Prostaglandin D2 induces apoptosis of human osteoclasts through ERK1/2 and Akt signaling pathways

In a recent study we have shown that prostaglandin D-2 (PGD(2)) induces human osteoclast (OC) apoptosis through the activation of the chemoattractant receptor homologous molecule expressed on T-helper type 2 cell (CRTH2) receptor and the intrinsic apoptotic pathway. However, the molecular mechanisms underlying this response remain elusive. The objective of this study is to investigate the intracellular signaling pathways mediating PGD(2)-induced OC apoptosis. OCs were generated by in vitro differentiation of human peripheral blood mononuclear cells (PBMCs), and then treated with or without the selective inhibitors of mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase, (MEK)-1/2, phosphatidylinosito13-kinase (PI3K) and NF-kappa B/I kappa B kinase-2 (IKK2) prior to the treatments of PGD(2) as well as its agonists and antagonists. Fluorogenic substrate assay and immunoblotting were performed to determine the caspase-3 activity and key proteins involved in Akt, ERK1/2 and NF-kappa B signaling pathways. Treatments with both PGD(2) and a CRTH2 agonist decreased ERK1/2 (Thr202/Tyr204) and Akt (Ser473) phosphorylation, whereas both treatments increased beta-arrestin-1 phosphorylation (Ser412) in the presence of naproxen, which was used to eliminate endogenous prostaglandin production. In the absence of naproxen, treatment with a CRTH2 antagonist increased both ERK1/2 and Akt phos-phorylations, and reduced the phosphorylation of beta-arrestin-1. Treatment of OCs with a selective MEK-1/2 inhibitor increased caspase-3 activity and OC apoptosis induced by both PGD(2) and a CRTH2 agonist Moreover, a CRTH2 antagonist diminished the selective MEK-1/2 inhibitor-induced increase in caspase-3 activity in the presence of endogenous prostaglandins. In addition, treatment of OCs with a selective PI3K inhibitor decreased ERK1/2 (Thr202/Tyr204) phosphorylation caused by PGD(2), whereas increased ERK1/2 (Thr202/Tyr204) phosphorylation by a CRTH2 antagonist was attenuated with a PI3K inhibitor treatment. The DP receptor was not implicated in any of the parameters evaluated. Treatment of OCs with PGD(2) as well as its receptor agonists and antagonists did not alter the phosphorylation of RelA/p65 (Ser536). Moreover, the caspase-3 activity was not altered in OCs treated with a selective IKK2/NF-kappa B inhibitor. In conclusion, endogenous or exogenous PGD(2) induces CRTH2-dependent apoptosis in human differentiated OCs; beta-arrestin-1, ERK1/2, and Akt, but not IKK2/NF-kappa B are probably implicated in the signaling pathways of this receptor in the model studied. (C) 2013 Elsevier Inc. All rights reserved.