Published online before print August 28, 2008, doi: 10.1161/CIRCRESAHA.108.179754
© 2008 American Heart Association, Inc.
Molecular Medicine |
Shp2 Negatively Regulates Growth in Cardiomyocytes by Controlling Focal Adhesion Kinase/Src and mTOR Pathways
From the Department of Internal Medicine (T.M.M., C.F.M.Z.C., A.M.S., P.K.P., M.J.A.S., K.G.F.) and Department of Medical Genetics (V.D.B.P., I.L.-C.), Faculty of Medical Sciences, State University of Campinas, Sao Paulo, Brazil.
Correspondence to Kleber G. Franchini, MD, PhD, Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Cidade Universitária "Zeferino Vaz", 13081-970, Campinas, Sao Paulo, Brazil. E-mail franchin{at}unicamp.br
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Abstract |
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The aim of this study was to investigate whether Shp2 (Src homology region 2, phosphatase 2) controls focal adhesion kinase (FAK) activity and its trophic actions in cardiomyocytes. We show that low phosphorylation levels of FAK in nonstretched neonatal rat ventricular myocytes (NRVMs) coincided with a relatively high basal association of FAK with Shp2 and Shp2 phosphatase activity. Cyclic stretch (15% above initial length) enhanced FAK phosphorylation at Tyr397 and reduced FAK/Shp2 association and phosphatase activity in anti-Shp2 precipitates. Recombinant Shp2 C-terminal protein tyrosine phosphatase domain (Shp2-PTP) interacted with nonphosphorylated recombinant FAK and dephosphorylated FAK immunoprecipitated from NRVMs. Depletion of Shp2 by specific small interfering RNA increased the phosphorylation of FAK Tyr397, Src Tyr418, AKT Ser473, TSC2 Thr1462, and S6 kinase Thr389 and induced hypertrophy of nonstretched NRVMs. Inhibition of FAK/Src activity by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine} abolished the phosphorylation of AKT, TSC2, and S6 kinase, as well as the hypertrophy of NRVMs induced by Shp2 depletion. Inhibition of mTOR (mammalian target of rapamycin) with rapamycin blunted the hypertrophy in NRVMs depleted of Shp2. NRVMs treated with PP2 or depleted of FAK by specific small interfering RNA were defective in FAK, Src, extracellular signal-regulated kinase, AKT, TSC2, and S6 kinase phosphorylation, as well as in the hypertrophic response to prolonged stretch. The stretch-induced hypertrophy of NRVMs was also prevented by rapamycin. These findings demonstrate that basal Shp2 tyrosine phosphatase activity controls the size of cardiomyocytes by downregulating a pathway that involves FAK/Src and mTOR signaling pathways.
Key Words: hypertrophy • cell signaling • cardiomyocytes • focal adhesion kinase
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Introduction |
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Cardiomyocytes respond to increases in functional demand by hypertrophic growth. This reactive hypertrophy involves concerted gene expression and the accumulation of myocyte proteins and organelles that are coordinated by signaling cascades activated by mechanical stress and a variety of soluble endocrine, paracrine, and autocrine factors.1
Focal adhesion kinase (FAK) is involved in the hypertrophic response of cardiomyocytes to biomechanical stress and agonists such as phenylephrine and endothelin.2–6 In cardiomyocytes, FAK is highly expressed, has a relatively low basal level of activity, and is promptly activated by hypertrophic stimuli.2–4,7 FAK overexpression upregulates marker genes associated with hypertrophy in cardiomyocytes,8 whereas a loss of FAK function impairs the upregulation of these genes in response to hypertrophic stimuli.4–6 The involvement of FAK in reactive hypertrophy has been confirmed in mice with cardiomyocyte-restricted FAK deletion or myocardial FAK silencing,9–11 but the mechanistic pathways that link FAK to hypertrophy in cardiomyocytes remain uncertain.
