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Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-alpha.

Câmara J, Jarai G - Fibrogenesis Tissue Repair (2010)

Bottom Line: TNF-alpha markedly enhances the effect of TGF-beta1 on EMT, whereas IL1beta shows only a very weak effect and CTGF has no significant effect.Our results also suggest that bone morphogenetic protein-4 is likely to have a context dependent effect during the EMT of HBECs, being able to induce the expression of EMT markers and, at the same time, to inhibit TGF-beta induced epithelial transdifferentiation.Our results can contribute to a better understanding of lung fibrosis and to the development of new therapeutic approaches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Novartis Institutes for BioMedical Research, Respiratory Disease Area, Wimblehurst Road, Horsham, RH12 5AB West Sussex, UK. gabor.jarai@novartis.com.

ABSTRACT

Background: Defective epithelial repair, excess fibroblasts and myofibroblasts, collagen overproduction and fibrosis occur in a number of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Pathological conversion of epithelial cells into fibroblasts (epithelial-mesenchymal transition, EMT) has been proposed as a mechanism for the increased fibroblast numbers and has been demonstrated to occur in lung alveolar epithelial cells. Whether other airway cell types also have the capability to undergo EMT has been less explored so far. A better understanding of the full extent of EMT in airways, and the underlying mechanisms, can provide important insights into airway disease pathology and enable the development of new therapies. The main aim of this study was to test whether primary human bronchial epithelial cells are able to undergo EMT in vitro and to investigate the effect of various profibrotic factors in the process.

Results: Our data demonstrate that primary human bronchial epithelial cells (HBECs) are able to undergo EMT in response to transforming growth factor-beta 1 (TGF-beta1), as revealed by typical morphological alterations and EMT marker progression at the RNA level by real-time quantitative polymerase chain reaction and, at the protein level, by western blot. By using pharmacological inhibitors we show that this is a Smad-dependent mechanism and is independent of extracellular signal-related kinase pathway activation. Additional cytokines and growth factors such as tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL1beta) and connective tissue growth factor (CTGF) were also tested, alone or in combination with TGF-beta1. TNF-alpha markedly enhances the effect of TGF-beta1 on EMT, whereas IL1beta shows only a very weak effect and CTGF has no significant effect. We have also found that cell-matrix contact, in particular to fibronectin, an ECM component upregulated in fibrotic lesions, potentiates EMT in both human alveolar epithelial cells and HBECs. Furthermore, we also show that the collagen discoidin domain receptor 1 (DDR1), generally expressed in epithelial cells, is downregulated during the EMT of bronchial epithelium whereas DDR2 is unaffected. Our results also suggest that bone morphogenetic protein-4 is likely to have a context dependent effect during the EMT of HBECs, being able to induce the expression of EMT markers and, at the same time, to inhibit TGF-beta induced epithelial transdifferentiation.

Conclusions: The results presented in this study provide additional insights into EMT, a potentially very important mechanism in fibrogenesis. We show that, in addition to alveolar epithelial type II cells, primary HBECs are also able to undergo EMT in vitro upon TGF-beta1 stimulation via a primarily Smad 2/3 dependent mechanism. The effect of TGF-beta1 is potentiated on fibronectin matrix and in the presence of TNF-alpha, representing a millieu reminiscent of fibrotic lesions. Our results can contribute to a better understanding of lung fibrosis and to the development of new therapeutic approaches.

No MeSH data available.


