Effects of H2O2 on mitogenic signaling
To examine the oxidation state of Prxs during mitogenic signaling, mouse C10 lung epithelial cells were collected in G0 by serum deprivation, and the formation of Prx-SO2
H in response to serum stimulation was assessed using an antibody specific for Prx-SO2
H was not detected above background levels in cells stimulated for 15 min with medium containing serum concentrations from 2 to 20%, a range that induces dose-dependent induction of tyrosine phosphorylation (, lanes 2–5), activation of the ERK1/2 and PI-3 kinase–Akt mitogenic signaling pathways, and expression of cyclin D1 (Ranjan et al., 2006
). These results indicate that normal mitogenic signaling does not require inactivation of Prxs by hyperoxidation, in agreement with a recent report on the role of PrxII in PDGF signaling (Choi et al., 2005
Figure 2. Hyperoxidation of Prxs in serum-stimulated cells correlates with inhibition of cell proliferation. (A) C10 cells synchronized by serum deprivation were stimulated with medium containing the indicated concentration of FBS for 15 min, and levels of phosphotyrosine (more ...)
To further explore Prx oxidation in cell cycle control, we adopted an experimental paradigm that utilizes a dose-dependent H2
generating system to evoke transient cell cycle arrest (Burch et al., 2004
). C10 cells were synchronized in G0 by serum deprivation and induced to reenter the cell cycle by adding medium containing 10% FBS with or without glucose oxidase (GOx). In complete medium with glucose and 10% FBS, GOx caused the dose-dependent production of H2
in a linear fashion for at least 8 h (). For example, in complete medium, 5.0 mU/ml GOx generated ~10 μM H2
During the first 6 h of serum stimulation, 1.0 or 2.5 mU/ml GOx had little effect on the expression of cyclin D1, whereas doses of 5.0 mU/ml or greater blocked expression of cyclin D1 (, lanes 7–9). In response to continuous exposure to 1.0 mU/ml GOx, the levels of activated ERK1/2 were similar to the serum control, cyclin D1 was expressed, and hyperoxidized 2-Cys Prxs were not observed (, lane 5), suggesting that C10 cells are able to metabolize considerable amounts of exogenous H2O2 during the G0–G1 transition without accumulating hyperoxidized 2-Cys Prxs. At 2.5 mU/ml, levels of phospho-ERK1/2 were unaffected, Prx-SO2H was barely detectable after 6 h of exposure, and cyclin D1 was expressed at nearly normal levels. In contrast, at 5.0 mU/ml, hyperoxidized Prx-SO2H accumulated to substantial levels and cyclin D1 was not expressed (, lane 7). Concentrations of GOx ≥10.0 mU/ml induced accumulation of hyperoxidized Prx-SO2H, caused hyperactivation of ERK1/2, and blocked expression of cyclin D1 (, lanes 8 and 9).
We previously showed that termination of ERK1/2 signaling after 3 h of exposure to the highest dose of GOx (15 mU/ml) restores expression of cyclin D1 but not cell proliferation (Burch et al., 2004
). Hence, prolonged activation of ERK1/2 is a useful marker of oxidant-induced arrest at the G0–G1 transition of the cell cycle. Although GOx influenced the levels of phospho-ERK1/2 in a dose-dependent manner as before, it did not induce phosphorylation of JNK in synchronized cells at any dose (, lanes 5–9). In asynchronous cells, activation of JNK in C10 cells by H2
is associated with cell death (Pantano et al., 2003
To determine if retroreduction of Prx-SO2
H prevented the accumulation of Prx-SO2
H, serum-stimulated cells were treated with 1-chloro-2,4-dinitrobenzene (DNCB), with or with out GOx. DNCB depletes cells of reduced glutathione (GSH) and blocks reduction of Trx by inhibiting TrxR (Arner et al., 1995
), thereby impairing the ability of Trx and GSH to participate in the retroreduction of Prx-SO2
H to catalytically active forms. Within 10 min, 5 μM DNCB caused a 90% reduction in GSH levels that persisted for at least 3 h (unpublished data).
