Requirement for Generation of H2O2 for Platelet-Derived Growth Factor Signal Transduction

Stimulation of rat vascular smooth muscle cells (VSMCs) by platelet-derived growth factor (PDGF) transiently increased the intracellular concentration of hydrogen peroxide (H2O2). This increase could be blunted by increasing the intracellular concentration of the scavenging enzyme catalase or by the chemical antioxidant N-acetylcysteine. The response of VSMCs to PDGF, which includes tyrosine phosphorylation, mitogen-activated protein kinase stimulation, DNA synthesis, and chemotaxis, was inhibited when the growth factor-stimulated rise in H2O2 concentration was blocked. These results suggest that H2O2 may act as a signal-transducing molecule, and they suggest a potential mechanism for the cardioprotective effects of antioxidants.

. lecular Biology Laboratory (EMBL) data library (ac-tIns the t h n f i a e n t e r o (ci)an dicald the band.,dnesrcue 32. For reverse transcriptase-polymerase chain reaction cession numbers AC X90568 and X90569, respec-In1 the cntieroA andte Ire bands, dee nsedt astructuresan (RT-PCR), human RNAs were prepared essentially tively  115 and 120 predicts that soleus titin is encoded by a kinase stimulation, DNA synthesis, and chemotaxis, was inhibited when the growth 101-kb mRNA, encompasses 33,000 residues, and factor-stimulated rise in H202 concentration was blocked. These results suggest that has a molecular mass of 3700 kD. H 02 may act as a signal-transducing molecule, and they suggest a potential mechanism 18 Structures 3, 391 (1995). inactivates tobacco catalase, leading to a rise ecules, it would apprear that a role for H)2, H2O2 into the extracellular space (5,6). We achieve the intracellular [H2O2j seen after increase in intracellular catalase activity.
investigated the possibility that a parallel PDGF stimulation required extracellular To show that the addition of exogenous between nitric oxide (NO) and H2O2 might [H2O2] in the 0.1I to 1.0 mM range (10). catalase did not result in its nonspecific exist. High concentrations of these diffusible This range of extracellular [H2021 also ap-binding to the outer surfaces of VSMCs, we reactive oxygen intermediates play a role in pears to induce the tyrosine phosphorylation loaded cells with catalase and then exposed host defense; however, at low concentra-of the p42 isoform of mitogen-activated pro-them to proteinase K. Although proteinase tions, NO modulates signal transduction of tein (MAP) kinase (Fig. IC). K (1 mg/ml) rapidly inactivated purified endothelial and neuronal cells. To evaluate We decreased [H2021 in VSMCs by in-catalase in solution, the enzymatic activity whether H2O2 could have a similar function creasing the amount of the peroxide-scavengof catalase-loaded cells was resistant to the in signal transduction, we studied VSMCs ing enzyme catalase. This enzyme rapidly deprotease (Fig. 2D). We' also stimulated for which low concentrations of exogenous grades H2O2 to water and molecular oxygen. VSMCs loaded with catalase with PDGF H2O2 are mitogenic (7). Infection of VSMCs with an adenovirus that and assayed for H2O2 levels. Catalase-load-PDGF stimulation of primary rat VSMCs encodes catalase (AdGCat) resulted in measur-ed VSMCs had a reduced peak level of DCF revealed an increase in intracellular [H2O2J able increases in intracellular catalase activity fluorescence after the addition of PDGF. as measured by the oxidation of the perox-( Fig. 2A) (11). The level of enzymatic activity The relative peak DCF fluorescence seen ide-sensitive fluorophore 2',7'-dichlorofluowas a function of the multiplicity of infection after growth factor addition was a function rescin (DCF). Microfluorometric study with (MOI), and no increase was seen when a of catalase concentrations (Fig. 2E). confocal microscopy showed that, compared control adenovirus encoding the Escherichia We assessed the effect of increased inwith quiescent cells, stimulation with the coli lacZ gene (Adj3gal) was used. Addition of tracellular catalase activity on PDGF signal PDGF-AB isoform (5 ng/ml) rapidly in-purified catalase to the culture medium result-transduction. PDGF-AB (5 ng/mI) induced creased DCF