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RECEPTORS AND SIGNAL TRANSDUCTION

ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKC-controlled ERK1/2 activity within the nucleus

Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida

Submitted 12 December 2005 ; accepted in final form 18 May 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS NOTE ADDED IN PROOF REFERENCES

 
High-affinity binding of angiotensin II (ANG II) to the ANG II type 1 receptor (AT₁R) results in the activation of ERK1/2 mitogen-activated protein kinases (MAPK). However, the precise mechanism of ANG II-induced ERK1/2 activation has not been fully characterized. Here, we investigated the signaling events leading to ANG II-induced ERK1/2 activation using a c-Src/Yes/Fyn tyrosine kinase-deficient mouse embryonic fibroblast (MEF) cell line stably transfected with the AT₁R (SYF/AT₁). ERK1/2 activation was reduced by 50% within these cells compared with wild-type controls (WT/AT₁). The remaining 50% of intracellular ERK1/2 activation was dependent upon heterotrimeric G protein and protein kinase C zeta (PKC) activation. Therefore, ANG II-induced ERK1/2 activation occurs via two independent mechanisms. We next investigated whether a loss of either c-Src/Yes/Fyn or PKC signaling affected ERK1/2 nuclear translocation and cell proliferation in response to ANG II. ANG II-induced cell proliferation was markedly reduced in SYF/AT₁ cells compared with WT/AT₁ cells (P < 0.01), but interestingly, ERK2 nuclear translocation was normal. ANG II-induced nuclear translocation of ERK2 was blocked via pretreatment of WT/AT₁ cells with a PKC pseudosubstrate. ANG II-induced cell proliferation was significantly reduced in PKC pseudosubstrate-treated WT/AT₁ cells (P < 0.01) and was completely blocked in SYF/AT₁ cells treated with this same compound. Thus ANG II-induced cell proliferation appears to be regulated by both ERK1/2-driven nuclear and cytoplasmic events. In response to ANG II, the ability of ERK1/2 to remain within the cytoplasm or translocate into the nucleus is controlled by c-Src/Yes/Fyn or heterotrimeric G protein/PKC signaling, respectively.

Src family tyrosine kinases; angiotensin II

Previous reports have implicated Src family kinases in ANG II-dependent ERK1/2 activation. ANG II-induced ERK1/2 activation was reduced in vascular smooth muscle cells (VSMC) isolated from c-Src knockout mice (–/–), as well as in rat VSMC over expressing a dominant negative form of c-Src (16). Work from our group demonstrated that c-Src is required for the activation of ERK2 via a Shc/Grb2/ERK2-dependent mechanism (32). In each of these reports, however, intracellular ERK1/2 activation was never completely reduced to levels present in non-ANG II-stimulated cells. It is not clear whether this inability to fully attenuate ERK1/2 activation was due to incomplete inhibition of Src kinases or whether ERK1/2 activation was occurring through Src kinase-independent signaling pathways as well.

A number of other proteins kinases have also been implicated in the activation of ERK1/2. The AT₁R is capable of transactivating tyrosine kinase receptors, including the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), which in turn activate ERK1/2 (10, 21, 23). In addition to receptor transactivation, numerous cytoplasmic kinases have been shown to mediate ERK1/2 activation. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) blocks ERK1/2 activation in EGF-treated preglomerular smooth muscle cells (2, 12). Furthermore, various isoforms of protein kinase C (PKC) have been shown to mediate intracellular ERK1/2 activation in response to different ligands (12, 13, 20, 24). It is not clear from these reports, though, whether the mechanism of ERK1/2 activation differs depending upon the receptor activated or whether ERK1/2 activation occurs simultaneously via multiple independent signaling mechanisms.

Here, we sought to determine whether AT₁R-generated ERK1/2 activation occurs via the concurrent activation of Src kinase-dependent and Src kinase-independent signaling pathways using a cell line devoid of Src kinases (SYF/AT₁). We found that ANG II-induced ERK1/2 activation is mediated by two distinct and independent signaling events: namely, a c-Src/Yes/Fyn-dependent and a heterotrimeric G protein/PKC-dependent mechanism. Furthermore, each pathway is responsible for the activation of roughly half of the total active ERK1/2 pool. These results were supported in multiple cell types, suggesting that this is a generalized mechanism for ANG II-induced ERK1/2 activation.

