RFX3 Modulation of FOXJ1 regulation of cilia genes in the human airway epithelium
© Didon et al.; licensee BioMed Central Ltd. 2013
Received: 28 January 2013
Accepted: 10 June 2013
Published: 3 July 2013
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.
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.
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.
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.
Cilia are typically classified as either motile or primary [1–3]. 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]. Motile cilia also occur as solitary structures, as sperm flagella, and in the embryonic node . Primary cilia are nonmotile, solitary structures that are present in many cell types, and often have sensory functions such as in the retina and renal tubules [8, 9].
The multiciliated cells of the human airway epithelium, accounting for 50 to 90% of the airway epithelial cell population [4, 5, 10, 11], perform the critical function of transporting mucus in a cephalad direction to remove inhaled environmental contaminants from the airways [4, 5]. Dysfunction of this transport apparatus, whether acquired or inherited, results in mucus congestion in the lower airways and serves as a nidus for recurrent infections [6, 12]. The airway multiciliated cells are derived from stem/progenitor cells within the basal cell population of the airway epithelium, cells capable of replicating and differentiating into secretory, intermediate and ciliated cells [13–15]. Generation of multiciliated cells in the adult airway epithelium is an ongoing process during airway epithelium homeostasis, and in response to injury [4, 5]. Substantial advances have been made in understanding the key regulators of motile and primary ciliated cell differentiation in model organisms such as mice, zebrafish and xenopus [16–20], but the transcriptional network regulating the differentiation of human airway basal progenitor cells into motile multiciliated cells is not well understood. Forkhead box J1 (FOXJ1) is one of the most well characterized transcription factors involved in motile ciliated cell differentiation in model organisms and has been suggested as a key regulator of the motile ciliated cell differentiation program [19–29]. 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].
With this background, we asked two questions. First, is FOXJ1 a key regulator of the multiciliated cell differentiation program of the human airway epithelium? Second, what roles do the transcriptional regulators NOTO and/or RFX play in human airway ciliated cell differentiation? To answer these questions, we capitalized on the recent development in our laboratory to culture pure populations of primary human airway epithelial basal cells obtained by bronchoscopy and airway epithelial brushing of healthy individuals . These basal cells have transcriptomes distinct from that of the airway differentiated cells, and are capable of differentiating into a multiciliated epithelium on air liquid interface cultures. The data demonstrate that in the human airway epithelium, FOXJ1 is a key regulator of multiciliated cell differentiation, that NOTO does not play a role in this process, and that RFX3 functions as transcriptional co-activator to FOXJ1, inducing expression of cilia genes involved in the differentiation towards the multiciliated cell lineage from basal progenitor cells.
Subjects were evaluated at the Department of Genetic Medicine Clinical Research Facility under the auspices of Weill Cornell and Rockefeller University NIH Clinical Translational Science Centers, using Weill Cornell Institutional Review Board clinical protocols approved for this study. Written informed consent was obtained.
Sampling the airway epithelium
Bronchoscopy was used to collect large airway epithelial cells by brushing the epithelium. The complete differentiated airway epithelium from 5 healthy, nonsmoking individuals was evaluated, and for 14 other individuals, the epithelium was cultured under conditions to obtain pure populations of basal cells as previously described [32–34] (see Additional file 1: Additional Methods for more detail).
Culture and immunohistochemistry characterization of basal cells
The basal cell culture and immunohistochemistry characterization protocol have been previously described . Cells were sub-cultured at day 7 to a density of 104 cells/cm2. Cells from passage 1 to 5 were characterized and used in this study (see Additional file 1: Additional Methods for more detail).
TaqMan quantitative real-time RT-PCR
TaqMan real-time RT-PCR was performed as previously described . Relative expression levels were determined with the average value of untransfected or EGFP-control plasmid transfected basal cells as the normalizer (see Additional file 1: Additional Methods for more detail).
