Ligand-independent trans-activation of the platelet-derived growth factor receptor by reactive oxygen species requires protein kinase C-delta and c-Src.

Reactive oxygen species are involved in the mitogenic signal transduction cascades initiated by several growth factors and play a critical role in mediating cardiovascular diseases. Interestingly, H(2)O(2) induces tyrosine phosphorylation and trans-activation of the platelet-derived growth factor receptor and the epidermal growth factor receptor in many cell lines including vascular smooth muscle cells. To investigate the molecular mechanism by which reactive oxygen species contribute to vascular diseases, we have examined a signal transduction cascade involved in H(2)O(2)-induced platelet-derived growth factor receptor activation in vascular smooth muscle cells. We found that H(2)O(2) induced a ligand-independent phosphorylation of the platelet-derived growth factor-beta receptor at Tyr(1021), a phospholipase C-gamma binding site, involving the requirement of protein kinase C-delta and c-Src that is distinct from a ligand-dependent autophosphorylation. Also, H(2)O(2) induced the association of protein kinase C-delta with the platelet-derived growth factor-beta receptor and c-Src in vascular smooth muscle cells. These findings will provide new mechanistic insights by which enhanced reactive oxygen species production in vascular smooth muscle cells induces unique alleys of signal transduction distinct from those induced by endogenous ligands leading to an abnormal vascular remodeling process.

Several cardiovascular diseases are characterized by a state of excess oxidative stress associated with enhanced production of reactive oxygen species (ROS) 1 within the arterial wall. ROS including superoxide anion, hydrogen peroxide (H 2 O 2 ), and hydroxyl radical are generated in a variety of cells stimulated with cytokines, growth factors, and agonists of G-protein-coupled receptors and are important chemical mediators that regulate signal transduction. Because it has recently become clear that ROS may mediate specific cellular functions such as cell growth, hypertrophy, and apoptosis, understanding the intracellular signaling of vascular smooth muscle cells (VSMCs) induced by ROS should provide further insight into the pathogenesis of cardiovascular diseases (1)(2)(3)(4).
We and others have demonstrated that ROS enhance the activation of non-receptor tyrosine kinases such as JAK2 (5,6), Src (7), and PYK2 (8), and receptor tyrosine kinases such as epidermal growth factor (EGF) receptor (9,10) and plateletderived growth factor (PDGF) receptor (11). ROS also activate certain proliferation-associated signaling pathways such as extracellular signal-regulated kinase (ERK) (12) and Akt (13) via a growth factor receptor-dependent manner in VSMCs (10,14). Moreover, PDGF has long been implicated in atherosclerosis, and the PDGF receptor stimulation leads to potent proliferation and migration of VSMCs (15)(16)(17). Therefore, the activation of PDGF receptor by ROS could specifically be involved in vascular remodeling associated with cardiovascular diseases.
It has been reported that ROS such as H 2 O 2 induces tyrosine phosphorylation of cellular proteins, which is strongly potentiated by a combination treatment with vanadate. The mechanism was believed to be attributed in part to the inhibition of tyrosine phosphatase or activation of tyrosine kinase or both (18). However, the precise mechanism by which ROS induce PDGF receptor activation remains unknown. PDGF initially activates the intrinsic tyrosine kinase of the receptors, leading to their dimerization and autophosphorylation. Many Src homology 2-containing proteins such as Ras-GTPase-activating protein, phospholipase C-␥ (PLC␥), phosphatidylinositol 3-kinase, and Grb-2 bind to specific tyrosine phosphorylation sites in the activated PDGF receptor (19). Among these proteins, PLC␥ is an enzyme that produces inositol triphosphate and diacylglycerol. These hydrolysis byproducts of PLC␥ mobilize Ca 2ϩ from intracellular stores and activate protein kinase C (PKC), respectively (20). PLC␥ binds to phosphorylated Tyr 1021 in the C-terminal tail of the PDGF␤ receptor, and the activation of PLC␥ is involved in both cell growth and chemotaxis in certain circumstances (19).