In the absence of GOx, DNCB blocked the ability of serum to induce expression of cyclin D1 but did not prevent phosphorylation of ERK1/2 (, lane 4) or cause the accumulation of hyperoxidized Prxs. In contrast, DNCB markedly sensitized 2-Cys Prxs to hyperoxidation by GOx (, compare lanes 6–9 with lanes 10–13), suggesting that Prx retroreduction pathways are active during cell cycle reentry. Although phospho-ERK1/2 levels were increased in cells treated with GOx and enhanced in cells treated with DNCB and GOx, only with DNCB were high concentrations of GOx able to induce phosphorylation of JNK (, lanes 10–13).
Cell proliferation was then examined in serum-stimulated cells treated with DNCB and/or GOx (). GOx and/or DNCB were added to serum-stimulated cells, and proliferation was examined over a 3-d period without changing the culture media. C10 cells exposed to 1.0 or 2.5 mU/ml GOx proliferated as well as untreated controls, whereas those exposed to doses of GOx ≥5.0 mU/ml failed to proliferate by 3 d (). Greater than 70% of cells arrested in response to all but the highest dose of GOx (15.0 mU/ml) remained viable for at least 3 d ( and not depicted). Caspase 3 was not activated in serum-stimulated cells at any dose of GOx, although it was readily activated after exposure to GOx by staurosporin (unpublished data), indicating that proapoptotic pathways were functional in arrested C10 cells. Cells treated with DNCB alone recovered slowly (), whereas cells treated with DNCB and any dose of GOx did not proliferate (not depicted).
Although DNCB sensitized Prxs to hyperoxidation by GOx, it did not sensitize Prxs to hyperoxidation in response to serum at any time point. Together, these studies indicate that formation of Prx-SO2H may not be required for mitogenic signaling during the G0–G1 transition of the cell cycle. In contrast, dose-response experiments with GOx revealed a sharp transition from unimpeded cell proliferation to cell cycle arrest that occurred between concentrations of 2.5 and 5.0 mU/ml, and that arrest was reflected in failure to express cyclin D1.
Oxidation of PrxI and -II and cell cycle progression
Transitions between dimers, decamers, and high molecular mass oligomers of Prxs are governed by oxidation state (Wood et al., 2002
; Moon et al., 2005
), phosphorylation during G2/M (Chang et al., 2002
; Jang et al., 2006
), and other parameters (for review see Wood et al., 2003b
). To study the oxidation state of 2-Cys Prxs under various conditions, an immunoblotting method was devised to detect the relative amounts of reduced or oxidized Prx (Prx-SH, Prx-SOH, or Prx-S-S-Prx) versus hyperoxidized Prx (Prx-SO2
H). With this method, it was possible to estimate the fraction of catalytically active PrxI and -II despite the limitation that the Prx-SO2
H antibody recognizes hyperoxidized PrxI and -II with equivalent efficiency.
When extracts were resolved by standard SDS-PAGE, total PrxI and -II levels detected by immunoblotting and quantified by densitometry varied less than ±8% during the first 6 h after serum stimulation, with or without GOx (). When probed first for Prx-SO2H and then for either PrxI or -II after stripping the membrane, immunoblotting produced reciprocal signals that reflected the fraction of PrxI or -II that was not catalytically inactivated versus the fraction that was inactivated by hyperoxidation. Using densitometry, the levels of reduced/oxidized PrxI (), reduced/oxidized PrxII (), and Prx-SO2H () were estimated as a function of GOx concentration after 3 h of exposure and after 3 h of recovery in fresh medium (). At 2.5 mU GOx/ml, >85% of PrxI was hyperoxidized after a 3-h exposure (, lane 6). After recovery, <50% of PrxI was hyperoxidized, and the reduction in Prx-SO2H levels () was accompanied by recovery of the signal for reduced PrxI (, lane 15; and ), confirming the activity of retroreduction pathways in C10 cells. PrxII appeared to be less sensitive to hyperoxidation than PrxI; at 2.5 mU/ml GOx (, lane 6), only ~25% of PrxII had been inactivated by 3 h (). At 10 or 15 mU/ml, both PrxI and -II were quantitatively hyperoxidized (, compare lanes 8 and 9 with lanes 17 and 18), and little signal for reduced PrxI and -II was regained after a 3-h recovery period (). In cells treated with GOx, expression of cyclin D1 was inversely correlated with the levels of Prx-SO2H ().