Basal cell cultures and exogenous FOXJ1 and RFX3 expression were assessed by Western analysis as previously described . Immobilized proteins were reacted with anti-KRT5; anti-KRT14; anti-MUC5AC; anti-DNAI1; anti-acetylated TUBA; anti-FOXJ1; anti-RFX3 and anti-GAPDH antibodies (see Additional file 1: Additional Methods for more detail).
Airway epithelium differentiation in air-liquid interface culture
To assess expression profiles during the differentiation of human basal cells to ciliated cells, ciliated cell differentiation was induced from basal cells (n = 3 subjects) in vitro using air-liquid interface (ALI) cultures as previously described  (see Additional file 1: Additional Methods for more detail).
Gene transfer to primary human airway basal cells
Human FOXJ1 and RFX3 cDNA were subcloned into expression plasmids to generate PGK.FOXJ1.IRES.EGFP, PGK.RFX3.IRES.EGFP, CMV.FLAG-FOXJ1 and CMV.FLAG-RFX3 expression plasmids. PGK.EGFP and CMV.EGFP expression plasmids were used as a control expression plasmid, respectively. Firefly luciferase (Luc) reporter gene plasmids driven by the direct upstream promoters of DNALI1, SPAG6, KRT14 and FOXJ1 were generated using standard cloning methods. DNAI1-Luc, TEKT1-Luc, RFX3-Luc, RFX2-Luc and Random sequence-Luc reporter gene plasmids were commercially available. A Renilla luciferase control reporter plasmid was used for normalization of transfection efficiency. The plasmids were transfected into primary human airway epithelial basal cells using lipofectamine LTX and promoter firefly luciferase activity was read in a luminometer. The data are reported as fold-induction (FOXJ1 compared to EGFP or FOXJ1 + RFX3 compared to FOXJ1) of at least three independent experiments read in triplicate, normalized to Renilla luciferase activity (see Additional file 1: Additional Methods for more detail).
FOXJ1- RFX3 interaction
To assess the interaction of human FOXJ1 and RFX3, 293A cells were transfected with PGK.FOXJ1 and CMV.FLAG-RFX3 expression plasmids. Proteins were immunoprecipitated using EZview Red Anti-FLAG M2 affinity gel and eluted with FLAG peptide (see Additional file 1: Additional Methods for more detail).
All data in this study are presented as mean ± standard error. Statistical comparisons between continuous variables were calculated using an unpaired, two-tailed t test with unequal variance. A p-value <0.05 was considered to be significant (see Additional file 1: Additional Methods for more detail).
Primary human airway epithelial basal cell cultures
Temporal expression of FOXJ1 and RFX3 during human airway ciliated cell differentiation
Assessment of expression of the putative cilia-associated transcriptional regulators FOXJ1, 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 . In contrast, NOTO, a suggested FOXJ1-upstream master regulator in mouse notochord ciliogenesis , was not expressed at any time-point (Figure 1D). Interestingly, NOTO was also not expressed in the complete airway epithelium, suggesting that the signaling network regulating airway epithelial motile ciliogenesis in the human airway epithelium is somewhat different than notochord ciliogenesis in mice and zebrafish (not shown).
