Hippo-Foxa2 signaling pathway plays a role in peripheral lung maturation and surfactant homeostasis Chaeuk Chunga,b,c, Tackhoon Kimb, Miju Kimb, Minchul Kimb, Hoogeun Songb, Tae-Shin Kimb, Eunjeong Seob, Sang-Hee Leea, Hanbyul Kima,b, Sang Kyum Kima,b, Geon Yood, Da-Hye Leeb, Deog-Su Hwangd, Tatsuo Kinashie, Jin-Man Kimf, and Dae-Sik Limb,1 bDepartment of Biological Sciences, National Creative Research Initiatives Center, Biomedical Research Center, aGraduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea; cDepartment of Pulmonary and Critical Care Medicine, and fDepartment of Pathology, Chungnam National University School of Medicine, Daejeon 301-721, Korea; dDepartment of Biological Sciences, Seoul National University, Seoul 151-742, Korea; and eDepartment of Molecular Genetics, Institute of Biomedical Science, Kansai Medical University, Osaka 570-8506, Japan Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 1, 2013 (received for review November 27, 2012) Respiratory distress syndrome (RDS), which is induced by insufficient be involved in surfactant production and homeostasis during late production of surfactant, is the leading cause of mortality in preterm gestation (1, 4–7). Mice containing a mutation of TTF-1 or a de- babies. Although several transcription factors are known to be letion of Foxa2 or C/EBPα display immature type II pneumocytes involved in surfactant protein expression, the molecular mechanisms and lack type I pneumocytes without an alteration in branching and signaling pathways upstream of these transcription factors have morphogenesis, resulting in RDS (8, 9). Foxa2, TTF-1, and C/EBPα remained elusive. Here, using mammalian Hippo kinases (Mst1/2, reciprocally interact to regulate transcriptional targets of surfac- mammalian sterile 20-like kinase 1/2) conditional knockout mice, we tant synthesis and peripheral lung maturation (5). Some upstream demonstrate that Mst1/2 kinases are critical for orchestration of regulators, such as Nfatc3 (nuclear factor of activated T cells, transcription factors involved in surfactant protein homeostasis and cytoplasmic 3) and protein kinase A were identified (10, 11), but prevention of RDS. Mice lacking Mst1/2 in the respiratory epithelium so far the mechanism regulating peripheral lung maturation and exhibited perinatal mortality with respiratory failure and their lungs surfactant homeostasis are poorly understood and complex. contained fewer type I pneumocytes and more immature type II Mammalian sterile 20-like kinase 1 and 2 (Mst1/2) are upstream BIOLOGY pneumocytes lacking microvilli, lamellar bodies, and surfactant pro- kinases involved in the Hippo pathway, the unique signaling path- DEVELOPMENTAL tein expression, pointing to peripheral lung immaturity and RDS. In way involved in organ development and cancer (12). Interestingly, contrast to previous findings of YAP (Yes-associated protein)-mediated Mst1/2- and 45kD WW domain protein (WW45)-knockout mice canonical Hippo signaling in the liver and intestine, loss of Mst1/2 show a tendency to accumulate undifferentiated progenitor cells kinases induced the defects in pneumocyte differentiation in- (13, 14). For example, liver-specificablationofMst1/2 causes an dependently of YAP hyperactivity. We instead found that Mst1/2 increase in the number of hepatocytes and facultative progenitor kinases stabilized and phosphorylated the transcription factor cells (oval cells) (15). Intestine-specific ablation of Mst1/2 also Foxa2 (forkhead box A2), which regulates pneumocyte maturation results in intestinal progenitor cell proliferation and subsequent and surfactant protein expression. Taken together, our results colonic tumorigenesis (16, 17). The phenotypes of these organs suggest that the mammalian Hippo kinases play crucial roles in are attributed to hyperactivity of Yes-associated protein (YAP), surfactant homeostasis and coordination of peripheral lung differ- an oncogenic downstream effector molecule of the Hippo path- entiation through regulation of Foxa2 rather than of YAP. way. These studies suggest the interesting possibility that Yap- mediated Hippo signaling pathway, the so-called canonical Hippo non-canonical Hippo pathway | lung development pathway, works as critical inhibitor of epithelial progenitor cell proliferation in epithelial organs. Whether this paradigm is ap- eripheral lung immaturity and deficiency of pulmonary sur- plicable to other epithelial organs is not yet clear. Recent studies Pfactant cause many intractable pulmonary diseases. Deficit of have also demonstrated the role of Mst1/2 kinases in a YAP- surfactant because of preterm birth or genetic disorders of surfac- independent mechanism, the so-called noncanonical Hippo path- tant homeostasis induce respiratory distress syndrome (RDS) in the way. For example, Mst1 suppresses lymphoma development by newborn period (1). Dysregulation of these genes also underlies the promoting faithful