Human granulocyte-macrophage colony-stimulating factor plus phorbol myristate acetate stimulate a promyelocytic cell line to produce an IL-1 inhibitor

We recently described an IL-1 inhibitor found in urine of febrile patients. It is a 26-kDa glycoprotein that acts by blocking the binding of IL-1 to its receptor. In a search for a cell source for the urinary IL-1 inhibitor, we tested three promyelocytic cell lines, H-161, AML-193, and HL-60, for their ability to produce this protein. Under normal culture conditions none of these cell lines produce detectable IL-1 inhibitory activity. The H-161 cells were treated with differentiation-inducing agents, i.e., sodium butyrate, hemin, retinoic acid, DMSO, vitamin D3, and PMA alone or in combination with IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, TNF-alpha, IFN-gamma, granulocyte-CSF, macrophage-CSF, granulocyte/macrophage-CSF (GM-CSF), and Con A and tested for the production of IL-1 inhibitor. Production of IL-1 inhibitor was detected in cell supernatant, when H-161 cells were differentiated to adherent macrophage-like cells under the influence of PMA followed by a second signal provided by GM-CSF. Treatment of the other two cell lines, AML-193 and HL-60, with PMA plus GM-CSF also yielded similar IL-1 inhibitor protein. Partial purified H-161-derived IL-1 inhibitor showed specific binding to IL-1R-bearing cells and blocked the binding of IL-1 to its receptor and is thus similar to the urinary-derived molecule. We conclude the GM-CSF provides a signal to adherent macrophage-like cells to become "inhibitory macrophages" and to produce a competitive inhibitor of IL-1.     

A transient rise in cytosolic calcium follows stimulation of quiescent cells with growth factors and is inhibitable with phorbol myristate acetate

We have used aequorin as an indicator for the intracellular free calcium ion concentration [( Ca++]i) of Swiss 3T3 fibroblasts. Estimated [Ca++]i of serum-deprived, subconfluent fibroblasts was 89 (+/-20) nM, almost twofold higher than that of subconfluent cells growing in serum, whose [Ca++]i was 50 (+/-19) nM. Serum, partially purified platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) stimulated DNA synthesis by the serum-deprived cells, whereas epidermal growth factor (EGF) did not. Serum immediately and transiently elevated the [Ca++]i of serum-deprived cells, which reached a maximal value of 5.3 microM at 18 s poststimulation but returned to near prestimulatory levels within 3 min. Moreover, no further changes in [Ca++]i were observed during 12 subsequent h of continuous recording. PDGF produced a peak rise in [Ca++]i to approximately 1.4 microM at 115 s after stimulation, and FGF to approximately 1.2 microM at 135 s after stimulation. EGF caused no change in [Ca++]i. The primary source of calcium for these transients was intracellular, since the magnitude of the serum-induced rise in [Ca++]i was reduced by only 30% in the absence of exogenous calcium. Phorbol 12-myristate 13-acetate (PMA) had no effect on resting [Ca++]i. When, however, quiescent cells were treated for 30 min with 100 nM PMA, serum-induced rises in [Ca++]i were reduced by sevenfold. PMA did not inhibit growth factor-induced DNA synthesis and was by itself partially mitogenic. We suggest that if calcium is involved as a cytoplasmic signal for mitogenic activation of quiescent fibroblasts, its action is early, transient, and can be partially substituted for by PMA. Activated protein kinase C may regulate growth factor-induced increases in [Ca++]i.     

