Cardiovascular Research

Cardiovascular Research 2005 67(1):50-59; doi:10.1016/j.cardiores.2005.03.002

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Stawowy, P. Articles by Graf, K. Search for Related Content PubMed PubMed Citation Articles by Stawowy, P. Articles by Graf, K. Social Bookmarking          What's this?

Copyright © 2005, European Society of Cardiology

Protein kinase C epsilon mediates angiotensin II-induced activation of β₁-integrins in cardiac fibroblasts

Philipp Stawowy^a, Christian Margeta^a, Florian Blaschke^a, Carsten Lindschau^b, Chantel Spencer-Hänsch^a, Michael Leitges^c, Giuseppe Biagini^d, Eckart Fleck^a and Kristof Graf^a,*

^aDepartment of Medicine-Cardiology, Deutsches Herzzentrum Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany
^bDepartment of Internal Medicine, Hannover Medical School, Hannover, Germany
^cMax Planck Institute for Experimental Endocrinology, Hannover, Germany
^dDepartment of Biomedical Sciences, University of Modena and Reggio Emilia, Italy

^* Corresponding author. Tel.: +49 30 4593 2413; fax: +49 30 4593 2415. Email address: graf{at}dhzb.de

Received 31 August 2004; revised 1 March 2005; accepted 2 March 2005

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Objective: Angiotensin II (AII) promotes cardiac fibrosis by increased extracellular matrix production and enhanced interaction of matrix proteins with their cellular receptors, including integrins. AII and other growth factors augment β₁-integrin-dependent adhesion and spreading by "inside-out signaling" without affecting the total number of integrin receptors. "Inside-out signaling" involves specific signaling pathways, including protein kinase C (PKC), leading to activation of β₁-integrins. In the present study we investigated the mechanisms involved in AII-increased adhesion of adult rat cardiac fibroblasts (CFBs), obtained from Sprague–Dawley rats, to collagen I mediated by β₁-integrin.

Methods and results: Treatment of CFBs with AII induced a concentration-dependent increase in adhesion to collagen I (2.2-fold, p<0.01) within 3–6 h of treatment. This effect was mediated by β₁-integrin via the angiotensin AT₁ receptor and was significantly reduced by protein kinase C inhibition. AII significantly induced phosphorylation of PKC epsilon (PKC), which is involved in β₁-integrin-dependent adhesion and motility, and phosphorylation of the cytoplasmatic tail of β₁-integrin at threonine 788/789, required for adhesion. Phosphorylation of β₁-integrin and an increase in adhesion was also induced by L--phosphatidylinositol-3,4,5-triphosphate (L--PIP₃), an activator of endogeneous PKC. AII failed to increase adhesion in myofibroblasts obtained from PKC (–/–) mice, but not in cells obtained from control mice. Co-immunoprecipitation and double immunofluorescence demonstrated that AII induced a close association of PKC with β₁-integrin in CFBs.

Conclusion: The present study demonstrates that AII increased β₁-integrin-dependent adhesion to collagen I in cardiac fibroblasts by inside-out signaling via PKC and phosphorylation of the cytoplasmatic tail of the β₁-integrin.

KEYWORDS Angiotensin II; Integrin; Collagen; Protein kinase C

---

This article is referred to in the Editorial by L. Hein (pages 6–8) in this issue.

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Integrin heterodimers, which belong to the family of transmembrane receptors, provide a direct adhesive link between the cells and their surrounding matrix, thereby participating in inside-out/outside-in signaling events required for cell functions [1,2]. In the heart the interaction of cardiac fibroblasts (CFBs) with their surrounding matrix is critical for repair mechanisms, including synthesis of matrix proteins, proliferation, collagen gel contraction and cell motility.

Angiotensin II (AII) has been shown to be critical for cardiac remodeling and cardiac fibrosis. AII induces fibronectin, laminin and TGF-β₁ mRNA via its angiotensin type 1-receptor (AT₁) in CFBs [3,4]. AT₁-receptor expression, angiotensin-converting enzyme (ACE) activity and accumulation of fibrillar collagens are increased in the healing rodent myocardium following myocardial infarction [5,6].

AII increases CFB mediated collagen gel contractions, which are partially mediated via β₁-integrins, _vβ₃-integrins and the adhesive protein osteopontin [7,8]. Increased expression of osteopontin, a ligand for β₁-integrins is associated with cardiac hypertrophy in humans and rodents [9] and parallels heart failure progression [10]. AII treatment of cardiac fibroblasts has been shown to increase the expression of _vβ₃-integrin in CFBs in vitro [11]. Thibault et al. recently described an increased receptor density and binding affinity of ₈β₁ integrins on CFBs following AII or TGF-β1 stimulation [12]. In contrast, Burgess and coworkers found an increase in β₁-integrins, but decreases of ₁ and ₂ integrins (₁β₁- and ₂β₁-integrin are the major collagen receptors in the heart) in CFBs taken from rats with hypertension or exercise induced hypertrophy [13]. The underlying cellular mechanisms leading to enhanced β₁ integrin function following AII treatment are not clearly defined in CFBs. There is evidence for an increased myocardial expression of integrins after chronic AII treatment (vβ3 and ₈β₁), however clustering, activation and conformational changes mediated by "inside-out signaling" might be a major effect of AII on β₁-integrins in CFBs [12–14]. It has been shown that enhanced inside-out signaling via protein kinase C (PKC) activation by AII is a potential mechanism for enhanced integrin-matrix interaction in vascular smooth muscle cells [15] and cardiac myocytes [16]. Several PKC isoforms are involved in the regulation of integrin function [17,18]. PKC epsilon (PKC), a novel PKC isoform, which is Ca²⁺ independent, is essential for adhesion and migration, as well as trafficking of the β₁-integrin chain to the cell surface [19,20]. In human glioma cells, PKC positively regulated β₁-integrin-dependent adhesion and spreading which is associated with increased focal adhesions and clustering of specific integrins [20].

