Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1

Relaxin/insulin-like family peptide receptor 1 (RXFP1) mediates relaxin’s antifibrotic effects and has reduced expression in the lung and skin of patients with fibrotic interstitial lung disease (fILD) including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). This may explain the failure of relaxin-based anti-fibrotic treatments in SSc, but the regulatory mechanisms controlling RXFP1 expression remain largely unknown. This study aimed to identify regulatory elements of RXFP1 that may function differentially in fibrotic fibroblasts. We identified and evaluated a distal regulatory region of RXFP1 in lung fibroblasts using a luciferase reporter system. Using serial deletions, an enhancer upregulating pGL3-promoter activity was localized to the distal region between -584 to -242bp from the distal transcription start site (TSS). This enhancer exhibited reduced activity in IPF and SSc lung fibroblasts. Bioinformatic analysis identified two clusters of activator protein 1 (AP-1) transcription factor binding sites within the enhancer. Site-directed mutagenesis of the binding sites confirmed that only one cluster reduced activity (-358 to -353 relative to distal TSS). Co-expression of FOS in lung fibroblasts further increased enhancer activity. In vitro complex formation with a labeled probe spanning the functional AP-1 site using nuclear proteins isolated from lung fibroblasts confirmed a specific DNA/protein complex formation. Application of antibodies against JUN and FOS resulted in the complex alteration, while antibodies to JUNB and FOSL1 did not. Analysis of AP-1 binding in 5 pairs of control and IPF lung fibroblasts detected positive binding more frequently in control fibroblasts. Expression of JUN and FOS was reduced and correlated positively with RXFP1 expression in IPF lungs. In conclusion, we identified a distal enhancer of RXFP1 with differential activity in fibrotic lung fibroblasts involving AP-1 transcription factors. Our study provides insight into RXFP1 downregulation in fILD and may support efforts to reevaluate relaxin-based therapeutics alongside upregulation of RXFP1 transcription.


Introduction
Pulmonary fibrosis is a hallmark of fibrotic interstitial lung diseases (fILD). Although the pathogenesis of fILD is not fully understood [1], fibroblast activation in the lungs of patients with fILD results in aberrant extracellular matrix (ECM) collagen accumulation [2]. Idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) are two of the most common types of fILD. IPF is a chronic and progressive disease associated with high morbidity and mortality [1,2]. In patients with SSc, fILD is the disease manifestation associated with the highest mortality [3]. Despite the increasing global burden of fILDs [4,5], our understanding of the mechanisms underlying the development and progression of fibrosis and our ability to target these pathogenic pathways is lacking.
RXFP1 expression is downregulated in whole lung tissue and lung fibroblasts from patients with fILD, including IPF and SSc [17][18][19][20]. In vitro studies of fibroblasts isolated from IPF and SSc lungs demonstrates minimal responsiveness to relaxin treatment in reducing extracellular matrix accumulation, but restoration of RXFP1 expression restores the anti-fibrotic effects in these cells [17]. However, transcriptional regulation of RXFP1 in fibroblasts is poorly understood.
Characterization of RXFP1 regulation will provide insight to therapeutic targets for restoring relaxin's anti-fibrotic effects in patients with fILD [14].
Activator protein 1 (AP-1) belongs to the superfamily of basic leucine zipper DNA-binding transcription factors. It exists as a dimer mainly consisting of two subfamilies: Fos and Jun subunits [21]. AP-1 targets the TPA response element (TRE, also known as the AP-1 site) that regulates gene expression in response to physiologic and pathologic functions [22]. This includes the transcriptional upregulation of genes important for tissue remodeling [23]. AP-1 also plays a central role in enhancer repertoires selection in fibroblasts, which are critical for tissue differentiation during development [24]. There is limited research to date investigating the role of AP-1 superfamily transcription factor regulation of RXFP1.
In this study, we sought to characterize the regulatory regions of the RXFP1 gene and to identify transcriptional elements important in its regulation. Through fine mapping of these regions, we identified a novel distal enhancer containing specific binding motifs for AP-1. We further demonstrated direct binding of AP-1 to the RXFP1 regulatory elements using in vitro models. Our study provides insight to the transcriptional regulation of RXFP1 in lung fibroblasts, which may have future implications for relaxin-based therapeutics.

Cell Culture
The study was approved and was determined to be "non-human" study by the Institutional Review Board at the University of Pittsburgh (STUDY18100070). Donor lungs were obtained from the CORE (Center for Organ Recovery and Education). IPF and SSc explanted lungs were recovered from patients who underwent lung transplantation at the University of Pittsburgh Medical Center. Lung fibroblast lines derived from these lungs were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum and 50 μg/mL penicillin-streptomycin (Thermo Fisher Scientific Inc.) at 37°C and 5% CO 2 .

