Summary Spontaneous pneumothoraces due to lung cyst rupture afflict patients with the rare disease Birt-Hogg-Dubé (BHD) syndrome caused by mutations of the tumor suppressor gene folliculin (FLCN) by unknown mechanism. BHD lungs exhibit increased alveolar epithelial cell apoptosis. We show that Flcn deletion in lung epithelium leads to cell apoptosis, alveolar enlargement and impaired lung function. FLCN loss also impairs alveolar epithelial barrier function. Flcn-null epithelial cell apoptosis is the result of impaired AMPK activation and increased cleaved caspase-3. AMPK activator LKB1 and E-cadherin are downregulated by Flcn loss and restored by its expression. Flcn-null cell survival is rescued by AICAR or constitutively active AMPK. AICAR also improves lung condition of Flcnf/f:SP-C-Cre mice. Our data show that Flcn regulates lung epithelial cell survival and alveolar size and suggest that lung cysts in BHD may result from an underlying defect in alveolar epithelial cell survival attributable to FLCN regulation of the E-cadherin-LKB1-AMPK axis.

The purpose of this study was to determine the expression and cellular functions of the epithelial NADPH oxidase DUOX1 during alveolar type II cell development. When human fetal lung cells (gestational age 11-22 wk) were cultured to confluency on permeable filters, exposure of cells to a hormone mixture (dexamethasone, 8-Br-cAMP, and IBMX, together referred to as DCI) resulted in differentiation of cells into a mature type II phenotype as assessed by expression of lamellar bodies, surfactant proteins, and transepithelial electrical parameters. After 6 days in culture in presence of DCI, transepithelial resistance (2,616 Ϯ 529 ⍀ ⅐ cm 2 ) and potential (Ϫ8.5 Ϯ 0.6 mV) indicated epithelial polarization. At the same time, treatment with DCI significantly increased the mRNA expression of DUOX1 (ϳ21-fold), its maturation factor DUOXA1 (ϳ12-fold), as well as DUOX protein (ϳ12-fold), which was localized near the apical cell pole in confluent cultures. For comparison, in fetal lung specimens, DUOX protein was not detectable at up to 27 wk of gestational age but was strongly upregulated after 32 wk. Function of DUOX1 was assessed by measuring H 2O2 and acid production. Rates of H2O2 production were increased by DCI treatment and blocked by small interfering RNA directed against DUOX1 or by diphenylene iodonium. DCI-treated cultures also showed increased intracellular acid production and acid release into the mucosal medium, and acid production was largely blocked by knockdown of DUOX1 mRNA. These data establish the regulated expression of DUOX1 during alveolar maturation, and indicate DUOX1 in alveolar H2O2 and acid secretion by differentiated type II cells.nicotinamide adenine dinucleotide phosphate oxidase; alveolar type II cells; hydrogen peroxide; intracellular pH THE NADPH OXIDASE IS A PROMINENT mechanism of innate defense best described in professional phagocytes where NADPH oxidase-derived superoxide (O 2•Ϫ ) in the phagosome is critically important for bacterial killing (42). A number of isoforms of the phagocytic NADPH oxidase have been identified in nonmyeloid cell types, resulting in the recognition of the NOX gene family consisting of five small (NOX1 to NOX5) and two large members (DUOX1 and DUOX2). All of these genes encode membrane proteins that share the structural characteristics of intracellular NADPH and FAD bindings sites and two membrane-embedded hemes that support electron transfer from intracellular NADPH to extracellular oxygen (5). NADPH oxidase activity further results in the production of intracellular H ϩ (from NADPH; Ref. 14). Two DUOX isoforms were initially identified and cloned from the thyroid (12, 15) where DUOX is expressed in the apical membrane and provides follicular H 2 O 2 for the oxidation of iodide to iodine during the synthesis of thyroid hormone (11). Apart from the thyroid, DUOX has been mainly found in epithelial tissues that are continuously exposed to microbes, where DUOX is considered to function as a component of the innate defense barrier. DUOX was found to be expressed i...

Yeast Atg1 initiates autophagy in response to nutrient limitation. The Ulk gene family encompasses the mammalian orthologs of yeast ATG1. We created mice deficient for both Ulk1 and Ulk2 and found that the mice die within 24 h of birth. When found alive, pups exhibited signs of respiratory distress. Histological sections of lungs of the Ulk1/2 DKO pups showed reduced airspaces with thickened septae. A similar defect was seen in Atg5-deficient pups as both Ulk1/2 DKO and Atg5 KO lungs show numerous glycogen-laden alveolar type II cells by electron microscopy, PAS staining, and increased levels of glycogen in lung homogenates. No abnormalities were noted in expression of genes encoding surfactant proteins but the ability to incorporate exogenous choline into phosphatidylcholine, the major phospholipid component of surfactant, was increased in comparison to controls. Despite this, there was a trend for total phospholipid levels in lung tissue to be lower in Ulk1/2 DKO and Atg5 KO compared with controls. Autophagy was abundant in lung epithelial cells from wild-type mice, but lacking in Atg5 KO and Ulk1/2 DKO mice at P1. Analysis of the autophagy signaling pathway showed the existence of a negative feedback loop between the ULK1 and 2 and MTORC1 and 2, in lung tissue. In the absence of autophagy, alveolar epithelial cells are unable to mobilize internal glycogen stores independently of surfactant maturation. Together, the data suggested that autophagy plays a vital role in lung structural maturation in support of perinatal adaptation to air breathing.

