Clara-like Cells Research Articles

The Pulmonary NEB ME Is a Complex Intraepithelial Unit.

Among the intrapulmonary myelinated vagal sensory airway receptors, pulmonary neuroepithelial bodies (NEBs) definitely reveal the most complex organisation. This updated review aims at delivering the broad and thorough knowledge of the system that is essential for understanding its physiological relevance.Complexly organised pulmonary NEBs are an integral part of the intrapulmonary airway epithelium of all air-breathing vertebrates. For decades, a quest has been going on to unravel the functional significance of these intriguing structures that appear to be modified in the course of many pulmonary diseases.The pulmonary NEB microenvironment (ME) is composed of organoid clusters of pulmonary neuroendocrine cells (PNECs) that are able to store and release neurotransmitters and are closely contacted by extensive (mainly afferent) nerve terminals, emphasising a potential receptor/effector role and probable signalling to the central nervous system. PNECs are largely shielded from the airway lumen by a special type of Clara cells, the Clara-like cells, with potential stem cell characteristics.Since pulmonary NEBs are widely dispersed in the airway epithelium, and represent less than one percent of the cells in the epithelial lining, investigating the NEB ME largely depends on its unequivocal microscopic identification. Nowadays, multidisciplinary approaches allow to combine functional morphological investigations in cryosections and cleared whole lungs, live cell imaging in lung vibratome slices, and selective gene expression analysis after laser microdissection of genetically tagged NEBs.So far, functional studies of the pulmonary NEB ME revealed that PNECs can be activated by various mechanical and chemical stimuli, resulting in a calciummediated release of neurotransmitters. A number of publications in the past decades have exposed NEBs as potential hypoxia sensors.In the past few years, combination of in vivo experiments and gene expression analysis unveiled the pulmonary NEB ME as a quiescent stem cell niche in healthy postnatal mouse lungs. The stem cell population was shown to be activated by transient mild inflammation, and silencing appears to involve bone morphogenetic protein signalling that may be mediated by vagal afferents.Today, it is clear that only an integrated approach that takes all current information into account will be able to explain the full role of the pulmonary NEB ME in health and disease.

Acute Lung Inflammation Induces Differential Gene Expression Changes in the Airway Stem Cell Niche of the Neuroepithelial Body Microenvironment

Own and literature data point out that the neuroepithelial body (NEB) microenvironment (ME) may harbor stem cell characteristics in healthy postnatal mouse lungs1. Furthermore, the NEB ME has been suggested to be implicated in airway development and adult airway epithelial repair after severe injury. The NEB ME consists of strongly innervated groups of pulmonary neuroepithelial endocrine cells that are specifically covered by a unique population of so‐called Clara‐like cells (CLCs). The airway epithelium, including the NEB ME, reveals a very low turnover rate in the absence of injury. We recently showed that CLCs can be selectively activated following lipopolysaccharide (LPS) induced mild lung inflammation2.A GAD67‐GFP mouse model, with GFP fluorescent NEB cells, allows selective laser microdissection (LMD) of the NEB ME in cryostat sections of postnatal mouse lungs, and is compatible with high‐end PCR techniques1. RNA was isolated from LMD‐collected samples, both of the NEB ME and control airway epithelium (CAE) of untreated controls (NEB MEctrl; CAEctrl) and of LPS‐challenged mice (NEB MELPS; CAELPS). A panel of more than 600 genes from a set of stem cell‐related PCR arrays 1 was used to evaluate gene expression changes.Both up‐ and downregulation (two‐fold threshold) of the gene expression levels, between the NEB ME and CAE and between LPS‐treated and control mice, were analyzed to find out which pathways might be involved in activation of the NEB ME. In LPS‐challenged mouse lungs, 231 (38.4%) of the 601 analyzed stem cell‐related genes showed a differential expression in the NEB MELPS compared to CAELPS, of which 141 (23.4%) were upregulated and 90 (15.0%) were downregulated in the NEB MELPS. Comparably, in healthy control animals1, 218 (36.3%) genes were differentially expressed in the NEB MEctrl/CAEctrl, of which 168 (28.0%) were upregulated and 50 (8.3%) were downregulated. Importantly, multiple genes showed a differential expression in the activated NEB MELPS compared to the non‐activated NEB MEctrl; 238 (39.6%) genes showed an at least two‐fold shift in expression, 136 (22.6%) of which were upregulated and 102 (17.0%) were downregulated in the NEB MELPS. Expression data further revealed that Stem Cell, Cancer Stem Cell and the TGFβ/BMP pathway arrays harbored the highest numbers of differentially expressed genes in the NEB MELPS/NEB MEctrl, and that several genes that are involved in development‐ and regeneration‐related pathways, such as BMP, Notch and Wnt, appeared to change expression following LPS treatment.These observations support the interpretation that, in our LPS‐induced lung inflammation model, the selective proliferation of CLCs in the NEB ME results from the activation of stem cells in this niche. The observed extensive differential expression of stem cell related genes is likely involved in regulating and maintaining a balance between stem cell silencing/proliferation and commitment of daughter cells to differentiate and restore airway epithelial integrity.Support or Funding InformationUA grant GOA BOF 2015 (30729 to DA)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Selective activation and proliferation of a quiescent stem cell population in the neuroepithelial body microenvironment

