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

Abstract

Background: 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. 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.

Methods: To 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.

Results: Basal 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.

Conclusion: FOXJ1 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.

Figure 1

Primary human airway epithelial basal/progenitor cell cultures. A. Immunocytochemical verification of basal cell phenotype. Airway basal cells from healthy nonsmokers (n = 14) were purified by culturing large airway epithelial cells obtained by bronchoscopy and brushing. After 7 days of culture, the cells were trypsinized and cytospin preparations were assessed for expression of cytokeratin 5 (KRT5; basal cell-specific marker); β-tubulin IV (TUBB4; marker of ciliated cells); mucin 5AC (MUC5AC; secretory cell marker); and N-cadherin (CDH2; marker for mesenchymal cells). B. TaqMan quantitative real-time RT-PCR assessment of mRNA expression of the basal cell markers KRT5, KRT14 and tumor protein p63 (TP63) in complete large airway epithelial cell population (n = 5 subjects) recovered by airway brushing (blue bars) compared to basal cell cultures (day 7; red bars. n = 3 subjects). C. Western analysis for the basal cell markers KRT5 and KRT14, the cilia markers dynein, axonemal, intermediate chain 1 (DNAI1) and acetylated α-tubulin (Ac-TUBA), the secretory cell marker secretoglobin family 1A member 1 (SCGB1A1) and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Lane 1 – differentiated airway epithelium obtained by brushing. Lane 2 - cell extracts from basal cell cultures (day 7). D. Forkhead box J1 (FOXJ1), regulatory factor X3 (RFX3), Not homeobox (NOTO) and GAPDH mRNA expression during basal cell differentiation on air liquid interface (ALI) over 28 days (n = 3 subjects per time point). The expression was assessed by TaqMan quantitative real-time RT-PCR and a gene was deemed “undetectable” if not detected after 40 cycles of assessed undiluted cDNA. B = basal cells not cultured on ALI. * p < 0.05; ** p < 0.01; *** p < 0.005 compare all to GAPDH.

Figure 2

Expression of the transcription factor FOXJ1 in primary human basal cells after transfection with a FOXJ1-expression plasmid. A. TaqMan quantitative real-time RT-PCR assessment of the relative FOXJ1 mRNA expression in untransfected basal cells (mock), compared to increasing amounts EGFP-transfected or FOXJ1-transfected basal cells. B. Western analysis assessment of FOXJ1 and GAPDH expression in untransfected basal cells and cells transfected with either the control EGFP or FOXJ1 expression plasmids. Data represents the mean expression level ± standard error of pooled data from three replicated individual experiments with cells from three different subjects. All assays were carried out 24 hr after transfection.

Figure 3

FOXJ1-mediated expression of cilia-related genes and enhancement of their promoters in primary human basal cells transfected with either control EGFP or FOXJ1 expression plasmids. All data is based on TaqMan quantitative real-time RT-PCR 48 hr after transfection and a gene was deemed “undetectable” if not detected after 40 cycles of assessed undiluted cDNA. A. Basal cell markers KRT5, KRT14 and TP63. B. Secretory cell markers MUC5AC, MUC5B and SCGB1A1. C. Ciliated cell-associated genes, including centrin 2 (CETN2); dynein, axonemal, heavy chain 11 (DNAH11), dynein, axonemal, intermediate chain 1 (DNAI1); dynein, axonemal, light intermediate chain 1 (DNALI1); EF-hand domain, C-terminal, containing 1 (EFHC1); sperm associated antigen 6 (SPAG6), tektin 1 (TEKT1), TEKT2 and tubulin, alpha 1a (TUBA1A). D. Shown is firefly luciferase activity in basal cells transfected with control EGFP or FOXJ1 expression plasmids together with firefly luciferase reporter gene plasmids driven by promoters of the ciliated cell-associated genes DNALI1, DNAI1, SPAG6 and TEKT1. Controls include the promoter for the basal cell marker KRT14 and a random sequence. The data were normalized for transfection efficiency per well by Renilla luciferase activity from co-transfected Renilla luciferase plasmid (pTK-RL). Bars represent mean ± standard error of pooled data from replicates of a minimum of three individual experiments with cells from three different subjects.

