NADPH oxidase is the major source of superoxide production in cardiovascular tissues. We reported previously that PG (prostaglandin) F2α caused hypertrophy of vascular smooth muscle cells by induction of NOX1, a catalytic subunit of NADPH oxidase. PGF2α-induced NOX1 expression was mediated by transactivation of the EGF (epidermal growth factor) receptor and subsequent activation of ERK (extracellular-signal-regulated kinase) 1/2, PI3K (phosphoinositide 3-kinase) and ATF-1 (activating transcription factor-1), a member of the CREB (cAMP-response-element-binding protein)/ATF family. As the receptor for PGF2α is known to activate PKC (protein kinase C), involvement of PKC in up-regulation of NOX1 expression was investigated in A7r5 cells. GF109203x, a non-selective inhibitor of PKC, dose-dependently suppressed the induction of NOX1 mRNA by PGF2α. Whereas an inhibitor of the conventional PKC, Gö 6976, and a PKCϵ translocation-inhibitor peptide had no effect, an inhibitor of PKCδ, rottlerin, significantly attenuated the PGF2α-induced increase in NOX1 mRNA. Gene silencing of PKCδ by RNA interference significantly suppressed the PGF2α-induced increase in NOX1 mRNA, as well as phosphorylation of the EGF receptor, ERK1/2 and ATF-1. Silencing of the PKCδ gene also attenuated the PDGF (platelet-derived growth factor)- induced increase in NOX1 mRNA and transactivation of the EGF receptor. Moreover, the augmented synthesis of the protein induced by PGF2α or PDGF was abolished by gene silencing of PKCδ. These results suggest that PKCδ-mediated transactivation of the EGF receptor is elicited not only by PGF2α, but also by PDGF, and that the subsequent activation of ERK1/2 and ATF-1 leads to up-regulation of NOX1 gene expression and ensuing hypertrophy in the vascular cell lineage.
Superoxide (O2−) and hydrogen peroxide (H2O2), the so-called reactive oxygen species, have been documented as intrinsic signalling molecules in cardiovascular tissues. The major source of O2− in vascular cells and cardiac myocytes is NADPH oxidase [1–3]. Among several homologues of the catalytic subunit of the phagocyte NADPH oxidase (gp91phox; NOX2) recently identified, NOX1 has been implicated in the proliferation and hypertrophy of VSMC (vascular smooth muscle cells). Expression of NOX1 is induced by various vasoactive factors, including angiotensin II, PDGF (platelet-derived growth factor), phorbol ester and FBS (fetal bovine serum) [4,5]. Previously, we have shown that PG (prostaglandin) F2α, one of the primary prostanoids that is generated in the vascular tissue, also induces NOX1 and causes hypertrophy of VSMC through the increased generation of O2− . A transcription factor of the CREB (cAMP-response-element-binding protein)/ATF (activating transcription factor) family, ATF-1, was determined to be essential for the up-regulation of NOX1 not only by PGF2α, but by PDGF, phorbol ester and FBS . In addition, PGF2α-induced NOX1 expression was mediated by the transactivation of the EGF (epidermal growth factor) receptor and subsequent activation of ERK (extracellular-signal-related kinase) 1/2, PI3K (phosphoinositide 3-kinase), and ATF-1 .
The specific receptor for PGF2α, FP, is a G-protein-coupled receptor  that activates phospholipase C and elicits mobilization of cytosolic Ca2+. Since phorbol ester induces NOX1 expression in VSMC [5,7], we sought to investigate whether PKC (protein kinase C) is involved in PGF2α-induced NOX1 expression. In the present paper we report that PKCδ mediates the up-regulation of NOX1 not only by PGF2α, but also by PDGF. The involvement of transactivation of the EGF receptor was indicated in this process.
GF109203x and rottlerin were purchased from Sigma (St. Louis, MO, U.S.A.). PGF2α was from Nacalai Tesque (Kyoto, Japan). Antibodies against the EGF receptor or phosphorylated EGF receptor were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.) and Biosource International (Nivelles, Belgium) respectively. Antibodies against ERK or phosphorylated ERK were purchased from New England Biolabs (Beverly, MA, U.S.A.). Horseradish-peroxidase-conjugated anti-rabbit or anti-mouse antibodies were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Gö 6976 and a PKCϵ translocation-inhibitor peptide were purchased from Merck (Tokyo, Japan). The homodimer PDGF-BB was obtained from PeproTech (London, UK). [α-32P]dCTP and [α-32P]UTP were from ICN Biomedicals (Costa Mesa, CA, U.S.A.). [35S]-EXPRESS Protein Labeling Mix was purchased from Perkin-Elmer Life Sciences (Boston, MA, U.S.A.). Hydroethidine was obtained from Polyscience (Warrington, PA, U.S.A.).