BackgroundThe microenvironment (ME) of neuroepithelial bodies (NEBs) harbors densely innervated groups of pulmonary neuroendocrine cells that are covered by Clara-like cells (CLCs) and is believed to be important during development and for adult airway epithelial repair after severe injury. Yet, little is known about its potential stem cell characteristics in healthy postnatal lungs.MethodsTransient mild lung inflammation was induced in mice via a single low-dose intratracheal instillation of lipopolysaccharide (LPS). Bronchoalveolar lavage fluid (BALF), collected 16 h after LPS instillation, was used to challenge the NEB ME in ex vivo lung slices of control mice. Proliferating cells in the NEB ME were identified and quantified following simultaneous LPS instillation and BrdU injection.ResultsThe applied LPS protocol induced very mild and transient lung injury. Challenge of lung slices with BALF of LPS-treated mice resulted in selective Ca2+-mediated activation of CLCs in the NEB ME of control mice. Forty-eight hours after LPS challenge, a remarkably selective and significant increase in the number of divided (BrdU-labeled) cells surrounding NEBs was observed in lung sections of LPS-challenged mice. Proliferating cells were identified as CLCs.ConclusionsA highly reproducible and minimally invasive lung inflammation model was validated for inducing selective activation of a quiescent stem cell population in the NEB ME. The model creates new opportunities for unraveling the cellular mechanisms/pathways regulating silencing, activation, proliferation and differentiation of this unique postnatal airway epithelial stem cell population.

The Neuroepithelial Body Microenvironment as a Postnatal Airway Epithelial Stem Cell Niche: Critical Role for BMP Signaling

Main objective was to develop models that enable exploring the stem cell characteristics of pulmonary neuroepithelial bodies (NEBs) in postnatal mouse lungs. The NEB microenvironment (ME) consists of innervated groups of pulmonary neuroepithelial endocrine cells, covered by socalled Clara‐like cells (CLCs). Epithelial cells of intrapulmonary conductive airways typically show a very low turn‐over rate in healthy postnatal lungs. Own and literature data point out that the NEB ME is important during development and for adult lung epithelial repair after total ablation, and that CLCs may act as stem cells under these circumstances. However, the absence of a model to physiologically induce selective cell proliferation in the healthy NEB ME, has so far hampered the identification of signaling processes to support their characterization as a quiescent postnatal stem cell niche involved in postnatal airway epithelial homeostasis.We developed a laser microdissection (LMD)‐based method that is compatible with high‐end PCR techniques to selectively isolate the NEB ME and compare its gene expression with control airway epithelium (CAE) [1]. To identify genes that may be involved in signaling mechanisms in the NEB ME, as a potential postnatal stem cell niche, pooled NEB and CAE samples were collected using LMD and gene expression was compared using a panel of stem cell related PCR Arrays. Expression of stem cell ‘signature’ genes in the NEB ME was confirmed and quantified by qPCR [1].Since inhibition of bone morphogenetic protein (BMP) signaling has been reported to induce proliferation of basal progenitor cells in the mouse trachea [2], and members of the BMP pathway showed a high expression in the NEB ME in multiple PCR arrays, their possible involvement in the stem cell characteristics of the NEB ME was further evaluated. Localization of BMP2, BMP7 and BMP receptors in the NEB ME could be confirmed by multiple immunofluorescent labeling.Additionally, mild transient acute lung injury (ALI), induced by a single low‐dose intratracheal lipopolysaccharide (LPS) challenge, was shown to induce a remarkable selective proliferation of CLCs in the NEB ME. It also resulted in a strongly decreased expression of BMP ligands in the NEB ME, indicating that stem cell activation by ALI might involve inhibition of the BMP pathway. Treating mice with LDN‐193189, a small molecule inhibitor of BMP type I receptors, recently revealed that compromising BMP signaling on its own appears to cause cell proliferation in the NEB ME that is reminiscent of what was seen after LPS treatment.In conclusion, a highly reproducible mild ALI model was generated for the selective activation of a quiescent postnatal airway stem cell population in the NEB ME, creating novel opportunities for unraveling the mechanisms that are involved. So far, combined LMD, gene and protein expression analysis, and specific inhibition of BMP signaling strongly suggest the involvement of BMP in silencing cell proliferation in the non‐activated stem cell niche of the NEB ME.Support or Funding InformationSupported by UA grant GOA BOF 2015 (30729 to DA).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Selective Activation and Proliferation of a Unique Stem Cell Population in the Neuroepithelial Body Microenvironment by Transient Acute Lung Injury

