Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo
Mari Kono, Ana E. Tucker, Jennifer Tran, Jennifer B. Bergner, Ewa M. Turner, Richard L. Proia
Mari Kono, Ana E. Tucker, Jennifer Tran, Jennifer B. Bergner, Ewa M. Turner, Richard L. Proia
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Technical Advance Inflammation

Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo

Abstract

Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S1P1 recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease.

Authors

Mari Kono, Ana E. Tucker, Jennifer Tran, Jennifer B. Bergner, Ewa M. Turner, Richard L. Proia

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Figure 2

S1P1 activation in MEFs.

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S1P1 activation in MEFs. (A) Validation of modified S1P1 signaling pathw...
(A) Validation of modified S1P1 signaling pathway in MEFs. S1P1 GFP signaling MEFs were cultured for 16 hours in medium containing 10% charcoal-stripped FBS and received either S1P (10–7 M) or vehicle (4 mg/ml BSA in PBS). Nuclei were stained with Hoechst, and MEF cultures were imaged under an inverted laser-scanning confocal microscope. The experiment was repeated twice in duplicate, and a representative result is shown. (B) Treatment of MEFs with RP-001. S1P1 GFP signaling MEFs were cultured for 16 hours in medium containing 10% charcoal-stripped FBS and received either S1P or RP-001. After 24 hours, nuclei were stained with Hoechst, and MEF cultures were imaged under an inverted laser-scanning confocal microscope. The experiment was performed in duplicate, and a representative result is shown. (C) Flow cytometry analysis of S1P1 GFP signaling MEFs. S1P1 GFP signaling MEFs were cultured for 16 hours in medium containing 10% charcoal-stripped FBS and various concentrations of S1P were added. After 24 hours, the number of GFP+ cells was determined by flow cytometry. The experiment was repeated twice. Data represent mean ± SEM (n = 3). (D) Endogenous S1P1 signaling pathway in MEFs. S1P1 GFP signaling MEFs were cultured for 16 hours in medium containing 0.1% FBS and received 1 μM of S1P1 receptor ligands (RP-001, S1P, or SEW2871) or vehicle (4 mg/ml BSA in PBS). After 10 minutes, the cell lysate was harvested and then Akt and phospho-Akt were identified by Western blotting (see complete unedited blot in the supplemental material). The experiment was performed in triplicate, and a representative result is shown. Scale bars: 100 μm.