Introduction
Cardiac fibrosis, as a consequence of remodeling processes, results from cardiac adaptations to the hemodynamic overload caused by heart failure and cardiac hypertrophy. Adaptive remodeling processes initiate structural alterations representing cell morphological changes and excessive deposition of extracellular matrix (ECM) (1–3). Substances in the ECM provide cardiomyocytes with structural, chemical and mechanical substrates, which are essential for normal development and responses to pathological signals. Cardiomyocytes can sense stimuli from the ECM, which results in structural or functional remodeling (4). Collagen, particularly type I, is a major component of the ECM, which forms networks in interstitial spaces among cells, and provides structural integrity and mechanical stretching of the heart (5). Therefore, type I collagen may be critical in cardiac fibrosis.
Transforming growth factor (TGF)-β is a key mediator in the pathogenesis of hypertrophic and dilative ventricular remodeling by stimulating cardiomyocyte growth and inducing interstitial fibrosis (6,7). In the pressure-overloaded human heart, the upregulation of TGF-β is correlated with the degree of fibrosis (8). The overexpression of TGF-β in transgenic mice has been found to result in atrial hypertrophy, which is characterized by interstitial fibrosis and hypertrophic growth of cardiomyocytes (9). Active TGF-β binds to the constitutively active type II receptor (TGFβRII) at the cell surface, and subsequently transphosphorylates the cytoplasmic domain of the type I receptor (TGFβRI) (10). As TGFβRI mediates the majority of the cellular responses to TGF-β1 and has a predominant role in intracellular signal transduction (11), the activation of TGF-β receptors propagates downstream intracellular signals through Smad proteins, which are essential components in the signaling pathway of TGF-β (10,12–14). Although TGF-β signaling is important in genes associated with the ECM, whether the ECM is involved in regulating TGF-β signaling remains to be elucidated.
In vivo, cardiomyocytes are intricately connected to the ECM, and stretch is transduced via the intercellular and ECM connections via proteins located on the cell surface and subcellular adhesion complexes, and via stretch-sensing proteins. Stretching and the expression of integrin are increased during cardiac fibrosis (15–17). Integrin represents a primary link between ECM ligands and directly initiates intracellular signaling cascades (18–21). Integrin can interact with TGF-β to enhance cardiac fibrosis (21), suggesting that collagen may be involved in TGF-β signaling, which regulates the homeostasis of cardiac fibrosis. Integrin can activate focal adhesion kinase (FAK), which results in cardiac structural and electrical changes (22). In addition, mechanical stretch is important in tissue morphogenesis and remodeling, and evokes various hypertrophic responses from cell adhesion receptors and the expression of TGF-β1, and activates mitogen-activated protein kinases (MAPKs) in cardiomyocytes (23–25). Accordingly, integrin and mechanosensitive stretch channels may potentially regulate TGF-β signaling. MAPKs have pathophysiological effects in cardiac remodeling (26). Our previous studies demonstrated that collagen can modulate calcium dynamics and electrical activities of cardiomyocytes, with activation of the renin-angiotensin and MAPK systems (27,28). Therefore, the aims of the present study were to evaluate the effects of collagen on TGF-β signaling and investigate the underlying mechanisms. The results of the present study demonstrated that collagen differentially modulated the expression levels of TGFβRI and TGFβRII and the activity of MAPKs through interactions of integrin with stretch signaling.