The microenvironment (ME) of pulmonary neuroepithelial bodies (NEBs) has been suggested to be implicated in airway development and adult airway epithelial repair after severe injury. Regardless the increasing evidence that NEBs act as a stem cell niche under these circumstances, little is known about the potential stem cell characteristics of the NEB ME in healthy postnatal lungs. The NEB ME consists of highly innervated groups of pulmonary neuroepithelial endocrine cells that are specifically covered by a unique population of Clara‐like cells (CLCs) with supposed stem cell characteristics. The relatively low number and widespread distribution of NEBs in the epithelium of intrapulmonary airways of postnatal animals has so far hampered the direct identification of mechanisms and signaling processes to support their characterization as a stem cell niche.A GAD67‐GFP mouse model, in which NEB cells selectively express GFP, permits their straightforward identification in cryostat sections of postnatal mouse lungs. Transient acute lung injury (ALI) was induced via intratracheal instillation of lipopolysaccharide (LPS; 1mg/kg bw). Lung function was monitored using a noninvasive unrestrained whole body plethysmograph. In one experiment, bronchoalveolar lavage fluid (BALF) was collected 24h after LPS instillation, and used to challenge the NEB ME in ex vivo lung slices of control mice. In another experiment, BrdU (10 mg/kg bw), as a marker for cells that divide during the observation window, was injected intraperitoneally at the time of LPS instillation and after 24h, the animals were euthanized after 48h, and the lungs were processed for freeze sectioning and BrdU immunostaining.Challenge of lung slices with BALF of ALI mice was shown to induce a reversible and reproducible selective Ca2+‐mediated activation of CLCs in the NEB ME of control mice. 48h after LPS challenge and BrdU injection, lung sections of ALI mice revealed a remarkable and highly significant increase in the number of BrdU‐labeled cells that surround NEBs, as compared to control lungs. As judged from sham‐treated mice, the instillation procedure itself also resulted in a modest increase in NEB‐associated BrdU‐labeled cells. Preliminary plethysmography data revealed that a single intratracheal LPS instillation results in short and transient ALI. Lung function parameters show the largest changes around six hours after treatment, and have restored to values that resemble those of the control group after 24h.In conclusion, mild transient ALI appears to result in the production of soluble mediators in the airways that selectively activate, and induce proliferation of, a stem cell population in the NEB ME. The changes that occur in the dividing cells will be further explored using gene expression profiling. The described workflow, together with our previously optimized LMD protocol, provides a solid base for unraveling molecular characteristics of the NEB ME.Support or Funding InformationHercules Foundation grant AUAH‐09‐001 (DA); UA grant GOA BOF 2015 (DA).

Bronchoalveolar sublineage specification of pluripotent stem cells: effect of dexamethasone plus cAMP-elevating agents and keratinocyte growth factor.

