ERK1/2 communicates GPCR and EGFR signaling pathways to promote CTGF-mediated hypertrophic cardiomyopathy upon Ang-II stimulation
Hypertrophic cardiomyopathy occurs along with pathological phenomena such as cardiac hypertrophy, myocardial fibrosis and cardiomyocyte activity. However, few of the specific molecular mechanisms underlying this pathological condition have been mentioned.
All target proteins and markers expression in the study was verified by PCR and western bloting. H9c2 cell morphology and behavior were analyzed using immunofluorescent and proliferation assays, respectively. And, the CTGF protein secreted in cell culture medium was detected by ELISA.
We found that high expression of CTGF and low expression of EGFR were regulated by ERK1/2 signaling pathway during the cardiac hypertrophy induced by Ang-II stimulation. CTGF interacted with EGFR, and the interaction is reduced with the stimulation of Ang-II. ERK1/2 serves as the center of signal control during the cardiac hypertrophy.
The ERK1/2 cooperates with GPCR and EGFR signaling, and promotes the occurrence and development of cardiac hypertrophy by regulating the expression and binding states of CTGF and EGFR. The study revealed a regulation model based on ERK1/2, suggesting that ERK1/2 signaling pathway may be an important control link for mitigation of hypertrophic cardiomyopathy treatment.
KeywordsERK1/2 CTGF EGFR Hypertrophic cardiomyopathy
Atrial natriuretic peptide
Angiotensin II receptor blockers
B-type natriuretic peptide
Connective tissue growth factor
Epidermal growth factor receptor
Enzyme linked immunosorbent assay
Extracellular regulated protein kinase 1/2
G protein coupled receptor
Polymerase chain reaction
Receptor tyrosine kinase
small interfering RNA
Tris-buffered saline with Tween-20
Cardiomyopathy is a group of lesions that cause progressive dysfunction of the heart due to structural changes in the inferior part of the heart and impaired myocardial wall function. Its clinical manifestations include cardiac enlargement, arrhythmia, embolism, and heart failure . Cardiomyopathy refers to myocardial disease with cardiac dysfunction, clinical types of dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, undifferentiated cardiomyopathy, and specific cardiomyopathy, of which DCM is the most common. DCM may be related to myocardial damage caused by abnormal metabolism of viruses and bacterial drugs . Viral myocarditis is considered to be the most important cause. The etiology and pathogenesis of this disease are still unclear. In addition to idiopathic and family hereditary, it is thought to be related to persistent viral infection and autoimmune-induced myocardial damage. The main pathological changes were enlargement of the heart chamber, thinning of the ventricular wall, and often accompanied by mural thrombus .
As a major kinase signal molecule in the cell, ERK1/2 plays an important role in numerous intracellular signaling pathways including G protein coupled receptor (GPCR) and receptor tyrosine kinase (RTK), and participates in numerous physiological and pathological events . There also been many reports on the relationship between the related signal pathways in inflammation-related cardiomyopathy and the processes of cardiomyocyte fibrosis [5, 6]. As we all know, ERK1/2 can not only be used as a downstream molecule of the classical epidermal growth factor receptor (EGFR) signaling pathway, but also can be activated by GPCR signaling pathway [7, 8]. However, no research has been conducted on the cooperative regulation between these two signals in cardiomyopathy.
Database analysis showed that EGFR showed low expression in DCM patients, while connective tissue growth factor (CTGF) showed high expression . As a secreted protein, CTGF plays an important role in cardiomyopathy. Previous studies have shown that it has a certain role in promoting myocardial fibrosis and myocardial cell apoptosis . However, studies based on tumor models have shown that CTGF can act as a ligand of EGFR, stimulate EGFR activation, and play a role in promoting tumor cell proliferation and even tumor cell EMT . This differs from the differential expression of CTGF and EGFR in DCM patients. However, their specific regulatory pattern in DCM and relationship with activation of ERK1/2 signaling molecules are unknown.
In this study, we studied the specific regulatory mechanisms in the cardiac hypertrophy cell model, revealed its specific molecular mechanism, and provided a certain theoretical basis and possible molecular targets for the treatment of DCM and other hypertrophic cardiomyopathy.
Ang-II stimulation promotes cardiomyocyte fibrosis and hypertrophy
In addition, myocardial fibrosis is also an important pathological phenomenon of DCM . In the study, protein expression levels of three important cardiomyocyte fibrin markers, α-SMA, Paxillin and Vinculin, were examined. The results showed that all three markers showed significant high expression when induced by Ang-II (Fig. 1d). Although the results of this experiment did not fully explain the occurrence of myocardial fibrosis, it did prove that a similar phenomenon occurred after the treatment. On the other hand, it demonstrated the reliability of the Ang-II-stimulated cell model for induced cardiomyocyte hypertrophy. Taken together above results, 10 nM Ang-II stimulation for 24 h was used as a cell model for cardiac hypertrophy in the subsequent relevant experimental treatment.
