The secreted kinase VLK is a regulator of lung organogenesis

Abstract

Vertebrate Lonesome Kinase (VLK) is a secreted protein and responsible for phosphorylation of various secretory pathway-resident and extracellular proteins. Mice harboring a global knockout of the VLK gene (Pkdcc) die at birth, demonstrating an essential role of VLK during development. Furthermore, these mice exhibited a strongly hypoplastic lung, shorter bones and cleft palate. Their early postnatal lethality was most likely due to a lung defect and resulting respiratory problems. These results suggested that mesenchymal cells are the main drivers of the observed phenotype, but this hypothesis was not experimentally tested in previous studies. In this thesis project, we generated a mesenchyme-specific Pkdcc knockout using mice expressing Cre recombinase under control of the Col1a2 promoter, and we focused the analysis on the lung. In addition, a preliminary characterization of the skin was performed. Homozygous deletion of Pkdcc in mesenchymal cells caused growth retardation, craniofacial abnormalities, a severe lung phenotype and early postnatal lethality. Therefore, we mainly characterized the lungs of CTRL and Pkdcc knockout mice at embryonic day 18.5. Loss of VLK resulted in a reduction of alveolar type II cells, which secrete surfactant and thereby reduce tension within the lung upon inhalation of air. Furthermore, bronchial epithelial cells were less abundant, and the wall area of the bronchi was reduced. Finally, an overall delay in epithelial cell differentiation was suggested by the high expression of the progenitor marker SOX9, which is normally down-regulated at this stage of development. These findings suggest a role of mesenchyme-derived VLK in the differentiation of lung epithelial cells. On the histological level, the lungs of Pkdcc knockout embryos appeared hypoplastic and exhibited a higher tissue density, which was not due to higher cell proliferation or an increased number of cells. Mass spectrometry-based proteomics using lung tissue from CTRL and Pkdcc knockout embryos revealed a higher abundance of several matrix proteins in the Pkdcc knockout mice, which was not a consequence of higher mRNA levels. In particular, fibromodulin (FMOD) showed a 5-fold higher abundance in the knockout, and matrilin-4 (MATN4) levels were 3-fold higher. FMOD is a small-leucine-rich proteoglycan that is involved in collagen fibrillogenesis and MATN4 is an adaptor protein linking different ECM proteins. The higher abundance of these proteins suggests that loss of mesenchymal VLK causes alterations in matrix organization and structure, thereby regulating epithelial cell differentiation in a non-cellautonomous manner. Using in vitro studies, we showed that secretion of MATN4 is dependent on an active kinase domain of VLK in HEK 293T cells and to a lesser extent in Pkdcc-/- mouse embryonic fibroblasts, suggesting that secretion of MATN4 is phosphorylation-dependent. Due to the early postnatal lethality, we could only investigate the consequences of a heterozygous loss of Pkdcc in adult lung and skin tissue. In the lung, there was a very mild increase in tissue density, II but lung function was not obviously affected as revealed by a methacholine challenge test. We could not detect differences in normal or wounded skin at the macroscopic or histological level. These results suggest that a 50% reduction in the levels of VLK protein is compatible with normal development and homeostasis of lung and skin. To study the consequences of a complete loss of VLK in adult lung, skin and tendon tissue, we established mice that should allow a tamoxifen-inducible Pkdcc knockout using Col1a2-CreER mice. This strategy allowed an efficient Pkdcc knockout in the tendon, but not in the skin or lung. In tail tendon stress tests, we could not observe a difference in the stress modulus or in the diameter of tail tendon fascicles. In summary, this work revealed an essential role of VLK during murine lung development. The loss of VLK affects several types of lung epithelial cells and the general lung structure density, suggesting a non-cell autonomous function of mesenchyme-derived VLK in the control of lung epithelial cell differentiation. Furthermore, our data point to important functions of VLK in other tissues and thus provide an important basis for future studies.