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Endocrinology, doi:10.1210/en.2007-0312
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Endocrinology Vol. 149, No. 2 461-465
Copyright © 2008 by The Endocrine Society

BRIEF COMMUNICATION

Caveolin-1 Down-Regulation Inhibits Insulin-Like Growth Factor-I Receptor Signal Transduction in H9C2 Rat Cardiomyoblasts

Barbara Salani, Lucia Briatore, Silvano Garibaldi, Renzo Cordera and Davide Maggi

Departments of Endocrinology and Medicine (B.S., L.B., R.C., D.M.) and Internal Medicine (S.G.), University of Genova, 5-16132 Genova, Italy

Address all correspondence and requests for reprints to: Davide Maggi, M.D., Ph.D., Department of Endocrinology and Medicine, Viale Benedetto XV, 6, University of Genova, 5-16132 Genova, Italy. E-mail: davide.maggi{at}unige.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Caveolin (Cav)-1, the major caveolar protein, directly interacts with IGF-I receptor (IGF-IR) and its intracellular substrates. To determine the role of Cav-1 in IGF-IR signaling, we transfected H9C2 cells with small interfering RNA specific for Cav-1-siRNA. The selective down-regulation of Cav-1 (90%) was associated with a smaller reduction of Cav-2, whereas Cav-3 expression was unaffected. A significant reduction of IGF-IR tyrosine phosphorylation in Cav-1-siRNA H9C2 cells was found compared with H9C2 control cells (Ctr-siRNA). The reduced IGF-IR autophosphorylation resulted in a decrease of insulin receptor substrate-1, Shc, and Akt activation. In addition, in Cav-1-siRNA H9C2 cells, IGF-I did not prevent apoptosis, suggesting that Cav-1 is required to mediate the antiapoptotic effect of IGF-I in cardiomyoblasts. The down-regulation of Cav-1 decreased IGF-IR activation and affected the ability of IGF-I to prevent apoptosis after serum withdrawal also in human umbilical vein endothelial cells. These results demonstrate that: 1) Cav-1 down-regulation negatively affects IGF-IR tyrosine phosphorylation; 2) this effect causes a reduced activation of insulin receptor substrate-1, Shc, and Akt; and 3) Cav-1 is involved in IGF-IR antiapoptotic signaling after serum deprivation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CAVEOLAE ARE invaginations of the plasma membrane implied in the regulation of vesicular transport and intracellular signaling (1, 2). Caveolin (Cav)-1 regulates the integrity of caveolae function and represents the principal component of the caveolar compartment (3) where several tyrosine kinase receptors are localized, including IGF-I receptor (IGF-IR) (4). IGF-I tyrosine phosphorylates Cav-1 and activates Shc and insulin receptor substrate-1 (IRS-1) (4, 5, 6) in caveolae (7, 8), supporting a role of these specialized membrane structures on the IGF-IR transduction pathway (4, 7, 8).

The role of Cav-1 in IGF-I signaling is controversial. Although in 3T3-L1 cells Cav-1 silencing by small interfering RNA (siRNA) does not affect IGF-I signaling as well as cell differentiation (9), we have previously demonstrated that Cav-1 regulates IGF-IR signaling in endothelial cells. In fact, down-regulation of Cav-1 abolishes insulin as well as IGF-I stimulated endothelial nitric oxide synthase phosphorylation in human umbilical vein endothelial cells (HUVECs), demonstrating the importance of Cav-1 in the IGF-IR transduction pathway (10).

Recently, it has been suggested that Cav-1 may be important for postnatal cardiovascular function, including endothelial function (11, 12).

Because IGF-I protects cardiac cells from serum withdrawal-induced apoptosis (13), we studied the effect of Cav-1 down-regulation on IGF-IR in the rat cardiomyoblast cell line H9C2. H9C2 cells constitutively also express Cav-2 and Cav-3, thus they represent an effective model to determine the specific role of Cav-1 in IGF-IR signaling.