Effect of FOXJ1 gene transfer to primary human basal cells
Role of RFX3
RFX3 acts as a transcriptional co-activator to FOXJ1 that enhances the expression of cilia-associated genes
Human RFX3 interaction with human FOXJ1
Auto-regulatory feed-back mechanism of FOXJ1 and RFX3
Role of RFX2
RFX-family members other than RFX3 have also been implicated in cilia homeostasis in various model organisms [18, 20, 26, 37]. Assessment of expression of the different RFX-transcription factor members during human airway basal cell to ciliated cell differentiation on air liquid interface cultures in vitro demonstrated that the expression patterns over time for RFX2 were also characteristic of genes participating in the ciliated cell differentiation program , in similarity with FOXJ1 and RFX3 (Figure 1D, Additional file 4: Figure S2A). We further assessed the ability of FOXJ1 and /or RFX3 to induce endogenous RFX2 mRNA expression in basal cells transfected with FOXJ1 and /or RFX3 expression plasmids, as well as induced RFX2 promoter activity in basal cells co-transfected with FOXJ1 and /or RFX3 expression plasmids and a plasmid carrying the firefly luciferase reporter gene driven by the RFX2 promoter (Additional file 4: Figure S2B,C). The data demonstrated increased RFX2 promoter activity (p < 0.0005) and mRNA expression (p < 0.04) in cells transfected with the FOXJ1 expression plasmid alone and no significant induction of either RFX2 promoter activity or mRNA expression in cells transfected with RFX3 alone, compared to cells transfected with the control EGFP plasmid. Cells transfected with RFX3 seemed to have elevated RFX2 mRNA expression, although to a variable degree and not significant. When RFX3 was co-transfected with FOXJ1, however, the FOXJ1 induced RFX2 promoter activity (p < 0.03) was further enhanced, suggesting a similar and potentially partially overlapping role for RFX2 and RFX3 downstream of FOXJ1 in human airway epithelial ciliogenesis.
Motile multiciliated cells, the dominant cell type in the airway epithelium, perform the critical function of transporting mucus and entrapped inhaled environmental contaminants up from the airways [4, 5]. Dysfunction of this mucociliary clearance apparatus during development or after injury results in airway mucus-plugging which constitutes a source of recurring infections. In this study, we hypothesized that in the human airway epithelium, the two transcription factors FOXJ1 and RFX3 cooperatively regulate the differentiation of cells from the basal progenitor cell lineage toward the ciliated cell linage.
Primary human airway basal cells from healthy nonsmokers induced expression of FOXJ1 and RFX3 while differentiating into a ciliated epithelium in air-liquid interface cultures. Expression of FOXJ1 in basal cells transfected and cultured on plastic induced promoter-activity from ciliated cell genes, but not basal cell gene promoters, as well as increased expression of RFX3 and a wide variety of ciliated cell genes, but not the expression of basal or secretory cell genes. In contrast, RFX3 induced FOXJ1 expression but did not induce expression of cilia-related genes, which suggest that a threshold amount of FOXJ1 is required to induce cilia gene expression. When RFX3 was expressed together with FOXJ1, however, the combination of the two factors up-regulated ciliated gene promoters and mRNA expression to a greater extent than FOXJ1 alone. Knockdown and chromatin immunoprecipitation (ChIP) experiments validating the effects of FOXJ1 and RFX3 on downstream cilia-associated genes would have been valuable, but due to technical limitations, including a baseline expression of the downstream target genes bordering or below detectable levels in basal cells, this was not feasible. A bioinformatic assessment of potential FOXJ1 and RFX3 binding sites in the promoters of the cilia-related target genes could not fully explain the FOXJ1 and RFX3 mediated transcriptional regulation of these genes (data not shown).
However, immunoprecipitation studies in 293A cells corroborated our findings and demonstrate an interaction between the human FOXJ1 and RFX3 proteins. Our data further suggest a positive auto-regulatory FOXJ1-RFX3 feedback mechanism where FOXJ1 is upstream of RFX3. Hence, our findings demonstrate that FOXJ1 is an important regulator of cilia-associated gene expression in primary human airway epithelial basal cells and that RFX3 acts as a co-activator to FOXJ1 in this process during basal/progenitor cell differentiation toward the multiciliated cell lineage.