Intracellularly, FAK is kept quiescent by intramolecular interactions mediated by its FERM (protein 4.1, ezrin, radixin, and moesin homology) N-terminal domain that locks the catalytic domain into an inactive configuration.12 Interaction with the C-terminal region of cardiac myosin heavy chain may be important in maintaining FAK quiescence in cardiomyocytes.13 The autoinhibition of FAK may be relieved by rupture of the intramolecular interaction between the FERM and the catalytic domain,14 with subsequent autophosphorylation at Tyr397 that recruits and activates Src.15 The FAK/Src complex phosphorylates various substrates that, in turn, activate several classes of signaling molecules.16 In cardiomyocytes, biomechanical stress increases FAK phosphorylation at Tyr397 and activates the FAK/Src complex.4,7
Tyrosine phosphatases provide an additional level for regulating FAK phosphorylation and activity.16 In agreement with this, the constitutive activation of Shp2 reduces whereas a lack of this protein increases FAK phosphorylation.17–19 In cardiomyocytes, Shp2 activation is involved in FAK tyrosine dephosphorylation and apoptosis after stimulation with cathepsin G.20 However, it is not known whether Shp2 regulates basal or stretch-induced FAK phosphorylation and signaling in these cells.
In this work, we investigated the influence of Shp2 on basal and stretch-induced activation of FAK by depleting the levels of Shp2 with small interfering (si)RNA and then examining the phosphorylation of the FAK/Src complex, extracellular signal-regulated kinase (ERK), AKT, TSC2, and S6 kinase (S6K). We also investigated the effect of Shp2 depletion on the size of neonatal rat ventricular myocytes (NRVMs). Experiments with pharmacological inhibitors suggested a role for FAK/Src signaling in the activation of a downstream pathway (consisting of AKT, TSC2, and mTOR [mammalian target of rapamycin]/S6K) that mediated the effects of Shp2 depletion and stretch on cardiomyocyte size. Overall, our results indicate that Shp2 negatively controls basal FAK phosphorylation and limits cell size by modulating the activity of the mTOR signaling network.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Culture and Stretching of NRVMs
Primary cultures of NRVMs from 1- to 2-day-old Wistar rats were prepared and stretched 15% above initial length in Bioflex collagen culture plates (Flexcell International), as previously reported.4 Nconatal rats were handled in compliance with the principles of laboratory animal care formulated by the Animal Care and Use Committee of the State University of Campinas.
Pull-Down Assay
Glutathione S-transferase (GST)-tagged Shp2-PTP (provided by Dr Eugene Y. Chin, Brown University Medical School, Providence, RI) and full-length FAK (Invitrogen) were used for pull-down assays. GST-conjugated glutathione beads were used as a negative control for nonspecific binding. The pull-down pellets were run on SDS-PAGE, and the membranes were blotted with anti-FAK antibody.
Phosphatase Activity
The protein tyrosine phosphatase activities of anti-Shp2 and anti-FAK immune complexes and recombinant Shp2-PTP were assayed based on the hydrolysis of pp60-cSrc phosphoregulatory peptide.
FAK Tyrosine Dephosphorylation Assay
The activity of Shp2-PTP toward phosphorylated FAK was assessed in anti-FAK immune complexes precipitated from NRVMs. FAK tyrosine dephosphorylation was examined in immunoblots with anti-phospho-specific (Tyr397) FAK antibody.
siRNA Synthesis and Transfection Protocol
siRNAs targeted to rat FAK or Shp2 or to GFP were designed, synthesized, and transfected in NRVMs as previously reported11 and as detailed in the online data supplement.
Immunoblotting
Samples of NRVMs were centrifuged and the soluble fraction was resuspended in Laemmli loading buffer before separation on 8% SDS–polyacrylamide gels. Proteins were transferred from the gels to nitrocellulose membranes that were then incubated with primary antibodies exposed to 125I-labeled protein A.
Immunoprecipitation
Samples of NRVMs were homogenized in solubilization buffer, and the supernatant was used for the assays. Equal amounts of total protein from NRVMs extracts were used for immunoprecipitation with specific antibodies and protein A-Sepharose 6MB.
Multiplex RT-PCR
Transcripts of cardiac β-myosin heavy chain and β-actin were analyzed by multiplex RT-PCR as described in the online data supplement.
