Abstract

Integrin-linked kinase (ILK) is an integrin-binding cytoplasmic protein that is involved in regulating numerous cellular processes and extracellular matrix accumulation. We reported that ILK may be involved in cellular senescence, but whether ILK is the cause of senescence or an accompanying phenomenon still remains to be explored. Here, RNA interference and gene transfer techniques were used to knock down and overexpress ILK in 3-month-old and 28-month-old rat primary cardiac fibroblasts. The results show that, in younger cells, ILK overexpression induces larger cell shapes, lower proliferation capacity, and higher levels of enzymatic β-galactosidase activity, and increases basal p53 and p21 protein levels, whereas knock-down of ILK prevents phenotypic changes typical of senescence in aging cells. In addition, ILK could induce the cytoskeleton proteins to organize into dense, thick bundles of filaments, which contribute to cellular enlargement and extracellular fibronectin assembly. The results indicate that ILK can accelerate the process of cellular senescence.

CELLULAR replicative senescence or cell senescence is a proliferative exhaustion found in somatic cells (1). When cultured in vitro, somatic cells have a finite life span to which they undergo a set number of cell divisions and subsequently stop dividing. This phenomenon is characteristic of many cell types from a variety of species (1). The senescent cells have specific characteristics such as enlarged cell shapes, shortened telomeres (2), and high expression of an enzymatic β-galactosidase (SA-β-Gal) activity at pH 6.0 (3). In addition, senescent cells are characterized by an irreversible G1 growth arrest involving the repression of genes that drive cell-cycle progression and the upregulation of cell-cycle inhibitors such as p53 and its transcriptional target, p21 (4). Senescent cells can also show select cell-specific functional changes, such as cell adhesion, migration, and the ability to produce extracellular matrix (ECM) (5–7). However, the underlying mechanisms for cell senescence are still unclear.

Integrin-linked kinase (ILK) is an integrin-binding cytoplasmic protein that is involved in the regulation of cell survival, cell cycle, adhesion, proliferation, cell shape, and ECM accumulation (8–17). Our previous studies have indicated that the expression of ILK in aging rats is increased in both renal glomerular mesangial cells and tubular epithelial cells, and is accompanied by the accumulation of fibronectin (Fn). These previous studies suggest that ILK might be closely correlated with the senescence process of the kidney (7).

Aging is a complex physiological process in which the function and structure of many organ systems become altered. Additionally, cardiovascular diseases are the leading cause of death worldwide, especially in elderly populations. One pathophysiological component of cardiovascular disease is myocardial fibrosis, which is primarily derived from cardiac fibroblasts. Cardiac fibroblasts are the major producers of cardiac ECM, which plays an important role in the senescent process within the heart. Hence, cardiac fibroblasts were selected as a target in this study to explore the relationship between ILK and the senescent process of cardiac fibroblasts. We separated and cultured 3-month-old and 28-month-old rat cardiac fibroblasts and investigated the expression of ILK. The results showed that ILK expression was increased in cardiac fibroblasts derived from aging rats. Subsequently, adenoviral expression vectors encoding the ILK gene and ILK-siRNA (small interference RNA) were used to infect cardiac fibroblasts, and these fibroblasts were inspected for changes in morphology, phenotype, proliferation, cell-cycle progression, cytoskeleton assembly, and extracellular Fn assembly. Our results indicated that high levels of ILK could change the arrangement and expressions of cytoskeleton proteins, induce cellular senescence-associated changes, and enhance Fn extracellular assembly in cardiac fibroblasts.

Methods

Reagents

All organic chemicals were of analytic grade and were obtained from Sigma-Aldrich (St. Louis, MO). Cell culture medium, fetal bovine serum, and supplements were obtained from Invitrogen (Carlsbad, CA). Monoclonal mouse anti-ILK antibody (Ab) 65.1 was generated as previously described (18) and used for immunofluorescence staining. Polyclonal rabbit anti-ILK antibody was purchased from Upstate Biotechnology Inc. (Charlottesville, VA). The polyclonal rabbit anti-p53 antibody, the mouse anti-p21 antibody, the mouse anti-smooth muscle alpha actin (α-SMA) antibody, the mouse antivimentin antibody, and the goat antiactin antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Texas Red–labeled phalloidin and FITC-labeled secondary antibodies were purchased from Santa Cruz Biotechnology, Inc.

Primary Culture of Rat Cardiac Fibroblasts

Cardiac fibroblasts from male Wistar rats were isolated by enzymatic digestion of ventricular tissue, as described previously (19,20). Briefly, the heart of six 3-month-old male Wistar rats weighing 200–250 g and of six 28-month-old rats weighing 470–530 g were removed, minced, and washed in phosphate-buffered saline (PBS) under sterile conditions. The tissues were digested at 37°C in digestion medium containing a mixture of collagenase V (100 mg/100 mL), trypsin (50 mg/100 mL), and pancreatin (60 mg/100 mL) for 10 minutes with constant shaking. Cells from the 4th to the 10th digestions, each of 10-minute duration, were collected and plated on 35 mm plates containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, penicillin at 100 U/mL, and streptomycin at 100 U/mL. After incubation at 37°C in a CO2 incubator for 150 minutes, unattached cells were discarded, and the attached cells (mostly fibroblasts) were washed and grown in the plating medium. Confluent cultures were passaged three or four times on 100 mm dishes. The cells were verified to be fibroblasts by immunocytochemistry using polyclonal antivimentin antibodies (21).

