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(Hypertension. 2008;51:232.)
© 2008 American Heart Association, Inc.

Original Articles

Novel Role of Protein Kinase C- Tyr³¹¹ Phosphorylation in Vascular Smooth Muscle Cell Hypertrophy by Angiotensin II

Hidekatsu Nakashima; Gerald D. Frank; Heigoro Shirai; Akinari Hinoki; Sadaharu Higuchi; Haruhiko Ohtsu; Kunie Eguchi; Archana Sanjay; Mary E. Reyland; Peter J. Dempsey; Tadashi Inagami; Satoru Eguchi

From the Cardiovascular Research Center and Department of Physiology (H.N., H.S., A.H., S.H., H.O., K.E., S.E.) and Department of Anatomy and Cell Biology (A.S.), Temple University School of Medicine, Philadelphia, Pa; the Department of Biochemistry (G.D.F., T.I.), Vanderbilt University School of Medicine, Nashville, Tenn; the Department of Craniofacial Biology, School of Dentistry, and Department Cell and Developmental Biology, School of Medicine (M.E.R.), University of Colorado Health Science Center, Aurora; and the Departments of Pediatrics and Molecular and Integrative Physiology (P.J.D.), University of Michigan, Ann Arbor.

Correspondence to Satoru Eguchi, Cardiovascular Research Center and Department of Physiology, Temple University School of Medicine, 3420 N Broad St, Philadelphia, PA 19140. E-mail seguchi{at}temple.edu

	Abstract

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We have shown previously that activation of protein kinase C- (PKC) is required for angiotensin II (Ang II)–induced migration of vascular smooth muscle cells (VSMCs). Here, we have hypothesized that PKC phosphorylation at Tyr³¹¹ plays a critical role in VSMC hypertrophy induced by Ang II. Immunoblotting was used to monitor PKC phosphorylation at Tyr³¹¹, and cell size and protein measurements were used to detect hypertrophy in VSMCs. PKC was rapidly (0.5 to 10.0 minutes) phosphorylated at Tyr³¹¹ by Ang II. This phosphorylation was markedly blocked by an Src family kinase inhibitor and dominant-negative Src but not by an epidermal growth factor receptor kinase inhibitor. Ang II-induced Akt phosphorylation and hypertrophic responses were significantly enhanced in VSMCs expressing PKC wild-type compared with VSMCs expressing control vector, whereas the enhancements were markedly diminished in VSMCs expressing a PKC Y311F mutant. Also, these responses were significantly inhibited in VSMCs expressing kinase-inactive PKC K376A compared with VSMCs expressing control vector. From these data, we conclude that not only PKC kinase activation but also the Src-dependent Tyr³¹¹ phosphorylation contributes to Akt activation and subsequent VSMC hypertrophy induced by Ang II, thus signifying a novel molecular mechanism for enhancement of cardiovascular diseases induced by Ang II.

Key Words: angiotensin II • AT₁ receptor • signal transduction • protein kinase C • Src • hypertrophy • vascular smooth muscle cells

	Introduction

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Angiotensin II (Ang II) plays a major role in vascular remodeling outside of its hemodynamic effects. In cultured vascular smooth muscle cells (VSMCs), cardiac myocytes, and cardiac fibroblasts, Ang II has been shown to promote hypertrophy and/or hyperplasia. There are 2 subtypes of Ang II receptors, AT₁ and AT₂, although the major physiological and pathophysiological actions of Ang II are facilitated through the AT₁ receptor. In VSMCs, activation of the AT₁ receptor coupled to G_q increases intracellular Ca²⁺ and activates protein kinase C (PKC).^1,2 In this regard, several PKC isoforms, including PKC, are believed to be activated by Ang II in VSMCs.^3–5 In addition, various tyrosine kinases and serine/threonine kinases are rapidly activated by Ang II and likely play important roles in mediating vascular remodeling induced by Ang II.^6,7 However, the detailed role of each PKC isoform in mediating Ang II-induced vascular remodeling, as well as the possible signal cross-talk with other kinases, has been insufficiently characterized.

Increasing evidence suggest that PKC is involved in many mechanisms promoting VSMC remodeling and dysfunction.^8–11 It was reported that PKC is activated by mechanical stress, and VSMCs from PKC-null mice migrate slower than control VSMCs.¹² Previously, we have shown that PKC kinase activity is required for activation of several tyrosine kinases by Ang II or reactive oxygen species in VSMCs.^4,13,14 Moreover, we have reported recently that PKC is required for activation of Rho, Rho-kinase and c-Jun NH₂-terminal kinase and subsequent migration of VSMCs by using kinase-inactive PKC overexpression.¹⁵ These data suggest an important role of PKC in mediating vascular remodeling induced by Ang II.

PKC is also phosphorylated on tyrosine residues in many cells, including VSMCs and cardiac myocytes.^13,16–18 Although there are multiple tyrosine phosphorylation sites on PKC, Tyr³¹¹ located between the regulatory and catalytic domains is of particular interest. This is because the Tyr³¹¹ phosphorylation has been linked to increased kinase activity in cells treated with H₂O₂.¹⁹ PKC phosphorylation at Tyr³¹¹ may also affect the selectivity of substrates.¹⁷ Taken together with the above information, we have tested the hypothesis that PKC Tyr³¹¹ phosphorylation plays a major role in Ang II-induced vascular hypertrophy. We found that PKC phosphorylation at Tyr³¹¹ was induced by Ang II through a Src family kinase and that this phosphorylation was involved in Akt activation and subsequent VSMC hypertrophy.