We also determined that the manner in which ERK1/2 is activated (i.e., via c-Src/Yes/Fyn- or PKC-dependent signaling) differentially affected ERK1/2 nuclear translocation but did not affect cell proliferation. PKC-dependent signaling influenced the translocation of ERK1/2 from the cytoplasm into the nucleus, whereas Src kinase-dependent signaling did not. However, both c-Src/Yes/Fyn- and PKC-dependent signaling did mediate a portion of ANG II-induced cell proliferation. As such, it appears that ANG II-induced cell proliferation is regulated by simultaneous ERK1/2-driven events within the nucleus and cytoplasm, controlled by either PKC or c-Src/Yes/Fyn, respectively. This study therefore provides valuable insight into signaling via the AT₁R and the intracellular events associated with ANG II-induced cell proliferation.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS NOTE ADDED IN PROOF REFERENCES

 
Generation of WT/AT₁ and SYF/AT₁ stable cell lines. Immortalized SYF and WT MEF cells were a gift from Dr. Philippe Soriano and were previously isolated from c-Src/Yes/Fyn triple knockout and WT mice, respectively, at embryonic day E9.5 (18, 37). Both cell lines lack endogenous AT₁R expression and were therefore transfected by our group with 20 µg of an hemagglutinin (HA)-tagged AT₁R wild-type cDNA plasmid as previously described (31). Two days after transfection, the cells were switched to medium supplemented with 500 µg/ml Zeocin (Invitrogen) to select for stable transfectants. Surviving colonies were ring-cloned, and binding assays were performed using ¹²⁵I-labeled [Sar¹,Ile⁸]ANG II (PerkinElmer Life Sciences) as described previously (1). Nonspecific binding was defined as binding in the presence of 1.0 µM cold ANG II. Scatchard analysis was used to identify respective WT/AT₁ and SYF/AT₁ clones in which the binding parameters were similar.

Cell culture. WT/AT₁ and SYF/AT₁ cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/l glucose supplemented with 10% fetal bovine serum (Hyclone), 1 mM sodium pyruvate, 10 U/ml penicillin, 10 µg/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES, and 100 µg/ml Zeocin. VSMC cells were cultured in the same media but without Zeocin. CHO/AT₁ and CHO/AT₁-M5 cells were cultured in F-12 media supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 U/ml penicillin, 10 µg/ml streptomycin, 1 mM sodium pyruvate, and 10 mM HEPES. WT/AT₁, SYF/AT₁, and VSMC were growth-arrested in serum-free DMEM for 48 h prior to experiments. CHO cells were growth-arrested in the same manner for 24 h. All cell culture reagents were obtained from Invitrogen.

Transient transfections. SYF/AT₁ cells were transiently transfected with 10 µg plasmid encoding green fluorescent protein (GFP)-ERK2 using 10 µl Lipofectin, following the manufacturer’s instructions (Invitrogen). SYF/AT₁ cells were also cotransfected with 10 µg of a Src-encoding plasmid (gift of S. Parsons; Univ. of Virginia) where indicated.

Pharmacological inhibitors. AG1295, AG1478, GDPS, Gö6976, Gö6983, LY294002, PD98059, PP2, Raf1 kinase inhibitor 1, and rottlerin were all obtained from Calbiochem and used at concentrations found to have maximum inhibitory effect (10, 13, 21, 23, 25, 33, 35, 39, 41). Sodium fluoride (Sigma), chelerythrine (LKT Labs), and the PKC myristoylated pseudosubstrate (PKC MP; Biomol) were also used at previously determined concentrations (17, 36, 46). SYF/AT₁ cells were permeabilized with 5 nM saponin (U.S. Biochemical) before treatment with GDPS (29). All other reagents were obtained from Sigma or Fisher. Cells were pretreated with inhibitor for the indicated time and stimulated with 10^–7 M ANG II as described.

siRNA treatment of WT/AT₁ cells. Short interfering RNA (siRNA) reagents were purchased from Santa Cruz Biotechnology. Cells were grown in 100-mm culture plates (Corning) to 80% confluency. Adherent cells were trypsinized and resuspended in serum-containing medium without antibiotics. Cells and medium were centrifuged at 500 g for 5 min, and pelleted cells were resuspended in fresh serum-containing medium without antibiotics. Cells were transferred to 6-well culture plates (Corning) and grown to 40–50% confluency. Transfection reagents were prepared as described in the online protocol (http://www.scbt.com/support/protocol_13.php) with the exception that the concentration of siRNA used was increased fourfold. Cells were next transfected for 48 h at 37°C with either control siRNA or PKC-specific siRNA in serum-containing medium without antibiotics. Cells were serum-starved for 48 h and treated with 10^–7 M ANG II for 0, 5, and 10 min. Cells were lysed, and whole-cell protein lysates were prepared. Cell lysates were separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane, and Western blots were performed with the indicated antibodies.

Immunoprecipitation. Cells were washed with 2 vol of ice-cold PBS containing 1 mM Na₃VO₄ and lysed in 0.8 ml ice-cold RIPA buffer containing protease inhibitors. Normalized lysates (0.5 mg/ml) were immunoprecipitated exactly as described (33). The immunoprecipitating anti-PLC antibody was from Santa Cruz Biotechnology. The immunoprecipitating anti-GFP antibody was from Cell Signaling Technology.

Western blot. Proteins were detected using enhanced chemiluminescence exactly as described (33). The cocktail of anti-ERK1/2(P) antibodies were from Promega and Santa Cruz Biotechnology. The anti-ERK1/2, the anti-MEK1/2, the anti-MEK1/2(P), and the anti-PKC antibodies were from Santa Cruz Biotechnology. The anti-phosphotyrosine antibody (PY20) was from BD Transduction Laboratories.