Figure 4

Expression of cilia-associated transcription factor regulatory factor X3 (RFX3) in basal cells after transfection with a FOXJ1 or RFX3-expression plasmid. A. TaqMan quantitative real-time RT-PCR assessment of the relative endogenous RFX3 mRNA expression in EGFP or FOXJ1-transfected basal cells 48 hr after transfection. B. Firefly luciferase activity in basal cells transfected with control EGFP or FOXJ1 expression plasmids together with firefly luciferase reporter gene plasmid driven by the RFX3 promoter 48 hr after transfection. C. TaqMan quantitative real-time RT-PCR assessment of the relative RFX3 mRNA expression in basal cells transfected with the control EGFP or RFX3 expression plasmid. D. Western analysis assessment of RFX3 and GAPDH expression in untransfected basal cells and cells transfected with either control EGFP or RFX3 expression plasmid. Data represents the mean ± standard error of pooled data from replicates of a minimum of three individual experiments with cells from three different subjects.

Figure 5

RFX3-mediated enhancement of FOXJ1-induced expression of ciliated cell-associated genes in primary human basal cells transfected with both a FOXJ1 and RFX3 expression plasmid. A. TaqMan quantitative real-time RT-PCR assessment of the relative mRNA expression of the ciliated cell-associated genes DNAI1, DNALI1, SPAG6, TEKT1 and TEKT2 in basal cells transfected with 50% and 100% FOXJ1 expression plasmids compared to basal cells co-transfected with 50% and 100% of FOXJ1 and RFX3 expression plasmids together for 48 hr after transfection. B. TaqMan quantitative real-time RT-PCR assessment of the basal cell markers KRT5, KRT14 and TP63. C. Firefly luciferase activity assessment of basal cells transfected with firefly luciferase reporter gene plasmids driven by promoters of the ciliated cell-associated genes DNALI1, DNAI1, SPAG6 and TEKT1; or the basal cell marker KRT14; or a random sequence as a negative control. Data were normalized for transfection efficiency per well by Renilla luciferase activity from co-transfected Renilla luciferase plasmid (pTK-RL) 48 hr after transfection. Bars represent mean ± standard error of pooled data from replicates of a minimum of three individual experiments from different subjects.

Figure 6

Human FOXJ1 and RFX3 protein-to-protein interaction. 293A cells were transfected with FOXJ1 and RFX3 expression plasmids. After 48 hr, cell lysates were prepared in lysis buffer and incubated overnight with Anti-FLAG M2 affinity gel. Input and immunoprecipitated fractions were resolved by SDS-PAGE and analyzed by Western blotting with antibody specific for FOXJ1, FLAG and GAPDH as a loading control. Lanes 1-4 - Before immunoprecipitation (input). Lanes 5-8 - After RFX3 targeted immunoprecipitation. Lane 1 - PGK.EGFP and CMV.empty (mock); lane 2 - PGK.FOXJ1 and CMV.empty (FOXJ1); lane 3 - CMV.FLAG-RFX3 and PGK.EGFP (RFX3); lane 4 - PGK.FOXJ1 and CMV.FLAG-RFX3 (FOXJ1 + RFX3); lane 5 - PGK.EGFP and CMV.empty (mock); lane 6 - PGK.FOXJ1 and CMV.empty (FOXJ1); lane 7 - CMV.FLAG-RFX3 and PGK.EGFP (RFX3); and lane 8 - PGK.FOXJ1 and CMV.FLAG-RFX3 (FOXJ1 + RFX3).

Figure 7

FOXJ1-RFX3 auto-regulatory feedback. A. Firefly luciferase activity in basal cells transfected with control EGFP, FOXJ1, RFX3 or FOXJ1+ RFX3 expression plasmids together with firefly luciferase reporter gene plasmids driven by the FOXJ1 and RFX3 promoters. The data were normalized for transfection efficiency per well by Renilla luciferase activity from co-transfected Renilla luciferase plasmid (pTK-RL) 48 hour after transfection. Bars represent mean ± standard error of pooled data from a minimum of three individual experiments assessed for 48 hr after transfection. B. TaqMan quantitative real-time RT-PCR assessment of the relative endogenous FOXJ1 mRNA expression in EGFP or RFX3-transfected basal cells for 48 hr after transfection. Bars represent mean ± standard error of pooled data from replicates of three individual experiments from different subjects.