Neuropeptide S (NPS) variants modify the signaling and risk effects of NPS Receptor 1 (NPSR1) variants in asthma

Abstract

Single nucleotide polymorphisms (SNPs) close to the gain-of-function substitution, Asn(107)Ile (rs324981, A>T), in Neuropeptide S Receptor 1 (NPSR1) have been associated with asthma. Furthermore, a functional SNP (rs4751440, G>C) in Neuropeptide S (NPS) encodes a Val(6)Leu substitution on the mature peptide that results in reduced bioactivity. We sought to examine the effects of different combinations of these NPS and NPSR1 variants on downstream signaling and genetic risk of asthma. In transfected cells, the magnitude of NPSR1-induced activation of cAMP/PKA signal transduction pathways and downstream gene expression was dependent on the combination of the NPS and NPSR1 variants with NPS-Val(6)/NPSR1-Ile(107) resulting in strongest and NPS-Leu(6)/NPSR1-Asn(107) in weakest effects, respectively. One or two copies of the NPS-Leu(6) (rs4751440) were associated with physician-diagnosed childhood asthma (OR: 0.67, 95%CI 0.49-0.92, p = 0.01) and together with two other linked NPS variants (rs1931704 and rs10830123) formed a protective haplotype (p = 0.008) in the Swedish birth cohort BAMSE (2033 children). NPS rs10830123 showed epistasis with NPSR1 rs324981 encoding Asn(107)Ile (p = 0.009) in BAMSE and with the linked NPSR1 rs17199659 (p = 0.005) in the German MAGIC/ISAAC II cohort (1454 children). In conclusion, NPS variants modify asthma risk and should be considered in genetic association studies of NPSR1 with asthma and other complex diseases.

Fig 1. NPS variants and CRE luciferase activity.

Dose-response curves of NPS-Val(6) and NPS-Leu(6) (10 pM– 100 μM) based on the cAMP response element (CRE) luciferase activity in human embryonic kidney epithelial (HEK293) cells transiently transfected with either NPSR1-Ile(107) or NPSR1-Asn(107) coding variants. One representative experiment is shown. The CRE-driven luciferase activity is expressed in mean arbitrary units ± SEM of triplicates 3 h after NPS stimulation.

Fig 2. Real time RT-PCR analysis of NPSR1 downstream target genes.

(A) Time-dependent mRNA expression of NR4A1, FOS, CXCL2, and CCL20 in human embryonic kidney epithelial (HEK293) cells transiently transfected with either NPSR1-Ile(107) or NPSR1-Asn(107) coding variants and stimulated with either NPS-Val(6) or NPS-Leu(6) NPS (100 nM) for 1, 3, 6, and 24 h. (B) Dose-response curves of NR4A1, FOS, IER3, and EGR1 in human SH-SY5Y stable cell line over-expressing NPSR1-Ile(107) stimulated with either NPS-Leu(6) or NPS V6 (0.0001–1 μM) for 3 h. The results are presented as fold-changes in comparison to the unstimulated cells. GAPDH was used as the endogenous reference, and data are expressed as mean of triplicate samples. The error bars represent 95% confidence intervals. In all experiments, results were calculated with the comparative C_t method.

Fig 3. Location and block structure of three genotyped SNPs in the NPS gene.

(A) The scale represents the relative positions of rs1931704 (G>A) in the upstream regulatory region of NPS, followed by rs10830123 (G>C) in the intronic region and rs4751440 (G>C) in the coding region. Allele changes are indicated by > and given on the positive chromosomal strand of the Human Reference Assembly. The arrow indicates NPS transcription from the positive strand. (B) Effects of NPS SNPs on the risk of physician-diagnosed asthma in carriers of either one or two copies of the alternative allele. Bars represent 95% confidence intervals (CI). OR: Odds Ratio. Given the allele and genotype frequencies, the dominant model which best fits the data is shown.

Fig 4. Gene-gene interactions between NPSR1 and NPS.

Location of six NPSR1 SNPs with significant interactions with NPS rs10830123 in the multiplicative model for epistasis in connection with their p-value for interaction and linkage disequilibrium. The most significant interaction signals in BAMSE (green) and MAGIC/ISAAC (orange) are marked in bold. Statistics on LD between the functional rs324981 and other interacting SNPs are provided as D’ and r².

Fig 5. A schematic model on the functional crosstalk between NPS and NPSR1.

(A) The non-synonymous NPS-Leu(6) substitution affects NPS bioactivity. Depending on the NPSR1-Asn(107) or NPSR1-Ile(107) genotype, binding of NPS can lead to strong or minimal signaling as well as differential expression of downstream target genes such as the transcription factors FOS and NR4A1. (B) Two hypothetical mechanisms in which the differential expression of FOS and NR4A1 can modulate the outcomes of the NPS/NPSR1 pathway. FOS and NR4A1 bind to their regulatory elements in NPSR1 creating a feed-back loop that affect cell surface expression of NPSR1 and availability, and also, affect a number of other downstream genes that modulate neuroinflammation and ultimately asthma risk.