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Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts.

Ghavami S, Cunnington RH, Gupta S, Yeganeh B, Filomeno KL, Freed DH, Chen S, Klonisch T, Halayko AJ, Ambrose E, Singal R, Dixon IM - Cell Death Dis (2015)

Bottom Line: In many other cellular systems, TGF-β(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown.Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells.These results support the hypothesis that TGF-β(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Physiology, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada [2] Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada [3] Department of Physiology and Institute of Cardiovascular Sciences, St. Boniface Research Centre, University of Manitoba, Winnipeg, Manitoba, Canada [4] Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Transforming growth factor-β(1) (TGF-β(1)) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β(1)-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β(1) to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β(1) promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β(1) in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

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Related in: MedlinePlus

TGF-β1-induced autophagy is a requisite of TGF-β1-induced pro-fibrosis in hATMyofbs. (a) Primary hATMyofbs were treated with TGF-β1 (10 ng/ml) in the presence of the autophagy inhibitors Baf-A1 (10 nM) and 3-MA (2.5 mM) for the indicated durations. Western blotting analysis revealed that inhibition of autophagy abrogated the fibrogenic effects of TGF-β1 (i.e., decreased collagen type Iα2 and fibronectin expression levels), whereas this treatment did not affect Smad2 or Smad3 phosphorylation. Equal protein loading was confirmed using GAPDH levels. Results are the means from three independent experiments using cells from three different donors. (b and c) Densitometric analysis of collagen Iα2 and fibronectin levels in hATMyofbs, which were stimulated with TGF-β1 or autophagy inhibitors (i.e., Baf-A1 (10 nM) (b) or 3-MA (2.5 mM) (c). Inhibition of autophagy significantly decreased collagen Iα2 and fibronection biosynthesis. Data are the means of three independent experiments using hATMyofbs from three different donors. For each experiment, collagen Iα2 and fibronectin levels were compared with those from time-matched controls and normalized to GAPDH levels. (d–f) Protein required for autophagy induction (Atg7) were stably knocked down in hATMyofbs. Atg7 knocked down cells and their correspondence scramble infected cells were treated with TGF-β1 (10 ng/ml) for 48 and 96 h. Whole-cell lysates were extracted and then collagen Iα2 and fibronectin expression levels were measured in the cell lysates. Protein loading was confirmed using GAPDH. (f) Densitometry analysis showed that Atg7 knockdown was associated with a significant (P<0.01) decrease of TGF-β1-induced fibronectin biosynthesis in hATMyofbs. (g and h) Atrg5 KD MEF were treated with TGF-β1 (10 ng/ml) for the indicated time points. LC3 lipidation, Atg5-12 conjugation fibronectin expression, total Smad 2/3 and Smad2 phosphorylation were measured in whole-cell lysates. Protein loading was confirmed using β-actin. (g and h) Densitometry analysis (h) revealed that ATG5 knockdown was associated with a significant (P<0.01) decrease in TGF-β1-induced fibronectin biosynthesis in MEF. (i and j) Autophagy induction increases TGF-β1-induced fibrogenic effects. hATMyofbs were pretreated with Rapaymcin (4 h, 1000 nM) and then co-treated with TGF-β1 (10 ng/ml) for the indicated duration. LC3 lipidation, collagen type1 α2 expression, fibronectin expression, total Smad 2/3 and Smad2 phosphorylation were measured in whole-cell lysates. Protein loading was confirmed using GAPDH. (j) Densitometry analysis showed that Rapamycin (1000 nM) significantly (P<0.01) increased TGF-β1-induced fibronectin and collagen type 1 α2 biosynthesis in hATMyofbs. **P<0.01
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fig2: TGF-β1-induced autophagy is a requisite of TGF-β1-induced pro-fibrosis in hATMyofbs. (a) Primary hATMyofbs were treated with TGF-β1 (10 ng/ml) in the presence of the autophagy inhibitors Baf-A1 (10 nM) and 3-MA (2.5 mM) for the indicated durations. Western blotting analysis revealed that inhibition of autophagy abrogated the fibrogenic effects of TGF-β1 (i.e., decreased collagen type Iα2 and fibronectin expression levels), whereas this treatment did not affect Smad2 or Smad3 phosphorylation. Equal protein loading was confirmed using GAPDH levels. Results are the means from three independent experiments using cells from three different donors. (b and c) Densitometric analysis of collagen Iα2 and fibronectin levels in hATMyofbs, which were stimulated with TGF-β1 or autophagy inhibitors (i.e., Baf-A1 (10 nM) (b) or 3-MA (2.5 mM) (c). Inhibition of autophagy significantly decreased collagen Iα2 and fibronection biosynthesis. Data are the means of three independent experiments using hATMyofbs from three different donors. For each experiment, collagen Iα2 and fibronectin levels were compared with those from time-matched controls and normalized to GAPDH levels. (d–f) Protein required for autophagy induction (Atg7) were stably knocked down in hATMyofbs. Atg7 knocked down cells and their correspondence scramble infected cells were treated with TGF-β1 (10 ng/ml) for 48 and 96 h. Whole-cell lysates were extracted and then collagen Iα2 and fibronectin expression levels were measured in the cell lysates. Protein loading was confirmed using GAPDH. (f) Densitometry analysis showed that Atg7 knockdown was associated with a significant (P<0.01) decrease of TGF-β1-induced fibronectin biosynthesis in hATMyofbs. (g and h) Atrg5 KD MEF were treated with TGF-β1 (10 ng/ml) for the indicated time points. LC3 lipidation, Atg5-12 conjugation fibronectin expression, total Smad 2/3 and Smad2 phosphorylation were measured in whole-cell lysates. Protein loading was confirmed using β-actin. (g and h) Densitometry analysis (h) revealed that ATG5 knockdown was associated with a significant (P<0.01) decrease in TGF-β1-induced fibronectin biosynthesis in MEF. (i and j) Autophagy induction increases TGF-β1-induced fibrogenic effects. hATMyofbs were pretreated with Rapaymcin (4 h, 1000 nM) and then co-treated with TGF-β1 (10 ng/ml) for the indicated duration. LC3 lipidation, collagen type1 α2 expression, fibronectin expression, total Smad 2/3 and Smad2 phosphorylation were measured in whole-cell lysates. Protein loading was confirmed using GAPDH. (j) Densitometry analysis showed that Rapamycin (1000 nM) significantly (P<0.01) increased TGF-β1-induced fibronectin and collagen type 1 α2 biosynthesis in hATMyofbs. **P<0.01

