Involvement of Phosphatidylcholine-specific Phospholipase C in Platelet-derived Growth Factor-induced Activation of the Mitogen-activated Protein Kinase Pathway in Rat-1 Fibroblasts

Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) in mouse embryo fibroblasts (MEFs).In order to investigate a role for phospholipase C-␥1 (PLC-␥1), we used targeted disruption of the Plcg1 gene in the mouse to develop Plcg1 ؉/؉ and Plcg1 ؊/؊ cell lines. Plcg1؉/؉ MEFs treated with PDGF showed a time-and dose-dependent increase in the production of total inositol phosphates that was substantially reduced in Plcg1 ؊/؊ cells. Plcg1 ؉/؉ cells also showed a PDGF-induced increase in PLD activity that had a similar dose dependence to the PLC response but was down-regulated after 15 min. Phospholipase D activity, however, was markedly reduced in Plcg1 ؊/؊ cells. The PDGF-induced inositol phosphate formation and the PLD activity that remained in the Plcg1 ؉/؉ and Plcg1 ؊/؊ cells following PDGF treatment, it is possible neither PLC nor PLD are necessary for this growth factor response. In summary, these data indicate that PLC-␥ is required for growth factor-induced activation of PLD in MEFs.

Section: Fig 7 Western Blot Of Map Kinase Tvii-targeted Plcg1mentioning

confidence: 62%

Analysis of Platelet-derived Growth Factor-induced Phospholipase D Activation in Mouse Embryo Fibroblasts Lacking Phospholipase C-γ1

Hess

Carpenter

et al. 1998

“…2. There have been recent reports in which D609 was found to not only exert effects upon PC-PLC but also PLD (44,45) and PI-PLC (46,47). These results do not preclude the possible role of PC-PLC or PLD in MMP-13 protein expression.…”

Section: Discussionmentioning

confidence: 99%

Serotonin-induced MMP-13 Production Is Mediated via Phospholipase C, Protein Kinase C, and ERK1/2 in Rat Uterine Smooth Muscle Cells

Shum

Melendez

Jeffrey

2002

Serotonin (5-hydroxytryptamine; 5-HT), acting via the 5-HT 2A receptor, up-regulates the transcription and production of interstitial collagenase (matrix metalloproteinase-13; MMP-13), a critical enzyme responsible for maintaining the integrity of the uterus, after parturition. Serotonin treatment of rat uterine myometrial smooth muscle cells induced inositol phosphate (IP) turnover, which was abolished by the 5-HT 2A receptorspecific antagonists ketanserin and spiperone. The phospholipase C (PLC) inhibitors U73122 and D609 attenuated serotonin-mediated-IP turnover with a corresponding inhibition of MMP-13 protein production. Subsequent recovery of both MMP-13 protein expression and IP generation was seen following the removal of D609. Protein kinase C (PKC) activators, the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol and phorbol myristate acetate (PMA), mimicked the effect of serotonin on MMP-13 protein expression; prolonged PMA treatment (which down-regulates PKC) lowered MMP-13 protein levels. The PKC-specific inhibitors bisindolylmaleimide I, calphostin C, CGP 41251, and the PKC␦-selective inhibitor rottlerin were able to suppress serotonin up-regulation of MMP-13. Furthermore, the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 blocked serotonin-dependent activation of p44/42 MAPK (pERK1/2), a downstream effector of PKC and also down-regulated MMP-13 protein expression. Similarly, calphostin C and rottlerin depressed activation of p44/42 MAPK. From these studies, serotonin, binding through the 5-HT 2A receptor, initiates a signaling cascade whereby stimulation of PLC leads to the activation of PKC and subsequently the ERK1/2 pathway, which ultimately results in MMP-13 production.The maintenance of the three-dimensional architecture of the mammalian uterus is carefully regulated during pregnancy and after parturition. Production of fibrillar collagens (Types I and III) is up-regulated 10-fold in the uterus during pregnancy (1, 2). These collagens possess a rigid superhelical structure, which is responsible for providing the tensile strength necessary to accommodate the force exerted by the growing fetus. After parturition, a precisely programmed non-necrotic and non-inflammatory process of connective tissue remodeling occurs, which results in the rapid and dramatic degradation of the collagen amassed and returns the uterus to reproductive competence (3). This transition is initiated by the enzyme interstitial collagenase (matrix metalloproteinase-13; MMP-13), 1 a member of the matrix metalloproteinase family. It acts as not only the rate-limiting step but also the committed step in the catalysis of the degradation of extracellular collagen (4, 5). The expression of MMP-13 occurs during postpartum involution and is not present either during the pregnancy or even within hours prior to parturition. The myometrial smooth muscle cell of the uterus has been identified not only as the source for MMP-13 (6) but also that of its natural substrate, collagen (7). Thus, the myometrial smooth muscle...

