in serum free medium. To assay for binding of endogenous Prdx1, 1000 μg of protein lysate was immunoprecipitated using anti-flag affinity matrix and incubated 3 hrs at 40C in an hypoxic chamber. The affinity matrix was harvested by centrifugation at 3000xg for 2 min, and washed with 0.5 ml of lysis buffer 3 times. The resin was re-suspended in 20 μl of reducing or non-reducing SDS-PAGE sample buffer, boiled 10 mins, and analyzed by Western blotting for MKP expression, Prdx1 and Prdx1Cys52SO3 binding. * = IP from untransfected cells. Co-IPs were also analyzed in the absence of β-mercaptoethanol. No Prdx1 dimer staining positive for SO2/3 were detected. #: detection of a higher molecular weight 2-Cys Prdx family member. Co-IP unbound fraction can be found in the supplemental material Fig. S3B. E and F. 293T HEK cells were cotransfected with Flag-MKP-1 or Flag-MKP-5 either with HA-Prdx1WT or HA-Prdx1CI, and treated with increasing amounts of H202 for 30 min in serum free medium. For co-IP, 1000μg of lysate was added to anti-flag affinity matrix and incubated for 1h at 40C. Following incubation, the affinity matrix was harvested as stated above, re-suspended in 20μl of reducing sample buffer, and boiled for 10 min. Binding of HA-Prdx1WT and HA-Prdx1CI was determined by Western blotting. Analysis of cell lysate for protein expression can be found in the supplemental material Fig. S3C.
Figure 4
Figure 4. Prdx1 protects MKP-5 from oxidation-induced oligomerization and inactivation
A and B. Flag-MKP-1 or Myc-MKP-5 were overexpressed together with untagged Prdx1 in 293T cells, using 1.0 μg of DNA. (Note: initial experiments included N-terminal tagged Prdx1 proteins. This enhanced slightly the MKP-1 oligomerization; we therefore used untagged Prdx1). Cells were treated for 30 min with increasing concentrations of H2O2 in serum-free medium, and protein lysates analyzed under non-reducing conditions by Western blotting for oligomerization of MKP-1 and MKP-5 (n= 3). C and D. As in A and B, with the exception of co-expressing untagged Prdx1-CI. (n=3) E-H, Western blot analysis of A-D for phosphorylation of p38MAPKα. Quantification include three independent experiments. Density of phosphorylated p38MAPKα Western blot bands were analyzed using Image J software (http://rsbweb.nih.gov/ij/) and normalized to densities of p38MAPKα protein bands. Lastly, values were normalized to density of untreated cells expressing exogenous Prdx1. P-values of p38MAPKα-phosphorylation for MKP-1 + WT-Prdx1: 0.011 (25μM H2O2), 0.002 (100μM H2O2), 0.004 (250μM H2O2). MKP-1 + CI-Prdx1: 0.01 (100μM H2O2). MKP-5 + WT-Prdx1: 0.04 (100μM H2O2), 0.013 (250μM H2O2). MKP-5 + CI-Prdx1: 0.014 (250μM H2O2). Western blots showing MKP-1 or MKP-5 co-expressed with Prdx1 WT or Prdx1 CI run under non-reducing conditions can be found in the supplemental information along with Western blot analysis under reducing conditions (Figs. 4SA and 4SB). I and J. 293T HEK cells were co-transfected with Flag-MKP-1 or Flag-MKP5 and with Prdx1 WT or Prdx1 CI, respectively, and treated with increasing amounts of H2O2 for 30 min in serum free medium. Lysates were run under reducing conditions, and analyzed by Western blotting for phosphorylation of p38MAPKα on Thr180 and Tyr182, expression of p38MAPKα, MKPs and Prdx1.