	Materials and Methods

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An expanded Methods section describing reagents, primary antibodies, cell culture, and statistical analysis is available at http://hyper.ahajournals.org.

Retrovirus Infection
Wild-type or Y311F PKC containing enhanced green fluorescent protein (GFP) at the C terminus²⁰ was cloned into the pBM-IRES-PURO vector, and high titer retroviral supernatants were generated.²¹ VSMCs were infected with retrovirus, and the infected VSMCs were selected as described previously.^22,23 To assess complete viral transformation after an antibiotic selection, in addition to the detection of the overexpression by immunoblotting, we routinely confirmed >99% infection efficiency of our retrovirus vectors by the GFP tagged to the mutants and detected under a fluorescent microscope.

Adenovirus Infection
The generation of adenovirus encoding wild-type and a kinase-inactive K376A PKC mutant construct and dominant-negative K295M+Y527F Src was described previously.^24,25 The titer (plaque-forming units per milliliter) of adenovirus was determined by Adeno-XTM Rapid Titer kit (BD Biosciences). VSMCs were infected with adenovirus for 2 days, as described previously.¹⁴ To assess complete viral transformation, we routinely confirmed >99% infection efficiency of our adenovirus vectors by GFP encoded by these vectors separately and detected under a fluorescent microscope.

Immunoblotting
Cell lysates were subjected to SDS-PAGE and transferred to a nitrocellulose membrane, as described previously.²⁶ The membranes were then exposed to primary antibodies overnight at 4°C. After incubation with the peroxidase-linked secondary antibody (Amersham Biosciences) with dilution between 1:1000 and 1:10 000 (depending on the primary antibody) for 1 hour at room temperature, the immunoreactive proteins were visualized by a chemiluminescence reaction kit (Pierce).

Protein Assay
VSMCs on 12-well culture plates were incubated with serum-free DMEM for 3 days in retrovirus-infected VSMCs. For adenovirus infection, VSMCs were incubated with serum-free DMEM for 1 day and infected with adenovirus at 100 multiplicity of infection in serum-free DMEM for 2 days. The cells were further incubated with or without 100 nmol/L of Ang II for 3 days. After aspiration of the medium, cells were washed twice with ice-cold Hanks’ balanced salt solution, and the total amount of cellular protein was measured as described previously.²⁷

Cell Volume Assay
After the pretreatments described in the protein assay, VSMCs were washed with Hanks’ balanced salt solution and trypsinized. The cells were then suspended in PBS, and the cell volume was measured by Z2 Coulter Particle Count and Size Analyzer (Beckman Coulter).²⁷

Proliferation Assay
After the pretreatments described in the protein assay, cell proliferation was measured using a CellTiter 96 Aqueous cell proliferation assay kit (Promega), following the manufacturer’s protocol, as described previously.²⁷ Basically, this assay measures cell viabilities on the PKC manipulation with or without Ang II stimulation for 3 days.

5-Bromodeoxyuridine Assay
After the adenovirus infection, as described for the protein assay, the cells were pretreated with or without 100 nmol/L of Ang II for 24 hours, and 5-bromodeoxyuridine incorporation was determined for an additional 24 hours by a 5-bromodeoxyuridine incorporation kit (Calbiochem) according to the manufacturer’s protocol.

	Results

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Phosphorylation of PKC at Tyr³¹¹ by Ang II Through the G_q-Coupled AT₁ Receptor
In 3-day serum-starved rat aortic VSMCs, Ang II (100 nmol/L) stimulated phosphorylation of PKC at Tyr³¹¹ in a rapid (within 30 seconds) and transient manner with a peak of 2 to 5 minutes (Figure 1). The phosphorylation returned to the baseline level at 40 minutes (Figure S1A). Thus, in subsequent experiments, unless otherwise stated, VSMCs were stimulated with Ang II for 2 minutes for evaluation of the PKC phosphorylation. Pretreatment with an AT₁ receptor antagonist, RNH6270, totally blocked PKC phosphorylation by Ang II (Figure S1B). The AT₁ receptor is mainly coupled to the heterotrimeric G protein G_q, whereas G protein–independent signal transduction by the AT₁ has been reported.² Thus, we determined whether G_q contributed to the Ang II-induced PKC phosphorylation. Pretreatment with a selective G_q inhibitor, YM-254890,^22,28 completely blocked PKC phosphorylation at Tyr³¹¹ by Ang II (Figure S1C), indicating that Ang II-induced phosphorylation of PKC at Tyr³¹¹ was mediated through G_q activation.

View larger version (38K): [in this window] [in a new window]   Figure 1. Phosphorylation of PKC at Tyr311 by Ang II. VSMCs were stimulated with 100 nmol/L of Ang II for the indicated time periods. The cell lysates were immunoblotted with phosphoselective antibody, which detects PKC Tyr311 phosphorylation and anti-PKC antibody. The PKC phosphorylation at Tyr311 was measured by densitometry, normalized to total PKC, and shown as mean±SEM (n=3). *P<0.05 vs the basal control.