In the present study, we demonstrate that β₁-integrin activation by AII involves activation of PKC and association of PKC with the β₁-integrin chain.

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
2.1. Materials
Culture plastic ware was from Beckton Dickinson and antibodies against rat ₁-, ₂-, ₅-integrin and β₁-integrin from Pharmingen and Chemicon. The rabbit polyclonal antibody against phospho β₁-integrin (Thr788/789) was from Biosource. Anti-phospho-PKC (Ser729), anti-PKC, anti-phospho-PKC (Ser657) were from Upstate. Anti-PKC, anti-PKC, anti-phospho-PKC (Thr507), anti-AT_1a receptor and anti-RACK1 were from Santa Cruz. Anti-vimentin and secondary antibodies for Western blot were purchased from Immunotech. Secondary antibodies and reagents for immunofluorescence were from Vector. AII was from Bachem. The AT₁ receptor blocker DUP 753 was provided by Merck. L--phosphatidylinositol-3,4,5-triphosphate (L--PIP₃) was purchased from Calbiochem. Matrix proteins and all other reagents and materials were purchased from Sigma-Aldrich, if not stated elsewhere.

2.2. Cell culture
Animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health (NIH publication No. 85-23, revised 1996). Adult CFBs were prepared from Sprague–Dawley rats and characterized as described previously [4,11]. Cells were grown in DMEM/low glucose with 10% FBS until they reached confluence at which time they were detached by accutase (PAA) treatment and split 1:4. All experiments were performed in the 2nd to 5th passage after starvation in serum free DMEM containing insulin (5 µg/mL), transferrin (5 µg/mL) and selenium (5 ng/mL) for 48 h. Thereafter cells were incubated with AII, inhibitors or vehicle in concentrations as indicated in the results section.

Mice lacking the epsilon isoform of PKC were generated as described elsewhere (Leitges et al., manuscript in preparation) [21]. Myofibroblasts were extracted from PKC (–/–) mice and from control wild type mice (Cv129/Bl6) with the identical genetic background. Myofibroblasts from mouse aorta were prepared by sequential enzymatic digestions with elastase type IV (Sigma, 0.5 mg/mL) and collagenase (Serva, 1.4 mg/mL) for 15 min and 60–90 min (0.5 mg/mL elastase and 2 mg/mL collagenase), respectively. Cells were cultivated in SmBM-medium with supplements SmGM-2 (Clonetics).

2.3. Adhesion
Adhesion assays were performed as described previously [15]. Adhesive substrates (rat collagen I) were added to 96 well plates (Nunc) and incubated overnight at 4 °C. Non specific binding was blocked with 1% BSA at 37 °C for 1 h.

2.4. Immunofluorescence
Cells were grown on plastic chamber slide coated with collagen I for 24 h and kept in serum free medium for another 24 h before treatment. After appropriate treatment, cells were rinsed in PBS and fixed in cold methanol at –20 °C. Cells were permeabilized in 0.1% Triton-X100/PBS, blocked with 10% normal goat serum and incubated with primary antibodies diluted in antibody dilution containing 3% bovine serum albumin, and 0.05% Tween 20. Primary antibodies were incubated overnight at 4 °C. Following washing FITC- and Texas Red-conjugated secondary antibodies were used. Microscopy was carried out with an Olympus BX61, using analySIS imagine software.

In double immunoflurorescence experiments species-specific antibodies were used to distinguish primary antibodies, as described previously [22].

2.5. Western blot analysis
Immunoblotting was done as described [23]. Proteins were extracted in RIPA-buffer containing freshly dissolved protease inhibitors. Up to 50 µg of proteins were mixed with sample buffer and applied on 8% SDS-polyacrylamide gel electrophoresis. Following migration, proteins were transferred onto nitrocellulose membranes (BioRad). The membranes were first incubated overnight at 4 °C with the primary antibody, followed by incubation with secondary antibodies. Peroxidase activity product was revealed with the enhanced chemiluminescence method (ECL, Amersham). Semiquantitative densitometry was performed with the National Institutes of Health (NIH) software program, ImageJ, and is expressed in arbitrary units (AU).

