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Overexpression of SerpinE2/protease nexin-1 Contribute to Pathological Cardiac Fibrosis via increasing Collagen Deposition

View Article: PubMed Central - PubMed

ABSTRACT

Although increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis, the molecular mechanisms of pressure overload or AngII -induced cardiac interstitial fibrosis remain elusive. In this study, serpinE2/protease nexin-1 was over-expressed in a cardiac fibrosis model induced by pressure-overloaded via transverse aortic constriction (TAC) in mouse. Knockdown of serpinE2 attenuates cardiac fibrosis in a mouse model of TAC. At meantime, the results showed that serpinE2 significantly were increased with collagen accumulations induced by AngII or TGF-β stimulation in vitro. Intriguingly, extracellular collagen in myocardial fibroblast was reduced by knockdown of serpinE2 compared with the control in vitro. In stark contrast, the addition of exogenous PN-1 up-regulated the content of collagen in myocardial fibroblast. The MEK1/2- ERK1/2 signaling probably promoted the expression of serpinE2 via transcription factors Elk1 in myocardial fibroblast. In conclusion, stress-induced the ERK1/2 signaling pathway activation up-regulated serpinE2 expression, consequently led accumulation of collagen protein, and contributed to cardiac fibrosis.

No MeSH data available.


ERK1/2 signal pathway via Elk1 in regulating serpinE2.(a) The change of p-ERK1/2 and Elk1 in TAC mouse heart. n = 6, *P < 0.05 vs. control. Full-length blots/gels are presented in Supplementary Figure 13 and 14. (b) The mRNA level of serpinE2 was decreased by transfection with siRNA Elk in CFs; The level of serpinE2 was decreased by transfection with siRNA Elk in the supernatants of fibroblast; The level of collagen was decreased by transfection with siRNA Elk in the supernatants of fibroblast. n = 6, *P < 0.05 vs. control. (c) ChIP testing in vivo binding of Elk1 to promoter of Rattus norvegicus genes of serpin family E member 2 (SerpinE2). Top: Schematic representation of the two upstream region of the serpinE2. Bottom: PCR products of Elk1-binding sites following immunoprecipitation with anti- Elk1 antibody. The anti-IgG antibody and H2O treatment were used as negative control. The anti- Elk1 antibody was used to target specific immunoprecipitation. ChIP analysis of Elk1 binding to the promoter between-962 and-716 (PCR products 246 bp). Elk1 binding to target Elk1 site 1 activates serpinE2 promoter activity. ChIP analysis of in vivo Elk1 site2 binding to the promoter between -349 and -53 bp (PCR products 296 bp). ChIP assay showed that Elk1 binding to this target Elk1 site 2 also did activate serpinE2 promoter activity. (d) Effect of knockdown of ERK1 on serpinE2 and the collagen content in supernatants of fibroblasts inducd by ANG-II. n = 6, *P < 0.05 vs. control, #P < 0.05 vs. ANG-II. (e) Effect of knockdown of ERK1 on serpinE2, collagen and p-ERK1/2 in cardiac fibroblasts inducd by ANG-II. n = 6, *P < 0.05 vs. control, #P < 0.05 vs. ANG-II. Full-length blots/gels are presented in Supplementary Figure 15.
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f7: ERK1/2 signal pathway via Elk1 in regulating serpinE2.(a) The change of p-ERK1/2 and Elk1 in TAC mouse heart. n = 6, *P < 0.05 vs. control. Full-length blots/gels are presented in Supplementary Figure 13 and 14. (b) The mRNA level of serpinE2 was decreased by transfection with siRNA Elk in CFs; The level of serpinE2 was decreased by transfection with siRNA Elk in the supernatants of fibroblast; The level of collagen was decreased by transfection with siRNA Elk in the supernatants of fibroblast. n = 6, *P < 0.05 vs. control. (c) ChIP testing in vivo binding of Elk1 to promoter of Rattus norvegicus genes of serpin family E member 2 (SerpinE2). Top: Schematic representation of the two upstream region of the serpinE2. Bottom: PCR products of Elk1-binding sites following immunoprecipitation with anti- Elk1 antibody. The anti-IgG antibody and H2O treatment were used as negative control. The anti- Elk1 antibody was used to target specific immunoprecipitation. ChIP analysis of Elk1 binding to the promoter between-962 and-716 (PCR products 246 bp). Elk1 binding to target Elk1 site 1 activates serpinE2 promoter activity. ChIP analysis of in vivo Elk1 site2 binding to the promoter between -349 and -53 bp (PCR products 296 bp). ChIP assay showed that Elk1 binding to this target Elk1 site 2 also did activate serpinE2 promoter activity. (d) Effect of knockdown of ERK1 on serpinE2 and the collagen content in supernatants of fibroblasts inducd by ANG-II. n = 6, *P < 0.05 vs. control, #P < 0.05 vs. ANG-II. (e) Effect of knockdown of ERK1 on serpinE2, collagen and p-ERK1/2 in cardiac fibroblasts inducd by ANG-II. n = 6, *P < 0.05 vs. control, #P < 0.05 vs. ANG-II. Full-length blots/gels are presented in Supplementary Figure 15.

