Calcium-Sensing Receptor as a Novel Target for the Treatment of Idiopathic Pulmonary Fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and no curative therapies. Fibroblast activation by transforming growth factor β1 (TGFβ1) and disrupted metabolic pathways, including the arginine-polyamine pathway, play crucial roles in IPF development. Polyamines are agonists of the calcium/cation-sensing receptor (CaSR), activation of which is detrimental for asthma and pulmonary hypertension, but its role in IPF is unknown. To address this question, we evaluated polyamine abundance using metabolomic analysis of IPF patient saliva. Furthermore, we examined CaSR functional expression in human lung fibroblasts (HLFs), assessed the anti-fibrotic effects of a CaSR antagonist, NPS2143, in TGFβ1-activated normal and IPF HLFs by RNA sequencing and immunofluorescence imaging, respectively; and NPS2143 effects on polyamine synthesis in HLFs by immunoassays. Our results demonstrate that polyamine metabolites are increased in IPF patient saliva. Polyamines activate fibroblast CaSR in vitro, elevating intracellular calcium concentration. CaSR inhibition reduced TGFβ1-induced polyamine and pro-fibrotic factor expression in normal and IPF HLFs. TGFβ1 directly stimulated polyamine release by HLFs, an effect that was blocked by NPS2143. This suggests that TGFβ1 promotes CaSR activation through increased polyamine expression, driving a pro-fibrotic response. By halting some polyamine-induced pro-fibrotic changes, CaSR antagonists exhibit disease-modifying potential in IPF onset and development.

Keywords: TGFβ1; arginine–polyamine pathway; calcium/cation-sensing receptor; idiopathic pulmonary fibrosis; negative allosteric modulator.

Figure 1

Arginine pathway metabolites are increased in idiopathic pulmonary fibrosis (IPF) patient saliva samples. (A) Diagram representing the arginine–polyamine metabolic pathway. (B) Principal component analysis (PCA) score plot between the main principal components (PC1 and PC2) shows a tight cluster of the IPF samples based on their metabolic profile compared to controls, with a 95% confidence ellipse drawn for each group (shown by the dotted lines). Assessment of PC1 and PC2, which account for 76.4% of the total variation between the control (HC) and IPF clusters, highlights the prominence of polyamines. (C) Associated box and whisker plots of metabolites in the arginine–polyamine pathway highlight the differences between healthy and IPF profiles. Y-axis: Log₁₀ normalization of metabolite intensity. Statistical analysis was performed with unpaired two-tailed Student t-test; * p < 0.05, ** p < 0.01. N = 6 controls; 6 IPF patients.

Figure 2

Calcium-sensing receptor (CaSR) is functionally expressed in normal human lung fibroblasts (NHLFs) and is activated by polyamines upregulated in IPF. (A) Representative image of CaSR expression in NHLF (green), nuclei (blue). Scale bar: 100 µm. (B) Summary data of intracellular calcium levels ([Ca²⁺]_i) in NHLF in response to: (Ci) 5 mM Ca²⁺ (divalent cation); (Di) 5 mM L-ornithine (basic amino acid); and (Ei) 1 mM spermine (polyamine). (B,Cii–Eii) Treatment with a CaSR negative allosteric modulator (CaSR NAM) prevents these increases in [Ca²⁺]_i. (C–E) Representative traces of NHLF [Ca²⁺]_i response to CaSR activators. Summary data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA (with Sidak’s post hoc test); *** p < 0.001, **** p < 0.0001. 5 mM Ca²⁺ (n = 3–6; 108 cells), 5 mM L-ornithine (n = 5–6; 109 cells), and 1 mM spermine (n = 3; 39 cells). n = 3–6 independent experiments. CaSR negative allosteric modulator: NAM, NPS2143 (1 μM).

