Regulation of cytosolic phospholipase A2 in rat endometrial stromal cells: the role of epidermal growth factor

The effect of epidermal growth factor on the levels of cytosolic phospholipase A2 mRNA and protein in cultured rat endometrial stromal cells isolated from uteri sensitized for the decidual cell reaction was examined. Treatment with epidermal growth factor increased the steady-state cytosolic phospholipase A2 mRNA and protein levels as demonstrated by Northern and Western blot analyses, respectively. Immunocytochemical analysis demonstrated an increase of cytosolic phospholipase A2 protein in most cells, as opposed to a small subpopulation of cells in culture. These results show that epidermal growth factor causes an increase in steady-state cytosolic phospholipase A2 mRNA and protein levels in rat endometrial stromal cells from uteri sensitized for the decidual cell reaction. Epidermal growth factor receptor ligands may regulate cytosolic phospholipase A2 and thus prostaglandin production in the endometrial stromal cells during implantation.     

Mechanism of group IVA cytosolic phospholipase A(2) activation by phosphorylation

Group IVA cytosolic phospholipase A2 (cPLA2) has been shown to play a critical role in the agonist-induced release of arachidonic acid. To understand the mechanism by which phosphorylation of Ser505 and Ser727 activates cPLA2, we systematically analyzed the effects of S505A, S505E, S727A, S727E, S505A/S727A, S505A/S727E, and S505E/S727E mutations on its enzyme activity and membrane affinity. In vitro membrane binding measurements showed that S505A has lower affinity than the wild type or S505E for phosphatidylcholine membranes, which is exclusively due to faster desorption of the membrane-bound S505A. In contrast, neither S727A nor S727E mutation had a significant effect on the phosphatidylcholine vesicle binding affinity of cPLA2. The difference in in vitro membrane affinity between wild type (or S505E) and S505A increased with the decrease in Ca2+ concentration, reaching >60-fold at 2.5 microm Ca2+. When HEK293 cells transfected with cPLA2 and mutants were stimulated with ionomycin, the wild type and S505E translocated to the perinuclear region and caused the arachidonic acid release at 0.4 microm Ca2+, whereas S505A showed no membrane translocation and little activity to release arachidonic acid. Further mutational analysis of hydrophobic residues in the active site rim (Ile399, Leu400, and Leu552) indicate that a main role of the Ser505 phosphorylation is to promote membrane penetration of these residues, presumably by inducing a conformational change of the protein. These enhanced hydrophobic interactions allow the sustained membrane interaction of cPLA2 in response to transient calcium increases. On the basis of these results, we propose a mechanism for cPLA2 activation by calcium and phosphorylation.     

Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells

The current study examined the signal transduction steps involved in the selective release of arachidonic acid (AA) induced by the addition of secretory phospholipase A2 (sPLA2) isotypes to bone marrow-derived mast cells (BMMC). Overexpression of sPLA2 receptors caused a marked increase in AA and PGD2 release after stimulation of BMMC, implicating sPLA2 receptors in this process. The hypothesis that the release of AA by sPLA2 involved activation of cytosolic PLA2 (cPLA2) was next tested. Addition of group IB PLA2 to BMMC caused a transient increase in cPLA2 activity and translocation of this activity to membrane fractions. Western analyses revealed that these changes in cPLA2 were accompanied by a time-dependent gel shift of cPLA2 induced by phosphorylation of cPLA2 at various sites. A noncatalytic ligand of the sPLA2 receptor, p-amino-phenyl-alpha-D-mannopyranoside BSA, also induced an increase in cPLA2 activity in BMMC. sPLA2 receptor ligands induced the phosphorylation of p44/p42 mitogen-activated protein kinase. Additionally, an inhibitor of p44/p42 mitogen-activated protein kinase (PD98059) significantly inhibited sPLA2-induced cPLA2 activation and AA release. sPLA2 receptor ligands also increased Ras activation while an inhibitor of tyrosine phosphorylation (herbimycin) inhibited the increase in cPLA2 activation and AA release. Addition of partially purified sPLA2 from BMMC enhanced cPLA2 activity and AA release. Similarly, overexpression of mouse groups IIA or V PLA2 in BMMC induced an increase in AA release. These data suggest that sPLA2 mediate the selective release of AA by binding to cell surface receptors and then inducing signal transduction events that lead to cPLA2 activation.     