Involvement of Src in PKC Tyr³¹¹ Phosphorylation by Ang II
Activation of the AT₁ receptor by Ang II leads to rapid transactivation of the epidermal growth factor (EGF) receptor, which seems to mediate many key components of downstream signal transduction in VSMCs,²⁹ whereas an Src family kinase has been implicated as a PKC Tyr³¹¹ kinase.¹⁷ To clarify the involvement of Src family kinase and/or EGF receptor transactivation in PKC phosphorylation, we pretreated VSMCs with PP2, an Src family kinase inhibitor, or AG1478, an EGF receptor family kinase inhibitor. Interestingly, Ang II-induced PKC Tyr³¹¹ phosphorylation was markedly blocked by PP2 (5 µmol/L), whereas AG1478 (1 µmol/L) had no inhibitory effect. As expected, AG1478, but not PP2, inhibited Ang II-induced EGF receptor transactivation as detected by its autophosphorylation at Tyr¹⁰⁶⁸ (Figure 2A). Also, PP3 (5 µmol/L), the inactive control chemical for PP2, had no inhibitory effect on Ang II-induced PKC Tyr³¹¹ phosphorylation (Figure S1D). To support these pharmacological experiments, the effect of dominant-negative Src was examined. Infection of adenovirus encoding dominant-negative Src, but not the control vector, markedly inhibited PKC Tyr³¹¹ phosphorylation induced by Ang II, whereas neither virus affected the EGF receptor transactivation (Figure 2B). Sufficient overexpression of the dominant-negative Src mutant, as compared with endogenous Src, was confirmed (Figure 2B). These data suggest that Src, but not the EGF receptor, mediates Ang II-induced PKC Tyr³¹¹ phosphorylation.

View larger version (28K): [in this window] [in a new window]   Figure 2. Involvement of Src in PKC Tyr311 phosphorylation induced by Ang II. A, VSMCs were pretreated with an Src family kinase inhibitor, PP2 (5 µmol/L), or an EGF receptor kinase inhibitor, AG1478 (1 µmol/L), for 30 minutes and stimulated with 100 nmol/L of Ang II for 2 minutes. B, VSMCs were infected with adenovirus encoding dominant-negative (dn) Src or control vector and stimulated with 100 nmol/L of Ang II for 2 minutes. A and B, The cell lysates were immunoblotted with phosphospecific antibodies, which detect PKC Tyr311 phosphorylation or EGF receptor autophosphorylation at Tyr1068, and with anti-PKC, anti-EGF receptor, and anti-Src antibodies, as indicated. The PKC phosphorylation at Tyr311 was measured by densitometry and shown as mean±SEM (n=3). *P<0.05 vs the basal control. P<0.05 vs the stimulated control.

Involvement of PKC Tyr³¹¹ Phosphorylation and PKC Kinase Activity in Ang II–Induced VSMC Hypertrophy
To verify the functional significance of the Tyr³¹¹ phosphorylation, we established VSMCs that overexpress wild-type PKC or a PKC Y311F mutant containing GFP at the C terminus using retrovirus infection (Figure 3A). In wild-type PKC expressing VSMCs, the Ang II-induced increase in cellular protein was significantly enhanced compared with the control VSMCs. However, the enhancement was much less in Y311F-expressing VSMCs (Figure 3B). Moreover, in wild-type PKC VSMCs, Ang II significantly increased the cell volume, whereas no enhancement was observed in Y311F mutant-expressing cells (Figure S2A). There was no significant change in cell number among these VSMCs stimulated by Ang II (Figure S2B). The confluence state of these VSMCs at the time of the measurements was <90%, because without a mitogen, the VSMC did not significantly proliferate after serum starvation. Also, 5-bromodeoxyuridine incorporation was not significantly changed by Ang II, regardless of wild-type PKC overexpression in VSMCs (Figure S2C). In addition, there was no enhancement of an apoptotic marker, cleaved caspase-3, detected in either control or PKC overexpressing VSMCs with 4 hours of Ang II stimulation (Figure S3).

View larger version (32K): [in this window] [in a new window]   Figure 3. PKC phosphorylation at Tyr311 contributes to VSMC hypertrophy induced by Ang II. A, VSMCs were infected with retrovirus encoding control vector, wild-type PKC tagged with GFP, or PKC Y311F mutant tagged with GFP. The cell lysates were immunoblotted with antibodies as indicated. An arrow denotes exogenously introduced GFP-tagged PKC. B, VSMCs infected with the above retrovirus were stimulated with 100 nmol/L of Ang II for 3 days. Afterward, cellular protein levels were measured by a protein assay kit. The data were presented as fold basal (mean±SEM; n=3). *P<0.05.

To investigate whether the kinase activity of PKC is also required for Ang II-induced protein synthesis in VSMCs, we infected VSMCs with an adenovirus encoding a kinase-inactive PKC mutant (K376A). In VSMCs expressing K376A, both Ang II–induced protein synthesis (Figure 4) and the increase in cell volume (Figure S4A) were significantly inhibited compared with control VSMCs. Again, there was no significant change in cell number among these VSMCs stimulated by Ang II (Figure S4B). These data suggest that PKC Tyr³¹¹ phosphorylation and PKC kinase activity are both required for Ang II–induced hypertrophy in VSMCs.

View larger version (43K): [in this window] [in a new window]   Figure 4. Kinase activity of PKC is required for Ang II–induced protein synthesis in VSMCs. VSMCs were infected with adenovirus encoding a kinase-inactive PKC mutant (K376A) or control empty vector and stimulated with 100 nmol/L of Ang II for 3 days. Afterward, cellular protein levels were measured by a protein assay kit. The data were presented as fold basal (mean±SEM; n=3). *P<0.05 vs the basal control. P<0.05 vs the stimulated control. Also, immunoblotting of PKC and GAPDH to confirm K376A overexpression was performed.

