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

Abstract

The expression of b-catenin-dependent genes can be increased through the Cre recombinase (Cre)-mediated elimination of the exon 3-encoded sequence. This mutant b-catenin is termed DE3, and promotes the expression of b-catenin-dependent genes. Our previous study used the DE3 model to demonstrate that persistent b-catenin activity inhibited bronchiolar Clara-to-ciliated cell differentiation. The present study was designed to evaluate the roles of b-catenin in regulating the tracheal progenitor cell hierarchy. However, initial experiments demonstrated that the tetracyclineresponsive 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 b-catenin reporter transgene TOPGal demonstrated that b-catenin-dependent gene expression led to the genotype-dependent tracheal and bronchiolar phenotype. Comparative analyses of wild-type or keratin 14-rtTA1/1 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.Keywords: Clara cell; doxycycline; Cre recombinase; b-catenin; stereology LUNG EPITHELIAL CELL LINEAGESThe tracheobronchial, bronchiolar, and alveolar compartments of murine airway epithelia contain distinct cell lineages. Within the tracheobronchial compartment, basal cells act as progenitors of secretory and ciliated cells during lung development (1). However, Clara-like cells are the progenitors of ciliated cells in the adult steady-state trachea (2). After treatment with naphthalene (NA), the basal cell becomes the progenitor of nascent Clara-like and ciliated cells (3-7). We suggested that signaling from Clara-like cells to basal cells limits the fate of adult basal cells to self-renewal (3).The bronchiolar epithelium is maintained by Clara cells (8, 9), whereas the alveolar epithelium is maintained by Type II cells (10). Tracing the lineage of bronchiolar Clara cells demonstrated that the airway and alveolar epithelia comprise distinct lineages in the steady state (11). Although injury may lead to bronchiolarization of the alveolar duct or alveolarization of the terminal bronchiole (12-14), the roles for a dual-lineage cell, termed the bronchoalveolar stem cell, (13) have been challenged (9, 15). The molecular mechanisms that establish these lineages have re...

Research on WISP1 in Lung Disease

“…These TOPGAL mice have provided a sensitive approach for dissecting the role of the canonical β-catenin pathway in lung development [56], injury [57], and repair [58,54], as well as airway epithelial lineage and stem cell studies [59]. Pharmacological approaches to dissect the contribution of WNTβ-catenin canonical signaling include activation by lithium chloride, a well-known inhibitor of GSK-3β [58], or inhibition by using ICG-001, a selective inhibitor of WNTβ-catenin-dependent transcription [60].…”

Lung Epithelial Healing: A Modified Seed and Soil Concept

“…We and others have shown that air-liquid-interface (ALI) cultures of primary mouse tracheal epithelial cells can be used to evaluate the cellular and molecular mechanisms regulating FBP function (20,29,38,39,(48)(49)(50). We showed that FBP initiate ALI cultures and that K5/K15/K14 triple-positive basal cells comprise 98% of the ALI culture on Culture Day 2 (38).…”

“…We showed that FBP viability and function were not altered by transduction or recombination of a marker allele (ROSA26-floxed STOP-LacZ) (39). Interestingly, Ad5-cre transduction of FBP harboring a floxed allele resulted in generation of a mosaic epithelium that was composed of wild-type and recombined cells (38,39). This mosaicism was fortuitous, as it allowed us to study FBP behavior in genetically modified cells under identical conditions.…”

“…Since each FBP generates only one of several possible cell types, it is not a TSC. The molecular signals that regulate FBP differentiation involve direct and indirect cell-cell interactions that function through b-catenin (38,39). A summary of these results is presented in the Vermont Stem Cell Meeting Report (manuscript in preparation).…”

“…These two differentiated cell types were generated at equal frequency (75). (Note that FBP-derived ALI cultures typically generate more ciliated cells than Clara-like cells [20,38,39].) Rimmed clone cell differentiation was also stimulated by tracheal cells that were not in contact with the rimmed clone cells.…”