Figure 3. Dose-dependent hyperoxidation of PrxI and -II by fluxes of hydrogen peroxide. Serum-starved C10 cells were stimulated with DME with 10% FBS containing the indicated concentration of GOx for 3 h. After exposure, cells were washed and allowed to recover (more ...)
Figure 4. Prx oxidation occurs before depletion of cellular GSH. Using the strip–reprobe immunoblotting method, densitometry was used to quantify the signals for the fraction of PrxI (A) or PrxII (B) that was not hyperoxidized as a function of GOx concentration (more ...)
To assess the relationship between Prx hyperoxidation and cellular redox status, GSH levels were measured as a function of GOx concentration after exposure and recovery (). A considerable drop in GSH levels was not observed at 3 h until concentrations of GOx exceeded 5.0 mU/ml, and at all concentrations of GOx, GSH levels increased after recovery in fresh medium (). These results agree well with a report that shows PrxII is hyperoxidized in response to levels of H2
that do not inhibit the TrxR–Trx system or deplete cells of GSH (Baty et al., 2005
). Hence, cells treated with 5.0 mU/ml GOx for 3 h that retained near normal levels of GSH underwent transient cell cycle arrest, whereas those treated with either 10 or 15 mU/ml GOx that accumulated hyperoxidized PrxI and -II that could not be reduced after 3 h of recovery (), perhaps because of low GSH levels (), were not able to proliferate.
Serum stimulation engages PrxI and -II in peroxide metabolism
When assessed under standard conditions, the total levels of PrxI and -II did not change during the first 6 h of serum stimulation (). When samples were denatured in the presence of SDS, but without reducing agents to preserve disulfide bonds, gel electrophoresis showed that both PrxI (, lane 1) and PrxII (lane 7) from serum-starved cells were partitioned between 23-kD Prx-SH/Prx-SOH monomers and 38-kD Prx-S-S-Prx homodimers. Upon addition of serum, the levels of PrxI (, lanes 2–6) and Prx II (lanes 8–12) monomers decreased, and PrxI and -II homodimers with intersubunit disulfide bonds increased (, lanes 2–6 and 8–12, respectively). After exposure to 15 mU/ml GOx, all dimers with intersubunit disulfide bonds were lost by 30 min, and only hyperoxidized PrxI and -II monomers were detected for the duration of the experiment (, lanes 13–17; and not depicted). Because homodimers with intersubunit disulfide bonds are produced only during peroxide catalysis (), these results indicate that PrxI and -II metabolize H2O2 produced in response to serum stimulation. Upon hyperoxidation, a condition in which intersubunit disulfide bonds cannot form, only Prx-SO2H monomers were observed, as expected.
Figure 5. Serum stimulation increases the levels of Prx-S-S-Prx dimers. At the indicated times, extracts were prepared from serum-stimulated C10 cells in the absence of reducing agents and resolved by gel electrophoresis in the presence of SDS. After transfer, (more ...)
Recruitment of hyperoxidized PrxII into high molecular mass oligomers
At 2.5 mU/ml GOx, 85% of PrxI was hyperoxidized, and yet cells expressed cyclin D1 and proliferated normally. In contrast, cells treated with 5.0 mU/ml GOx did not express cyclin D1 or proliferate. To better understand this difference, native gel electrophoresis was used to examine the effect of GOx on the oligomerization state of PrxI and -II. When cell extracts were resolved by electrophoresis in the absence of reducing agents and SDS, immunoblotting indicated that PrxI was organized exclusively in complexes >660 kD (unpublished data). In contrast, PrxII was detected in two sets of bands that we refer to as A–A′ and B–B′ (). Compared with the mobility of native molecular mass markers, A–A′ migrated with an apparent molecular mass of ~66 kD and B–B′ with a mass of ~140 kD. Although similar PrxII complexes have been observed in other cell types (Moon et al., 2005
), the precise constituents of these complexes are not known.
Figure 6. Hyperoxidation of PrxII induces structural transitions that correlate with cell cycle arrest. (A and B) Serum-starved C10 cells (time 0) were stimulated with medium containing 10% FBS and the indicated concentration of GOx with or without 5 μM (more ...)
