Ethanol stimulates shape change in human platelets by activation of phosphoinositide-specific phospholipase C

Administration of ethanol to human platelets resulted in a rapid shape change which was maximal within 30 s. Ethanol did not cause aggregation or secretion of ATP at any time and inhibited aggregation induced by collagen. In platelets that were loaded with the intracellular calcium indicator fura2, ethanol induced a rapid mobilization of calcium from internal, thrombin-sensitive pools. Cytosolic calcium increased to a maximum within 5 s and decreased slowly over the ensuing 5 min to near basal levels. The mobilization of calcium by ethanol coincided with the rapid formation of phosphatidic acid and a decrease in the level of phosphatidylinositol 4,5-bisphosphate, as measured in 32P-labeled platelets. In platelets labeled with myo-[2-3H]inositol, ethanol caused a 20-30% increase in the levels of inositol (1,4,5)-trisphosphate and inositol bisphosphate within 10 s. Ethanol also induced the transient phosphorylation of myosin light chain (20 kDa) and a 40 kDa protein, a known substrate for protein kinase C. The results indicate that ethanol activates phosphoinositide-specific phospholipase C in human platelets. The subsequent mobilization of intracellular calcium and activation of protein kinase C can account for the shape change induced by ethanol.     

The mitogenic signaling pathway of fibroblast growth factor is not mediated through polyphosphoinositide hydrolysis and protein kinase C activation in hamster fibroblasts

Basic or acidic fibroblast growth factor (FGF), alone, was found to be as potent as alpha-thrombin to reinitiate DNA synthesis in G0-arrested Chinese hamster lung fibroblasts (CCL39). Basic FGF at 50 ng/ml or thrombin at 1 unit/ml rapidly initiated early events such as cytoplasmic alkalinization (0.2-0.3 pH units), rise in cytoplasmic Ca2+, phosphorylation of ribosomal protein S6 and increased c-myc expression, followed by a 30-40-fold increase in labeled nuclei. Whereas thrombin is a potent activator of phospholipase C as judged by the rapid release of inositol trisphosphate, inositol bisphosphate and by the massive accumulation of total inositol phosphate (IP) in the presence of 20 mM Li+, FGF failed to induce the breakdown of polyphosphoinositides in quiescent CCL39 cells. Indeed, no inositol trisphosphate nor inositol bisphosphate could be detected in response to FGF; in presence of Li+ the total IP release never exceeded 8% of the IP released by the action of thrombin. Two additional findings indicated that FGF and thrombin activate different signaling pathways. First, we found that, in contrast to thrombin, the FGF-induced rise in the cytoplasmic free Ca2+ concentration measured by quin-2 fluorescence, is strictly dependent upon the presence of Ca2+ in the external medium. Second, we found that FGF failed to activate protein kinase C as judged by the epidermal growth factor-receptor binding assay. Treatment of the cells with either thrombin or phorbol esters, rapidly inhibited 125I-labeled epidermal growth factor binding (50-60%). Basic or acidic FGF had no effect. We conclude that: the FGF-receptor signaling pathway is not coupled to phospholipase C activation, and early mitogenic events and reinitiation of DNA synthesis can be initiated independently of inositol lipid breakdown and protein kinase C activation.     

Inhibition by cyclic AMP of guanine nucleotide-induced activation of phosphoinositide-specific phospholipase C in human platelets

Phosphoinositide-specific phospholipase C (PLC) activity of human platelet membranes was activated by the nonhydrolyzable guanine nucleotide GTP gamma S. This activation did not occur in either membranes prepared from dibutyryl cyclic AMP-pretreated platelets (A-membranes) or those prepared from untreated cells and subsequently incubated with cyclic AMP (cAMP) (B-membranes). This cAMP-mediated inhibition was abolished in the presence of inhibitors of cAMP-dependent protein kinase (A-kinase), suggesting that the inhibition was due to phosphorylation of (a) protein component(s). No significant differences were observed in the basal PLC activity and the extent of pertussis toxin-catalyzed ADP-ribosylation among control membranes and the two types of phosphorylated membranes (A- and B-membranes). GTP-binding activities of Gs, Gi and GTP-binding proteins of lower molecular masses were not altered by the phosphorylation of the membranes. These findings suggest that a GTP-binding protein is involved in the GTP gamma S-mediated activation of PLC and that cAMP (plus A-kinase) inhibits this activation by phosphorylating a membrane protein (probably a 240-kDa protein), rather than the GTP-binding protein or PLC itself. It is likely that this phosphorylation uncouples the GTP-binding protein from PLC.     