In Cav-1 down-regulated H9C2 cells, activation of IGF-IR as well as phosphorylation of its proximal downstream substrates IRS-1 and Shc are greatly reduced. In addition, Akt kinase phosphorylation is decreased. In particular, the silencing of Cav-1 impairs the ability of IGF-IR to prevent serum withdrawal-induced apoptosis. Here, we show that in H9C2 cells and HUVECs, IGF-IR signal transduction is Cav-1 dependent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells culture and transfection
The H9C2 cell line was obtained from American Type Culture Collection (Manassas, VA). H9C2 cells were cultured in DMEM at 37 C in 5% CO2, and the medium was changed every 2–3 d.

HUVECs (Cambrex Corp., East Rutherford, NJ) were cultured in endothelial basal media-2 supplemented with growth factors, 2 mM glutamine, 2% fetal bovine serum, 100 U/ml penicillin G, and 100 mg/ml streptomycin sulfate, and used between passages 7 and 8.

Cav-1 gene (GenBank accession no. NM001753) silencing was performed using a mix of two siRNAs directed against the following target sequences: 5'-AACAGGGCAACATCTACAAGC-3' (A), and 5'-AACCAGAAGGGACACACAGTT-3' (B). Sequence corresponding to the control (Ctr) siRNA was as follows: 5'-AAAGAGCGACTTTACACACTT-3'.

H9C2 cells and HUVECs were grown to 80% confluence and transfected with siRNA (200 nM) using Oligofectamine (Invitrogen Corp., Carlsbad, CA). After 5 h, transfection mixtures were replaced with regular medium. Forty-eight hours after transfection, H9C2 cells and HUVECs, serum starved for 24 h, were stimulated for 0, 5, and 15 min with IGF-I (100 nM) and lysed. Alternatively 48 h after transfection, H9C2 cells and HUVECs siRNA transfected cells were cultured in serum-free medium with or without IGF-I (100 nM) for other 24 h. Cells were then subjected to fluorescein isothiocyanate (FITC) annexin V/propidium iodide (PI) double staining and fluorescence-activated cell sorter analysis to evaluate apoptosis.

Immunoblot
H9C2 cells were lysed in radioimmunoprecipitation assay buffer, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM sodium orthovanadate, and 0.1 µM phenylmethylsulfonyl fluoride. Total cellular proteins were separated on SDS-PAGE and transferred to nitrocellulose. The immunoblots were performed as described by Repetto et al. (10). Bands of interest were measured by densitometry using the National Institutes of Health (NIH) Image software (Bethesda, MD), and statistical analysis was performed with Prism software (GraphPad Software Inc., San Diego, CA).

Anti-Cav-1, Cav-2, and Cav-3 antibodies were purchased from BD Transduction Laboratories (Franklin Lakes, NJ). Anti-IGF-IR and phosphotyrosine antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-IRS-1, Akt, Shc, MAPK, phospho-Akt (ser473), phospho-Shc (tyr317), and phospho-MAPK antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA).

Flow cytometric detection of apoptosis
Double staining for FITC-annexin V binding and for cellular DNA using PI was performed as follows. After washing twice with PBS, 1 x 106 cells were resuspended in binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2]. FITC-Annexin V was added to a final concentration of 1 µg/ml Annexin V. A 0.1 volume of PI (10 µg/ml in binding buffer) was added, resulting in a final concentration of 1 µg PI/ml cell suspension. The mixture was incubated for 10 min in the dark at room temperature, and cellular fluorescence was then measured by flow cytometry with a FACScan apparatus (BD).

Statistical analysis
All experiments were performed at least three times. Data are expressed as the mean ± SD. Statistical differences were assessed by one-way ANOVA. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
H9C2 cells constitutively express Cav-1, Cav-2, and Cav-3. To down-regulate Cav-1 in the rat H9C2 cell line, we used siRNA. Cav-1 siRNA specifically reduced Cav-1 expression by 90% as compared with Ctr cells, whereas the Cav-2 amount was slightly decreased. Cav-3, IGF-IR, as well actin expression were unchanged (Fig. 1Go).


