Anne Hinds

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

T1alpha, 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 T1alpha is expressed in the foregut endoderm before the lung buds, T1alpha mRNA and protein levels increase substantially in late fetuses when expression is restricted to alveolar type I cells. We generated T1alpha null mutant mice to study the role of T1alpha 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 T1alpha 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 T1alpha and/or abnormal morphogenesis of type I cells alter the proliferation rate of distal lung cells, probably by disruption of epithelial-mesenchymal signaling.

Neuroepithelial body microenvironment is a niche for a distinct subset of Clara-like precursors in the developing airways

Clara cells of mammalian airways have multiple functions and are morphologically heterogeneous. Although Notch signaling is essential for the development of these cells, it is unclear how Notch influences Clara cell specification and if diversity is established among Clara cell precursors. Here we identify expression of the secretoglobin Scgb3a2 and Notch activation as early events in a program of secretory cell fate determination in developing murine airways. We show that Scgb3a2 expression in vivo is Notch-dependent at early stages and ectopically induced by constitutive Notch1 activation, and also that in vitro Notch signaling together with the pan-airway transcription factor Ttf1 (Nkx2.1) synergistically regulate secretoglobin gene transcription. Furthermore, we identified a subpopulation of secretory precursors juxtaposed to presumptive neuroepithelial bodies (NEBs), distinguished by their strong Scgb3a2 and uroplakin 3a (Upk3a) signals and reduced Ccsp (Scgb1a1) expression. Genetic ablation of Ascl1 prevented NEB formation and selectively interfered with the formation of this subpopulation of cells. Lineage labeling of Upk3a-expressing cells during development showed that these cells remain largely uncommitted during embryonic development and contribute to Clara and ciliated cells in the adult lung. Together, our findings suggest a role for Notch in the induction of a Clara cell-specific program of gene expression, and reveals that the NEB microenvironment in the developing airways is a niche for a distinct subset of Clara-like precursors.

The α-Isoform of Caveolin-1 Is a Marker of Vasculogenesis in Early Lung Development

Caveolin-1 is a scaffolding protein component of caveolae, membrane invaginations involved in endocytosis, signal transduction, trans- and intracellular trafficking, and protein sorting. In adult lung, caveolae and caveolin-1 are present in alveolar endothelium and Type I epithelial cells but rarely in Type II cells. We have analyzed patterns of caveolin-1 expression during mouse lung development. Two caveolin-1 mRNAs, full-length and a 5′ variant that will translate mainly into caveolin-1 α and -β isoforms, are detected by RT-PCR at embryonic day 12 (E12) and afterwards in the developing and adult lung. Immunostaining analysis, starting at E10, shows caveolin-1α localized in primitive blood vessels of the forming lung, in an overlapping pattern to the endothelial marker PECAM-1, and later in all blood vessels. Caveolin-1 α is not detected in fetal or neonatal lung epithelium but is detected in adult epithelial Type I cells. Caveolin-1 was previously shown to be expressed in alveolar Type I cells. These data suggest that expression of caveolin-1 isoforms is differentially regulated in endothelial and epithelial cells during lung development. Caveolin-1α is an early marker for lung vasculogenesis, primarily expressed in developing blood vessels. When the lung is fully differentiated postnatally, caveolin-1 α is also expressed in alveolar Type I cells.

Alterations in gene expression in T1α null lung: a model of deficient alveolar sac development

BackgroundDevelopment of lung alveolar sacs of normal structure and size at late gestation is necessary for the gas exchange process that sustains respiration at birth. Mice lacking the lung differentiation gene T1α [T1α(-/-)] fail to form expanded alveolar sacs, resulting in respiratory failure at birth. Since little is known about the molecular pathways driving alveolar sacculation, we used expression microarrays to identify genes altered in the abnormal lungs and, by inference, may play roles in normal lung morphogenesis.ResultsAltered expression of genes related to cell-cell interaction, such as ephrinA3, are observed in T1α(-/-) at E18.5. At term, FosB, Egr1, MPK-1 and Nur77, which can function as negative regulators of the cell-cycle, are down-regulated. This is consistent with the hyperproliferation of peripheral lung cells in term T1α (-/-) lungs reported earlier. Biochemical assays show that neither PCNA nor p21 are altered at E18.5. At term in contrast, PCNA is increased, and p21 is decreased.ConclusionThis global analysis has identified a number of candidate genes that are significantly altered in lungs in which sacculation is abnormal. Many genes identified were not previously associated with lung development and may participate in formation of alveolar sacs prenatally.