Mentions: Phosphorylation of ERK1/2 was translocated to the nucleus and activates transcription factors such as Elk1, NF-κB, c-fos and GATA4 etc28. In our study, ERK1/2 were activated by TAC and Elk1 was also significantly up-regulated in TAC heart (P < 0.05) (Fig. 7a). Among these transcription factors, Elk1 has a close relationship with collagen synthesis2930. In our results, knockdown of Elk1 significantly reduced mRNA expression of serpinE2 and the content of serpinE2 in the supernatants of fibroblast (Fig. 7b), suggesting the participation of p-ERK1/2 in ANG-II-induced serpinE2 expression via ERK-dependent transcription of Elk1, which was supported by the notion that the level of collagen was also decreased by transfection with siRNA Elk1 in the supernatants of fibroblast (Fig. 7b). We then subsequently conducted ChIP to further verify the protein-DNA interactions that occur inside the nucleus of CFs cells. More important was that there are 2 optimal and conserved Elk1 binding sites in the promoter region of the serpinE2 gene. The data in Fig. 7c, suggest that Elk1 is able to bind to the serpinE2 promoter, and the binding site located in −962 to −716 bp and −349 to −53 bp upstream of serpinE2 promoter (Fig. 7c). SerpinE2 expression and the collagen was selectively knocked down by siRNA-ERK1 transfection in supernatants of CFs induced by AngII (Fig. 7d). Transfection of siRNA-ERK1 caused an obviously reducdion of ERK1 in CFs (Fig. 7e). Transfection of siRNA-ERK1 prevents the AngII-induced up-regulation expression of serpinE2, collagen and p-ERK level. These results suggested that ERK1/2 signal pathway regulated serpinE2 via Elk1 transcription activator.