Densitometric analysis. Western blots were scanned and densitized using UnScanIt Gel Analysis (Silk Scientific). The average pixel value minus background was obtained for each cell type and normalized to the average pixel value for the respective non-ANG II-treated cells. All data are expressed as means ± SE.

Immunofluorescence. GFP-ERK2 plasmid was kindly provided by Dr. Philip J. S. Stork (15). WT/AT₁ and SYF/AT₁ cells were plated onto four-chambered slides (Lab-Tek) and grown to 50% confluency. The cells were washed one time with PBS (pH 7.4) to remove dead cells and debris. The cells were transfected for 5 h with 10 µg of GFP-ERK2 plasmid using Lipofectin (Invitrogen) and following the manufacturer’s instructions. Serum-free medium was replaced with serum-containing medium and incubated at 37°C for 2 days. Cells were washed twice in PBS and then starved for 48 h in serum-free medium. Following starvation, all cells were ligand treated with 10^–7 M ANG II for 0, 5, or 10 min. The cells were rinsed once in PBS and fixed in 4% paraformaldehyde for 10 min. Slide chambers were removed, and the slides were dipped twice into chilled PBS. Excess PBS was drained from each slide, and a glass coverslip was mounted to each slide using Vectashield and DAPI mounting medium (Vector Labs). The edges of the slide were sealed with nail polish sealer (Maybelline). Slides were viewed on a Zeiss Axioplan II fluorescence microscope.

Quantification of nuclear and cytoplasmic fluorescence. Fluorescence in the nucleus or cytoplasm was quantified using the NIH Image J Program. Pixel intensity readings were taken from either the nucleus or cytoplasm of multiple cells within each field. Values were averaged for each treatment group representative of three independent experiments. Nuclear fluorescence was then normalized to cytoplasmic fluorescence by dividing the average nuclear pixel intensity by the average cytoplasmic pixel intensity from each treatment group.

Measurement of cellular ATP levels. WT/AT₁ and SYF/AT₁ cells were plated onto 100-mm culture dishes and grown to 80% confluency. Cells were serum-starved for 48 h and treated with 10^–7 M ANG II. Cellular ATP levels were assessed using the ViaLight HS proliferation/cytotoxicity kit (Cambrex) following the manufacturer’s protocol. A luminometer (Monolight model 2030) was used to measure bioluminescence.

Measurement of formazan production. WT/AT₁ and SYF/AT₁ cells were plated onto 96-well plates and grown to 80% confluency. Cells were starved for 48 h in serum-free medium and treated with 10^–7 M ANG II as indicated. Formazan production was measured using the CellTiter 96 Aqueous One Solution Reagent (Promega) following the manufacturer’s instructions. Production of formazan was proportional to increased absorbance at 490 nm as measured by spectrophotometry.

Cell count. WT/AT₁ and SYF/AT₁ cells were plated onto 100-mm culture dishes and grown to 80% confluency. Cells were serum-starved and treated with 10^–7 M ANG II as indicated. Both cell types were counted using a hemocytometer as described (35).

Statistical analysis. Data were analyzed by two-way ANOVA. All data passed a normality test as well as equal variance test. Pair-wise comparisons were made following the Holm-Sidak method. All data are expressed as mean ± SE. Single asterisks indicate P < 0.05, and double asterisks indicate P < 0.01.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS NOTE ADDED IN PROOF REFERENCES

 
Roughly 50% of ANG II-induced ERK1/2 activation is mediated by Src family tyrosine kinase-dependent signaling. Previous reports have suggested that c-Src is a critical mediator of intracellular ERK1/2 activation (16, 32, 39). We first sought to determine the contribution that Src kinases have on ERK1/2 activation, specifically in response to ANG II using c-Src/Yes/Fyn-deficient (SYF) and wild-type (WT) MEF cells. These SYF fibroblasts were previously isolated at day E9.5 from a developing c-Src/Yes/Fyn-deficient mouse embryo and have been shown to be completely devoid of these proteins (18, 37). MEF cells containing functional c-Src/Yes/Fyn were also isolated from WT littermates and served as controls (WT cells).

