Expression of RXFP1 Is Decreased in Idiopathic Pulmonary Fibrosis. Implications for Relaxin-based Therapies

Abstract

Rationale: Relaxin is a hormone that has been considered as a potential therapy for patients with fibrotic diseases.

Objectives: To gauge the potential efficacy of relaxin-based therapies in idiopathic pulmonary fibrosis (IPF), we studied gene expression for relaxin/insulin-like family peptide receptor 1 (RXFP1) in IPF lungs and controls.

Methods: We analyzed gene expression data obtained from the Lung Tissue Research Consortium and correlated RXFP1 gene expression data with cross-sectional clinical and demographic data. We also employed ex vivo donor and IPF lung fibroblasts to test RXFP1 expression in vitro. We tested CGEN25009, a relaxin-like peptide, in lung fibroblasts and in bleomycin injury.

Measurements and main results: We found that RXFP1 is significantly decreased in IPF. In patients with IPF, the magnitude of RXFP1 gene expression correlated directly with diffusing capacity of the lung for carbon monoxide (P < 0.0001). Significantly less RXFP1 was detected in vitro in IPF fibroblasts than in donor controls. Transforming growth factor-β decreased RXFP1 in both donor and IPF lung fibroblasts. CGEN25009 was effective at decreasing bleomycin-induced, acid-soluble collagen deposition in vivo. The relaxin-like actions of CGEN25009 were abrogated by RXFP1 silencing in vitro, and, in comparison with donor lung fibroblasts, IPF lung fibroblasts exhibited decreased sensitivity to the relaxin-like effects of CGEN25009.

Conclusions: IPF is characterized by the loss of RXFP1 expression. RXFP1 expression is directly associated with pulmonary function in patients with IPF. The relaxin-like effects of CGEN25009 in vitro are dependent on expression of RXFP1. Our data suggest that patients with IPF with the highest RXFP1 expression would be predicted to be most sensitive to relaxin-based therapies.

Keywords: RXFP1; pulmonary fibrosis; relaxin; transforming growth factor-β.

Figure 1.

Relaxin/insulin-like family peptide receptor 1 (RXFP1) expression is decreased in pulmonary fibrosis. (A) Gene expression data from the Lung Tissue Research Consortium (107 control subjects and 134 patients with idiopathic pulmonary fibrosis [IPF]) were analyzed for the relaxin receptor, RXFP1, and relaxin 2 (RLN2). RXFP1 was downregulated 2.9-fold in IPF (*P < 0.0001 by unpaired t test), and RLN2 was significantly increased in IPF but with a fold change of 1.17 (**P < 0.003 by unpaired t test). (B) Whole-lung homogenates from patients with IPF and donor control subjects were subjected to immunoblotting for RXFP1 as described in the Methods section of the main text. (C) Band intensity of RXFP1 was normalized to β-actin using ImageJ software (National Institutes of Health, Bethesda, MD) for the immunoblot shown in B as described in the Methods section (*P < 0.0029 by Mann-Whitney U test; n = 4). (D) Immunoblot for RXFP1 from systemic sclerosis (SSc) lungs and donor controls. (E) ImageJ quantification of RXFP1 band intensity for the immunoblot shown in D (*P = 0.05 by Mann-Whitney U test; n = 3). (F) Immunoblots for RXFP1 from whole-lung homogenates derived from mouse lungs 14 days after bleomycin injury (n = 6 per group). (G) ImageJ quantification of RXFP1 band intensity for the immunoblots shown in F (*P = 0.078 by Mann-Whitney U test; n = 6). (H) Mice were injured with bleomycin and then treated with CGEN25009 beginning at Day 7 or Day 12 after injury. Lungs were collected at Day 21 after injury, and collagen was quantified by performing Sircol assays (Biocolor, Carrickfergus, UK). Using a generalized linear model with bootstrapping and a robust variance estimator, we found that treatment of bleomycin-injured animals with CGEN25009 was associated with a significant decrease in acid-soluble collagen (mean effect, −15.8; *^,†P < 0.001). (I) Quantitative reverse transcription–polymerase chain reaction for Col1a1, performed on flow-sorted tdTomato⁺ cells from bleomycin-injured mice treated with CGEN25009 (n = 7) or vehicle (n = 6) beginning at Day 12 after injury. Data shown in I–K were analyzed by using the Kolmogorov-Smirnov test, and the bars represent the median ± range. (J) Fibronectin (*P < 0.008). (K) Acta2 (α-smooth muscle actin [α-SMA]) (*P < 0.04). (L) Immunoblots of whole-lung homogenates for α-SMA from bleomycin-injured mice treated with vehicle (n = 5) or CGEN25009 (n = 7). (M) Densitometry results for immunoblots shown in L (*P = 0.0025 by Mann-Whitney U test). (N) Flow cytometric analysis for cell-specific markers of cells isolated from the bronchoalveolar lavage (BAL) of bleomycin-injured mice treated with CGEN25009 (n = 7) or vehicle (n = 6) beginning at Day 12 after injury: Ly6C (neutrophils), CD68 (macrophages), CD3 (T cells), and B220 (B cells). Data were analyzed by Mann-Whitney U test. P values are indicated in the figure. All bars except those in I–K represent the mean ± SEM. Acta2 = actin, α2; Col1a1 = collagen type I, α1; Fn1 = fibronectin 1.

Figure 2.