2.6. Coimmunoprecipitations
Cell lysates were prepared as described above for Western blotting. For each immunoprecipitation, 4 µg of the appropriate antibody, 300 µg of proteins and 30 µl of protein A/G agarose beads (Pharmacia) were incubated for 3 h at 4 °C. For control immunoprecipitation, the primary antibody was replaced by goat anti mouse IgG+IgM (H+L). Immunoprecipitates were rinsed three times in immunoprecipitation buffer. The immunoprecipitates were subjected to immunoblotting as described above.

2.7. Immunohistochemistry
Immunohistochemistry was performed with formalin-fixated myocardial section obtained from 3 month old Sprague–Dawley rats following the ABC method (Zymed), as published [10,11].

2.8. Statistics
Analysis of variance (ANOVA), and paired or unpaired t-test were performed for statistical analysis, as appropriate. A p value less than 0.05 was considered to be statistically significant. Data were expressed as mean ± SEM, if not stated elsewhere.

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
3.1. AII increased adhesion of cardiac fibroblasts to collagen I via the AT₁ receptor and activation of PKC
AII increased adhesion of CFBs to collagen I in a concentration-dependent fashion (Fig. 1A). AII-induced a concentration-dependent increase of adhesion to collagen I (2.2-fold, p<0.01), within 24 h treatment. Time course experiments with two different concentrations of AII (0.1 and 1 µmol/L) demonstrated that adhesion increased within 2 h after addition of AII and reached its maximum after 5 h (Fig. 1B) The effect of AII (1 µmol/L) was not seen after pretreatment of cells with the AT₁ receptor blocker DUP 753 (100 µmol/L, 1 h before AII). In contrast, the AT₂ receptor blocker PD 123319 (100 µmol/L) failed to affect adhesion (Fig. 1A). In parallel experiments conincubation was performed with the broad-spectrum PKC inhibitor calphostin C (0.2 µmol/L, 1 h before AII) and following preincubation with phorbol-myristate acetate (PMA, 1 µmol/L) for 24 h, which down regulates cellular PKC activity. Both ways of inhibition significantly reduced the effect of AII on adhesion to collagen I (Fig. 1C).

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 (A) AII induced increased adhesion of CFBs to collagen I. Adhesion of CFBs (20,000/well) to collagen I (20 µg/ml) coated plates were performed with CFBs treated with different concentrations of AII for 24 h and in the presence of the AT1 receptor blocker DUP753 (100 µmol/L) and the AT2 receptor blocker PD 123319 (100 µmol/L). (B) Time course experiments were performed with CFBs treated with AII 0.1 µmol/L (filled circle), AII 1 µmol/L (hollow circle) or diluent (triangle). Results are expressed as mean ± SEM, n=9–12. *p<0.05 vs. control (co), #p<0.05 vs. AII, results are expressed as mean ± SEM, n=12–16. (C) Blocking experiments with antibodies against β1-integrin (β1ab) and 5-integrin (5ab) were performed with untreated cells and cells treated with AII (1 µmol/L) and adhesion was tested on collagen I. CFBs were incubated for 30 min with antibodies before adhesion was started. Control (co) experiments were performed with non-specific IgGs. *p<0.05 vs. control (co); results are expressed as mean ± SEM, n=9. (D) The AII-induced adhesion to collagen I is mediated via PKC. CFBs were incubated with AII (1 µmol/L) for 24 h alone, or in presence of the PKC inhibitor calphostin C (Cal C, 0.2 µmol/L)) or after 24 h treatment with PMA (1 µmol/L). *p<0.05 vs. control (co), #p<0.05 vs. AII; results are expressed as mean ± SEM, n=12.

 
To investigate whether this effect was mediated by increased status or quantity of β₁ integrins, blocking experiments with antibodies against β₁-integrin (HA2/5, 25 µg/mL) and as control against ₅ integrin (HM8, 25 µg/mL) were performed with untreated cells and cells treated with AII (1 µmol/L, Fig. 2C and D). Blocking experiments demonstrated that adhesion to collagen I was reduced by HA2/5 by approx. 75% (p<0.01), but not by HM8 in treated cells or controls. Adhesion to fibronectin was significantly reduced by HM8, since it targets the fibronectin receptor ₅β₁-integrin (data not shown). Quantitation of β₁-, ₁- and ₂–using flow cytometry and Western blot in three different experiments demonstrated no significant changes in the cellular levels of these integrins after treatment with AII (1 µmol/L) for 24 h (data not shown).

View larger version (44K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 (A) CFBs stimulated with AII (1 µmol/L) up to 120 minutes demonstrate an increase of phophorylated β1-integrin as shown in this representative Western blot. Actin was determined to demonstrate protein loading. (B) Various concentrations of AII significantly enhanced phosphorylation of β1 integrin. The figure on the right summarizes densitometry readings from four different experiments *p<0.05 vs. control (co). (C) AII-induced phosphorylation was significantly reduced by conincubation with calphostin C (0.2 µmol/L; Cal C) or after pretreatment with PMA (1 µmol/L). Parallel blots for β1-integrin total protein revealed stable protein concentrations throughout the whole experiment. The right figure demonstrates the results of the densitometry readings of four experiments. *p<0.05 vs. control (co), #p<0.05 vs. AII; results are expressed as mean ± SEM.