RGS16 function is regulated by epidermal growth factor receptor-mediated tyrosine phosphorylation

Galpha(i)-coupled receptor stimulation results in epidermal growth factor receptor (EGFR) phosphorylation and MAPK activation. Regulators of G protein signaling (RGS proteins) inhibit G protein-dependent signal transduction by accelerating Galpha(i) GTP hydrolysis, shortening the duration of G protein effector stimulation. RGS16 contains two conserved tyrosine residues in the RGS box, Tyr(168) and Tyr(177), which are predicted sites of phosphorylation. RGS16 underwent phosphorylation in response to m2 muscarinic receptor or EGFR stimulation in HEK 293T or COS-7 cells, which required EGFR kinase activity. Mutational analysis suggested that RGS16 was phosphorylated on both tyrosine residues (Tyr(168) Tyr(177)) after EGF stimulation. RGS16 co-immunoprecipitated with EGFR, and the interaction did not require EGFR activation. Purified EGFR phosphorylated only recombinant RGS16 wild-type or Y177F in vitro, implying that EGFR-mediated phosphorylation depended on residue Tyr(168). Phosphorylated RGS16 demonstrated enhanced GTPase accelerating (GAP) activity on Galpha(i). Mutation of Tyr(168) to phenylalanine resulted in a 30% diminution in RGS16 GAP activity but completely eliminated its ability to regulate G(i)-mediated MAPK activation or adenylyl cyclase inhibition in HEK 293T cells. In contrast, mutation of Tyr(177) to phenylalanine had no effect on RGS16 GAP activity but also abolished its regulation of G(i)-mediated signal transduction in these cells. These data suggest that tyrosine phosphorylation regulates RGS16 function and that EGFR may potentially inhibit Galpha(i)-dependent MAPK activation in a feedback loop by enhancing RGS16 activity through tyrosine phosphorylation.     

Tyrosine phosphorylation of phospholipase C-II in vitro by the epidermal growth factor receptor

In a number of cell lines, epidermal growth factor (EGF) rapidly stimulates the breakdown of inositol phospholipids. Phosphatidylinositol-specific phospholipase C (PLC), therefore, plays an important role in this biological response to EGF, but the mechanism by which EGF-receptor complexes modulate the activation of PLC is not understood. We have previously suggested that tyrosine phosphorylation of PLC or an unknown PLC-associated protein by the EGF receptor is involved in the activation process (Wahl, M. I., Daniel, T. O., and Carpenter, G. (1988) Science 241, 968-970) and have recently shown by immunoprecipitation that the addition of EGF to 32P-labeled cells increases tyrosine and serine phosphorylation of PLC-II (Wahl, M. I., Nishibe, S., Suh, P.-G., Rhee, S. G., and Carpenter, G. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1568-1572). In this communication we demonstrate that PLC-II (Mr = 145,000) purified from bovine brain can be phosphorylated in vitro in an EGF-dependent manner by the tyrosine kinase activity of the purified EGF receptor. While PLC-II is an efficient phosphorylation substrate for the purified EGF receptor, PLC-I is a poor substrate and PLC-III is not phosphorylated to any detectable extent. Though all three PLC isozymes possess typical tyrosine phosphorylation sequences, the EGF receptor is surprisingly selective in vitro for the phosphorylation of PLC-II. High performance liquid chromatography comparison of tryptic phosphotyrosyl peptides from PLC-II phosphorylated in vivo and in vitro indicated a similar pattern of multiple tyrosine phosphorylation sites. These findings show that the EGF receptor can directly phosphorylate PLC-II in an efficient and selective manner.     

Epidermal growth factor induces tyrosine phosphorylation of epidermal growth factor receptors not occupied with ligand in intact A431 cells.

The mechanism of epidermal growth factor receptor (EGF-R) autophosphorylation in intact A431 cells was studied. We detected epidermal growth factor (EGF) induced tyrosine phosphorylation of EGF-R not occupied with ligand. Cell monolayers were subjected to irradiation after incubation with photoreactive derivative of EGF and uncoupled EGF was extracted by acidic treatment. Subsequent immunoprecipitation with antiphosphotyrosine antibodies resulted in precipitation of both EGF-R complexes with EGF and EGF-R with unoccupied ligand-binding site. The fact of precipitation of EGF-R with unoccupied ligand-binding site in conjunction with our finding of rapid dephosphorylation of EGF-R after EGF extraction by acidic treatment, strongly supports the interpretation that cross-phosphorylation of EGF-R may take place in intact cells.     