Relaxin/insulin-like family peptide receptor 1 (RXFP1) gene expression correlates directly with diffusing capacity of the lung for carbon monoxide (Dl_CO) in idiopathic pulmonary fibrosis (IPF) and is associated with a distinct gene expression profile. (A) Considering only the patients with IPF from the Lung Tissue Research Consortium, we organized patients into four quartiles based on increasing expression of RXFP1 and compared their demographic and clinical data (P < 0.0001 for trend by one-way analysis of variance; n = 33–34/quartile). (B) Sex representation in each quartile. Women are represented in yellow and men in purple, showing no significant difference based on sex across quartiles (P = not significant by test χ² for trend). (C) No significant effects were observed with regard to age. (D and E) Pearson correlation coefficients of FVC and Dl_CO as a function of square root (sqrt)–transformed RXFP1 gene expression for IPF, non-IPF interstitial lung disease (non-IPF ILD), and the two together. R² and P values are indicated in the figure. (F) Differentially expressed genes in patients with IPF based on RXFP1 expression are represented by a heatmap. Each column represents a patient, and each row represents a gene. The color bar represents fold change. Between the upper two and lower two quartiles, 5,371 genes were differentially expressed. All bars represent the mean ± SEM.

Figure 3.

Transforming growth factor (TGF)-β decreases expression of relaxin/insulin-like family peptide receptor 1 (RXFP1) in human lung fibroblasts. Human lung fibroblasts were isolated from patients with idiopathic pulmonary fibrosis (IPF) and donor control subjects as described in the Methods section in the main text. Cells were cultured in the presence or absence of TGF-β. (A) Immunoblotting was performed for RXFP1 and β-actin. Decreased expression of RXFP1 in normal and IPF lung fibroblasts was observed in response to TGF-β. IPF lung fibroblasts also showed decreased expression of RXFP1 at baseline compared with normal lung fibroblasts. (B) Quantification of RXFP1 band intensity using ImageJ software (*^,†P < 0.005 by two-way analysis of variance [ANOVA]; n = 3 per group). (C) Quantitative reverse transcription–polymerase chain reaction was performed for RXFP1 and peptidylprolyl isomerase A (PPIA) under the same conditions as described in B. In response to TGF-β, mRNA for RXFP1 was significantly decreased in donor lung fibroblasts, and, at baseline, RXFP1 mRNA was significantly decreased in IPF fibroblasts compared with donor fibroblasts (*^,†P < 0.005 by two-way ANOVA; n = 3 per group). In two of three lines tested, mRNA for RXFP1 in IPF fibroblasts following TGF-β was undetectable. (D) Donor lung fibroblasts were stimulated with TGF-β at different concentrations, and the lysates were processed for immunoblotting for RXFP1. (E) Quantification of immunoblots shown in D by using ImageJ software (P < 0.0004 by one-way ANOVA for left-to-right trend). (F) Donor lung fibroblasts were stimulated with TGF-β in the presence of the activin-like kinase receptor 5 inhibitor SB431542 to inhibit Smad signaling. RXFP1 expression was significantly decreased following TGF-β, and this response was completely abrogated by preincubation with SB431542. (G) Quantification of RXFP1 band from immunoblots shown in F (*P < 0.05 by two-way ANOVA; n = 3). (H) Donor lung fibroblasts were stimulated first with CGEN25009, followed by TGF-β. Cells were then lysed and processed for immunoblotting for RXFP1, collagen I, α-smooth muscle actin (α-SMA), and β-actin. Quantification was performed using ImageJ software for (I) RXFP1, (J) collagen I, and (K) α-smooth muscle actin. For I–K, *^,†P < 0.05 by two-way ANOVA. All bars represent the mean ± SEM.

Figure 4.

Sensitivity of fibroblasts to CGEN25009 is dependent on relaxin/insulin-like family peptide receptor 1 (RXFP1) expression. Donor lung fibroblasts were incubated with nontargeting short interfering RNA (siRNA) oligonucleotides (siControl) or RXFP1-targeting siRNA oligonucleotides (siRXFP1) and then stimulated with the relaxin-like peptide CGEN25009. (A) Immunoblots for RXFP1, phosphorylated myosin light chain 20 (P-MLC20) and total MLC20, collagen I, and α-smooth muscle actin (α-SMA). Quantification of band intensity was performed, and data were analyzed by two-way analysis of variance. (B) RXFP1 (*P < 0.05, siControl + vehicle vs. siRXFP1 + vehicle; n = 3). (C) P-MLC20/total MLC20 (*P < 0.05 for siControl + vehicle vs. siControl + CGEN25009; ^†P < 0.05 for siControl + vehicle vs. siRXFP1 + vehicle; n = 3). (D) Collagen I (*P < 0.05 for siControl + vehicle vs. siControl + CGEN25009; ^†P < 0.05 for siControl + vehicle vs. siRXFP1 + vehicle; n = 3). (E) α-SMA (*P < 0.05 for siControl + vehicle vs. siControl+CGEN25009; n = 3). (F) Donor and IPF lung fibroblasts were incubated with escalating concentrations of CGEN25009 and subjected to immunoblotting for phosphorylated MLC20 and total MLC20. (G) Dose–response curves for IPF (n = 3) and donor (n = 3) lung fibroblasts as a logarithmic function of the concentration of CGEN25009, showing an increased half-maximal inhibitory concentration for CGEN25009 in IPF lung fibroblasts compared with normal controls (n = 3). (H) IPF fibroblasts were infected with control (green fluorescent protein [GFP]) or RXFP1-expressing lentiviral particles and then stimulated with escalating concentrations of CGEN25009. Lysates were subjected to immunoblotting for phosphorylated and total MLC20, RXFP1, and β-actin. (I) Dose–response curves for RXFP1-infected and GFP-infected lung fibroblasts as a logarithmic function of the concentration of CGEN25009 (n = 3). All bars represent the mean ± SEM. IPF = idiopathic pulmonary fibrosis.