 
3.2. AII induced phosphorylation of β₁ integrin and PKC
Wennerberg et al. could demonstrate that the phosphorylation of threonines 788 and 789 within the cytoplasmic tail of the β₁-integrin is critical for the adhesion of GD25 cells [24]. AII induced an increase of phosphorylation of β₁-integrin at threonines 788 and 789 which was detectable 20 min after beginning of treatment with 1 µmol/L AII (Fig. 2A). Increased signal for phosphorylation of β₁-integrin was detectable up to 12–24 h (data not shown). AII (1 µmol/L) induced a two-fold increase in phosphorylation (p<0.05) as determined by Western blotting and consecutive image analysis (Fig. 2B). The effect of AII was significantly inhibited by PKC inhibition, using calphostin C (0.2 µmol/L) or pretreatment with PMA (1 µmol/L) for 24 h, indicating that a PKC isoform is involved in the phosphorylation of β₁-integrin (Fig. 2C). The effect of AII on phosphorylation of β₁-integrin was also abolished in the presence of the AT₁ receptor blocker DUP 753 (data not shown).

PKC seems to be essential for adhesion, migration and trafficking of the β₁-integrins in some tumor cell lines [19,20]. Since PKC inhibition was able to prevent AII-induced adhesion and phosphorylation of cardiac β₁-integrin, we investigated whether AII induced phosphorylation of PKC (Fig. 3) and whether an activator of PKC, L--phosphatidylinositol-3,4,5-triphosphate (L--PIP₃) [25] can mimic the effect of AII.

View larger version (47K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A) CFBs stimulated with AII (1 µmol/L) demonstrate an increase of phophorylated PKC after 20 min as demonstrated in this representative Western blot. Phosphorylation of MAP-kinase was determined to demonstrate the intracellular signal activation after addition of AII as positive control. AII induced a typical phopshorylation of MAP-kinase within 10 min. Actin was determined to demonstrate protein loading. (B) Various concentrations of AII significantly enhanced phosphorylation of PKC determined 60 min after addition of AII. Total PKC protein levels were not affected with AII. The figure on the right summarizes densitometry readings of four different experiments *p<0.05 vs. control (co).

 
AII induced a phosphorylation of PKC which was detectable already after 10 min in cardiac fibroblasts (Fig. 2A). Increased signal for phosphorylation of PKC was detectable up to 12–24 h (data not shown). AII (1 µmol/L) induced a nearly three-fold increase in phosphorylation (p<0.05) as determined by Western blotting and consecutive image analysis (Fig. 3B). Incubation of CFBs with L--PIP₃ (1–5 µmol/L), an activator of PKC, for 60 min induced a significant phosphorylation of PKC and β₁-integrin (p<0.05, Fig. 4A) and a significant increase in adhesion of CFBs, determined 6 h after stimulation with L--PIP₃ (p<0.05, Fig. 4B), which indicates that PKC might play a role for AII induced activation of β₁-integrins. In parallel experiments phosphorylation of PKC and PKC were investigated which did not show a significant response after AII stimulation in cardiac fibroblasts (data not shown).

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 (A) CFBs were stimulated with L--PIP3 (0.1–5 µmol/L) for 1 h and phosphorylation of β1-integrin and PKC was determined as demonstrated in the representative Western blots. The figures summarizes densitometry readings from three different experiments *p<0.05 vs. control (co). (B) Treatment with L--PIP3 (1–5 µmol/L) increased adhesion of CFBs to collagen I. Adhesion of CFBs (20,000/well) to collagen I (20 µg/mL) was inhibited in presence of calphostin C (0.2 µmol/L; Cal C) or after pretreatment with PMA (1 µmol/L); *p<0.05 vs. control (co), #p<0.05 vs. L--PIP3; results are expressed as mean ± SEM, n=6.

 
Furthermore, we performed experiments with myofibroblasts obtained from aortae of PKC (–/–) mice and control wildtype mice, PKC (+/+). Cells from both, PKC (–/–) and PKC (+/+) mice, showed a typical fibroblast like shape and were positive for vimentin and alpha-smooth muscle actin (data not shown). Western blot confirmed the absence of PKC (Fig. 5C, second lane) at the 95kD band, whereas expression of β₁-integrin and the AT₁-receptor protein was comparable in both, PKC (+/+) and PKC (–/–) myofibroblasts (Fig. 5C). In PKC (+/+) cells AII treatment for 24 h induced a significant increase in adhesion to collagen I, which was abolished in presence of calphostin C (0.2 µmol/L) or pretreatment with PMA (0.1 µmol/L) or DUP753 (100 µmol/L, Fig. 5A). In PKC (–/–) cells basal adhesion was not significantly disturbed, but AII treatment did not induce a significant increase of adhesion (Fig. 5B). These experiments demonstrate that the presence of PKC is essential for AII mediated activation of the β₁-integrin in fibroblasts.