Respiratory progenitors can be efficiently generated from pluripotent stem cells (PSCs). However, further targeted differentiation into bronchoalveolar sublineages is still in its infancy, and distinct specifying effects of key differentiation factors are not well explored. Focusing on airway epithelial Clara cell generation, we analyzed the effect of the glucocorticoid dexamethasone plus cAMP-elevating agents (DCI) on the differentiation of murine embryonic and induced pluripotent stem cells (iPSCs) into bronchoalveolar epithelial lineages, and whether keratinocyte growth factor (KGF) might further influence lineage decisions. We demonstrate that DCI strongly induce expression of the Clara cell marker Clara cell secretory protein (CCSP). While KGF synergistically supports the inducing effect of DCI on alveolar markers with increased expression of surfactant protein (SP)-C and SP-B, an inhibitory effect on CCSP expression was shown. In contrast, neither KGF nor DCI seem to have an inducing effect on ciliated cell markers. Furthermore, the use of iPSCs from transgenic mice with CCSP promoter-dependent lacZ expression or a knockin of a YFP reporter cassette in the CCSP locus enabled detection of derivatives with Clara cell typical features. Collectively, DCI was shown to support bronchoalveolar specification of mouse PSCs, in particular Clara-like cells, and KGF to inhibit bronchial epithelial differentiation. The targeted in vitro generation of Clara cells with their important function in airway protection and regeneration will enable the evaluation of innovative cellular therapies in animal models of lung diseases.

Epithelial Cell Differentiation of Human Mesenchymal Stromal Cells in Decellularized Lung Scaffolds

Identification of appropriate donor cell types is important for lung cell therapy and for lung regeneration. Previous studies have indicated that mesenchymal stromal cells derived from human bone marrow (hBM-MSCs) and from human adipose tissue (hAT-MSCs) may have the ability to trans-differentiate into lung epithelial cells. However, these data remain controversial. Herein, the ability of hBM-MSCs and hAT-MSCs to repopulate acellular rodent lung tissue was evaluated. hBM-MSCs and hAT-MSCs were isolated from bone marrow aspirate and lipoaspirate, respectively. Rat lungs were decellularized with CHAPS detergent, followed by seeding the matrix with hBM-MSCs and hAT-MSCs. Under appropriate culture conditions, both human MSC populations attached to and proliferated within the lung tissue scaffold. In addition, cells were capable of type 2 pneumocyte differentiation, as assessed by marker expression of surfactant protein C (pro-SPC) at the protein and the RNA level, and by the presence of lamellar bodies by transmission electron microscopy. Additionally, hAT-MSCs contributed to Clara-like cells that lined the airways in the lung scaffolds, whereas the hBM-MSCs did not. We also tested the differentiation potential of MSCs on different extracellular matrix components in vitro, and found that protein substrate influences MSC epithelial differentiation. Together our data show the capacity for human MSCs to differentiate toward lung epithelial phenotypes, and the possibility of using these cells for lung cell therapies and tissue engineering.

Functional expression of the multimodal extracellular calcium-sensing receptor in pulmonary neuroendocrine cells

The Ca(2+)-sensing receptor (CaSR) is the master regulator of whole-body extracellular free ionized [Ca(2+)]o. In addition to sensing [Ca(2+)]o, CaSR integrates inputs from a variety of different physiological stimuli. The CaSR is also expressed in many regions outside the [Ca(2+)]o homeostatic system, including the fetal lung where it plays a crucial role in lung development. Here, we show that neuroepithelial bodies (NEBs) of the postnatal mouse lung express a functional CaSR. NEBs are densely innervated groups of neuroendocrine epithelial cells in the lung representing complex sensory receptors in the airways and exhibiting stem cell characteristics. qRT-PCR performed on laser microdissected samples from GAD67-GFP mouse lung cryosections revealed exclusive expression of the CaSR in the NEB microenvironment. CaSR immunoreactivity was present at NEB cells from postnatal day 14 onwards. Confocal imaging of lung slices revealed that NEB cells responded to an increase of [Ca(2+)]o with a rise in intracellular Ca(2+) ([Ca(2+)]i); an effect mimicked by several membrane-impermeant CaSR agonists (e.g. the calcimimetic R-568) and that was blocked by the calcilytic Calhex-231. Block of TRPC channels attenuated the CaSR-dependent increases in [Ca(2+)]i, suggesting that Ca(2+) influx through TRPC channels contributes to the total [Ca(2+)]i signal evoked by the CaSR in NEBs. CaSR also regulated baseline [Ca(2+)]i in NEBs and, through paracrine signaling from Clara-like cells, coordinated intercellular communication in the NEB microenvironment. These data suggest that the NEB CaSR integrates multiple signals converging on this complex chemosensory unit, and is a key regulator of this intrapulmonary airway stem cell niche.