Ang-II stimulation promotes CTGF expression and reduces EGFR expression
Ang-II stimulates activation of ERK1/2 signal and is mediated by GPCR
For further experiments, we used U0126 and Ang-II receptor inhibitors ARBs, respectively, to detect changes in cellular hypertrophy by actin staining. Under U0126 or ARBs treatment, Ang-II stimulation did not result in hypertrophy in H9c2 cardiomyocytes, which was significantly different from the Ang-II-stimulated hypertrophic model control group (Fig. 4b). Similarly, western blotting results also showed that Ang-II stimulation did not increase the expression of ANP and BNP when treated with U0126 or ARBs (Fig. 4a and c). At the same time, we also noted that Ang-II stimulation did not increase p-ERK1/2 in the presence of ARBs, and that the increased Ang-II-induced CTGF expression disappeared (Fig. 4c). These results indicate that increased expression of p-ERK1/2 and CTGF is regulated by Ang-II receptor, a GPCR. In addition, cell viability experiments showed that the downstream signal of p-ERK1/2 promotes cell activity, and high cell activity represents an increase in cell proliferation (Fig. 4d). The balance of proliferation and apoptosis of cardiomyocytes is also closely related to the occurrence and development of cardiac hypertrophy.
Reduced interaction between CTGF and EGFR stimulated by Ang-II
The expression of CTGF and EGFR under the stimulation of Ang-II is affected by the activity of ERK1/2
DCM is a kind of cardiomyopathy, and it occurs along with pathological phenomena such as cardiac hypertrophy, myocardial fibrosis and cardiomyocyte activity . In response to this disease, a series of molecular mechanisms were studied based on the cell model of myocardial hypertrophy and fibrosis. The main results are as follows: 1) During the cardiac hypertrophy induced by Ang-II stimulation, CTGF was highly expressed and EGFR was significantly down-regulated; 2) Increased CTGF and decreased EGFR were regulated by ERK1/2 signaling pathway; 3) CTGF interacted with EGFR, and the interaction is reduced with the stimulation of Ang-II; 4) ERK1/2 serves as the center of signal regulation. On the one hand, the signal of AngII receptors is carried by ERK1/2, on the other hand, it regulates the expression of EGFR and plays a role in cooperative regulation.
The high expression of CTGF in DCM tissue has been reported in many previous studies [13, 14]. However, few of the specific molecular mechanisms underlying this pathological condition have been mentioned. Studies have shown that CTGF plays an important role in the process of ECM responses. Studies have also shown that CTGF significantly improved left ventricular (LV) systolic and diastolic function in PKCε mice, and slowed the progression of LV dilatation . These studies have shown that CTGF plays an important role in the development of cardiac hypertrophy. It is in view of this that we have studied the upstream molecular signals that regulate CTGF overexpression. The experimental results demonstrate that CTGF is highly expressed in response to Ang-II stimulation, and that this change in expression is regulated by the ERK1/2 signaling pathway. This also indirectly shows that the ERK1/2 signaling pathway plays a key regulatory role in the development of cardiac hypertrophy.
As early as 1999, studies have found that CTGF can interact with TGFβ . In the 2013 study, researchers demonstrated that CTGF can bind to EGFR and inhibit EGFR-mediated downstream phosphorylation events . However, no studies have reported the changes and effects of their combination in the cardiomyopathy model. On the one hand, our study also demonstrated that CTGF and EGFR can interact with each other in a cardiomyocyte model. On the other hand, in view of the differential expression of CTGF and EGFR under Ang-II stimulation, we also demonstrated that this interaction is regulated by Ang-II. Ang-II stimulation promotes the expression of CTGF through the activation of ERK1/2 signaling pathway, while the expression of EGFR decreases. Increased expression of CTGF promotes the development of cellular hypertrophy, while a decrease in EGFR reduces the interaction with CTGF to release more CTGF to promote the development of cardiac hypertrophy.
ERK1/2 plays an important role in the occurrence and development of tumors as an important signaling pathway molecule and phospho-kinase in cells . There are many researches in this area. In these studies, most of them are ERK1/2 that receives tyrosine kinase receptors signals, which in turn activate [18, 19]. Our study found that Ang-II stimulation can activate ERK1/2 signaling, which is a type of GPCR. At the same time, ERK1/2 feedback inhibited EGFR expression. This provides the possibility for ERK1/2 to coordinate GPCR and EGFR signaling during the cardiac hypertrophy. Our experimental results also confirmed this possibility, and this regulatory pattern directly regulates the expression and mode of action of downstream CTGF. This further expands the importance of ERK1/2 in the development of DCM disease.