Plasmids and Cloning
Polymerase chain reaction (PCR) products were gel purified using Qiagen QlAquick gel purification columns (Qiagen) according to the manufacturer's instructions. The PCR products were cloned using promoter-less pGL3-basic (pB) vector or pGL3-promoter (pP) vector containing a SV40 promoter (Promega Corporation) and Gibson Assembly (New England BioLabs). The relative location and size of RXFP1 DNA in each luciferase reporter plasmid are listed in Supplemental Table 1.

Dual Luciferase Assay
Fibroblasts were seeded at 5,000 cells/well in 24 well cell culture plates and cultured overnight prior to transfection with either the pGL3-RXFP1 reporter plasmids alone (0.4μg/well) or co-transfection with a transcription factor expression plasmid (0.3μg pGL3-RXFP1 reporter and 0.1μg expression plasmid per well). A Renilla luciferase vector (pGL4.74 [hRluc/TK]) was used as a control (0.001μg/well, Promega) for transfection efficiency. Plasmids were transfected into primary lung fibroblasts using Lipofectamine 2000 according to the manufacturers' instruction. At 40 hours post-transfection, the cells were washed with PBS, lysed in 1 × passive lysis buffer and analyzed using the Dual-Luciferase Reporter Assay System (Promega Corporation) and a SpectraMax L Microplate Reader (Molecular Devices, LLC.). Relative expression levels of pGL-RXFP1 reporters were normalized against pB or pP vector luciferase activity.

6
Prediction of putative promoter and TATA element DNA sequences upstream of both distal and proximal transcriptional start sites (TSS) were used to identify putative promoter and TATA elements. The Neural Network Promoter Prediction method (http://www.fruitfly.org/seq_tools/promoter.html) was used with a minimum promoter score of 0.85 [25]. The location of each identified element was determined based on the corresponding TSS.

Site-Directed Mutagenesis
Site-directed mutagenesis of the AP-1 binding sites in the distal enhancer reporter plasmids were performed using the Q5® Site-Directed Mutagenesis Kit (New England BioLabs).

Nuclear Protein Extraction
Nuclear proteins were prepared using fibroblasts at 80-90% confluency and the Nuclear Extract Kit (Active Motif), according to the manufacturer's protocol. and JUNB (C-11) from Santa Cruz Biotechnology with nuclear proteins for 10 minutes on ice and 10 minutes at room temperature prior to the binding reaction described above. Rabbit IgG (Cell Signaling) was used as a negative control.

Chromatin Immunoprecipitation (ChIP) Assay
ChIP assay was performed as described [26,27]. Briefly, lung fibroblasts were grown on 100-mm tissue culture dishes to 90% confluence. Cells were cross-linked with 1% formaldehyde for 10 minutes and harvested for fragmentation using sonication. The chromatin fragments were immunoprecipitated with 3μg of the indicated antibodies for c-JUN (Cell signaling) and rabbit normal IgG (Cell signaling). The precipitated fragments were washed five times and analyzed by PCR using a primer pair (F: 5'-AAACACTGGACTGGGTTTGG-3' and R: 5'-GGAAAGTAGGCCCCTTGAGA-3') spanning the putative AP-1 binding site 2 on the RXFP1 enhancer. ChIP assay was performed using rabbit IgG as a negative control. Densitometry analysis of the PCR amplification was performed using ImageJ [28] Positive pulldown of bound DNA sequences was determined using the IgG as a control. (fPKM) and were square root transformed for normality prior to analysis.

Statistical Analysis
All data were expressed as the mean ± SD. Student's t-test was used for two-way comparisons. Gene expression levels of control and IPF groups were compared using the Mann-Whitney U test. Correlation of FOS and JUN gene expression levels with RXFP1 expression levels was analyzed using linear regression modeling as described [30]. All analyses were performed in Prism GraphPad version 7.05 and a p value < 0.05 significance threshold was used. . This is designated as the "Long" form of RXFP1 (https://www.gencodegenes.org/). As shown in Figure 1A, there are multiple splicing variants associated with Short RXFP1 [31], while only one transcript is associated with the Long form.

