The asthma candidate gene NPSR1 mediates isoform specific downstream signalling.

BACKGROUND: Neuropeptide S Receptor 1 (NPSR1, GPRA, GPR154) was first identified as an asthma candidate gene through positional cloning and has since been replicated as an asthma and allergy susceptibility gene in several independent association studies. In humans, NPSR1 encodes two G protein-coupled receptor variants, NPSR1-A and NPSR1-B, with unique intracellular C-termini. Both isoforms show distinct expression pattern in asthmatic airways. Although NPSR1-A has been extensively studied, functional differences and properties of NPSR1-B have not yet been clearly examined. Our objective was to investigate downstream signalling properties of NPSR1-B and functional differences between NPSR1-A and NPSR1-B. METHODS: HEK-293 cells transiently overexpressing NPSR1-A or NPSR1-B were stimulated with the ligand neuropeptide S (NPS) and downstream signalling effects were monitored by genome-scale affymetrix expression-arrays. The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²âº assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays. RESULTS: NPSR1-B signalled through the same pathways and regulated the same genes as NPSR1-A, but NPSR1-B yielded lower induction on effector genes than NPSR1-A, with one notable exception, CD69, a marker of regulatory T cells. CONCLUSIONS: We conclude that NPSR1-B is regulating essentially identical set of genes as NPSR1-A, with few, but possibly important exceptions, and that NPSR1-A induces stronger signalling effects than NPSR1-B. Our findings suggest an isoform-specific link to pathogenetic processes in asthma and allergy.

Downstream target genes of the neuropeptide S-NPSR1 pathway.

The neuropeptide S (NPS)-NPS receptor 1 (NPSR1) pathway has recently been implicated in the pathogenesis of asthma. The purpose of this study was to identify downstream gene targets regulated by NPSR1 upon NPS stimulation. A total of 104 genes were found significantly up-regulated and 42 down-regulated by microarray analysis 6 h after NPS administration. By Gene Ontology enrichment analysis, the categories 'cell proliferation', 'morphogenesis' and 'immune response' were among the most altered. A TMM microarray database comparison suggested a common co-regulated pathway, which includes JUN/FOS oncogene homologs, early growth response genes, nuclear receptor subfamily 4 members and dual specificity phosphatases. The expression of four up-regulated genes, matrix metallopeptidase 10 (MMP10), INHBA (activin A), interleukin 8 (IL8) and EPH receptor A2 (EPHA2), exhibited a significant NPS dose-response relationship as confirmed by quantitative reverse-transcriptase-PCR and for MMP10 by immunoassay. Immunohistochemical analyses revealed that MMP10 and TIMP metallopeptidase inhibitor 3 (TIMP3) were both strongly expressed in bronchial epithelium, and macrophages and eosinophils expressed MMP10 in asthmatic sputum samples. Because remodeling of airway epithelium is a feature of chronic asthma, the up-regulation of MMP10 and TIMP3 by NPS-NPSR1 signaling may be of relevance in the pathogenesis of asthma.

Neuropeptide S and G protein-coupled receptor 154 modulate macrophage immune responses.

G protein-coupled receptor 154 (GPR154) is a recently discovered asthma susceptibility gene upregulated in the airways of asthma patients. We previously observed increased pulmonary mRNA expression of the murine ortholog Gpr154 in a mouse model of ovalbumin (OVA)-induced inflammation. However, the expression profile of GPR154 in leukocytes and the cellular functions of the receptor and its endogenous agonist neuropeptide S (NPS) have remained unidentified. Here, we characterized the mRNA expression of NPS and GPR154 by using real-time RT-PCR in fractionated human blood cells and in peripheral blood mononuclear cells (PBMCs) with monocyte or T cell activation. The expression of GPR154 in leukocytes was further confirmed by immunoblotting experiments and immunohistochemical staining of human sputum samples. Additionally, we characterized the expression of GPR154 in the lung tissue samples and in the bronchoalveolar lavage (BAL) fluid of OVA sensitized and challenged BALB/c mice. In human blood and sputum cells, monocyte/macrophages and eosinophils were identified as GPR154-positive cells. In PBMCs, monocyte activation with LPS but not T cell activation with anti-CD3/CD28 antibodies resulted in increased NPS and GPR154 expression. In the lung tissue samples and in the BAL fluid of OVA-challenged mice, GPR154 expression was upregulated in alveolar macrophages in comparison to controls. In the mouse macrophage RAW 264.7 cell line, NPS-stimulated Galphas- and Galphaq-dependent phagocytosis of Escherichia coli. The results show that GPR154 is upregulated in macrophages after antigen challenge and that NPS is capable of inducing phagocytosis of unopsonized bacteria.