Angiotensin converting enzyme 2 is primarily epithelial and is developmentally regulated in the mouse lung

Angiotensin converting enzyme (ACE) 2 is a carboxypeptidase that shares 42% amino acid homology with ACE. Little is known about the regulation or pattern of expression of ACE2 in the mouse lung, including its definitive cellular distribution or developmental changes. Based on Northern blot and RT‐PCR data, we report two distinct transcripts of ACE2 in the mouse lung and kidney and describe a 5′ exon 1a previously unidentified in the mouse. Western blots show multiple isoforms of ACE2, with predominance of a 75–80 kDa protein in the mouse lung versus a 120 kDa form in the mouse kidney. Immunohistochemistry localizes ACE2 protein to Clara cells, type II cells, and endothelium and smooth muscle of small and medium vessels in the mouse lung. ACE2 mRNA levels peak at embryonic day 18.5 in the mouse lung, and immunostaining demonstrates protein primarily in the bronchiolar epithelium at that developmental time point. In murine cell lines ACE2 is strongly expressed in the Clara cell line mtCC, as opposed to the low mRNA expression detected in E10 (type I‐like alveolar epithelial cell line), MLE‐15 (type II alveolar epithelial cell line), MFLM‐4 (fetal pulmonary vasculature cell line), and BUMPT‐7 (renal proximal tubule cell line). In summary, murine pulmonary ACE2 appears to be primarily epithelial, is developmentally regulated, and has two transcripts that include a previously undescribed exon. J. Cell. Biochem. 101:1278–1291, 2007.

ERM is expressed by alveolar epithelial cells in adult mouse lung and regulates caveolin‐1 transcription in mouse lung epithelial cell lines

We previously identified an Ets cis‐element in the mouse caveolin‐1 promoter that is selectively activated in lung epithelial (E10), but not lung endothelial murine lung endothelial cell line (MFLM‐4), cell lines and therefore appears important for differential, cell‐specific caveolin‐1 transcription. In the present study, we demonstrate that immunostaining of adult mouse lung detects the ETS protein Ets‐related molecule (ERM PEA3) in distal lung epithelium in alveolar type I and II cells, but not in bronchial epithelium or lung endothelial cells. We tested ERM and polyomavirus enhancer activator 3 (PEA3) for their ability to increase endogenous caveolin‐1 transcripts and to activate caveolin‐1 promoter fragments containing the −865 Ets cis‐element. Chromatin immunoprecipitation (ChIP) assays show that both ERM and PEA3 bind to the caveolin‐1 promoter in murine E10, but not MFLM‐4, cells. Normalized luciferase activities show that only ERM activates the caveolin‐1 promoter in E10 cells, but neither protein enhances promoter activity in MFLM‐4 cells. Mutation of the Ets site blocks ERM‐mediated promoter activation in E10 cells. Furthermore, overexpression of ERM increases the cellular content of caveolin‐1 mRNA and protein, in E10, but not MFLM‐4, cells. The effects of PEA3 on the cellular content of endogenous caveolin‐1 expression are variable. These results demonstrate that ERM is involved in caveolin‐1 regulation in a murine lung epithelial, but not lung endothelial cell line. We conclude that transcriptional regulation of caveolin‐1 differs markedly between lung epithelial and endothelial cell lines, perhaps explaining why the onset of caveolin‐1 expression differs in epithelial and endothelial cells during lung development. J. Cell. Biochem. 102: 13–27, 2007.