In this study, we explored the mechanisms involved in the activation of PDGF␤ receptor by H 2 O 2 stimulation in VSMCs. We found that H 2 O 2 enhances the Tyr 1021 phosphorylation of PDGF␤ receptor distinct from the ligand-dependent manner. Furthermore, the activation of non-receptor kinases, c-Src and PKC␦, and their complex formation are required for H 2 O 2 -induced PDGF␤ receptor activation. Taken together, our findings indicate that H 2 O 2 activates the PDGF␤ receptor via signal transduction pathways utilizing Src and PKC␦, and the requirement of c-Src/PKC␦ distinguishes H 2 O 2 -induced PDGF␤ receptor activation from the ligand-induced activation by the autophosphorylation mechanism in VSMCs. The activation of this particular signaling pathway involved in the regulation of cell growth and migration may explain the significance of ROS in cardiovascular diseases.
Cell Culture-VSMCs were prepared from the thoracic aorta of Sprague-Dawley rats by the explant method as described previously (21). Subcultured cells from passages 3-12 were used in the experiments and showed 99% positive immunostaining with smooth muscle ␣-actin antibody (Sigma).
Replication-deficient Adenovirus Generation-The generation and characterization of adenovirus encoding dominant negative mutant of PKC␦ were as described previously (22). The dominant negative PKC␦ cDNA contained a lysine to arginine mutation in the ATP binding domain at amino acid position 376. The recombinant adenovirus was plaque-purified, expanded, and titered in HEK293 cells using the agarose gel overlay method. VSMCs were infected with adenovirus for 2 days as described previously (6).
Immunoprecipitation and Immunoblot Analysis-After stimulation, VSMCs were lysed with ice-cold immunoprecipitation buffer (150 mM NaCl, 50 mM HEPES, pH 7.5, 1% Triton X-100, 1 mM EDTA, 10 mM NaF, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10% glycerol, 10 mg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). The cell lysates were centrifuged, and supernatant was immunoprecipitated with the antibody and A/G-agarose for 16 h at 4°C as described previously. Cell lysates or immune complex were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted using an ECL detection kit (Amersham Biosciences) as described previously (23).
Reproducibility of Results-Unless stated otherwise, results are representative of at least three separated experiments yielding similar results.  1A). In contrast, PDGF-BB caused rapid Tyr 1021 phosphorylation of PDGF␤ receptor as early as 1 min and sustained the phosphorylation up to 10 min (Fig. 1B). When the cell lysates were immunoprecipitated with anti-PDGF␤ receptor antibody and immunoblotted with anti-phosphotyrosine antibody, H 2 O 2 also induced tyrosine phosphorylation of PDGF␤ receptor maximally at 10 -30 min. Moreover, H 2 O 2 enhanced PLC␥ association with the PDGF␤ receptor in a similar time course as the H 2 O 2 -induced tyrosine phosphorylation of PDGF␤ receptor (Fig. 1C).  (Fig. 2).

H 2 O 2 Stimulates Tyrosine Phosphorylation of PDGF␤
Role of PDGF␤ Receptor Kinase Activity in Tyrosine Phosphorylation of PDGF␤ Receptor-To evaluate whether PDGF␤ receptor kinase activity contributes to the tyrosine phosphorylation of PDGF␤ receptor induced by H 2 O 2 , we used AG1295, a potent and selective inhibitor of this kinase (28). AG1295 (12.5-50 M) strongly inhibited PDGF-BB-induced Tyr 1021 phosphorylation of PDGF␤ receptor in VSMCs (Fig. 3A). In contrast, AG1295 had no significant effect on H 2 O 2 -induced Tyr 1021 phosphorylation of the PDGF␤ receptor (Fig. 3B). Also, AG1295 did not alter H 2 O 2 -induced tyrosine phosphorylation of the PDGF␤ receptor when the receptor was immunoprecipitated and immunoblotted with anti-phosphotyrosine antibody (data not shown).  PKC␤ isoform inhibitor, did not alter H 2 O 2 -induced Try 1021 phosphorylation of the PDGF␤ receptor. Similar inhibition by rottlerin but not by Go6976 was observed in the immunoprecipitation of the PDGF␤ receptor combined with immunoblotting with anti-phosphotyrosine antibody. In contrast, rottlerin had no effect on PDGF-BB-mediated PDGF␤ receptor tyrosine phosphorylation (Fig. 4, A and B).  5). To further explore the possibility that PKC␦ may associate with the PDGF␤ receptor in response to H 2 O 2 , co-immunoprecipitation experiments were performed. When H 2 O 2 -stimulated cell lysates were immunoprecipitated with anti-PDGF␤ receptor antibody and immunoblotted with anti-PKC␦ antibody, PKC␦ was detected in the PDGF␤ receptor immunoprecipitates. In the reciprocal experiments, PDGF␤ receptor was detected in anti-PKC␦ immunoprecipitates from H 2 O 2 -stimulated VSMCs (Fig. 6). These data suggest that H 2 O 2 -induced tyrosine phosphorylation of the PDGF␤ receptor requires the activation of PKC␦ and its association with the PDGF␤ receptor.

