T1α, a lung type I cell differentiation gene, is required for normal lung cell proliferation and alveolus formation at birth

T1α, a differentiation gene of lung alveolar epithelial type I cells, is developmentally regulated and encodes an apical membrane protein of unknown function. Morphological differentiation of type I cells to form the air-blood barrier starts in the last few days of gestation and continues postnatally. Although T1α is expressed in the foregut endoderm before the lung buds, T1α mRNA and protein levels increase substantially in late fetuses when expression is restricted to alveolar type I cells. We generated T1α null mutant mice to study the role of T1α in lung development and differentiation and to gain insight into its potential function. Homozygous null mice die at birth of respiratory failure, and their lungs cannot be inflated to normal volumes. Distal lung morphology is altered. In the absence of T1α protein, type I cell differentiation is blocked, as indicated by smaller airspaces, many fewer attenuated type I cells, and reduced levels of aquaporin-5 mRNA and protein, a type I cell water channel. Abundant secreted surfactant in the narrowed airspaces, normal levels of surfactant protein mRNAs, and normal patterns and numbers of cells expressing surfactant protein-B suggest that differentiation of type II cells, also alveolar epithelial cells, is normal. Anomalous proliferation of the mesenchyme and epithelium at birth with unchanged numbers of apoptotic cells suggests that loss of T1α and/or abnormal morphogenesis of type I cells alter the proliferation rate of distal lung cells, probably by disruption of epithelial-mesenchymal signaling.     

Epithelial laminin alpha5 is necessary for distal epithelial cell maturation, VEGF production, and alveolization in the developing murine lung

Laminin alpha5 is prominent in the basement membrane of alveolar walls, airways, and pleura in developing and adult lung. Targeted deletion of laminin alpha5 in mice causes developmental defects in multiple organs, but embryonic lethality has precluded examination of the latter stages of lung development. To identify roles for laminin alpha5 in lung development, we have generated an inducible lung epithelial cell-specific Lama5 null (SP-CLama5(fl/-)) mouse through use of the Cre/loxP system, the human surfactant protein C promoter, and the reverse tetracycline transactivator. SP-CLama5(fl/-) embryos exposed to doxycycline from E6.5 died a few hours after birth. Compared to control littermates, SP-CLama5(fl/-) lungs had dilated, enlarged distal airspaces, but basement membrane ultrastructure was preserved. Distal epithelial cell differentiation was perturbed, with a marked reduction of alveolar type II cells and a virtual absence of type I cells. Cell proliferation was reduced and apoptosis was increased. Capillary density was diminished, and this was associated with a decrease in total lung VEGF production. Overall, these findings indicate that epithelial laminin alpha5, independent of its structural function, is necessary for murine lung development, and suggest a role for laminin alpha5 in signaling pathways that promote alveolar epithelial cell differentiation and VEGF expression.     

Type II epithelial cells are critical target for hyperoxia-mediated impairment of postnatal lung development

Type II epithelial cells are essential for lung development and remodeling, as they are precursors for type I cells and can produce vascular mitogens. Although type II cell proliferation takes place after hyperoxia, it is unclear why alveolar remodeling occurs normally in adults whereas it is permanently disrupted in newborns. Using a line of transgenic mice whose type II cells could be identified by their expression of enhanced green fluorescent protein and endogenous expression of surfactant proteins, we investigated the age-dependent effects of hyperoxia on type II cell proliferation and alveolar repair. In adult mice, type II cell proliferation was low during room air and hyperoxia exposure but increased during recovery in room air and then declined to control levels by day 7. Eight weeks later, type II cell number and alveolar compliance were indistinguishable from those in room air controls. In newborn mice, type II cell proliferation markedly increased between birth and postnatal day 7 before declining by postnatal day 14. Exposure to hyperoxia between postnatal days 1 and 4 inhibited type II cell proliferation, which resumed during recovery and was aberrantly elevated on postnatal day 14. Eight weeks later, recovered mice had 70% fewer type II cells and 30% increased lung compliance compared with control animals. Recovered mice also had higher levels of T1alpha, a protein expressed by type I cells, with minimal changes detected in genes expressed by vascular cells. These data suggest that perinatal hyperoxia adversely affects alveolar development by disrupting the proper timing of type II cell proliferation and differentiation into type I cells.     