View larger version (54K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Wildtype mouse myofibroblasts (A), and from PKC (–/–) mice (B) were stimulated with AII for 24 h. In cells obtained from wildtype PKC (+/+) mice, AII increased adhesion to collagen I, which was inhibited in presence of calphostin C and the AT1-receptor blocker DUP 753. The figures summarizes data from three different experiments (n=9) *p<0.05 vs. control (co). (C) The presence of PKC, β1-integrin, and the AT1-receptor was determined by Western blot. PKC protein was not detectable in myofibroblasts from PKC (–/–) mice, but in PKC (+/+), whereas expression for β1-integrin, and the AT1-receptor was comparable in both cell types.

 
Immunofluorescence experiments with antibodies against β₁-integrins and PKC were performed with CFBs before and 24 h after treatment with AII (Fig. 6A–F). AII (0.1 µmol/L for 1 h) caused stress fiber formation and focal contacts in CFBs (Fig. 6C, F) compared to untreated CFBs (Fig. 6B, E). Antibodies against β₁-integrins (Fig. 6C) and PKC (Fig. 6F) produced a specific staining at focal contacts and stress fibers as well in the perinuclear area, which indicates a potential association of these proteins in CFBs stimulated with AII.

View larger version (93K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Representative immunfluorescence experiments with CFBs untreated (A, B, D, E) and after treatment with AII (1 µmol/L) for 24 h (C, F). CFBs were stained with non-specific mouse IgGs as control (A), with a polyclonal rabbit-PKC (B, C), with a polyclonal rabbit-anti-β1-integrin (E, F), and vimentin (D). Unstimulated CFBs express diffuse immunoreactivity for PKC and β1 integrin (B, E). After addition of AII focal immunoreactivity along stress fibers was observed for both proteins (C, D). Experiments demonstrated herein were repeated twice with CFBs obtained from a different preparation.

 
In addition, we performed double-immunofluorescence experiments with antibodies against phosphorylated PKC and β₁-integrin in CFBs after 1 h treatment with AII (0.1 µmol/L, Fig. 7A), which demonstrated a close cellular association of both proteins after AII treatment. This was supported by immunoprecipitation experiments performed with CFBs treated with AII (Fig. 7B). Immunoblots demonstrated a significant association of PKC with β₁-integrin after 0.5 and 1 h (Fig. 7B).

View larger version (73K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 (A) Representative micrographs from double immunfluorescence experiments with anti-β1-integrin and anti-PKC antibodies demonstrate immunoreactivity after treatment with AII (1 µmol/L) for 1 h. Merging of both fluorescence activities indicate a close association of β1-integrin and PKC after treatment with AII. The bar represents 50 µm. (B) The figure demonstrates a representative blot obtained after immunoprecipitation with anti-PKC and anti-β1 integrin antibodies and immunoblotting against anti-PKC and anti-β1 integrin. Cells were treated with AII (1 µmol/L) as indicated and Western blotting performed with anti-β1 integrin, anti-PKC or actin. All experiments demonstrated in this figure were at least repeated twice with CFBs obtained from a different preparation.

 
Myocardial sections obtained from the normally developed left ventricles of rat myocardium demonstrated a specific immunoreactivity for vimentin, PKC and β₁-integrins in perivascular regions, typical areas for cardiac fibroblasts (Fig. 8).

View larger version (149K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Myocardial sections from the left ventricle of normal rat hearts demonstrate immunoreactivity for PKC (B), vimentin (C) and β1-integrin (D) in perivascular areas typical for cardiac fibroblasts. Control was performed with non-specific mouse IgGs (A).

 

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
The interstitial matrix of the myocardium is predominantly composed of fibronectin and type I collagen, which is upregulated after myocardial infarction, fibrosis and in experimental left ventricular hypertrophy [5,26–28]. Since adhesion, de-adhesion and spreading are integral parts in order to achieve the ability of cells to move and migrate, these mechanisms are integral parts of cardiac remodeling mechanisms. AII is one of the important factors controlling the change of the myocardium towards fibrosis and heart failure.

In the present study we demonstrate that AII induced a significant increase of β₁-integrin-dependent adhesion of CFBs to collagen I via activation of PKC and phosphorylation of β₁-integrin. The effect of AII was mediated by an AT₁ receptor-dependent mechanism. The effect of AII was associated with phosphorylation of PKC and the cytoplasmatic tail of the β₁-integrin at threonine 788/789. Immunofluorescence, double-immunofluorescence, as wells as co-immunoprecipitation experiments suggest that AII induced a close cellular association of PKC and β₁-integrin, mainly localized to focal contacts, stress fibers and in the perinuclear region. Whether the proteins associate directly or via an adaptor protein can not be answered by the present study.

It was demonstrated in a glioma cell line that PKC associates with a specific anchoring molecule termed RACK1 (receptors for activated C-kinase 1), which associates with the β₁-integrin chain [20]. It has been shown that activated PKCs bind to RACKs, which enable their intracellular translocation and transmission of function [29]. We found that RACK1 is abundant in cardiac fibroblasts (data not shown). It is likely, that this anchoring molecule is involved in the phosphorylation of the β₁-integrin cytoplasmatic domain as well.

