Abstract
L-type prostaglandin synthase (L-PGDS) produces PGD(2), a lipid mediator involved in neuromodulation and inflammation. Here, we show that L-PGDS and arrestin-3 (Arr3) interact directly and can be co-immunoprecipitated endogenously from MG-63 osteoblasts. Perinuclear L-PGDS/Arr3 co-localization is observed in PGD(2)-producing MG-63 cells and is induced by the addition of the L-PGDS substrate or co-expression of COX-2 in HEK293 cells. Inhibition of L-PGDS activity in MG-63 cells triggers redistribution of Arr3 and L-PGDS to the cytoplasm. Perinuclear localization of L-PGDS is detected in wild-type mouse embryonic fibroblasts (MEFs) but is more diffused in MEFs-arr-2(-/-)-arr-3(-/-). Arrestin-3 promotes PGD(2) production by L-PGDS in vitro. IL-1β-induced PGD(2) production is significantly lower in MEFs-arr-2(-/-)-arr-3(-/-) than in wild-type MEFs but can be rescued by expressing Arr2 or Arr3. A peptide corresponding to amino acids 86-100 of arrestin-3 derived from its L-PGDS binding domain stimulates L-PGDS-mediated PGD(2) production in vitro and in MG-63 cells. We report the first characterization of an interactor/modulator of a PGD(2) synthase and the identification of a new function for arrestin, which may open new opportunities for improving therapies for the treatment of inflammatory diseases.
Highlights
Quebec. 2 Supported by fellowships from the Canadian Arthritis Network for part of this work. 3 Holds a Canada Research Chair in Molecular Endocrinology. 4 Recipient of “Chercheur boursier senior” salary support from the Fonds de la recherche en santedu Quebec and the recipient of the Andre -Lussier Research Chair in Rheumatology
L-prostaglandin D2 synthase (PGDS) Directly Interacts with Arrestin-3—lipocalintype PGDS (L-PGDS) was identified as a potential Arr3 partner during a yeast two-hybrid screen of a human HeLa cell Matchmaker cDNA library using Arr3 as bait
No growth was observed in yeast cells transformed with pAS2.1-Arr3 and pGADGH-hematopoietic PGDS (H-PGDS), the other PGD2 synthase, suggesting that Arr3 interacts with L
Summary
Quebec. 2 Supported by fellowships from the Canadian Arthritis Network for part of this work. 3 Holds a Canada Research Chair in Molecular Endocrinology. 4 Recipient of “Chercheur boursier senior” salary support from the Fonds de la recherche en santedu Quebec and the recipient of the Andre -Lussier Research Chair in Rheumatology. Immunofluorescence Staining and Confocal Microscopy— For co-localization experiments, HEK293 or MEF cells were plated and transfected as described above with Arr3-GFP, COX-2, and/or L-PGDS-HA where indicated.
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