Construction of Recombinant Adenoviruses

The adenoviral expressing vector encoding a full-length ILK was generated based on a previously described protocol (22,23). Briefly, complementary DNA encoding the full-length ILK was inserted into the EcoRI/XbaI sites of the pAdTrack-cytomegalovirus (CMV) shuttle vector. The shuttle vector plasmid was linearized with PmeI, purified by phenol and chloroform extraction and ethanol precipitation, and mixed with super-coiled pADEasy-1. We transformed the vectors into Escherichia coli BJ5183 by electroporation with a Bio-Rad Gene Pulser electroporator (Hercules, CA). The bacteria were immediately placed in 1 mL of LB Broth, Lennox (tryptone at 10 g/L, yeast extract at 5 g/L, NaCl at 5 g/L) (Fisher, Pittsburgh, PA) and grown at 37°C for 1 hour. The bacteria were then inoculated onto agar containing LB Broth supplemented with kanamycin at 50 μg/mL. After 16–20 hours growth, colonies were picked and grown in 2 mL of LB Broth containing kanamycin at 50 μg/mL. Clones were screened by digestions with the restriction endonucleases PacI and BamHI. The positive plasmids were transformed into DH10B cells by electroporation for large-scale amplification. The plasmid DNA was digested with PacI, ethanol-precipitated, and was used to transfect HEK293 cells with Lipofectamine PLUS (Invitrogen). The transfected cells were harvested 10 days after transfection. The cells were lysed by three cycles of freezing in a methanol and dry ice bath and rapidly thawing at 37°C, and the lysate containing the recombinant adenovirus was collected. The control adenoviral expression vector encoding β-Gal was kindly provided by Tong-Chuan He and Bert Vogelstein (Howard Hughes Medical Institute, the Johns Hopkins Oncology Center, Baltimore, MD) (22,23).

siRNA Experiments

The siRNA was designed to target the following rat ILK sequence (GenBank, NM_133409): GGA CAC ATT CTG GAA GGG G. The corresponding primers are as follows, sense: 5′-AAG GAC ACA TTC TGG AAG GGG CCT GTC TC-3′, and antisense: 5′-AAC CCC TTC CAG AAT GTG TCC CCT GTC TC-3′ to produce siRNA-ILK by using the Silencer siRNA construction kit (Ambion, Inc., Austin, TX). A control siRNA (siRNA-con) of noncorrelated rat sequence (sense: 5′-AAG AAG AAG TCG TGC TGC TTC CCT GTC TC-3′, antisense: 5′-AAG AAG CAG CAC GAC TTC TTC CCT GTC TC-3′) was also designed. Each sequence was blasted to assess specificity. Transient transfections of primary rat cardiac fibroblasts from 3-month-old rats and 28-month-old rats were carried out by using 2.5 μL of Lipofectamine reagent for 48 hours, according to the manufacturer's guidelines.

Adenovirus Infection in Cardiac Fibroblasts

The recombinant adenoviral expression vectors (green fluorescent protein [GFP]-ILK) or the control adenoviral expression vectors encoding β-Gal (GFP-β-Gal) were used to infect cardiac fibroblasts in 3-month-old rats and 28-month-old rats. The infection efficiency was monitored by the expression of GFP encoded by the viral vectors, and it typically reached 80%–90% within 48 hours.

Cellular Shape Changes

Cardiac fibroblasts from 3-month-old and 28-month-old rats were infected by adenoviral expressing vectors containing GFP-β-Gal, GFP-ILK, siRNA-ILK, or siRNA-con. After 48 hours, cellular morphology was observed by confocal fluorescence or phase-contrast light transmission microscopy. The planar area of cells was automatically calculated by LaserPix software (Bio-Rad Laboratories, Hercules, CA). The planar area was expressed as mean ± standard deviation (square microns).

Senescence-Associated-β-Gal Staining

Cells were washed in PBS, fixed for 3–5 minutes (room temperature) in 2% formaldehyde/0.2% glutaraldehyde, washed, and incubated with fresh senescence-associated β-Gal stain solution at a concentration of 1 mg/mL for 24 hours at room temperature (7). After washing with PBS, the percentages cells staining positive for SA-β-Gal were counted. Positive cells had blue sedimentation in their cytoplasm.

Cellular Immunofluorescence Staining

Immunofluorescence staining was performed using a standard protocol. Briefly, cells cultured on coverslips were washed twice with cold PBS and fixed with 3.7% paraformaldehyde for 10 minutes at room temperature. Following three extensive washings with PBS containing 0.1% Triton X-100, the cells were blocked with 10% normal goat serum in PBS buffer for 20 minutes at room temperature, then incubated with the specific primary Abs overnight at 4°C, incubated with species-specific fluorescein rhodamine-conjugated or fluorescein isothiocyanate-conjugated secondary Abs for 60 minutes at room temperature. Stained cell monolayers were observed using a Bio-Rad Radiance 2000 laser confocal microscope.

Cell Proliferation Capacity Analyses

Cells were seeded into 96-well plates (2 × 103 cells per well), then synchronized for 24 hours. After incubation for 24, 48, and 72 hours, 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl -2H-tetrazolium bromide (MTT) (2 mg/mL, 20 μL) was added into each well and incubated for 4 hours. Dimethyl sulfoxide (100 μL) was subsequently added to each well to dissolve the formazan crystals. The absorption at 570 nm was measured.

