The Role of Bone Morphogenetic Protein Signalling in Adult Lung Health and Disease

Bone morphogenetic protein (BMP) signalling is essential for correct lung morphogenesis. The pathway controls branching morphogenesis in the nascent lung and is also involved in the establishment of correct epithelial cell distribution throughout the airways. In the adult lung, BMP signalling is reactivated during airway injury and inflammation and there is evidence of aberrant signalling in lung cancer and in chronic lung diseases such as asthma and fibrosis. However, little is known about the role of BMP signalling in healthy adult airways. Furthermore the effect of incorrect BMP pathway expression during epithelial repair and inflammatory and malignant lung diseases remains elusive. The aims of this project were to characterise BMP pathway expression in the descending airways of large animal models and to investigate the role of BMP signalling during adult airway homeostasis, recovery and disease. To investigate the role of BMP signalling in malignant lung disease we used a heterogeneous lung cancer cell line DLKP. We explored the effect of the BMP pathway on epithelial-to-mesenchymal (EMT) progression and phenotypic plasticity between the tumour subpopulations. BMP-4 treatment induced mesenchymal-like projections in the DLKP-SQ clones and significant morphological changes in the DLKP-M clones towards the stem-cell like colonies of DLKP-I populations. Elevated N-cadherin and Vimentin protein expression was also evident in the clones following BMP-4 treatment. By stably transfecting an E-cadherin gene in these E-cadherin-null cells we demonstrated the distinct phenotypic differences between these tumour subpopulations. Healthy porcine and rhesus macaque lungs were used to characterise BMP pathway expression throughout the airways. Active BMP signalling was present in the descending airway epithelium and a gradient in pathway activation along the proximal-distal axis of the lungs was observed [1]. Using a primary porcine tracheal in vitro explant model the BMP signalling gradient was investigated further and it was found that by modulating BMP signalling, using exogenous BMP-4 stimulation, the epithelial phenotype of the porcine tracheal cells was altered. Finally, to assess the role of BMP signalling during airway inflammation and repair an established non-human primate model of allergic airway disease was used. A reduction in pSMAD1/5/8 expression was present in the epithelium of asthmatic monkeys compared to healthy controls. In addition, following a period of six months in filtered air to facilitate airway repair, there was a significant increase in Proliferating Cell Nuclear Antigen (PCNA), BMP Receptor 1a (BMPRIa) and pSMAD1/5/8 throughout the asthmatic airways. Taken together, these data suggest that not only is the developmental pathway re-activated during inflammatory airway disease but that basal BMP pathway expression is important for maintaining healthy airways. Overall these data highlight the presence and importance of BMP signalling gradients in healthy adult airways and further implicate BMP signalling in the pathogenesis of inflammatory and malignant airway diseases. [1] T.M Lynn, E.L Molloy, J.C Masterson, S.F. Glynn, R.W. Costello, M.V. Avdalovic, E.S Schelegle, L.A. Miller, D.M Hyde, S O’Dea. ‘SMAD signalling in descending airways of healthy versus asthmatic rhesus macaques highlights a relationship between inflammation and BMPs’. AJRCMB, In Press

The Role of Bone Morphogenetic Protein Signalling in Adult Lung Health and Disease

Bone morphogenetic protein (BMP) signalling is essential for correct lung morphogenesis. The pathway controls branching morphogenesis in the nascent lung and is also involved in the establishment of correct epithelial cell distribution throughout the airways. In the adult lung, BMP signalling is reactivated during airway injury and inflammation and there is evidence of aberrant signalling in lung cancer and in chronic lung diseases such as asthma and fibrosis. However, little is known about the role of BMP signalling in healthy adult airways. Furthermore the effect of incorrect BMP pathway expression during epithelial repair and inflammatory and malignant lung diseases remains elusive. The aims of this project were to characterise BMP pathway expression in the descending airways of large animal models and to investigate the role of BMP signalling during adult airway homeostasis, recovery and disease. To investigate the role of BMP signalling in malignant lung disease we used a heterogeneous lung cancer cell line DLKP. We explored the effect of the BMP pathway on epithelial-to-mesenchymal (EMT) progression and phenotypic plasticity between the tumour subpopulations. BMP-4 treatment induced mesenchymal-like projections in the DLKP-SQ clones and significant morphological changes in the DLKP-M clones towards the stem-cell like colonies of DLKP-I populations. Elevated N-cadherin and Vimentin protein expression was also evident in the clones following BMP-4 treatment. By stably transfecting an E-cadherin gene in these E-cadherin-null cells we demonstrated the distinct phenotypic differences between these tumour subpopulations. Healthy porcine and rhesus macaque lungs were used to characterise BMP pathway expression throughout the airways. Active BMP signalling was present in the descending airway epithelium and a gradient in pathway activation along the proximal-distal axis of the lungs was observed [1]. Using a primary porcine tracheal in vitro explant model the BMP signalling gradient was investigated further and it was found that by modulating BMP signalling, using exogenous BMP-4 stimulation, the epithelial phenotype of the porcine tracheal cells was altered. Finally, to assess the role of BMP signalling during airway inflammation and repair an established non-human primate model of allergic airway disease was used. A reduction in pSMAD1/5/8 expression was present in the epithelium of asthmatic monkeys compared to healthy controls. In addition, following a period of six months in filtered air to facilitate airway repair, there was a significant increase in Proliferating Cell Nuclear Antigen (PCNA), BMP Receptor 1a (BMPRIa) and pSMAD1/5/8 throughout the asthmatic airways. Taken together, these data suggest that not only is the developmental pathway re-activated during inflammatory airway disease but that basal BMP pathway expression is important for maintaining healthy airways. Overall these data highlight the presence and importance of BMP signalling gradients in healthy adult airways and further implicate BMP signalling in the pathogenesis of inflammatory and malignant airway diseases. [1] T.M Lynn, E.L Molloy, J.C Masterson, S.F. Glynn, R.W. Costello, M.V. Avdalovic, E.S Schelegle, L.A. Miller, D.M Hyde, S O’Dea. ‘SMAD signalling in descending airways of healthy versus asthmatic rhesus macaques highlights a relationship between inflammation and BMPs’. AJRCMB, In Press

The Role of Bone Morphogenetic Protein Signalling in Adult Lung Health and Disease

Bone morphogenetic protein (BMP) signalling is essential for correct lung morphogenesis. The pathway controls branching morphogenesis in the nascent lung and is also involved in the establishment of correct epithelial cell distribution throughout the airways. In the adult lung, BMP signalling is reactivated during airway injury and inflammation and there is evidence of aberrant signalling in lung cancer and in chronic lung diseases such as asthma and fibrosis. However, little is known about the role of BMP signalling in healthy adult airways. Furthermore the effect of incorrect BMP pathway expression during epithelial repair and inflammatory and malignant lung diseases remains elusive. The aims of this project were to characterise BMP pathway expression in the descending airways of large animal models and to investigate the role of BMP signalling during adult airway homeostasis, recovery and disease. To investigate the role of BMP signalling in malignant lung disease we used a heterogeneous lung cancer cell line DLKP. We explored the effect of the BMP pathway on epithelial-to-mesenchymal (EMT) progression and phenotypic plasticity between the tumour subpopulations. BMP-4 treatment induced mesenchymal-like projections in the DLKP-SQ clones and significant morphological changes in the DLKP-M clones towards the stem-cell like colonies of DLKP-I populations. Elevated N-cadherin and Vimentin protein expression was also evident in the clones following BMP-4 treatment. By stably transfecting an E-cadherin gene in these E-cadherin-null cells we demonstrated the distinct phenotypic differences between these tumour subpopulations. Healthy porcine and rhesus macaque lungs were used to characterise BMP pathway expression throughout the airways. Active BMP signalling was present in the descending airway epithelium and a gradient in pathway activation along the proximal-distal axis of the lungs was observed [1]. Using a primary porcine tracheal in vitro explant model the BMP signalling gradient was investigated further and it was found that by modulating BMP signalling, using exogenous BMP-4 stimulation, the epithelial phenotype of the porcine tracheal cells was altered. Finally, to assess the role of BMP signalling during airway inflammation and repair an established non-human primate model of allergic airway disease was used. A reduction in pSMAD1/5/8 expression was present in the epithelium of asthmatic monkeys compared to healthy controls. In addition, following a period of six months in filtered air to facilitate airway repair, there was a significant increase in Proliferating Cell Nuclear Antigen (PCNA), BMP Receptor 1a (BMPRIa) and pSMAD1/5/8 throughout the asthmatic airways. Taken together, these data suggest that not only is the developmental pathway re-activated during inflammatory airway disease but that basal BMP pathway expression is important for maintaining healthy airways. Overall these data highlight the presence and importance of BMP signalling gradients in healthy adult airways and further implicate BMP signalling in the pathogenesis of inflammatory and malignant airway diseases. [1] T.M Lynn, E.L Molloy, J.C Masterson, S.F. Glynn, R.W. Costello, M.V. Avdalovic, E.S Schelegle, L.A. Miller, D.M Hyde, S O’Dea. ‘SMAD signalling in descending airways of healthy versus asthmatic rhesus macaques highlights a relationship between inflammation and BMPs’. AJRCMB, In Press

The Role of Bone Morphogenetic Protein Signalling in Adult Lung Health and Disease

Bone morphogenetic protein (BMP) signalling is essential for correct lung morphogenesis. The pathway controls branching morphogenesis in the nascent lung and is also involved in the establishment of correct epithelial cell distribution throughout the airways. In the adult lung, BMP signalling is reactivated during airway injury and inflammation and there is evidence of aberrant signalling in lung cancer and in chronic lung diseases such as asthma and fibrosis. However, little is known about the role of BMP signalling in healthy adult airways. Furthermore the effect of incorrect BMP pathway expression during epithelial repair and inflammatory and malignant lung diseases remains elusive. The aims of this project were to characterise BMP pathway expression in the descending airways of large animal models and to investigate the role of BMP signalling during adult airway homeostasis, recovery and disease. To investigate the role of BMP signalling in malignant lung disease we used a heterogeneous lung cancer cell line DLKP. We explored the effect of the BMP pathway on epithelial-to-mesenchymal (EMT) progression and phenotypic plasticity between the tumour subpopulations. BMP-4 treatment induced mesenchymal-like projections in the DLKP-SQ clones and significant morphological changes in the DLKP-M clones towards the stem-cell like colonies of DLKP-I populations. Elevated N-cadherin and Vimentin protein expression was also evident in the clones following BMP-4 treatment. By stably transfecting an E-cadherin gene in these E-cadherin-null cells we demonstrated the distinct phenotypic differences between these tumour subpopulations. Healthy porcine and rhesus macaque lungs were used to characterise BMP pathway expression throughout the airways. Active BMP signalling was present in the descending airway epithelium and a gradient in pathway activation along the proximal-distal axis of the lungs was observed [1]. Using a primary porcine tracheal in vitro explant model the BMP signalling gradient was investigated further and it was found that by modulating BMP signalling, using exogenous BMP-4 stimulation, the epithelial phenotype of the porcine tracheal cells was altered. Finally, to assess the role of BMP signalling during airway inflammation and repair an established non-human primate model of allergic airway disease was used. A reduction in pSMAD1/5/8 expression was present in the epithelium of asthmatic monkeys compared to healthy controls. In addition, following a period of six months in filtered air to facilitate airway repair, there was a significant increase in Proliferating Cell Nuclear Antigen (PCNA), BMP Receptor 1a (BMPRIa) and pSMAD1/5/8 throughout the asthmatic airways. Taken together, these data suggest that not only is the developmental pathway re-activated during inflammatory airway disease but that basal BMP pathway expression is important for maintaining healthy airways. Overall these data highlight the presence and importance of BMP signalling gradients in healthy adult airways and further implicate BMP signalling in the pathogenesis of inflammatory and malignant airway diseases. [1] T.M Lynn, E.L Molloy, J.C Masterson, S.F. Glynn, R.W. Costello, M.V. Avdalovic, E.S Schelegle, L.A. Miller, D.M Hyde, S O’Dea. ‘SMAD signalling in descending airways of healthy versus asthmatic rhesus macaques highlights a relationship between inflammation and BMPs’. AJRCMB, In Press

The Role of Bone Morphogenetic Protein Signalling in Adult Lung Health and Disease

