Maxime A. Gallant

Prostaglandin D2 Receptors Control Osteoclastogenesis and the Activity of Human Osteoclasts

We recently showed that human osteoblasts synthesize prostaglandin D2 (PGD2) and express both DP and CRTH2 receptors. Activation of the DP receptor decreased osteoprotegerin production, whereas activation of the CRTH2 receptor induced osteoblast chemotaxis and decreased RANKL expression. Our objectives in this study were to determine the presence, distribution, and action of these receptors in the functions of human osteoclasts and in osteoclastogenesis. Immunohistochemistry was used to detect the presence of DP and CRTH2 in in vitro–differentiated human osteoclasts in culture and in osteoclasts in situ. The effects of the activation of PGD2 receptors on the cytoskeleton were determined by fluorescence microscopy. Specific agonists and antagonists allowed the study of the roles of these receptors on bone resorption and osteoclast differentiation. Our results show that in vitro–differentiated human osteoclasts and authentic fetal osteoclasts express both DP and CRTH2 receptors, as shown by immunocytochemistry. Similar results were obtained in osteoclasts from normal, osteoporotic, pagetic, and osteoarthritic adult bone tissues. Stimulation of osteoclasts with PGD2 induced a robust reorganization of the cytoskeleton with a decrease in the number of cells presenting actin rings and an increase of lamellipodia, effects mediated by the DP and CRTH2 receptors, respectively. PGD2 showed an inhibitory effect on bone resorption activity acting through the DP receptor. In vitro osteoclastogenesis from peripheral blood mononuclear cells cultured in the presence of RANKL and macrophage‐colony stimulating factor was decreased by activation of either DP or CRTH2 receptors. These results suggest that PGD2 receptors could be useful targets in certain bone diseases because their specific activation/inhibition leads to a decrease in osteoclastogenesis and to inhibition of bone resorption by osteoclasts.

Inverse agonist and pharmacochaperone properties of MK-0524 on the prostanoid DP1 receptor.

Prostaglandin D2 (PGD2) acts through two G protein-coupled receptors (GPCRs), the prostanoid DP receptor and CRTH2 also known as DP1 and DP2, respectively. Several previously characterized GPCR antagonists are now classified as inverse agonists and a number of GPCR ligands are known to display pharmacochaperone activity towards a given receptor. Here, we demonstrate that a DP1 specific antagonist, MK-0524 (also known as laropiprant), decreased basal levels of intracellular cAMP produced by DP1, a Gαs-coupled receptor, in HEK293 cells. This reduction in cAMP levels was not altered by pertussis toxin treatment, indicating that MK-0524 did not induce coupling of DP1 to Gαi/o proteins and that this ligand is a DP1 inverse agonist. Basal ERK1/2 activation by DP1 was not modulated by MK-0524. Interestingly, treatment of HEK293 cells expressing Flag-tagged DP1 with MK-0524 promoted DP1 cell surface expression time-dependently to reach a maximum increase of 50% compared to control after 24 h. In contrast, PGD2 induced the internalization of 75% of cell surface DP1 after the same time of stimulation. The increase in DP1 cell surface targeting by MK-0524 was inhibited by Brefeldin A, an inhibitor of transport from the endoplasmic reticulum-Golgi to the plasma membrane. Confocal microscopy confirmed that a large population of DP1 remained trapped intracellularly and co-localized with calnexin, an endoplasmic reticulum marker. Redistribution of DP1 from intracellular compartments to the plasma membrane was observed following treatment with MK-0524 for 24 h. Furthermore, MK-0524 promoted the interaction between DP1 and the ANKRD13C protein, which we showed previously to display chaperone-like effects towards the receptor. We thus report that MK-0524 is an inverse agonist and a pharmacochaperone of DP1. Our findings may have important implications during therapeutic treatments with MK-0524 and for the development of new molecules targeting DP1.

