The role of Trop2 in fetal lung development

Abstract

Adequate fetal lung growth in utero is vital for survival at birth. If infants are born without adequately developed lungs due to either preterm birth, which occurs before the lungs have had sufficient time to develop, or to abnormal lung development, they can suffer respiratory failure. A thorough understanding of the mechanisms involved in lung development is therefore required to better prevent and treat these disorders. During fetal life, many factors coordinate to promote normal lung growth and development, including local factors, circulating factors and mechanical factors. One of the major factors regulating fetal lung growth and development during late gestation, is the degree to which the developing lungs are expanded by lung liquid. Although many studies have demonstrated the importance of lung expansion for fetal lung growth the underlying mechanisms have remained elusive despite extensive research. However, the effect of lung expansion is likely to be mediated by alterations in the expression of genes encoding local factors that regulate lung growth and development. Trop2 was identified in our laboratory as a gene that had increased expression following an increase in lung expansion and was considered a potential candidate for mediating the growth response induced by increased lung expansion. The global aim of this thesis has been to investigate the role of the gene Trop2 and the protein that it encodes, TROP2, in regulating fetal lung cell proliferation and migration. TROP2 is a trans-membrane protein and known oncogene that is highly up-regulated in the majority of human cancers and has been linked to increased metastasis and decreased survival. As organogenesis and tumourigenesis are thought to share common regulatory pathways we hypothesised that Trop2 would also regulate cell proliferation and migration during fetal lung development. The data in Chapter 3 demonstrated that Trop2 gene expression was correlated to cell proliferation in the fetal sheep lung during normal development and following altered lung growth induced by alterations in lung expansion and circulating glucocorticoid levels. The TROP2 protein was localized to those cell types which proliferate in response to increase lung expansion; fibroblasts, type II alveolar epithelial cells (AECs) and endothelial cells. It was also present in airway epithelial cells and smooth muscle cells. This is the first study to show that fibroblasts, myofibroblasts and smooth muscle cells express Trop2. Most importantly, TROP2 was co-localised to proliferating fetal lung cells. The data presented in Chapter 3 therefore provided a significant amount of circumstantial evidence that a relationship exists between Trop2 expression and lung cell proliferation and thus supported our hypothesis that Trop2 may be an important regulator of cell proliferation in the developing lungs. In Chapter 4, I was able to successfully establish primary cultures of distal lung epithelial cells and fibroblasts from fetal rats. The isolated epithelial cells were labeled positive for surfactant protein C, which is indicative of the type-II AEC phenotype, but markers of type-I AECs were also detected by real-time PCR, suggesting that the epithelial cells may be adopting an intermediate cell phenotype in culture. The isolated fetal lung fibroblasts differentiated into a myofibroblast-like phenotype in culture which is common in cultured fibroblasts from many organs. Both distal lung epithelial and fibroblast cell cultures maintained the ability to proliferate and to express Trop2. In addition, the proportion of cells containing the TROP2 protein correlated to the proportion of epithelial cells and fibroblasts that were proliferating in culture, supporting the hypothesis that TROP2 regulates fetal lung cell proliferation. The primary cultures systems developed in Chapter 4 allowed the further investigation of the role of TROP2 in regulating lung cell proliferation and migration. In Chapter 5, Trop2 was successfully knocked-down in the cultured fetal lung myofibroblast-like cells using siRNA technology. The knock-down of Trop2 in those cells resulted in decreased cell proliferation, decreased cell migration and altered cell morphology. When combined with the in vivo correlations from Chapter 3, this data suggests that Trop2 is likely to regulate lung growth and development at least in part by regulating fetal lung fibroblast/myofibroblast proliferation and migration. The reduction in cell proliferation and migration following knock-down of Trop2 in myofibroblast-like cells is likely due to the alterations in cytoskeletal filaments and adhesion proteins. Cell migration involves various steps, such as extension of the lamellipodia, adhesion of the protruding lamellipodia, then contraction of actin and disassembly of cell adhesions at the rear of the cell, causing translocation of the cell body towards the lamellipodia. This process involves the coordination of many fibres and proteins, including actin, vimentin, cortactin and vinculin. In Chapter 5 the knock-down of Trop2 in myofibroblast-like cells altered the distribution of vimentin, cortactin, actin and vinculin suggesting that TROP2 is a master regulator of the cell migration and proliferation machinery in fetal lung myofibroblasts. This study suggests that TROP2 may regulate the migration of fetal lung myofibroblasts along the developing alveolar septa. The data presented here makes Trop2 an attractive candidate for coordinating fetal lung growth and maturation of alveolar septa. In conclusion, the studies presented in this thesis increase our understanding of the likely role of the Trop2 gene in fetal lung development. Trop2 regulates the proliferation, the adhesion and migration apparatus and migration ability, of primary fetal lung myofibroblast-like cells in culture. This suggests that Trop2 is likely to be regulating fetal lung growth in vivo and may regulate the migration of fetal lung myofibroblasts along the developing alveolar septa. Understanding the molecular pathways that underlie fetal lung growth and development will ultimately enable the development of better treatments and preventative strategies for respiratory disorders at birth.