Cell-Cycle Analysis

Cardiac fibroblasts of 3-month-old or 28-month-old rats were cultured as shown above; the cultures were then synchronized for 24 hours. After 48 hours, 1 × 106 (each sample) cells were collected with 0.25% trypsin, washed twice with cold PBS, and fixed with 70% alcohol in PBS for 12 hours at 4°C. Cells were stained for 30–60 minutes at 4°C in propidium iodide solution (RNase at 100 μg/mL, propidium iodide solution at 100 μg/mL) after washing twice with PBS. Stained cells were analyzed in a flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Western Blot and Northern Blot Analyses

Messenger RNA (mRNA) and protein expression of ILK, vimentin, α-SMA, p53, and p21 were assessed by Northern blot and Western blot. Cells (5 × 106) were homogenized in 0.2 mL of ice-cold RIPA extraction buffer. Western blot analysis for protein expression was carried out by using standard protocols. Then, total RNA was isolated from cells using TRIzol reagent (Gibco BRL). Northern blot analysis for mRNA expression was also carried out by using standard protocols (7).

Biochemical Determination of Fn Assembly

The extracellular assembly of Fn was quantitatively determined by a biochemical method described previously by Wu and colleagues (18). Cardiac fibroblasts (3 × 106) were seeded in 100-mm tissue culture plates in complete medium as described above and incubated for 48 hours. The cell monolayers were then washed three times with PBS containing 1 mM 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF) and harvested with a rubber policeman. The ECM fraction was isolated by sequential extraction of the cells with: (i) 3% Triton X-100 in PBS containing 1 mM AEBSF; (ii) DNase I at 100 μg/mL in 50 mM Tris, pH 7.4, 10 mM MnCl2, 1 M NaCl, and 1 mM AEBSF; and (iii) 2% deoxycholate in Tris, pH 8.8, and 1 mM AEBSF. Fibronectin in the deoxycholate-insoluble ECM fraction was analyzed by Western blot with monoclonal anti-Fn Ab according to the method described above.

Statistical Analysis

All data analyses were performed with SPSS (SPSS Inc., Chicago, IL). Parametric data are reported as mean ± standard deviation. Student's t test was used for statistical analysis between two groups. Comparison among groups was conducted by using analysis of variance (ANOVA). Probability values <.05 were considered significant.

Results

ILK Expression in Cardiac Fibroblasts From Young and Aging Rats

The expression of ILK was detected in cardiac fibroblasts from 3-month-old (young) and 28-month-old (old) male Wistar rats by means of immunofluorescent staining, Northern blot, and Western blot analysis. The results showed that ILK was localized to the cytoplasm, and increased in aging cells (Figure 1A). Northern blot and Western blot analysis revealed that ILK mRNA expression and protein abundance were also increased in aging cells (Figure 1, B and C). In cardiac fibroblasts from young and old rats, the ratios of ILK mRNA and protein to β-actin were 0.34 ± 0.06 versus 0.62 ± 0.10 (p <.01), and 0.31 ± 0.07 versus 0.59 ± 0.09 (p <.01), respectively. The results showed that aging cells contained approximately 2-fold more ILK mRNA and protein than young cells did, indicating that ILK may play an important role in the aging process of cardiac fibroblasts.

Overexpression of ILK Induces the Senescent-Like Change of Cardiac Fibroblasts

To examine the effects of ILK, young and old cardiac fibroblasts were infected with GFP-ILK. The infected fibroblasts showed higher levels of ILK protein (Figure 2A) than did control cells and cells infected with GFP-β-Gal and (p <.05).

After being infected with GFP-β-Gal and GFP-ILK, both the young and the old cells with overexpression of ILK presented changes typical of senescence, such as flattened and enlarged cell shapes (Figure 2B, p <.01; control fibroblasts were transfected with a GFP vector as a detection marker).

SA-β-Gal is a hallmark of cell replicative senescence or physiologic age (3). Hence, we assayed β-Gal activity of young and old cardiac fibroblasts transfected with the GFP-β-Gal or GFP-ILK gene. The results indicated that the percentage of cells staining positive for SA-β-Gal was significantly higher in the old cells than in the young ones (Figure 2C) (12.17 ± 4.71% vs 71.00 ± 11.14%, p <.01). The percentage of cells staining positive for SA-β-Gal was also higher in cells transfected with GFP-ILK than in those transfected with GFP-β-Gal, regardless of age (Figure 2C). In young cardiac fibroblasts, cells infected with GFP-ILK showed 26.0 ± 5.55% of cells staining, whereas infection with GFP-β-Gal showed 10.0 ± 4.43% (p <.01). In aging cardiac fibroblasts, the percentage of cells staining positive for SA-β-Gal was also higher for the cells infected with GFP-ILK than with GFP-β-Gal (92.33 ± 6.23% vs 74.17 ± 11.44%, respectively; p <.01).

The MTT assay measured cellular proliferative capacity in young versus old and GFP-β-Gal- versus GFP-ILK-infected fibroblasts at 24, 48, and 72 hours (Figure 2D). The cellular proliferative capacity of aging cells at 24 hours (0.49 ± 0.04 vs 0.45 ± 0.06, p <.05), 48 hours (0.71 ± 0.06 vs 0.58 ± 0.04, p <.01), and 72 hours (1.0 ± 0.09 vs 0.81 ± 0.07, p <.01) was significantly lower than that of young cells. In both cells, the proliferative capacity of cells infected with the ILK gene was also significantly lower than that in cells infected with GFP-β-Gal, indicating that overexpression of ILK can induce loss of replicative potential over time.

Taken together, these results indicate that overexpression of ILK could induce premature senescence (such as the enlarged cell shapes, the higher expression of SA-β-Gal, and the reduction of cellular proliferation capacity) in cardiac fibroblasts.