It has been demonstrated that both Akt and extracellular signal regulated kinase (ERK) 1/2 are involved in Ang II-induced VSMC hypertrophy.^30–32 To assess the functional role of PKC Tyr³¹¹ phosphorylation and kinase activity in Ang II–induced hypertrophic signaling, we examined Akt and ERK 1/2 activation in the above-treated cells. In wild-type PKC expressing VSMCs, Ang II–induced Akt phosphorylation was markedly enhanced, whereas no enhancement of Akt phosphorylation by Ang II was seen in Y311F-expressing VSMCs (Figure 5A). In contrast, neither wild-type nor Y311F expression altered Ang II–induced ERK phosphorylation in VSMCs. Also, PKC Tyr³¹¹ phosphorylation by Ang II precedes the Akt phosphorylation. We have demonstrated previously that K376A PKC had no inhibitory effect on ERK 1/2 phosphorylation induced by Ang II in VSMCs.¹⁵ In contrast, Akt phosphorylation induced by Ang II was markedly inhibited in K376A-expressing VSMCs (Figure 5B). These data suggest that Ang II–induced VSMC hypertrophy is positively regulated by PKC kinase activation and Tyr³¹¹ phosphorylation through their involvement with Akt activation but not ERK activation.

View larger version (69K): [in this window] [in a new window]   Figure 5. PKC kinase activity and Tyr311 phosphorylation are required for Ang II–induced Akt activation of VSMCs expressing PKC mutants. A, The retrovirus-infected VSMCs (vector, wild-type PKC, or Y311F mutant) were stimulated with 100 nmol/L of Ang II for the indicated time periods. An arrow indicates GFP-tagged PKC position. B, The adenovirus-infected VSMCs (vector or K376A mutant) were stimulated with 100 nmol/L of Ang II for the indicated time periods. A and B, Cell lysates were immunoblotted with antibodies as indicated. The Akt Ser473 phosphorylation signal was measured by densitometry and shown as mean±SEM (n=3). *P<0.05 vs the basal control. P<0.05 vs the stimulated control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major novel findings of this study revealed that PKC activation associated with Tyr³¹¹ phosphorylation mediates Ang II–induced VSMC hypertrophy through a mechanism involving Akt. Also, Ang II seems to induce PKC Tyr³¹¹ phosphorylation through the G_q-coupled AT₁ receptor via Src. These findings provide a new signaling mechanism by which the AT₁ receptor activation leads to PKC-mediated vascular remodeling and may serve as a potential therapeutic target toward cardiovascular diseases.

Ang II–induced rapid PKC Tyr³¹¹ phosphorylation has been reported¹⁸ with a slightly more sustained time course, which may be because of a shorter serum starvation than in the present study. However, the upstream regulators of PKC Tyr³¹¹ phosphorylation have not yet been identified. The present study using a selective G_q inhibitor indicates that the phosphorylation is mediated through G_q activation. This is in agreement with our recent observation suggesting that Ang II–induced PKC Tyr³¹¹ phosphorylation in VSMCs requires intracellular Ca²⁺ elevation.³³ Because the AT₁ receptor is the dominant receptor expressed in our cultured VSMCs,³⁴ we have not evaluated the possible confounding of these signal transductions by the AT₂ receptor. Increasing evidence suggests the counterregulatory functions of the AT₂ receptors toward the AT₁ receptor-dependent functions, including vascular hypertrophy, as well as hyperplasia in vivo.^35,36 Therefore, it will be interesting to further characterize a possible signal cross-talk of the PKC regulation between these subtype receptors in vivo.

Here, we report that PKC phosphorylation at Tyr³¹¹ by Ang II is at least in part Src dependent in VSMCs. Supporting this finding is the fact that several others have reported that PKC Tyr³¹¹ phosphorylation in select cell types depended on Src family kinases when stimulated with various non-G protein–coupled receptor agonists.^19,37,38 Also, Src family kinases have been shown to be complexed with PKC in several cell types, including VSMCs.^13,38–40 However, possible contribution of other Src family kinases (Fyn and yes) expressed in VSMCs⁴¹ in Ang II–induced PKC Tyr³¹¹ phosphorylation remains to be determined. Although we have not studied Ang II–induced Src phosphorylation, such as at the positive regulatory Tyr⁴¹⁶ residue, the Src inhibitor PP2 used in this study has been shown to block this phosphorylation effectively in VSMCs.^42,43 In addition, our data suggest that the involvement of EGF receptor transactivation in the PKC phosphorylation by Ang II is unlikely. However, the EGF receptor kinase inhibitor AG1478, if used at 10 times more concentration than in the present study, partially attenuated c-Src phosphorylation at Tyr⁴¹⁶ induced by Ang II in VSMCs.⁴² Thus, further careful evaluation may be necessary regarding the possible partial but minor involvement of the EGF receptor transactivation in this PKC cascade.

We have previously used a PKC inhibitor, rottlerin, to elucidate the role of this PKC isoform in signal transductions of the AT₁ receptor in VSMCs.^4,15 However, we have not used this inhibitor in the present study because of the reported off-target effects,⁴⁴ which would be inappropriate for our long-term hypertrophic experiments. In PKC-deficient VSMCs, cytoskeletal signaling, reorganization, and subsequent migration in response to mechanical stress were diminished.¹² Also, an overexpression study using the wild-type PKC suggested that PKC mediates p38 mitogen-activated protein kinase activation induced by high glucose in VSMCs.⁴⁵ However, by using the K376A mutant, as well as rottlerin, our previous studies have shown that PKC kinase activity is essential for Ang II–induced activation of a select set of protein kinases, which include JAK2, Rho-kinase, p21-activated kinase, and c-Jun NH₂-terminal kinase but not ERK or p38 mitogen-activated protein kinase.^14,15,33 Thus, involvement of PKC in p38 mitogen-activated protein kinase activation may be agonist dependent.

It has been reported that PKC-deficient mouse VSMCs are resistant to apoptotic responses compared with control VSMCs.⁴⁶ Overexpression of PKC in VSMC cell lines also results in G1 arrest and apoptosis.^10,47 These apoptotic or necrotic changes, if they occur, could be associated with enlargement of cell volume.⁴⁸ However, this scenario is quite unlikely in our present study, because there was no difference in caspase-3 cleavage or cell viability with PKC overexpression regardless of Ang II stimulation, as shown in Figure S3.

Here, we further revealed Akt as a PKC-dependent kinase in VSMCs stimulated by Ang II, which plays a significant role in VSMC hypertrophy.³¹ To support our notion, a similar link between PKC and Akt was observed in thrombin-induced nuclear factor B activation in endothelial cells.⁴⁹ In addition, other mechanisms may coordinately regulate VSMC hypertrophy on PKC activation by Ang II, such as expression of Smad3 and transforming growth factor-β,¹¹ and the Tyr³¹¹ phosphorylation of PKC.

Interestingly, our data suggest that Ang II–induced PKC Tyr³¹¹ phosphorylation is also required for enhanced Akt activation and VSMC hypertrophy observed in VSMCs overexpressing wild-type PKC. However, the PKC Y311F mutant did not show a dominant-negative effect to inhibit Akt activation below the vector-transfected cells, and one of the hypertrophic responses was still slightly greater than the control cells, demonstrating distinct characteristics of the PKC mutants. The kinase-inactive mutant, K376A, not only loses its wild-type ability to positively regulate Akt activation and subsequent hypertrophy but also competes with endogenous PKC and, thus, acts as a dominant-negative PKC inhibitor. Y311F mutant also loses most of its own hypertrophic characteristics, whereas it is unable to interfere with the endogenous wild-type PKC. Although PKC Tyr³¹¹ phosphorylation has been proposed to enhance kinase activity, recent findings suggest additional roles of the Tyr³¹¹ phosphorylation in mediating unique functions of this PKC isoform.^17,50 The Tyr³¹¹ phosphorylation may be the additional component required for the complex formation among PKC, Akt, and its upstream kinase, 3-phosphoinositide–dependent kinase 1/3-phosphoinositide-dependent kinase 1 and subsequent Akt activation, which seems to require the PKC kinase activity.⁵¹ Taken together, it is attractive to speculate that the PKC phosphorylation may contribute to Akt activation and subsequent hypertrophy independent from the kinase activity. To support this notion, we observed a comparable autophosphorylation of Y311F PKC at Ser^643/676 phosphorylation to that of wild-type in Ang II–stimulated VSMCs (unpublished observation), thus reflecting the kinase activity remains intact in the Y311F mutant.

In the present study, we have not used a standard protein synthesis assay measuring a radiolabeled leucine incorporation. However, we believe that our 2 distinct methods used here measure the hypertrophic effects of Ang II just as sufficiently and perhaps more directly. Our data demonstrating hypertrophic responses by Ang II stimulation in VSMCs are consistent with highly sited past articles using intact aortic segments⁵² and cultured aortic VSMCs.⁵³ Moreover, no significant enhancement of DNA synthesis was observed in Ang II-stimulated VSMCs regardless of PKC overexpression. However, because our data rely on overexpression strategies, a future study should be conducted by using specific RNA silencing to evaluate the overall roles of PKC in mediating VSMC hypertrophy induced by Ang II. In addition, slight distinctions in control cell responses between Figures 3B and 4A may be caused by distinct control vectors used, as well as by a selection of the permanently infected cells in the retroviral experiment. It is also unlikely that the PKC Y311F mutant affects other PKC isoforms expressed in VSMCs nonspecifically, because this residue is unique to PKC. Other than the data shown in Figure 3A, we and others have demonstrated previously the specificities of the PKC mutants used in the present study.^{14,50,54–56}

A recent study using proteomic analysis of PKC-deficient VSMCs revealed that >30 proteins are altered, including enzymes related to glucose and lipid metabolism, thus highlighting the critical role of PKC in the development of cardiovascular diseases.⁹ PKC activation increases O₂ derived free radical generation from mitochondria and thereby promotes a pro-oxidant state.⁵⁷ Therefore, it will be interesting to expand the present findings by evaluating the regulation of proteins, such as pyruvate dehydrogenase and heat shock proteins, which are likely involved in atherosclerosis, as well as metabolic disorders.^58,59

Perspectives
We found that, in addition to PKC kinase activity, PKC phosphorylation at Tyr³¹¹ seems to be required for Akt activation and subsequent VSMC hypertrophy induced by Ang II, which is considered a potential mechanism of atherosclerosis and restenosis after vascular injury. However, our findings are limited within cell culture experiments. Therefore, further clarification of the signal transduction in vivo could contribute to a better understanding of the molecular mechanism of cardiovascular diseases, as well as to the development of better strategies for their treatment.

	Acknowledgments

 
Sources of Funding

This work was supported by National Institutes of Health grants HL076770 (S.E.), HL076575 (G.D.F.), and DE015648 (M.E.R.); by American Heart Association Established Investigator Award 0740042N (S.E.), and by W.W. Smith Charitable Trust grant H0605 (S.E.).

Disclosures

None.

	Footnotes

 
The first 2 authors contributed equally to this work.