Both the SYF and WT cells do not endogenously express the AT₁R (data not shown). Therefore, the AT₁R was stably transfected into both cell types to constitute ANG II signaling. These AT₁R stable cell lines have been named SYF/AT₁ and WT/AT₁, respectively. Saturation binding studies were then performed to identify respective SYF/AT₁ and WT/AT₁ clones in which the binding parameters were similar. SYF/AT₁ (clone 16) and WT/AT₁ (clone 2) both had a K_D of 0.4 nM and a B_max of 140–150 fmol/mg protein (Supplemental Fig. S1; the online version of this article contains supplemental data.). To demonstrate that these two cell lines were similar in all aspects other than the levels of c-Src/Yes/Fyn, we first examined the expression levels of Jak2 and STAT3, two non-Src kinase-dependent genes. We found that these two genes were expressed at similar levels in the two cell types (Supplemental Fig. S2, A and B). We next examined the ability of ANG II to activate PLC1 (Supplemental Fig. S2C). We found that both cell types were capable of increasing PLC₁ tyrosine phosphorylation levels to roughly equal levels, an indication that the AT₁ receptor can signal similarly in both cell types when examining signaling events independent of c-Src/Yes/Fyn. Finally, we chose to examine the ability of PDGF to activate ERK1/2 in both cell types, as previous work has shown that PDGF activates ERK1/2 in these cells in a c-Src/Yes-Fyn-independent manner (18, 37). We found that PDGF-mediated ERK1/2 phosphorylation was achieved to about the same extent in both the SYF/AT₁ and WT/AT₁ cells, indicating that the cellular machinery required to activate ERK1/2 is functioning equally in both cell types (Supplemental Fig. S2D). Collectively, these data suggest that the WT/AT₁ and SYF/AT₁ cells are similar in all aspects except for c-Src/Yes/Fyn expression and signaling pathways that are dependent on these three proteins.

We next assessed ANG II-induced ERK1/2 activation in WT/AT₁ and SYF/AT₁ cells. Cells were stimulated with 10^–7 M ANG II for 0, 5, and 10 min, and ERK1/2 activity was assessed via Western blot analysis. ANG II-dependent ERK1/2 activation was decreased in SYF/AT₁ cells compared with WT/AT₁ cells after 5 and 10 min of ANG II treatment (Fig. 1A). Furthermore, ANG II-induced ERK1/2 activation was reduced in WT/AT₁ cells pretreated with the Src family kinase inhibitor, PP2, to levels comparative to ANG II-stimulated SYF/AT₁ cells (Fig. 1B). Finally, ERK1/2 activation in the SYF/AT₁ cells was partially restored by transiently transfecting these cells with a c-Src expression plasmid (Fig. 1C). Taken together, these results demonstrate that Src kinases mediate a portion of ANG II-induced ERK1/2 activation.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Quantification of ERK1/2 activation in response to ANG II in WT/AT1 and SYF/AT1 cells. A: wild-type cells (WT/AT1) and c-Src/Yes/Fyn tyrosine kinase-deficient mouse embryonic fibroblast (MEF) cells stably transfected with the AT1R (SYF/AT1) cells were treated with 10–7 M ANG II for 0, 5, and 10 min, and active ERK1/2 levels were assessed via Western blot analysis using the indicated antibodies. Data are representative of eight Western blots. B: WT/AT1 and SYF/AT1 cells were pretreated with 30 µM PP2 or DMSO for 60 min, then stimulated with 10–7 M ANG II for 0, 5, and 10 min. Active ERK1/2 levels were assessed via Western blot analysis using the indicated antibodies. Data are representative of three Western blots. C: SYF/AT1 cells were transfected with a plasmid encoding GFP-ERK2. Cells were also cotransfected with a Src-encoding plasmid or empty vector control as indicated. Cells were stimulated with 10–7 M ANG II for 0, 5, and 10 min. Whole-cell lysates were immunoprecipitated with GFP antibody, and Western blot was performed for phosphorylated ERK2 to identify any changes in ERK2 activation occurring in cells transfected with both GFP-ERK2 and Src. D: quantification of active ERK2 amounts from A. Fold changes in active ERK2 in response to ANG II treatment were calculated by dividing average ERK2 pixel density in ANG II-treated cells by average pixel density in nontreated controls.

ANG II-dependent ERK1/2 activation requires either c-Src/Yes/Fyn or heterotrimeric G protein activation. Heterotrimeric G proteins have previously been shown to activate ERK1/2 in response to ANG II (35). We next wanted to test whether the remaining 50% of ANG II-induced ERK1/2 activation observed in the SYF/AT₁ cells was mediated entirely by heterotrimeric G protein signaling. SYF/AT₁ cells were permeabilized using saponin and then pretreated with the heterotrimeric G protein inhibitor, GDPS (29). The -phosphate group of this compound has been replaced with a sulfate group, hindering the ability of heterotrimeric G proteins to exchange GDP for GTP and subsequently become activated. Permeabilized SYF/AT₁ cells treated with vehicle control served as controls. Cells were then stimulated with 10^–7 M ANG II, and ERK1/2 activation was assessed via Western blot. SYF/AT₁ cells permeabilized with saponin and treated with vehicle control still demonstrated ERK2 activation in response to ANG II (Fig. 2A). However, ERK2 activation was attenuated in GDPS-treated cells since there was not a statistically significant increase in ERK2 phosphorylation levels in ANG II-stimulated cells compared with untreated controls. These data suggest that ANG II-induced activation of ERK1/2 occurring independent of c-Src/Yes/Fyn requires heterotrimeric G protein activation.