Wennerberg et al. have demonstrated that phosphorylation of threonines residues within the cytoplasmic tail of the β₁-integrin are critical events for adhesion, FAK activation and cell spreading in GD25 cells [24]. We observed that AII induced phosphorylation of these threonine residues in the cytoplasmatic tail of the β₁-integrin in a concentration and time-dependent manner, which was abolished by PKC inhibition. Time course of phosphorylation events went along with the increase of adhesion observed in CFBs after AII treatment. The study indicates that PKC activation, and especially activation of PKC, is crucial to increase adhesion. To further investigate the role of PKC, we used a pharmacological activator, L--PIP₃ [25], which activates PKC. This agent activated the phosphorylation of PKC and β₁-integrins in CFBs and induced a significant increase of adhesion of CFBs. Finally we performed adhesion experiments with myofibroblasts obtained from PKC (–/–) mice which did not show an increase of adhesion after AII treatment, although the AT₁ receptor is expressed in these cells. In contrast, myofibroblasts from wildtype control mice had a preserved response to AII resulting in a significant increase of adhesion to collagen I. These experiments underline the essential role of PKC for activation of β₁-integrins by AII.

Our results are in good accordance with studies obtained with cell lines and tumor cells demonstrating that PKC isoforms are involved in the regulation of integrin function [17,18]. Especially, PKC seems to be essential for adhesion, migration and trafficking of the β₁-integrins [19,20], as recently reported. We have observed that activation of β₁-integrin heterodimers after AII treatment was without substantial quantitative increase in vascular cells [15,30]. In the present study Western blot and flow cytometry studies did not present a significant change in β₁-integrin levels up to 24 h after AII treatment. However we did not exclude that longer treatment periods might increase the levels of β₁-integrin. The present data indicate that "inside-out" signaling induced by AII leads to activation of β₁-integrins, whereas increased protein levels of integrin receptors might reflect a response to chronic stimulation in cardiac cells [14]. Our present observation that integrin function is modulated without changes in the expression pattern is supported by a recent study which demonstrated that hypoxia activated β₁-integrin-mediated functions in human smooth muscle cells by increasing the avidity [31]. The binding of an antibody to a specific β₁-integrin activation epitope was increased after hypoxia [31,32]. Unfortunately, this antibody could not be used in rat CFBs, used in this study.

Even though no data quantification could be performed due to differences in protein loadings, a functional interaction of β₁-integrins with PKC triggered by AII is further supported by their coimmunoprecipitation, as demonstrated in the present study.

In functional assays the PKC inhibitors generally completely inhibited AII-induced adhesion of CFBs, whereas phosphorylation levels of the of PKC and β₁-integrin proteins were not entirely abolished, as detected by immunoblotting. However, based on the experiments published by Wennerberg et al. [24], who demonstrated the importance of Thr^788/789 for adhesion using β₁-integrin mutants, we think that part of the phosphorylation response seen with the antibody against phosphorylated β₁-integrin might not only reflect the phosphorylation of Thr^788/789 alone. This includes the possibility of PKC independent phosphorylation of the cytoplasmatic tail of β₁-integrin.

Nevertheless, the present data indicate that after phosphorylation of the β₁-integrin-chain the avidity of the integrin receptors for collagen I increase and that this mechanism contributes to the profibrotic cellular events driven by the renin-angiotensin-system in the heart. Protein kinase C epsilon seems to play a center role for the activation of β₁-integrin containing collagen I receptors.

	Acknowledgements

 
We thank Brigitte Wollert-Wulf and Heike Meyborg for excellent technical assistance.

This work was supported in part by a grant of the Deutsche Forschungsgemeinschaft (GR 1368/2-2) to KG and a grant of the Philip Morris Research Foundation to EF and KG. CM was supported by the Deutscher Akademischer Austauschdienst (DAAD).