In extracts of serum-starved cells, bands A and B were the predominant form of PrxII (, lane 1). Addition of DNCB or FBS alone for 3 h did not change the mobility of PrxII on native gels (, lanes 2 and 3), but together DNCB and FBS increased the signal of band B′ (lane 4). Because FBS and DNCB do not induce Prx hyperoxidation (), changes in band B may reflect structural transitions during formation of PrxII-S-S-PrxII dimers during peroxide metabolism (), in agreement with studies that show PrxII metabolizes H2
produced in response to growth factors (Choi et al., 2005
) and terminates H2
-activated signaling by phospholipase D1 (Xiao et al., 2005
In response to exposure to 1.0 or 2.5 mU/ml GOx, band B′ increased in abundance relative to band B, perhaps reflecting increased engagement of the PrxII 140-kD complex in peroxide metabolism (, lanes 5 and 6). At concentrations of GOx of 5.0 mU/ml or higher, bands B and B′ disappeared, band A decreased, and band A′ appeared (, lanes 7–9). As observed in , DNCB shifted the dose response for the A–A′ and B–B′ complexes to lower concentrations of GOx (, lanes 10–14).
When reprobed for Prx-SO2H, little hyperoxidized PrxII was observed for cells treated with 1.0 mU/ml GOx (, lane 5), whereas hyperoxidized Prx-SO2H was observed to comigrate with band B′ in extracts from cells treated with 2.5 mU/ml GOx (, lane 6). At concentrations of GOx ≥5.0 mU/ml, Prx-SO2H was incorporated into several discrete high molecular mass complexes (HMCs) with apparent molecular masses >500 kD and considerable levels of A′ accumulated (, lanes 7–9). Recruitment of Prx-SO2H into HMCs correlated with loss of signal from the PrxII B–B'complex (, lanes 7–9).
PrxII complexes accumulate during cell proliferation
In time course experiments, the A–A′ and B–B′ complexes responded to serum stimulation and cell proliferation and, during recovery from exposure, to 5.0 mU/ml GOx. The levels of the 140-kD B–B′ complex fluctuated during the first 12 h of serum stimulation (, lanes 1–6) and increased markedly in abundance as cells reached confluence 48–96 h later (lanes 8–10). As cells reached confluence, increases in the A–A′ also were observed (, lanes 8–10). Serum stimulation and cell proliferation for >3 d caused no change in the signal for total Prx-SO2H detected under reducing and denaturing conditions or Prx-SO2H in HMCs detected by native gel electrophoresis (, lanes 2–10). The PrxII complexes were largely unaffected by exposing cells to 2.5 mU/ml GOx for the first 3 h of serum stimulation (, lanes 1–9), even though substantial levels of Prx-SO2H were observed under these conditions (, lanes 1–4) and the cultures took slightly longer to reach confluence. Note that 2.5 mU/ml GOx did not increase HMCs containing Prx-SO2H.
At 5.0 mU/ml GOx, the B–B′ complex was not observed during the 3-h exposure, HMCs containing Prx-SO2H increased in abundance, and cyclin D1 was not expressed (, lanes 1 and 2). After GOx was removed at 3 h, total Prx-SO2H levels were reduced over time, and Prx-SO2H in HMCs returned to background levels (, lanes 3–9). As signal for Prx-SO2H diminished in HMCs, A′ was lost, the B–B′ complex reappeared, and cyclin D1 was expressed (, lanes 5–9). By 96 h, the HMCs and PrxII A–A′ and B–B′ complexes observed by native gel electrophoresis were identical in extracts from cells exposed to all three conditions, even though proliferation to confluence was delayed in cells treated with 5.0 mU/ml GOx (e.g., total cellular protein at 72 h was ~50% of the 10% FBS control).
Localization of hyperoxidized 2-Cys Prxs
Immunofluorescence confocal microscopy was used to localize Prx-SO2H within C10 cells treated with various doses of GOx. In all cells, the Prx-SO2H antibody reacted with the cell nucleus, but this signal did not correlate with the level of Prx hyperoxidation detected by immunoblotting. In cells treated with 1.0 mU/ml GOx for 3 h, immunostaining was occasionally observed in small patches at the cell periphery (), and this pattern was more obvious in cells treated with 2.5 mU/ml GOx (). At 5.0 mU/ml, GOx staining was observed in a filamentous pattern in the cell cytoplasm (). Prx-SO2H in cytoplasmic filaments was particularly evident in cells treated with 10.0 mU/ml GOx, and at 15 mU/ml GOx, staining was prominent around the cell periphery (). At higher doses of GOx, the peripheral Prx-SO2H staining pattern correlated with changes in morphology that included a considerable increase in cell diameter. A filamentous cytoplasmic staining pattern for Prx-SO2H was not observed in asynchronous cells at any dose of GOx ( and not depicted).