Overexpression of SerpinE2/protease nexin-1 Contribute to Pathological Cardiac Fibrosis via increasing Collagen Deposition
ERK1/2 signal pathway via Elk1 in regulating serpinE2.(a) The change of p-ERK1/2 and Elk1 in TAC mouse heart. n = 6, *P < 0.05 vs. control. Full-length blots/gels are presented in Supplementary Figure 13 and 14. (b) The mRNA level of serpinE2 was decreased by transfection with siRNA Elk in CFs; The level of serpinE2 was decreased by transfection with siRNA Elk in the supernatants of fibroblast; The level of collagen was decreased by transfection with siRNA Elk in the supernatants of fibroblast. n = 6, *P < 0.05 vs. control. (c) ChIP testing in vivo binding of Elk1 to promoter of Rattus norvegicus genes of serpin family E member 2 (SerpinE2). Top: Schematic representation of the two upstream region of the serpinE2. Bottom: PCR products of Elk1-binding sites following immunoprecipitation with anti- Elk1 antibody. The anti-IgG antibody and H2O treatment were used as negative control. The anti- Elk1 antibody was used to target specific immunoprecipitation. ChIP analysis of Elk1 binding to the promoter between-962 and-716 (PCR products 246 bp). Elk1 binding to target Elk1 site 1 activates serpinE2 promoter activity. ChIP analysis of in vivo Elk1 site2 binding to the promoter between -349 and -53 bp (PCR products 296 bp). ChIP assay showed that Elk1 binding to this target Elk1 site 2 also did activate serpinE2 promoter activity. (d) Effect of knockdown of ERK1 on serpinE2 and the collagen content in supernatants of fibroblasts inducd by ANG-II. n = 6, *P < 0.05 vs. control, #P < 0.05 vs. ANG-II. (e) Effect of knockdown of ERK1 on serpinE2, collagen and p-ERK1/2 in cardiac fibroblasts inducd by ANG-II. n = 6, *P < 0.05 vs. control, #P < 0.05 vs. ANG-II. Full-length blots/gels are presented in Supplementary Figure 15.
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f7: ERK1/2 signal pathway via Elk1 in regulating serpinE2.(a) The change of p-ERK1/2 and Elk1 in TAC mouse heart. n = 6, *P < 0.05 vs. control. Full-length blots/gels are presented in Supplementary Figure 13 and 14. (b) The mRNA level of serpinE2 was decreased by transfection with siRNA Elk in CFs; The level of serpinE2 was decreased by transfection with siRNA Elk in the supernatants of fibroblast; The level of collagen was decreased by transfection with siRNA Elk in the supernatants of fibroblast. n = 6, *P < 0.05 vs. control. (c) ChIP testing in vivo binding of Elk1 to promoter of Rattus norvegicus genes of serpin family E member 2 (SerpinE2). Top: Schematic representation of the two upstream region of the serpinE2. Bottom: PCR products of Elk1-binding sites following immunoprecipitation with anti- Elk1 antibody. The anti-IgG antibody and H2O treatment were used as negative control. The anti- Elk1 antibody was used to target specific immunoprecipitation. ChIP analysis of Elk1 binding to the promoter between-962 and-716 (PCR products 246 bp). Elk1 binding to target Elk1 site 1 activates serpinE2 promoter activity. ChIP analysis of in vivo Elk1 site2 binding to the promoter between -349 and -53 bp (PCR products 296 bp). ChIP assay showed that Elk1 binding to this target Elk1 site 2 also did activate serpinE2 promoter activity. (d) Effect of knockdown of ERK1 on serpinE2 and the collagen content in supernatants of fibroblasts inducd by ANG-II. n = 6, *P < 0.05 vs. control, #P < 0.05 vs. ANG-II. (e) Effect of knockdown of ERK1 on serpinE2, collagen and p-ERK1/2 in cardiac fibroblasts inducd by ANG-II. n = 6, *P < 0.05 vs. control, #P < 0.05 vs. ANG-II. Full-length blots/gels are presented in Supplementary Figure 15.
Mentions: Phosphorylation of ERK1/2 was translocated to the nucleus and activates transcription factors such as Elk1, NF-κB, c-fos and GATA4 etc28. In our study, ERK1/2 were activated by TAC and Elk1 was also significantly up-regulated in TAC heart (P < 0.05) (Fig. 7a). Among these transcription factors, Elk1 has a close relationship with collagen synthesis2930. In our results, knockdown of Elk1 significantly reduced mRNA expression of serpinE2 and the content of serpinE2 in the supernatants of fibroblast (Fig. 7b), suggesting the participation of p-ERK1/2 in ANG-II-induced serpinE2 expression via ERK-dependent transcription of Elk1, which was supported by the notion that the level of collagen was also decreased by transfection with siRNA Elk1 in the supernatants of fibroblast (Fig. 7b). We then subsequently conducted ChIP to further verify the protein-DNA interactions that occur inside the nucleus of CFs cells. More important was that there are 2 optimal and conserved Elk1 binding sites in the promoter region of the serpinE2 gene. The data in Fig. 7c, suggest that Elk1 is able to bind to the serpinE2 promoter, and the binding site located in −962 to −716 bp and −349 to −53 bp upstream of serpinE2 promoter (Fig. 7c). SerpinE2 expression and the collagen was selectively knocked down by siRNA-ERK1 transfection in supernatants of CFs induced by AngII (Fig. 7d). Transfection of siRNA-ERK1 caused an obviously reducdion of ERK1 in CFs (Fig. 7e). Transfection of siRNA-ERK1 prevents the AngII-induced up-regulation expression of serpinE2, collagen and p-ERK level. These results suggested that ERK1/2 signal pathway regulated serpinE2 via Elk1 transcription activator.

View Article: PubMed Central - PubMed

ABSTRACT

Although increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis, the molecular mechanisms of pressure overload or AngII -induced cardiac interstitial fibrosis remain elusive. In this study, serpinE2/protease nexin-1 was over-expressed in a cardiac fibrosis model induced by pressure-overloaded via transverse aortic constriction (TAC) in mouse. Knockdown of serpinE2 attenuates cardiac fibrosis in a mouse model of TAC. At meantime, the results showed that serpinE2 significantly were increased with collagen accumulations induced by AngII or TGF-&beta; stimulation in vitro. Intriguingly, extracellular collagen in myocardial fibroblast was reduced by knockdown of serpinE2 compared with the control in vitro. In stark contrast, the addition of exogenous PN-1 up-regulated the content of collagen in myocardial fibroblast. The MEK1/2- ERK1/2 signaling probably promoted the expression of serpinE2 via transcription factors Elk1 in myocardial fibroblast. In conclusion, stress-induced the ERK1/2 signaling pathway activation up-regulated serpinE2 expression, consequently led accumulation of collagen protein, and contributed to cardiac fibrosis.

No MeSH data available.