fluorescence by 50to 100-fold. ed in even greater increases in intracellular a rapid increase in tyrosine phosphorylation The PDGF-stimulated increase in [H2O2] iS VSMC catalase activity. Because catalase is a of numerous proteins (Fig. 3A). As catalase transient (Fig. IA), with [H202j peaking 60-kD protein which in solution forms hoactivity increased, the PDGF-induced stimwithin the first few minutes after growth motetramers, it seems unlikely that the pro-ulation of tyrosine phosphorylation was corfactor addition and then returning rapidly tein simply diffuses into VSMCs. Indeed, al-respondingly reduced in a dose-dependent toward basal levels. This time course is sim-though loading of VSMCs with catalase by fashion. At the highest amount of intracelilar to that described for PDGF-induced ty-addition of the purified enzyme to the medilular catalase activity, an amount that alrosine phosphorylation (8). Exogenously um resulted in a time-dependent increase in most completely blocks the growth factoradded H2O2 or other oxidant stresses can VSMC intracellular catalase activity, this was induced rise in [H2O2J, stimulation of tyinduce tyrosine phosphorylation in several not observed in several other cell lines tested rosine phosphorylation by PDGF was alcell types (9). We observed similar effects in such as human umbilical vein endothelial most completely inhibited (Fig. 3A). VSMCs. Increasing extracellular [H2O2] cells (HUVECs) or HeLa cells (Fig. 2B). Up-The addition of catalase even in large from 0.01 to 10 mM resulted in significant take of catalase into VSMCs did not occur at amounts (3000 U/ml) for prolonged periods increases in the total amount of tyrosine-40C, suggesting that it is an energy-dependent of time (>5 days) produced no visible effects phosphorylated proteins (Fig. 1 B). To and perhaps receptor-mediated process (10). on VSMCs and did not affect viability as The increase in intracellular catalase activity assessed by trypan blue exclusion, although was dependent on the concentration of extra-catalase-loaded cells grew at approximately A B H2O (MM) cellular catalase (Fig. 2C). At the highest half the rate of control cells (10). In addi- donor sodium nitroprusside (SNP) was add-Ad.Cat affected cell morphology and in gensates was decreased in cells containing high ed to the medium there was a rise in the eral reduced the responsiveness of VSMCs to catalase activity (Fig. 4F, insert). concentration of cyclic guanosine 3',5'growth factors (10). Nonetheless, MAP ki-We tested whether other reactive oxygen monophosphate (cGMP) equal to or greater nase phosphorylation after PDGF addition intermediate scavengers would have effects than that of control cells (Fig. 3B). This was inhibited to a greater extent in Ad.Catsimilar to those of catalase. When VSMCs indicates that the H202 and NO pathways infected compared with similarly infected were treated with the chemical antioxidant are separable and that increased intracellular Ad.jgal cells (Fig. 4C). In accordance with N-acetylcysteine (NAC), we noted a concatalase does not have a toxic or global effect the amount of MAP kinase tyrosine phoscentration-dependent reduction in PDGFon all signal transduction pathways.
phorylation, MAP kinase enzymatic activity stimulated DCF fluorescence (Fig. 5A). We next identified specific proteins whose [as assessed by phosphorylation of a myelin NAC caused a concentration-dependent regrowth factor-stimulated tyrosine phosphobasic protein (MBP) substrate] varied in reladuction in PDGF-stimulated tyrosine phosrylation was regulated by endogenous H202 tion to catalase activity (Fig. 4D). Consisphorylation (Fig. 5B). Similarly, millimolar release. Stimulation of VSMCs with PDGF tent with the reduction in MAP kinase amounts of NAC resulted in a reduction of induced an increase in the amount of typhosphorylation and activity, PDGF-in-PDGF-stimulated MAP kinase phosphorylarosine-phosphorylated p44 and p42 isoforms duced [3H]thymidine incorporation was retion and thymidine uptake (10). of MAP kinase. As intracellular catalase acduced in the presence of increased amounts The mechanism by which H202 partictivity increased, both basal and PDGF-stimuof catalase (Fig. 4E).