Tyrosine phosphorylation by the epidermal growth factor receptor kinase induces functional alterations in microtubule-associated protein 2

We have examined the effect of tyrosine phosphorylation of microtubule-associated protein 2 (MAP2) by the epidermal growth factor (EGF) receptor kinase on its functions. Incubation of MAP2 with the EGF receptor in the presence of ATP resulted in a great decrease in the ability of MAP2 to promote tubulin polymerization. Under a variety of conditions, the decrease in the ability correlated with the extent of phosphorylation of MAP2. Furthermore, another function of MAP2, the actin filament cross-linking activity, was also inactivated by the incubation of MAP2 with the EGF receptor and ATP. The loss of this activity also correlated well with the extent of phosphorylation. These data indicate that tyrosine phosphorylation of MAP2 by the EGF receptor kinase inactivates both the tubulin polymerizing activity and actin filament cross-linking activity of MAP2. Thus, this study has clearly shown that tyrosine phosphorylation could modify the function of a cytoskeletal protein.     

A nonmitogenic analogue of epidermal growth factor induces early responses mediated by epidermal growth factor

Cyanogen bromide-cleaved epidermal growth factor (CNBr-EGF) binds to EGF receptors with reduced affinity compared to the native hormone but fails to induce DNA synthesis. However, at similar receptor occupancy, CNBr-EGF is as potent as EGF in activating early cell responses to the hormone. The phosphorylation of membrane proteins, the stimulation of Na+-K+-ATPase as reflected by the ouabain-sensitive uptake of 86Rb of fibroblasts, changes in the organization of microfilaments and in cell-morphology, and the activation of the enzyme ornithine-decarboxylase are all induced by CNBr-EGF as well as EGF Our results are consistent with the notion that EGF-induced phosphorylation could act as a "second messenger" for the action of various EGF-induced responses such as activation of Na+-K+-ATPase, changes in the cytoskeleton and cell morphology, and the activation of the enzyme ornithine decarboxylase. However, the stimulation of phosphorylation of membrane proteins and other early responses are either not required or necessary but insufficient for the induction of DNA synthesis. Suboptimal concentrations of EGF together with CNBr-EGF stimulate DNA synthesis in human fibroblasts. Other growth factors such as insulin, fibroblast growth factor, and prostaglandin F2 alpha, which potentiate the mitogenic response of EGF, do not effect the response to CNBr-EGF. This suggests that the restoration of the mitogenic properties of CNBr-EGF by suboptimal doses of EGF occurs at the level of EGF receptors or during their processing.     

Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3

Receptor tyrosine kinase regulation of phospholipase C-epsilon (PLC-epsilon), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-gamma1 and PLC-epsilon, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-epsilon, and Rap2B-dependent translocation of PLC-epsilon to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-gamma1 but not of PLC-epsilon. Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-gamma1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-epsilon.     

Regulation of cytosolic phospholipase A2 phosphorylation by proteolytic cleavage of annexin A1 in activated mast cells

Annexin A1 (ANXA1) is cleaved at the N terminal in some activated cells, such as macrophages, neutrophils, and epithelial cells. We previously observed that ANXA1 was proteolytically cleaved in lung extracts prepared from a murine OVA-induced asthma model. However, the cleavage and regulatory mechanisms of ANXA1 in the allergic response remain unclear. In this study, we found that ANXA1 was cleaved in both Ag-induced activated rat basophilic leukemia 2H3 (RBL-2H3) cells and bone marrow-derived mast cells. This cleavage event was inhibited when intracellular Ca(2+) signaling was blocked. ANXA1-knockdown RBL-2H3 cells produced a greater amount of eicosanoids with simultaneous upregulation of cytosolic phospholipase A(2) (cPLA(2)) activity. However, there were no changes in degranulation activity or cytokine production in the knockdown cells. We also found that cPLA(2) interacted with either full-length or cleaved ANXA1 in activated mast cells. cPLA(2) mainly interacted with full-length ANXA1 in the cytosol and cleaved ANXA1 in the membrane fraction. In addition, introduction of a cleavage-resistant ANXA1 mutant had inhibitory effects on both the phosphorylation of cPLA(2) and release of eicosanoids during the activation of RBL-2H3 cells and bone marrow-derived mast cells. These data suggest that cleavage of ANXA1 causes proinflammatory reactions by increasing the phosphorylation of cPLA(2) and production of eicosanoids during mast-cell activation.     

