Activation of Cytosolic Phospholipase A2 by Platelet-derived Growth Factor Is Essential for Cyclooxygenase-2-dependent Prostaglandin E2 Synthesis in Mouse Osteoblasts Cultured with Interleukin-1

Abstract

The synthesis of prostaglandins (PGs) is regulated by the arachidonic acid release by phospholipase A 2 (PLA 2 ) and its conversion to PGs by cyclooxygenase (COX). In the present study, we examined the regulation of PG synthesis by interleukin (IL)-1␣ in primary mouse osteoblastic cells isolated from mouse calvaria. Although IL-1␣ greatly enhanced cox-2 mRNA expression and its protein levels, PGE 2 was not produced until 24 h. When arachidonic acid was added to osteoblastic cells precultured with IL-1␣ for 24 h, PGE 2 was produced within 10 min. Of several growth factors tested, platelet-derived growth factor (PDGF) specifically initiated the rapid synthesis of PGE 2 , which was markedly suppressed by a selective inhibitor of cox-2 (NS-398). In mouse osteoblastic cells, cytosolic PLA 2 (cPLA 2 ) mRNA and its protein were constitutively expressed and increased approximately 2-fold by IL-1␣, but secretory PLA 2 mRNA was not detected. PDGF rapidly stimulated PLA 2 activity, which was blocked completely by a cPLA 2 inhibitor (arachidonyltrifluoromethyl ketone). The PDGF-induced cPLA 2 activation was accompanied by phosphorylation of its protein. These results indicate that cox-2 induction by IL-1␣ is not sufficient, but cPLA 2 activation by PDGF is crucial for IL-1␣-induced PGE 2 synthesis in mouse osteoblasts. Prostaglandins (PGs)1 are potent regulators of bone metabolism that are produced mainly by osteoblasts in the bone tissue. Among several forms of PG, PGE 2 is the major product of osteoblasts, and its production is regulated by several growth factors and cytokines including interleukin-1 (IL-1), parathyroid hormone, basic fibroblast growth factor (bFGF), and PGE 2 itself (1-6). PGE 2 is a complicated regulator of bone metabolism, potently stimulating bone resorption in vitro (7) and stimulating both bone formation and bone resorption in vivo (8). PGE 2 production is involved in the mechanism of bone resorption induced by IL-1 in vitro (7, 9). PG synthesis is regulated by two successive metabolic steps, the release of arachidonic acid from membranous phospholipids and its conversion to prostanoids. Phospholipase A 2 (PLA 2 ), the enzyme responsible for arachidonic acid release, consists of two forms, secretory PLA 2 (sPLA 2 ) and cytosolic PLA 2 (cPLA 2 ) (10 -13). Secretory PLA 2 requires millimolar levels of calcium for its activation and is divided into several groups, among which pancreatic (type I) and nonpancreatic (type II) subtypes are well characterized (10). Type II sPLA 2 is widely distributed in several tissues and inflammatory exudates. On the other hand, cPLA 2 is an intracellular enzyme that is found in various cells and tissues including macrophages, mast cells, platelets, kidney, and brain (10,14,15). This enzyme preferentially hydrolyzes arachidonic acid at the S n 2 position of membranous phospholipids and requires micromolar levels of calcium for its activation. Both the phosphorylation and Ca 2ϩ -dependent translocation of cPLA 2 to the membranes are essential for its activation (16 -20).C...

Regulation of Cyclooxygenase-2 pathway by HER2 receptor

“…The observed NDF-mediated stimulation of COX-2 expression in colorectal cancer cells was functional, as there was a signi®cant increase in the levels of accumulated PGE2 in the culture medium in NDF-treated CaCo-2 cells compared to the PGE2 levels in the control culture medium ( Figure 6C). However the observed increase in the COX-2 protein was not accompanied by concomitant increase in the accumulation of PGE2 in the medium but a delay was observed with maximum levels reaching at 24 h. The delay in the accumulation of PGE2 in the medium could be due to culturing of the cells in the low serum conditions and such conditions are known to reduce the activity of the other component enzymes in the PGE2 pathway and delay PGE2 secretion (Chen et al, 1997). The observed stimulation of PGE2 secretion in NDF-treated colorectal cancer cells was mediated through COX-2, as it could be e ectively blocked by a speci®c COX-2 inhibitor, NS-398 (Ciardiello et al, 1991) (see Figure 9D).…”

The Functions of Five Distinct Mammalian Phospholipase A2s in Regulating Arachidonic Acid Release

“…Our present studies have led to confirmation that cPLA 2 is certainly involved in IL-1-induced delayed AA release but only when FCS is also present. The requirement for both inflammatory (IL-1) and growth (FCS) stimuli for cPLA 2 activation may explain why inflammatory cytokines failed to activate cPLA 2 during the delayed response in some previous studies performed in the presence of low FCS concentrations (36,37). Proinflammatory stimuli that induce delayed AA metabolism, such as IL-1, tumor necrosis factor, and lipopolysaccharide, strongly activate members of the mitogen-activated protein kinase family (60), which in turn phosphorylate cPLA 2 at Ser 505 to increase its intrinsic activity (29,61), without accompanying Ca 2ϩ signaling.…”

“…The AA thus liberated is supplied to constitutive COX-1 and 5-lipoxygenase to be converted into prostanoids and leukotrienes, respectively (30 -33). cPLA 2 has also been implicated in delayed, COX-2-dependent prostanoid generation lasting for hours despite the absence of Ca 2ϩ signaling in this setting (15,19,34,35), although in some experimental systems cPLA 2 failed to supply AA to COX-2 during the delayed response unless the cells were exposed to a secondary Ca 2ϩ -mobilizing stimulus (36,37). Increased expression of cPLA 2 induced by proinflammatory stimuli has been reported to be linked to an ongoing delayed response (19,34,35,38) or to priming for increased immediate response (21,36,37,39,40).…”

Membrane-Bound Prostaglandin E Synthase-1-Mediated Prostaglandin E2 Production by Osteoblast Plays a Critical Role in Lipopolysaccharide-Induced Bone Loss Associated with Inflammation

“…Two isoforms of the COX enzyme, COX-1 and COX-2, have been identified, and previous studies have shown that COX-1 is a constitutive enzyme and COX-2 is an enzyme induced by various stimuli. In osteoblasts, the expression of COX-2, but not COX-1, is markedly induced by IL-1 and LPS (13). The terminal step for PGE 2 synthesis is catalyzed by PGES for the conversion of PGH 2 to PGE 2 .…”

“…PGE 2 synthesis is regulated by three metabolic steps: the release of arachidonic acid from the membranous phospholipids by phospholipase A 2 (PLA 2 ), the conversion of arachidonic acid to PGH 2 by cyclooxygenase (COX), and the synthesis of PGE 2 by PGE synthase (PGES) (10 -12). We have reported that the purpose of cytosolic PLA 2 (cPLA 2 ) expression in osteoblasts is to release arachidonic acid following PGE 2 production (13,14). Two isoforms of the COX enzyme, COX-1 and COX-2, have been identified, and previous studies have shown that COX-1 is a constitutive enzyme and COX-2 is an enzyme induced by various stimuli.…”