View larger version (37K): [in this window] [in a new window]   Fig. 2. ANG II-induced ERK1/2 activation is partially dependent upon heterotrimeric G proteins. A and B: SYF/AT1 cells were pretreated with either 2 mM GDPS for 20 min or 2 mM NaF for 1 h, respectively, and stimulated with 10–7 M ANG II for 0, 5, and 10 min. C: CHO/AT1 and CHO/AT1-M5 cells were first pretreated with 30 µM PP2 or DMSO for 1 h before ANG II treatment. ERK2 phosphorylation was determined via Western blot using the indicated antibodies. A–C: total ERK protein loading was demonstrated in each case by stripping the membrane and reprobing with the anti-ERK1/2 antibodies (bottom panels). All Western blots are representative of three independent experiments.

To further demonstrate that heterotrimeric G proteins mediate all c-Src/Yes/Fyn-independent ERK1/2 activation in response to ANG II, we utilized a previously generated CHO cell line that was stably transfected with a mutant AT₁R that is uncoupled from heterotrimeric G protein activation (denoted as CHO/AT₁-M5 cells) (9). These cells still retain their ability to activate tyrosine kinases such as c-Src/Yes/Fyn. CHO cells stably expressing wild-type AT₁R with similar affinity and abundance (denoted as CHO/AT₁) were used as controls. Both cell types were stimulated with 10^–7 M ANG II, and ERK1/2 activation was assessed via Western blot. ANG II-induced ERK1/2 activation in CHO/AT₁ cells reached maximum levels after 5 min of ANG II treatment (Fig. 2C). ERK1/2 activation also occurred in CHO/AT₁-M5 cells in response to ANG II, but maximum ERK1/2 activation was significantly reduced compared with CHO/AT₁ cells. Therefore, we confirm that ANG II-induced ERK1/2 activation is partially dependent upon heterotrimeric G protein activation. Furthermore, pretreatment of CHO/AT₁-M5 cells with PP2 completely blocked ANG II-induced ERK1/2 activation, indicating that the remaining 50% of ERK1/2 activation is dependent upon Src kinases.

ANG II-dependent ERK1/2 activation occurring independent of c-Src/Yes/Fyn requires PKC activation. Protein kinase C isoforms have been shown to be activated downstream of heterotrimeric G proteins and have also been shown to be important for ERK1/2 activation (12, 13, 20, 24). We next wanted to identify the specific PKC isoforms responsible for activating ERK1/2 in a c-Src/Yes/Fyn-independent manner. SYF/AT₁ cells were first treated with the broad-range PKC inhibitor, chelerythrine. Cells were then stimulated with 10^–7 M ANG II, and ERK1/2 activation assessed by Western blot. ERK1/2 activation was eliminated in SYF/AT₁ cells treated with chelerythrine relative to DMSO-treated controls (Fig. 3A). Pretreatment with inhibitors specific for other proteins known to activate ERK1/2, including the PDGFR, the EGFR, phosphatidylinositol 3'-kinase (PI3K), and Raf1, did not cause a statistically significant reduction in ANG II-induced ERK1/2 activation in the SYF/AT₁ cell (Supplemental Fig. S3, A–D). As such, these data confirm that Src kinase-independent ERK1/2 activation in response to ANG II specifically requires PKC.

View larger version (40K): [in this window] [in a new window]   Fig. 3. ANG II-induced ERK1/2 activation is partially dependent upon PKC. A: SYF/AT1 cells were pretreated with DMSO or 30 µM chelerythrine for 60 min, then stimulated with 10–7 M ANG II for 0, 5, and 10 min. ERK1/2 activation was assessed via Western blot using the indicated antibodies (top). Total ERK1/2 protein loading was demonstrated by stripping the membrane and reprobing with the indicated antibodies (bottom). These data are representative of three independent experiments. B: the effect of inhibitor treatment on the percent maximum ANG II-induced ERK2 activation. Maximum ANG II-induced ERK2 activation was quantified from A and from Supplemental Fig. S3, A–C, in the presence (+) or absence (–) of inhibitor. Phospho-ERK2 bands were densitized. Values were then normalized to ERK2 activation in ANG II-stimulated cells not pretreated with inhibitor, then multiplied by 100. Gö6983 and chelerythrine pretreatment significantly reduced ERK1/2 activation (P < 0.01) compared with vehicle-treated controls.

We next directly assessed the effect of PKC inhibition on ANG II-induced ERK1/2 activation. WT/AT₁ and SYF/AT₁ cells were first pretreated with a PKC MP, a potent and specific inhibitor for PKC (46). Both cell types were then stimulated with 10^–7 M ANG II, and ERK1/2 activation assessed via Western blot analysis. ERK1/2 activation in WT/AT₁ cells was significantly reduced with the addition of the PKC MP, whereas ERK1/2 activation in SYF/AT₁ cells treated with PKC MP was reduced to levels found in non-ligand-treated cells (Fig. 4, A and B). Interestingly, treatment of WT/AT₁ cells with PKC MP reduced ANG II-induced ERK1/2 activation to levels present in ANG II-stimulated SYF/AT₁ cells.