Differential tyrosine phosphorylation of phospholipase D isozymes by hydrogen peroxide and the epidermal growth factor in A431 epidermoid carcinoma cells.

The regulatory mechanism through which the phospholipase D (PLD) isoforms PLD1 and PLD2 are activated is poorly understood. We investigated the possibility that the PLD isozymes are differentially regulated in response to pharmacologic stimulants in cells. In this report, we demonstrate for the first time that H2O2 and EGF differentially induce tyrosine phosphorylation of the PLD isozymes in A431 cells, which express both PLD1 and PLD2. H2O2 induced tyrosine phosphorylation of PLD1 and PLD2, whereas EGF only caused the tyrosine phosphorylation of PLD2. Both agents also induced phosphorylation of the EGF receptor. Interestingly, the PLD isozymes were associated with the EGF receptor and PKC-alpha in a ligand independent manner. Activation of PLD by H2O2 and EGF nearly correlated with tyrosine phosphorylation of the protein in PLD1 immune complexes. Activation of PLD by both agents was inhibited by the PKC inhibitor, Ro 31-8220, and by the down-regulation of PKC. Pretreatment of the cells with the tyrosine kinase inhibitor tyrphostin AG1478 resulted in inhibition of the H2O2 and EGF-induced tyrosine phosphorylation and PLD activation. These results indicate that H2O2 and EGF induce differential tyrosine phosphorylation of PLD isozymes. Also, the activation of PLD by these agonists involves tyrosine phosphorylation and PKC activation.     

Secretory phospholipase A2 mediates cooperative prostaglandin generation by growth factor and cytokine independently of preceding cytosolic phospholipase A2 expression in rat gastric epithelial cells

Transforming growth factor (TGF)-alpha and interleukin (IL)-1beta are responsible for the healing of gastric lesions through, in part, prostaglandin (PG) generation. We examined the contribution of cytosolic and secretory phospholipase A(2)s (cPLA(2) and sPLA(2)) to the PG generation by rat gastric epithelial cells in response to both stimuli. Stimulation with TGF-alpha for 24 h increased cPLA(2) and cyclooxygenase (COX)-2 markedly, PGE(2) slightly, and type IIA sPLA(2) and COX-1 not at all, whereas IL-1beta increased sPLA(2) only. Both stimuli synergistically increased PGE(2), sPLA(2), and the two COXs but not cPLA(2). The onset of the PGE(2) generation paralleled the sPLA(2) release but was apparently preceded by increases in cPLA(2) and the two COXs. The increase in PGE(2) was impaired by inhibitors for sPLA(2) and COX-2 but not COX-1. cPLA(2) inhibitors suppressed PGE(2) generation by TGF-alpha alone but not augmentation of PGE(2) generation or sPLA(2) release by IL-1beta in combination with TGF-alpha. Furthermore, despite an increase in cPLA(2) including its phosphorylated form (phosphoserine), -induced arachidonic acid liberation was impaired in the TGF-alpha/IL-1beta-stimulated cells, in which p11, a putative cPLA(2) inhibitory molecule, was also increased and co-immunoprecipitated with cPLA(2). These results suggest that synergistic stimulation of sPLA(2) and COX-2 expression by TGF-alpha and IL-1beta results in an increase in PGE(2). Presumably, the preceding cPLA(2) expression is not involved in the PGE(2) generation, because of impairment of its hydrolytic activity in the stimulated cells.     

Secretory phospholipase A(2) inhibits epidermal growth factor-induced receptor activation

Secretory phospholipase A(2) (sPLA(2)) plays important roles in mediating various cellular processes, including cell proliferation, differentiation, apoptosis, and inflammatory response. In this study, we demonstrated that a basic sPLA(2) inhibits epidermal growth factor (EGF)-induced EGF receptor activation, as determined by autophosphorylation of EGF receptor, EGF-activated phospholipase D (PLD) activity, and phospholipase C-gamma(1) (PLC-gamma(1)) tyrosine phosphorylation in a human epidermoid carcinoma cell line, A-431. Treatment of cells with exogenous neutral sphingomyelinase (SMase) or a cell permeable ceramide analog, C(2)-ceramide, also caused similar inhibitory effects on EGF-induced activation of EGF receptor, tyrosine phosphorylation of PLC-gamma(1), and the activation of PLD. sPLA(2)-induced inhibition of EGF receptor was associated with arachidonic acid release, which was followed by an increase in intracellular ceramide formation. Both sPLA(2) and exogenous C(2)-ceramide are able to inhibit the proliferation of A-431. The data presented indicate for the first time that sPLA(2) downregulates the EGF receptor-mediated intracellular signal transduction that may be mediated by arachidonic acid and/or ceramide.     