Figure 1
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  		FIG. 1. Effect of Cav-1 down-regulation on Cav-2, Cav-3, IGF-IR, IRS-1, and actin protein expression after siRNA transfection. H9C2 cells were transfected with Cav-1-siRNA and scrambled Ctr siRNA. Seventy-two hours from the transfection, serum-starved cells were immunoblotted with the anti-Cav-1, Cav-2, Cav-3, IGF-IR, and actin antibodies. The blots represent three independent experiments.

 
Down-regulation of Cav-1 reduced the ability of IGF-I to stimulate IGF-IR autophosphorylation. As shown in Fig. 2Go, in H9C2 Ctr cells, IGF-I caused IGF-IR tyrosine phosphorylation with a peak after 5 min (9 ± 1-fold of increase over the basal). In Cav-1-siRNA H9C2 cells, this peak of activation was completely lost (3.6 ± 1.2) (P < 0.01 compared with Ctr cells).


Figure 2
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  		FIG. 2. IGF-IR time course of activation after Cav-1 down-regulation. A, H9C2 cells were transfected with Cav-1-siRNA and scrambled Ctr siRNA as described in Materials and Methods. Seventy-two hours from the transfection, serum-starved cells were treated with IGF-I (100 nM) for the indicated times and lysed. Total cell lysates were immunoprecipitated with an antiphosphotyrosine antibody. Immunoprecipitated proteins were resolved on 8% SDS-PAGE, transferred onto nitrocellulose, blotted with an antibody directed against IGF-IR (IGF-IR-P), and developed by enhanced chemiluminescence. B, Bands obtained by Western blot analysis were quantified by densitometry using NIH Image software and were expressed as fold of increase under the basal. Vertical bars represent means ± SD of three independent experiments. *, P < 0.01.

 
We next examined the ability of IGF-IR to tyrosine phosphorylate its proximal downstream targets IRS-1 and Shc. After Cav-1 siRNA treatment, tyrosine phosphorylation of IRS-1 and p66 Shc subunit in response to IGF-I was severely reduced. This effect did not depend on a reduced expression of IRS-1 and p66 Shc that was superimposable in Cav-1-siRNA H9C2 cells compared with Ctr cells (Fig. 3Go). Akt serine phosphorylation, but not Akt amount, was significantly reduced by Cav-1-siRNA transfection. After Cav-1 down-regulation, IRS-1, Shc-p66, and Akt phosphorylation, in the presence IGF-I, was significantly reduced at 5 and 15 min (Fig. 4Go). In contrast, MAPK activation was lower (Fig. 3Go) but without a statistically significant difference (Fig. 4Go).


Figure 3
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  		FIG. 3. Effect of Cav-1 down-regulation on IRS-1, Akt, Shc, and MAPK phosphorylation after IGF-I treatment. H9C2 cells were transfected with siRNA for Cav-1 and with scrambled Ctr siRNA as described in Fig. 2Go. Total cell lysates were immunoprecipitated with a specific antibody against phosphotyrosine and blotted with an antibody directed against IRS-1 (IRS-1-P-Tyr). Alternatively, total cell lysates were separated on SDS-PAGE, transferred onto nitrocellulose, and blotted with an antibody directed against total IRS-1, phospho-Shc (Shc-P), total Shc, phospho-Akt (Akt-P), total Akt, phopsho-MAPKs (MAPK-P), total MAPKs, and developed by enhanced chemiluminescence. The blots represent three independent experiments.

 

Figure 4
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  		FIG. 4. Densitometric analysis of bands corresponding to phosphorylated IRS-1, Shc, Akt, and MAPKs in H9C2 cells after Cav-1 down-regulation. Bands corresponding to phosphotyrosine IRS-1 (A), phospho-Shc (B), phospho-Akt (C), and phopsho-MAPKs (D) after Western blot analysis were quantified by densitometry using NIH Image software and were expressed as fold of increase under the basal. Vertical bars represent means ± SD of three independent experiments. #, P < 0.05. *, P < 0.01.