Cell culture and Northern blot analysis
A7r5 cells, obtained from American Type Culture Collection (U.S.A.), were seeded in 10 cm dishes (1×106 cells/dish) and cultured for 24 h in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10 % (v/v) FBS. Cells were incubated for a further 48 h in DMEM containing 0.5% (v/v) FBS and were pre-treated with or without various inhibitors for 1 h and subsequently exposed to 100 nM PGF2α for 24 h. Northern blot analysis was performed as described previously [6,7].
Western blot analysis
Synthesis of anti-PKCδ dsRNAs (double-stranded RNAs)
Anti-PKCδ dsRNAs were designed against nucleotides 381–410 [PKCδ-RNAi (RNA interference)-1] and 888–917 (PKCδ-RNAi-2) of the rat PKCδ mRNA sequence . Sense or antisense oligonucleotides containing the hairpin sequence, the terminator sequence and overhanging sequences were synthesized, amplified by PCR and inserted into the pPUR-KE expression vector, which contains a tRNAVal promoter. Establishment of A7r5 clones stably expressing an anti-PKCδ dsRNA was performed, essentially as described previously . Measurements of O2− production and protein synthesis were performed using hydroethidine and 35S-labelled methionine as described previously .
Values were expressed as the means±S.E.M. Statistical analysis was performed with Student's t test. For multiple treatment groups, one-way ANOVA followed by Bonferroni's t test was applied.
GF109203x suppresses PGF2α-induced NOX1 expression
PGF2α exerts its biological actions through binding to its specific receptor FP . FP is coupled to phospholipase C and elicits mobilization of cytosolic Ca2+ and activation of PKC in VSMC or ventricular myocytes [11,12]. Since phorbol ester induces the expression of NOX1 in VSMC [5,7], we first examined whether PKC is involved in the induction of NOX1 by PGF2α. Pre-treatment of A7r5 cells with GF109203x, an isoform-non-selective inhibitor of PKC, dose-dependently reduced the PGF2α-induced increase in NOX1 mRNA (Figure 1).
GF109203x suppresses PGF2α-induced NOX1 expression
Rottlerin, but not Gö 6976 or a PKCϵ translocation-inhibitor peptide, suppresses PGF2α-induced NOX1 expression
We next investigated which PKC isoform is involved in NOX1 induction. As phorbol ester was shown previously to induce NOX1 expression in VSMC [5,7], an isoform of either a conventional PKC (cPKC) or a novel PKC (nPKC) which requires DAG (diacylglycerol) for its activation, is a likely candidate. In fact, no effect from a peptide inhibitor or a dominant-negative form of PKCζ, an atypical PKC that does not require DAG for its activation, was observed on PGF2α-induced NOX1 expression (results not shown). Since an inhibitor of cPKC, Gö 6976, did not affect NOX1 induction even at 1 μM (Figure 2A), the PKC isoform involved in PGF2α-induced NOX1 expression appeared to be a member of the nPKCs. Among nPKCs, transcripts for PKCδ and PKCϵ, but not for PKCη, were detected in A7r5 cells by RT (reverse transciptase)-PCR (results not shown). Therefore, the effects of a selective inhibitor of PKCδ, rottlerin , and a peptide that inhibits translocation of PKCϵ were examined. As shown in Figure 2(B), rottlerin significantly suppressed the PGF2α-induced increase in NOX1 mRNA at concentrations higher than 5 μM, whereas the PKCϵ translocation-inhibitor peptide had no effect (Figure 2C). These results suggest that PKCδ is the isoform involved in NOX1 induction by PGF2α.
Rottlerin but not Gö 6976 or a PKCϵ translocation-inhibitor peptide suppresses PGF2α-induced NOX1 expression
Gene silencing of PKCδ
To confirm the role of PKCδ in the up-regulation of NOX1 by PGF2α, dsRNAs targeted at the rat PKCδ mRNA sequence were introduced into A7r5 cells. Following single-cell cloning of the transfectants, two clones, PKCδ-RNAi-1 and PKCδ-RNAi-2, which stably expressed the dsRNA targeted at the two independent sites of the mRNA sequence, were isolated (Figure 3A). In these clones, protein levels of PKCδ, but not of PKCϵ, were reduced compared with the mock-transfected cells (Figure 3B).
Gene silencing of PKCδ by RNAi
Gene silencing of PKCδ attenuates transactivation of the EGF receptor by PGF2α and downstream signalling
Involvement of PKCδ in transactivation of the EGF receptor has been documented in [14,15]. Previously, we demonstrated that PGF2α-induced NOX1 expression is mediated by transactivation of the EGF receptor and subsequent activation of ERK1/2, PI3K and ATF-1 [7,8]. Based on these findings, we first examined whether gene silencing of PKCδ affects phosphorylation of these proteins. As shown in Figure 4(A), phosphorylation of the EGF receptor was significantly attenuated in PKCδ knocked-down clones. Similarly, the PGF2α-induced phosphorylation of ERK1/2 and ATF-1 was markedly suppressed in these clones (Figures 4B and 4C).