ANKRD13C Acts as a Molecular Chaperone for G Protein-coupled Receptors

Although the mechanisms that regulate folding and maturation of newly synthesized G protein-coupled receptors are crucial for their function, they remain poorly characterized. By yeast two-hybrid screening, we have isolated ANKRD13C, a protein of unknown function, as an interacting partner for the DP receptor for prostaglandin D2. In the present study we report the characterization of this novel protein as a regulator of DP biogenesis and trafficking in the biosynthetic pathway. Co-localization by confocal microscopy with an endoplasmic reticulum (ER) marker, subcellular fractionation experiments, and demonstration of the interaction between ANKRD13C and the cytoplasmic C terminus of DP suggest that ANKRD13C is a protein associated with the cytosolic side of ER membranes. Co-expression of ANKRD13C with DP initially increased receptor protein levels, whereas siRNA-mediated knockdown of endogenous ANKRD13C decreased them. Pulse-chase experiments indicated that ANKRD13C can promote the biogenesis of DP by inhibiting the degradation of newly synthesized receptors. However, a prolonged interaction between ANKRD13C and DP resulted in ER retention of misfolded/unassembled forms of the receptor and to their proteasome-mediated degradation. ANKRD13C also regulated the expression of other GPCRs tested (CRTH2, thromboxane A2 (TPα), and β2-adrenergic receptor), whereas it did not affect the expression of green fluorescent protein, GRK2 (G protein-coupled receptor kinase 2), and VSVG (vesicular stomatitis virus glycoprotein), showing specificity toward G protein-coupled receptors. Altogether, these results suggest that ANKRD13C acts as a molecular chaperone for G protein-coupled receptors, regulating their biogenesis and exit from the ER.

Characterization of C-terminal tail determinants involved in CRTH2 receptor trafficking: identification of a recycling motif.

The molecular mechanisms regulating the trafficking of the CRTH2 receptor are poorly understood. In the present study, we characterize C-terminal tail determinants involved in the agonist-induced trafficking of the CRTH2 receptor for prostaglandin D(2). Our results showed that progressive deletion of C-terminal tail residues from amino acid 395 up to 337 gradually impaired CRTH2 internalization by approximately 50% as measured by ELISA in HEK293 cells. Surprisingly, further deletion of the C-tail to amino acid 328 or 317 resulted in receptor mutants displaying internalization similar to the wild-type receptor. Individual mutations of Asp(330), Ser(331), Glu(332), and Leu(333) to Ala in the C-tail of the full length receptor resulted in a 45% increase in internalization of the receptor mutants relative to the wild-type receptor. Pretreatment with the recycling inhibitor monensin increased internalization of the wild-type receptor but did not affect that of the D330A, S331A, E332A and L333A mutants, indicating that these residues are part of a recycling motif. Further experiments revealed that Asp(330), Ser(331) and Glu(332) are not only involved in receptor recycling, but are also required for promotion of CRTH2 internalization by GRK2 and GRK5. Site-directed mutagenesis identified Thr(347) as a major site for PKC-induced internalization of the receptor. Confocal microscopy revealed that arrestin-3 dissociated from the receptor after agonist stimulation and internalization, suggesting that CRTH2 is a class A G protein-coupled receptor. Our study identified specific amino acids in the CRTH2 receptor C-tail implicated in the agonist-induced internalization and the recycling of the receptor.

Increased Concentrations of Prostaglandin D2 During Post-Fracture Bone Remodeling

Objective. To test the hypothesis that increased concentrations of prostaglandin D2 (PGD2) correlate with bone remodeling. Studies using isolated bone cells indicate that PGD2 may be implicated in the regulation of bone homeostasis, with a positive influence on bone anabolism. We studied patients with traumatic fractures and age- and sex-matched healthy controls as an in vivo model of increased bone remodeling. Methods. Thirty-five patients with bone fracture and matched controls were recruited. Urine and sera samples were collected. Urinary 11ß-PGF2α, a PGD2 metabolite, and PGE2 metabolites (PGEM), serum lipocalin-type PGD2 synthase (L-PGDS), bone alkaline phosphatase (bone ALP), and crosslinked C-telopeptides of type I collagen (CTX) were measured. Results. At 5–6 weeks post-fracture, 11ß-PGF2α, L-PGDS, bone ALP, and CTX were significantly increased in the fracture patients compared to controls. PGEM levels were not different between groups. Levels of 11ß-PGF2α and bone ALP were positively correlated, suggesting that PGD2 may be implicated in fracture repair. Conclusion. These results support our working hypothesis that PGD2 could be implicated in the control of bone anabolism in humans.

An Interaction between L-prostaglandin D Synthase and Arrestin Increases PGD2 Production

L-type prostaglandin synthase (L-PGDS) produces PGD2, 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 PGD2-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 PGD2 production by L-PGDS in vitro. IL-1β-induced PGD2 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 PGD2 production in vitro and in MG-63 cells. We report the first characterization of an interactor/modulator of a PGD2 synthase and the identification of a new function for arrestin, which may open new opportunities for improving therapies for the treatment of inflammatory diseases.