 
To determine the role of Cav-1 in IGF-I biological action, we evaluated whether Cav-1 could be required to maintain antiapoptotic effects of IGF-I on H9C2 serum withdrawal-induced apoptosis. Cav-1-siRNA and Ctr-siRNA H9C2 cells were cultured in serum-free medium for 24 h in the presence or absence of IGF-I (100 nM). IGF-I reduced significantly the number of Ctr-siRNA H9C2 cells undergoing apoptosis in response to serum withdrawal (Fig. 5Go). This effect was completely absent in Cav-1-siRNA H9C2 cells. To confirm these findings in a different cell type, we silenced Cav-1 in HUVECs. Cav-1 down-regulation (~90%) reduced IGF-IR tyrosine phosphorylation and, consequently, IGF-I antiapoptotic action after induced apoptosis (Fig. 6Go).


Figure 5
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  		FIG. 5. Apoptosis analysis in H9C2 siRNA transfected cells after serum withdrawal. H9C2 cells were transfected by Cav1-siRNA and with scrambled Ctr siRNA. Forty-eight hours after transfection, cells were cultured in serum-free medium for another 24 h in the presence (W/O Serum/IGF-I) and absence (W/O Serum) of IGF-I (100 nM). *, P < 0.01.

 

Figure 6
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  		FIG. 6. A, IGF-IR time course of activation after Cav-1 down-regulation in HUVECs. HUVECs were transfected with Cav-1-siRNA and scrambled Ctr siRNA as described in Materials and Methods. Seventy-two hours from the transfection, serum-starved cells were treated with IGF-I (100 nM) for the indicated times and lysed. Total cell lysates were immunoprecipitated with an antiphosphotyrosine antibody and blotted with an antibody directed against IGF-IR (IGF-IR-P), or blotted directly with an antibody directed against Cav-1 and developed by enhanced chemiluminescence. Bands obtained by Western blot analysis corresponding to IGF-IR phosphorylation were quantified by densitometry using NIH Image software and were expressed as fold of increase under the basal. Vertical bars represent means ± SD of three independent experiments. *, P < 0.01. B, Apoptosis analysis in human umbilical vein endothelial siRNA transfected cells after serum withdrawal treatment. Forty-eight hours after transfection with siRNA, HUVECs were cultured in serum-free medium for another 24 h in the presence (W/O Serum/IGF-I) and absence (W/O Serum) of IGF-I (100 nM). *, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we acutely down-regulated the expression of Cav-1 in H9C2 cells to evaluate the direct role of this protein on IGF-I signaling pathway. Here, for the first time, we provide evidence that the specific down-regulation of Cav-1 impairs IGF-IR tyrosine autophosphorylation with a decreased activation of the downstream substrates IRS-1, Shc, and Akt, whereas MAPK activation remained almost unchanged.

IGF-IR colocalizes with Cav-1 in the lipid rafts enriched fractions on plasma membrane (4). We have previously shown that: 1) activated IGF-IR causes Cav-1 tyrosine phosphorylation and coimmunoprecipitation with IRS-1 (7), and 2) the activation of Shc in caveolae and the recruitment of MAPKs to these plasma membrane regions (8).

Contrasting results have been reported suggesting that disruption of caveolae in 3T3-L1 obtained by methylcyclodextrin treatment (14) or Cav-1 siRNA stable silencing (9) have no effect on IGF-IR and IRS-1 activation. However, it should be considered that the disruption of caveolae by cholesterol depletion is an efficient but invasive method (15). Stable transfection with a plasmid siRNA vector as well as the creation of knockout (KO) mice do not exclude the possibility of adaptive cellular mechanisms to compensate the absence of Cav-1. In addition, it could be hypothesized that Cav-1 exerts its action on the IGF-IR pathway only in a cell type-specific manner.

The observation that Cav-1 KO mice developed cardiomyopathies (16, 17) and showed an impaired angiogenic response to exogenous stimuli (18) suggest that H9C2 cardiomyoblasts and HUVECs represent a good model to study the role of Cav-1.