Cytochemistry
Cells were fixed in 4% (wt/vol) paraformaldehyde and stained with tetramethylrhodamine isothiocyanate-phalloidin (diluted 1:50 in PBS), as previously described.4 The cells were mounted in Vectashield and examined by fluorescence microscopy (LEICA DM4500B). Cell images were used for quantification of the surface area of cardiomyocytes as detailed in the online data supplement.
Statistical Analysis
The densitometric readings of the immunoblots are expressed as the percentage change relative to the controls for the number of independent experiments (n) indicated, each done with separate primary cultures (
70 neonatal rat hearts were used per culture). Cell surface area was obtained by the analysis of individual cardiomyocytes (at least 250 cells counted for each treatment) and presented as the means±SEM. Statistical comparisons of the actual densitometric readings and cell surface area were done with ANOVA and Bonferroni’s multiple-range test. Values of P<0.05 indicated statistical significance.
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Results |
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Stretch Modulates FAK/Shp2 Association in NRVMs
As reported previously,4–6 stretch induced FAK phosphorylation at Tyr397 but did not change FAK protein expression in NRVMs (Figure 1A). Immunoblots of GAPDH were used as internal controls for protein loading. High amounts of Shp2 were found in the anti-FAK immune complex from nonstretched cells, indicating a basal association between FAK and Shp2 in NRVMs (Figure 1B). The specificity of the FAK/Shp2 coimmunoprecipitation assay was confirmed in assays with beads only (Figure II in the online data supplement). Cyclic stretch lasting for 10 to 60 minutes markedly reduced the amount of Shp2 in the immune complex of anti-FAK antibody but did not change the amount of FAK or Shp2 expressed in NRVMs. The fraction of Shp2 associated with FAK was estimated by comparing the amount of Shp2 immunoprecipitated by an excess of anti-FAK antibody with the amount of Shp2 in the anti-Shp2 immune complex. Shp2 precipitated by anti-FAK accounted for
20% of the total Shp2 immunoprecipitated with anti-Shp2 antibody (Figure 1C). Binding assays with recombinant full-length FAK and GST-tagged-Shp2-PTP or GST showed that FAK coprecipitated with GST-Shp2-PTP but not with GST (Figure 1D), suggesting a direct interaction between nonphosphorylated FAK and Shp2-PTP.
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Recombinant Shp2-PTP Dephosphorylates FAK In Vitro
Control experiments were done to determine the activity of recombinant Shp2-PTP (Figure 2A and 2B). The phosphatase activity of Shp2-PTP toward pp60-cSrc phosphoregulatory peptide increased with the length of incubation and with the amount of enzyme. Shp2-PTP dephosphorylated FAK immunoprecipitated from nonstretched and stretched cells (Figure 2C). Similar amounts of FAK were immunoprecipitated from nonstretched and stretched NRVMs.
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Stretch Regulates Shp2 Phosphatase Activity in NRVMs
Figure 2D shows that the anti-Shp2 immune complex from stretched NRVMs had a lower protein tyrosine phosphatase activity (70% at 10 minutes) compared to that from nonstretched cells. Reductions in phosphatase activity, although less intense than those seen in 10-minute-stretched cells, were still detected in immunoprecipitates from NRVMs stretched for 30 and 60 minutes. The amount of immunoprecipitated Shp2 was similar in samples from nonstretched or stretched NRVMs. The protein tyrosine phosphatase activity was also reduced in the anti-FAK immunoprecipitates from stretch in comparison to that from nonstretched cells (Figure 2E).
Phosphorylation of Shp2 at Tyr542 is required for the adapter function of Shp2 and may directly regulate its phosphatase activity.21 Cyclic stretch increased the amount of Shp2 phosphorylated at Tyr542, despite the reduction in phosphatase activity of the anti-Shp2 immune complex (Figure 2F).
