Dissection of signaling networks regulating stem cell maintenance and asymmetric cell division in developing and adult lung epithelia
Final Report Abstract
The primary objective of the project was a better understanding of the molecular signaling network that determines the balance between self-renewal and differentiation of progenitor cells of endodermally derived lung epithelia. We wanted to clarify the specific roles of individual components of the notch-signaling pathway for symmetric and asymmetric cell divisions and cellular differentiation and for the generation of stem cells of the lung epithelium, which constitutes a major part of the lung parenchyma. To this end we analyzed the role of Numb and Numblike, which were assumed to act as critical factors for asymmetric cell division, for lung development and lung diseases. We generated SPCrtTApos/tetO-Cre-pos/Numb-lox-/-/Numblike-/- mouse mutants, which were found to be viable despite a complete loss of Numb and Numblike in the distal lung epithelium. Conditional deletion of Numb-lox-/-/Numblike-/- in the distal lung epithelium under basal conditions leads to morphological changes of epithelial cells accompanied by a basal-to-apicolateral translocation of E-Cadherin suggesting a potential function of Numb/Numblike in the lung epithelium polarity and integrity. Interestingly, adult double homozygous mutants show a significant decrease of about 30% of SPC+ Alveolar Type Il cells (ATII) compared to littermate control animals. After injury of the respiratory epithelium with bleomycin, Numb/Numblike deficient mice were protected against lung fibrosis. The molecular processes that regulate these changes are under investigation. To gain insights in the molecular processes controlled by Numb and Numblike we identified novel interaction partners by SILAC (Stable isotope labeling in cells). To monitor the fate of Bronchioalveolar stem cells (BASCs), which were proposed to represent an organ resident stem cell population of the distal lung, we developed a novel cell tracing system allowing genetic tagging of cells that are defined by the co-expression of two different marker genes. The technology is built on a new split-system based on (I) separate, CCSP and SPC driven expression of inactive N- and C-terminal effector fragments and (II) intein-mediated reconstitution of functional effector proteins. In vitro, split-iCre and split-tTA elements reconstitute with high efficiency and show substantial re-activation of recombination or trans-activation activity. Subsequently, these elements were targeted to the endogenous SPC (N-term.) or CCSP locus (C-term.) to establish the BASC split-iCre/split-tTA mouse models. All knock-in strains show specific expression in the appropriate cell type. Finally, by crossing our BASC mouse models to corresponding reporter strains (Rosa26stopflox lacZ tetObi-lacZ/huGFP) we were able to trace the BASC population, demonstrating that the splitsystem permits reconstitution of functional effector protein in CCSP+/SPC+ cells in vivo. So far our preliminary results suggest that BASCs do not contribute to the maintenance and repair of lung epithelia under physiological conditions and after mild injury. We are currently analyzing BASC tracer mice subjected to more severe injury (elastase instillation, lung lobectomy).
Publications
- (2010). Identification of right heart-enriched genes in a murine model of chronic outflow tract obstruction. J. Mol. Cell. Cardiol. 49, 598-605
grosse Kreymborg, K. Uchida, S., Gellert, P., Schneider, A., Boettger, T., Voswinckel, R., Wietelmann, A., Szibor, M., Weissmann, N., Ghofrani, A. H., Schermuly, R., Schranz, D., Seeger, W,. Braun, T.
- (2011). FGF-2 dependent induction of smooth muscle cell migration during arteriogenesis is mediated by Rapt. Art. Thromb. Vasc. Biol. 31, 2297-2305
Pöling, J., Szibor, M., Rees, W., Gajawada, P., Khochfar, Z., Lörchner, H., Kubin, T., Warnecke, H., Braun, T.
- (2012). Inhibition of Notch2 by Numb/Numblike controls myocardial compaction in the heart. Cardiovasc. Res. 96, 276-285
Yang, J.-W. Bücker, S. Jungblut, B., Böttger, T., Cinnamon, Y., Tchorz, J., Müller, M., Bettler, B., Harvey, R., Sun, Q.-Y., Schneider, A., Braun, T.
(See online at https://doi.org/10.1093/cvr/cvs250) - Phosphodiesterase-4 promotes proliferation and angiogenesis of lung cancer by crosstalk with HIF. Oncogene. 2012 Apr 23
Pullamsetti, S. S., Banat, G. A., Schmall, A., Szibor, M., Pomagruk, D., Hänze, J., Kolosionek, E., Wilhelm, J., Braun, T., Grimminger, F., Seeger, W., Schermuly, R. T., Savai, R.
(See online at https://dx.doi.org/10.1038/onc.2012.136) - (2013). Overexpression of Twinkle-helicase protects cardiomyocytes from genotoxic stress caused by reactive oxygen species. Proc. Natl. Acad. Sci. 110, 19408-19413
Pohjoismäki, J.L.O., Williams, S.L., Boettger, T., Goffart, S., Kim, J., Suomalainen, A., Moraes, C.T., Braun, T.
(See online at https://doi.org/10.1073/pnas.1303046110) - (2013). Quantitative Proteome Analysis of Alveolar Type-II Cells Reveals a Connection of lntegrin Receptor Subunits Beta 2/6 and WNT Signaling. J Proteome Res. 12, 5598-55608
Mukhametshina, R. T., Ruhs, A., Singh, I., Hasan, D., Contreras, A., Mehta, A., Nikam, V. S., Ahlbrecht, K., Carraro, G., Cabrera-Fuentes, H. A., Jiang, D., Voswinckel, R., Seeger, W., Bellusci, S., Scharffetter-Kochanek, K., Bagaeva, T. V., Preissner, K. T., Boettger, T., Braun, T., Krüger, M., Barreto, G.
(See online at https://doi.org/10.1021/pr400573k) - (2013). Transient Inhibition of FGFR2b-Ligands Signaling Leads to Irreversible Loss of Cellular 13-Catenin Organization and Signaling in AER during Mouse Limb Development. PLoS One. e76248
Danopoulos, S., Parsa, S., Al Alam, D., Tabatabai, R., Baptista, S., Tiozzo, C., Carraro, G., Wheeler, M., Barreto, G., Braun, T., Li, X., Hajihosseini, M. K., Bellusci, S.
(See online at https://doi.org/10.1371/journal.pone.0076248) - Hmga2 is required for canonical WNT signaling during lung development. BMC Biol. 2014 Mar 24;12(1):21
Singh, I., Metha, A., Contreras, A., Carraro, G., Wheeler, M., Cabrera-Fuentes, H. A., Bellusci, S., Seeger, W., Braun, T., and Barreto, G.
(See online at https://doi.org/10.1186/1741-7007-12-21)