Overexpression of ILK Upregulates the p53 and p21 Proteins and Arrests Cardiac Fibroblasts in G1 Phase

It is well known that, after completing a finite number of divisions, cell growth is arrested in G1 phase, and the cells cannot enter the S phase of the cell cycle. This is termed cell replicative senescence. To investigate the cellular mechanism for the reduction of cellular proliferative capacity in ILK overexpressing cells and to clarify whether ILK could induce the expressions of p53 and p21 (cell-cycle repressors), young and old cardiac fibroblasts were infected with either GFP-β-Gal or GFP-ILK. After 48 hours, the abundance of p53 protein in aging cells was 2.4-fold that of young cells (0.35 ± 0.06 vs 0.83 ± 0.04, p <.01), and the p21 protein in aging cells was 2-fold that of young cells (0.31 ± 0.04 vs 0.61 ± 0.10, p <.01). In both young and aging cells, both p53 and p21 protein was significantly higher in cells infected with GFP-ILK as compared to cells infected with GFP-β-Gal (Figure 3). Under the same conditions, the cell-cycle progression was assayed by flow cytometry. The percentages of young and old cells at G1 phase were 66.57 ± 2.46% and 71.66 ± 3.58%, respectively (Table 1). In both types of fibroblasts, infection of GFP-ILK increased the percentage of cells in G1 phase as compared to GFP-β-Gal-infected fibroblasts (69.07 ± 1.02% vs 65.90 ± 2.03%, p <.05 in young fibroblasts; 85.14 ± 3.84% vs 75.16 ± 2.94% in old fibroblasts). Our results suggest that ILK overexpression can arrest cells in G1 phase.

These results shows that overexpression of ILK in cardiac fibroblasts can induce increased expression of both p53 and p21 proteins and facilitate cell-cycle arrest in G1 phase, which indicates that the increases in ILK has an important role in the aging-related growth reduction.

Silencing ILK Expression Can Prevent Senescence-Like Phenotypes in Cardiac Fibroblasts

Based on the results mentioned above, overexpression of ILK could induce a senescence-like phenotype and inhibit the cellular proliferation capacity in young cardiac fibroblasts. This result leaves an interesting question of whether silencing of ILK can prevent these effects on aging cells.

The siRNA targeting rat ILK sequence was designed and transfected into cardiac fibroblasts. To investigate its efficacy, cardiac fibroblasts were infected with either siRNA-ILK or irrelevant nonspecific siRNA (siRNA-con). ILK expression was assessed by Western blot. ILK protein expressions were markedly lower in cardiac fibroblasts transfected with siRNA-ILK than in both control cells and cells transfected with nonspecific siRNA-con (Figure 4A), suggesting that the siRNA-ILK was effective and specific in silencing ILK expression in cardiac fibroblasts.

It was found that, in old cardiac fibroblasts infected with siRNA-ILK, the SA-β-Gal positive staining rate was significantly reduced compared to that in both control cells and cells infected with siRNA-con (30 ± 6% vs 78 ± 10% and 74 ± 9%, p <.01). However, in young cells, the SA-β-Gal positive staining rate of the three groups was similar (control group: 19 ± 6%; siRNA-con group: 17 ± 5%; siRNA-ILK group: 18 ± 5.5%, p >.05, Figure 4B), indicating that the inhibition of ILK can prevent the senescence-like phenotype of old fibroblasts. Regardless of whether the fibroblasts were transfected with siRNA-ILK, they did not show any significant changes in cellular proliferation capacity or morphology (data not shown).

In addition it was found that, in aging cardiac fibroblasts transfected with siRNA-ILK, the G1 phase arrest of the cell cycle was prevented through the reduction of p53 and p21 expression. In young and old cardiac fibroblasts transfected with siRNA-ILK, the percentages of cells at G1 phase and the expressions of p21 and p53 protein were significantly lower than those of controls or siRNA-con groups (Table 2, Figure 4C). These results disclose that the inhibition of ILK can attenuate the G1 phase arrest through downregulation of the expression levels of the cell-cycle inhibitors p21 and p53.

ILK Mediates the Cytoskeletal Proteins and Enhances Fn Extracellular Assembly in Old Cardiac Fibroblasts

The senescent fibroblasts have markedly specific characteristics such as enlarged cell shapes and enhanced ECM assembly, both of which may be affected by cytoskeletal proteins. α-SMA and vimentin are major cytoskeletal proteins in cardiac fibroblasts that contribute to the progression toward senescent characteristics.

Young and old cardiac fibroblasts were infected with adenoviral expressing vectors GFP-β-Gal, GFP-ILK, siRNA-ILK, or siRNA-con. α-SMA protein abundances and mRNA expression were detected by immunofluorescence staining, Western blot, and Northern blot. In young cardiac fibroblasts infected with GFP-ILK, the fluorescence intensity of α-SMA in cells was significantly higher, and was accompanied by more densely organized bundles of long filaments than were cells transfected with GFP-β-Gal or young control cells; however, they resembled old cells (Figure 5A). The results of Northern blot and Western blot analysis were consistent with those of immunofluorescence staining (Figure 5B and C).

We analyzed the ability of the cells expressing different levels of ILK to assemble an Fn matrix. The ILK-overexpressing young cells infected with GFP-ILK assembled an extensive Fn matrix resembling that formed by old cardiac fibroblasts, whereas control infectants (GFP-β-Gal) assembled a small amount of Fn matrix that was indistinguishable from that of young controls (Figure 5D and E). Quantitative determination revealed a more than 6-fold induction of Fn assembly in the extracellular compartment in ILK-overexpressing cells compared with control and GFP-β-Gal-transfected cells.