Identification of a functional promoter associated with distinct
To determine whether a functional promoter is associated with each of the two forms of RXFP1, we analyzed the core promoter regions of each transcript using a pGL3 luciferase reporter system and primary lung fibroblasts isolated from donor lungs, as controls, and IPF lungs.
A 233bp DNA element spanning -142 to +90 of the distal TSS (hg38, chr4:158,315,311) for the Long form (distal promoter), and a 194bp fragment covering -145 to +48 of the proximal TSS (hg38, chr4:158,521,714) for the Short form (proximal promoter) were tested (hereafter, all sequence locations are numbered relative to its corresponding TSS). As shown in Figure 1B, the distal promoter showed increased activity compared with pB vector, a promoter-less vector for testing promoter activity of targeted sequences, in both control and IPF lung fibroblasts (p=0.004 and 0.002, respectively). In contrast, the reporter activities for the proximal promoter in both control and IPF fibroblasts were reduced compared with the pB vector.
We further analyzed the two promoter regions for chromatin characteristics associated with active transcriptional regulation including H3K4Me1, H3K27Ac, H3K4Me3 and DNAse sensitivity clusters using the Encyclopedia of DNA Elements (ENCODE) histone ChIP data tracts in the UCSC genome browser ( Figure 1C). Consistent with the reporter assay, only the distal promoter region was associated with positive transcriptional regulation signals, indicating that this was the only functional core promoter for the RXFP1 gene in lung fibroblasts.

Differential distal promoter activities between control and fibrotic lung fibroblasts
Given the lack of promoter activity in the core proximal regulatory region, we extended our search for potential regulatory elements to both the proximal (PE) and distal (DE) regulatory regions. We analyzed the likelihood of a functional promoter by identifying a TATA box in a 3.1kb region (-2158bp to +971bp) and a 1.4kb region (-1202bp to +161bp) within the DE and PE regions, respectively. These regions possess potential regulatory functions based on the UCSC genome browser. Consistent with the lack of proximal promoter activity, there was no TATA box within 200bp upstream of the proximal TSS. However, a potential site was identified in the proximal region at -1095 to -1114. For the distal region, a TATA box was identified at -16 to +3 in addition to another site between -1946bp to -1927bp (Figure 2A).
These extended regions were further characterized in lung fibroblasts from control, IPF, and SSc patients for promoter activity using pB. Similar to the proximal core promoter, there was no increased activity for the PE in any fibroblast lines compared to the pB vector ( Figure 2B).
We performed serial deletions of the 1.4kb PE to rule out any repressor element interfering with promoter activity. Deleting 274 bp or 700 bp upstream sequences did not result in any significant promoter activity increase compared to the pB vector ( Figure 2C), further supporting that only the distal regulatory region has promoter function.

Localization of an enhancer region upstream of the distal promoter with differential activities in control and fibrotic fibroblasts
Since the extended distal region retained promoter activities among control and fibrotic fibroblasts, we tested whether the extended distal region was associated with enhancer function using pGL3promoter, which contains a SV40 promoter and used for testing enhancer activity of targeted sequences. Using control fibroblasts, we consistently observed greater than 50-fold enhancer activity in the distal region while there was no activity for the proximal extended region compared to the pP vector ( Figure 2D). The distal enhancer activity was significantly reduced in This was confirmed with the additional deletion of 1233bp 3' sequences (DE-D2toD3) of the D2 clone that fully restored the 3.1kb enhancer activity (106 ± 6%).

Fine mapping of the distal enhancer region
The distal enhancer partially overlaps with a region of dense transcription factor binding sites (TFBS, https://genome.ucsc.edu/)( Figure 3A). Therefore, we constructed a 608bp (−675 to −68) clone based on the TF binding cluster and designated it as pP-TFBS. Direct comparison of the distal RXFP1 enhancer (pP-D2toD3) and the TFBS element showed similar enhancer activities in control and SSc lung fibroblasts ( Figure 3B).
The enhancer activities were significantly reduced in SSc compared to control fibroblasts.
We performed serial deletion using the pP-TFBS to further map the enhancer region ( Figure 3C). increased activity compared to TFBS-D1 (p=<0.001). Thus, the enhancer appears to reside in this 343bp region (Figure 3D).

Distal enhancer activity is partially mediated through AP-1
To identify transcription factors that may mediate the enhancer activity, we mined the UCSC genome browser and identified binding sites for multiple transcription factors (supplemental Figure 1). Since AP-1 is known to be an important transcription factor in extracellular matrix metabolism [23], and also has multiple known binding sites, we searched for an AP-1 binding site within our 343bp enhancer region using PROMO [32]. Two clusters of AP-1 binding sites were identified at -525 to -520 (site 1) and -358 to -353 (site 2) (Figure 4A). To   A number of studies support relaxin as a potent anti-fibrotic agent [7-11, 33, 34]. Relaxin enhances the degradation of ECM in tissues by upregulating members of the matrix metalloproteinase (MMP) family [35]. The failed clinical studies for relaxin-based treatments in SSc patients [12] may be related to reduced expression of RXFP1 in fibroblasts of these patients, which would abrogate their responsiveness to relaxin [17][18][19]31]. Patients with IPF and SSc with higher RXFP1 expression in their lungs have better pulmonary function, supporting the pathophysiologic relevance of this locus in fILDs [17]. In vitro silencing of RXFP1 results in insensitivity to exogenous relaxin, an effect which is reversed by enhancement of RXFP1 expression in both control and IPF lung fibroblasts [17]. In this context, upregulation of RXFP1 may serve as a therapeutic option that would help to restore the responsiveness to relaxin-based therapies in fibrotic tissues [36]. Our study suggests that transcriptional modulation of RXFP1 in fibroblasts from patients with fILD may be one of the strategies to restore RXFP1 expression and the responsiveness to relaxin-based antifibrotic therapies in patients with IPF and SSc.