Transcription factor and microRNA interactions in lung cells: an inhibitory link between NK2 homeobox 1, miR-200c and the developmental and oncogenic factors Nfib and Myb

BackgroundThe transcription factor NK2 homeobox 1 (Nkx2-1) plays essential roles in epithelial cell proliferation and differentiation in mouse and human lung development and tumorigenesis. A better understanding of genes and pathways downstream of Nkx2-1 will clarify the multiple roles of this critical lung factor. Nkx2-1 regulates directly or indirectly numerous protein-coding genes; however, there is a paucity of information about Nkx2-1-regulated microRNAs (miRNAs).Methods and resultsBy miRNA array analyses of mouse epithelial cell lines in which endogenous Nkx2-1 was knocked-down, we revealed that 29 miRNAs were negatively regulated including miR-200c, and 39 miRNAs were positively regulated by Nkx2-1 including miR-1195. Mouse lungs lacking functional phosphorylated Nkx2-1 showed increased expression of miR-200c and alterations in the expression of other top regulated miRNAs. Moreover, chromatin immunoprecipitation assays showed binding of NKX2-1 protein to regulatory regions of these miRNAs. Promoter reporter assays indicated that 1kb of the miR-200c 5′ flanking region was transcriptionally active but did not mediate Nkx2-1- repression of miR-200c expression. 3′UTR reporter assays support a direct regulation of the predicted targets Nfib and Myb by miR-200c.ConclusionsThese studies suggest that Nkx2-1 controls the expression of specific miRNAs in lung epithelial cells. In particular, we identified a regulatory link between Nkx2-1, the known tumor suppressor miR-200c, and the developmental and oncogenic transcription factors Nfib and Myb, adding new players to the regulatory mechanisms driven by Nkx2-1 in lung epithelial cells that may have implications in lung development and tumorigenesis.

Expression of Piwi protein MIWI2 defines a distinct population of multiciliated cells

P-element-induced wimpy testes (Piwi) proteins are known for suppressing retrotransposon activation in the mammalian germline. However, whether Piwi protein or Piwi-dependent functions occur in the mammalian soma is unclear. Contrary to germline-restricted expression, we observed that Piwi-like Miwi2 mRNA is indeed expressed in epithelial cells of the lung in adult mice and that it is induced during pneumonia. Further investigation revealed that MIWI2 protein localized to the cytoplasm of a discrete population of multiciliated airway epithelial cells. Isolation and next-generation sequencing of MIWI2-positive multiciliated cells revealed that they are phenotypically distinct from neighboring MIWI2-negative multiciliated cells. Mice lacking MIWI2 exhibited an altered balance of airway epithelial cells, demonstrating fewer multiciliated cells and an increase in club cells. During pneumococcal pneumonia, Miwi2-deficient mice exhibited increased expression of inflammatory mediators and increased immune cell recruitment, leading to enhanced bacterial clearance. Taken together, our data delineate MIWI2-dependent functions outside of the germline and demonstrate the presence of distinct subsets of airway multiciliated cells that can be discriminated by MIWI2 expression. By demonstrating roles for MIWI2 in airway cell identity and pulmonary innate immunity, these studies elucidate unanticipated physiological functions for Piwi proteins in somatic tissues.

Retinoic acid signaling is essential for airway smooth muscle homeostasis

Airway smooth muscle (ASM) is a dynamic and complex tissue involved in regulation of bronchomotor tone, but the molecular events essential for the maintenance of ASM homeostasis are not well understood. Observational and genome-wide association studies in humans have linked airway function to the nutritional status of vitamin A and its bioactive metabolite retinoic acid (RA). Here, we provide evidence that ongoing RA signaling is critical for the regulation of adult ASM phenotype. By using dietary, pharmacologic, and genetic models in mice and humans, we show that (a) RA signaling is active in adult ASM in the normal lung, (b) RA-deficient ASM cells are hypertrophic, hypercontractile, profibrotic, but not hyperproliferative, (c) TGF-β signaling, known to cause ASM hypertrophy and airway fibrosis in human obstructive lung diseases, is hyperactivated in RA-deficient ASM, (d) pharmacologic and genetic inhibition of the TGF-β activity in ASM prevents the development of the aberrant phenotype induced by RA deficiency, and (e) the consequences of transient RA deficiency in ASM are long-lasting. These results indicate that RA signaling actively maintains adult ASM homeostasis, and disruption of RA signaling leads to aberrant ASM phenotypes similar to those seen in human chronic airway diseases such as asthma.