Tumour necrosis factor (cachectin) induces phospholipase A2 activity and synthesis of a phospholipase A2-activating protein in endothelial cells.

Tumour necrosis factor (TNF) is an important mediator of endotoxin-induced vascular collapse and other inflammatory reactions. Eicosanoids have been implicated in the pathogeensis of these responses. In order to explore further the potential interactions between TNF and eicosanoid metabolism in eliciting vascular responses, we studied the effects of TNF on the bovine endothelial cell line CPAE. TNF induced cellular retraction observed by light microscope. This morphological change was monitored by the passage of iodinated protein A between adjacent cells and by release of [3H]arachidonic acid metabolites from cells. Both the morphological and functional responses were abrogated by inhibition of eicosanoid synthesis with BW755c. The release of [3H]arachidonic acid metabolites appeared to be mediated by a transient increase in phospholipase A2 activity. Phospholipase C activity was not affected by TNF. The maximal increase in phospholipase A2 activity occurred at 5 min following the addition of TNF. Phospholipase A2 activation, [3H]arachidonic acid-metabolite synthesis and passage of iodinated protein A, required both RNA and protein synthesis and were associated with an increase in the synthesis of a recently described phospholipase A2-activating protein. The Bordetella pertussis toxin, islet-activating protein, also inhibited the increase in phospholipase A2 activity, the release of [3H]arachidonic acid metabolites and the passage of iodinated protein A, suggesting that the TNF receptor-ligand interaction resulting in cellular retraction, phospholipase A2 activation and eicosanoid synthesis, is coupled through the Ni guanine nucleotide regulatory protein in these cells.     

Truncation of annexin A1 is a regulatory lever for linking epidermal growth factor signaling with cytosolic phospholipase A2 in normal and malignant squamous epithelial cells

Regulation of cell growth and apoptosis is one of the pleiotropic functions of annexin A1 (ANXA1). Although previous reports on the overexpression of ANXA1 in many human cancers and on growth suppression and/or induction of apoptosis by ANXA1 may indicate the tumor-suppressive nature of ANXA1, molecular mechanisms of the function of ANXA1 remain largely unknown. Here we provide evidence that ANXA1 mechanistically links the epidermal growth factor-triggered growth signal pathway with cytosolic phospholipase A(2) (cPLA(2)), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human keratinocytes (NHK). Ca(2+)-dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA(2), resulting in inhibition of cPLA(2) activity, which is essential for the growth of NHK. On exposure of NHK to epidermal growth factor, ANXA1 was cleaved solely at Trp(12), and this cleavage was executed by cathepsin D. In squamous cancer cells, this pathway was shown to be constitutively activated. The newly found mechanistic intersection may be a promising target for establishing new measures against human cancer and other cell growth disorders.     

A role for G-proteins in the epidermal growth factor stimulation of phospholipase A2 in rat kidney mesangial cells

We have previously demonstrated phospholipase C (PLC) independent activation of phospholipase A₂(PLA₂) by epidermal growth factor (EGF) in glomerular mesangial cells in culture. In the current study using glass beads to permeabilize [³H]- or [¹⁴C]-arachidonate labelled mesangial cells we demonstrate that guanine nucleotides modulate the EGF-mediated stimulation of arachidonic acid release (75% inhibition with 100 M GDPS and 108% augmentation with 100 M GTPS). GTPS alone stimulated both the release of free arachidonic acid and production of diacylglycerol (DAG), while EGF itself neither stimulated DAG nor augmented the DAG response to GTPS. These findings suggest the intermediacy of a G-protein in PLC-independent stimulation of PLA₂ by a growth factor, and provide a model system for determining the relationship between G-protein intermediacy and the intrinsic tyrosine kinase activity of the growth factor receptor.Abbreviations EGF Epidermal Growth Factor - PLC phospholipase C - PLA₂ phospholipase A₂ - DAG Diacylglycerol - NEFA non-esterified fatty acid - GTPS guanosine-5-0-[3-thio]triphosphate - GDP\S guanosine-5-0-[2-thio]diphosphate     