Role of PKC␦ in PDGF␤ Receptor
Role of c-Src in PDGF␤ Receptor Tyrosine Phosphorylation Induced by H 2 O 2 -It has been previously reported that the Src family kinases are involved in signaling events evoked by ROS (5, 7). To determine the role of Src family kinases in H 2 O 2induced PDGF␤ receptor activation, we used PP2, a pyrazolopyrimidine that interacts specifically with Src family kinases together with PP3, a negative control for PP2 (32,33). PP2 but not PP3 completely inhibited H 2 O 2 -induced Tyr 1021 phosphorylation of the PDGF␤ receptor in VSMCs (Fig. 7). These data suggest that H 2 O 2 -induced tyrosine phosphorylation of PDGF␤ receptor is mediated by Src kinase activation.
PKC␦ has been shown to become tyrosine-phosphorylated in COS-7 cells exposed to H 2 O 2 (29). Also, PKC␦ can be tyrosinephosphorylated by the Src family kinases (34 -36). Thus, we determined whether PKC␦ was phosphorylated on tyrosine by H 2 O 2 stimulation in VSMCs. H 2 O 2 -induced tyrosine phosphorylation of PKC␦ was initially detected at 3 min and continued up to 10 min (Fig. 8A). H 2 O 2 -induced tyrosine phosphorylation of PKC␦ was inhibited by pretreatment with PP2 but not with PP3 (Fig. 8B). We further examined the activation of c-Src and its possible association with PKC␦ in response to H 2 O 2 . Previous studies showed that full catalytic activity of c-Src requires phosphorylation at Tyr 418 (37). We found that H 2 O 2 stimulated Tyr 418 phosphorylation of c-Src as early as 3 min, peaking at 5 min, whereas it decreased at 10 min. Also, H 2 O 2 stimulated a prominent increase in the association of PKC␦ with c-Src in VSMCs. The complex formation between PKC␦ and c-Src was detected within 3 min and plateaued by 5 min (Fig. 8C). These data indicate that the activation of c-Src and its association with PKC␦ by H 2 O 2 may lead to the activation of PKC␦ and subsequent tyrosine phosphorylation of the PDGF␤ receptor in VSMCs.

DISCUSSION
The principal findings of this study are that H 2 O 2 stimulates Tyr 1021 phosphorylation of the PDGF␤ receptor in VSMCs and that this phosphorylation requires the activation of two protein kinases, PKC␦ and c-Src, but not the intrinsic kinase activity of the PDGF␤ receptor. These findings will provide new mechanistic insights by which enhanced ROS production in VSMCs induces unique alleys of signal transduction distinct from those induced by endogenous ligands leading to an abnormal vascular remodeling process.