Nano-TiO2 particles impair adhesion of airway epithelial cells to fibronectin

Titanium dioxide engineered nanoparticles (nano-TiO2) are widely used in the manufacturing of a number of products. Due to their size (<100nm), when inhaled they may be deposited in the distal lung regions and damage Clara cells. We investigated the mechanisms by which short-term (1-h) incubation of human airway Clara-like (H441) cells to nano-TiO2 (6nm in diameter) alters the ability of H441 cells to adhere to extracellular matrix. Our results show that 1h post-incubation, there was a 3-fold increase of extracellular H2O2, increased intracellular oxidative stress as demonstrated by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) oxidation, and a 5-fold increase of phosphor-ERK1/2 as measured by Western blotting. These changes were accompanied by a 25% decrease of H441 adherence to fibronectin (p<0.05 compared to vehicle incubated H441 cells). Pretreatment with the ERK1/2 inhibitor U0126 for 3h, partially prevented this effect. In conclusion, short-term exposure of H441 cells to nano-TiO2 appears to reduce adherence to fibronectin due to oxidative stress and activation of ERK1/2.

Evaluation of a Common Variant of the Gene Encoding Clara Cell 10 kd Protein (CC10) as a Candidate Determinant for Asthma Severity and Steroid Responsiveness Among Chinese Children

Objective. The gene (SCGB1A1) encoding Clara cell 10-kDa protein (CC10), a steroid-inducible immune modulator, is a candidate gene for asthma, but the evidence is equivocal. The potential influence of a common variant on asthma severity and serum CC10 levels during acute exacerbation and after corticosteroid treatment in Chinese case–control children and its functional relevance was investigated. Methods. Genotyping of a non-coding variant G+38A was performed in 489 children, of whom 277 had asthma with varying severity, and 212 healthy controls. Associations were tested for asthma, asthma severity, and responsiveness to steroid treatment. The transcriptional activity of this variant was examined in a Clara-like cell line (H358) using transient transfection assays. Results. Significant association was observed for the combined GA and AA genotypes of the CC10 G+38A variant and an increased risk of asthma [odds ratio (OR), 2.62, p < .001]. This association was correlated with asthma severity (moderate: OR, 2.85, p < .001; near-fatal: OR, 4.81, p < .001). Also, patients with the GA and AA genotypes showed significantly lower serum CC10 (p < .01) and provocation concentration causing a 20% fall (PC20) in forced expiratory volume in 1 s (FEV1) (p < .0001) when compared with those with the GG. After glucocorticoid treatment, the CC10 levels were significantly increased in asthmatic patients with GG (p < .0001), but not those with the GA and AA genotypes. Moreover, a lower dexamethasone-induced reporter (luciferase) activity was observed for H358 cells transiently transfected with the G38A risk allele (A) compared with wild-type allele (G). Conclusions. These findings suggest that the CC10 G+38A variant may contribute to the severity of asthma and lower level of steroid responsiveness.

Neuroepithelial Bodies as Mechanotransducers in the Intrapulmonary Airway Epithelium

In rodent lungs, a major part of the myelinated vagal airway afferents selectively contacts pulmonary neuroepithelial bodies (NEBs). Because most myelinated vagal airway afferents concern physiologically characterized mechanoreceptors, the present study aimed at unraveling the potential involvement of NEB cells in transducing mechanosensory information from the airways to the central nervous system. Physiological studies were performed using confocal Ca(2+) imaging of airway epithelium in murine lung slices. Mechanical stimulation by short-term application of a mild hypoosmotic solution (230 mosmol) resulted in a selective, fast, reversible, and reproducible Ca(2+) rise in NEB cells. Other airway epithelial cells could only be activated using more severe hypoosmotic stimuli (< 200 mosmol). NEB cells selectively expressed the Ca(2+)-permeable osmo- and mechanosensitive transient receptor potential canonical channel 5 (TRPC5) in their apical membranes, whereas immunoreactivity for TRP vanilloid-4 and TRP melastatin-3 was abundant in virtually all other airway epithelial cells. Hypoosmotic activation of NEB cells was prevented by GsMTx-4, an inhibitor of mechanosensitive ion channels, and by SKF96365, an inhibitor of TRPC channels. Short application of gadolinium, reported to activate TRPC5 channels, evoked a transient Ca(2+) rise in NEB cells. Osmomechanical activation of NEB cells gave rise to a typical delayed activation of Clara-like cells due to the release of ATP from NEB cells. Because ATP may activate the NEB-associated P2X(2/3) ATP receptor expressing myelinated vagal afferents, the current observations strongly suggest that pulmonary NEB cells are fully equipped to initiate mechanosensory signal transduction to the central nervous system via a purinergic signaling pathway.