Figure 5
Figure 5. Prdx1 promotes MKP-5 catalytic activity and protects it from H2O2-induced stress
Time courses of 160 μM DiFMUP hydrolysis by recombinant MKP-1 and MKP-5 phosphatase activity was measured in the presence of Prdx1 and H2O2. An equimolar ratio of MKP:Prdx1 recombinant protein was added to 1X reaction buffer in 1.5ml eppendorf tubes at room temperature. Concentrations of MKPs were kept the same in each reaction. H2O2 was diluted in sterile H2O, and added to the recombinant proteins and allowed to incubate at room temperature for 10 min. The proteins were pipetted into a black, clear bottom 96 well plate, followed by the addition of 160 μM of DiFMUP substrate. The plate was covered in foil, and read at ~360/460nm on a fluorescence plate reader every 10 min for 2 h. X-axis represent time in minutes, y-axis represent flourescent signal generated DiFMUP hydrolysis. A. Comparison of MKP-1 and MKP-5 catalytic activity. B. Left panel: MKP-1 activity over time in the presence of increasing concentrations of H2O2. Right panel: same as in left panel in the presence of Prdx1. C. Left panel: MKP-5 activity over time in the presence of increasing concentrations of H2O2. Right panel: same as left panel, in the presence of Prdx1. Statistical analysis: Utilization of substrate was measured for MKP-1 and MKP-5 at three levels of H2O2 concentration (0, 0.05, 0.15 mM) in the absence or presence of Prdx1. Prdx1 treatment effects over time on MKP activity were modeled in a random effects model, allowing for fixed effects of Prdx1, time in minutes. Random intercepts and experimental effects were included. The six tests of differences in treatment effects (MKP activity without Prdx1 compared to MKP activity in the presence of Prdx1) over time were all statistically significant at the α = 0.05 level. *: p<0001, **: p=0.0121, ***: p<0.0001; #: p<0.0001, ##: p<0.0001, ###: p<0.0001. Moreover, differences in H2O2 concentration effects over time were also tested within Prdx1 treated samples: for MKP1, H2O2 differences over time: p < 0.0001; for MKP5, H2O2 differences over time: p = 0.0002.
Figure 6
Figure 6. Prdx1 prevents ROS-induced senescence in MCF-7 cells by promoting MKP-5 activity
A and B. MCF-7 cells were infected with retrovirus for Flag tagged MKP-1 WT, MKP-1 C258S, MKP-5 WT or MKP-5 C408S and 7 days later with lentivirus for shPrdx1 or EV. After 5 days of selection, cells were plated overnight at 35,000 cells/well in 6 well plates and treated with H2O2 every day for 4 days in DMEM containing 10% FBS. Following treatment, cells were allowed to recover for 10 days in fresh medium, and stained for SA-β-gal activity as previously stated. Blue cells were quantified as described in Fig. 2. Plot is a representative of two independent experiments. MCF-7 cells expressing MKP-1 and MKP-5 WT and CI mutants were plated in 6 cm dishes overnight at 8.0 X 104, and then treated with 5μM of MG132 proteasome inhibitor overnight. The following day, medium was removed and the plates washed in 1X sterile PBS two times, and incubated in serum free medium for 120 minutes. Following equilibration in serum-free medium, cells were treated with 25 μM H2O2 treatment for 2 h. Cells were lysed with 150 μl of lysis buffer, and protein lysates were analyzed by Western blotting for p38MAPKα phosphorylation on Thr180/Tyr182, JNK phosphorylation on Thr183/Tyr185, p38MAPKδ levels, and actin protein levels. Arrow indicates slower migrating band, which could be phosphorylated p38MAPKδ. C. Cells from A were plated in soft agar, and colonies were counted.
Figure 7
Figure 7. The peroxidatic cysteine Cys52 of Prdx1 is a sensor in ROS-signaling
Under normal ROS homeostasis, Prdx1 promotes both MKP-1 and MKP-5 activity. However, under increased ROS, Prdx1-Cys52-SO3 forms less Prdx1/MKP-1 complexes leading to MKP-1 inactivation. This is comparable to data we obtained for PTEN(7). Conversely, in the presence of MKP-5, Prdx1-Cys52-SO3 binds to MKP-5 and preserves MKP-5 activity. We therefore speculate that MKP-1, which in some instances prefers JNK over p38MAPKα as a substrate (52) is inactivated under high oxidative stress (because of the dissociation from Prdx1) to allow JNK activity. MKP-5 on the other hand favors, depending on the cellular context, p38MAPKα over JNK as a substrate (53). This way, MKP-5 activation by Prdx1 is thereby preventing p38MAPKα signaling in H2O2-induced senescence. The net outcome of all this may be that Prdx1 in a H2O2-dose dependent manner prevents oxidative p38MAPKαmediated stress-induced senescence to promote JNK mediated signaling.

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