Related in: MedlinePlus

Transforming growth factor (TGF)-β1 induces epithelial mesenchymal transition (EMT) in human bronchial epithelial cells (HBECs). HBECs were incubated with TGF-β1 (5 ng/ml) for 72 h in order to induce EMT. (a) Quantitative polymerase chain reaction analysis shows that TGF-β1 induces the mRNA expression of collagen I α 1 and matrix metalloprotease (MMP)2 and downregulates the epithelial marker E-cad. The relative expression level of each gene was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in the same sample. Statistical significance was determined by Student's t test; *P < 0.05, **P < 0.01, ***P < 0.001. (b) Western-blot analysis shows that TGF-β1 induces an upregulation of mesenchymal proteins (N-cad, vimentin, MMP-2) and of the myofibroblast protein α-smooth muscle actin and a downregulation of the epithelial marker E-cad in two different culture media. It also reveals an upregulation of phosho-Smad2 with TGF-β1 stimulation. An antibody against GAPDH was used as loading control. All further experiments were performed in the presence of BEGM medium unless indicated otherwise.
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Figure 1: Transforming growth factor (TGF)-β1 induces epithelial mesenchymal transition (EMT) in human bronchial epithelial cells (HBECs). HBECs were incubated with TGF-β1 (5 ng/ml) for 72 h in order to induce EMT. (a) Quantitative polymerase chain reaction analysis shows that TGF-β1 induces the mRNA expression of collagen I α 1 and matrix metalloprotease (MMP)2 and downregulates the epithelial marker E-cad. The relative expression level of each gene was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in the same sample. Statistical significance was determined by Student's t test; *P < 0.05, **P < 0.01, ***P < 0.001. (b) Western-blot analysis shows that TGF-β1 induces an upregulation of mesenchymal proteins (N-cad, vimentin, MMP-2) and of the myofibroblast protein α-smooth muscle actin and a downregulation of the epithelial marker E-cad in two different culture media. It also reveals an upregulation of phosho-Smad2 with TGF-β1 stimulation. An antibody against GAPDH was used as loading control. All further experiments were performed in the presence of BEGM medium unless indicated otherwise.

Mentions: A549 cells (ATCC) were maintained in DMEM (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and human bronchial epithelial cells (HBECs; Lonza, Basel, Switzerland) were maintained in BEGM medium (Lonza), both at 37°C in the presence of 5% CO2 in a humidified incubator. Except for the experiments shown in Figure 1, all further experiments with HBECs were performed in BEGM medium only, unless indicated otherwise.


Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-alpha.

Câmara J, Jarai G - Fibrogenesis Tissue Repair (2010)

Transforming growth factor (TGF)-β1 induces epithelial mesenchymal transition (EMT) in human bronchial epithelial cells (HBECs). HBECs were incubated with TGF-β1 (5 ng/ml) for 72 h in order to induce EMT. (a) Quantitative polymerase chain reaction analysis shows that TGF-β1 induces the mRNA expression of collagen I α 1 and matrix metalloprotease (MMP)2 and downregulates the epithelial marker E-cad. The relative expression level of each gene was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in the same sample. Statistical significance was determined by Student's t test; *P < 0.05, **P < 0.01, ***P < 0.001. (b) Western-blot analysis shows that TGF-β1 induces an upregulation of mesenchymal proteins (N-cad, vimentin, MMP-2) and of the myofibroblast protein α-smooth muscle actin and a downregulation of the epithelial marker E-cad in two different culture media. It also reveals an upregulation of phosho-Smad2 with TGF-β1 stimulation. An antibody against GAPDH was used as loading control. All further experiments were performed in the presence of BEGM medium unless indicated otherwise.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821296&req=5

Figure 1: Transforming growth factor (TGF)-β1 induces epithelial mesenchymal transition (EMT) in human bronchial epithelial cells (HBECs). HBECs were incubated with TGF-β1 (5 ng/ml) for 72 h in order to induce EMT. (a) Quantitative polymerase chain reaction analysis shows that TGF-β1 induces the mRNA expression of collagen I α 1 and matrix metalloprotease (MMP)2 and downregulates the epithelial marker E-cad. The relative expression level of each gene was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in the same sample. Statistical significance was determined by Student's t test; *P < 0.05, **P < 0.01, ***P < 0.001. (b) Western-blot analysis shows that TGF-β1 induces an upregulation of mesenchymal proteins (N-cad, vimentin, MMP-2) and of the myofibroblast protein α-smooth muscle actin and a downregulation of the epithelial marker E-cad in two different culture media. It also reveals an upregulation of phosho-Smad2 with TGF-β1 stimulation. An antibody against GAPDH was used as loading control. All further experiments were performed in the presence of BEGM medium unless indicated otherwise.
Mentions: A549 cells (ATCC) were maintained in DMEM (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and human bronchial epithelial cells (HBECs; Lonza, Basel, Switzerland) were maintained in BEGM medium (Lonza), both at 37°C in the presence of 5% CO2 in a humidified incubator. Except for the experiments shown in Figure 1, all further experiments with HBECs were performed in BEGM medium only, unless indicated otherwise.