Shp2 Silencing Increases FAK Phosphorylation in Nonstretched NRVMs
To assess the functional importance of Shp2 in FAK signaling in vivo, NRVMs were depleted of Shp2 with specific siRNA. The efficiency of siRNA transfection in NRVMs was probed with fluorescent oligonucleotide. An intracellular fluorescent signal was observed in perinuclear foci of NRVMs transfected with fluorescent oligonucleotide (Figure 3A). Cells transfected with nonfluorescent siRNA targeted to Shp2 were used as controls (Figure 3B). The efficiency of fluorescent oligonucleotide transfection in NRVMs was routinely at least 80% (supplemental Figure III). Transfection of siRNA targeted to Shp2 in NRVMs reduced Shp2 by 80%, whereas no change in Shp2 expression was seen in cells treated with Lipofectamine alone or irrelevant siRNA targeted to GFP (Figure 3C). Shp2 depletion was paralleled by a marked reduction in the phosphatase activity of the anti-Shp2 immune complex (Figure 3D). The specificity of siRNA targeted to Shp2 was initially tested by comparing the extent of Shp2 silencing and the phenotypic changes induced by siRNAs targeted to distinct regions of Shp2 mRNA (supplemental Figure I). The specificity of the siRNA targeted to Shp2 was further confirmed by the lack of change in the expression of PTP-1B, a close family member of Shp2 (Figure 3E), and unrelated FAK.
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Shp2 silencing markedly increased FAK Tyr397 phosphorylation in nonstretched NRVMs, but did not potentiate FAK phosphorylation in stretched cells (Figure 3F). Lipofectamine or siRNA targeted to GFP did not affect basal FAK phosphorylation (supplemental Figure IV). To test whether the influence of Shp2 depletion on FAK phosphorylation was dependent on Shp2 catalytic activity additional experiments were done with the phosphatase inhibitor TFMS [bis(4-trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene].22 Assays with anti-Shp2 immune complex confirmed the ability of TFMS to inhibit the catalytic activity of Shp2 in vivo (supplemental Figure V). As shown in Figure 3G, treatment with TFMS markedly increased FAK phosphorylation at Tyr397 in nonstretched NRVMs. However, as with Shp2 depletion, treatment with TFMS did not potentiate the effect of stretch on FAK phosphorylation.
In addition to FAK Tyr397, Shp2 silencing increased FAK tyrosine phosphorylation at Tyr861 and Tyr925, and Src phosphorylation at Tyr418 (Figure 4A), suggesting that depletion of Shp2 promotes activation of the FAK/Src complex. To further assess the functional consequences of an Shp2 deficiency on downstream signaling, we examined the phosphorylation of the ERK, AKT, and mTOR pathways in nonstretched NRVMs treated with siRNA targeted to Shp2. Figure 4B shows that depletion of Shp2 enhanced AKT Ser473, TSC2 Thr1462, and S6K Thr389 phosphorylation in nonstretched NRVMs, whereas no consistent change was seen in ERK Thr202/Tyr204 phosphorylation. Figure 4C shows that treatment with PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine} abolished the phosphorylation of AKT, TSC2 and S6K in NRVMs depleted of Shp2.
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Shp2 Depletion Induces Hypertrophic Growth of NRVMs
We next sought to determine whether the depletion of Shp2 might result in the hypertrophy of cardiomyocytes. Treatment with siRNA targeted to GFP did not change the overall morphology of NRVMs, as compared to untreated cells (supplemental Figure VI). Figure 5A through 5E shows that the depletion of Shp2 increased the size of the cardiomyocytes by 2.7-fold. In addition, the depletion of Shp2 with siRNA increased the expression of β-myosin heavy chain (β-MHC) in nonstretched NRVMs (Figure 5F). Treatment with PP2 abolished the increases in β-MHC expression and cell size induced by the depletion of Shp2, indicating that these effects were dependent on FAK/Src signaling. In contrast, treatment with PP2 did not alter β-MHC expression or cell size in NRVMs transfected with siRNA targeted to GFP.
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We then treated cells with rapamycin to examine the requirement for the mTOR pathway in NRVM hypertrophy induced by Shp2 depletion. Treatment with rapamycin significantly attenuated the hypertrophic growth of NRVMs depleted of Shp2 (Figure 5G through 5K), indicating that Shp2 negatively regulates cell size upstream to mTOR.
