RFX3 Modulation of FOXJ1 regulation of cilia genes in the human airway epithelium

Lukas Didon,Neil R Hackett,Rachel K Zwick,Ion Wa Chao,Rui Wang,Matthew S Walters,Ronald G Crystal

doi:10.1186/1465-9921-14-70

Abstract

BackgroundCiliated cells play a central role in cleansing the airways of inhaled contaminants. They are derived from basal cells that include the airway stem/progenitor cells. In animal models, the transcription factor FOXJ1 has been shown to induce differentiation to the ciliated cell lineage, and the RFX transcription factor-family has been shown to be necessary for, but not sufficient to induce, correct cilia development.MethodsTo test the hypothesis that FOXJ1 and RFX3 cooperatively induce expression of ciliated genes in the differentiation process of basal progenitor cells toward a ciliated cell linage in the human airway epithelium, primary human airway basal cells were assessed under conditions of in vitro differentiation induced by plasmid-mediated gene transfer of FOXJ1 and/or RFX3. TaqMan PCR was used to quantify mRNA levels of basal, secretory, and cilia-associated genes.ResultsBasal cells, when cultured in air-liquid interface, differentiated into a ciliated epithelium, expressing FOXJ1 and RFX3. Transfection of FOXJ1 into resting basal cells activated promoters and induced expression of ciliated cell genes as well as both FOXJ1 and RFX3, but not basal cell genes. Transfection of RFX3 induced expression of RFX3 but not FOXJ1, nor the expression of cilia-related genes. The combination of FOXJ1 + RFX3 enhanced ciliated gene promoter activity and mRNA expression beyond that due to FOXJ1 alone. Corroborating immunoprecipitation studies demonstrated an interaction between FOXJ1 and RFX3.ConclusionFOXJ1 is an important regulator of cilia gene expression during ciliated cell differentiation, with RFX3 as a transcriptional co-activator to FOXJ1, helping to induce the expression of cilia genes in the process of ciliated cell differentiation of basal/progenitor cells.

Highlights

Ciliated cells play a central role in cleansing the airways of inhaled contaminants
Primary human airway epithelial basal cell cultures To test the hypothesis that Forkhead box J1 (FOXJ1) is a key regulator of the motile multiciliated cell differentiation program in the human airway epithelium, we first established primary human airway epithelial basal cell cultures [32]
Temporal expression of FOXJ1 and RFX3 during human airway ciliated cell differentiation Assessment of expression of the putative cilia-associated transcriptional regulators FOXJ1, Not homeobox (NOTO) and RFX3 during human airway basal cell to ciliated cell differentiation on air liquid interface cultures (ALI) in vitro demonstrated that the expression pattern over time for both FOXJ1 and RFX3 was characteristic of genes participating in the ciliated cell differentiation program [36]

Summary

Introduction

Ciliated cells play a central role in cleansing the airways of inhaled contaminants They are derived from basal cells that include the airway stem/progenitor cells. Motile multiciliated cells typically contain 100– 300 specialized cilia that are able to beat in a coordinated fashion and move liquid across the cell surface [4,5] These cilia are found in only a few cell types in humans, including the airway epithelium lining the lung and sinuses, ependymal cells lining the ventricles of the brain and spinal canal, and the epithelium of the oviducts and epididymal ducts [5,6]. To a variable degree, depending on the species, organ and whether multi – or monociliated, the role of FOXJ1 in the differentiation of motile ciliated cells is modulated by the transcription factor Not homeobox (NOTO) and the regulatory factor X (RFX)-family [16,20,24,30,31]

Methods

Results

Discussion

Conclusion