Transgenic extracellular superoxide dismutase protects postnatal alveolar epithelial proliferation and development during hyperoxia

Transgenic (TG) human (h) extracellular superoxide dismutase (EC-SOD) targeted to type II cells protects postnatal newborn mouse lung development against hyperoxia by unknown mechanisms. Because alveolar development depends on timely proliferation of type II epithelium and differentiation to type I epithelium, we measured proliferation in bronchiolar and alveolar (surfactant protein C-positive) epithelium in air and 95% O2-exposed wild-type (WT) and TG hEC-SOD newborn mice at postnatal days 3, 5, and 7 (P3-P7), traversing the transition from saccular to alveolar stages. We found that TG hEC-SOD ameliorated the 95% O2-impaired bromodeoxyuridine uptake in alveolar and bronchiolar epithelium at P3, but not at P5 and P7, when overall epithelial proliferation rates were lower in air-exposed WT mice. Mouse EC-, CuZn-, and Mn-SOD expression were unaffected by hyperoxia or genotype. TG mice had less DNA damage than 95% O2-exposed WT mice at P3, measured by TdT-mediated dUTP nick end labeling (P < 0.05). Hyperoxia induced cell-cycle inhibitory protein p21cip/waf mRNA at P3, WT > TG, P = 0.06. 95% O2 impaired apical expression of type I cell alpha protein (T1alpha) in WT but not in TG mice at P3 and increased T1alpha in WT and TG mice at P7. Reducing the 95% O2-induced impairment of epithelial proliferation at a critical window of lung development was associated with protection against DNA damage and preservation of apical T1alpha expression at P3.     

Increased lung expansion alters lung growth but not alveolar epithelial cell differentiation in newborn lambs

Although increased lung expansion markedly alters lung growth and epithelial cell differentiation during fetal life, the effect of increasing lung expansion after birth is unknown. We hypothesized that increased basal lung expansion, caused by ventilating newborn lambs with a positive end-expiratory pressure (PEEP), would stimulate lung growth and alter alveolar epithelial cell (AEC) proportions and decrease surfactant protein mRNA levels. Two groups of lambs were sedated and ventilated with either 0 cmH(2)O PEEP (controls, n = 5) or 10 cmH(2)O PEEP (n = 5) for 48 h beginning at 15 +/- 1 days after normal term birth. A further group of nonventilated 2-wk-old lambs was used for comparison. We determined wet and dry lung weights, DNA and protein content, a labeling index for proliferating cells, surfactant protein mRNA expression, and proportions of AECs using electron microscopy. Although ventilating lambs for 48 h with 10 cmH(2)O PEEP did not affect total lung DNA or protein, it significantly increased the proportion of proliferating cells in the lung when compared with nonventilated 2-wk-old controls and lambs ventilated with 0 cmH(2)O PEEP (control: 2.6 +/- 0.5%; 0 PEEP: 1.9 +/- 0.3%; 10 PEEP: 3.5 +/- 0.3%). In contrast, no differences were observed in AEC proportions or surfactant protein mRNA levels between either of the ventilated groups. This study demonstrates that increases in end-expiratory lung volumes, induced by the application of PEEP, lead to increased lung growth in mechanically ventilated 2-wk-old lambs but do not alter the proportions of AECs.     

Distribution and surfactant association of carcinoembryonic cell adhesion molecule 6 in human lung

Carcinoembryonic cell adhesion molecule 6 (CEACAM6) is a glycosylated, glycophosphatidylinositol-anchored protein expressed in epithelial cells of various primate tissues. It binds gram-negative bacteria and is overexpressed in human cancers. CEACAM6 is associated with lamellar bodies of cultured type II cells of human fetal lung and protects surfactant function in vitro. In this study, we characterized CEACAM6 expression in vivo in human lung. CEACAM6 was present in lung lavage of premature infants at birth and increased progressively in intubated infants with lung disease. Of surfactant-associated CEACAM6, ～80% was the fully glycosylated, 90-kDa form that contains the glycophosphatidylinositol anchor, and the concentration (3.9% of phospholipid for adult lung) was comparable to that for surfactant proteins (SP)-A/B/C. We examined the affinity of CEACAM6 by purification of surfactant on density gradient centrifugation; concentrations of CEACAM6 and SP-B per phospholipid were unchanged, whereas levels of total protein and SP-A decreased by 60%. CEACAM6 mRNA content decreased progressively from upper trachea to peripheral fetal lung, whereas protein levels were similar in all regions of adult lung, suggesting proximal-to-distal developmental expression in lung epithelium. In adult lung, most type I cells and ～50% of type II cells were immunopositive. We conclude that CEACAM6 is expressed by alveolar and airway epithelial cells of human lung and is secreted into lung-lining fluid, where fully glycosylated protein binds to surfactant. Production appears to be upregulated during neonatal lung disease, perhaps related to roles of CEACAM6 in surfactant function, cell proliferation, and innate immune defense.     