Mentions: TGF-β1 is but one of an array of factors shown to be involved in the induction of cardiac fibrosis, as demonstrated in overexpression and knockout models.28, 29, 30 As atrial fibrillation is a serious clinical problem with high incidence in society31 and is linked to fibrosis of atrial tissues,32 we investigated whether or not there was an association between TGF-β1-induced fibrosis and autophagy in hATMyofbs. Primary hATMyofbs constitute a clinically relevant model for the study of TGF-β1-induced fibrosis and autophagy. Our results show that TGF-β1 (10 ng/ml) induces significant increases in the synthesis of collagen type Iα2 and fibronectin in the presence of LC3β II lipidation and increases Smad2 and Smad3 phosphorylation and p62 degradation (Figures 1a and b). We also showed that TGF-β1 treatment significantly did not affect the viability of hATMyofb cells (Figure 1c) while conversely it is associated with a significant induction of their proliferation at 72 and 120 h after treatments when compared with 48-h cultures (Figure 1d; P≤0.01). Fibrillar collagen type I and fibronectin deposition was also confirmed using transmission electron microscopic (TEM) images from hATMyofbs, which were stimulated with TGF-β1 at 10 ng/ml for 96 h (Figures 1e and f). Figures 1e and f indicate an increase of fiber deposition after TGF-β1 treatment. TGF-β1 stimulation increased collagen Iα2 secretion from hATMyofb cells compared with time-matched control cells, which also proved TGF-β1-induced fibrosis (western blotting analysis in Figure 1g). Possible TGF-β1 autophagy induction was further investigated using TEM images from hATMyofbs stimulated with TGF-β1 at 10 ng/ml for 96 h. Figures 1h and i clearly show autophagosome and autophagolysosmes in hATMyofbs stimulated with TGF-β1. Moreover, our immunocytochemistry data indicate punctate LC3β II staining and lysosomal activation in hATMyofbs treated with TGF-β1 (Figure 1j), with a significant (P<0.001) increase in the number of cells. In this case, LC3β and lysotracker were co-localized (Figure 1k), which is consistent with the autophagy activation in these cells. To obtain more quantitative assessment of the induction of autophagy, we used bafilomycin-A1 (Baf-A1; 10 nM) to block the fusion of autophagosomes and lysosomes and assess the presence of autophagy flux.13, 33, 34 As shown in Figure 2a, we demonstrate autophagic flux, for example, autophagosome delivery to lysosomes and autophagolysosome formation, by co-treating hATMyofbs with TGF-β1 and Baf-A1 for 48 and 96 h. TGF-β1-induced accumulation of LC3-II was enhanced in the presence of Baf-A1, which supports the suggestion that TGF-β1 enhances autophagosome synthesis.


Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts.

Ghavami S, Cunnington RH, Gupta S, Yeganeh B, Filomeno KL, Freed DH, Chen S, Klonisch T, Halayko AJ, Ambrose E, Singal R, Dixon IM - Cell Death Dis (2015)

TGF-β1-induced autophagy is a requisite of TGF-β1-induced pro-fibrosis in hATMyofbs. (a) Primary hATMyofbs were treated with TGF-β1 (10 ng/ml) in the presence of the autophagy inhibitors Baf-A1 (10 nM) and 3-MA (2.5 mM) for the indicated durations. Western blotting analysis revealed that inhibition of autophagy abrogated the fibrogenic effects of TGF-β1 (i.e., decreased collagen type Iα2 and fibronectin expression levels), whereas this treatment did not affect Smad2 or Smad3 phosphorylation. Equal protein loading was confirmed using GAPDH levels. Results are the means from three independent experiments using cells from three different donors. (b and c) Densitometric analysis of collagen Iα2 and fibronectin levels in hATMyofbs, which were stimulated with TGF-β1 or autophagy inhibitors (i.e., Baf-A1 (10 nM) (b) or 3-MA (2.5 mM) (c). Inhibition of autophagy significantly decreased collagen Iα2 and fibronection biosynthesis. Data are the means of three independent experiments using hATMyofbs from three different donors. For each experiment, collagen Iα2 and fibronectin levels were compared with those from time-matched controls and normalized to GAPDH levels. (d–f) Protein required for autophagy induction (Atg7) were stably knocked down in hATMyofbs. Atg7 knocked down cells and their correspondence scramble infected cells were treated with TGF-β1 (10 ng/ml) for 48 and 96 h. Whole-cell lysates were extracted and then collagen Iα2 and fibronectin expression levels were measured in the cell lysates. Protein loading was confirmed using GAPDH. (f) Densitometry analysis showed that Atg7 knockdown was associated with a significant (P<0.01) decrease of TGF-β1-induced fibronectin biosynthesis in hATMyofbs. (g and h) Atrg5 KD MEF were treated with TGF-β1 (10 ng/ml) for the indicated time points. LC3 lipidation, Atg5-12 conjugation fibronectin expression, total Smad 2/3 and Smad2 phosphorylation were measured in whole-cell lysates. Protein loading was confirmed using β-actin. (g and h) Densitometry analysis (h) revealed that ATG5 knockdown was associated with a significant (P<0.01) decrease in TGF-β1-induced fibronectin biosynthesis in MEF. (i and j) Autophagy induction increases TGF-β1-induced fibrogenic effects. hATMyofbs were pretreated with Rapaymcin (4 h, 1000 nM) and then co-treated with TGF-β1 (10 ng/ml) for the indicated duration. LC3 lipidation, collagen type1 α2 expression, fibronectin expression, total Smad 2/3 and Smad2 phosphorylation were measured in whole-cell lysates. Protein loading was confirmed using GAPDH. (j) Densitometry analysis showed that Rapamycin (1000 nM) significantly (P<0.01) increased TGF-β1-induced fibronectin and collagen type 1 α2 biosynthesis in hATMyofbs. **P<0.01
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fig2: TGF-β1-induced autophagy is a requisite of TGF-β1-induced pro-fibrosis in hATMyofbs. (a) Primary hATMyofbs were treated with TGF-β1 (10 ng/ml) in the presence of the autophagy inhibitors Baf-A1 (10 nM) and 3-MA (2.5 mM) for the indicated durations. Western blotting analysis revealed that inhibition of autophagy abrogated the fibrogenic effects of TGF-β1 (i.e., decreased collagen type Iα2 and fibronectin expression levels), whereas this treatment did not affect Smad2 or Smad3 phosphorylation. Equal protein loading was confirmed using GAPDH levels. Results are the means from three independent experiments using cells from three different donors. (b and c) Densitometric analysis of collagen Iα2 and fibronectin levels in hATMyofbs, which were stimulated with TGF-β1 or autophagy inhibitors (i.e., Baf-A1 (10 nM) (b) or 3-MA (2.5 mM) (c). Inhibition of autophagy significantly decreased collagen Iα2 and fibronection biosynthesis. Data are the means of three independent experiments using hATMyofbs from three different donors. For each experiment, collagen Iα2 and fibronectin levels were compared with those from time-matched controls and normalized to GAPDH levels. (d–f) Protein required for autophagy induction (Atg7) were stably knocked down in hATMyofbs. Atg7 knocked down cells and their correspondence scramble infected cells were treated with TGF-β1 (10 ng/ml) for 48 and 96 h. Whole-cell lysates were extracted and then collagen Iα2 and fibronectin expression levels were measured in the cell lysates. Protein loading was confirmed using GAPDH. (f) Densitometry analysis showed that Atg7 knockdown was associated with a significant (P<0.01) decrease of TGF-β1-induced fibronectin biosynthesis