Figure 3

Enrichment analysis of differentially expressed genes (DEGs) from RNA sequencing (RNAseq) in primary human lung fibroblasts treated with vehicle (0.01% DMSO), CaSR NAM (1 μM NPS2143), TGFβ1 (5 ng/mL), and TGFβ1+ CaSR NAM for 72 h. The MA plots show the log₂-fold change between experimental groups (M) against the mean expression across all the samples (A) for each gene. Statistically significant differentially expressed genes (DEGs) for each comparison are shown in red. The x-axis shows the mean of normalized gene counts; the y-axis shows log₂ fold-changes in expression (positive values represent upregulated genes, while negative values denote downregulation). (A) MA plot of RNAseq showing the DEGs in TGFβ1 treated cells relative to unstimulated cells (vehicle control) cells. Red dots represent statistically significant DEGs (Down-DEGs: 1999; Up-DEGs: 2757), while black dots represent non-significantly altered genes. (B) No significant difference in gene expression was observed when the CaSR NAM-treated cells were compared with vehicle control cells. (C) MA plot showing the DEGs in cells co-treated with TGFβ1 + CaSR NAM relative to TGFβ1-treated cells. Red dots represent statistically significant DEGs (Down-DEG: 2608; Up-DEG: 2150). (D,E) Annotation of key Reactome pathways based on the enrichment analysis of the statistically significant DEGs. (D) Key Reactome pathways upregulated by TGFβ1 relative to vehicle control. (E) Key Reactome pathways downregulated by CaSR NAM in the presence of the fibrotic stimulus, TGFβ1, relative to TGFβ1 treatment alone. Benjamini–Hochberg p-value adjustment was performed for all statistical tests; level of controlled false positive rate was set to 0.05. * p < 0.05, **** p < 0.0001. N = 3 donors. NAM: Calcium-sensing receptor (CaSR) Negative Allosteric Modulator treatment; TGFβ1: Transforming growth factor β1 (TGFβ1) treatment; TGFβ1 + NAM: TGFβ1 and CaSR Negative Allosteric Modulator co-treatment.

Figure 4

TGFβ1 upregulates genes involved in proline and polyamine metabolism, an effect abrogated by the CaSR NAM, NPS2143, in normal human lung fibroblasts. Fibroblasts were treated with vehicle (0.01% DMSO), CaSR NAM (1 μM NPS2143), TGFβ1 (5 ng/mL), and TGFβ1 + CaSR NAM for 72 h. (A) Heatmap generated from RNA sequencing data showing the log₂ ratio of differentially expressed genes (versus vehicle) implicated in these metabolic pathways. (B) RNA sequencing data show exogenous TGFβ1 treatment upregulates genes associated with proline synthesis and polyamine metabolism, while co-treatment with the CaSR NAM, NPS2143 downregulates gene expression, except for GLUL, ALDH4A1, ARG2, and ALDH18A1. (C) CaSR NAM restores TGFβ1-induced increase in polyamine metabolism to baseline. The genes in red are upregulated by TGFβ1 and downregulated by TGFβ1 and CaSR NAM co-treatment; genes in black were differentially expressed in the presence of TGFβ1, but CaSR NAM co-treatment had no effect on their expression; genes in grey were not affected by either treatment. Genes encoding glutaminase: GLS, glutamine synthase: GLUL, pyrroline 5-carboxylate synthase: ALDH18A1, pyrroline 5-carboxylate dehydrogenase: ALDH4A1, pyrroline 5-carboxylate reductase/proline synthase: PYCR, proline oxidase: PRODH, arginase: ARG, ornithine aminotransferase: OAT, antizyme inhibitor: AZIN, antizyme: OAZ1, ornithine decarboxylase: ODC, spermidine synthase: SRM, spermine synthase: SMS, and spermine oxidase: SMOX, polyamine transporter-Solute Carrier Family 3 Member 2 (SLC3A2) are involved in these pathways. Data in the violin plots represent median fold change, truncated at minimum and maximum values. Benjamini–Hochberg p-value adjustment was performed for all statistical tests; level of controlled false positive rate was set to 0.05. * p < 0.05, ** p < 0.01, *** p < 0.001. N = 3 donors. CaSR negative allosteric modulator (NAM); NPS2143 (1 μM); Vehicle (VEH).