FAK/Src Complex Mediates Stretch-Induced Hypertrophy in NRVMs
Because FAK/Src signaling is suspected to play a role in hypertrophy caused by biomechanical stimuli, we investigated whether signaling via the mTOR pathway is involved in stretch-induced hypertrophy. To assess the role of FAK in stretch-induced hypertrophy, FAK function was disrupted with the FAK/Src inhibitor PP2 or with siRNA targeted to FAK. The optimization of FAK silencing by siRNA in cardiomyocytes is described elsewhere.11 The treatment of NRVMs with siRNA targeted to FAK reduced FAK protein expression by 80% (Figure 6A). Treatment with PP2 or the depletion of FAK abolished the stretch-induced FAK phosphorylation at Tyr397 and Src phosphorylation at Tyr418. Cyclic stretch stimulated the phosphorylation of ERK Thr202/Tyr204, AKT Ser473, TSC2 Thr1462, and S6K Thr389 in NRVMs (Figure 6B), whereas a deficiency in FAK signaling abrogated the stretch-induced phosphorylation of ERK, AKT and S6K. Although the disruption of FAK signaling did not affect the shape and size of nonstretched cells (data not shown), treatment with PP2 or FAK depletion markedly attenuated the stretch-induced hypertrophy in NRVMs (Figure 6C through 6F and 6H). Rapamycin abolished the stretch-induced hypertrophy, indicating an involvement of the mTOR signaling pathway in this phenomenon downstream to FAK/Src signaling (Figure 6G and 6H).
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Discussion |
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The results of this study indicate a role for the tyrosine phosphatase Shp2 in regulating the basal activity of FAK in cardiomyocytes. The relevance of this effect was underscored by the demonstration that Shp2 depletion induced the expression of β-MHC and a hypertrophic morphology in nonstretched NRVMs by a mechanism dependent on FAK/Src and mTOR activation.
Control of FAK Phosphorylation by Shp2 in Cardiomyocytes
Phosphorylation at Tyr397 positively regulates FAK activity and signaling.4,7,16 As shown here, FAK phosphorylation at Tyr397 was enhanced in NRVMs depleted of Shp2, supporting a view that Shp2 regulates basal FAK activity in cardiomyocytes. Further, Shp2 depletion enhanced Src Tyr418 and FAK Tyr861/Tyr925 phosphorylation, suggesting that the depletion of this protein was sufficient to bypass the mechanisms that activate the FAK/Src complex in response to biomechanical stress and soluble factors. In agreement with these data, FAK hyperphosphorylation has been observed in fibroblasts depleted of Shp2.17–19 However, other studies have reported hypophosphorylation of FAK and Src in Shp2 deficient fibroblasts.23,24 The reason for these discrepancies is unclear but may be related to differences in the regulation of FAK and Src in distinct cell types and experimental models.
An essential question raised by our data is how Shp2 negatively regulates basal FAK activity. In this regard, the finding that FAK phosphorylation was increased in NRVMs treated with the phosphatase inhibitor TFMS indicates that the control of basal FAK phosphorylation requires Shp2 catalytic activity. This notion is supported by the finding that FAK can be dephosphorylated by Shp2-PTP, which might also explain the correlation between the relatively high levels of phosphatase activity and the low levels of FAK phosphorylation in nonstretched NRVMs. Additionally, we found that Shp2-PTP may physically interact with nonphosphorylated FAK and that in nonstretched cells Shp2 is associated with FAK, again suggesting the possibility that FAK and Shp2 may form a complex that regulates basal FAK phosphorylation in cardiomyocytes.
An additional question in this model is what mechanisms maintain basal Shp2 activity in nonstretched NRVMs. This aspect was not examined in this study, but, because Shp2 catalytic activity is positively controlled by the binding of its N-terminal SH2 domain to phosphotyrosyl protein ligands, it is possible that interaction with basally phosphorylated protein partners such as Gab2, for instance, might sustain a relatively high basal level of Shp2 activity in nonstretched cardiomyocytes.21 However, further studies are necessary to address this issue.