Direct and indirect roles for β-catenin in facultative basal progenitor cell differentiation.

The conducting airway epithelium is maintained and repaired by endogenous progenitor cells. Dysregulated progenitor cell proliferation and differentiation is thought to contribute to epithelial dysplasia in chronic lung disease. Thus modification of progenitor cell function is an attractive therapeutic goal and one that would be facilitated by knowledge of the molecular pathways that regulate their behavior. We modeled the human tracheobronchial epithelium using primary mouse tracheal epithelial cell cultures that were differentiated by exposure to the air-liquid-interface (ALI). A basal cell subset, termed facultative basal cell progenitors (FBP), initiate these cultures and are the progenitor for tracheal-specific secretory cells, the Clara-like cell, and ciliated cells. To test the hypothesis that β-catenin is necessary for FBP function, ALI cultures were generated from mice homozygous for the Ctnb(flox(E2-6)) allele. In this model, exons 2-6 of the β-catenin gene are flanked by LoxP sites, allowing conditional knockout of β-catenin. The β-catenin locus was modified through transduction with Adenovirus-5-encoding Cre recombinase. This approach generated a mosaic epithelium, comprised of β-catenin wild-type and β-catenin knockout cells. Dual immunostaining and quantitative histomorphometric analyses demonstrated that β-catenin played a direct role in FBP-to-ciliated cell differentiation and that it regulated cell-cell interactions that were necessary for FBP-to-Clara-like cell differentiation. β-catenin was also necessary for FBP proliferation and long-term FBP viability. We conclude that β-catenin is a critical determinant of FBP function and suggest that dysregulation of the β-catenin signaling pathway may contribute to disease pathology.

Roles for β-Catenin and Doxycycline in the Regulation of Respiratory Epithelial Cell Frequency and Function

The expression of β-catenin-dependent genes can be increased through the Cre recombinase (Cre)-mediated elimination of the exon 3-encoded sequence. This mutant β-catenin is termed DE3, and promotes the expression of β-catenin-dependent genes. Our previous study used the DE3 model to demonstrate that persistent β-catenin activity inhibited bronchiolar Clara-to-ciliated cell differentiation. The present study was designed to evaluate the roles of β-catenin in regulating the tracheal progenitor cell hierarchy. However, initial experiments demonstrated that the tetracycline-responsive element-Cre transgene (TRE-Cre) was active in the absence of a reverse tetracycline transactivator driver or inducer, doxycycline (Dox). This spurious TRE-Cre transgene activity was not detected using the ROSA26-floxed STOP-LacZ reporter. To determine if the phenotype was a consequence of genotype or treatment with Dox, tracheal and lung specimens were evaluated using quantitative histomorphometric techniques. Analyses of uninduced mice demonstrated a significant effect of genotype on tracheal epithelial cell mass, involving basal, Clara-like cell types. The bronchial and bronchiolar Clara cell mass was also decreased. Paradoxically, an effect on ciliated cell mass was not detected. Activation of the β-catenin reporter transgene TOPGal demonstrated that β-catenin-dependent gene expression led to the genotype-dependent tracheal and bronchiolar phenotype. Comparative analyses of wild-type or keratin 14-rtTA(+/0)/TRE-cre(+/0)/DE3(+/+) mice receiving standard or Dox chow demonstrated an effect of treatment with Dox on basal, Clara-like, and Clara cell masses. We discuss these results in terms of cautionary notes and with regard to alterations of progenitor cell hierarchies in response to low-level injury.