Figure 7. Organization of cytoplasmic Prx-SO2H in response to oxidative stress. C10 cells were plated on coverslips and synchronized in G0 by serum deprivation. After serum stimulation for 3 h, with or without exposure to the indicated concentration of GOx (mU/ml), (more ...)
Ectopic expression of HA-PrxI and -PrxII and oxidant-induced arrest
Up-regulation of PrxI is thought to counteract the effects of enhanced oxidant production in tumor cells and thereby promote cell survival and proliferation (Chang et al., 2005
; Park et al., 2006
). To test the effects of Prx expression on responses to GOx, we generated expression vectors for HA-tagged PrxI, PrxII, and PrxII-ΔC, a robust mutant of PrxII that is 100-fold less sensitive to inactivation by H2
(Koo et al., 2002
; Wood et al., 2003a
). HA-PrxI interacts with endogenous PrxI in coimmunoprecipitation experiments, and HA-PrxI and -PrxII are hyperoxidized in response to GOx and reduced during recovery (unpublished data), indicating that HA-tagged Prxs function in peroxide metabolism in a manner similar to their endogenous counterparts. C10 cells were first transfected with expression constructs, and 24 h later the cultures were trypsinized and cells were plated at identical cell densities and synchronized by serum deprivation for 72 h as before. The transfected and serum-starved cell cultures were then treated with 5.0 mU/ml GOx as before.
In synchronized cells, immunoblotting showed HA-PrxI (, lanes 10–12) and HA-PrxII (lanes 13–15) were expressed at levels about fourfold that of their endogenous counterparts. Because of addition of the HA epitope tag and deletion of the PrxII C-terminal domain, HA-PrxII-ΔC comigrated with endogenous PrxII. As compared with untransfected cells (, lane 3) or vector controls (lane 6), expression of catalase (lane 9) and the robust PrxII-ΔC mutant (lane 18) reduced but did not eliminate Prx-SO2H levels generated in response to GOx during a 3-h exposure, with 3 h of recovery period as before. HA-PrxI (, lane 12) and HA-PrxII (, lane 15) were hyperoxidized under these conditions and thereby increased the total cellular levels of Prx-SO2H as measured by densitometry (). After recovery, expression of HA-PrxI or -PrxII did not reduce the levels of phospho-ERK1/2 or promote expression of cyclin D1 (). Although cells expressing catalase (, lanes 7–9) or PrxII-ΔC (, lanes 16–18) showed lower levels of total Prx-SO2H and pERK1/2 after recovery, cells had not expressed cyclin D1 or resumed proliferation by this time. Expression of HA-PrxI or -PrxII did not affect expression of cyclin D1 in response to serum alone (, lanes 11 and 14). When cells treated with 5.0 mU/ml GOx were examined after 72 h of recovery, cells expressing HA-PrxI and -PrxII proliferated in a manner similar to vector controls, whereas cells expressing PrxII-ΔC resumed proliferation earlier during recovery (). Thus, as in serum-stimulated cells, the accumulation of Prx-SO2H in cells overexpressing PrxI or -II was correlated with delays in cell cycle progression during recovery.
Figure 8. Elevated expression of PrxI and -II does not prevent cell cycle arrest in response to oxidative stress. (A) C10 cells were transfected with the indicated expression vectors or vector control, synchronized by serum deprivation, and treated with 5.0 mU/ml (more ...)
To confirm that HA-PrxII-ΔC was cytoprotective, stable cell lines were generated and treated with 5.0 mU GOx/ml continuously for 16 h. Flow cytometry showed that after 16 h ~30% of control cells exhibited a sub-G1 DNA content, whereas in comparison, ~10% of the cell population expressing HA-PrxII was detected in the sub-G1 fraction. In contrast, <0.5% of cells expressing PrxII-ΔC were detected in the dead cell fraction (unpublished data).