Received September 13, 2007; first decision September 30, 2007; accepted December 10, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Touyz RM, Schiffrin EL. Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular smooth muscle cells. Pharmacol Rev. 2000; 52: 639–672.[Abstract/Free Full Text]

2. Mehta PK, Griendling KK. Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol. 2006; 292: C82–C97.[CrossRef][Medline] [Order article via Infotrieve]

3. Liao DF, Monia B, Dean N, Berk BC. Protein kinase C-zeta mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells. J Biol Chem. 1997; 272: 6146–6150.[Abstract/Free Full Text]

4. Frank GD, Saito S, Motley ED, Sasaki T, Ohba M, Kuroki T, Inagami T, Eguchi S. Requirement of Ca(2+) and PKCdelta for Janus kinase 2 activation by angiotensin II: involvement of PYK2. Mol Endocrinol. 2002; 16: 367–377.[Abstract/Free Full Text]

5. Nakashima H, Suzuki H, Ohtsu H, Chao JY, Utsunomiya H, Frank GD, Eguchi S. Angiotensin II regulates vascular and endothelial dysfunction: recent topics of Angiotensin II type-1 receptor signaling in the vasculature. Curr Vasc Pharmacol. 2006; 4: 67–78.[CrossRef][Medline] [Order article via Infotrieve]

6. Yin G, Yan C, Berk BC. Angiotensin II signaling pathways mediated by tyrosine kinases. Int J Biochem Cell Biol. 2003; 35: 780–783.[CrossRef][Medline] [Order article via Infotrieve]

7. Higuchi S, Ohtsu H, Suzuki H, Shirai H, Frank GD, Eguchi S. Angiotensin II signal transduction through the AT1 receptor: novel insights into mechanisms and pathophysiology. Clin Sci (Lond). 2007; 112: 417–428.[Medline] [Order article via Infotrieve]

8. Wakino S, Kintscher U, Liu Z, Kim S, Yin F, Ohba M, Kuroki T, Schonthal AH, Hsueh WA, Law RE. Peroxisome proliferator-activated receptor gamma ligands inhibit mitogenic induction of p21(Cip1) by modulating the protein kinase Cdelta pathway in vascular smooth muscle cells. J Biol Chem. 2001; 276: 47650–47657.[Abstract/Free Full Text]

9. Mayr M, Siow R, Chung YL, Mayr U, Griffiths JR, Xu Q. Proteomic and metabolomic analysis of vascular smooth muscle cells: role of PKCdelta. Circ Res. 2004; 94: e87–e96.[Abstract/Free Full Text]

10. Ryer EJ, Sakakibara K, Wang C, Sarkar D, Fisher PB, Faries PL, Kent KC, Liu B. Protein kinase C delta induces apoptosis of vascular smooth muscle cells through induction of the tumor suppressor p53 by both p38-dependent and p38-independent mechanisms. J Biol Chem. 2005; 280: 35310–35317.[Abstract/Free Full Text]

11. Ryer EJ, Hom RP, Sakakibara K, Nakayama KI, Nakayama K, Faries PL, Liu B, Kent KC. PKCdelta is necessary for Smad3 expression and transforming growth factor beta-induced fibronectin synthesis in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2006; 26: 780–786.[Abstract/Free Full Text]

12. Li C, Wernig F, Leitges M, Hu Y, Xu Q. Mechanical stress-activated PKCdelta regulates smooth muscle cell migration. FASEB J. 2003; 17: 2106–2108.[Medline] [Order article via Infotrieve]

13. Saito S, Frank GD, Mifune M, Ohba M, Utsunomiya H, Motley ED, Inagami T, Eguchi S. Ligand-independent trans-activation of the platelet-derived growth factor receptor by reactive oxygen species requires protein kinase C-delta and c-Src. J Biol Chem. 2002; 277: 44695–44700.[Abstract/Free Full Text]

14. Frank GD, Mifune M, Inagami T, Ohba M, Sasaki T, Higashiyama S, Dempsey PJ, Eguchi S. Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-delta. Mol Cell Biol. 2003; 23: 1581–1589.[Abstract/Free Full Text]

15. Ohtsu H, Mifune M, Frank GD, Saito S, Inagami T, Kim-Mitsuyama S, Takuwa Y, Sasaki T, Rothstein JD, Suzuki H, Nakashima H, Woolfolk EA, Motley ED, Eguchi S. Signal-crosstalk between Rho/ROCK and c-Jun NH2-terminal kinase mediates migration of vascular smooth muscle cells stimulated by angiotensin II. Arterioscler Thromb Vasc Biol. 2005; 25: 1831–1836.[Abstract/Free Full Text]

16. Jackson DN, Foster DA. The enigmatic protein kinase Cdelta: complex roles in cell proliferation and survival. FASEB J. 2004; 18: 627–636.[Abstract/Free Full Text]

17. Steinberg SF. Distinctive activation mechanisms and functions for protein kinase Cdelta. Biochem J. 2004; 384: 449–459.[CrossRef][Medline] [Order article via Infotrieve]

18. Tan M, Xu X, Ohba M, Cui MZ. Angiotensin II-induced protein kinase D activation is regulated by protein kinase Cdelta and mediated via the angiotensin II type 1 receptor in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2004; 24: 2271–2276.[Abstract/Free Full Text]

19. Konishi H, Yamauchi E, Taniguchi H, Yamamoto T, Matsuzaki H, Takemura Y, Ohmae K, Kikkawa U, Nishizuka Y. Phosphorylation sites of protein kinase C delta in H2O2-treated cells and its activation by tyrosine kinase in vitro. Proc Natl Acad Sci U S A. 2001; 98: 6587–6592.[Abstract/Free Full Text]