Materials and methods
Cell culture
The protocol was approved by the Department of Medical Research of Cathay General Hospital, Taipei, Taiwan (no. CGH-MR-10014). HL-1 cells derived from mouse atrial cardiac muscle cells (29) were provided by Dr Claycomb, Louisiana State University Health Sciences Center (New Orleans, USA). The cells for various experiments were cultured in a humidified atmosphere of 5% CO2 at 37°C in Claycomb medium (JRH Biosciences, Lenexa, KS, USA). As described previously, the HL-1 cells were cultured with or without (control) type I rat tail tendon collagen (10 µg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 24 h following cell seeding (27). To investigate the roles of integrin and stretch, the HL-1 cells were pretreated with either monoclonal hamster anti-rat β1 integrin antibody (β1 integrin; 10 µg/ml; clone Ha2/5; #555002; BD Pharmingen, San Diego, CA, USA), to inhibit the in vitro adhesion of CD29-expressing cells to type I collagen, or the stretch-activated ion channel inhibitor, gadolinium (50 µM; Sigma-Aldrich) for 30 min prior to collagen incubation (30,31). The cells were plated at a density of 5×105 cells/well in 6-well culture plates.
Western blot analysis
The HL-1 cells were homogenized and lysed in RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS) and protease inhibitor cocktails (Sigma-Aldrich). The protein concentrations were determined using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein samples (40 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis under reducing conditions and electrophoretically transferred onto equilibrated polyvinylidenedifluoride membranes (Amersham Biosciences, Upsulla, Sweden). The blots were probed with the following primary antibodies overnight at 4°C: Rabbit anti-FAK (cat. no. 3285; 1:1,000), rabbit anti-phosphorylated FAK (cat. no. 3283; 1:1,000), rabbit anti-extracellular signal-regulated kinase 1/2 (ERK1/2; cat. no. 9102; 1:1,000), rabbit anti-phosphorylated-ERK1/2 (cat. no. 4370; 1:1,000) rabbit anti-c-Jun N-terminal kinase (JNK; cat. no. 9258; 1:1,000), mouse anti-phosphorylated-JNK (cat. no. 9255; 1:1,000), rabbit anti-p38 (cat. no. 9212; 1:1,000), rabbit anti-phosphorylated-p38 (cat. no. 9211; 1:1,000), rabbit anti-Smad2/3 (cat. no. 8685; 1:1,000), rabbit anti-phosphorylated-Smad2/3 (cat. no. 8828; 1:1,000), rabbit anti-TGFβRII (cat. no. 11888; 1:500) (all Cell Signaling Technology, Inc., Danvers, MA, USA); rabbit anti-TGFβRI (cat. no. SAB1300113, Sigma-Aldrich; 1:500); and horseradish peroxidase-conjugated goat anti-rabbit (cat. no. AP132P; 1:20,000) or goat anti-mouse immunglobulin G (cat. no. AP124P; 1:8,000) secondary antibodies (EMD Millipore, Billerica, MA, USA) for 1 h at room temperature. The bound antibodies were detected with an enhanced chemiluminescence detection system (EMD Millipore) and analyzed with AlphaEaseFC version 4.0 software (Alpha Innotech Corporation, San Leandro, CA, USA). The targeted bands were normalized to mouse anti-cardiac α-sarcomeric actin (cat. no. A2172; Sigma-Aldrich; 1:2,000) to confirm equal protein loading.
Cell proliferation assay
Cell proliferation was analyzed using a BrdU incorporation assay (BrdU Cell Proliferation Assay kit, Cell Signaling Technologies, Inc.). The HL-1 cells were seeded at 1×104 cells/well in 96-well plates and left to attach overnight. The cells were then incubated with or without type I rat tail tendon collagen (10 µg/ml) for 24 h. The HL-1 cells were pretreated with β1 integrin antibody (10 µg/ml), gadolinium (50 µM), p38 MAPK inhibitor (SB203580; 3 µM; Merck Millipore, Darmstadt, Germany), ERK inhibitor (PD98059; 50 µM; Sigma-Aldrich) or JNK inhibitor (SP600125; 50 µM; Calbiochem, San Diego, CA, USA) for 30 min prior to collagen incubation. BrdU solution was added 4 h prior to the end of cell treatment. Following fixing of cells and denaturing of DNA, BrdU detection antibody and horseradish peroxidase-conjugated secondary antibody were added. Finally, the HRP substrate, 3,3′,5,5′-tetramethylbenzidine, was added to develop color, and BrdU incorporation was quantified by reading the absorbance at 540 nm.