ipates in PDGF signal transduction is unlated tyrosine phosphorylation of MAP kinase Increased amounts of intracellular catalase clear. Exogenously added H202 can reverswere inhibited (Fig. 4A). These results were activity also inhibited the ability of PDGF to ibly inactivate protein tyrosine phosphanot due to changes in the steady-state amount stimulate chemotaxis of VSMCs (Fig. 4F). A tases, and enzymatic activity can be subseof MAP kinase protein in catalase-treated -50% reduction in migration was observed at quently restored by thiol donors (13). As cells (Fig. 4B). Qualitatively similar results the highest level of catalase activity. The such, the growth factor-stimulated rise in were obtained after adenoviral gene transfer. molecular basis of PDGF-induced migration [H202] may serve to transiently inactivate Infection with Ad.Cat (200 MOI) produced is not fully understood, but it might involve intracellular tyrosine phosphatases, allowintracellular catalase activity that was equivthe cytoskeletal-associated protein paxillin, ing for a temporary alteration in the kinasealent to that observed with 72 hours of cata-whose tyrosine phosphorylation is stimulated phosphatase balance. The magnitude of the lase loading at an intermediate dose (300 by PDGF in several cell types including rise in intracellular [H)2] may therefore be U/ml) of extracellular catalase (Fig. 2, A and VSMCs (12). The amount of tyrosine-phosan important determinant of signal trans-C). Such infection with either Ad.Bgal or phorylated paxillin in PDGF-stimulated lyduction. This is supported by three observations. Exogenous H202 stimulates MAP alrsiultdwt9DG9safncino5A ma1 growth factor, fibroblast growth factor, stimulated fluorescence averaged from 60 to 200 and proteinase K diluted and removed. The cells were or angioten in I all produce a rise in units. Although DOE is oxidized by both H202 and subsequently lysed in buffer A. Residual catalase acor angiotensin~~~~~~~~~~~h ydroxyl radicals, the lack of fluorescence in PDGFtivity was measured as described (20).
[H202] with a correlation existing between stimulated catalase-loaded cells suggests that the 22. Confluent VSMCs with and without catalase loading the magnitude and duration of an increase fluorescent signal after the addition of growth factor were stimulated for 30 min with 10 mM SNP. Cells in [H202] and the level of tyrosine phos-1.is predominantly derived from H202 (see Fig. 2E).
were subsequently lysed with 6% trichloroacetic acid 1.VSMCs deprived of serum for 3 days were stimulated (TCA). Lysates were neutralized, and equal amounts phorylation (10). These latter observations with the indicated concentration of H202 or PDGF-AB (2 x 1 05 cells) were used to determine amounts of also strengthen the case that in many ways (5 Offermanns, J. Immunol. 152,250(1994)]. Myelin secrte atalse,and the amount of catalase cipitated proteins were divided in equal portions, and basic protein (MBP) phosphorylation was used as an secrete catalase,~~~~~~~~~t yrosine-phosphory~latedl proteins from 50 mg of ly-index of MAP kinase activity, where 100% activity in serum increases in certain disease states sate were subjected to SDS-polyacrylamide gel elecrepresents the activity derived from PDGF-stimulat- (14). Thus, growth of VSMCs might be in-trophoresis (SDS-PAGE) (4 to 20% gels) and trans-ed cells in the absence of catalase loading.
fluenced by extracellular catalase in vivo.
ferred to a nitrocellulose filter. When indicated, filters 24. Confluent VSMCs were maintained in media without were probed with either an antibody to phosphotyserum for3 days and then, as indicated, stimulated with Recent epidemiological studies suggest a car-rosine (RC20), a broadly reactive antibody to MAP PDGF-AB (5 ng/ml). After 18 hours, cells in quadruplidioprotective effect of antioxidants (15