Moreover, in old cardiac fibroblasts, the fluorescence intensity of α-SMA in cells transfected with siRNA-ILK was significantly lower than that of cells transfected with siRNA-con or old control cells, and was similar to that of young cells (Figure 6A). The results of Northern blot and Western blot analysis were consistent with those of immunofluorescence staining (Figure 6B and C). As shown in Figure 6D and E, the immunofluorescence staining and Western blot analysis revealed that the silencing of ILK reduced Fn protein expression in old cardiac fibroblasts to almost the expression levels found in young cells. Under the same conditions, no significant change in Fn protein was found in cardiac fibroblasts transfected with siRNA-con or in control cells. These results imply that ILK not only induces the organization and expression alteration of cytoskeletal protein α-SMA expression, but perhaps more importantly, it preferentially promotes the extracellular assembly of Fn.

Vimentin is a biologic marker of fibroblasts that contributes to the cell shape. However, studies have shown that the expression of vimentin is increased, and the arrangement of filaments is altered, in senescent human fibroblasts (6,24). ILK mediates interactions between multiple cytoskeletal proteins, controlling cytoskeleton changes. Then, by means of ILK gene transfection, we investigated vimentin alterations in cells with overexpression or silencing of ILK. Figure 7A shows that, in young cardiac fibroblasts, ILK-transfected cells had densely organized bundles of long vimentin filaments, which extended from the perinuclear region to the peripheries of the cytoplasm as they do in old control cells. Conversely, the young control and GFP-β-Gal-transfected cells exhibited smaller cell shapes and a fur-like network of thin and short vimentin filaments. Northern and Western blot analysis also showed greater abundance in GFP-ILK–transfected cells and old control cells compared with young control and GFP-β-Gal-transfected cells (Figure 7B and C). Meanwhile, in old cardiac fibroblasts transfected with siRNA-ILK, the vimentin filaments became slender, which was a similar morphology to young fibroblasts, but the shapes were not significantly different from the old control and siRNA-con-transfected cells (Figure 8A). The level of vimentin mRNA and protein in old cells transfected with siRNA-ILK was similar to that of young control cells, and were 2-fold lower than the old control and siRNA-con cells (Figure 8B and C). These results demonstrated that high levels of ILK in old cardiac fibroblasts can alter the dense filament arrangement and overexpression of vimentin, which correlates with the senescence-related shape alteration.

Discussion

ILK is an integrin-binding cytoplasmic protein that is involved in the regulation of a number of cellular processes and ECM accumulation. Studies have demonstrated that ILK plays a central role in the activation or repression of genes encoding proteins involved in the regulation of cell survival, cell cycle, cell adhesion, and proliferation (8–11). Our previous study demonstrated that ILK might be closely correlated with the senescent process of cells and tissue from the kidney (7).

As well as with the senescent process in the kidney, senescence is associated with marked changes in cardiac structure and morphology such as cardiomyocyte loss, hypertrophy of the remaining cells, and the development of fibrosis. Fibrosis is found in nondiseased old hearts, and therefore can be independently associated with the aging process (25,26). As cardiac function declines with advancing age, ECM collagen and Fn influence diastolic stiffness. So the present work demonstrates that in the old cardiac fibroblasts, the increase of ILK is associated with the senescence-related phenotype and that overexpression or silencing of ILK could significantly alter cytoskeleton organization, cell senescence-associated alterations, and Fn extracellular assembly in cardiac fibroblasts.

First, in our study, we found that there were significant differences between young and aging cardiac fibroblasts of rats. Aging cardiac fibroblasts contained approximately 2-fold more ILK protein and mRNA than did young cardiac fibroblasts. Aging cardiac fibroblasts presented enlarged cell shapes, low proliferation capacity, high percentages of G1 phase cells, high enzymatic β-Gal activity, and upregulation of the cell-cycle inhibitor p53 and its transcriptional target p21, as compared to young cardiac fibroblasts. The results strongly suggested that rat cardiac fibroblasts could undergo senescence and the processes of cell senescence could be closely related to ILK.

Although high density of ILK expression has been found in cardiac fibroblasts, it was unknown whether ILK may have an accompanying role or an essential role in the aging process of cardiac fibroblasts. So, this study was designed to explore the relationship between ILK expression and the alteration of cell senescence. The adenoviral expressing vector encoding ILK and siRNA-ILK was used to infect young and old cardiac fibroblasts. The results showed that young cells overexpressing ILK exhibited senescent cell-like alterations, and aging cells overexpressing ILK presented higher levels of senescence. In the young cardiac fibroblasts, overexpression of ILK could flatten and enlarge cell shapes, increase the percentage of cells staining positive for SA-β-Gal, upregulate p53 and p21 proteins, and arrest cells in the G1 phase. Meanwhile, in the old cardiac fibroblasts, siRNA was used to silence ILK expression. Results showed that these techniques were effective and specific to decreased ILK expression. Inhibition of ILK could attenuate senescent phenotypes of old cardiac fibroblasts, such as reducing the SA-β-Gal positive staining rate, lessening the expression of p53 and p21, and preventing the G1 phase arrest of cell cycle; however, the cell shape in ILK knock-down cardiac fibroblasts was not significantly changed. Taken together, it is reasonable to suggest that there may be an association between decreased ILK expression and cell senescence prevention. The high level of ILK may result in unregulated cell proliferation and contribute, at least in part, to the cell senescence of old cardiac fibroblasts.