GM-CSF mediates alveolar macrophage proliferation and type II cell hypertrophy in SP-D gene-targeted mice

Mice deficient in surfactant protein (SP) D develop increased surfactant pool sizes and dramatic changes in alveolar macrophages and type II cells. To test the hypothesis that granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates alveolar macrophage proliferation and activation and the type II cell hypertrophy seen in SP-D null mice, we bred SP-D and GM-CSF gene-targeted mice to obtain littermate double null, single null, and wild-type mice. Bronchoalveolar lavage levels of phospholipid, protein, SP-D, SP-A, and GM-CSF were measured from 1 to 4 mo. There was an approximately additive accumulation of phospholipid, total protein, and SP-A at each time point. Microscopy showed normal macrophage number and morphology in GM-CSF null mice, numerous giant foamy macrophages and hypertrophic type II cells in SP-D null mice, and large but not foamy macrophages and mostly normal type II cells in double null mice. These results suggest that the mechanisms underlying the alveolar surfactant accumulation in the SP-D-deficient and GM-CSF-deficient mice are different and that GM-CSF mediates some of the macrophage and type II cell changes seen in SP-D null mice.     

Integrins beta1, alpha6, and alpha3 contribute to mechanical strain-induced differentiation of fetal lung type II epithelial cells via distinct mechanisms

Mechanical forces regulate lung maturation in the fetus by promoting type II epithelial differentiation. However, the cell surface receptors that transduce these mechanical cues into cellular responses remain largely unknown. When distal lung type II epithelial cells isolated from embryonic day 19 rat fetuses were cultured on flexible plates coated with laminin, fibronectin, vitronectin, collagen, or elastin and exposed to a level of mechanical strain (5%) similar to that observed in utero, transmembrane signaling responses were induced under all conditions, as measured by ERK activation. However, mechanical stress maximally increased expression of the type II cell differentiation marker surfactant protein C when cells were cultured on laminin substrates. Strain-induced alveolar epithelial differentiation was inhibited by interfering with cell binding to laminin using soluble laminin peptides (IKVIV or YIGSR) or blocking antibodies against integrin beta1, alpha3, or alpha6. Additional studies were carried out with substrates coated directly with different nonactivating anti-integrin antibodies. Blocking integrin beta1 and alpha6 binding sites inhibited both cell adhesion and differentiation, whereas inhibition of alpha3 prevented differentiation without altering cell attachment. These data demonstrate that various integrins contribute to mechanical control of type II lung epithelial cell differentiation on laminin substrates. However, they may act via distinct mechanisms, including some that are independent of their cell anchoring role.     

Mesenchymal maintenance of distal epithelial cell phenotype during late fetal lung development

Classical tissue recombination experiments have reported that at early gestation both tracheal and distal lung epithelium have the plasticity to respond to mesenchymal signals. Herein we examined the role of epithelial-mesenchymal interactions in maintaining epithelial differentiation at late (E19-E21, term = 22 days) fetal gestation in the rat. Isolated distal lung epithelial cells were recombined with mesenchymal cells from lung, skin, and intestine, and the homotypic or heterotypic recombinant cell aggregates were cultured for up to 5 days. Recombining lung epithelial cells with mesenchyme from various sources induced a morphological pattern that was specific to the type of inducing mesenchyme. In situ analysis of surfactant protein (SP)-C, SP-B, and Clara cell secretory protein (CCSP) expression, as well as SP-C and CCSP promoter transactivation experiments, revealed that distal lung epithelium requires lung mesenchyme to maintain the alveolar, but not bronchiolar, phenotype. Incubation of lung recombinants with an anti-FGF7 antibody resulted in a partial inhibition of mesenchyme-induced SP-C promoter transactivation. Immunoreactivity for Delta and Lunatic fringe, components of the Notch pathway that regulates cell differentiation, was downregulated in the heterotypic recombinants. In contrast, Hes1 mRNA expression was increased in these recombinants. Cumulatively, these results suggest that at late fetal gestation, distal lung epithelial cells are not fully committed to a specific phenotype and still have the plasticity to respond to various signals. Their alveolar phenotype is likely maintained by Notch/Notch ligand interactions and mesenchymal factors, including FGF7.     