in hATMyofbs. (g and h) Atrg5 KD MEF were treated with TGF-β1 (10 ng/ml) for the indicated time points. LC3 lipidation, Atg5-12 conjugation fibronectin expression, total Smad 2/3 and Smad2 phosphorylation were measured in whole-cell lysates. Protein loading was confirmed using β-actin. (g and h) Densitometry analysis (h) revealed that ATG5 knockdown was associated with a significant (P<0.01) decrease in TGF-β1-induced fibronectin biosynthesis in MEF. (i and j) Autophagy induction increases TGF-β1-induced fibrogenic effects. hATMyofbs were pretreated with Rapaymcin (4 h, 1000 nM) and then co-treated with TGF-β1 (10 ng/ml) for the indicated duration. LC3 lipidation, collagen type1 α2 expression, fibronectin expression, total Smad 2/3 and Smad2 phosphorylation were measured in whole-cell lysates. Protein loading was confirmed using GAPDH. (j) Densitometry analysis showed that Rapamycin (1000 nM) significantly (P<0.01) increased TGF-β1-induced fibronectin and collagen type 1 α2 biosynthesis in hATMyofbs. **P<0.01
Mentions: TGF-β1 is but one of an array of factors shown to be involved in the induction of cardiac fibrosis, as demonstrated in overexpression and knockout models.28, 29, 30 As atrial fibrillation is a serious clinical problem with high incidence in society31 and is linked to fibrosis of atrial tissues,32 we investigated whether or not there was an association between TGF-β1-induced fibrosis and autophagy in hATMyofbs. Primary hATMyofbs constitute a clinically relevant model for the study of TGF-β1-induced fibrosis and autophagy. Our results show that TGF-β1 (10 ng/ml) induces significant increases in the synthesis of collagen type Iα2 and fibronectin in the presence of LC3β II lipidation and increases Smad2 and Smad3 phosphorylation and p62 degradation (Figures 1a and b). We also showed that TGF-β1 treatment significantly did not affect the viability of hATMyofb cells (Figure 1c) while conversely it is associated with a significant induction of their proliferation at 72 and 120 h after treatments when compared with 48-h cultures (Figure 1d; P≤0.01). Fibrillar collagen type I and fibronectin deposition was also confirmed using transmission electron microscopic (TEM) images from hATMyofbs, which were stimulated with TGF-β1 at 10 ng/ml for 96 h (Figures 1e and f). Figures 1e and f indicate an increase of fiber deposition after TGF-β1 treatment. TGF-β1 stimulation increased collagen Iα2 secretion from hATMyofb cells compared with time-matched control cells, which also proved TGF-β1-induced fibrosis (western blotting analysis in Figure 1g). Possible TGF-β1 autophagy induction was further investigated using TEM images from hATMyofbs stimulated with TGF-β1 at 10 ng/ml for 96 h. Figures 1h and i clearly show autophagosome and autophagolysosmes in hATMyofbs stimulated with TGF-β1. Moreover, our immunocytochemistry data indicate punctate LC3β II staining and lysosomal activation in hATMyofbs treated with TGF-β1 (Figure 1j), with a significant (P<0.001) increase in the number of cells. In this case, LC3β and lysotracker were co-localized (Figure 1k), which is consistent with the autophagy activation in these cells. To obtain more quantitative assessment of the induction of autophagy, we used bafilomycin-A1 (Baf-A1; 10 nM) to block the fusion of autophagosomes and lysosomes and assess the presence of autophagy flux.13, 33, 34 As shown in Figure 2a, we demonstrate autophagic flux, for example, autophagosome delivery to lysosomes and autophagolysosome formation, by co-treating hATMyofbs with TGF-β1 and Baf-A1 for 48 and 96 h. TGF-β1-induced accumulation of LC3-II was enhanced in the presence of Baf-A1, which supports the suggestion that TGF-β1 enhances autophagosome synthesis.

Bottom Line: In many other cellular systems, TGF-β(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown.Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells.These results support the hypothesis that TGF-β(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Physiology, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada [2] Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada [3] Department of Physiology and Institute of Cardiovascular Sciences, St. Boniface Research Centre, University of Manitoba, Winnipeg, Manitoba, Canada [4] Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Transforming growth factor-β(1) (TGF-β(1)) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β(1)-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β(1) to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β(1) promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β(1) in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

Show MeSH
Related in: MedlinePlus