Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose
Chemokine-like receptor 1 (CMKLR1) is a G protein-coupled receptor (GPCR) expressed by inflammatory monocytes and up-regulated in fibroblast-like synoviocytes (FLSs), both of which are pathogenic in osteoarthritis (OA) and rheumatoid arthritis (RA). Its cognate ligand, chemerin, is a chemoattractant for invading inflammatory cells and is present in the synovial lining of arthritis patients. In OA and RA, chemerin/CMKLR1 signaling contributes to disease pathogenesis via recruitment of inflammatory leukocytes and stimulates breakdown of cartilage matrix by metalloproteases.
CMKLR1, like most GPCRs, is desensitized by recruitment of G protein receptor kinases (GRKs) and β-arrestins. Due to the significant role of chemerin/CMKLR1 interaction in human inflammatory arthritis, we examined the mechanism of CMKLR1 regulation by G protein receptor kinases (GRK) 2, 3, and 6 and β-arrestin-2.
Methods
De-identified, healthy, OA, and RA human FLSs were cultured for measurement of relative gene expression by qRT-PCR of CMKLR1, GRK2, GRK3 and GRK6 as compared to housekeeping gene IDUA (ΔCt).
A modified Tango assay was used to measure β-arrestin-2 recruitment to CMKLR1. HTLA cells were over-expressed with CMKLR1 and GRK2, GRK3, or GRK6 via calcium phosphate precipitation and stimulated with increasing concentrations of chemerin. After 24 hours, luminescence was measured on a Promega Glomax Multi+ Detection System.
Peritoneal activated monocytes/macrophages from C57Bl/6 wildtype (WT) and transgenic mice deficient in GRK2, GRK3, GRK6, or β-arrestin-2 (-/-) were studied ex vivo for CMKLR1 receptor internalization and migration. Calcein-labeled monocyte migration to media alone, SDF-1, or chemerin was examined using the FalconTM HTS FluoroBlok 96-Multiwell Insert System (3 µm pore-size) and a Fluoroskan Ascent Microplate Fluorometer. To measure CMKLR1 internalization, receptor surface expression was measured by flow cytometry at 30 sec, 1 min, 5 min and 10 min post-stimulation with chemerin and mean fluorescence intensity (MFI) normalized relative to 0 min on F4/80 positive cells.
Results
Relative gene expression of CMKLR1 is increased in both OA and RA FLS samples compared to normal controls. In addition, RA FLS show increased GRK2, -3, and -6 relative gene expression compared to controls. β-arrestin-2 recruitment to ligand-activated CMKLR1 is preferentially mediated by GRK6 as compared to GRK2 and -3. Additionally, GRK6-/- and β-arrestin-2 -/- murine peritoneal macrophages have enhanced chemotaxis to chemerin, as well as significantly decreased receptor internalization after ligand stimulation when compared to controls.
Conclusion
Chemerin/CMKLR1 signaling has an important role in the pathogenesis of OA and RA through the activation of cells within the joint, as well as recruiting proinflammatory monocytes to sites of inflammation. Chemerin-induced migration of proinflammatory monocytes and CMKLR1 receptor internalization is predominantly regulated by GRK6 phosphorylation and β-arrestin-2 recruitment.
Disclosure:
D. S. Serafin,
None;
M. F. Sassano,
None;
D. Mattox,
None;
R. Timoshchenko,
None;
M. J. Billard,
None;
D. P. Siderovski,
None;
B. Roth,
None;
T. K. Tarrant,
None.
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