In order to furtherinvestigate the mechanism underlying the significant reduction of PP1/2A activities and elevated p-Erk1/2 level during cellularsenescence, ROS generation was measured during cellular senescenceby FACS analysis after incubating both the young and mid-oldHDF cells with H2-DCFDA. As shown in Fig. 5A, the mid-old cellscontained about 20 times more ROS than the young cells, andthe ROS generation by both could efficiently be inhibited bypretreatment of the cells with 10 mM NAC. When young HDF cells were treatedonce with 1 mM H2O2, Erk1/2 phosphorylation was markedly increasedat 10 min, however, the increase was greatly attenuated in 40min. At the same time, when the cells were pretreated with 10 mM NAC for 1hr, phosphorylation of Erk1/2 protein was efficientlyinhibited (Fig. 5B). To discern whether H2O2 was responsiblefor the reduction of PP1/2A activities or not, the enzyme activitieswere measured after treatment of the young cells once with 200µM H2O2. As seen in Fig. 5C, the enzyme activities weretransiently inhibited in 10 min, but started to recover 40 minlater and finally returned to the control level in 8 h. To furtherinvestigate the regulation of p-Erk1/2 level and activity ofPP1/2A by H2O2 treatment, we measured the changes of the abovetwo parameters by immunoblot analysis and the activity assay,respectively. As shown in Fig. 5D, when the young cells were repeatedly treated with 200 µM H2O2 at every 8 h, PP1/2Aactivities remained significantly inhibited after 72 h; control HDF young cells had specific activity of 28.31 ± 0.97 pmol/min/µg protein, and it changed to 24.63 ±2.35, 30.50 ± 3.81, 29.06 ± 2.08, 25.38 ±
1.54, and 23.63 ± 2.68 pmol/min/µg protein at 5min, 24, 48, 72, and 80 h, respectively.
Concomitant with thechanges of PP1/2A activities, phosphorylation of Erk1/2 proteinswas simultaneously affected by H2O2 treatment. These data indicatethat repeated damage of the young cells by H2O2 exposure persistently inhibited PP1/2A activities, resulting in the accompanying inductionof p-Erk1/2 levels.
To elucidate whether decrease of PP1/2A activities in the mid-oldcells was due to the oxidation of cysteine residues in the enzyme molecules or not, thiol-specific reducing agents ,such as DTT,b-mercaptoethanol, and NAC, were added to the lysates of the young HDF cells, which had been pretreated with H2O2 for 10 min. Asshown in the left panel of Fig. 6, the control PP1/2A activitywas decreased from 27.96 ± 0.76 to 24.04 ± 0.63 pmol/min/µg protein (p < 0.005) by the treatment withH2O2. However, the inhibition was reversed to 33.38 ±0.66, 29.79 ± 1.26, and 27.63 ± 1.32 pmol/min/µgprotein by the addition of DTT, b-mercaptoethanol, and NAC, respectively. All three reagents significantly reactivated PP1/2A activityas compared with that of the H2O2 alone treatment (+, p <0.002, and #, p < 0.02). However, only DTT, but not b-mercaptoethanoland NAC, significantly increased PP1/2A activity of the youngcells more than the control (p < 0.005). Interestingly, whenthe assay was carried out with mid-old cell lysates, whose PP1/2Aactivity had already been reduced, most likely due to accumulatedH2O2 during senescence, b-mercaptoethanol could significantlyincrease PP1/2A activity, as compared with the control (22.04± 0.80 versus 18.71 ± 0.76 pmol/min/µg protein,p < 0.005, right panel in Fig. 6), thus strongly suggestingthat the high level of ROS found in senescent cells oxidizedthiol residues, which are important for phosphatase activity.
Fig. 5. Reactive oxygen species generated during cellular senescence-regulated PP1/2A activities and p-Erk1/2 levels. (A) Marked difference of ROS level between young and mid-old HDF cells, measured by FACS analysis. The young and mid-old cells were seeded into culture dishes up to 25 and 70%, respectively. After 24 h, the monolayer was re-fed with
complete medium and then treated with H2DCF-DA 10 min prior to cell harvest. The cells were pretreated with NAC 1 h prior to harvest and treated with H2DCF-DA for 10 min before the harvest. Changes of fluorescence by generated ROS were measured by FACS analysis. Note 19-fold (4.25 versus 79.82) higher ROS in the mid-old cells than the young cells. (B) Treatment of HDF young cells with 1 mM H2O2 significantly increased Erk1/2 phosphorylation, but it was efficiently blocked by NAC pretreatment. Control HDF cells (lane 1) were treated with 1 mM H2O2 for 10 (lane 2) and for 40 min (lane 4), pretreated for 1 h with 10 mM NAC, and then treated with H2O2 for 10 (lane 3) and for 40 min (lane 5).
The cells were harvested, and p-Erk1/2 expression level was measured by immunoblot analysis. (C) To investigate whether ROS regulated PP1/2A activity or not, HDF young cells were treated once with 200 µM H2O2. The enzyme activity was transiently inhibited in 10 min; however, it was recovered in 40 min and then returned to control level in 8 h. D, simultaneous regulation of PP1/2A activities and Erk1/2 phosphorylation of young HDF cells by repetitive treatment with H2O2. Young HDF cells were treated with 200 µM H2O2 every 8 h and then harvested after 0 min (lane 1), 5 min (lane 2), 24 h (lane 3), 48 h (lane 4), 72 h (lane 5), and 80 h. The cell lysates were used for p-Erk1/2 immunoblot analysis (upper panel) and PP1/2A activity assay (lower panel). Note the reciprocal changes of p-Erk1/2 (increase) and the PP1/2A activities (decrease) at 5 min. The reciprocal change was consistent by repeated treatments for 72 h.
Fig. 6. Protein Phosphatase 1 and 2A Activities were Restored by Thiol-specific Reducing Agents in Mid-old HDF Cells. In the case of the young HDF cells, the cells were pretreated with 1 mM H2O2. However, the mid-old cells were not pretreated, because the PP1/2A activity was already reduced, most likely due to accumulated H2O2 during senescence. The cell lysates were prepared within 10 min in the phosphate-free condition.
By using the phosphopeptide as a substrate, PP1/2A activity was measured with the control (C) and H2O2-treated cell lysates by the described method. The assay was also performed with either DTT (1 mM), b-mercaptoethanol (2ME, 0.1%), or NAC (10 mM) added to the cell lysates of the H2O2-treated cell lysates. Recovery of the senescent-induced PP1/2A activity was also evaluated with the mid-old cell lysates by addition of 0.1% b-mercaptoethanol to the control cell lysate. All the samples were triplicated, and the assays were performed more than three times.