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Cellular repressor of E1A-stimulated genes attenuates cardiac hypertrophy and fibrosis.

Bian Z, Cai J, Shen DF, Chen L, Yan L, Tang Q, Li H - J. Cell. Mol. Med. (2008)

Bottom Line: It has been proposed that CREG acts as a ligand that enhances differentiation and/or reduces cell proliferation.Constitutive over-expression of human CREG in the murine heart attenuated the hypertrophic response, markedly reduced inflammation.These beneficial effects were associated with attenuation of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase 1 (MEK-ERK1)/2-dependent signalling cascade.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, PR China.

ABSTRACT
Cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein of 220 amino acids. It has been proposed that CREG acts as a ligand that enhances differentiation and/or reduces cell proliferation. CREG has been shown previously to attenuate cardiac hypertrophy in vitro. However, such a role has not been determined in vivo. In the present study, we tested the hypothesis that overexpression of CREG in the murine heart would protect against cardiac hypertrophy and fibrosis in vivo. The effects of constitutive human CREG expression on cardiac hypertrophy were investigated using both in vitro and in vivo models. Cardiac hypertrophy was produced by aortic banding and infusion of angiotensin II in CREG transgenic mice and control animals. The extent of cardiac hypertrophy was quantitated by two-dimensional and M-mode echocardiography as well as by molecular and pathological analyses of heart samples. Constitutive over-expression of human CREG in the murine heart attenuated the hypertrophic response, markedly reduced inflammation. Cardiac function was also preserved in hearts with increased CREG levels in response to hypertrophic stimuli. These beneficial effects were associated with attenuation of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase 1 (MEK-ERK1)/2-dependent signalling cascade. In addition, CREG expression blocked fibrosis and collagen synthesis through blocking MEK-ERK1/2-dependent Smad 2/3 activation in vitro and in vivo. Therefore, the expression of CREG improves cardiac functions and inhibits cardiac hypertrophy, inflammation and fibrosis through blocking MEK-ERK1/2-dependent signalling.

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The effect of CREG on TGF-β/Smad signalling. (A and B) Representative blots of Smad-2 phos-phorylation and Smad-2/3 transloca-tion from indicated groups 8 weeks after AB (n= 3) or 4 weeks of Ang II infusion (n= 4). The results were reproducible in three separate experiments (CE, cytoplasmic extract; NE, nuclear extract). (C) Representative blots of Smad-2 phosphorylation and Smad-2/3 translocation induced by TGF-β1 in cardiac fibroblasts after infection with different adenovirus. (D) The effect of ERK1/2 activation on collagen synthesis along with promoter activities of COL1A2 and CTGF. Cells were infected with or without indicated adenovirus for 24 hrs, and then incubated with 15 ng/ml TGF-β1 for up to 48 hrs. [3H] proline incorporation and luciferase assay were performed as described in ‘Materials and methods’. *P < 0.01 was obtained for control group. The results were reproducible in three separate experiments.
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fig05: The effect of CREG on TGF-β/Smad signalling. (A and B) Representative blots of Smad-2 phos-phorylation and Smad-2/3 transloca-tion from indicated groups 8 weeks after AB (n= 3) or 4 weeks of Ang II infusion (n= 4). The results were reproducible in three separate experiments (CE, cytoplasmic extract; NE, nuclear extract). (C) Representative blots of Smad-2 phosphorylation and Smad-2/3 translocation induced by TGF-β1 in cardiac fibroblasts after infection with different adenovirus. (D) The effect of ERK1/2 activation on collagen synthesis along with promoter activities of COL1A2 and CTGF. Cells were infected with or without indicated adenovirus for 24 hrs, and then incubated with 15 ng/ml TGF-β1 for up to 48 hrs. [3H] proline incorporation and luciferase assay were performed as described in ‘Materials and methods’. *P < 0.01 was obtained for control group. The results were reproducible in three separate experiments.

Mentions: TGF-β1 induces collagen synthesis via activation of a number of transcription factors, including Smads [14, 15]. To further elucidate the cellular mechanisms underlying the antifibrotic effects of CREG, we assessed the regulatory role of CREG on Smad cascade activation. The increased level of Smad 2 phosphorylation and Smad 2/3 nuclear translocation was attenuated in TG mice in response to hypertrophic stimuli (Fig. 5A and B). We then infected cardiac fibroblasts with Ad-CREG or Ad-shCREG and treated with TGF-β1. Western blot analyses revealed significant phosphorylation of Smad 2 and translocation of Smad 2/3 without any significant alterations in the expression of Smad 2 protein after TGF-β1 treatment. Ad-CREG infection, however, almost completely suppressed Smad 2 phosphorylation as well as Smad 2/3 nuclear translocation (Fig. 5C). Importantly, Ad-shCREG infection enhanced TGF-β1s effects. To further examine the mechanisms involved, we used confluent cardiac fibroblasts infected with Ad-GFP, Ad-caERK1/2, or Ad-dnERK1/2. Activation of ERK1/2 by infection with Ad-caERK1/2 revealed a significant increase in, whereas blocking ERK1/2 activity by Ad-dnERK1/2 infection almost completely abrogated, collagen synthesis and COL1A2 or CTGF promoter activities in response to TGF-β1 (Fig. 5D). These findings suggest that CREG blocks collagen synthesis by disrupting MEK-ERK1/2-dependent TGF-β-Smad signalling.