In vitro establishment is not a sufficient prerequisite for transformation by activated ras oncogenes

Activated ras genes transform REF52 cells only at low frequencies and adenovirus early region 1A collaborates with ras oncogenes to convert REF52 cells to a tumorigenic phenotype. While failure to transform did not result from an absence of ras gene expression, E1A appeared to enhance expression of transfected ras genes by approximately tenfold. However, enhanced ras expression alone does not account for collaboration by E1A since overexpression of T24 Ha-ras p21 induced morphological crisis and cell growth arrest rather than stable transformation. These results indicate that E1A contributes complementing biochemical activities that enable ras genes to transform REF52 and suggest that the role of E1A in primary cell transformation may extend beyond facilitating in vitro establishment.     

Inhibition of human skin fibroblast proliferation by histamine and phorbol esters is mediated by protein kinase C

The proliferation of human skin fibroblasts in culture was examined using a [3H]thymidine incorporation assay. Histamine inhibited thymidine incorporation with an IC50 of about 0.2 microM. This effect was blocked by the H1 receptor antagonist mepyramine but not by the H2 receptor antagonist cimetidine. Protein kinase C activators, including several phorbol esters and mezerine, also inhibited thymidine incorporation. The IC50 for beta-phorbol 12,13-didecanoate was less than 0.1 nM. The alpha-isomer of this compound was inactive. Long-term treatment of cells with the beta-isomer eliminated the ability of both histamine and phorbol ester to inhibit thymidine incorporation, presumably due to downregulation of protein kinase C. Our results suggest that histamine H1 receptors are linked to activation of protein kinase C and that activation of this enzyme leads to an inhibition of cell proliferation.     

Activation of phospholipase D by ras proteins is independent of protein kinase C

Growth factors activate phospholipases, causing the generation of diverse lipid metabolites with second messenger function. Among them, the phosphatidylcholine-preferring phospholipase D (PLD) has attracted great interest, since in addition to the transient activation by growth factors stimulation, it is constitutively activated in some of the src- and ras-transformed cells investigated. To establish further the functional relationship of ras oncogenes with PLD, we have investigated its mechanism of regulation. Growth factors such as PDGF or FGF activate the PC-PLD enzyme by a common, PKC-dependent mechanism. By contrast, ras oncogenes activate the PC-PLD enzyme by a PKC-independent mechanism. These results suggest the existence of at least two mechanisms for PLD activation, and ras oncogenes contribute to one of them. © 1996 Wiley-Liss, Inc.     

Evidence for a role of phosphatidylcholine-hydrolysing phospholipase C in the regulation of protein kinase C by ras and src oncogenes.

The products of ras and src oncogenes are thought to be important components in pathways regulating cell proliferation and differentiation. In fibroblasts transformed by these oncogenes, increased diacylglycerol levels have been found which most probably arise from activation of the turnover of phosphatidylcholine. Diacylglycerol is a key activator of protein kinase C whose role in cell growth and transformation has been proposed. We demonstrate here by using immunochemical techniques that transformation by ras or src oncogenes is associated with permanent translocation of protein kinase C to the cytoplasmic membrane. However, no down-regulation of the enzyme is observed despite its permanent activation in these transformants. Importantly, the lack of down-regulation observed in ras and src transformed cell lines is mimicked by chronic treatment of NIH 3T3 fibroblasts with exogenous Bacillus cereus phosphatidylcholine-hydrolysing phospholipase C, but not with phorbol myristate acetate or exogenous Bacillus thuringiensis phosphatidylinositol-hydrolysing phospholipase C. These results strongly suggest that diacylglycerol derived from phosphatidylcholine but not from phosphoinositide turnover is responsible for the atypical regulation of protein kinase C in cell lines transformed by ras and src oncogenes.     

Transformation by Rho exchange factor oncogenes is mediated by activation of an integrin-dependent pathway.

Constitutive activation of growth factor receptor signaling pathways leads to uncontrolled growth, but why tumor cells become anchorage independent is less clear. The fact that integrins transmit signals required for cell growth suggests that constitutive activation of steps downstream from integrins mediates anchorage independence. Since the small GTPase Rho may mediate integrin signal transduction, the effects of serum and the Rho nucleotide exchange factor oncogenes dbl and lbc on cell growth and signaling pathways were examined. Our data show that these oncogenes induce anchorage-independent but serum-dependent growth and stimulation of signaling pathways. These results show, therefore, that anchorage-independent growth results from constitutive activation of integrin-dependent signaling events. They also support the view that Rho is a functionally important mediator of integrin signaling.     