Our results also suggest that a reduction in Shp2 activity toward FAK has a permissive role in the biomechanically induced activation of FAK in NRVMs. Accordingly, stretch-induced FAK phosphorylation was paralleled by reductions in Shp2 tyrosine phosphatase activity and in the association between FAK and Shp2. This suggests the possibility that FAK activation by mechanical stress in cardiomyocytes may depend on the downregulation of Shp2 phosphatase activity and the dissociation of FAK from Shp2. In this regard, the activation of FAK in cardiomyocytes requires the relocation of this protein to Z-discs and costameres by an intricate process that involves activation of the RhoA/ROCK signaling complex and the rearrangement of cytoskeletal filaments.4,5,13,25 Interestingly, Shp2 activity is markedly increased in cells when RhoA is downregulated or when stress fibers are disrupted.19,26,27 In contrast, the binding of Src to FAK reduces the susceptibility of FAK to tyrosine phosphatases.28 Together, these data suggest the possibility that FAK/Src association, the activation of RhoA/ROCK signaling and the rearrangement of cytoskeletal proteins may reduce the susceptibility of FAK to Shp2 phosphatase activity, tune down Shp2 catalytic activity, and promote the dissociation of FAK from Shp2, thereby contributing to activation of the FAK/Src complex in stretched cardiomyocytes. Once again, further studies are necessary to substantiate this hypothesis.
Given that the phosphorylation of tyrosine residues in the C-terminal domain of Shp2 is suspected to regulate Shp2 phosphatase activity, we expected to find a reduction in the phosphorylation of Shp2 Tyr542 in samples from stretched NRVMs. Instead, we found enhanced phosphorylation of Shp2 Tyr542 in samples from stretched NRVMs, despite the reduction in the phosphatase activity of Shp2 immunoprecipitates. The reason for this paradox is unclear, although there is evidence that Shp2 phosphorylation is not an absolute requirement for Shp2 catalytic activity.29 Hence, the phosphorylation of Shp2 may not necessarily provide an accurate indication of Shp2 phosphatase activity in our experimental model.
Shp2 and Cardiomyocyte Hypertrophy
A major new finding of this study is that the depletion of Shp2 induces the expression of β-MHC and cardiomyocyte hypertrophy. This implies that Shp2 may negatively regulate pathways involved in controlling cell size. In this regard, our results identified a pathway by which FAK/Src, AKT, and mTOR may interact to stimulate cardiomyocyte hypertrophy after Shp2 depletion. The depletion of Shp2 enhanced AKT, TSC2, and S6K phosphorylation by a mechanism dependent on FAK/Src activation, as suggested by the lack of phosphorylation of these signaling proteins in NRVMs treated with PP2. Additionally, pharmacological inhibition of the FAK/Src and mTOR pathways suppressed the hypertrophy induced by Shp2 depletion. These data substantiate the recent demonstration that Shp2 negatively regulates growth in fibroblasts by interfering with the mTOR/S6K pathway.30 The data are also consistent with evidence for a link between these signaling molecules and the regulation of hypertrophy in cardiomyocytes. For example, FAK controls AKT via interaction with the p85 subunit of phosphatidylinositol 3-kinase,7,16 and AKT regulates mTOR primarily through the phosphorylation of TSC2 at Thr1462.31 FAK also directly interacts with and inhibits TSC2, thereby influencing cell size through regulation of the mTOR/S6K pathway.32 An increase in cardiomyocyte size has been observed following the stimulation of AKT by the upstream activator of phosphatidylinositol 3-kinase and after inhibition of PTEN, the enzyme that degrades inositol 3,4,5-trisphosphate.33 In addition, a constitutively active mutant of AKT increases cardiomyocyte size.34 The hypertrophy caused by the expression of this active AKT is attenuated by rapamycin, which indicates that mTOR is the principal effector.34 Rapamycin also blocks left ventricular hypertrophy induced by mechanical stress.35