Cellular repressor of E1A-stimulated genes attenuates cardiac hypertrophy and fibrosis.

Bian Z, Cai J, Shen DF, Chen L, Yan L, Tang Q, Li H - J. Cell. Mol. Med. (2008)

The effect of CREG on TGF-β/Smad signalling. (A and B) Representative blots of Smad-2 phos-phorylation and Smad-2/3 transloca-tion from indicated groups 8 weeks after AB (n= 3) or 4 weeks of Ang II infusion (n= 4). The results were reproducible in three separate experiments (CE, cytoplasmic extract; NE, nuclear extract). (C) Representative blots of Smad-2 phosphorylation and Smad-2/3 translocation induced by TGF-β1 in cardiac fibroblasts after infection with different adenovirus. (D) The effect of ERK1/2 activation on collagen synthesis along with promoter activities of COL1A2 and CTGF. Cells were infected with or without indicated adenovirus for 24 hrs, and then incubated with 15 ng/ml TGF-β1 for up to 48 hrs. [3H] proline incorporation and luciferase assay were performed as described in ‘Materials and methods’. *P < 0.01 was obtained for control group. The results were reproducible in three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4496144&req=5

fig05: The effect of CREG on TGF-β/Smad signalling. (A and B) Representative blots of Smad-2 phos-phorylation and Smad-2/3 transloca-tion from indicated groups 8 weeks after AB (n= 3) or 4 weeks of Ang II infusion (n= 4). The results were reproducible in three separate experiments (CE, cytoplasmic extract; NE, nuclear extract). (C) Representative blots of Smad-2 phosphorylation and Smad-2/3 translocation induced by TGF-β1 in cardiac fibroblasts after infection with different adenovirus. (D) The effect of ERK1/2 activation on collagen synthesis along with promoter activities of COL1A2 and CTGF. Cells were infected with or without indicated adenovirus for 24 hrs, and then incubated with 15 ng/ml TGF-β1 for up to 48 hrs. [3H] proline incorporation and luciferase assay were performed as described in ‘Materials and methods’. *P < 0.01 was obtained for control group. The results were reproducible in three separate experiments.
Mentions: TGF-β1 induces collagen synthesis via activation of a number of transcription factors, including Smads [14, 15]. To further elucidate the cellular mechanisms underlying the antifibrotic effects of CREG, we assessed the regulatory role of CREG on Smad cascade activation. The increased level of Smad 2 phosphorylation and Smad 2/3 nuclear translocation was attenuated in TG mice in response to hypertrophic stimuli (Fig. 5A and B). We then infected cardiac fibroblasts with Ad-CREG or Ad-shCREG and treated with TGF-β1. Western blot analyses revealed significant phosphorylation of Smad 2 and translocation of Smad 2/3 without any significant alterations in the expression of Smad 2 protein after TGF-β1 treatment. Ad-CREG infection, however, almost completely suppressed Smad 2 phosphorylation as well as Smad 2/3 nuclear translocation (Fig. 5C). Importantly, Ad-shCREG infection enhanced TGF-β1s effects. To further examine the mechanisms involved, we used confluent cardiac fibroblasts infected with Ad-GFP, Ad-caERK1/2, or Ad-dnERK1/2. Activation of ERK1/2 by infection with Ad-caERK1/2 revealed a significant increase in, whereas blocking ERK1/2 activity by Ad-dnERK1/2 infection almost completely abrogated, collagen synthesis and COL1A2 or CTGF promoter activities in response to TGF-β1 (Fig. 5D). These findings suggest that CREG blocks collagen synthesis by disrupting MEK-ERK1/2-dependent TGF-β-Smad signalling.

Bottom Line: It has been proposed that CREG acts as a ligand that enhances differentiation and/or reduces cell proliferation.Constitutive over-expression of human CREG in the murine heart attenuated the hypertrophic response, markedly reduced inflammation.These beneficial effects were associated with attenuation of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase 1 (MEK-ERK1)/2-dependent signalling cascade.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, PR China.

ABSTRACT
Cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein of 220 amino acids. It has been proposed that CREG acts as a ligand that enhances differentiation and/or reduces cell proliferation. CREG has been shown previously to attenuate cardiac hypertrophy in vitro. However, such a role has not been determined in vivo. In the present study, we tested the hypothesis that overexpression of CREG in the murine heart would protect against cardiac hypertrophy and fibrosis in vivo. The effects of constitutive human CREG expression on cardiac hypertrophy were investigated using both in vitro and in vivo models. Cardiac hypertrophy was produced by aortic banding and infusion of angiotensin II in CREG transgenic mice and control animals. The extent of cardiac hypertrophy was quantitated by two-dimensional and M-mode echocardiography as well as by molecular and pathological analyses of heart samples. Constitutive over-expression of human CREG in the murine heart attenuated the hypertrophic response, markedly reduced inflammation. Cardiac function was also preserved in hearts with increased CREG levels in response to hypertrophic stimuli. These beneficial effects were associated with attenuation of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase 1 (MEK-ERK1)/2-dependent signalling cascade. In addition, CREG expression blocked fibrosis and collagen synthesis through blocking MEK-ERK1/2-dependent Smad 2/3 activation in vitro and in vivo. Therefore, the expression of CREG improves cardiac functions and inhibits cardiac hypertrophy, inflammation and fibrosis through blocking MEK-ERK1/2-dependent signalling.

Show MeSH
Related in: MedlinePlus