ILK is a key component of the cell–ECM adhesion structure, and can connect intracellular cytoskeleton and signaling proteins. A number of studies have demonstrated that the senescent cells exhibited alterations of regulatory system of gene transcription (27–29) and ECM proteins (30,31), and the organization of the cytoskeleton (6,24). The regulatory system of gene transcription, the ECM proteins, and the organization of the cytoskeleton are paratactic. These findings suggested that the various changes in senescent cells might be closely related to actin cytoskeleton organization, focal adhesions between cells and between the cell and the ECM, and the related downstream signal transductions. As expected, ILK expression was closely related to the cytoskeleton arrangement and expression in our study. As overexpressing ILK in young cardiac fibroblasts, the expressions of α-SMA and vimentin were increased, and their arrangement was altered, and extracellular Fn was assembled, which were in parallel with the senescence-associated cellular alterations, while knock-down of ILK in old cells could markedly prevent those changes. Other studies revealed that senescent cells exhibited unusual large, thick bundles of cytoskeleton proteins (6). However, the study by Nishio and colleagues (24) was more interesting, showing that young fibroblasts displayed senescent cell morphology by means of transfection with vimentin cytoskeleton. A number of studies (32–35) have shown that the alterations of cytoskeleton organization can bring about cellular conformation alterations, the signals of which were then transferred into the nucleus leading to the alterations in gene transcription. Additionally, signalling from cytoskeletal alterations can be transferred to the cellular membrane and ECM, and can induce changes in cell shape, phenotype, cell adhesion, and migration. Our results strongly suggest that ILK not only induces the organization and expression of cytoskeletal protein, but perhaps more importantly, it preferentially promotes the extracellular assembly of Fn in the senescent process of cardiac fibroblasts.

Conclusion

The present study has explored the relationship between ILK and senescence of cardiac fibroblasts. From primary culture of cardiac fibroblasts of young and aging rats, we found that higher levels of ILK were expressed in old cardiac fibroblasts compared with young cells, which contributed to the cellular replicative senescence. Our experimental data also indicated that ILK could alter cytoskeleton organization and enhance Fn extracellular assembly.

Decision Editor: Huber R. Warner, PhD

Figure 1.

Expressions of integrin-linked kinase (ILK) in young and aging cardiac fibroblasts. A, Indirect immunofluorescence staining (×600); B, messenger RNA expression; C, protein abundance. (*p <.01, n = 6; Young: cells of 3-month-old rats; Old: cells of 28-month-old rats)

Figure 1.

Expressions of integrin-linked kinase (ILK) in young and aging cardiac fibroblasts. A, Indirect immunofluorescence staining (×600); B, messenger RNA expression; C, protein abundance. (*p <.01, n = 6; Young: cells of 3-month-old rats; Old: cells of 28-month-old rats)

Figure 2.

Integrin-linked kinase (ILK) protein overexpression of young and old cardiac fibroblasts induced senescence-like change. A, ILK protein expression detected by Western blot in young and old cardiac fibroblasts transfected with green fluorescent protein (GFP)-ILK and GFP- beta-galactosidase (β-Gal) (*p <.01, young GFP-ILK vs young control cells and young GFP-β-Gal; old GFP-ILK vs old control cells and old GFP-β-Gal; n = 6). B, Enlarged and flattened shapes of young and old cardiac fibroblasts transfected with GFP-ILK (×600). Bar graph of the planar area summarizes data obtained in 36 cells (*p <.05 vs young control cells and young GFP-β-Gal; #p <.05 vs old control cells and old GFP-β-Gal). C, Cells staining positive for enzymatic (SA)-β-Gal showed blue color sedimentation in the cytoplasm (×100). Bar graph of percentage of cells staining positive for SA-β-Gal transfected with and without GFP-ILK summarizes data obtained from 500 cells (*p <.01 vs control cells and cells transfected with GFP-β-Gal). D, Cellular proliferation of those cells was detected with 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl -2H-tetrazolium bromide (MTT) assay (#p <.05, *p <.01 vs control cells and cells transfected with GFP-β-Gal; n = 6). Comparison among groups was conducted with analysis of variance. OD = optical density

Figure 2.

Integrin-linked kinase (ILK) protein overexpression of young and old cardiac fibroblasts induced senescence-like change. A, ILK protein expression detected by Western blot in young and old cardiac fibroblasts transfected with green fluorescent protein (GFP)-ILK and GFP- beta-galactosidase (β-Gal) (*p <.01, young GFP-ILK vs young control cells and young GFP-β-Gal; old GFP-ILK vs old control cells and old GFP-β-Gal; n = 6). B, Enlarged and flattened shapes of young and old cardiac fibroblasts transfected with GFP-ILK (×600). Bar graph of the planar area summarizes data obtained in 36 cells (*p <.05 vs young control cells and young GFP-β-Gal; #p <.05 vs old control cells and old GFP-β-Gal). C, Cells staining positive for enzymatic (SA)-β-Gal showed blue color sedimentation in the cytoplasm (×100). Bar graph of percentage of cells staining positive for SA-β-Gal transfected with and without GFP-ILK summarizes data obtained from 500 cells (*p <.01 vs control cells and cells transfected with GFP-β-Gal). D, Cellular proliferation of those cells was detected with 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl -2H-tetrazolium bromide (MTT) assay (#p <.05, *p <.01 vs control cells and cells transfected with GFP-β-Gal; n = 6). Comparison among groups was conducted with analysis of variance. OD = optical density

Figure 3.