Phospholipase C-protein kinase C mediated phospholipase D activation pathway is involved in tamoxifen induced apoptosis

Tamoxifen (TAM) is the endocrine therapeutic agent the most widely used in the treatment of breast cancer, and it operates primarily through the induction of apoptosis. In this study, we attempted to elucidate the non-ER mediated mechanism behind TAM treatment, involving the phospholipase C-protein kinase C (PLC-PKC) mediated phospholipase D (PLD) activation pathway, using multimodality methods. In TAM treated MCF7 cells, the PLC and PLD protein and mRNA levels increased. Phosphatidylethanol (PEt) and diacylglycerol (DAG) generation also increased, showing increased activity of PLD and PLCgamma1. Translocation of PKCalpha, from cytosol to membrane, was observed in TAM treated cells. By showing that both PKC and PLC inhibitors could reduce the effects of TAM-induced PLD activation, we confirmed the role of PKC and PLC as upstream regulators of PLD. Finally, we demonstrated that TAM treatment reduced the viability of MCF7 cells and brought about rapid cell death. From these results, we confirmed the hypothesis that TAM induces apoptosis in breast cancer cells, and that the signal transduction pathway, involving PLD, PLC, and PKC, constitutes one of the possible mechanisms underlying the non-ER mediated effects associated with TAM.     

Glycerol-3-phospho-D-myo-inositol 4-phosphate (Gro-PIP) is an inhibitor of phosphoinositide-specific phospholipase C

The deacylated forms of the phosphoinositides were used to determine whether the guinea pig uterus phosphoinositide-specific phospholipase C (PI-PLC I, Mr 60,000) required fatty acids at the sn-1 and sn-2 positions for the hydrolysis of the sn-3 phosphodiester bond. L-alpha-Glycerophospho-D-myo-inositol 4-phosphate (Gro-PIP), but not glycerol 3-phosphate (Gro-3-P), L-alpha-glycerophospho-D-myo-inositol (Gro-PI), or L-alpha-glycerophospho-D-myo-inositol 4,5-bisphosphate (Gro-PIP2), inhibited PI-PLC I in a concentration-dependent manner. Assays performed with 10 microM [3H]phosphatidylinositol ([3H]PI), 10 microM [3H]phosphatidylinositol 4-phosphate ([3H]PIP) or 10 microM [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) as substrates, with increasing [Gro-PIP] revealed an IC50 = 380 microM. Kinetic studies with increasing [3H]PI substrate concentrations in the presence of 100 microM and 300 microM Gro-PIP demonstrated that Gro-PIP exhibited competitive inhibition; Kis = 40 microM. Ca2+ concentrations over the range 1.1 microM to 1 mM did not effect inhibition, suggesting that Gro-PIP inhibition of [3H]PI hydrolysis was calcium-independent. To determine whether Gro-PIP was a substrate, 20 microM and 500 microM [3H]Gro-PIP were incubated with PI-PLC I. Anion-exchange HPLC analysis revealed no [3H]IP2 product formation, indicating that [3H]Gro-PIP was not hydrolyzed. Assays performed with [3H]PI and [3H]PIP substrates in the presence of 500 microM [3H]Gro-PIP revealed approx. 75% less [3H]inositol 1-phosphate ([3H]IP1) and [3H]inositol 1,4-bisphosphate ([3H]IP2) product formation, respectively, indicating that [3H]Gro-PIP inhibited the hydrolysis of the substrates by PI-PLC I. These data suggest that Gro-PIP does not serve as a substrate, and that it inhibits PI-PLC I by competitive inhibition in a Ca2(+)-independent fashion.     

Direct activation of phospholipase C-gamma by fibroblast growth factor receptor is not required for mesoderm induction in Xenopus animal caps.

Members of the fibroblast growth factor (FGF) family induce mesoderm formation in explants of Xenopus embryonic ectoderm (animal caps). Recent studies have been directed at determining signaling pathways downstream of the FGF receptor that are important in mesoderm induction. We have recently shown that a point mutation in the FGF receptor changing tyrosine 766 to phenylalanine (Y/F mutation) abolishes phospholipase C-gamma (PLC-gamma) activation in mammalian cells. To explore the role of PLC-gamma activation in FGF-stimulated mesoderm induction, we constructed two chimeric receptors, each consisting of the extracellular portion of the platelet-derived growth factor beta receptor, with one having the transmembrane and intracellular portions of the wild-type FGF receptor 1 (PR-FR wt) and the other having the corresponding region of the Y/F766 mutant FGF receptor 1 (PR-FR Y/F766). When expressed in Xenopus oocytes, only PR-FR wt was able to mediate PLC gamma phosphorylation, inositol-1,4,5-trisphosphate accumulation, and calcium efflux in response to platelet-derived growth factor stimulation. However, both receptors mediated mesoderm induction in Xenopus animal caps as measured by cap elongation, muscle-specific actin mRNA induction, and skeletal muscle formation. These results demonstrate that PLC gamma activation by the FGF receptor is not required for FGF-stimulated mesoderm induction.     