Cell adhesion assay
The HL-1 cells were trypsinized and seeded at a density of 2×104 cells/well in a 96-well plate following treatment with type I collagen (10 µg/ml) and β1 integrin antibody (10 µg/ml). Subsequent to attachment for 2.5 h in a 37°C incubator, the unattached cells were removed by washing with phosphate-buffered saline. The cells were then stained with 0.1% crystal violet in 2% ethanol in 0.1 M borate (pH 9) for 15 min. Cell adhesion levels were evaluated by reading the absorbance of crystal violet at 540 nm following the addition of 100 ml pure methanol to solubilize the dye.
Statistical analysis
Continuous variables are expressed as the mean ± standard error of the mean. The different groups of HL-1 cells were compared using a paired t-test or repeated one-way analysis of variance with a Tukey's post-hoc test. The effects of the β1 integrin antibody or gadolinium on collagen were evaluated using an unpaired t-test. All statistical tests were performed using SigmaStat 3.5 (Systat Software, Inc., San Jose, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Effects of collagen and β1 integrin in modulating the expression of TGF-β receptors and FAK
Compared with the control, the collagen (10 µg/ml)-treated HL-1 cells exhibited significantly decreased expression levels of TGFβRI (16±5%) and TGFβRII (21±5%) and upregulated FAK phosphorylation, (42±9%). The β1 integrin antibody (10 µg/ml) did not alter the expression levels of TGFβRI, TGFβRII or the phosphorylation of FAK in the HL-1 cells. In the presence of the β1 integrin antibody, collagen downregulated the expression of TGFβRI, however, no effects on the expression of TGFβRII or the phosphorylation of FAK were observed (Fig. 1A). Therefore, the β1 integrin antibody attenuated the collagen-associated down-regulation in the expression of TGFβRII and phosphorylation of FAK, but did not alter the collagen-associated downregulation of TGFβRI.
Effects of collagen and β1 integrin in the modulation of MAPK phosphorylation
As shown in Fig. 1B, the collagen (10 µg/ml)-treated HL-1 cells exhibited increased phosphorylation of p38 (25±7%) and ERK1/2 (20±6%), but decreased the phosphorylation of JNK (13±5%). The β1 integrin antibody increased the phosphorylation of p38 by 55±15% in the HL-1 cells, but did not affect the phosphorylation of ERK1/2 or JNK. Therefore, β1 integrin was expected to increase the level of phosphorylated p38. In the presence of the β1 integrin antibody, collagen did not alter the phosphorylation of p38, however, the phosphorylation of ERK1/2 was increased and that of JNK was decreased. Accordingly, the collagen-induced changes in the levels of phosphorylated ERK1/2 and JNK were not altered by the presence of the β1 integrin antibody, which suggested that integrin may not completely modulate the collagen effects of collagen on MAPKs.
Effect of mechanical stretch in collagen-mediated expression of the TGF-β receptor and Smad2/3
Compared with the control, gadolinium (50 µM) decreased the expression of TGFβRI by 38±12% and TGFβRII by 26±6%. In the presence of gadolinium, collagen did not alter the expression levels of TGFβRI or TGFβRII. However, gadolinium decreased the collagen-induced downregulation of TGFβRI by 25±7%. Collagen decreased the phosphorylation of Smad2/3 by 17±5%, compared with the control. Gadolinium (50 µM) decreased the phosphorylation of Smad2/3 in the HL-1 cells by 24±6%, and decreased the phosphorylation of Smad2/3 by 23±6% in the presence of collagen (Fig. 2A).