Treatment of A431 cells with epidermal growth factor (EGF) induces desensitization of EGF-stimulated phosphatidylinositol turnover

Epidermal growth factor (EGF) stimulates the turnover of phosphoinositides in A431 cells. In cells that were pretreated with EGF for 30 min at 37 degrees C and then washed to remove surface-bound hormone, a 70-100% decrease in the EGF-stimulated production of inositol monophosphate, inositol bisphosphate, and inositol triphosphate was noted when the cells were exposed to the agonist a second time. Since only a 15% decrease in receptor number was observed in these pretreated cells, the loss of responsiveness to EGF for the production of inositol phosphates could not be attributed to a down-regulation of the EGF receptors. These data suggest that pretreatment of A431 cells with high concentrations of EGF leads to a desensitization of the EGF receptor. This desensitization of the receptor by EGF is apparent within 10-15 min of the addition of EGF and is maximal by 30 min. The desensitization appears to be homologous in nature since pretreatment of cells with EGF did not diminish their responsiveness to bradykinin; and conversely, pretreatment with bradykinin did not diminish the subsequent responsiveness of the cells to EGF. Desensitization to EGF was observed in cells in which protein kinase C had been down-regulated by prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate, implying that EGF receptor desensitization is independent of protein kinase C. The desensitizing effects of EGF on growth factor-induced phosphatidylinositol turnover could be prevented by pretreatment of the cells with the calmodulin antagonist trifluoperazine, suggesting that calmodulin may be involved in the regulation of EGF receptor sensitivity.     

Localization and regulation of cytosolic phospholipase A(2)

Liberation of arachidonic acid by cytosolic phospholipase A(2) (cPLA(2)) upon cell activation is often the initial and rate-limiting step in leukotriene and prostaglandin biosynthesis. This review discusses the essential features of cPLA(2) isoforms and addresses intriguing insights into the catalytic and regulatory mechanisms. Gene expression, posttranslational modification and subcellular localization can regulate these isoforms. Translocation of cPLA(2)alpha from the cytosol to the perinuclear region in response to calcium transients is critical for the immediate arachidonic acid release. Therefore, particular emphasis is placed on the mechanism of the translocation and the role of the proteins and lipids implicated in this process. The regional distribution and cellular localization of cPLA(2) may help to better understand its function as an arachidonic acid supplier to downstream enzymes and as a regulator of specific cellular processes.     

5-HT2A receptor induces ERK phosphorylation and proliferation through ADAM-17 tumor necrosis factor-alpha-converting enzyme (TACE) activation and heparin-bound epidermal growth factor-like growth factor (HB-EGF) shedding in mesangial cells

In this study, we present multiple lines of evidence to support a critical role for heparin-bound EGF (epidermal growth factor)-like growth factor (HB-EGF) and tumor necrosis factor-alpha-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT(2A) receptor in renal mesangial cells. 5-hydroxy-tryptamine (5-HT) resulted in rapid activation of TACE, HB-EGF shedding, EGFR activation, ERK phosphorylation, and longer term increases in DNA content in mesangial cells. ERK phosphorylation was attenuated by 1) neutralizing EGFR antibodies and the EGFR kinase inhibitor, AG1478, 2) neutralizing HB-EGF, but not amphiregulin, antibodies, heparin, or CM197, and 3) pharmacological inhibitors of matrix-degrading metalloproteinases or TACE small interfering RNA. Exogenously administered HB-EGF stimulated ERK phosphorylation. Additionally, TACE was co-immunoprecipitated with HB-EGF. Small interfering RNA against TACE also blocked 5-HT-induced increases in ERK phosphorylation, HB-EGF shedding, and DNA content. In aggregate, this work supports a pathway map that can be depicted as follows: 5-HT --> 5-HT(2A) receptor --> TACE --> HB-EGF shedding --> EGFR --> ERK --> increased DNA content. To our knowledge, this is the first time that TACE has been implicated in 5-HT-induced EGFR transactivation or in proliferation induced by a G protein-coupled receptor in native cells in culture.