CXCL5, a member of the CXC family of chemokines, contributes to neutrophil recruitment during lung inflammation, but its regulation is poorly understood. Because the T cell-derived cytokine IL-17A enhances host defense by triggering production of chemokines, particularly in combination with TNF-α, we hypothesized that IL-17A would enhance TNF-α–induced expression of CXCL5. Intratracheal coadministration of IL-17A and TNF-α in mice induced production of CXCL1, CXCL2, and CXCL5, which was associated with increased neutrophil influx in the lung at 8 and 24 h. The synergistic effects of TNF-α and IL17A were greatly attenuated in Cxcl5−/− mice at 24 h, but not 8 h, after exposure, a time when CXCL5 expression was at its peak in wild-type mice. Bone marrow chimeras produced using Cxcl5−/− donors and recipients demonstrated that lung-resident cells were the source of CXCL5. Using differentiated alveolar epithelial type II (ATII) cells derived from human fetal lung, we found that IL-17A enhanced TNF-α–induced CXCL5 transcription and stabilized TNF-α–induced CXCL5 transcripts. Whereas expression of CXCL5 required activation of NF-κB, IL-17A did not increase TNF-α–induced NF-κB activation. Apical costimulation of IL-17A and TNF-α provoked apical secretion of CXCL5 by human ATII cells in a transwell system, whereas basolateral costimulation led to both apical and basolateral secretion of CXCL5. The observation that human ATII cells secrete CXCL5 in a polarized fashion may represent a mechanism to recruit neutrophils in host defense in a fashion that discriminates the site of initial injury.

Abstract. To investigate the mechanism of thyroid hormone action on pulmonary surfactant synthesis, we characterized the effect of triiodothyronine on phosphatidylcholine synthesis in cultured fetal rabbit lung. Since glucocorticoids stimulate surfactant synthesis and reduce the incidence of Respiratory Distress Syndrome in premature infants, we also examined the interaction of triiodothyronine and dexamethasone. no further stimulation and the triiodothyronine effect was partially reversed within 24 h. Half-maximal stimulation of choline incorporation occurred at a triiodothyronine concentration (0.10 nM) very similar to the dissociation constant for triiodothyronine binding to nuclear receptor (0.11 nM). The relative potencies of thyroid hormone analogs for nuclear binding and stimulation of phosphatidylcholine synthesis were also similar: triiodothyroacetic acid > triiodothyronine-proprionic acid > L-triiodothyronine D-triiodothyronine > thyroxine > 3,5-diethyl-3'-isopropyl-DL-thyronine 3,5-dimethyl-3'-isopropyl-L-thyronine -reverse triiodothyronine. The effect of triiodothyronine was blocked by the presence of either actinomycin D or cycloheximide, inhibitors of ribonucleic acid and protein synthesis, respectively.We conclude that triiodothyronine stimulates phosphatidylcholine synthesis by a process involving nuclear receptors and de novo ribonucleic acid and protein synthesis. These findings support the concept that endogenous triiodothyronine has a physiologic role in lung maturation and suggest that combined antenatal therapy with thyroid hormone and glucocorticoid may be useful for prevention of Respiratory Distress Syndrome in the premature infant.

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ABSTRACT. We studied P-adrenergic receptors and responses in human fetal lung (15-25 wk gestation) maintained in explant culture with and without added dexamethasone. To determine P-adrenergic receptor concentration, we performed radioligand binding assays with [lZ5I]-iodocyanopindolol. We also examined the ability of isoproterenol to stimulate cAMP generation as a measure of response to P-adrenergic receptor occupancy. In control cultures, P-receptor concentration increased significantly from d 0 to 3 of culture and thereafter remained stable. The kd (-24 pM) of [1251]-iodocyanopindolol did not change with time in culture. The ability of isoproterenol to stimulate cAMP generation over basal levels increased in controls throughout the 5 d in explant culture. Addition of dexamethasone (10 nM) to the culture medium partially blocked the increase in ,f3-receptor concentration and decreased both cAMP content and generation (basal and stimulated) in a dose-dependent manner (median effective concentration -1 nM). In these same explants, dexamethasone increased the activity of fatty acid synthetase, an enzyme important in surfactant synthesis, more than 2-fold. Our results indicate that P-adrenergic receptors and isoproterenol stimulation of cAMP generation increase spontaneously in human fetal lung grown in explant culture. Dexamethasone, which accelerates other aspects of human lung development in vitro, decreases P-adrenergic receptor concentration and inhibits P-adrenergic responses. (Pediatr Res 28: 190-195,1990) Abbreviations ICYP, [1251]-iodocyanopindololThere is increasing clinical and experimental evidence that /Iadrenergic responses play a key role in the adaptation of the fetal lung to extrauterine life (I). In vivo and in vitro, P-adrenergic agonists and cAMP analogues stimulate surfactant release and the synthesis of surfactant apoprotein and lipid. P-adrenergic receptors have been identified in human fetal lung during early gestation (2, 3) and the receptor concentration has been shown to increase between 15 and 23 wk gestation (3). In addition, lung P-adrenergic receptors are functionally linked to adenylate cy- Although the effects of explant culture and glucocorticoids on the P-adrenergic response cascade in human fetal lung have not been previously studied, several groups have demonstrated enhancement of morphologic maturation and increased surfactant synthesis in fetal lung of different species in vivo and in explant culture after glucocorticoid treatment (4). Maternal dexamethasone treatment increases the concentration of pulmonary Padrenergic receptors in fetal rabbit (5, 6). In explant cultures of fetal rabbit (7-9) and rat (10) lung and in a cell line derived from adult human lung (1 I), glucocorticoids increase the concentration of P-adrenergic receptors. Additionally, maternal glucocorticoid treatment increased high affinity agonist P-receptor complexes in fetal rabbit lung (7), a functional alteration found previously in some adult tissues (12). In this study, we measured the concentration...