Expression of p53 and p21 proteins in cardiac fibroblasts. Young and old rat cardiac fibroblasts were transfected with adenoviral expressing vectors green fluorescent protein-beta galactosidase (GFP-β-Gal) or GFP-integrin-linked kinase (ILK). After 48 hours, p53 and p21 proteins were detected with Western blot. A, Western blot results of p21 and p53 proteins. B and C, Relative p21 and p53 protein abundances normalized to β-actin (*p <.01 vs young control cells and young GFP-β-Gal; #p <.01 vs old control cells and old GFP-β-Gal; n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Figure 3.

Expression of p53 and p21 proteins in cardiac fibroblasts. Young and old rat cardiac fibroblasts were transfected with adenoviral expressing vectors green fluorescent protein-beta galactosidase (GFP-β-Gal) or GFP-integrin-linked kinase (ILK). After 48 hours, p53 and p21 proteins were detected with Western blot. A, Western blot results of p21 and p53 proteins. B and C, Relative p21 and p53 protein abundances normalized to β-actin (*p <.01 vs young control cells and young GFP-β-Gal; #p <.01 vs old control cells and old GFP-β-Gal; n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Figure 4.

Inhibition of integrin-linked kinase (ILK) expression in cardiac fibroblasts by small interference RNA (siRNA). A, ILK protein expression detected by Western blot in young and old cardiac fibroblasts transfected by siRNA and siRNA-con (*p <.01, young siRNA-ILK vs young control cells and young siRNA-con; old siRNA-ILK vs old control cells and old siRNA-con; n = 6). B, Effect of siRNA on enzymatic beta galactosidase (SA-β-Gal) staining of cardiac fibroblasts in 500 cells (p <.01 vs old control cells and old siRNA-con). C, Downregulation of the proteins p21 and p53 in cardiac fibroblasts infected with siRNA-ILK detected with Western blot (*p <.01 vs young control cells and young siRNA-con; #p <.01 vs old control cells and old siRNA-con; n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Figure 4.

Inhibition of integrin-linked kinase (ILK) expression in cardiac fibroblasts by small interference RNA (siRNA). A, ILK protein expression detected by Western blot in young and old cardiac fibroblasts transfected by siRNA and siRNA-con (*p <.01, young siRNA-ILK vs young control cells and young siRNA-con; old siRNA-ILK vs old control cells and old siRNA-con; n = 6). B, Effect of siRNA on enzymatic beta galactosidase (SA-β-Gal) staining of cardiac fibroblasts in 500 cells (p <.01 vs old control cells and old siRNA-con). C, Downregulation of the proteins p21 and p53 in cardiac fibroblasts infected with siRNA-ILK detected with Western blot (*p <.01 vs young control cells and young siRNA-con; #p <.01 vs old control cells and old siRNA-con; n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Figure 5.

Overexpression of integrin-linked kinase (ILK) induced smooth muscle alpha actin (α-SMA) protein abundance and enhanced fibronectin (Fn) extracellular assembly in young cardiac fibroblasts. Cardiac fibroblasts from 3-month-old rats were transfected with adenoviral expressing vectors green fluorescent protein-beta galactosidase (GFP-β-Gal) and GFP-ILK. After 48 hours, α-SMA protein and messenger RNA expression were detected by means of immunofluorescence staining (A, ×600), Northern blot (B), and Western blot (C). Extracellular Fn expression was detected by means of immunofluorescence staining (D, ×600) and Western blot (E). (*p <.05, n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Figure 5.

Overexpression of integrin-linked kinase (ILK) induced smooth muscle alpha actin (α-SMA) protein abundance and enhanced fibronectin (Fn) extracellular assembly in young cardiac fibroblasts. Cardiac fibroblasts from 3-month-old rats were transfected with adenoviral expressing vectors green fluorescent protein-beta galactosidase (GFP-β-Gal) and GFP-ILK. After 48 hours, α-SMA protein and messenger RNA expression were detected by means of immunofluorescence staining (A, ×600), Northern blot (B), and Western blot (C). Extracellular Fn expression was detected by means of immunofluorescence staining (D, ×600) and Western blot (E). (*p <.05, n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Figure 6.

Silencing of integrin-linked kinase (ILK) reduced smooth muscle alpha actin (α-SMA) protein and attenuated the fibronectin (Fn) extracellular assembly in young cardiac fibroblasts. Cardiac fibroblasts from 28-month-old rats were transfected with control small interference RNA (siRNA-con) and siRNA-ILK. After 48 hours, α-SMA protein and messenger RNA expression were detected by means of immunofluorescence staining (A, ×600), Northern blot (B), and Western blot (C). Extracellular Fn expression was detected by means of immunofluorescence staining (D, ×600) and Western blot (E). (*p <.05, n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Figure 6.

Silencing of integrin-linked kinase (ILK) reduced smooth muscle alpha actin (α-SMA) protein and attenuated the fibronectin (Fn) extracellular assembly in young cardiac fibroblasts. Cardiac fibroblasts from 28-month-old rats were transfected with control small interference RNA (siRNA-con) and siRNA-ILK. After 48 hours, α-SMA protein and messenger RNA expression were detected by means of immunofluorescence staining (A, ×600), Northern blot (B), and Western blot (C). Extracellular Fn expression was detected by means of immunofluorescence staining (D, ×600) and Western blot (E). (*p <.05, n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Figure 7.

Overexpression of integrin-linked kinase (ILK) enhanced the vimentin protein expression and changed vimentin filaments to densely bundled filaments in young cardiac fibroblasts. Cardiac fibroblasts from 3-month-old rats were transfected with adenoviral expressing vectors green fluorescent protein-beta galactosidase (GFP-β-Gal) and GFP-ILK. After 48 hours, vimentin protein and messenger RNA expression were detected by means of immunofluorescence staining (A, ×400), Northern blot (B), and Western blot (C). (*p <.01 vs young control and GFP-β-Gal–transfected young cells, n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Figure 7.

