Hippo-Foxa2 signaling pathway plays a role in peripheral lung maturation and surfactant homeostasis

Abstract

Respiratory distress syndrome (RDS), which is induced by insufficient production of surfactant, is the leading cause of mortality in preterm babies. Although several transcription factors are known to be involved in surfactant protein expression, the molecular mechanisms and signaling pathways upstream of these transcription factors have remained elusive. Here, using mammalian Hippo kinases (Mst1/2, mammalian sterile 20-like kinase 1/2) conditional knockout mice, we demonstrate that Mst1/2 kinases are critical for orchestration of transcription factors involved in surfactant protein homeostasis and prevention of RDS. Mice lacking Mst1/2 in the respiratory epithelium exhibited perinatal mortality with respiratory failure and their lungs contained fewer type I pneumocytes and more immature type II pneumocytes lacking microvilli, lamellar bodies, and surfactant protein expression, pointing to peripheral lung immaturity and RDS. In contrast to previous findings of YAP (Yes-associated protein)-mediated canonical Hippo signaling in the liver and intestine, loss of Mst1/2 kinases induced the defects in pneumocyte differentiation independently of YAP hyperactivity. We instead found that Mst1/2 kinases stabilized and phosphorylated the transcription factor Foxa2 (forkhead box A2), which regulates pneumocyte maturation and surfactant protein expression. Taken together, our results suggest that the mammalian Hippo kinases play crucial roles in surfactant homeostasis and coordination of peripheral lung differentiation through regulation of Foxa2 rather than of YAP.non-canonical Hippo pathway | lung development P eripheral lung immaturity and deficiency of pulmonary surfactant cause many intractable pulmonary diseases. Deficit of surfactant because of preterm birth or genetic disorders of surfactant homeostasis induce respiratory distress syndrome (RDS) in the newborn period (1). Dysregulation of these genes also underlies the pathogenesis of many chronic lung diseases that have been considered idiopathic, such as interstitial lung disease, pulmonary alveolar proteinosis, and others (2). Elucidating the mechanism underlying regulation of surfactant would be very helpful for understanding the molecular basis of refractory lung diseases of both infants and adults.Alveoli are composed of type I and type II pneumocytes. The type II pneumocyte is the progenitor cell of the type I pneumocyte and plays a central role in physiological pulmonary functions, especially surfactant production (3). Type I pneumocytes contact alveolar capillaries and participate in gas exchange. Lung morphogenesis in mice is a dynamic process that can be divided into embryonic [embryonic day (E) 9-11.5], pseudoglandular (E11.5-16.5), canalicular (E16.5-17.5), saccular [E17.5 to postnatal (PN) day 5], and alveolar (PN5-28) stages. During canalicular and saccular stages in late gestation, peripheral lungs containing type I and II pneumocytes fully differentiate in preparation for postnatal respiration (4).Many transcription factors, including Foxa2 (forkhead...

A conserved MST1/2–YAP axis mediates Hippo signaling during lung growth

“…These mice died at various time points after birth; most of them perished before they reached 3 weeks of age although a few survived beyond that (Fig, 1I, J, K). These findings are largely similar to reports on Mst1 f/f ; Mst2 −/− mice that also carry Nkx2.1-Cre (Chung et al, 2013). In that study, a conditional allele of Mst1 ( Mst1 f ) (Katagiri et al, 2009) and a null allele of Mst2 ( Mst2 − ) (Oh et al, 2009) were utilized to assess the functional consequence of Mst1/2 removal in the lung.…”

“…Consistent with the expected difference in Cre activity, MST2 protein reduction is more complete and upregulation of YAP targets more pronounced in lungs in which Mst1/2 are removed by Shh-Cre than those induced by Nkx2.1-Cre in our investigation. Of note, a recent study (Chung et al, 2013) reported reduced YAP, CTGF and FOXA2 protein levels in lungs derived from Mst1 f/f ; Mst2 −/− mice that carry Nkx2.1-Cre . It is unclear how this discrepancy has arisen.…”

“…For instance, removal of Yap in the lung epithelium results in defective lung development (Mahoney et al, 2014), supporting an essential role of Hippo signaling in controlling lung growth. However, this issue is complicated by a recent report (Chung et al, 2013) in which the MST1/2 kinases were shown to regulate the transcription factor FOXA2 and not YAP during lung development. In this study, we employ multiple mouse Cre lines as well as lung cell lines to investigate the interactions between MST1/2 and YAP in controlling lung growth.…”

Hippo pathway in lung development

“…The lung development failure causing reduced airspaces in mutant lungs leaded to mice neonatal death and the mutant mice showed a phenotype mimicking RDS in preterm infants. One study demonstrated Mst1/2 regulated lung development through Foxa2 independently of Yap/Taz signaling (12). However, the other studies indicated that Hippo components Mst1/2 and Mob1 regulated lung development through Hippo effector Yap/Taz, which means a canonical Hippo-Yap signaling pathway was involved (13,15,16).…”

“…These genetic studies will complement cell-based assays in delineating the interactions of Hippo pathway components in the respiratory system. Interestingly, even though complete deletion of Sav1 in mice leads to embryonic lethality, lungspecific Sav1 knockout mice are viable and fertile without apparent gross abnormalities (12).…”