The finding that the AKT and mTOR pathways may be upregulated by FAK/Src activation after Shp2 depletion prompted us to investigate whether these pathways were also involved in hypertrophy in response to prolonged stretch. Consistent with the notion that FAK/Src signaling is critical for the hypertrophic response to prolonged stretch, our results showed that deficient FAK/Src signaling (produced by FAK depletion or treatment with PP2) markedly attenuated the hypertrophy of NRVMs induced by prolonged cyclic stretch. Additionally, AKT, TSC2, and S6K were phosphorylated in response to stretch by a mechanism mediated by FAK/Src activation. Rapamycin blunted the stretch-induced hypertrophy of NRVMs. Together, these findings highlight a potentially critical role for AKT and mTOR as downstream mediators of the prohypertrophic actions of the FAK/Src signaling complex. Of note, FAK/Src activation following Shp2 depletion leads to increases in cell size and in the expression of β-MHC, which generally is associated with pathological hypertrophy. Accordingly, previous studies have indicated that persistent activation of FAK may be related to the expression of markers of pathological hypertrophy and deterioration of myocardium.4,5,11 In line with this, persistent activation of AKT and mTOR has been shown to promote pathological hypertrophy,34 whereas short-term and low levels of AKT and mTOR pathways activation may function preferentially as regulators of physiological hypertrophy.36
Our results suggest that AKT mediates the effects of FAK/Src signaling on mTOR. However, we cannot exclude the existence of additional mediators between the FAK/Src and mTOR pathways. Indeed, previous studies have implicated ERK as a downstream mediator of FAK/Src signaling induced by hypertrophic stimuli in cardiomyocytes.5,7,10 In agreement with this, we observed that cyclic stretch resulted in ERK1/2 activation by a mechanism mediated by FAK/Src signaling. The hypertrophic effect of ERK activation can be mediated by numerous downstream targets, including the inactivation of TSC2 by phosphorylating sites that differ from the AKT target sites.37 ERK may therefore act synergistically with AKT to activate mTOR and to mediate the hypertrophic response to mechanical stress. A schematic representation of the proposed growth pathway of FAK and Shp2 is shown in Figure 7. However, there was no consistent change in ERK activity in NRVMs depleted of Shp2, suggesting that ERK did not contribute to the hypertrophy induced by Shp2 depletion. In contrast to these findings, Shp2 depletion has been reported to reduce the basal activity of ERK and to attenuate its activation by hypertrophic stimuli in cardiomyocytes.38 The reason for this apparent paradox is unknown, but the simplest explanation is that it is related to differences in the experimental models and stimuli used in the various studies.
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Overall, the results of this study indicate that Shp2 may be an essential component of the signaling pathways involved in cardiomyocyte hypertrophy. Interestingly, despite the recent demonstration of dilated cardiomyopathy in Shp2 null mice,38 Shp2 loss-of-function in Leopard syndrome is accompanied by hypertrophic cardiomyopathy.39 Conversely, only mild changes in myocardial mass have been reported in patients and mice with Shp2 gain-of-function in Noonan syndrome.40,41 These observations imply that a reduced or increased activity of Shp2 may determine hypertrophic or hypotrophic phenotypes, respectively. It will be interesting to determine whether signaling mediated by the FAK/Src and mTOR pathways plays a role in the pathogenesis of the cardiac changes associated with the Leopard and Noonan syndromes.
In conclusion, Shp2 negatively regulates the basal activity of the FAK/Src signaling complex in cardiomyocytes. This regulation, which is effected by modulating the mTOR complex downstream, has a central role in controlling cardiomyocyte size under initial conditions and during hypertrophy in response to biomechanical stress. The relative importance of Shp2 activity in the pathophysiology of cardiac hypertrophy and heart failure remains to be determined.
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Acknowledgments |
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We thank Dr Stephen Hyslop (Department of Pharmacology, State University of Campinas) for comments and valuable suggestions.
Sources of Funding
Funding for this work was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grants 2006/54878-3, 2006/55920-3, and 2004/10167-0 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grants 305604/2006-6 and 474650/2006-5.
Disclosures
None.
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Footnotes |
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Original received December 10, 2007; resubmission received May 19, 2008; revised resubmission received August 17, 2008; accepted August 20, 2008.
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