BackgroundClaudins are transmembrane proteins expressed in tight junctions that prevent paracellular transport of extracellular fluid and a variety of other substances. However, the expression profile of Claudin-6 (Cldn6) in the developing lung has not been characterized.Methods and resultsCldn6 expression was determined during important periods of lung organogenesis by microarray analysis, qPCR and immunofluorescence. Expression patterns were confirmed to peak at E12.5 and diminish as lung development progressed. Immunofluorescence revealed that Cldn6 was detected in cells that also express TTF-1 and FoxA2, two critical transcriptional regulators of pulmonary branching morphogenesis. Cldn6 was also observed in cells that express Sox2 and Sox9, factors that influence cell differentiation in the proximal and distal lung, respectively. In order to assess transcriptional control of Cldn6, 0.5, 1.0, and 2.0-kb of the proximal murine Cldn6 promoter was ligated into a luciferase reporter and co-transfected with expression vectors for TTF-1 or two of its important transcriptional co-regulators, FoxA2 and Gata-6. In almost every instance, TTF-1, FoxA2, and Gata-6 activated gene transcription in cell lines characteristic of proximal airway epithelium (Beas2B) and distal alveolar epithelium (A-549).ConclusionsThese data revealed for the first time that Cldn6 might be an important tight junctional component expressed by pulmonary epithelium during lung organogenesis. Furthermore, Cldn6-mediated aspects of cell differentiation may describe mechanisms of lung perturbation coincident with impaired cell junctions and abnormal membrane permeability.
These data reveal that tight junctional proteins such as Cldn6 are differentially regulated by tobacco-smoke exposure and that Cldns are potentially targeted when epithelial cells respond to tobacco smoke. Further research may show that Cldns expressed in tight junctions between parenchymal cells contribute to impaired structural integrity of the lung coincident with smoking.
Claudin 6 (Cldn6) is a tetraspanin protein expressed by barrier epithelial cells. In order to assess the effects of persistent tight junctions involving Cldn6 during lung development, a doxycycline (dox)-inducible conditional transgenic mouse was generated that up-regulates Cldn6 in the distal lung. Pups had unlimited access to dox from conception until sacrifice date at embryonic day (E) 18.5. Quantitative PCR, immunoblotting, and immunohistochemistry revealed significantly elevated Cldn6 expression in transgenic mice compared to non-transgenic controls. There were no differences in terms of lung size, lung weight, or whole body weight at the time of necropsy. Histological evaluations led to the discovery that E18.5 Cldn6 transgenic pups appeared to be in the early canalicular stage of development coincident with fewer, thickened respiratory airspaces. In contrast, controls appeared to have entered the saccular stage characterized by thin airspace walls and spherical architecture. Immunostaining for transcriptional regulators including TTF-1 and FoxA2 was conducted to assess cell differentiation and specific cell types were identified via staining for pro-surfactant protein C (alveolar type II epithelial cells) or Clara Cell Secretory Protein (cub or Clara cells). Lastly, cell turnover was qualitatively measured via staining for cell proliferation or apoptosis. These data suggest that Cldn6 is an important junctional protein potentially involved in the programming of epithelial cells during lung development. Furthermore, genetic down-regulation of Cldn6 as development proceeds may influence differentiation observed in the transition from the canalicular to the saccular lung. KEY WORDS: claudin 6, lung, transgenic, mouseLung development is a complex process of highly organized and dynamic events. Tubular branching of the lung airways characterizes the pseudoglandular stage of lung organogenesis and the subsequent canalicular stage coincides with pulmonary epithelial cell differentiation that results in the formation of the air-blood barrier (Burri, 1984;Torday, 1992;Copland and Post, 2004). Numerous signaling and transcriptional control pathways critically influence the precise deposition of specialized cell types along the proximaldistal pulmonary axis.Tight junctions (TJs) are increasingly recognized as potentially critical modulators spatial epithelial cell programming. TJs begin to form at cell-cell contacts as the respiratory epithelium develops into a complex monolayer (Crapo et al., 1983;Ward and Nicholas, 1984). TJs are critical in the developing lung as they provide the means of compartmentalization required of barrier derivation. TJs Int. J. Dev. Biol. 59: 479-485 (2015) doi: 10.1387/ijdb.150086pr Abbreviations used in this paper: CCSP, clara cell secretory protein; Cldn6, claudin 6; Dox, doxycycline; FoxA2, Forkhead Box A2; PCNA, proliferating cell nuclear antigen; SP-C, surfactant protein C; TJ, tight junction; TTF-1, thyroid transcription factor-1.are an assembly of resident integral prot...
Smoking is a major risk factor for several chronic diseases including chronic obstructive pulmonary disease (COPD), a condition involving both emphysema and inflammation of the airways. COPD severity directly relates to abnormalities of pulmonary epithelial cells. Claudins contribute to tight junctions that prevent paracellular transport of extracellular fluid and diverse substances and Claudin 6 (Cldn6) is a protein expressed prominently in the lung parenchyma. To determine whether Cldn6 was differentially influenced by tobacco smoke, Cldn6 was evaluated by q‐PCR, immunoblitting, and immunofluorescence following exposure to tobacco smoke. Q‐PCR and immunoblotting revealed that Cldn6 was increased in alveolar type II‐like epithelial cells (A549) but not in Bease2B cells, a cell line derived from proximal airway epithelium. Luciferase assays incorporating 0.5kb, 1.0kb, or 2.0kb of the Cldn6 promoter also revealed increased transcription of the gene when cells were exposed to cigarette smoke extract. Cldn6 was also markedly increased in the lungs of Balb/C mice exposed to tobacco smoke delivered by an automated smoke machine (InExpose, Scireq, Canada) compared to animals exposed to room air. These data reveal for the first time that tight junctional proteins are differentially regulated by tobacco smoke exposure and that Cldns are potentially involved as neighboring epithelial cells respond to tobacco smoke. Further research may show that complexes between cells contribute to impaired structural integrity of the lung coincident with smoking. Grant Funding Source: Supported by the Flight Attendant’s Medical Research Institute (FAMRI) and a BYU MEG Award (PRR).
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