Regulation of complement factor H synthesis in U-937 cells by phorbol myristate acetate, lipopolysaccharide, and IL-1

The capacity of the human monocyte cell line U-937 to synthesize complement factor H was examined. The kinetics of secretion of factor H into cell culture supernatant were followed by a sensitive solid phase RIA capable of measuring 0.1 ng of protein. Daily secretion of factor H was low and almost linear and was approximately 3 ng of factor H per 10(6) cells. Factor H synthesis was inhibited by cycloheximide but returned to the levels seen in untreated cultures after removal of the inhibitor. LPS and IL-1 both effected a time- and dose-dependent enhancement of factor H synthesis. Induction of U-937 cells with PMA to differentiate into macrophage-like cells also resulted in increased factor H synthesis. RIA of cell lysates, immunofluorescence microscopy, as well as FACS analysis all revealed that factor H Ag was also associated with the U-937 cell membrane. The population of U-937 cells bearing membrane-associated factor H was decreased from 77 to 43% after stimulation for 48 h with LPS (1 microgram/ml). [35S]Methionine metabolic labeling and SDS-PAGE analysis of factor H immunoprecipitates from unstimulated and stimulated culture supernatants and cell lysates demonstrated a major polypeptide, m.w. 150,000, and a minor component, m.w. 42,000. Western blot analysis of factor H in fresh normal plasma also detected both 150,000 and 42,000 m.w. factor H proteins. This is in agreement with the recent demonstration of a 4.4- and 1.8-kb mRNA for factor H in human liver. These data demonstrate that U-937 cells synthesize factor H that is structurally and antigenically similar to factor H in normal plasma. The exact nature of the membrane-bound factor H and its functions and mechanism of attachment to the cell membrane remain to be elucidated.     

Chondrocyte activation by interleukin-1: analysis of the synergistic properties of fibroblast growth factor and phorbol myristate acetate

Following activation, monolayers of lapine articular chondrocytes secreted into their culture media large amounts of prostaglandin E2 (PGE2) and the neutral metalloproteinases collagenase and gelatinase. Partially purified preparations of synovial "chondrocyte activating factors" (CAF), which contain interleukin-1 (IL-1), generally proved stronger activators of chondrocytes than recombinant, human, IL-1 alpha (rHIL-1 alpha) or IL-1 beta (rHIL-1 beta). The presence of synergistic cytokines within the synovial material provides one possible explanation of this discrepancy. As first reported by K. Phadke (1987, Biochem. Biophys. Res. Commun. 142, 448-453) fibroblast growth factor (FGF) synergized with rHIL-1 in promoting the synthesis of neutral metalloproteinases. In our hands FGF alone did not induce neutral metalloproteinases and increased PGE2 synthesis only modestly. However, at doses from 1 ng/ml to 1 microgram/ml, FGF progressively enhanced the synthesis of PGE2, collagenase, and gelatinase by chondrocytes responding to rHIL-1. Acidic and basic FGF synergized equally well with both rHIL-1 alpha and rHIL-1 beta. Phorbol myristate acetate (PMA), but not the Ca2+-ionophore A23187, could substitute for FGF as a synergist. PMA alone was a poor inducer of collagenase or gelatinase but, unlike FGF, it greatly enhanced the synthesis of PGE2 by chondrocytes. Dot-blot analyses with a cDNA probe to collagenase mRNA confirmed that partially purified synovial CAF induced collagenase mRNA more effectively than rHIL-1, with rHIL-1 alpha being superior to rHIL-1 beta in this regard. The synergistic effects of both FGF and PMA upon IL-1-mediated collagenase induction were associated with increased abundance of collagenase mRNA.     

Phosphorylation of Nck in response to a variety of receptors, phorbol myristate acetate, and cyclic AMP.