Expression analysis of the regenerating gene (Reg) family members Reg‐IIIβ and Reg‐IIIγ in the mouse during development

The regenerating gene/regenerating islet-derived (Reg) family is a group of small secretory proteins. Within this family, Reg type-III (Reg-III) consists of: Reg-IIIα, -β, -γ, and -δ. To elucidate the physiological relevance of Reg-III, we examined the localization and ontogeny of Reg-IIIβ and Reg-IIIγ in mice at different time points spanning from embryonic day 13.5 to 7 weeks old, using in situ hybridization and immunohistochemistry. Our results showed that Reg-IIIβ was expressed in specific subsets of primary sensory neurons and motor neurons, and that expression was transient during the embryonic and perinatal periods. Reg-IIIβ expression was also observed in absorptive epithelial cells of the intestine. In contrast, Reg-IIIγ expression was mainly observed in epithelial cells of the airways and intestine, but not in the nervous system, and expression levels showed a gradually increasing pattern along with development. In the airways Reg-IIIγ was expressed in goblet and Clara-like cells, whereas in the intestine Reg-IIIγ was expressed in the absorptive epithelial cells and Paneth cells, and was found to be expressed in development before these organs had been exposed to the outside world. The present findings imply that Reg-IIIβ and Reg-IIIγ expression is regulated along divergent pathways. Furthermore, we also suggest that expression of Reg-IIIγ in the airway and intestinal epithelia may occur to protect these organs from exposure to antigens or other factors (e.g., microbes) in the outer world, whereas the transient expression of Reg-IIIβ in the nervous system may be associated with the development of the peripheral nervous system including such processes as myelination.

Perspectives of purinergic signaling in stem cell differentiation and tissue regeneration

Replacement of lost or dysfunctional tissues by stem cells has recently raised many investigations on therapeutic applications. Purinergic signaling has been shown to regulate proliferation, differentiation, cell death, and successful engraftment of stem cells originated from diverse origins. Adenosine triphosphate release occurs in a controlled way by exocytosis, transporters, and lysosomes or in large amounts from damaged cells, which is then subsequently degraded into adenosine. Paracrine and autocrine mechanisms induced by immune responses present critical factors for the success of stem cell therapy. While P1 receptors generally exert beneficial effects including anti-inflammatory activity, P2 receptor-mediated actions depend on the subtype of stimulated receptors and localization of tissue repair. Pro-inflammatory actions and excitatory tissue damages mainly result from P2X7 receptor activation, while other purinergic receptor subtypes participate in proliferation and differentiation, thereby providing adequate niches for stem cell engraftment and novel mechanisms for cell therapy and endogenous tissue repair. Therapeutic applications based on regulation of purinergic signaling are foreseen for kidney and heart muscle regeneration, Clara-like cell replacement for pulmonary and bronchial epithelial cells as well as for induction of neurogenesis in case of neurodegenerative diseases.

β-Catenin Dosage Is a Critical Determinant of Tracheal Basal Cell Fate Determination

The purpose of this study was to determine whether β-catenin regulates basal cell fate determination in the mouse trachea. Analysis of TOPGal transgene reporter activity and Wnt/β-catenin pathway gene expression suggested a role for β-catenin in basal cell proliferation and differentiation after naphthalene-mediated Clara-like and ciliated cell depletion. However, these basal cell activities occurred simultaneously, limiting precise determination of the role(s) played by β-catenin. This issue was overcome by analysis of β-catenin signaling in tracheal air-liquid interface cultures. The cultures could be divided into two phases: basal cell proliferation and basal cell differentiation. A role for β-catenin in basal cell proliferation was indicated by activation of the TOPGal transgene on proliferation days 3 to 5 and by transient expression of Myc (alias c-myc). Another peak of TOPGal transgene activity was detected on differentiation days 2 to 10 and was associated with the expression of Axin 2. These results suggest a role for β-catenin in basal to ciliated and basal to Clara-like cell differentiation. Genetic stabilization of β-catenin in basal cells shortened the period of basal cell proliferation but had a minor effect on this process. Persistent β-catenin signaling regulated basal cell fate by driving the generation of ciliated cells and preventing the production of Clara-like cells.