AP-1 is ubiquitously expressed in different cells and tissues and plays important roles in
multiple cellular processes including proliferation, differentiation, senescence, and cell death [21,37]. The AP-1 superfamily consists of four subfamilies, including FOS, JUN, ATF, and MAF, which exert their functions as homo-or hetero-dimers formed through their basic leucine-zipper (bZIP) motifs. The dimers formed with different AP-1 proteins are often associated with differential transcriptional regulation of target genes [38]. In general, the dimer of FOS and JUN is associated with positive gene regulation, while other family members such as JUNB act as negative transcriptional regulators [38]. Context dependent regulation by AP-1 transcription factors is also reported [37,39]. AP-1 transcription factors can also preferentially bind to distal enhancers instead of promoters in regulating target genes [40], supporting the finding from this study. We identified FOS and JUN as positive regulators for the RXFP1 gene distal enhancer in lung fibroblasts. We also found that their expression levels were reduced in IPF lungs. By upregulating these transcription factors in IPF fibroblasts we may be able to restore RXFP1 expression and thus responsiveness to relaxin-based therapeutics in fibrotic fibroblasts.
Conversely, FOSL2, a member of the AP-1 FOS subfamily has been shown to exert profibrotic effects. Transgenic Fosl2 mice develop spontaneous lung fibrosis with Fosl2expressing macrophages promoting lung fibrosis [41,42]. Interestingly, in the LGRC expression dataset, expression levels of FOSL2 and RXFP1 were negatively correlated (data not shown).
Therefore, the differential effects on lung fibrosis between JUN and FOS from this study in fibroblasts and the FOSL2 expression in mice macrophages illustrates the complexity of AP-1 family functions in lung fibrosis. Additionally, we found that miR-144-3p downregulates RXFP1 expression through 3'-untranslated region and JUN was required for constitutive miR-144-3p expression in lung fibroblasts, suggesting distinct function may be associated with the same AP-1 factor depending on their partners for dimerization. Although it is out of the scope of this study, systematic analysis of different AP-1 members in regulating, positively or negatively, RXFP1 expression is important for understanding the transcriptional regulation of this gene. The lack of regulatory functions in the proximal region is surprising, and may be due to the distal enhancer regulating the proximal region over a long range, for example through chromatin conformation changes [43]. As reviewed by Bejjani and colleagues, genome-wide analysis has shown that AP-1 commonly binds the distal enhancers and regulates distant genes [40]. Analysis of the chromatin architecture in the RXFP1 locus will be essential to determining whether AP-1 mediates distant control of the weak proximal regulatory region of RXFP1 through this mechanism. In addition, reduced AP-1 binding to the RXFP1 enhancer in IPF fibroblasts maybe due to the masking of AP-1 binding site by differential DNA methylation in this locus in IPF fibroblasts. Therefore, characterization of the epigenetic changes in fibrotic fibroblasts are warranted.
Our study does have some limitations. First, the analysis of RXFP1 regulatory elements was mainly performed using primary lung fibroblasts from control, IPF and SSc lungs. We showed reduced direct binding of JUN to the RXFP1 enhancer in lung fibroblasts using ChIP assay and positive correlation of JUN and FOS expression with RXFP1 in IPF whole lung expression.
However, the expression in whole lung may mask the cell-type specific expression differences of these genes. Second, we analyzed the in vivo binding of AP-1 to the RXFP1 enhancer using 5 pairs of control and IPF lung fibroblast lines. Since the fibroblasts isolated from lungs are often heterogeneous [44], analysis in additional independent fibrotic and control fibroblast lines is warranted. Third, the AP-1 family consists of a large number of different transcription factors with some distinct and similar functions [21,37]. We only focused our analysis on JUN and FOS.
Comprehensive analysis of other AP-1 family members in fibroblast RXFP1 regulation is important.
In conclusion, we identified a distal enhancer of RXFP1 with differential activity in fibrotic lung fibroblasts involving AP-1 transcription factors. Our study provides insight into the reduced expression of RXFP1 in patients with IPF and may support efforts to restore the effectiveness of relaxin-based therapeutics in fILD through the upregulation of RXFP1 transcription.