	Notes

 
Time for primary review 32 days

	References

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 

1. Clark E., Brugge J. Integrins and signal transduction pathway: the road taken. Science (1995) 268:233–239.[Abstract/Free Full Text]
2. Hynes R. Integrins: versatility, modulation and signaling in cell adhesion. Cell (1992) 69:11–25.[CrossRef][Web of Science][Medline]
3. Fisher S., Absher M. Norepinephrine and AII stimulate secretion of TGF-ß by neonatal rat cardiac fibroblasts in vitro. Am J Physiol (1995) 268:C910–C917.[Web of Science][Medline]
4. Iwami K., Ashizawa N., Yung S., Graf K., Hsueh W. Comparison of angiotensin II with other growth factors on EGR-1 and matrix gene expression in cardiac fibroblasts. Am J Physiol (1996) 270:H 2100–H 2107.[Medline]
5. Cleutjens J.P., Verluyten M.J., Smiths J.F., Daemen M.J. Collagen remodeling after myocardial infarction in the rat heart. Am J Pathol (1995) 147:325–338.[Abstract]
6. Weber K.T. Extracellular matrix remodeling in heart failure. A role for de novo angiotensin II generation. Circulation (1997) 96:4065–4082.[Free Full Text]
7. Burgess M.L., Carver W.E., Terracio L., Wilson S.P., Wilson M.A., Borg T.K. Integrin-mediated collagen gel contraction by cardiac fibroblasts. Effects of angiotensin II. Circ Res (1994) 74:291–298.[Abstract/Free Full Text]
8. Ashizawa N., Graf K., Do Y., Nunohiro T., Giachelli C., Meehan W., et al. Osteopontin is produced by rat cardiac fibroblasts and mediates AII-induced DNA synthesis and collagen gel contraction. J Clin Invest (1996) 98:2218–2227.[Web of Science][Medline]
9. Graf K., Do Y.S., Ashizawa N., Meehan W.P., Giachelli C.M., Marboe C.C., et al. Myocardial osteopontin expression is associated with left ventricular hypertrophy. Circulation (1997) 96:3063–3071.[Abstract/Free Full Text]
10. Stawowy P., Blaschke F., Pfautsch P., Goetze S., Lippek F., Wollert-Wulf B., et al. Increased myocardial expression of osteopontin in patients with advanced heart failure. Eur J Heart Fail (2002) 4:139–146.[CrossRef][Web of Science][Medline]
11. Graf K., Neuss M., Stawowy P., Hsueh W.A., Fleck E., Law R.E. Angiotensin II and alpha(v)beta(3) integrin expression in rat neonatal cardiac fibroblasts. Hypertension (2000) 35:978–984.[Abstract/Free Full Text]
12. Thibault G., Lacombe M.J., Schnapp L.M., Lacasse A., Bouzeghrane F., Lapalme G. Upregulation of alpha(8)beta(1)-integrin in cardiac fibroblast by angiotensin II and transforming growth factor-beta1. Am J Physiol Cell Physiol (2001) 281:C1457–C1467.[Abstract/Free Full Text]
13. Burgess M.L., Terracio L., Hirozane T., Borg T.K. Differential integrin expression by cardiac fibroblasts from hypertensive and exercise-trained rat hearts. Cardiovasc Pathol (2002) 11:78–87.[CrossRef][Web of Science][Medline]
14. Kawano H., Cody R.J., Graf K., Goetze S., Kawano Y., Schnee J., et al. Angiotensin II enhances integrin and alpha-actinin expression in adult rat cardiac fibroblasts. Hypertension (2000) 35:273–279.[Abstract/Free Full Text]
15. Kappert K., Schmidt G., Doerr G., Wollert-Wulf B., Fleck E., Graf K. Angiotensin II and PDGF-BB stimulate beta(1)-integrin-mediated adhesion and spreading in human VSMCs. Hypertension (2000) 35:255–261.[Abstract/Free Full Text]
16. Ross R.S., Borg T.K. Integrins and the myocardium. Circ Res (2001) 88(11):1112–1119.[Abstract/Free Full Text]
17. Laudanna C., Mochly-Rosen D., Liron T., Constantin G., Butcher E.C. Evidence of zeta protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis. J Biol Chem (1998) 273:30306–30315.[Abstract/Free Full Text]
18. Ng T., Shima D., Squire A., Bastiaens P.I., Gschmeissner S., Humphries M.J., et al. PKCalpha regulates beta1 integrin-dependent cell motility through association and control of integrin traffic. EMBO J (1999) 18:3909–3923.[CrossRef][Web of Science][Medline]
19. Ivaska J., Whelan R.D., Watson R., Parker P.J. PKC epsilon controls the traffic of beta1 integrins in motile cells. EMBO J (2002) 21:3608–3619.[CrossRef][Web of Science][Medline]
20. Besson A., Wilson T.L., Yong V.W. The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility. J Biol Chem (2002) 277:22073–22084.[Abstract/Free Full Text]
21. Gruber T., Thuille N., Hermann-Kleiter N., Leitges M., Baier G. Protein kinase C epsilon is dispensable for TCR/CD3-signaling. Mol Immunol (2005) 42:305–310.[CrossRef][Web of Science][Medline]
22. Stawowy P., Margeta C., Kallisch H., Seidah N.G., Chretien M., Fleck E., et al. Regulation of matrix metalloproteinase MT1-MMP/MMP-2 in cardiac fibroblasts by TGF-beta1 involves furin-convertase. Cardiovasc Res (2004) 63:87–97.[Abstract/Free Full Text]
23. Stawowy P., Blaschke F., Kilimnik A., Goetze S., Kallisch H., Chretien M., et al. Proprotein convertase PC5 regulation by PDGF-BB involves PI3-kinase/p70(s6)-kinase activation in vascular smooth muscle cells. Hypertension (2002) 39:399–404.[Abstract/Free Full Text]
24. Wennerberg K., Armulik A., Sakai T., Karlsson M., Fassler R., Schaefer E.M., et al. The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation. Mol Cell Biol (2000) 20:5758–5765.[Abstract/Free Full Text]
25. Derman M.P., Toker A., Hartwig J.H., Spokes K., Falck J.R., Chen C.S., et al. The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C. J Biol Chem (1997) 272:6465–6470.[Abstract/Free Full Text]
26. Burgess M.L., Buggy J., Price R.L., Abel F.L., Terracio L., Samarel A.M., et al. Exercise- and hypertension-induced collagen changes are related to left ventricular function in rat hearts. Am J Physiol (1996) 270:H151–H159.[Web of Science][Medline]
27. Brilla C.G., Pick R., Tan L.B., Janicki J.S., Weber K.T. Remodeling of the rat right and left ventricles in experimental hypertension. Circ Res (1990) 67:1355–1364.[Abstract/Free Full Text]
28. Weber K.T., Sun Y., Tyagi S.C., Cleutjens J.P. Collagen network of the myocardium: function, structural remodeling and regulatory mechanisms. J Mol Cell Cardiol (1994) 26:279–292.[CrossRef][Web of Science][Medline]
29. Mochly-Rosen D. Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science (1995) 268:247–251.[Abstract/Free Full Text]
30. Blaschke F., Stawowy P., Kappert K., Goetze S., Kintscher U., Wollert-Wulf B., et al. Angiotensin II-augmented migration of VSMCs towards PDGF-BB involves Pyk2 and ERK 1/2 activation. Basic Res Cardiol (2002) 97:334–342.[CrossRef][Web of Science][Medline]
31. Blaschke F., Stawowy P., Goetze S., Hintz O., Grafe M., Kintscher U., et al. Hypoxia activates beta(1)-integrin via ERK 1/2 and p38 MAP kinase in human vascular smooth muscle cells. Biochem Biophys Res Commun (2002) 296:890–896.[CrossRef][Web of Science][Medline]
32. Lenter M., Uhlig H., Hamann A., Jeno P., Imhof B., Vestweber D. A monoclonal antibody against an activation epitope on mouse integrin chain beta 1 blocks adhesion of lymphocytes to the endothelial integrin alpha 6 beta 1. Proc Natl Acad Sci U S A (1993) 90:9051–9055.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				C. K. Thodeti, B. Matthews, A. Ravi, A. Mammoto, K. Ghosh, A. L. Bracha, and D. E. Ingber TRPV4 Channels Mediate Cyclic Strain-Induced Endothelial Cell Reorientation Through Integrin-to-Integrin Signaling Circ. Res., May 8, 2009; 104(9): 1123 - 1130. [Abstract] [Full Text] [PDF]