In this study, we have shown that H 2 O 2 stimulates phosphorylation of the PDGF␤ receptor on Tyr residues, one of which was identified as Tyr 1021 in VSMCs. These results are in good agreement with a recent publication (38) showing that H 2 O 2 induced tyrosine phosphorylation of the PDGF␤ receptor as well. Also, our finding is further supported by the fact that PLC␥ is recruited to the PDGF␤ receptor after H 2 O 2 stimulation and because Tyr 1021 phosphorylation is known to provide a high affinity binding site for PLC␥ (19) Trans-activation of growth factor receptors such as EGF or PDGF receptor by stimuli that do not directly interact with the receptor is a current exciting topic of signal transduction research. Recently, an attractive mechanism for the trans-activation of the EGF receptor was discovered. This mechanism involves metalloprotease-dependent production of a mature ligand from its precursor (24 -27). Although the ligands for the PDGF␤ receptor expressed in VSMCs do not require this metalloprotease-dependent step to become an active ligand, VSMCs may also express a newly identified ligand for PDGF receptors such as PDGF-C or PDGF-D. The proform of these ligands contains a proteolytic cleavage site (39). Because we have shown that angiotensin II-induced EGF receptor transactivation requires both ROS production and a metalloproteasedependent ligand production (10, 26), we examined whether a metalloprotease function is required for PDGF␤ receptor or EGF receptor trans-activation by H 2 O 2 in this study. We confirmed that EGF receptor trans-activation by H 2 O 2 requires a metalloprotease in this study; however, PDGF␤ receptor activation appears to be independent from this mechanism.
Because the ligand-independent activation of this tyrosine kinase receptor may not lead to activation of intrinsic tyrosine kinase, we further asked whether the Tyr 1021 phosphorylation of PDGF␤ receptor requires its intrinsic tyrosine kinase activity by using a selective PDGF receptor kinase inhibitor, AG1295. Our results clearly demonstrate that AG1295 markedly inhibits PDGF-BB-induced Tyr 1021 phosphorylation of the PDGF␤ receptor but not the phosphorylation induced by H 2 O 2 . These data further confirm that the H 2 O 2 -induced PDGF␤ receptor activation is ligand-independent and suggest that the Tyr 1021 phosphorylation of PDGF␤ receptor might involve the inhibition of tyrosine phosphatase as previously proposed (18) or the activation of other tyrosine kinases, which may phosphorylate the receptor.
PKC␦ is one of the PKC isoforms shown to be activated by H 2 O 2 and is abundant in VSMCs (29,30). We have previously shown the requirement of PKC␦ in mediating the activation of non-receptor tyrosine kinases, PYK2 and JAK2, by angiotensin II in VSMCs and thereby established the specificities of PKC␦ inhibitors, rottlerin and a dominant negative adenovirus, used in this study (6). Here, we have shown for the first time that PKC␦ is indispensable for H 2 O 2 -induced but not PDGF-induced tyrosine phosphorylation of PDGF␤ receptor in VSMCs. This is further supported by the fact that there is an inducible complex formation between PKC␦ and the PDGF␤ receptor in response to H 2 O 2 . It has been shown that H 2 O 2 induces the association between PKC␣ and the PDGF receptor in Swiss 3T3 fibroblasts (40). Although we did not measure the PKC␣ association in this study, the participation of PKC␣ in H 2 O 2 -induced tyrosine phosphorylation of the PDGF␤ receptor is unlikely because of the results using a PKC␣ and PKC␤ inhibitors. We have previously shown that the inhibitor at the concentration used in this study is effective for the PKC␣-dependent event in VSMCs that was further confirmed by overexpression of wild type PKC␣ in VSMCs (41).
This study suggests the involvement of c-Src in H 2 O 2induced tyrosine phosphorylation of the PDGF␤ receptor in VSMCs. Because PP2 inhibits PKC␦ tyrosine phosphorylation, c-Src probably acts upstream of PKC␦-dependent PDGF␤ receptor phosphorylation by ROS. However, a recent report using VSMCs showed that angiotensin II stimulates ROS-dependent PDGF␤ receptor tyrosine phosphorylation that is insensitive to a Src kinase inhibitor PP1 (11). This may be attributed to a different agonist (angiotensin II) used or an inhibitor for Src (PP1) used in the study.
In conclusion, we demonstrated here that ROS induced a ligand-independent tyrosine phosphorylation of PDGF␤ receptor involving PKC␦ and c-Src that is distinct from PDGFinduced PDGF␤ receptor activation. Because both ROS and PDGF receptor signal transduction are strongly implicated in vascular diseases, our data add new mechanistic insight into these diseases associated with enhanced ROS action.