Gene silencing of PKCδ attenuates PGF2α-induced transactivation of the EGF receptor and activation of downstream signalling pathways
Gene silencing of PKCδ attenuates PGF2α-induced NOX1 expression, production of O2− and protein synthesis
Gene silencing of PKCδ attenuates PGF2α-induced NOX1 expression, O2− production and protein synthesis
We next examined the effects of gene silencing of PKCδ on the level of NOX1 mRNA. As shown in Figure 5(A), induction of NOX1 mRNA by PGF2α was almost completely abolished in PKCδ knocked-down clones. In addition, PGF2α-induced O2− production as well as protein synthesis were abolished in these clones (Figures 5B and 5C). These results suggest that PKCδ is essential for the PGF2α-induced up-regulation of NOX1 and resultant hypertrophy of VSMC.
Gene silencing of PKCδ attenuates transactivation of the EGF receptor and activation of ERK1/2 by PDGF
In the previous investigation, we demonstrated that silencing of the ATF-1 gene significantly reduced the induction of NOX1 by PDGF [7,8]. We therefore examined whether gene silencing of PKCδ affects up-regulation of NOX1 by PDGF. As shown in Figure 6(A), PDGF elicited phosphorylation of the EGF receptor in mock-transfected cells. In PKCδ knocked-down clones, phosphorylation of the EGF receptor was significantly attenuated, indicating that PKCδ is also involved in the transactivation of the EGF receptor by PDGF. In accord with these findings, phosphorylation of ERK1/2 induced by PDGF was significantly suppressed in these clones (Figure 6B).
Gene silencing of PKCδ attenuated PDGF-induced transactivation of the EGF receptor and activation of ERK1/2
Gene silencing of PKCδ attenuates PDGF-induced NOX1 expression and protein synthesis
Induction of NOX1 mRNA by PDGF was almost completely abolished in PKCδ knocked-down clones (Figure 7A). Furthermore, PDGF-induced protein synthesis was not observed in these clones (Figure 7B), suggesting that PKCδ is essential for the up-regulation of NOX1 and the ensuing vascular hypertrophy elicited not only by PGF2α, but also by PDGF.
Gene silencing of PKCδ attenuates PDGF-induced NOX1 expression and protein synthesis
The major lines of evidence provided in the present study are that: (i) a selective inhibitor of PKCδ, rottlerin, attenuated the induction of NOX1 by PGF2α, whereas an inhibitor of cPKC, Gö 6976, and a PKCϵ translocation-inhibitor peptide had no effect; (ii) RNAi targeted at the PKCδ mRNA suppressed PGF2α- or PDGF-induced phosphorylation of the EGF receptor; (iii) RNAi targeted at PKCδ suppressed the up-regulation of NOX1 induced by PGF2α as well as PDGF; (iv) RNAi targeted at PKCδ abolished the increased protein synthesis induced by PGF2α or PDGF. Based on these findings and those of our earlier studies [7,8], it is reasonable to conclude that PKCδ-mediated transactivation of the EGF receptor and subsequent activation of ERK1/2, PI3K and ATF-1 constitute the signalling pathways that lead to up-regulation of NOX1, not only by PGF2α, but also by PDGF (summarized in Figure 8). To our knowledge, the present study provides the first evidence for the possible involvement of PKCδ in hypertrophy of VSMC.
Diagram illustrating the proposed signalling pathways that lead to up-regulation of NOX1 in VSMC
The specific receptor for PGF2α, FP, is coupled to a G-protein and activates phospholipase C to elicit the mobilization of cytosolic Ca2+ . Some of the effects of PGF2α are, however, mediated through the transactivation of the EGF receptor. In fact, the PGF2α-induced increase in NOX1 mRNA involved the transactivation of the EGF receptor and subsequent activation of ERK1/2, PI3K and ATF-1 . However, the identity of the molecule that transduces the signals from FP to the EGF receptor remains unknown. The present study provides the first evidence for the functional role of PKCδ in transducing signals from FP to the EGF receptor to activate the ERK1/2 and ATF-1 signalling pathways.