Bottom Line: TNF-alpha markedly enhances the effect of TGF-beta1 on EMT, whereas IL1beta shows only a very weak effect and CTGF has no significant effect.Our results also suggest that bone morphogenetic protein-4 is likely to have a context dependent effect during the EMT of HBECs, being able to induce the expression of EMT markers and, at the same time, to inhibit TGF-beta induced epithelial transdifferentiation.Our results can contribute to a better understanding of lung fibrosis and to the development of new therapeutic approaches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Novartis Institutes for BioMedical Research, Respiratory Disease Area, Wimblehurst Road, Horsham, RH12 5AB West Sussex, UK. gabor.jarai@novartis.com.

ABSTRACT

Background: Defective epithelial repair, excess fibroblasts and myofibroblasts, collagen overproduction and fibrosis occur in a number of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Pathological conversion of epithelial cells into fibroblasts (epithelial-mesenchymal transition, EMT) has been proposed as a mechanism for the increased fibroblast numbers and has been demonstrated to occur in lung alveolar epithelial cells. Whether other airway cell types also have the capability to undergo EMT has been less explored so far. A better understanding of the full extent of EMT in airways, and the underlying mechanisms, can provide important insights into airway disease pathology and enable the development of new therapies. The main aim of this study was to test whether primary human bronchial epithelial cells are able to undergo EMT in vitro and to investigate the effect of various profibrotic factors in the process.

Results: Our data demonstrate that primary human bronchial epithelial cells (HBECs) are able to undergo EMT in response to transforming growth factor-beta 1 (TGF-beta1), as revealed by typical morphological alterations and EMT marker progression at the RNA level by real-time quantitative polymerase chain reaction and, at the protein level, by western blot. By using pharmacological inhibitors we show that this is a Smad-dependent mechanism and is independent of extracellular signal-related kinase pathway activation. Additional cytokines and growth factors such as tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL1beta) and connective tissue growth factor (CTGF) were also tested, alone or in combination with TGF-beta1. TNF-alpha markedly enhances the effect of TGF-beta1 on EMT, whereas IL1beta shows only a very weak effect and CTGF has no significant effect. We have also found that cell-matrix contact, in particular to fibronectin, an ECM component upregulated in fibrotic lesions, potentiates EMT in both human alveolar epithelial cells and HBECs. Furthermore, we also show that the collagen discoidin domain receptor 1 (DDR1), generally expressed in epithelial cells, is downregulated during the EMT of bronchial epithelium whereas DDR2 is unaffected. Our results also suggest that bone morphogenetic protein-4 is likely to have a context dependent effect during the EMT of HBECs, being able to induce the expression of EMT markers and, at the same time, to inhibit TGF-beta induced epithelial transdifferentiation.

Conclusions: The results presented in this study provide additional insights into EMT, a potentially very important mechanism in fibrogenesis. We show that, in addition to alveolar epithelial type II cells, primary HBECs are also able to undergo EMT in vitro upon TGF-beta1 stimulation via a primarily Smad 2/3 dependent mechanism. The effect of TGF-beta1 is potentiated on fibronectin matrix and in the presence of TNF-alpha, representing a millieu reminiscent of fibrotic lesions. Our results can contribute to a better understanding of lung fibrosis and to the development of new therapeutic approaches.

No MeSH data available.


Related in: MedlinePlus