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The asthma candidate gene NPSR1 mediates isoform specific downstream signalling.

Pietras CO, Vendelin J, Anedda F, Bruce S, Adner M, Sundman L, Pulkkinen V, Alenius H, D'Amato M, Söderhäll C, Kere J - BMC Pulm Med (2011)

Bottom Line: The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²⁺ assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

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 Ca²⁺ 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.

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Reporter assays on NPS-NPSR1 isoform specific activation of cAMP/PKA, MAPK/JNK and MAPK/ERK pathways. NPS stimulated NPSR1-A or -B overexpressing HEK-293 cells were assayed for transcription factor complex activation of pathways related to GPCR signalling. The results demonstrate that NPSR1-A is a more efficient activator, with a more than 6 fold change for the cAMP/PKA pathway. Data shown as means relative to an empty vector control ± SEM. * Indicates significant (p < 0.05) difference between NPSR1-A and -B.
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Figure 6: Reporter assays on NPS-NPSR1 isoform specific activation of cAMP/PKA, MAPK/JNK and MAPK/ERK pathways. NPS stimulated NPSR1-A or -B overexpressing HEK-293 cells were assayed for transcription factor complex activation of pathways related to GPCR signalling. The results demonstrate that NPSR1-A is a more efficient activator, with a more than 6 fold change for the cAMP/PKA pathway. Data shown as means relative to an empty vector control ± SEM. * Indicates significant (p < 0.05) difference between NPSR1-A and -B.

Mentions: To further investigate differences seen in gene regulation between NPSR1-A and NPSR1-B, we studied activation of transcription factors representing three signalling pathways; cAMP/PKA, MAPK/JNK and MAPK/ERK. In these experiments, HEK-293 cells were transiently transfected with NPSR1-A, -B or empty pCMV vector together with an inducible transcription factor responsive luciferase construct. Cells were stimulated with NPS (1 μM) for 6 h and assayed for the relative luciferase activity. We observed that NPSR1-A induced a stronger response than NPSR1-B of the cAMP/PKA pathway (six fold) and one and a half (ERK) to two fold stronger (JNK) activator of the MAPK pathways (Figure 6). The results further supported the expression-array experiment and illustrated that NPSR1-A yields a stronger activation of transcription factor complexes.


The asthma candidate gene NPSR1 mediates isoform specific downstream signalling.

Pietras CO, Vendelin J, Anedda F, Bruce S, Adner M, Sundman L, Pulkkinen V, Alenius H, D'Amato M, Söderhäll C, Kere J - BMC Pulm Med (2011)

Reporter assays on NPS-NPSR1 isoform specific activation of cAMP/PKA, MAPK/JNK and MAPK/ERK pathways. NPS stimulated NPSR1-A or -B overexpressing HEK-293 cells were assayed for transcription factor complex activation of pathways related to GPCR signalling. The results demonstrate that NPSR1-A is a more efficient activator, with a more than 6 fold change for the cAMP/PKA pathway. Data shown as means relative to an empty vector control ± SEM. * Indicates significant (p < 0.05) difference between NPSR1-A and -B.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3142248&req=5

Figure 6: Reporter assays on NPS-NPSR1 isoform specific activation of cAMP/PKA, MAPK/JNK and MAPK/ERK pathways. NPS stimulated NPSR1-A or -B overexpressing HEK-293 cells were assayed for transcription factor complex activation of pathways related to GPCR signalling. The results demonstrate that NPSR1-A is a more efficient activator, with a more than 6 fold change for the cAMP/PKA pathway. Data shown as means relative to an empty vector control ± SEM. * Indicates significant (p < 0.05) difference between NPSR1-A and -B.
Mentions: To further investigate differences seen in gene regulation between NPSR1-A and NPSR1-B, we studied activation of transcription factors representing three signalling pathways; cAMP/PKA, MAPK/JNK and MAPK/ERK. In these experiments, HEK-293 cells were transiently transfected with NPSR1-A, -B or empty pCMV vector together with an inducible transcription factor responsive luciferase construct. Cells were stimulated with NPS (1 μM) for 6 h and assayed for the relative luciferase activity. We observed that NPSR1-A induced a stronger response than NPSR1-B of the cAMP/PKA pathway (six fold) and one and a half (ERK) to two fold stronger (JNK) activator of the MAPK pathways (Figure 6). The results further supported the expression-array experiment and illustrated that NPSR1-A yields a stronger activation of transcription factor complexes.

Bottom Line: The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²⁺ assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

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 Ca²⁺ 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.

Show MeSH
Related in: MedlinePlus