Bradykinin-induced activation of phospholipase A2 is independent of the activation of polyphosphoinositide-hydrolyzing phospholipase C

This study evaluates the role of phosphatidylinositol 4,5-bisphosphate (PIP2) and its metabolites as possible mediators in the activation of phospholipases A2 in porcine aortic endothelial cells. We compared the time courses of bradykinin-induced turnover of phosphoinositides and the appearance of unesterified arachidonic acid (uAA) and eicosanoids. The metabolism of phosphoinositides was examined in cells prelabeled with [3H]inositol, which has a similar distribution as the endogenous inositol lipids. At 37 degrees C, bradykinin induced a rapid rise in lysophosphatidylinositol (lyso-PI) and inositol 1,4,5-trisphosphate (IP3) as well as a decrease in PIP2. Lyso-PI formation was detected at 10 s, as early as PIP2 degradation and IP3 formation. This suggests that the activation of PIP2-hydrolyzing phospholipase C and PI-hydrolyzing phospholipase A2 are simultaneous. However, at 30 degrees C, lyso-PI formation was detected in the absence of an increase in IP3 indicating that the activation of phospholipase A2 does not require the accumulation of IP3. The time course of formation of uAA and eicosanoids were examined in [3H]arachidonic acid-prelabeled cells. The 3H radioactivity was distributed among the phospholipid classes and subclasses the same as the endogenous phospholipids. Bradykinin stimulated the intracellular accumulation of uAA, detectable at 5 s, earlier than that of 1,2-diacylglycerol and phosphatidic acid. Such immediate formation of uAA further supports the notion that activation of phospholipase A2 is a very early event during the interaction of bradykinin with porcine endothelial cells, and that PIP2 hydrolysis is not prerequisite for the initial activation of phospholipase A2.     

C3-induced 3LL cell proliferation is mediated by C kinase

It has been demonstrated that the third component of complement (C3)(1) and its peptides increase normal and tumour cell proliferation. However, the signal cascade responsible for this phenomenon is still unknown. In this study, we elucidate some of the mechanisms involved in the signalling of C3 stimulation of cell proliferation. We have first investigated the in and out traffic of C3 peptides, then we have identified the subcellular localisation of internalised C3 and, finally, we have explored the role of protein phosphorylation in C3 traffic and in the proliferation of the Lewis lung carcinoma (3LL) cells. Our results indicate that traffic of C3 is not dependent on cytoskeletal integrity and requires protein kinase C-dependent phosphorylation. In addition, proliferation of 3LL cells stimulated by C3 depends on both C3 internalisation and protein-kinase C phosphorylation.     

A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity

A fourth type of rat phosphoinositide-specific phospholipase C (PLC IV) has been cloned for cDNA and sequenced. PLC IV is distinct from the other three types of rat PLC (PLC I, II, and III) with respect to primary structure and tissue distribution of its mRNAs. PLC IV contains two homologous regions included commonly in PLC I, II, and III and is most similar to PLC II (identity: 50.2%). PLC IV, in common with PLC II, has a sequence homologous to the N-terminal regulatory domains of nonreceptor tyrosine kinases of the src-family of oncogenes. Using an Escherichia coli expression system, we succeeded in producing active PLC IV in E. coli crude extracts. Various truncation experiments of the PLC IV cDNA revealed that the src-related domain is not necessary for catalytic activity while both domains homologous among PLC I-IV are essential. PLC IV is expressed in various rat tissues and abundant in spleen, suggesting that PLC IV plays a fundamental role in cellular functions such as growth and secretion.     

Carbon nanoparticle-induced lung epithelial cell proliferation is mediated by receptor-dependent Akt activation

Treatment of lung epithelial cells with different kinds of nano-sized particles leads to cell proliferation. Because bigger particles fail to induce this reaction, it is suggested that the special surface properties, due to the extremely small size of these kinds of materials, is the common principle responsible for this specific cell reaction. Here the activation of the protein kinase B (Akt) signaling cascade by carbon nanoparticles was investigated with regard to its relevance for proliferation. Kinetics and dose-response experiments demonstrated that Akt is specifically activated by nanoparticulate carbon particles in rat alveolar type II epithelial cells as well as in human bronchial epithelial cells. This pathway appeared to be dependent on epidermal growth factor receptor and beta(1)-integrins. The activation of Akt by these receptors is known to be a feature of adhesion-dependent signaling. However, intracellular proteins described in this context (focal adhesion kinase pp125(FAK) and integrin-linked kinase) were not activated, indicating a specific signaling mechanism. Inhibitor studies demonstrate that nanoparticle-induced proliferation is mediated by phosphoinositide 3-kinases and Akt. Moreover, overexpression of mutant Akt, as well as pretreatment with an Akt inhibitor, reduced nanoparticle-specific ERK1/2 phosphorylation, which is decisive for nanoparticle-induced proliferation. With this report, we describe the activation of a pathway by carbon nanoparticles that was so far known to be triggered by ligand receptor binding or on cell adhesion to extracellular matrix proteins.