Overexpression of integrin-linked kinase (ILK) enhanced the vimentin protein expression and changed vimentin filaments to densely bundled filaments in young cardiac fibroblasts. Cardiac fibroblasts from 3-month-old rats were transfected with adenoviral expressing vectors green fluorescent protein-beta galactosidase (GFP-β-Gal) and GFP-ILK. After 48 hours, vimentin protein and messenger RNA expression were detected by means of immunofluorescence staining (A, ×400), Northern blot (B), and Western blot (C). (*p <.01 vs young control and GFP-β-Gal–transfected young cells, n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Figure 8.

Silencing of integrin-linked kinase (ILK) reduced the vimentin protein expression and changed vimentin filaments to thin filaments or fur-like irregular networks in old cardiac fibroblasts. Cardiac fibroblasts from 28-month-old rats were transfected with control small interference RNA (siRNA-con) and siRNA-ILK. After 48 hours, vimentin protein and messenger RNA expression were detected by means of immunofluorescence staining (A, ×400), Northern blot (B), and Western blot (C). (*p <.01 vs old control and siRNA-con-transfected old cells; n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Figure 8.

Silencing of integrin-linked kinase (ILK) reduced the vimentin protein expression and changed vimentin filaments to thin filaments or fur-like irregular networks in old cardiac fibroblasts. Cardiac fibroblasts from 28-month-old rats were transfected with control small interference RNA (siRNA-con) and siRNA-ILK. After 48 hours, vimentin protein and messenger RNA expression were detected by means of immunofluorescence staining (A, ×400), Northern blot (B), and Western blot (C). (*p <.01 vs old control and siRNA-con-transfected old cells; n = 6). Comparison among groups was conducted with analysis of variance (ANOVA)

Table 1.

Percentages of Cells at G1 Phase and S Phase in Cardiac Fibroblasts Infected With GFP-ILK.

 G1 Phase (%)
 
 S Phase (%)
 
 
Groups 3-Month-Old Rats 28-Month-Old Rats 3-Month-Old Rats 28-Month-Old Rats 
Control 66.57 ± 2.46 71.66 ± 3.58 23.40 ± 1.62 16.29 ± 1.74 
GFP-β-Gal 65.90 ± 2.03 75.16 ± 2.94 27.88 ± 3.48 14.97 ± 1.62 
GFP-ILK 69.07 ± 1.02* 85.14 ± 3.84* 21.15 ± 1.05* 11.81 ± 1.06* 
 G1 Phase (%)
 
 S Phase (%)
 
 
Groups 3-Month-Old Rats 28-Month-Old Rats 3-Month-Old Rats 28-Month-Old Rats 
Control 66.57 ± 2.46 71.66 ± 3.58 23.40 ± 1.62 16.29 ± 1.74 
GFP-β-Gal 65.90 ± 2.03 75.16 ± 2.94 27.88 ± 3.48 14.97 ± 1.62 
GFP-ILK 69.07 ± 1.02* 85.14 ± 3.84* 21.15 ± 1.05* 11.81 ± 1.06* 

Notes: Values are means ± standard deviation (n = 6); comparison among groups was conducted by analysis of variance.

*p <.05 versus control and GFP-β-Gal-transfected cells.

GFP-β-Gal = green fluorescent protein-β-galactosidase; GFP-ILK = green fluorescent protein-integrin-linked kinase.

Table 2.

Percentages of Cells at G1 Phase and S Phase in Cardiac Fibroblasts Infected With siRNA-ILK.

 G1 Phase (%)
 
 S Phase (%)
 
 
Groups 3-Month-Old Rats 28-Month-Old Rats 3-Month-Old Rats 28-Month-Old Rats 
Control 62.11 ± 1.20 72.10 ± 1.18 32.25 ± 1.73 15.11 ± 0.90 
siRNA-con 65.88 ± 1.15 71.32 ± 1.55 34.05 ± 1.76 14.16 ± 1.21 
siRNA-ILK 62.46 ± 1.73 65.57 ± 3.0* 27.96 ± 1.35* 20.79 ± 1.44* 
 G1 Phase (%)
 
 S Phase (%)
 
 
Groups 3-Month-Old Rats 28-Month-Old Rats 3-Month-Old Rats 28-Month-Old Rats 
Control 62.11 ± 1.20 72.10 ± 1.18 32.25 ± 1.73 15.11 ± 0.90 
siRNA-con 65.88 ± 1.15 71.32 ± 1.55 34.05 ± 1.76 14.16 ± 1.21 
siRNA-ILK 62.46 ± 1.73 65.57 ± 3.0* 27.96 ± 1.35* 20.79 ± 1.44* 

Notes: Values are means ± standard deviation (n = 6); comparison among groups was conducted by analysis of variance.

*p <.05 vs control cells and siRNA-con-transfected cells.

siRNA-con = a control siRNA group; siRNA-ILK = an ILK siRNA group.

This work was supported by the Creative Research Group Fund of the National Natural Science Foundation of China (30121005), the Main National Basic Research Program of China (2007CB507400), grants from the National Natural Scientific Foundation of China (30270616, 30300161, 30370534), and the Postdoctoral Science Foundation of China (2003033190). Dr. C. Wu is supported by National Institutes of Health Grants GM65188 and DK54639.

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