20. DeVries TA, Neville MC, Reyland ME. Nuclear import of PKCdelta is required for apoptosis: identification of a novel nuclear import sequence. EMBO J. 2002; 21: 6050–6060.[CrossRef][Medline] [Order article via Infotrieve]

21. Sanderson MP, Erickson SN, Gough PJ, Garton KJ, Wille PT, Raines EW, Dunbar AJ, Dempsey PJ. ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx. J Biol Chem. 2005; 280: 1826–1837.[Abstract/Free Full Text]

22. Mifune M, Ohtsu H, Suzuki H, Nakashima H, Brailoiu E, Dun NJ, Frank GD, Inagami T, Higashiyama S, Thomas WG, Eckhart AD, Dempsey PJ, Eguchi S. G protein coupling and second messenger generation are indispensable for metalloprotease-dependent, heparin-binding epidermal growth factor shedding through angiotensin II type-1 receptor. J Biol Chem. 2005; 280: 26592–26599.[Abstract/Free Full Text]

23. Ohtsu H, Dempsey PJ, Frank GD, Brailoiu E, Higuchi S, Suzuki H, Nakashima H, Eguchi K, Eguchi S. ADAM17 mediates epidermal growth factor receptor transactivation and vascular smooth muscle cell hypertrophy induced by angiotensin II. Arterioscler Thromb Vasc Biol. 2006; 26: e133–e137.[Abstract/Free Full Text]

24. Ohba M, Ishino K, Kashiwagi M, Kawabe S, Chida K, Huh NH, Kuroki T. Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C. Mol Cell Biol. 1998; 18: 5199–5207.[Abstract/Free Full Text]

25. Miyazaki T, Sanjay A, Neff L, Tanaka S, Horne WC, Baron R. Src kinase activity is essential for osteoclast function. J Biol Chem. 2004; 279: 17660–17666.[Abstract/Free Full Text]

26. Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, Inagami T. Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem. 1998; 273: 8890–8896.[Abstract/Free Full Text]

27. Suzuki H, Eguchi K, Ohtsu H, Higuchi S, Dhobale S, Frank GD, Motley ED, Eguchi S. Activation of endothelial nitric oxide synthase by the angiotensin II type 1 receptor. Endocrinology. 2006; 147: 5914–5920.[Abstract/Free Full Text]

28. Takasaki J, Saito T, Taniguchi M, Kawasaki T, Moritani Y, Hayashi K, Kobori M. A novel G_q/11-selective inhibitor. J Biol Chem. 2004; 279: 47438–47445.[Abstract/Free Full Text]

29. Eguchi S, Frank GD, Mifune M, Inagami T. Metalloprotease-dependent ErbB ligand shedding in mediating EGFR transactivation and vascular remodelling. Biochem Soc Trans. 2003; 31: 1198–1202.[Medline] [Order article via Infotrieve]

30. Ushio-Fukai M, Alexander RW, Akers M, Griendling KK. p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy. J Biol Chem. 1998; 273: 15022–15029.[Abstract/Free Full Text]

31. Ushio-Fukai M, Alexander RW, Akers M, Yin Q, Fujio Y, Walsh K, Griendling KK. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem. 1999; 274: 22699–22704.[Abstract/Free Full Text]

32. Ohtsu H, Suzuki H, Nakashima H, Dhobale S, Frank GD, Motley ED, Eguchi S. Angiotensin II signal transduction through small GTP-binding proteins: mechanism and significance in vascular smooth muscle cells. Hypertension. 2006; 48: 534–540.[Free Full Text]

33. Woolfolk EA, Eguchi S, Ohtsu H, Nakashima H, Ueno H, Gerthoffer WT, Motley ED. Angiotensin II-induced activation of P21-activated kinase 1 requires Ca2+ and protein kinase C {delta} in vascular smooth muscle cells. Am J Physiol Cell Physiol. 2005; 289: C1286–C1294.[Abstract/Free Full Text]

34. Eguchi S, Matsumoto T, Motley ED, Utsunomiya H, Inagami T. Identification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells. Possible requirement of Gq-mediated p21ras activation coupled to a Ca2+/calmodulin-sensitive tyrosine kinase. J Biol Chem. 1996; 271: 14169–14175.[Abstract/Free Full Text]

35. Carey RM. Cardiovascular and renal regulation by the angiotensin type 2 receptor: the AT2 receptor comes of age. Hypertension. 2005; 45: 840–844.[Free Full Text]

36. Johren O, Dendorfer A, Dominiak P. Cardiovascular and renal function of angiotensin II type-2 receptors. Cardiovasc Res. 2004; 62: 460–467.[Abstract/Free Full Text]

37. Blake RA, Garcia-Paramio P, Parker PJ, Courtneidge SA. Src promotes PKCdelta degradation. Cell Growth Differ. 1999; 10: 231–241.[Abstract/Free Full Text]

38. Rybin VO, Guo J, Sabri A, Elouardighi H, Schaefer E, Steinberg SF. Stimulus-specific differences in protein kinase C delta localization and activation mechanisms in cardiomyocytes. J Biol Chem. 2004; 279: 19350–19361.[Abstract/Free Full Text]

39. Song JS, Swann PG, Szallasi Z, Blank U, Blumberg PM, Rivera J. Tyrosine phosphorylation-dependent and -independent associations of protein kinase C-delta with Src family kinases in the RBL-2H3 mast cell line: regulation of Src family kinase activity by protein kinase C-delta. Oncogene. 1998; 16: 3357–3368.[CrossRef][Medline] [Order article via Infotrieve]