Context-Dependent Differentiation of Multipotential Keratin 14–Expressing Tracheal Basal Cells

Multipotential (MP) differentiation is one characteristic of a tissue-specific stem cell (TSC). Lineage tracing of tracheobronchial basal cells after naphthalene (NA) injury or in the postnatal period demonstrated that basal cells were MP progenitors for Clara-like and ciliated cells. These studies, as well as reports of spatially restricted, label-retaining basal cells, and MP differentiation by human bronchial cells support the hypothesis that a TSC maintained and repaired the tracheobronchial epithelium. However, differences in basal cell phenotype (keratin [K] 5+ versus K14+), age (postnatal versus adult), health status (normal versus injured), and injury type (acid, detergent, NA) limited comparisons among studies and thus diminished the strength of the TSC argument. The finding that K14 was up-regulated after NA injury was a caveat to our previous analysis of reparative (r)K14-expressing cells (EC). Thus, the present study lineage traced steady-state (s)K14EC and evaluated differentiation potential in the normal and repairing epithelium. We showed that sK14EC were unipotential in the normal epithelium and MP after NA, sK14EC-dervied clones were not restricted to putative TSC niches, sK14EC cells were a direct progenitor for Clara-like and ciliated cells, MP-sK14EC clones accumulated over time, and sK14EC-derived Clara-like cells were progenitors for ciliated cells.

α1-Antitrypsin Inhibits Epithelial Na+ Transport In Vitro and In Vivo

A variety of studies have shown that Na(+) reabsorption across epithelial cells depends on the protease-antiprotease balance. Herein, we investigate the mechanisms by which alpha(1)-antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloride-sensitive epithelial Na(+) channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml = 1 microM) decreased ENaC currents across Xenopus laevis oocytes injected with human alpha,beta,gamma-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. A1AT also decreased ENaC single-channel activity when added in the pipette but not in the bath solutions of ENaC-expressing oocytes patched in the cell-attached mode. Incubation of A1AT with peroxynitrite (ONOO(-)), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO(-)-treated A1AT (1 microM) in C57BL/6 mice also decreased Na(+)-dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO(-)-treated A1AT decreased their ability to cleave a substrate of serine proteases. A1AT had no effect on amiloride-sensitive currents of oocytes injected with hENaC bearing Liddle mutations, presumably because these channels remain at the surface longer than the wild-type channels. These data indicate that A1AT may be an important modulator of ENaC activity and of Na(+)-dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.

Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of complex sensory airway receptors involved in the regulation of breathing. Together with their surrounding Clara-like cells, they exhibit stem cell potential through their capacity to regenerate depopulated areas of the epithelium following lung injury. We have employed confocal live cell imaging microscopy and novel electrophysiological techniques in a new ex vivo lung slice model to unravel potential purinergic signaling pathways within the NEB microenvironment. Quinacrine histochemistry indicated high amounts of vesicular ATP in NEB cells. Using a "reporter-patching" method adapted to create a uniquely sensitive and selective biosensor for the direct detection of ATP release from NEBs ex vivo, we demonstrated quantal ATP release from NEBs following their depolarization. Enhancing enzymatic extracellular ATP hydrolysis or inhibiting P2 receptors confirmed the central role of ATP in paracrine interactions between NEB cells and Clara-like cells. Combined calcium imaging, pharmacology, and immunohistochemistry showed that ligand-binding to functional P2Y(2) receptors underpins the activation of Clara-like cells. Hence, NEB cells communicate with their cellular neighbors in the NEB microenvironment by releasing ATP, which rapidly evokes purinergic activation of surrounding Clara-like cells. Besides ATP acting on the P2X(3) receptor expressing vagal sensory nerve terminals between NEB cells, local paracrine purinergic signaling within this potential stem cell niche may be important to both normal airway function, airway epithelial regeneration after injury, and/or the pathogenesis of small cell lung carcinomas.

Functional Live Cell Imaging of the Pulmonary Neuroepithelial Body Microenvironment

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine cells invariably accompanied by Clara-like cells. Together with NEBs, Clara-like cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal live cell imaging of physiological reactions in identified NEBs and surrounding epithelial cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated cells, Clara cells, and Clara-like cells in live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB cells and ciliated cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intracellular free calcium concentration ([Ca2+](i)) in NEBs and surrounding airway epithelial cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extracellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+](i) increase in NEB cells, while Clara-like cells displayed a delayed (+/- 4 s) [Ca2+](i) increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.