Mechanisms for proteinase-activated receptor 1-triggered prostaglandin E₂ generation in mouse osteoblastic MC3T3-E1 cells

Abstract

We analyzed signaling mechanisms for prostaglandin E2 (PGE2) production following activation of proteinase-activated receptor-1 (PAR1), a thrombin receptor, in preosteoblastic MC3T3-E1 cells. PAR1 stimulation caused PGE2 release, an effect suppressed by inhibitors of COX-1, COX-2, iPLA2, cPLA2, MAP kinases (MAPKs), Src, EGF receptor (EGFR) tyrosine kinase (EGFR-TK) and matrix metalloproteinase (MMP), but not by an intracellular Ca2+ chelator or inhibitors of PI3 kinase, protein kinase C (PKC) and NF-κB. PAR1 activation induced phosphorylation of MAPKs and upregulation of COX-2. The phosphorylation of p38 MAPK was suppressed by inhibitors of Src and EGFR-TK. The COX-2 upregulation was dependent on ERK, p38, EGFR-TK, Src, and COX-2 itself. PAR1 activation also induced MEK-dependent phosphorylation of cAMP response element binding protein (CREB). All inhibitors of EP1, EP2, EP3 and EP4 receptors suppressed the PAR1-triggered PGE2 release. Exogenously applied PGE2 facilitated PAR1-triggered COX-2 upregulation, but it alone had no effect. Together, the PAR1-mediated PGE2 production in MC3T3-E1 cells appears to involve iPLA2 and cPLA2 for arachidonic acid release, and the MEK/ERK/CREB and Src/MMP/EGFR/p38 pathways for COX-2 upregulation, which is facilitated by endogenous PGE2 formed by COX-2. These signaling mechanisms might underlie the role of the thrombin/PAR1/PGE2 system in the early stage of the bone healing.

PACAP and VIP signaling in chondrogenesis and osteogenesis

“…during adipogenesis [85] or in osteoblast cells [84], furthermore CREB phosphorylation is regulated by the MAPK system in MC3T3 cells [86]. Additionally, intracellular Ca 2+ concentration can be elevated by PACAP [87] or VIP [88], resulting in an activation of classical PKCs and ERK both influencing osteoblast differentiation [89].…”

Diversification of PAR signaling through receptor crosstalk

“…However, in most cells and tissues, COX-1 is constitutive and is responsible for the maintenance of PGs physiological homeostasis, whereas the expression of COX-2 is up-regulated in inflammatory cells and inflamed tissue [ 90 ]. Although most of the work in this field is focused on thrombin induction of COX-2 [ 91 ], the activation of PAR1 and PAR2 triggers PGE2 release by a mechanism involving both, COX-1 and COX-2 in mouse osteoblasts [ 92 ], and in the tracheal and bronchial smooth muscle [ 89 ]. Phospholipase A2 (PLA2) is responsible for the cleavage of phospholipids containing arachidonic acid, which will in turn activate COX enzymes.…”

“…The activation of PLA2 by intracellular Ca 2+ depends on the type of activated receptor and the cellular context: since PAR1-activated osteoblasts are insensitive to the Ca +2 chelator BAPTA/AM, and PKC inhibitors, whereas PAR2-activated lung epithelial cells require this stimulation for transiently activating ERK, and subsequently stimulating PLA2 (Fig. 2 C) [ 89 , 92 ]. On the other hand, as expected, the induction of COX-2 is delayed, and is mediated by ERK1/2, p38 MAPK, Src and EGF receptors (Fig.…”

Mechanisms of Bone Remodeling Disorder in Hemophilia

“…54 The PAR-1-triggered PGE2 generation in osteoblasts involves PLA2, which releases arachidonic acid from the membrane, and PAR-1 leads to the upregulation of COX-2 via activation of the MAPKs pathway. 55 A genetic study established the pivotal role of COX-2 in maintaining bone density, in which Cox2 knockout mice showed low bone mass. 56 PGE2, a metabolite of arachidonic acid under the action of the COX, is important to maintain the balance of bone.…”