				S. S. Palaniyandi, L. Sun, J. C. B. Ferreira, and D. Mochly-Rosen Protein kinase C in heart failure: a therapeutic target? Cardiovasc Res, May 1, 2009; 82(2): 229 - 239. [Abstract] [Full Text] [PDF]

				K. R. Legate and R. Fassler Mechanisms that regulate adaptor binding to {beta}-integrin cytoplasmic tails J. Cell Sci., January 15, 2009; 122(2): 187 - 198. [Abstract] [Full Text] [PDF]

				K. Bourd-Boittin, H. Le Pabic, D. Bonnier, A. L'Helgoualc'h, and N. Theret RACK1, a New ADAM12 Interacting Protein: CONTRIBUTION TO LIVER FIBROGENESIS J. Biol. Chem., September 19, 2008; 283(38): 26000 - 26009. [Abstract] [Full Text] [PDF]

				K. Graf Pulmonary hyperplasia and the two sides of PKC{zeta} Cardiovasc Res, June 1, 2008; 78(3): 409 - 410. [Full Text] [PDF]

				L. Huang, H.-C. Cheng, R. Isom, C.-S. Chen, R. A. Levine, and B. U. Pauli Protein Kinase C{epsilon} Mediates Polymeric Fibronectin Assembly on the Surface of Blood-borne Rat Breast Cancer Cells to Promote Pulmonary Metastasis J. Biol. Chem., March 21, 2008; 283(12): 7616 - 7627. [Abstract] [Full Text] [PDF]

				R. Lubrano, F. Soscia, M. Elli, F. Ventriglia, C. Raggi, E. Travasso, S. Scateni, V. Di Maio, P. Versacci, R. Masciangelo, et al. Renal and Cardiovascular Effects of Angiotensin-Converting Enzyme Inhibitor Plus Angiotensin II Receptor Antagonist Therapy in Children With Proteinuria Pediatrics, September 1, 2006; 118(3): e833 - e838. [Abstract] [Full Text] [PDF]

				S. Stanisavljevic, T. Ignjatovic, P. A. Deddish, V. Brovkovych, K. Zhang, E. G. Erdos, and R. A. Skidgel Angiotensin I-Converting Enzyme Inhibitors Block Protein Kinase C{epsilon} by Activating Bradykinin B1 Receptors in Human Endothelial Cells J. Pharmacol. Exp. Ther., March 1, 2006; 316(3): 1153 - 1158. [Abstract] [Full Text] [PDF]

				L. Hein Angiotensin II and cell-matrix adhesion: PKC{varepsilon} is essential Cardiovasc Res, July 1, 2005; 67(1): 6 - 8. [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Stawowy, P. Articles by Graf, K. Search for Related Content PubMed PubMed Citation Articles by Stawowy, P. Articles by Graf, K. Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics