FGF10 signaling in distal/alveolar epithelial progenitor cells - role in lung fibrosis
Final Report Abstract
Our research proposal can be divided into two major parts: one focusing on the role of Fgf10/Fgfr2b signaling during embryonic development and one dealing with the role of Fgf10/Fgfr2b signaling in lung fibrosis. We have initially validated our experimental approach to carry gain and loss of function of Fgf10/Fgfr2b signaling both during embryonic development and in the adult mouse. Using a mass spectrometry-based proteomics approach, we demonstrated how Fgf7 and Fgf10 lead to proliferation vs. migration, respectively, of epithelial cells. We showed that phosphorylation of a particular tyrosine residue of Fgfr2b controls the trafficking route of the receptor after internalization, allowing receptor recycling at the cell surface and sustained Akt and Shc phosphorylation. In addition to these results, we also identified specific phosphorylation sites for Adenomatous polyposis coli (Apc), a cytoplasmic protein which is part of the b-catenin degradation complex. It is clear from our limb data that one of the early consequence of decreasing Fgfr2b ligand signaling is the irreversible loss of b-catenin signaling in the epithelium. We are now validating that this is indeed also the case in the lung. To achieve this, we first determined the activation domains of Wnt signaling using three different Wnt reporter lines. We are also investigating the role of Apc in this context. We also further reported the role microRNA142 in controlling directly Apc expression in the lung mesenchyme, therefore controlling the amplification of mesenchymal progenitor cells. Using quantitative proteome analysis of adult alveolar type II cells (these cells are cellular targets for Fgf10 in the adult lung), we established an unsuspected link between integrin receptor subunits beta 2/6 and Wnt signaling. We finally reported that the transcription factor Eya1 controls cell polarity, spindle orientation, cell fate and Notch signaling in distal embryonic lung epithelium. These activities are also found downstream of Fgf10 in the epithelium. However, the relationship between Fgf10 and Eya1 is still under investigation. The second part of the project dealt with the role of Fgf10/Fgfr2b signaling in lung fibrosis using inflammatory-driven (bleomycin) and non-inflammatory-driven (Hermansky-Pudlak gene ko and amiodarone) animal models. We first demonstrated that Hsp1/2 double mutants developed fibrosis with subpleural onset at 3 months and full blown fibrosis at 9 months. The validation of the amiodarone model in mice is currently being carried out. We also evaluated the expression of Fgfr2b ligands in human lung specimen from IPF patients and organ donor (control). Our results suggest that an abundance of mesenchymal cells in end-stage IPF lungs leads to a shift towards mesenchymal dominant FGF signaling possibly sustained by a global lack of MAPK inhibitors (SPRY2, SPRY4). We also found that the Fgf10/Fgfr1b signaling axis may be recruited during repair after bleomycin-induced lung injury in wild type mice. Surprisingly, neither congenital deficiency in Fgf7, Fgf10 or Fgfr2b, nor attenuation of Fgfr2b ligands signaling during injury led to an overall worsening in fibrotic response or a difference in the rate of spontaneous repair in bleomycin-injured mice.
Publications
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(2010) Epithelial stress and apoptosis underlie Hermanky-Pudlak syndrome associated interstitial pneumonia. AJRCCM 182, 207-219
Mahavadi, P., Korfei, M., Henneke, I., Liebisch, G., Schmitz, G., Gochuico, B.R., Markart, P., Bellusci, S., Seeger, W., Ruppert, C., Guenther, A.
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(2010) Signaling by FGFR2b controls the regenerative capacity of adult mouse incisors. Development, 137 (22) 3743-52
Parsa, S., Kuremoto, K., Seidel, K., Tabatabai, R., MacKenzie, B., Yamaza, T., Akiyama, K., Branch, J., Koh, C.J., Al Alam, D., Klein, O.D., and Bellusci, S.
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(2011) Contrasting expression of canonical Wnt signaling reporters TOPGAL, BATGAL and Axin2LacZ during murine lung development and repair. PLoS One, 6(8): e23139
Al Alam, D., Green, M., Tabatabai, R., Parsa, S., Danopoulos, S., Sala, F.G., Branch, J., El Agha, E., Tiozzo, C., Voswinckel, R., Jesudason, E.C., Warburton, D. and Bellusci, S.
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(2011) Eya1 controls cell polarity, spindle orientation, cell fate and Notch signaling in distal embryonic lung epithelium. Development, 138 (7) 1395-407
El-Hashash, A.H., Turcatel, G., Al Alam, D., Buckley, S., Tokumitsu, H., Bellusci, S. and Warburton, D.
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(2012) Characterization of a novel Fibroblast growth factor 10 (Fgf10) knock-in mouse line to target mesenchymal progenitors during embryonic development. PLoS One. 7(6):e38452
El Agha, E., Al Alam, D., Carraro, G., MacKenzie, B., Goth, K., Langhe, S.P., Voswinckel, R., Hajihosseini, M.K., Rehan, V.K. and Bellusci, S.
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(2012) Parabronchial smooth muscle constitutes an airway epithelial stem cell niche in the mouse lung after injury. Journal of Clinical Investigation
Volckaert, T., Dill, E., Campbell, A., Tiozzo, C., Majka, S., Bellusci , S, and De Langhe, SP
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(2012) Transient Inhibition of FGFR2b Signaling Leads to Irreversible Loss of Cellular ß-catenin Organization and Signaling in AER During Mouse Limb Development. PLoS One, 8(10):e76248
Soula Danopoulos, Sara Parsa, Denise Al Alam, Reza Tabatabai, Sheryl Baptista, Caterina Tiozzo, Gianni Carraro, Matthew Wheeler, Guillermo Barreto, Thomas Braun, Mohammad K. Hajihosseini and Saverio Bellusci
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(2013) Functional proteomics defines the molecular switch underlying FGF receptor trafficking and cellular outputs. Mol Cell. 51(6):707-22
Francavilla C, Rigbolt KT, Emdal KB, Carraro G, Vernet E, Bekker-Jensen DB, Streicher W, Wikström M, Sundström M, Bellusci S, Cavallaro U, Blagoev B, Olsen JV
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(2014) Fgf10-positive cells represent a multipotent progenitor cell population during lung development and postnatally. Development 141(2):296-306
El Agha, E., Herold, S. Al Alam, D., Quantius, J., MacKenzie, B., Carraro, C., Minoo, P., Seeger, W., and Bellusci, S.
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(2014) miR142-3p balances proliferation and differentiation of lung mesenchymal cells. Development 141(6):1272-81
Carraro G., Shrestha, A., Rostkovious, J., Contreras, A., Chao, CM, El Agha, E., MacKenzie, B., Dilai, S., Guidolin, D., Taketo MM, Kumar, M., Seeger, W., Barreto, G and Bellusci, S.