The 47-kDa protein coimmunoprecipitated with phospholipase C (PLC)-gamma 1 by anti-PLC-gamma 1 monoclonal antibodies is proved to be Nck, a protein composed almost exclusively of one SH2 and three SH3 domains. Nck and PLC-gamma 1 are recognized by certain anti-PLC-gamma 1 monoclonal antibodies because Nck and PLC-gamma 1 share an epitope that likely is located in their SH3 domains. Nck is widely distributed in rat tissues, with an especially high level of expression in testes. The expression levels of Nck remains unchanged during the development of rat brain, whereas PLC-gamma 1 decreases during the same developmental period. Stimulation of A431 cells with epidermal growth factor elicits the tight association of Nck with the epidermal growth factor receptor and phosphorylation of Nck on both serine and tyrosine residues. The phosphorylation of Nck is also enhanced in response to stimulation of the nerve growth factor receptor in PC12 cells, the T-cell receptor complex in Jurkat cells, the membrane immunoglobulin M in Daudi cells, and the low-affinity immunoglobulin G receptor (Fc gamma RII) in U937 cells. The phosphorylation of Nck was also enhanced following treatment of A431 cells with phorbol 12-myristate 13-acetate or forskolin. These results suggest that Nck is a target for a variety of protein kinases that might modulate the postulated role of Nck as an adaptor for the physical and functional coordination of signalling proteins.     

Tumor promoters and epidermal growth factor stimulate anchorage-independent growth of adenovirus-tranformed rat embryo cells.

Previous studies indicated that the potent tumor promoter 12--0--tetradecanoyl-phorbol-13-acetate (TPA) enhances transformation of rat embryo cells (2 degrees RE) by a mutant of human Ad5 (H5ts125). This study examines the effect of TPA, its structural analogs and epidermal growth factor (EGF) on anchorage-independent growth of a cloned population of H5ts125-transformed 2 degrees RE cells (clone E11). Both TPA and EGF (approximately 10(-8) M) induced a 3--5 fold increase in agar cloning efficiency of E11 cells. In addition, macroscopic colonies appeared earlier and were larger and more diffuse. The TPA analogs phorbol--12,13--didecanoate (PDD) and ingenol--3,20--dibenzoate also enhanced growth in agar of E11 cells, whereas phorbol, 4 alpha PDD and 4--0--meTPA, which are inactive as tumor promoters, failed to enhance agar growth. In contrast to the results obtained with E11 cells, TPA, PDD or ingenol--3,20--bidenzoate failed to induce growth in agar of normal 2 degrees RE cells. Dexamethasone (10(-5)--10(-6) M), trans retinoic acid (10(-5)--10(-6) M) and the protease inhibitors leupeptin, antipain and elastatinol did not inhibit the ability of TPA to enhance the growth of E11 cells in agar. The TPA-enhanced anchorage independence was a stable property, since subclones of E11 cells isolated from TPA-agar plates had a higher agar cloning efficiency than the parental E11 cells when retested in the absence of TPA. This effect of TPA does not appear to reflect simple selection of a subpopulation of cells. When the parental E11 cells were first cloned in monolayer culture in the absence of TPA, all ten randomly picked clones showed enhanced growth in agar in the presence of TPA. In addition, prior growth of E11 cells in monolayer culture in the presence of TPA did not enhance their subsequent growth in agar. This system therefore provides an example in which TPA appears to enhance the acquisition of a stable cell property, and thus may be a useful model for studying mechanisms of tumor promotion and progression.     

sn-1,2-Dioctanoylglycerol. A cell-permeable diacylglycerol that mimics phorbol diester action on the epidermal growth factor receptor and mitogenesis