Effect of mechanical stretch on the collagen-mediated phosphorylation of MAPKs
Compared with the control cells, the gadolinium (50 µM)-treated HL-1 cells exhibited decreased JNK phosphorylation by 37±6%, however, the phosphorylation of p38 or ERK1/2 were not affected. In the presence of gadolinium, collagen did not alter the phosphorylation of p38, ERK or JNK. However, gadolinium further decreased the collagen-induced downregulated phosphorylation of JNK by 25±8%, and reversed the collagen-induced upregulated phosphorylation of p38 and ERK1/2, compared with the collagen-treated HL-1 cells (Fig. 2B).
Effects of β1 integrin and mechanical stretch on the collagen-mediated expression of TGF-β receptor and Smad2/3
As shown in Fig. 3A, the β1 integrin antibody (10 µg/ml) and gadolinium (50 µM) decreased the expression of TGFβRII by 32±6% (P<0.05, vs. control), however, no change in the expression of TGFβRI was observed in the HL-1 cells. In the presence of the β1 integrin antibody and gadolinium, collagen downregulated TGFβRI by 66±23% and upregulated TGFβRII by 26±8%. Compared with the gadolinium-treated cells (Fig. 2A), the HL-1 cells treated with the β1 integrin antibody and gadolinium (Fig. 3A) increased the expression of TGFβRI by 149±77% (P<0.05), however, the expression of TGFβRII was not affected (data not shown). The phosphorylation of Smad2/3 in the cells treated with the β1 integrin antibody and gadolinium did not differ from that of the control. The effects of the β1 integrin antibody and gadolinium on the phosphorylation of Smad2/3 were similar to those on the expression of TGFβRI.
Effects of β1 integrin and mechanical stretch in the collagen-mediated expression of MAPKs
Compared with the control HL-1 cells, β1 integrin antibody (10 µg/ml) and gadolinium (50 µM) decreased the phosphorylation of JNK by 45±8% (P<0.005), but did not alter the phosphorylation of p38 or ERK1/2 (Fig. 3B). In the presence of β1 integrin antibody and gadolinium, no changes in the phosphorylation of p38, ERK1/2 or JNK were observed, compared with the gadolinium-treated cells and the cells concomitantly treated with collagen.
Effects of β1 integrin and mechanical stretch on the proliferation and adhesion of collagen-treated HL-1 cells
To investigate the role of integrin and mechanical stretch on cell proliferation, the collagen (10 µg/ml)-treated HL-1 cells were incubated with or without β1 integrin antibody (10 µg/ml) or gadolinium (50 µM). As shown in Fig. 4A, collagen increased cell proliferation, and this collagen-induced cell proliferation was inhibited by β1 integrin antibody, but not by gadolinium. In addition, collagen increased cell adhesion by 16±7%, which was also inhibited by β1 integrin antibody (Fig. 4B). These results suggested that collagen promoted cardiomyocyte proliferation and adhesion through β1 integrin signaling.
Effect of MAPK inhibition on the proliferation of collagen-treated HL-1 cells
Compared with the control HL-1 cells, SB203580 (3 µM) increased cell proliferation by 33±11%, whereas PD98059 (50 µM) and SP600125 (50 µM) did not alter cell proliferation (Fig. 5). However, in the presence of collagen, PD98059 and SP600125, but not SB203580, inhibited the collagen-induced increase in cell proliferation.
Discussion
The present study is the first, to the best of our knowledge, to demonstrate that collagen directly modulated the expression of TGF-β receptors and their downstream signaling molecules. TGF-β is an important cytokine in the fibrotic responses of several organs, including the heart (6,7). The present study demonstrated that collagen accumulation during fibrosis can downregulate the expression of TGF-β receptors. TGF-β enhances the production of the ECM from cardiac fibroblasts, which can induce fibrosis (6,32). Although the underlying mechanisms remain to be fully elucidated, sustained TGF-β exposure has been shown to suppress the expression of TGF-β receptors (33). Therefore, an increase or decrease of collagen leads to the downregulation or upregulation of TGF-β receptor signaling, which may, at least in part, result in the known feedback loop between TGF-β and TGF-β receptors. In addition, the ECM has been reported to modulate MAPKs in cell function and differentiation (28,34,35). Collagen was found to increase the phosphorylation of p38 and ERK1/2 in the present study, which is compatible with previous findings in cardiac remodeling (36,37).