40. Brandt DT, Goerke A, Heuer M, Gimona M, Leitges M, Kremmer E, Lammers R, Haller H, Mischak H. Protein kinase C delta induces Src kinase activity via activation of the protein tyrosine phosphatase PTP alpha. J Biol Chem. 2003; 278: 34073–34078.[Abstract/Free Full Text]

41. Ishida M, Ishida T, Thomas SM, Berk BC. Activation of extracellular signal-regulated kinases (ERK1/2) by angiotensin II is dependent on c-Src in vascular smooth muscle cells. Circ Res. 1998; 82: 7–12.[Abstract/Free Full Text]

42. Touyz RM, Wu XH, He G, Salomon S, Schiffrin EL. Increased angiotensin II-mediated Src signaling via epidermal growth factor receptor transactivation is associated with decreased C-terminal Src kinase activity in vascular smooth muscle cells from spontaneously hypertensive rats. Hypertension. 2002; 39: 479–485.[Abstract/Free Full Text]

43. Che Q, Carmines PK. Src family kinase involvement in rat preglomerular microvascular contractile and [Ca2+]i responses to ANG II. Am J Physiol Renal Physiol. 2005; 288: F658–664.[Abstract/Free Full Text]

44. Soltoff SP. Rottlerin: an inappropriate and ineffective inhibitor of PKCdelta. Trends Pharmacol Sci. 2007; 28: 453–458.[CrossRef][Medline] [Order article via Infotrieve]

45. Igarashi M, Wakasaki H, Takahara N, Ishii H, Jiang ZY, Yamauchi T, Kuboki K, Meier M, Rhodes CJ, King GL. Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J Clin Invest. 1999; 103: 185–195.[Medline] [Order article via Infotrieve]

46. Leitges M, Mayr M, Braun U, Mayr U, Li C, Pfister G, Ghaffari-Tabrizi N, Baier G, Hu Y, Xu Q. Exacerbated vein graft arteriosclerosis in protein kinase Cdelta-null mice. J Clin Invest. 2001; 108: 1505–1512.[CrossRef][Medline] [Order article via Infotrieve]

47. Fukumoto S, Nishizawa Y, Hosoi M, Koyama H, Yamakawa K, Ohno S, Morii H. Protein kinase C delta inhibits the proliferation of vascular smooth muscle cells by suppressing G1 cyclin expression. J Biol Chem. 1997; 272: 13816–13822.[Abstract/Free Full Text]

48. Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun. 2005; 73: 1907–1916.[Free Full Text]

49. Rahman A, True AL, Anwar KN, Ye RD, Voyno-Yasenetskaya TA, Malik AB. Galpha(q) and Gbetagamma regulate PAR-1 signaling of thrombin-induced NF-kappaB activation and ICAM-1 transcription in endothelial cells. Circ Res. 2002; 91: 398–405.[Abstract/Free Full Text]

50. Kajimoto T, Shirai Y, Sakai N, Yamamoto T, Matsuzaki H, Kikkawa U, Saito N. Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase Cdelta in the Golgi complex. J Biol Chem. 2004; 279: 12668–12676.[Abstract/Free Full Text]

51. Brand C, Cipok M, Attali V, Bak A, Sampson SR. Protein kinase Cdelta participates in insulin-induced activation of PKB via PDK1. Biochem Biophys Res Commun. 2006; 349: 954–962.[CrossRef][Medline] [Order article via Infotrieve]

52. Holycross BJ, Peach MJ, Owens GK. Angiotensin II stimulates increased protein synthesis, not increased DNA synthesis, in intact rat aortic segments, in vitro. J Vasc Res. 1993; 30: 80–86.[Medline] [Order article via Infotrieve]

53. Geisterfer AA, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res. 1988; 62: 749–756.[Abstract/Free Full Text]

54. Fujii T, Garcia-Bermejo ML, Bernabo JL, Caamano J, Ohba M, Kuroki T, Li L, Yuspa SH, Kazanietz MG. Involvement of protein kinase C delta (PKCdelta) in phorbol ester-induced apoptosis in LNCaP prostate cancer cells. Lack of proteolytic cleavage of PKCdelta. J Biol Chem. 2000; 275: 7574–7582.[Abstract/Free Full Text]

55. Matassa AA, Carpenter L, Biden TJ, Humphries MJ, Reyland ME. PKCdelta is required for mitochondrial-dependent apoptosis in salivary epithelial cells. J Biol Chem. 2001; 276: 29719–29728.[Abstract/Free Full Text]

56. Motley ED, Eguchi K, Patterson MM, Palmer PD, Suzuki H, Eguchi S. Mechanism of endothelial nitric oxide synthase phosphorylation and activation by thrombin. Hypertension. 2007; 49: 577–583.[Abstract/Free Full Text]

57. Mayr M, Madhu B, Xu Q. Proteomics and metabolomics combined in cardiovascular research. Trends Cardiovasc Med. 2007; 17: 43–48.[CrossRef][Medline] [Order article via Infotrieve]

58. Kanwar RK, Kanwar JR, Wang D, Ormrod DJ, Krissansen GW. Temporal expression of heat shock proteins 60 and 70 at lesion-prone sites during atherogenesis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol. 2001; 21: 1991–1997.[Abstract/Free Full Text]

59. Mayr M, Mayr U, Chung YL, Yin X, Griffiths JR, Xu Q. Vascular proteomics: linking proteomic and metabolomic changes. Proteomics. 2004; 4: 3751–3761.[CrossRef][Medline] [Order article via Infotrieve]

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