Characterization of GPRA, a novel G protein-coupled receptor related to asthma.

We recently identified a novel positional asthma susceptibility gene, GPRA, which belongs to the G protein-coupled receptor family. In the present studies, we show that isoform specific activation of GPRA-A with its agonist, Neuropeptide S (NPS) resulted in significant inhibition of cell growth. GPRA has several variants due to extensive alternative splicing. We observed that only the full-length variants, GPRA-A and GPRA-B, with 7 transmembrane topology are transported into the plasma membrane, while the truncated proteins retain intracellular compartments. To clarify disease mechanism, we studied co-expression of the variants without finding any indication that truncated variants would inhibit the receptor transport into the plasma membrane. By using in situ hybridization and immunohistochemistry, we detected ubiquitous expression of GPRA-B, and frequent expression of GPRA-A in the epithelia of several organs including bronchi and gastrointestinal tract. Furthermore, we observed aberrant mRNA and protein expression levels of GPRA in the asthmatic bronchi. Finally, we demonstrate that GPRA and NPS are co-expressed in bronchial epithelium. In summary, this study provides evidence that GPRA might have functional relevance in modulating asthma by increased expression levels in the relevant tissues under diseased state and by potential inhibitory effect of GPRA-A activation on cell growth.

Novel sulfhydryl-reactive compounds orazipone and OR-1958 inhibit cytokine production and histamine release in rat and human mast cells.

Orazipone (OR-1384) and OR-1958 are novel anti-inflammatory sulfhydryl reactive compounds with potential applications in the treatment of chronic obstructive lung disease and colitis. Mast cells are potent immune system cells which can be found in abundant numbers in mucosa of lung and gut. We have studied whether the anti-inflammatory effect of these compounds could be mediated through inhibition of the function of mast cells and compared their effects with the glucocorticoid budesonide. Human mast cell line (HMC-1) cells were activated using a combination of a calcium ionophore and a phorbol ester and the production of cytokines was measured using ELISA assay. Tumour necrosis factor-alpha mRNA levels were assessed using a semiquantitative reverse transcriptase polymerase chain reaction assay. Histamine release was studied in rat peritoneal mast cells. Orazipone, OR-1958 and budesonide inhibited significantly and dose dependently tumour necrosis factor-alpha production in HMC-1 cells with IC50-values of 20, 10, and 0.25 microM, respectively. Polymerase chain reaction studies showed that OR-1958 attenuated the activation-induced increase of tumour necrosis factor-alpha mRNA in HMC-1 cells. OR-1958 and, to a lesser extent, orazipone inhibited dose dependently compound 48/80-induced histamine release from rat peritoneal mast cells in a reversible manner. In contrast, budesonide did not appreciably affect the histamine release. Both orazipone and OR-1958 inhibit efficiently mast cell functions and therefore could prove useful in the treatment of diseases associated with inappropriate mast cell activation.

Characterization of a common susceptibility locus for asthma-related traits.

Susceptibility to asthma depends on variation at an unknown number of genetic loci. To identify susceptibility genes on chromosome 7p, we adopted a hierarchical genotyping design, leading to the identification of a 133-kilobase risk-conferring segment containing two genes. One of these coded for an orphan G protein-coupled receptor named GPRA (G protein-coupled receptor for asthma susceptibility), which showed distinct distribution of protein isoforms between bronchial biopsies from healthy and asthmatic individuals. In three cohorts from Finland and Canada, single nucleotide polymorphism-tagged haplotypes associated with high serum immunoglobulin E or asthma. The murine ortholog of GPRA was up-regulated in a mouse model of ovalbumin-induced inflammation. Together, these data implicate GPRA in the pathogenesis of atopy and asthma.