“…Since the main pathway of SM biosynthesis in mammalian cells is catalyzed by SM synthase, and this occurs primarily through the transfer of the phosphorylcholine group from phosphatidylcholine to ceramide, the reaction products are both SM and diacylglycerol (38,45,47,64). Although the signaling pathways that mediate the proliferating effect of bFGF have not been fully elucidated, there is evidence that different growth factors, upon activation of their receptor tyrosine kinases, induce a PC-specific phospholipase C instrumental to their mitogenic effect (65)(66)(67). The evidence that (a) an extra-Golgi SM synthase is up-regulated by and necessary for bFGF growth stimulation of astrocytes, (b) the PC-specific phospholipase C inhibitor D609 exerts an inhibitory effect on the bFGF-induced stimulation of both SM biosynthesis and astrocyte proliferation, and (c) neosynthesized phosphatidylcholine is the major phosphocholine donor for SM synthesis in glial cells (68,69) raises the intriguing possibility, recently proposed by Luberto and Hannun (23), that the PC-specific phospholipase C involved in cell proliferation may, in part, be a SM synthase.…”

Section: Discussionmentioning

confidence: 99%

Basic Fibroblast Growth Factor-induced Proliferation of Primary Astrocytes

Riboni

Viani

Bassi

et al. 2001

We recently reported that the marked decrease in cellular ceramide in primary astrocytes is an early event associated with the mitogenic activity of basic fibroblast growth factor (bFGF) (Riboni, L., Viani, P., Bassi, R., Stabieini, A., and Tettamanti, G. (2000) GLIA 32, 137-145). Here we show that a rapid activation of sphingomyelin biosynthesis appears to be the major mechanism responsible for the fall in ceramide levels induced by bFGF. When quiescent astrocytes were treated with bFGF, an increased amount of newly synthesized ceramide (from either L-[ 3 H]serine or [ 3 H]sphingosine) was directed toward the biosynthesis of sphingomyelin. Conversely, bFGF did not appear to affect ceramide levels by other metabolic pathways involved in ceramide turnover such as sphingomyelin degradation and ceramide biosynthesis, degradation, and glucosylation. Enzymatic studies demonstrating a relevant and rapid increase in sphingomyelin synthase activity after bFGF treatment have provided a convincing explanation for the activation of sphingomyelin biosynthesis. The bFGF-induced increase in sphingomyelin synthase appears to depend on a post-translational activation mechanism. Moreover, in the presence of brefeldin A, the activation of sphingomyelin biosynthesis was abolished, suggesting that the enzyme is located in a compartment other than the Golgi apparatus. Also the phosphatidylcholine-specific phospholipase C inhibitor D609 exerted a potent inhibitory effect on sphingomyelin biosynthesis. Finally, we demonstrate that inhibition of sphingomyelin biosynthesis by brefeldin A or D609 led to a significant inhibition of bFGF-stimulated mitogenesis. All this supports that, in primary astrocytes, the early activation of sphingomyelin synthase is involved in the bFGF signaling pathway leading to proliferation.Ceramide, a key sphingolipid metabolite in both the biosynthesis and degradation of complex sphingolipids, is involved in the signal transduction of different extracellular stimuli that lead to cell proliferation, cell differentiation, cell cycle arrest, and apoptotic cell death (reviewed in Refs. 1-3). In all these events, the ceramide concentration, possibly at specific subcellular sites, is crucial. There is evidence that different activators such as cytokines, growth factors, and hormones elicit their biological effect by modulating the activity of sphingomyelinases, ceramide synthase, or neutral ceramidase (reviewed in Refs. 1 and 3-5) and thus affect ceramide levels.A role of ceramide as an intracellular mediator of specific extracellular agents has been recognized also in cells from the central nervous system (reviewed in Refs. 6 -8). In neuronal and glial cells, the administration of differentiating or apoptotic agents results in increased cellular levels of ceramide, which, in turn, participate in the cascade of events producing the final effects (9 -12). In a recent study (13), we demonstrated that ceramide plays a role in the growth control of glial cells by basic fibroblast growth factor (bFGF), 1 a factor stimul...