View larger version (23K): [in this window] [in a new window]   Fig. 4. PKC mediates ANG II-induced ERK1/2 activation independent of c-Src/Yes/Fyn. A: WT/AT1 and SYF/AT1 cells were pretreated with 1 µM PKC myristoylated pseudosubstrate (MP) for 1 h, then stimulated with ANG II for 0, 5, and 10 min. ERK1/2 activation was then assessed via Western blot analysis with the indicated antibodies (top). Total ERK1/2 protein loading was demonstrated by stripping the membrane and reprobing with the indicated antibodies (bottom). B: phosphorylated ERK2 amounts from A were quantified via densitometric analysis and expressed as a fold change relative to unstimulated controls. C: WT/AT1 cells were transfected with either PKC siRNA or control siRNA. All cells were then treated with 10–7 M ANG II for 0, 5, 10 min, and ERK1/2 activation assessed via Western blot analysis with the indicated antibodies (1st panel). Total ERK1/2 protein loading was demonstrated by stripping the membrane and reprobing with the indicated antibodies (2nd panel). PKC protein knockdown was confirmed by Western blot analysis with the indicated antibody (3rd panel). We confirmed that PKC siRNA had no effect on PKC protein levels (4th panel). D: the percentage of maximum ERK2 phosphorylation from C was determined by dividing by ERK2 activation in ANG II and control siRNA treated cells and multiplying by 100.

Collectively, these data suggest that PKC partially mediates ANG II-induced ERK1/2 activation.

ANG II-dependent ERK1/2 activation in VSMC is independently mediated by both a c-Src/Yes/Fyn- and a heterotrimeric G protein/PKC-dependent mechanism. We next wanted to determine whether ANG II-induced ERK1/2 activation is mediated by both c-Src/Yes/Fyn and heterotrimeric G protein/PKC signaling in cells that endogenously express the AT₁R. We chose to utilize primary cultures of VSMC isolated from rat aortas. c-Src/Yes/Fyn or PKC activity was then blocked using pharmacological inhibitors. VSMC were pretreated with either PP2, PKC MP, or PKC MP and PP2 in combination. The cells were then stimulated with 10^–7 M ANG II for 0, 5, and 10 min. Whole-cell lysates were prepared, and Western blot was performed with anti-ERK1/2(P)-pAbs to identify changes in ERK1/2 activation. We found that ANG II-induced ERK1/2 activation occurred after 5 min of ANG II treatment in VSMC (Fig. 5A). ERK1/2 activation was significantly reduced in VSMC treated with either PP2 or the PKC pseudosubstrate alone, and treatment with either of these inhibitors alone resulted in about a 50% reduction in ERK2 phosphorylation (Fig. 5B). Furthermore, ERK1/2 activation was completely reduced in cells treated with both PP2 and the PKC pseudosubstrate in combination. Collectively, these data confirm that the mechanisms of intracellular ERK1/2 activation are the same in our AT₁R-transfected MEF cells as in VSMC, which endogenously express the AT₁R, and occur via c-Src/Yes/Fyn- or heterotrimeric G protein/PKC-dependent signaling.

View larger version (31K): [in this window] [in a new window]   Fig. 5. ANG II-induced ERK1/2 activation is mediated by c-Src/Yes/Fyn- and PKC-dependent signaling in vascular smooth muscle cells (VSMC). A: VSMC were pretreated with either DMSO, 30 µM PP2, 1 µM PKC MP, or both PP2 and PKC MP in combination. All inhibitor treatment times were 1 h. ERK1/2 activation was then measured via Western blot analysis using the indicated antibodies (top). Total ERK1/2 protein loading was demonstrated by stripping the membrane and reprobing with the indicated antibodies (bottom). B: three representative Western blots of A were scanned and densitized, and the percent maximum ERK2 phosphorylation was calculated by dividing by active ERK2 amounts in non-inhibitor-treated cells after 5 min of ANG II stimulation and multiplying by 100.

Nuclear translocation of ERK2 is influenced by PKC-dependent signaling and not by c-Src/Yes/Fyn.

ANG II-dependent nuclear translocation of ERK1/2 was examined in both the WT/AT₁ and SYF/AT₁ cells. WT/AT₁ and SYF/AT₁ cells were transfected with a GFP-ERK2 plasmid to track ERK2 movement in response to ANG II treatment. Cells were then stimulated with 10^–7 M ANG II, fixed, and DAPI stained to visualize the nucleus. In the absence of ANG II, GFP-ERK2 was distributed fairly evenly between both the nucleus and cytoplasm in WT/AT₁ and SYF/AT₁ cells (Fig. 6, A and F). DAPI counterstain of Fig. 6, A and F, and merging of the GFP and DAPI images confirmed these findings (Fig. 6, C and I). In response to ANG II, ERK1/2 nuclear accumulation was markedly increased in both the WT/AT₁ and SYF/AT₁ cells (Fig. 6, B and G), and this finding was confirmed by DAPI counterstain (Fig. 6, D and J). As such, it appeared that ERK1/2 nuclear translocation was present in both ANG II-stimulated WT/AT₁ and SYF/AT₁ cells. Quantification of nuclear fluorescence relative to cytoplasmic fluorescence revealed a significant increase in ERK1/2 nuclear fluorescence in both WT/AT₁ and SYF/AT₁ cells stimulated with ANG II, indicative of increased nuclear translocation (Fig. 6, E and L). However, there was no statistically significant difference in nuclear fluorescence between ANG II-stimulated SYF/AT₁ cells compared with similarly treated WT/AT₁ controls. As such, it appears that c-Src/Yes/Fyn do not influence ERK1/2 nuclear translocation.

