Pertussis toxin-sensitive and insensitive G proteins mediate ALFL4-stimulated release of inositol phosphates, diglycerides and arachidonic acid in MC3T3-E1 osteoblasts

In Ihis study, we have measured the effects of the G protein activator AIF;on the formation of inositol phosphates. diglycerides and arachidonic acid in MC3T3-El osteoblasth. We have shown that AIF, induces a similar fold elevation in the levels of all three of these second messengers. AIF,also decreased the specific radioactivity of [‘Hlarachidonic acid in the diglyceride pool. These results suggest that A1F4activated a phosphatidylinositolspecific phospholipase C to liberate diglycerides and stimulated the metabolism of diglycerides to release arachidonic acid. The G protein inhibitor pertussis toxin was demonstrated to reduce the AIF,-induced release of inositol phosphates or [jH]arachidonic acid by 20-30%. However, incubation of cells with cholera toxin, a G protein activator, resulted in an amplification of the hasal and AIF,--stimulated liberation of inositol phosphates, diglycerides and [“Hlarachidonic acid (following deesterification from diglycerides). Therefore, this \tudy provides evidence that arachidonic acid availability for prostaglandin synthesis in osteoblasts may be regulated by the activity of a G protein coupled to a phosphatidylinositol-specific phospholipase C. Prostaglandins may play important role(s) in the regulation of bone metabolism and remodeling, since they have been demonstrated to stimulate both the formation and resorption of bone (1). However, during the invasion of bone by malignant cells or immune cells that mediate inilammatory responses. elevated levels of prostaglandins art: released from bone cells in reponse to cytokines like PDGF or monokines such as tumor necrosis factor ITNF) or interleukin 1 (11-l). Abnormally high levels of prostaglandins are thought to be at least partially responsible for the accelerated resorption of bone that is caused by cancer or inflammation (2-7). Therefore. the attenuation of prostaglandin synthesis in bone may be central to the therapeutic control of some forms of pathological osteolysis. Recently, we have demonstrated that TNFalpha stimulates prostaglandin synthesis in the osteoblast cell line MC3T3-El by increasing the availability of arachidonic acid substrate (8). TNF-alpha was shown to activate both a phospholipasr C which selectively hydrolyzes phosphatidylinositol (over other phospholipids) to release diglyceride and diglyceride-metabolizing lipases to release arachidonic acid (8). These findings suggested that changes in the activity of a PI-specific phospholipase C may modulate the levels of free arachidonic acid and availability for prostaglandin synthesis in bone cells. The activity of PI-specific phospholipase C has been shown to be regulated by GTP-binding proteins (9-1 I ). We have used the compound AIF.,-. which has been shown to activate a G protein coupled to a PI-specific phospholipase C ( 12), to study the role of G protein in the regulation of arachidonic acid release via this pathway in the mouse osteoblast cell line. MC3T3El. We present evidence that an activator of G proteins stimulates arachidonic acid release by increasing the metabolism of PI by phospholipase C and that of diglyceride by diglyceride lipases. These findings provide evidence for a new and potentially important regulatory site in the pathogenesis 01‘ prostaglandindependent bone resorption.