The present study found that the collagen-induced down-regulation of TGFβRII was inhibited by the integrin antibody. A previous study demonstrated that the interaction of type I collagen with α2β1 integrin causes downregulation of the TGF-β receptor via the activation of FAK and its diverse downstream signals, and this was found to be abrogated by treatment that inactivated FAK (35). The integrin antibody decreased the collagen-activated phosphorylation of FAK, suggesting that FAK uniquely mediates matrix-integrin interactions in cellular processes. However, integrin did not regulate the effects of collagen on TGFβRI, suggesting that collagen differentially modulates the expression of TGFβRI and TGFβRII.
The ECM has a regulatory role in several cellular processes, including cell growth, adhesion, division and differentiation. However, whether collagen regulates cardiomyocyte proliferation and adhesion remains to be elucidated. The ECM secreted from cardiac fibroblasts has been reported to promote cardiomyocyte proliferation through β1 integrin signaling (38). Integrins are a major class of ECM receptors for cell adhesion (20). The present study showed that collagen-binding β1 integrin is involved in mediating cell proliferation and adhesion, which suggested that β1 integrin may be responsible for cardiomyocyte proliferation during cardiac fibrosis. Although the inhibition of p38 enables the proliferation of cardiomyocytes (39), however, collagen-induced cell proliferation was inhibited by the inhibition of ERK in the present study. These results suggested that integrin and MAPK signaling in the cardiomyocytes may have been responsible for the effects of collagen on cardiomyocyte proliferation.
Mechanical stretch induces the release of TGF-β and is coupled with intracellular signals, which are responsible for cardiac hypertrophy (40,41). In the present study, it was found that gadolinium enhanced the downregulation of the TGFβRI, but not TGFβRII. The activation of TGFβRI leads to the phosphorylation of Smad2 and Smad3, and regulates transcription (13,14). In the present study, collagen down-regulated the phosphorylation of Smad2/3 in parallel with the expression of TGFβRI, which suggested that collagen was involved in the Smad-dependent pathway through TGFβRI. The MAPK pathway may be involved in the mechanical stretching-induced hypertrophic response (42,43). In the present study, it was found that gadolinium decreased the collagen-induced phosphorylation of p38 and ERK1/2. As gadolinium only reversed the effects of collagen on the activity of MAPKs in cells without the presence of the β1-integrin antibody, it is possible that collagen activated the stretch-induced MAPK signaling pathway independently of integrin-FAK signaling (44). However, collagen and gadolinium showed synergistic effects in inhibiting the expression of TGFβRI and phosphorylation of Smad2/3. These results suggested the presence of cross-talk between collagen and mechanical stretch in the TGFβRI/Smad signaling pathways. The present study demonstrated that collagen regulated the expression of TGF-β receptors and phosphorylation of MAPKs through at least two distinct mechanisms. Collagen regulated the expression of TGFβRII through cross-talk between integrin-FAK signaling and mechanical stretch, whereas the collagen-induced expression of TGFβRI and activation of MAPKs were predominantly mediated by mechanical stretch. In addition, collagen may modulate the phosphorylation of JNK beyond the mechanisms identified in the present study. In the present in vitro study, an atrial cell line from mice was used, and these highly purified cells were placed on an artificial collagen matrix, which may or may not be reflective of human atrial cells. The findings suggested that coordinated actions occurred among the ECM, integrin and mechanical stretch in response to cardiac fibrosis.
In conclusion, the present study demonstrated that collagen was involved in altering intracellular signaling in a positive and negative manner. The observations indicated that collagen modulated the expression of TGF-β receptors and the phosphorylation of MAPKs through interactions with integrin and mechanical stretch.