View larger version (35K): [in this window] [in a new window]   Fig. 6. Nuclear translocation of active ERK2 is unaffected by the loss of c-Src/Yes/Fyn. A–D and F–K: WT/AT1 or SYF/AT1 cells were transfected with a GFP-ERK2 plasmid and then stimulated with ANG II for 0 and 10 min. Nuclear translocation of ERK2 was assessed by fluorescent microscopy. Cells that were imaged from independent fields are outlined in white; fields from within the same experimental treatment group are shown as a composite image within the same panel. A and B: GFP-ERK2 images in nontreated and ANG II-treated WT/AT1 cells. E: nuclear fluorescence from A–B was quantified and normalized to cytoplasmic fluorescence. F–H: GFP-ERK2 images in nontreated and ANG II-treated SYF/AT1 cells in the presence or absence of 1 µM PKC MP (1 h). C and D: Merging of images A and B, respectively, with DAPI-stained images. I–K: merging of images F–H, respectively, with DAPI-stained images. L: nuclear fluorescence from F–H was quantified and normalized to cytoplasmic fluorescence. All images are representative of the entire field and were taken at x40 magnification. Bar in A represents 15 µm. Shown is one of two independent results.

View larger version (22K): [in this window] [in a new window]   Fig. 7. ANG II-induced cell proliferation is attenuated by blocking c-Src/Yes/Fyn- and PKC-dependent signaling. A: WT/AT1 and SYF/AT1 cells were stimulated with ANG II for 0 and 4 h. Cellular ATP levels were then measured. Data are expressed as fold change in ATP in ANG II-stimulated cells relative to nontreated controls. B: WT/AT1 and SYF/AT1 cells were stimulated with ANG II for 0 and 5 h. Formazan production was then measured. Data are expressed as fold change in formazan in ANG II-stimulated cells relative to nontreated controls. C: WT/AT1 and SYF/AT1 cells were stimulated with ANG II for 0 and 24 h. Some cells were pretreated with 1 µM PKC MP for 1 h. All cells were then detached and counted using a hemocytometer. All data are the mean of three independent experiments.

ANG II-induced cell proliferation is significantly reduced in cells lacking c-Src/Yes/Fyn and in cells pretreated with PKC MP.

Here, we assessed ANG II-induced cell proliferation via three different methodologies. We first measured cellular ATP levels, since ATP amounts have previously been reported to be indicators of cell number (7). WT/AT₁ and SYF/AT₁ cells were treated with 10^–7 M ANG II, and intracellular ATP levels were measured. After 4 h of ANG II treatment, ATP levels had already increased over threefold in WT/AT₁ cells (Fig. 7A). ATP levels in SYF/AT₁ cells exhibited a significant increase in response to ANG II, but the ATP levels within these cells were significantly reduced compared with ANG II-treated WT/AT₁ control cells. Measurement of ANG II-induced formazan production, another indicator of cell proliferation (4), confirmed these results. Formazan production was significantly increased in WT/AT₁ cells after 5 h of ANG II treatment (Fig. 7B). Although formazan production also increased significantly in SYF/AT₁ cells, it was significantly reduced compared with levels in WT/AT₁ cells. Collectively, these data suggest that c-Src/Yes/Fyn influence ANG II-induced cell proliferation.

We further assessed ANG II-induced cell proliferation by direct cell count. WT/AT₁ and SYF/AT₁ cells were treated with 10^–7 M ANG II for 0 and 24 h. Cells were detached and counted. WT/AT₁ cell number was increased by over threefold when treated with ANG II, relative to nontreated controls (Fig. 7C). SYF/AT₁ cell number was also significantly increased, but this increase was significantly reduced compared with ANG II-treated WT/AT₁ cells. As such, these data show that ANG II-induced cell proliferation was markedly reduced in SYF/AT₁ cells lacking c-Src/Yes/Fyn-dependent signaling.

