Abstract
Lysophosphatidic acid (LPA) is a lipid-derived signaling molecule that plays key roles in diverse biological processes including inflammation and uterine remodeling. Although the function of LPA and its receptors has been extensively studied using knock-out mice, the temporal-spatial expression of LPA receptors is less well-characterized. To gain further insight into the dynamic regulation of LPA receptor 3 (Lpar3) expression in vivo by bioluminescence imaging, we generated and characterized mice transgenic for a putative Lpar3 promoter fragment. A non-coding region of the Lpar3 gene immediately upstream of the start site was subcloned adjacent to the luciferase gene. Promoter activity was determined by in vitro luciferase assays, in vivo bioluminescent imaging or by semi-quantitative real-time PCR. The air-pouch model was used to investigate Lpar3 promoter activity in the context of inflammation. The putative Lpar3 promoter fragment behaved similarly to the endogenous promoter in vitro and in vivo. In male mice, elevated levels of Lpar3-induced luciferase activity were observed in the testis. In female mice, the basal level of luciferase activity in the uterus significantly increased during pseudopregnancy. Moreover, luciferase activity was upregulated by TNF-α in the air-pouch model. We report the identification of a functional Lpar3 promoter fragment and the generation of a transgenic mouse model to investigate the regulation of Lpar3 promoter activity non-invasively in vivo by bioluminescence imaging. This mouse model is a valuable tool for reproductive biology and inflammation research as well as other biological processes in which this receptor is involved.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Anliker B, Chun J (2004) Cell surface receptors in lysophospholipid signaling. Semin Cell Dev Biol 15:457–465
Bourgoin SG, Zhao C (2010) Autotaxin and lysophospholipids in rheumatoid arthritis. Curr Opin Investig Drugs 11:515–526
Contag CH, Spilman SD, Contag PR, Oshiro M, Eames B, Dennery P, Stevenson DK, Benaron DA (1997) Visualizing gene expression in living mammals using a bioluminescent reporter. Photochem Photobiol 66:523–531
Contos JJ, Ishii I, Chun J (2000) Lysophosphatidic acid receptors. Mol Pharmacol 58:1188–1196
Hama K, Aoki J, Bandoh K, Inoue A, Endo T, Amano T, Suzuki H, Arai H (2006) Lysophosphatidic receptor, LPA3, is positively and negatively regulated by progesterone and estrogen in the mouse uterus. Life Sci 79:1736–1740
Ishii I, Fukushima N, Ye X, Chun J (2004) Lysophospholipid receptors: signaling and biology. Annu Rev Biochem 73:321–354
Lalancette-Hebert M, Phaneuf D, Soucy G, Weng YC, Kriz J (2009) Live imaging of toll-like receptor 2 response in cerebral ischaemia reveals a role of olfactory bulb microglia as modulators of inflammation. Brain 132:940–954
Ye X, Hama K, Contos JJ, Anliker B, Inoue A, Skinner MK, Suzuki H, Amano T, Kennedy G, Arai H, Aoki J, Chun J (2005) LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing. Nature 435:104–108
Ye X, Skinner MK, Kennedy G, Chun J (2008) Age-dependent loss of sperm production in mice via impaired lysophosphatidic acid signaling. Biol Reprod 79:328–336
Zhao C, Fernandes MJ, Prestwich GD, Turgeon M, Di Battista J, Clair T, Poubelle PE, Bourgoin SG (2008) Regulation of lysophosphatidic acid receptor expression and function in human synoviocytes: implications for rheumatoid arthritis? Mol Pharmacol 73:587–600
Zhao C, Sardella A, Chun J, Poubelle PE, Fernandes MJ, Bourgoin SG (2011) TNF-α promotes LPA1- and LPA3-mediated recruitment of leukocytes in vivo through CXCR2 ligand chemokines. J Lipid Res 52:1307–1318
Acknowledgments
We thank Dr. J. Kriz (Department of Anatomy and Physiology, Laval University, Quebec, Canada) for providing the pIRES-PROM-Luc2-AcGFP plasmid vector. We would also like to thank Dr Robert Viger for insightful discussions and Catherine Brousseau and Johanne Ouellet for their assistance with the immunofluorescence experiments. This work was supported by a Grant from The Arthritis Society to Sylvain G. Bourgoin, Maria J. Fernandes and Patrice Poubelle. Maria J. Fernandes is a recipient of an Investigator award from The Arthritis Society.