Laminin-311 (Laminin-6) fiber assembly by type I-like alveolar cells.

Two epithelial cell types cover the alveolar surface of the lung. Type II alveolar epithelial cells produce surfactant and, during development or following wounding, give rise to type I cells that are involved in gas exchange and alveolar fluid homeostasis. In culture, freshly isolated alveolar type II cells assume a more squamous (type I-like) appearance within 4 days after plating. They assemble numerous focal adhesions that associate with the actin cytoskeleton at the cell margins. These alveolar epithelial cells lose expression of type II cell markers including SP-C and after 4 days in culture express the type I cell marker T1alpha. Those cells that express T1alpha also deposit fibers of laminin-311 in their matrix. The latter appears to be related to their development of a type I phenotype because freshly isolated, primary type I cells also assemble laminin-311-rich fibers in vitro. A beta1 integrin antibody antagonist inhibits the assembly of laminin-311 matrix fibers. Moreover, the formation of laminin fibers is dependent on the activity of the small GTPases and is perturbed by ML-7, a myosin light chain kinase inhibitor. In summary, our data indicate that assembly of laminin-311 fibers by lung epithelial cells is integrin and actin cytoskeleton dependent, and that these fibers are characteristic of type I alveolar cells.     

Neuregulin-ErbB4 signaling in the developing lung alveolus: a brief review

Lung immaturity is the major cause of morbidity and mortality in premature infants, especially those born <28 weeks gestation. Proper lung development from 23–28 weeks requires coordinated cell proliferation and differentiation. Infants born at this age are at high risk for respiratory distress syndrome (RDS), a lung disease characterized by insufficient surfactant production due to immaturity of the alveoli and its constituent cells in the lung. The ErbB4 receptor and its stimulation by neuregulin (NRG) plays a critical role in surfactant synthesis by alveolar type II epithelial cells. In this review, we first provide an introduction to normal human alveolar development, followed by a discussion of the neuregulin and ErbB4-mediated mechanisms regulating alveolar development and surfactant production.     

GM-CSF mediates alveolar epithelial type II cell changes, but not emphysema-like pathology, in SP-D-deficient mice

Surfactant protein D (SP-D) is a member of the collectin subfamily of C-type lectins, pattern recognition proteins participating in the innate immune response. Gene-targeted mice deficient in SP-D develop abnormalities in surfactant homeostasis, hyperplasia of alveolar epithelial type II cells, and emphysema-like pathology. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is required for terminal differentiation and subsequent activation of alveolar macrophages, including the expression of matrix metalloproteinases and reactive oxygen species, factors thought to contribute to lung remodeling. Type II cells also express the GM-CSF receptor. Thus we hypothesized GM-CSF might mediate some or all of the cellular and structural abnormalities in the lungs of SP-D-deficient mice. To test this, SP-D (D-G+) and GM-CSF (D+G-) single knockout mice as well as double knockout mice deficient for both SP-D and GM-CSF (D-G-) were analyzed by design-based stereology. Compared with wild type, D-G+ as well as D+G- mice showed decreased alveolar numbers, increased alveolar sizes, and decreased alveolar epithelial surface areas. These emphysema-like changes were present to a greater extent in D-G- mice. D-G+ mice developed type II cell hyperplasia and hypertrophy with increased intracellular surfactant pools, whereas D+G- mice had smaller type II cells with decreased intracellular surfactant pools. In contrast to the emphysematous changes, the type II cell alterations were mostly corrected in D-G- mice. These results indicate that GM-CSF-dependent macrophage activity is not necessary for emphysema development in SP-D-deficient mice, but that type II cell metabolism and proliferation are, either directly or indirectly, regulated by GM-CSF in this model.     