Finally, we assessed the effect of PKC inhibition of ANG II-induced cell proliferation. Both WT/AT₁ and SYF/AT₁ cells were pretreated with PKC MP and then stimulated with 10^–7 M ANG II. Changes in cell number were again assessed via a direct cell count. ANG II-induced cell proliferation was attenuated in WT/AT₁ cells pretreated with PKC MP compared with DMSO-treated controls (Fig. 7C). ANG II-induced cell proliferation was not significantly different in PKC MP-pretreated WT/AT₁ cells and SYF/AT₁ cells stimulated with ANG II. Furthermore, ANG II-induced cell proliferation was completely blocked in SYF/AT₁ cells pretreated with PKC MP. Thus both PKC and c-Src/Yes/Fyn mediate ANG II-induced cell proliferation through the activation of ERK1/2, although the mechanisms by which this occurs appear to be different, since ERK1/2 translocates into the nucleus in response to activation by PKC and remains in the cytoplasm when activated via c-Src/Yes/Fyn.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS NOTE ADDED IN PROOF REFERENCES

 
A diverse set of signaling pathways have been implicated in ERK1/2 activation, but the precise mechanisms of ERK1/2 activation in response to ANG II are not fully understood (30, 42). Furthermore, the cellular outcome associated with the activation of ERK1/2 via different signaling cascades is unclear. Are these signaling pathways functionally redundant, or does the activation of ERK1/2 by one pathway result in a different cellular outcome than when ERK1/2 is activated by another signaling cascade?

Here, we utilized MEF cells completely devoid of c-Src/Yes/Fyn. In doing so, we have completely eliminated all Src kinase function and the possibility that ERK1/2 activation can be mediated via any of these very similar family members. We found that while c-Src/Yes/Fyn tyrosine kinases do play a role in the activation of ERK1/2 as previously reported, ERK1/2 activation is not completely dependent on these proteins and persists at reduced levels in their absence. Interestingly, c-Src/Yes/Fyn are capable of activating only about 50% of intracellular ERK1/2. This seems to be a generalized phenomenon in other cells types as well, including CHO and RASM cells. An explanation for these results is that the remaining 50% of intracellular ERK1/2 is activated by c-Src/Yes/Fyn-independent mechanisms. We subsequently confirmed this, as we found that 50% of ANG II-induced ERK1/2 activation involved heterotrimeric G protein and PKC-dependent signaling. In summary, ANG II-induced ERK1/2 activation occurs via two specific mechanisms that work independent of one another.

Although both pathways activate an equal portion of ERK1/2 and contribute to cell proliferation, the mechanism whereby each pathway independently mediates this effect appears to be different. It had previously been thought that ERK1/2 must translocate into the nucleus to initiate events necessary for cell proliferation to occur, including the transcription of early response genes such as c-fos (5, 6, 30). Interestingly, we found that the loss of c-Src/Yes/Fyn had no effect on the ability of ERK1/2 to translocate into the nucleus. ERK1/2 was able to enter the nucleus in the absence of c-Src/Yes/Fyn; however, cell proliferation was still markedly reduced. An explanation for these findings is that ERK1/2 activated via c-Src/Yes/Fyn-dependent signaling acts upon cytoplasmic proteins to mediate proliferation, whereas ERK1/2 activated via PKC-dependent signaling translocates into the nucleus to directly mediate transcriptional events. This hypothesis was validated in an accompanying work by our group (11a).

The utility of having two mechanisms that dually activate ERK1/2 in response to stimulation of the AT₁R is intriguing. Both c-Src/Yes/Fyn and heterotrimeric G protein-dependent signaling appear to have an additive effect on ERK1/2 activation and subsequent cell proliferation in response to ANG II. Within adult mammalian systems, ANG II-induced cell proliferation is associated with abnormal cell proliferation during cardiovascular diseases and cancer, and to date, has not been implicated in cell growth and proliferation during a nondisease state (8, 39, 40, 43–45). As such, this study may have therapeutic merit, since local inhibition of both heterotrimeric G protein/PKC signaling as well as c-Src/Yes/Fyn-dependent signaling may be necessary to completely block ANG II-induced cell proliferation during disease states.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS NOTE ADDED IN PROOF REFERENCES

 
This work was supported by a Biomedical Research Support Program for Medical Schools Award to the University of Florida College of Medicine by the Howard Hughes Medical Institute, an American Heart Association Florida/Puerto Rico Affiliate Grant-in-Aid (0555359B), and National Institutes of Health Awards K01-DK-60471 and R01-HL-67277. M. D. Godeny was supported by a Pre-Doctoral Research Fellowship from the Florida/Puerto Rico Affiliate of the American Heart Association (0515138B).

	NOTE ADDED IN PROOF

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS NOTE ADDED IN PROOF REFERENCES

 
In the Articles in PresS version of this manuscript, the composite images representing Fig. 6 were not identified as such. We regret this error. Here, in this final-published version, Fig. 6 clearly shows these details, and the legend clearly states "Cells that were imaged from independent fields are outlined in white; fields from within the same experimental treatment group are shown as a composite image within the same panel."

	ACKNOWLEDGMENTS

 
We are indebted to Dr. Philippe Soriano for providing us with the mouse embryonic fibroblasts and Dr. Kenneth Bernstein for the CHO cells. We also thank Dr. Philip J. S. Stork for kindly providing us with the GFP-ERK2 plasmid.

	FOOTNOTES

 

Address for reprint requests and other correspondence: P. P. Sayeski, Dept. of Physiology and Functional Genomics, Univ. of Florida College of Medicine, P.O. Box 100274, Gainesville, FL 32610 (e-mail: psayeski{at}phys.med.ufl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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