Ciliated cell differentiation in model organs
Much of our knowledge of motile monocilium and multiciliated cell differentiation is based on studies of the mono motile ciliated cells of the mouse node and its homologues in zebrafish and xenopus [16–19]. FOXJ1 is one of the most well characterized transcription factors involved in motile ciliated cell differentiation, where FOXJ1 is known to regulate programs promoting basal body docking and axoneme formation [19–29]. Also the RFX-family of transcription factors has been shown to be a major regulator of ciliogenesis in multiple organisms, controlling the expression of the many essential genes required for making cilia [38–42] and has recently been shown to co-regulate cilia gene expression together with FOXJ1 in Drosophila . Beckers et al  demonstrated evidence that the murine nothochord motile monocilium differentiation program is regulated by the transcription factor NOTO, acting upstream of both FOXJ1 and RFX3. The novel nuclear protein multicilin was also recently shown to drive the expression of both FOXJ1 and multiciliated cell specific genes in frog and mouse multiciliated epithelia . Several investigators, using murine models and transformed human cell lines, have demonstrated that FOXJ1 is a key regulator of airway epithelial ciliated cell differentiation [21, 22, 27, 29, 44–50]. In the airway epithelium, Notch signaling had been demonstrated to be the most upstream cue for transcriptional events during the differentiation of multiciliated and other airway epithelial cell types .
Murine models of airway development and cellular differentiation have been critical to our basic understanding of the mucociliary clearance apparatus during development or after injury [21, 51], but the human airways differ from those of mice to some extent . The networks regulating cell commitment in the airway epithelium in humans are therefore likely to differ somewhat from those of mice and other animal models.
Human airway epithelial multiciliated cell differentiation
The human FOXJ1 gene was first cloned over 10 yr ago [52, 53], yet the function of FOXJ1 in the human airway epithelium remains relatively unknown. Studies have addressed whether mutations in the FOXJ1 gene could be linked to primary ciliary dyskinesia [54, 55], although this hypothesis has not been supported. The activity of the human FOXJ1 promoter has been demonstrated to be specific to ciliated cells in the mouse airway epithelium in vivo. The mouse FOXJ1 promoter has been shown to be active in primary human airway epithelial cells and the human airway epithelial cell line H441, but not in the human fibroblast cell line HT1080 . The murine and human FOXJ1 gene have been transfected into the transformed human airway epithelial BEAS-2B cell line and in the human embryonic kidney (HEK-293) cell line [21, 45, 57, 58]. Expression of murine FOXJ1 in BEAS-2B cells failed to induce cilia development , but consistent with the findings in the present study, murine FOXJ1 induced SPAG6 promoter activity in these cells .
FOXJ1 and RFX3 are highly expressed in the small airway epithelium , consistent with the concept that FOXJ1 and RFX3 together may play a pivotal role in maintaining homeostasis of the multiciliated cell population in the adult human airway epithelium. In the current study, we found that NOTO is not expressed in the human airway epithelium nor is its expression induced by ALI-mediated airway epithelial differentiation in vitro. Hence, in contrast to murine notochord ciliogenesis, FOXJ1 and RFX3 are not regulated upstream by NOTO in the human airway epithelium. We can only speculate on the identity of these players, but possible candidates include Notch, multicilin, FOXA2 and SOX2 [41, 43, 59–61].
Most evidence suggests that the basal cells of the human airway epithelium contain the pluripotent progenitor cell population, capable of replicating and differentiating into intermediate, secretory, and multiciliated cells [13–15, 32]. The present study demonstrates that, under conditions that normally prevent cellular differentiation, FOXJ1 ± RFX3 plays a significant role in modulating differentiation steps in multiciliated cell differentiation from the basal cell lineage. These findings also support the concept that airway epithelial basal cells contain the progenitor cell population capable of differentiating into multiciliated cells.
These studies were supported, in part, by P50 HL084936, UL1 RR024996, and UL1 RR024143. LD was supported, in part, by the Swedish Research Council – medicine (2009-7395), the Wenner-Gren Foundations, the Sweden America Foundation, The Swedish Society of Medicine and the Foundation BLANCEFLOR Boncompagni-Ludovisi, née Bildt. RW is supported, in part, by T32 HL094284.
We thank G. Wang and J. Salit for helpful advice; and N. Mohamed and D.N. McCarthy for help in preparing this manuscript.
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