Evidence for multiple roles for grainyhead-like 2 in the establishment and maintenance of human mucociliary airway epithelium

Abstract

Most of the airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated basal progenitor cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia, there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in coordinating multiple cellular processes required for epithelial morphogenesis, differentiation, remodeling, and repair. However, only a few target genes have been identified, and little is known about GRHL function in the adult lung. Here we focus on the role of GRHL2 in primary human bronchial epithelial cells, both as undifferentiated progenitors and as they differentiate in air–liquid interface culture into an organized mucociliary epithelium with transepithelial resistance. Using a dominant-negative protein or shRNA to inhibit GRHL2, we follow changes in epithelial phenotype and gene transcription using RNA sequencing or microarray analysis. We identify several hundreds of genes that are directly or indirectly regulated by GRHL2 in both undifferentiated cells and air–liquid interface cultures. Using ChIP sequencing to map sites of GRHL2 binding in the basal cells, we identify 7,687 potential primary targets and confirm that GRHL2 binding is strongly enriched near GRHL2-regulated genes. Taken together, the results support the hypothesis that GRHL2 plays a key role in regulating many physiological functions of human airway epithelium, including those involving cell morphogenesis, adhesion, and motility.

GRHL2 coordinates regeneration of a polarized mucociliary epithelium from basal stem cells

“…4 E, the expression of genes encoding transcription factors that are known to promote ciliogenesis (Multicilin [Mcidas], Rfx2, and Myb) are all significantly down-regulated in Grhl2 mutant spheres. This is consistent with our previous ChIP-Seq evidence that these genes are direct targets for Grhl2 (Gao et al, 2013). Transcripts of the ciliated cell-specific gene, Foxj1, are also reduced, although this gene does not have associated Grhl2-binding sites.…”

“…Specifically, recent studies have implicated Notch3 in promoting the differentiation of adult basal stem cells into parabasal progenitors and Notch1 and 2 in fate selection between secretory and ciliated linages (Rock et al, 2011b;Danahay et al, 2015;Mori et al, 2015;Pardo-Saganta et al, 2015). ChIP-Seq has shown Grhl2 occupancy of binding sites near NOT CH1, NOT CH3, JAG1, and JAG2 (Gao et al, 2013). We therefore assayed the level of expression of various Notch pathway components in organoid cultures and found that in mutant spheres Notch1, Jag1, and Jag2 were significantly up-regulated, whereas Notch3 and Dll3 were downregulated compared with wild type.…”

“…These genes are therefore potentially part of a network that regulates and integrates airway epithelial morphogenesis and differentiation from basal progenitors (Gao et al, 2013). However, the functions of most of these genes in airway epithelial cell biology are not known.…”

“…Previously, we addressed the role of Grhl2 in human airway epithelium using Krt5 + Trp63 + primary BCs in culture (Gao et al, 2013). We found that expression of dominant-negative Grhl2 protein inhibited both the ability of daughter cells to form a polarized epithelium with barrier function and their differentiation.…”

“…Processes regulated by these factors include apical-basal polarity, intercellular adhesion, barrier function, and motility, as well as lineage choice and differentiation of progenitor populations (Wilanowski et al, 2002;Paré et al, 2012;Senga et al, 2012;Gao et al, 2013;Li et al, 2013). Epithelial tissues in which vertebrate grainyhead-like genes are known to be important include skin, developing neural tube, placental trophoblast, hepatic biliary cells, inner ear, and embryonic kidney (nephric duct; Ting et al, 2005;Chalmers et al, 2006;Werth et al, 2010; Pseudostratified airway epithelium of the lung is composed of polarized ciliated and secretory cells maintained by basal stem/progenitor cells.…”