In here, we have demonstrated the ERK1/2 signaling pathway cooperates with GPCR and EGFR signaling, and promotes the occurrence and development of cardiac hypertrophy by regulating the expression and binding states of CTGF and EGFR in the cell model induced by Ang-II stimulation. In addition to confirming the role of CTGF during cardiac hypertrophy and possible therapeutic targets, this study also revealed a coordinated regulation model based on ERK1/2, suggesting that ERK1/2 signaling pathway may be an important control link for mitigation of cardiac hypertrophy treatment.
Cell line and cell culture
The H9c2 rat cardiomyocytes were purchased from the ATCC Biological Resources Center. After resuscitation cells were cultured in Dulbecco, s modified Eagle, s medium (DMEM) containing 10%FBS (Gibco, Life Technologies), 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
Plasmids and siRNAs
Full-length EGFR was amplified from cDNA. The polymerase chain reaction (PCR) products were cloned into the pCMV-N-Flag (Beyotime, Nantong, China). The cells were seeded in 6-well plates, cultured to 80~90% confluence, and then transiently transfected with the plasmid by using Lipofectamine 3000 (Invitrogen) according to the reverse transfection method provided by the manufacturer.
Duplex oligonucleotides were chemically synthesized and purified by GenePharma (Shanghai, China). The small interfering RNA (siRNA) duplexes used were CTGF, #1, 5′- CACCGCAATACCTTCTGCAGGCTGGACAAGAGATCCAGCCTGCAGAAGGTATTGTTTTTTG-3′. Cells were transfected with siRNA duplexes using Lipofectamine 3000 (Invitrogen) according to the reverse transfection method provided by the manufacturer.
The collected cells were prepared for total RNA extraction using using Trizol reagent (Invitrogen, USA). The cDNAs were synthesized using a reverse transcription kit (Quant One Step RT-PCR kit, TIANGEN, China), and real-time PCR was performed using SYBR green master mix (Quant one step qRT-PCR Kit, TIANGEN, China) on a MyiQTM2 (BIORAD, USA). β-actin was used to normalize the real-time PCR data. The following primer sequences were used: CTGF, 5′- GGCCTCTTCTGCGATTTCG-3'and 5′-GGCCTCTTCTGCGATTTCG-3′; EGFR, 5′-GGCCTCTTCTGCGATTTCG-3′ and 5′-GCAGCTTGACCCTTCTCGG-3′;β-actin, 5′-ATGGAGGGGAATACAGCCC-3′ and 5′-TTCTTTGCAGCTCCTTCGTT-3′.
The cells were lysed in radioimmune precipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail (Roche, Shanghai, China) to prepare the protein sample. Protein concentration was measured using Enhanced BCA Protein Assay Kit (Beyotime Biotechnology, China) and 40 μg of protein were loaded and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred to PVDF membranes and then blocked with 5% BSA in Tris-buffered saline with Tween-20 (TBST) before immunodetection with the following antibodies: BNP (1:1000, CST, USA), ANP (1:1000, CST, USA), Paxillin (1:1000, CST, USA), Vinculin (1:1000, CST, USA), CTGF (1:1000, CST, USA), ERK (1:1000, CST, USA), p-ERK (1:1000, CST, USA), β-actin (1:1000, CST, USA). After Primary antibody incubation for 16 h, the PVDF membranes were washed by TBST buffer before incubated with secondary antibody for 1.5 h. Specific binding was visualized by ECL reaction. The western blot bands were quantified using Image J Software (version 1.41).
Cells were fixed with 4% paraformaldehyde (PFA). Then, the cells were incubated with primary antibody against β-actin, followed by washing and incubation with AF594-conjugated goat anti-rabbit secondary antibodies (1:250; Earthox, USA) for 2 h at 37 °C. The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Fluorescent images were visualized and captured using an inverted fluorescence microscope (Olympus, Tokyo, Japan). Images were visualized and captured using a phase contrast microscope (Olympus, Tokyo, Japan).
Cell viability assay
Cells were seeded in a 96-well plate as a density of 5000 cells/well for 24 h. Cell viability was measured using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Japan), according to the manufacturer’s instructions. Absorbance was determined using the Multi-Mode Microplate Reader (PerkinElmer, Finland).
Cells culture supernatant was prepared for CTGF (Mouse CTGF ELISA Kit, Abcam, Cambridge, UK) detection according to the manufacturers’ protocol.
Data are presented as means ± SD. Statistical comparisons were performed by either Student’s 2-tailed t test or ANOVA with Tukey multiple comparison post-test, as appropriate. A P value of less than 0.05 was considered significant.
LX and LL designed the study. LX, LQ, NY, and ZC performed the experiments. LX and QS performed the statistical analysis. LQ, QS and LL drafted the manuscript. NY and WJ supervised the experimental work. All authors read and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China [No. 81170209] to Wei Jin. The grant provided for this study is primarily used for the design of the study, the collection, analysis, and interpretation of data or writing the manuscript in any way.
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The authors declare that they have no competing interests.
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