Transfection of Cav-1 siRNA in the H9C2 cell line significantly reduced the Cav-1 total amount. It has been shown that Cav-1 down-regulation reduces Cav-2 expression (19, 20). Accordingly, we observed a significant decrease of Cav-2 expression, whereas the Cav-3 amount remained unaffected, suggesting the presence of distinct biosynthetic pathways for these proteins. Moreover, the finding that Cav-3 is not sufficient to compensate for the lack of Cav-1 in IGF-I signaling suggests a specific role of Cav-1 in myoblast cells compared with Cav-3 (21).

Here, we have demonstrated that Cav-1 is required for the activation of IGF-IR in the presence of IGF-I. Because Cav-1 takes part in other growth factor receptor signaling in cardiovascular cells, it could represent a key molecule in the cross talk between IGF-IR and other receptors.

IGF-I stimulates IRS-1 tyrosine phosphorylation (22). In IGF-I treated Cav-1-siRNA H9C2 cells, we found a reduced IRS-1 tyrosine phosphorylation. We propose that reduced IGF-IR tyrosine phosphorylation in IGF-I treated Cav-1-siRNA H9C2 cells could down-regulate IRS-1 activation.

We have previously shown that IGF-IR activation causes IRS-1, p52/Shc, p46/Shc, and p66/Shc activation in lipid rafts/caveolae (7). Although H9C2 cells express all Shc isoforms (data not shown), IGF-I phosphorylated p66/Shc only (23). Accordingly, with data obtained in mice with IGF-IR deficiency, the reduced phosphorylation of p66/Shc after Cav-1 silencing could be a direct consequence of the decreased phosphorylation of the receptor. However, MAPK activation was not affected by Cav-1 silencing. Because MAPKs are downstream effectors of redundant pathways (24), it is possible that other IGF-IR substrates could activate MAPKs in the absence of Cav-1.

It has been proposed that Cav-1 acts as negative regulator of MAPK cascade (25). However, it has also been shown that Cav-1 KO mice did not present any difference in the basal activation of MAPKs compared with wild-type mice (20). In addition, the vascular endothelial growth factor signaling pathway also is not affected, at the level of MAPKs, by Cav-1 down-regulation (26). All these findings demonstrate that the role of Cav-1 on MAPK regulation is not completely clarified.

We found a decrease activation of Akt in siRNA-Cav-1 H9C2 cells compared with Ctr cells. Akt is a key step in the IGF-I signaling pathway that protects cardiomyocytes from death (27). We found that after serum withdrawal, siRNA-Cav-1 H9C2 cells did not respond to IGF-I survival stimulus.

Cav-1 is a key regulator of vascular permeability, vascular tone, as well as angiogenesis (18, 20, 28). IGF-I is a vascular protective factor with antiapoptotic properties (29). Thus, we verified whether Cav-1 was important for IGF-IR activation also in endothelial cells, silencing Cav-1 in HUVECs.

We previously demonstrated in HUVECs that Cav-1 down-regulation reduced the IGF-I induced activation of endothelial nitric oxide synthase (10), an antiapoptotic factor.

Accordingly, with this finding we found that Cav-1 silencing decreased IGF-IR autophosphorylation and inhibited the antiapoptotic effect of IGF-I after serum deprivation.

We demonstrate that Cav-1 down-regulation affected IGF-IR signaling, not only in cardiomyoblasts, but also in endothelial cells reducing IGF-I function.

In conclusion, we suggest that Cav-1 contributes to IGF-I mediated cardiovascular protection (21, 30).


   Acknowledgments
 
We thank Mrs. Maria Rosa Dagnino for her skillful administrative assistance.


   Footnotes
 
This work was supported by grants from Ministero dell’Universita e della Ricerca, Fondo degli Investimenti per la Ricerca di Base, Fondazione Cassa di Risparmio di Genova e Imperia, and University of Genova.

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 26, 2007

Abbreviations: Cav, Caveolin; Ctr, control; FITC, fluorescein isothiocyanate; HUVEC, human umbilical vein endothelial cell; IGF-IR, IGF-I receptor; IRS-1, insulin receptor substrate-1; KO, knockout; PI, propidium iodide; siRNA, small interfering RNA.

Received March 7, 2007.

Accepted for publication November 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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