chromosome segregation, independently of pathogenesis of many chronic lung diseases that have been con- YAP (18). Mst1/2 kinases also execute a variety of functions with sidered idiopathic, such as interstitial lung disease, pulmonary al- multiple partners, such as Foxo1, Foxo3, Akt, and Aurora B, in veolar proteinosis, and others (2). Elucidating the mechanism different biological contexts (19–22). underlying regulation of surfactant would be very helpful for un- We have now generated conditional double-knockout mice derstanding the molecular basis of refractory lung diseases of both (dKO) in which Mst1 and Mst2 were deleted in the developing lung infants and adults. epithelium. Our results revealed that Mst1/2 acts through modu- Alveoli are composed of type I and type II pneumocytes. The type lation of Foxa2 to play a critical role in the regulation of peripheral II pneumocyte is the progenitor cell of the type I pneumocyte and lung maturation and surfactant homeostasis with use of these mice. 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 Author contributions: C.C., T. Kim, Minchul Kim, H.S., T.-S.K., E.S., and D.-S.L. designed research; C.C., Miju Kim, H.S., T.-S.K., H.K., and D.-H.L. performed research; D.S.H., mice is a dynamic process that can be divided into embryonic T. Kinashi, and J.-M.K. contributed new reagents/analytic tools; C.C., T. Kim, Minchul [embryonic day (E) 9–11.5], pseudoglandular (E11.5–16.5), cana- Kim, E.S., S.-H.L., S.K.K., and J.-M.K. analyzed data; and C.C., T. Kim, G.Y., and D.-S.L. licular (E16.5–17.5), saccular [E17.5 to postnatal (PN) day 5], and wrote the paper. alveolar (PN5–28) stages. During canalicular and saccular stages in The authors declare no conflict of interest. late gestation, peripheral lungs containing type I and II pneumo- This article is a PNAS Direct Submission. cytes fully differentiate in preparation for postnatal respiration (4). Freely available online through the PNAS open access option. Many transcription factors, including Foxa2 (forkhead box A2), 1To whom correspondence should be addressed. E-mail: [email protected]. TTF-1 (thyroid transcription factor-1)/Nkx2.1 (NK2 homeobox 1), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and C/EBPα (CCAAT/enhancer binding protein-α), are known to 1073/pnas.1220603110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1220603110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 Results emphysematous lesions (Fig. 1C and Fig. S3A), suggesting the Lung-Specific Mst1/2-dKO Mice Succumb to RDS and Die in the Mst1/2-dKO mice died because of respiratory failure. Perinatal Period. Mst1/2-null mice lacking both Mst1 and Mst2 We then determined the onset of aberrant phenotype from E15.5 genes begin dying in utero at embryonic day 8.5 because of defects to E18.5. Until E17.5, there were no significant differences between in placental development and yolk sac/embryonic vascular pat- the lungs of Mst1/2-dKO mice and control mice (Fig. 1B). At that terning (21). To investigate the role of Mst1/2 in lung development, time, embryonic markers such as Sox9 (sex-determining region Y fl fl − − we crossed Mst1 / ;Mst2 / mice with Nkx2.1-Cre mice to generate box 9, a marker for distal lung progenitor cells) and Sox2 (marker fl fl − − the lung-specific Mst1/2-knockout mouse, Mst1 / ;Mst2 / ;Nkx2.1- for proximal lung progenitor cells) were also normally expressed in − − Cre, hereafter termed Mst1/2-dKO. Mst2 / mice did not show any Mst1/2 dKO lungs (23) (Fig.1D,andFig. S4 A and B). However, noticeable developmental or immunological defects (15). We alveoli of Mst1/2-dKO lungs at E18.5 and PN1 showed a re- confirmed the efficient deletion of Mst1 in the Mst2-null lung tis- markably reduced aerated space compacted with immature-looking sues (Fig. S1 A and B). LacZ staining showed that Nkx2.1 Cre was cuboidal cells (Fig. 1 B and C). At around PN5, Mst1/2-dKO lungs expressed in lung epithelial cells (Fig. S1C). Mst1/2-dKO mice were displayed spontaneous infiltration of inflammatory cells and hya- born at normal Mendelian ratio. Lung size and dry lung weight line membrane formation, which are characteristics typical of RDS were comparable between Mst1/2 dKO mice and control mice at in preterm infants (1), at which point the destruction of lung be- E15.5 and E18.5 (Fig. 1A and Fig. S2). However, Mst1/2-dKO mice came aggravated (Fig. 1C). No bacteria and fungi were detected in exhibited a markedly sick appearance at birth and almost all mice lung sections or in cytospins of bronchoalveolar lavage fluid, ruling died within the first 15 d after birth (Fig. S1D). At PN10, Mst1/ out the possibility of infection (Fig. 1C and Fig. S3B). 2-dKO lung showed severe accumulation of inflammatory cells We determined whether Mst1 and Mst2 affect cell proliferation in the alveolar space and bronchiole with multiple atelectasis and and apoptosis by performing BrdU labeling and TUNEL assays, fl fl − − Fig.