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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-induced epithelial mesenchymal transition (EMT) in human bronchial epithelial cells (HBECs) is Smad-dependent. HBECs that have been pre-incubated with the activin receptor-like kinase (ALK)-5 inhibitor SB431542 (SB, 10 μM) 1 h before the addition of TGF-β1 (5 ng/ml) are unable to undergo EMT after 5 days of differentiation. By contrast, mitogen-activated protein kinase inhibition by PD98059, a MEK inhibitor (PD, 10 μM), only partially inhibits EMT. (a) Representative phase contrast images (10× magnification, scale bar = 10 μM) show that HBECs in the presence of TGF-β1 lose cell-cell contact, become more sparse and change into an elongated fibroblastoid morphology. This effect is inhibited after pre-incubation of the cells with the ALK-4,-5,-7 inhibitor SB but not by the MEK inhibitor PD. (b) Western-blot analysis shows that SB completely abolishes TGF-β1 gene regulation of E-cad and the phosho-Smad2 signal is fully inhibited in the presence of SB. An antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (c) Western-blot analysis shows that the upregulation of matrix metalloprotease (MMP)-2 by TGF-β1 is partially inhibited by the presence of bone morphogenetic proteins (BMP)4 confirming the quantitative polymerase chain reaction (qPCR) results shown in (d). Phosho-Smad1/5/8 shows active BMP signalling in the presence of BMP4 which is inhibited in the presence of TGF-β1. An antibody against GAPDH was used as loading control. (d) q(PCR) analysis shows that SB inhibits gene expression changes of collagen I α 1, MMP2, MMP-9 and E-cad induced by TGF-β1 (top 2 panels). BMP4 (50 ng/ml) induces a significant upregulation of collagen I in the presence of TGF-β1, as compared to TGF-β1 alone (top left panel) and has no significant effect on E-cad expression either alone or in combination with TGF-β1 (top right panel). In contrast, the upregulation of MMP-2 and MMP-9 expression in the presence of TGF-β1 is significantly inhibited by concomitant BMP4 treatment (bottom 2 panels). The relative expression level of each gene was normalized to GAPDH mRNA in the same sample. Statistical significance was determined by one-way ANOVA followed by Tukey test; ***P < 0.001.
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Figure 2: Transforming growth factor (TGF)-β1-induced epithelial mesenchymal transition (EMT) in human bronchial epithelial cells (HBECs) is Smad-dependent. HBECs that have been pre-incubated with the activin receptor-like kinase (ALK)-5 inhibitor SB431542 (SB, 10 μM) 1 h before the addition of TGF-β1 (5 ng/ml) are unable to undergo EMT after 5 days of differentiation. By contrast, mitogen-activated protein kinase inhibition by PD98059, a MEK inhibitor (PD, 10 μM), only partially inhibits EMT. (a) Representative phase contrast images (10× magnification, scale bar = 10 μM) show that HBECs in the presence of TGF-β1 lose cell-cell contact, become more sparse and change into an elongated fibroblastoid morphology. This effect is inhibited after pre-incubation of the cells with the ALK-4,-5,-7 inhibitor SB but not by the MEK inhibitor PD. (b) Western-blot analysis shows that SB completely abolishes TGF-β1 gene regulation of E-cad and the phosho-Smad2 signal is fully inhibited in the presence of SB. An antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (c) Western-blot analysis shows that the upregulation of matrix metalloprotease (MMP)-2 by TGF-β1 is partially inhibited by the presence of bone morphogenetic proteins (BMP)4 confirming the quantitative polymerase chain reaction (qPCR) results shown in (d). Phosho-Smad1/5/8 shows active BMP signalling in the presence of BMP4 which is inhibited in the presence of TGF-β1. An antibody against GAPDH was used as loading control. (d) q(PCR) analysis shows that SB inhibits gene expression changes of collagen I α 1, MMP2, MMP-9 and E-cad induced by TGF-β1 (top 2 panels). BMP4 (50 ng/ml) induces a significant upregulation of collagen I in the presence of TGF-β1, as compared to TGF-β1 alone (top left panel) and has no significant effect on E-cad expression either alone or in combination with TGF-β1 (top right panel). In contrast, the upregulation of MMP-2 and MMP-9 expression in the presence of TGF-β1 is significantly inhibited by concomitant BMP4 treatment (bottom 2 panels). The relative expression level of each gene was normalized to GAPDH mRNA in the same sample. Statistical significance was determined by one-way ANOVA followed by Tukey test; ***P < 0.001.

Mentions: A549 and HBECs were incubated with pharmacological inhibitors for 1 h prior to the addition of TGF-β1 (5 ng/ml) for EMT induction for 48 h or 3-5 days, respectively (see legends for Figures 1,2,3,4,5 for further description). The Smad pathway inhibitor SB431542 (10 μM, Sigma-Aldrich) and the ERK pathway inhibitor PD98059 (10 μM, Calbiochem) were used.