Involvement of PKCδ in the transactivation of the EGF receptor was reported in ATP-stimulated VSMC  and in angiotensin-II-stimulated hepatic C9 cells . PKCδ is known to activate MMPs (matrix metalloproteinases) that cleave heparin-binding EGF [16,17]. The Src/Pyk2 (proline-rich tyrosine kinase 2) complex, implicated in the transactivation of the EGF receptor, is also activated by a PKCδ-dependent mechanism . These findings suggest that activation of both MMPs and Src/Pyk2 by PKCδ takes part in transactivation of the EGF receptor. In our previous study, inhibitors of Src and MMPs significantly suppressed the PGF2α-induced increase in NOX1 mRNA . Accordingly, PKCδ appears to activate both Src/Pyk2 and MMPs to elicit activation of the EGF receptor and downstream signalling pathways, which leads to the up-regulation of NOX1 gene expression.
This is the first report indicating the involvement of PKCδ in the PDGF-induced transactivation of the EGF receptor. In murine B82L fibroblasts and NIH-3T3 cells, transactivation of the EGF receptor by PDGF was documented [18,19]. Migration of fibroblasts and activation of p21-activated kinase, induced by PDGF, were dependent on the EGF receptor expressed in these cells. While the biological significance of the cross-talk between the PDGF-β receptor and the EGF receptor has been suggested, the precise mechanism of transactivation is still poorly understood. There is a report postulating heterodimer formation between the PDGF-β receptor and the EGF receptor in unstimulated VSMC . PDGF-β receptor dimers form in response to binding of PDGF to its receptor, and breakdown of the heterodimers by treatment with a Src inhibitor abolishes PDGF-induced transactivation of the EGF receptor . The present study in PKCδ knocked-down cells clearly demonstrates that PKCδ is an essential component for transactivation of the EGF receptor, not only by a G-protein-coupled receptor, FP, but also by the PDGF-β receptor, a receptor tyrosine kinase. It may be that PKCδ is involved in stabilization of the PDGF-β–EGF-receptor heterodimer through activation of Src, which phosphorylates one of the receptors, or a protein that links the receptors .
However, elucidating the different roles of PKCδ in VSMC seems to be somewhat elusive. Recent proteomic and metabolomic analysis of VSMC obtained from PKCδ-deficient mice demonstrated that PKCδ is crucial in regulating glucose and lipid metabolism, controlling the cellular redox state and maintaining VSMC differentiation . In contrast, another study indicated that PKCδ mediates mechanical-stress-induced de-differentiation and migration of VSMC, a key event in vascular remodelling . Angiotensin-II-induced activation of protein kinase D is also regulated by PKCδ in VSMC . We have demonstrated in the present study that increased protein synthesis induced by PGF2α or PDGF was abolished in cells in which expression of the PKCδ gene is knocked-down by RNAi. In this context, the role of PKCδ in the up-regulation of NOX1 supports the notion that PKCδ is involved in vascular remodelling by mediating the proliferation and hypertrophy of VSMC.
In the previous study, we reported that inhibitors of the mitochondrial respiratory chain suppressed the phosphorylation of ATF-1 and NOX1 gene expression induced by PGF2α, PDGF, FBS or phorbol ester . Previous studies have demonstrated that PKCδ is targeted to mitochondria upon activation by phorbol ester, H2O2, or a δ-opioid agonist [24–26]. The translocation of PKCδ to mitochondria elicits changes in the mitochondrial membrane potential and initiates the apoptotic pathway or confers cardioprotection to the ischaemic myocardium [24–26]. However, a close link between mitochondria and EGF receptor signalling has been reported. A-kinase anchor protein 121, a protein anchored to the outer membrane of the mitochondria, traps and inhibits protein tyrosine phosphatase D1, a Src-associated phosphatase that activates EGF receptor signalling . This implies that tethering or redistributing the signalling molecule to mitochondria modulates the Src-dependent EGF receptor transduction pathway. If mitochondria are involved in transducing signals from PKCδ to the EGF receptor, the organelle may play an important role in regulating superoxide generation by NOX1/NADPH oxidase. Further studies may cast light on the role of mitochondria in the regulation of gene expression mediated by the PKCδ–EGF receptor signalling pathway.
This work was supported in part by a Grant-in-Aid for Young Scientists (B)-14770036 from The Ministry of Education, Culture, Sports, Science and Technology of Japan. We are grateful to Dr J. Moscat, Centro de Biología Molecular Severo Ochoa, Consejo Superiòr de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain, for providing expression plasmids for dominant-negative and constitutive-active forms of PKCζ. We also thank Dr T. Nishinaka, of Kyoto Prefectural University of Medicine, Kyoto, Japan and Dr E. Funakoshi, of the Faculty of Pharmaceutical Sciences, Setsunan University, Japan for their valuable discussion and advice.
activating transcription factor-1
Dulbecco's modified Eagle's medium
epidermal growth factor
extracellular-signal-regulated protein kinase
fetal bovine serum
platelet-derived growth factor
protein kinase C
vascular smooth muscle cells
These authors made an equal contribution to this work.