Decreased expression of the type I isozyme of cAMP-dependent protein kinase in tumor cell lines of lung epithelial origin

A spontaneous transformant derived from a mouse lung epithelial cell line exhibited decreased cAMP-dependent protein kinase (PKA) activity. DEAE column chromatography demonstrated that this was caused by specific loss of the type I PKA isozyme (PKA I). Western immunoblot analysis indicated that indeed several mouse lung tumor-derived cell lines and spontaneous transformants of immortalized, nontumorigenic lung cell lines contained less PKA I regulatory subunit (RI) protein than normal cell lines. PKA II regulatory subunit protein differed only slightly among cell lines and showed no conspicuous trend between normal and neoplastic cells. The decrease in RI was apparently concomitant with decreased catalytic (C) subunit levels in neoplastic cells since no free catalytic subunit activity was detected by DEAE chromatography. Northern blot analysis using RI alpha and C alpha cDNA probes showed that the levels of RI alpha and C alpha mRNAs paralleled their intracellular protein concentrations; neoplastic cell lines contained significantly less RI alpha and C alpha mRNAs than the normal cell line. The decreased expression of both RI and C subunits therefore results in a net decrease of PKA I in neoplastic lung cells, an isozymic difference which may account for the differential effects of cAMP analogs on cell growth and differentiation in normal and neoplastic cells.     

Surfactant in respiratory distress syndrome and lung injury

A deficiency in alveolar surfactant due to immaturity of alveolar type II epithelial cells causes respiratory distress syndrome (RDS). In contrast to animals, the fetal maturation of surfactant in human lungs takes place before term, exceptionally large quantities of surfactant accumulating in the amniotic fluid. The antenatal development of surfactant secretion is very variable but corresponds closely to the risk of RDS. The variation in SP-A and SP-B genes, race, sex and perinatal complications influence susceptibility to RDS. Surfactant therapy has improved the prognosis of RDS remarkably. Abnormalities in alveolar or airway surfactant characterize many lung and airway diseases. In the acute respiratory distress syndrome, deficiencies in surfactant components (phospholipids, SP-B, SP-A) are evident, and may be caused by pro-inflammatory cytokines (IL-1, TNF) that decrease surfactant components. The resultant atelectasis localizes the disease, possibly allowing healing (regeneration, increase in surfactant). In the immature fetus, cytokines accelerate the differentiation of surfactant, preventing RDS. After birth, however, persistent inflammation is associated with low SP-A and chronic lung disease. A future challenge is to understand how to inhibit or redirect the inflammatory response from tissue destruction and poor growth towards normal lung development and regeneration.     

Expression profile of IGF system during lung injury and recovery in rats exposed to hyperoxia: a possible role of IGF-1 in alveolar epithelial cell proliferation and differentiation

Although several studies have shown that an induction of insulin-like growth factor (IGF) components occurs during hyperoxia-mediated lung injury, the role of these components in tissue repair is not well known. The present study aimed to elucidate the role of IGF system components in normal tissue remodeling. We used a rat model of lung injury and remodeling by exposing rats to > 95% oxygen for 48 h and allowing them to recover in room air for up to 7 days. The mRNA expression of IGF-I, IGF-II, and IGF-1 receptor (IGF-1R) increased during injury. However, the protein levels of these components remained elevated until day 3 of the recovery and were highly abundant in alveolar type II cells. Among IGF binding proteins (IGFBPs), IGFBP-5 mRNA expression increased during injury and at all the recovery time points. IGFBP-2 and -3 mRNA were also elevated during injury phase. In an in vitro model of cell differentiation, the expression of IGF-I and IGF-II increased during trans-differentiation of alveolar epithelial type II cells into type-I like cells. The addition of anti-IGF-1R and anti-IGF-I antibodies inhibited the cell proliferation and trans-differentiation to some extent, as evident by cell morphology and the expression of type I and type II cell markers. These findings demonstrate that the IGF signaling pathway plays a critical role in proliferation and differentiation of alveolar epithelium during tissue remodeling.