Figure 5

CaSR negative allosteric modulator, NPS2143, attenuates TGFβ1-induced upregulation of pro-fibrotic genes in normal human lung fibroblasts. NHLFs were treated with vehicle (0.01% DMSO), CaSR NAM (1 μM NPS2143), TGFβ1 (5 ng/mL), and TGFβ1 + CaSR NAM for 72 h. (A) Heatmap generated from RNA sequencing data showing the log₂ ratio of differentially expressed genes (versus vehicle). Exogenous TGFβ1 application upregulates genes related to fibroblast contractility, collagen synthesis, and extracellular matrix (ECM) remodeling. (B) CaSR NAM reduces the expression of genes associated with fibroblast contractility, collagen synthesis and assembly; and (C) ECM remodeling and maintenance. Genes encoding Transforming Growth Factor-β1: TGFB1, α-smooth muscle actin: ACTA2, smooth muscle-22α: TAGLN, ras homolog family member A: RHOA, collagen: COL, prolyl 4-hydroxylase, alpha polypeptide II: P4HA2, fibronectin: FN1, elastin: ELN, matrix-metalloproteinases: MMP, tissue inhibitors of metalloproteinases: TIMP, and serine protease inhibitor E1: SERPINE1 are involved in these pathways. Data in the violin plots represent median fold change, truncated at minimum and maximum values. Benjamini–Hochberg p-value adjustment was performed in all statistical tests; level of controlled false positive rate was set to 0.05. * p_adj <0.05, ** p_adj <0.01, *** p_adj <0.001, **** p_adj <0.0001. N = 3 donors. CaSR negative allosteric modulator: NAM, NPS2143 (1 μM).

Figure 6

CaSR is expressed in normal human lung fibroblasts (NHLFs) and IPF human lung fibroblasts (IPF HLFs). (A) Representative images showing CaSR expression when NHLFs were treated with vehicle (0.01% DMSO), CaSR NAM (1 μM NPS2143), TGFβ1 (5 ng/mL), and TGFβ1 + CaSR NAM for 72 h. (B) Image quantification analysis with bespoke software, StrataQuest, shows that TGFβ1 treatment increases CaSR expression in NHLFs. (C) Representative images showing CaSR expression in IPF HLFs in the same treatment conditions. (D) Image quantification with StrataQuest shows CaSR expression is maintained in IPF HLFs in all treatment conditions. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA (with Sidak’s post hoc test); * p < 0.05. N = 3 donors; n = 3 independent experiments. Scale bar: 100 µm. CaSR negative allosteric modulator: NAM.

Figure 7

TGFβ1-induced polyamine expression is abrogated by CaSR NAM in normal and IPF human lung fibroblasts (HLFs). (A,B) Ornithine secretion by normal HLFs and IPF HLFs is increased by TGFβ1 treatment, and the response is attenuated in the presence of CaSR NAM. (C,D) Low concentration of spermine is secreted by normal HLFs and IPF HLFs, with levels remaining relatively unchanged across the treatment conditions. (E,F) Intracellular spermine concentration is increased by exogenous TGFβ1 treatment in normal HLFs and IPF HLFs; co-treatment with CaSR NAM restores vehicle control levels of the polyamine. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA (with Sidak’s post hoc test); * p < 0.05, ** p < 0.01, *** p < 0.001. N = 3 donors; n = 3–6 independent experiments. CaSR negative allosteric modulator: NAM, NPS2143 (1 μM).

Figure 8

CaSR NAM reduces TGFβ1-induced pro-fibrotic changes in IPF human lung fibroblasts (HLFs). IPF HLFs were treated with vehicle (0.01% DMSO), CaSR NAM (1 μM NPS2143), TGFβ1 (5 ng/mL), and TGFβ1 + CaSR NAM for 72 h. (A) Representative images showing that TGFβ1 treatment increases alpha smooth muscle actin (αSMA) expression and stress-fibre formation (green), a response abrogated by the CaSR NAM. (B) Image quantification analysis shows that TGFβ1-induced increase in αSMA expression is halved in the presence of CaSR NAM. (C) Representative images showing diffuse Collagen 1 expression in IPF HLFs treated with TGFβ1. Co-treatment with CaSR NAM appears to reduce cell area (see Figure S7). (D) Image quantification confirms increased collagen 1 expression in IPF HLFs treated with TGFβ1. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA (with Sidak’s post hoc test); * p < 0.05, **** p < 0.0001. N = 3 donors; n = 3 independent experiments. Scale bar: 100 µm. CaSR negative allosteric modulator: NAM.