Author information
Authors and Affiliations
Corresponding authors
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplemental Fig. 1
Bioluminescent imaging of organs extracted from FVB/N-Tg(Lpar3-Luc2-AcGFP)Bouul male mice and comparison with the activity of the endogenous Lpar3 promoter. Organs isolated from male mice line FVB/N-Tg(Lpar3-Luc2-AcGFP)273Bouul (top row), FVB/N-Tg(Lpar3-Luc2-AcGFP)275Bouul (middle row) or FVB/N-Tg(Lpar3-Luc2-AcGFP)278Bouul (bottom row) were imaged and analyzed as described in Fig. 2. A graphic representation of the luminescent signals detected is shown (graphs on left, total counts/organ). Total RNA was also extracted from the organs of the same mice and analyzed by real time PCR with Lpar3-specific primers. The expression level of Lpar3 for each organ is presented in the graphs (on right) as a percentage of the expression of Lpar3 mRNA in the heart that was given an arbitrary reference value of 100 %. The values used to calculate this percentage were obtained by subtracting the mean of the Ct values for Lpar3 from the mean of the Ct values for GAPDH in each organ. The data are expressed as mean ± SEM from 2 (n = 2, mouse line FVB/N-Tg(Lpar3-Luc2-AcGFP)273Bouul) or 3 experiments (n = 3, mouse lines FVB/N-Tg(Lpar3-Luc2-AcGFP)275Bouul and FVB/N-Tg(Lpar3-Luc2-AcGFP)278Bouul). (TIFF 465 kb)
Supplemental Fig. 2
Bioluminescent imaging of organs extracted from FVB/N-Tg(Lpar3-Luc2-AcGFP)Bouul female mice and comparison with the activity of the endogenous Lpar3 promoter and effect of pseudopregnancy on the activity of the Lpar3 promoter fragment in vivo. (A) A graphic representation of the luminescent signals detected in female mouse of line FVB/N-Tg(Lpar3-Luc2-AcGFP)273Bouul (left panel). Total RNA was extracted from the organs of female mice and analyzed by real time PCR with Lpar3-specific primers (right panel). The expression level of Lpar3 for each organ is presented as a percentage of the expression of Lpar3 mRNA in the heart that was given an arbitrary reference value of 100 %. The values used to calculate this percentage were obtained by subtracting the mean of the Ct values for Lpar3 from the mean of the Ct values for GAPDH in each organ. The data are expressed as mean ± SEM from 2 experiments (n = 2). (B) Uteri were harvested from non-pregnant and pseudopregnant mice of line FVB/N-Tg(Lpar3-Luc2-AcGFP)273Bouul at 3.5 days post-coitus and total RNA isolated for analysis by real time PCR with Lpar3 and Luc2 specific primers. The expression of Lpar3 and Luc2 mRNA (Ct value for Lpar3 – Ct value for GAPDH) was expressed as a fold-increase above non-pregnant controls (n = 3). (TIFF 304 kb)
Supplemental Fig. 3
Effect of TNF-α treatment on the activity of the Lpar3 promoter in vivo. TNF-α (50 ng/pouch) was injected into the air pouch of mice of line FVB/N-Tg(Lpar3-Luc2-AcGFP) 273Bouul. (A) Total RNA was extracted from the air pouch tissues 16 h post-injection and analyzed by real time PCR with Lpar3 and Luc2 specific primers. The expression of the endogenous Lpar3 mRNA and the mRNA of the Luc2 reporter gene (Ct value for Lpar3 – Ct value for GAPDH) was expressed as a fold-increase above non-treated controls (given an arbitrary value of 1). The data are presented as mean ± SEM from three independent experiments (n = 3). (B) Air pouch tissues from mice in (A) were lysed and luciferase reporter activity (RLU: relative luciferase units) measured with a luminometer. The data are presented as mean ± SEM from three independent experiments (n = 3). Statistical comparisons were determined by T-test (* p < 0.05; ** p < 0.01). (TIFF 165 kb)
Rights and permissions
About this article
Cite this article
Zhao, C., Sardella, A., Davis, L. et al. A transgenic mouse model for the in vivo bioluminescence imaging of the expression of the lysophosphatidic acid receptor 3: relevance for inflammation and uterine physiology research. Transgenic Res 24, 625–634 (2015). https://doi.org/10.1007/s11248-015-9882-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-015-9882-8