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-induced epithelial mesenchymal transition (EMT) in human bronchial epithelial cells (HBECs) is Smad-dependent. HBECs that have been pre-incubated with the activin receptor-like kinase (ALK)-5 inhibitor SB431542 (SB, 10 μM) 1 h before the addition of TGF-β1 (5 ng/ml) are unable to undergo EMT after 5 days of differentiation. By contrast, mitogen-activated protein kinase inhibition by PD98059, a MEK inhibitor (PD, 10 μM), only partially inhibits EMT. (a) Representative phase contrast images (10× magnification, scale bar = 10 μM) show that HBECs in the presence of TGF-β1 lose cell-cell contact, become more sparse and change into an elongated fibroblastoid morphology. This effect is inhibited after pre-incubation of the cells with the ALK-4,-5,-7 inhibitor SB but not by the MEK inhibitor PD. (b) Western-blot analysis shows that SB completely abolishes TGF-β1 gene regulation of E-cad and the phosho-Smad2 signal is fully inhibited in the presence of SB. An antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (c) Western-blot analysis shows that the upregulation of matrix metalloprotease (MMP)-2 by TGF-β1 is partially inhibited by the presence of bone morphogenetic proteins (BMP)4 confirming the quantitative polymerase chain reaction (qPCR) results shown in (d). Phosho-Smad1/5/8 shows active BMP signalling in the presence of BMP4 which is inhibited in the presence of TGF-β1. An antibody against GAPDH was used as loading control. (d) q(PCR) analysis shows that SB inhibits gene expression changes of collagen I α 1, MMP2, MMP-9 and E-cad induced by TGF-β1 (top 2 panels). BMP4 (50 ng/ml) induces a significant upregulation of collagen I in the presence of TGF-β1, as compared to TGF-β1 alone (top left panel) and has no significant effect on E-cad expression either alone or in combination with TGF-β1 (top right panel). In contrast, the upregulation of MMP-2 and MMP-9 expression in the presence of TGF-β1 is significantly inhibited by concomitant BMP4 treatment (bottom 2 panels). The relative expression level of each gene was normalized to GAPDH mRNA in the same sample. Statistical significance was determined by one-way ANOVA followed by Tukey test; ***P < 0.001.
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Related In: Results  -  Collection

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Figure 2: Transforming growth factor (TGF)-β1-induced epithelial mesenchymal transition (EMT) in human bronchial epithelial cells (HBECs) is Smad-dependent. HBECs that have been pre-incubated with the activin receptor-like kinase (ALK)-5 inhibitor SB431542 (SB, 10 μM) 1 h before the addition of TGF-β1 (5 ng/ml) are unable to undergo EMT after 5 days of differentiation. By contrast, mitogen-activated protein kinase inhibition by PD98059, a MEK inhibitor (PD, 10 μM), only partially inhibits EMT. (a) Representative phase contrast images (10× magnification, scale bar = 10 μM) show that HBECs in the presence of TGF-β1 lose cell-cell contact, become more sparse and change into an elongated fibroblastoid morphology. This effect is inhibited after pre-incubation of the cells with the ALK-4,-5,-7 inhibitor SB but not by the MEK inhibitor PD. (b) Western-blot analysis shows that SB completely abolishes TGF-β1 gene regulation of E-cad and the phosho-Smad2 signal is fully inhibited in the presence of SB. An antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (c) Western-blot analysis shows that the upregulation of matrix metalloprotease (MMP)-2 by TGF-β1 is partially inhibited by the presence of bone morphogenetic proteins (BMP)4 confirming the quantitative polymerase chain reaction (qPCR) results shown in (d). Phosho-Smad1/5/8 shows active BMP signalling in the presence of BMP4 which is inhibited in the presence of TGF-β1. An antibody against GAPDH was used as loading control. (d) q(PCR) analysis shows that SB inhibits gene expression changes of collagen I α 1, MMP2, MMP-9 and E-cad induced by TGF-β1 (top 2 panels). BMP4 (50 ng/ml) induces a significant upregulation of collagen I in the presence of TGF-β1, as compared to TGF-β1 alone (top left panel) and has no significant effect on E-cad expression either alone or in combination with TGF-β1 (top right panel). In contrast, the upregulation of MMP-2 and MMP-9 expression in the presence of TGF-β1 is significantly inhibited by concomitant BMP4 treatment (bottom 2 panels). The relative expression level of each gene was normalized to GAPDH mRNA in the same sample. Statistical significance was determined by one-way ANOVA followed by Tukey test; ***P < 0.001.
Mentions: A549 and HBECs were incubated with pharmacological inhibitors for 1 h prior to the addition of TGF-β1 (5 ng/ml) for EMT induction for 48 h or 3-5 days, respectively (see legends for Figures 1,2,3,4,5 for further description). The Smad pathway inhibitor SB431542 (10 μM, Sigma-Aldrich) and the ERK pathway inhibitor PD98059 (10 μM, Calbiochem) were used.

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