Abstract
Background: The cGMP-dependent type 2 protein kinase, encoded by the prkg2 gene, is highly expressed in alveolar type 2 epithelial (AT2) cells. It is unclear whether prkg2 regulates AT2 cell homeostasis and re-alveolarization of injured lungs. This study aimed to investigate the role of prkg2 in the regulation of the fate of AT2 in vitro. Methods: Primary AT2 cells of wild-type (wt) and prkg2-/- mice were co-cultured with fibroblasts as three-dimensional organoids. The colony formation was analyzed between days 4 and 12 post-seeding. EdU assay was used to detect cells with active DNA synthesis. AT1 and AT2 cells in organoids were visualized with anti-podoplanin and anti-surfactant protein C antibodies, respectively. Results: Prkg2-/- AT2 cells developed a greater number of organoids than wt controls. However, compared to wt organoids, a lower number of AT2 but a greater number of AT1 cells were visualized. In addition, a lower number of proliferated cells (EdU+) were observed in prkg2-/- organoids compared to wt controls. The numbers of organoids and EdU+ cells were significantly reduced in protein kinase A (PKA) inhibitor H89-treated wt and prkg2-/- cultures. Organoids and EdU+ cells were increased by lipopolysaccharides (LPS) in both wt and prkg2-/- groups. The increase in the proportion of AT1 and AT2 cells in organoids was only seen in wt controls. Conclusions: Prkg2 may regulate the lineage of AT2 cells, which is affected by endotoxins and the interactive PKA signaling pathway.