Bone morphogenetic protein (BMP) signalling is essential for correct lung morphogenesis. The pathway controls branching morphogenesis in the nascent lung and is also involved in the establishment of correct epithelial cell distribution throughout the airways. In the adult lung, BMP signalling is reactivated during airway injury and inflammation and there is evidence of aberrant signalling in lung cancer and in chronic lung diseases such as asthma and fibrosis. However, little is known about the role of BMP signalling in healthy adult airways. Furthermore the effect of incorrect BMP pathway expression during epithelial repair and inflammatory and malignant lung diseases remains elusive. The aims of this project were to characterise BMP pathway expression in the descending airways of large animal models and to investigate the role of BMP signalling during adult airway homeostasis, recovery and disease. To investigate the role of BMP signalling in malignant lung disease we used a heterogeneous lung cancer cell line DLKP. We explored the effect of the BMP pathway on epithelial-to-mesenchymal (EMT) progression and phenotypic plasticity between the tumour subpopulations. BMP-4 treatment induced mesenchymal-like projections in the DLKP-SQ clones and significant morphological changes in the DLKP-M clones towards the stem-cell like colonies of DLKP-I populations. Elevated N-cadherin and Vimentin protein expression was also evident in the clones following BMP-4 treatment. By stably transfecting an E-cadherin gene in these E-cadherin-null cells we demonstrated the distinct phenotypic differences between these tumour subpopulations. Healthy porcine and rhesus macaque lungs were used to characterise BMP pathway expression throughout the airways. Active BMP signalling was present in the descending airway epithelium and a gradient in pathway activation along the proximal-distal axis of the lungs was observed [1]. Using a primary porcine tracheal in vitro explant model the BMP signalling gradient was investigated further and it was found that by modulating BMP signalling, using exogenous BMP-4 stimulation, the epithelial phenotype of the porcine tracheal cells was altered. Finally, to assess the role of BMP signalling during airway inflammation and repair an established non-human primate model of allergic airway disease was used. A reduction in pSMAD1/5/8 expression was present in the epithelium of asthmatic monkeys compared to healthy controls. In addition, following a period of six months in filtered air to facilitate airway repair, there was a significant increase in Proliferating Cell Nuclear Antigen (PCNA), BMP Receptor 1a (BMPRIa) and pSMAD1/5/8 throughout the asthmatic airways. Taken together, these data suggest that not only is the developmental pathway re-activated during inflammatory airway disease but that basal BMP pathway expression is important for maintaining healthy airways. Overall these data highlight the presence and importance of BMP signalling gradients in healthy adult airways and further implicate BMP signalling in the pathogenesis of inflammatory and malignant airway diseases. [1] T.M Lynn, E.L Molloy, J.C Masterson, S.F. Glynn, R.W. Costello, M.V. Avdalovic, E.S Schelegle, L.A. Miller, D.M Hyde, S O’Dea. ‘SMAD signalling in descending airways of healthy versus asthmatic rhesus macaques highlights a relationship between inflammation and BMPs’. AJRCMB, In Press

SMAD Signaling in the Airways of Healthy Rhesus Macaques versus Rhesus Macaques with Asthma Highlights a Relationship Between Inflammation and Bone Morphogenetic Proteins

Bone morphogenetic protein (BMP) signaling is important for correct lung morphogenesis, and there is evidence of BMP signaling reactivation in lung diseases. However, little is known about BMP signaling patterns in healthy airway homeostasis and inflammatory airway disease and during epithelial repair. In this study, a rhesus macaque (Macaca mulatta) model of allergic airway disease was used to investigate BMP signaling throughout the airways in health, disease, and regeneration. Stereologic quantification of immunofluorescent images was used to determine the expression of BMP receptor (BMPR) Ia and phosphorylated SMAD (pSMAD) 1/5/8 in the airway epithelium. A pSMAD 1/5/8 expression gradient was found along the airways of healthy juvenile rhesus macaques (n = 3, P , 0.005). Membrane-localized BMPRIa expression was also present in the epithelium of the healthy animals. After exposure to house dust mite allergen and ozone, significant down-regulation of nuclear pSMAD 1/5/8 occurs in the epithelium. When the animals were provided with a recovery period in filtered air, proliferating cell nuclear antigen, pSMAD 1/5/8, and membrane-localized BMPRIa expression were significantly increased in the epithelium of conducting airways (P , 0.005). Furthermore, in the asthmatic airways, altered BMPRIa localization was evident. Because of the elevated eosinophil presence in these airways, we investigated the effect of eosinophil-derived proteins on BMPRIa trafficking in epithelial cells. Eosinophil-derived proteins (eosinophil-derived neurotoxin, eosinophil peroxidase, and major basic protein) induced transient nuclear translocation of membrane-bound BMPRIa. This work mapping SMAD signaling in the airways of nonhuman primates highlights a potential mechanistic relationship between inflammatory mediators and BMP signaling and provides evidence that basal expression of the BMP signaling pathway may be important for maintaining healthy airways