The cell-permeable diacylglycerol, sn-1,2-dioctanoylglycerol (DiC8), is shown to mimic the effect of tumor promoting phorbol diesters on epidermal growth factor (EGF) binding and action in intact cells. DiC8 inhibited the binding of [3H]phorbol dibutyrate to A431 cell monolayers indicating that the diacylglycerol interacts with the phorbol diester receptor. At 0.3 microM, DiC8 half-maximally inhibited the high affinity binding of 125I-EGF to A431 human epidermoid carcinoma cells. Scatchard analysis indicated that the inhibition of 125I-EGF binding was very similar to that observed in the presence of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). DiC8 also mimicked the action of PMA to increase the phosphorylation state of the EGF receptor in 32P-labeled cells. Phosphoamino acid analysis demonstrated that DiC8 and PMA caused an increase in the level of EGF-receptor phosphoserine and phosphothreonine, whereas EGF caused an increase in the level of phosphoserine, phosphothreonine, and phosphotyrosine. Phosphopeptide mapping of the EGF receptor showed that DiC8 and PMA enhanced the phosphorylation of the same tryptic peptides. DiC8 inhibited the EGF-dependent tyrosine phosphorylation of the EGF receptor in A431 cells in a similar manner to that observed with PMA. In further experiments with quiescent Swiss 3T3 fibroblasts, DiC8 mimicked the ability of PMA to stimulate the incorporation of [methyl-3H]thymidine synergistically with low concentrations of EGF. This result indicates that DiC8 will mimic the long-term effects of PMA to regulate mitogenesis and raises the possibility that it may be active in two stage carcinogenesis. As both DiC8 and PMA stimulate the Ca2+- and phospholipid-dependent protein kinase (C-kinase) in vitro, the results support the hypothesis that the activation of C-kinase is a critical component of phorbol diester action on EGF receptor modulation and cell proliferation.     

Bacterial lipopolysaccharide, phorbol myristate acetate, and muramyl dipeptide stimulate the expression of a human monocyte surface antigen, Mo3e

Exposure of mononuclear phagocytes to bacterial lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or muramyl dipeptide (MDP) is known to stimulate a variety of cellular activities that include increases in phagocytosis, oxidative metabolism, synthesis and secretion of monokines, and cytotoxicity of microbes and tumor cells. We now report that culture of human peripheral blood monocytes in medium containing LPS, phorbol compounds, or MDP also results in the acquired expression of a plasma membrane antigen. Mo3e, as identified by a murine monoclonal antibody. Mo3e is barely detectable (by immunofluorescence flow cytometry) on freshly isolated monocytes, but is expressed in high antigen density after exposure of cells to E. coli, Salmonella minnesota, or Serratia marcescens LPS (at concentrations exceeding 0.1 ng/ml), PMA (and other biologically active phorbol compounds) (0.5 to 1 X 10(-8) M), or MDP (0.01 to 1 X 10(-6) M). Mo3e expression stimulated by LPS is prevented by pretreatment of LPS with polymyxin B, suggesting that the lipid A portion of LPS is responsible for Mo3e induction (polymyxin B has no effect on Mo3e expression stimulated by PMA or MDP). Culture of monocytes in medium containing protein synthesis inhibitors (or at 4 degrees C) blocks the acquisition of Mo3e. Recombinant IFN-gamma, which is also known to "activate" mononuclear phagocytes, does not stimulate Mo3e expression, although both LPS and IFN induce enhanced expression of monocyte Ia antigen. Analogous to their stimulatory effect on monocytes, LPS and PMA induce Mo3e expression by the human monocytic cell line, U-937. On the basis of these observations, Mo3e may represent an immunologic marker for monocyte activation stimulated in vitro by LPS, PMA (and related compounds), and MDP.     

Processing and secretion of tumor necrosis factor alpha in endotoxin-treated Mono Mac 6 cells are dependent on phorbol myristate acetate.

Lipopolysaccharide (LPS, endotoxin) is a potent stimulator of tumor necrosis factor alpha (TNF alpha) synthesis and secretion in mouse macrophage tumor cells (Golenbock, D. T., Hampton, R. Y., Qureshi, N., Takayama, K., and Raetz, C. H. R. (1991) J. Biol. Chem. 266, 19490-19498). In contrast, addition of LPS (10 ng/ml) to human monomyelocytic (Mono Mac 6) cells induces very little production of TNF alpha, as judged by immunoassay of the growth medium. When 30 ng/ml 4-beta-phorbol-12-myristate 13-acetate (PMA) is added together with LPS, large amounts of TNF alpha are secreted. PMA alone is inactive. Maximal TNF alpha levels in the medium are achieved at 1 ng/ml of LPS. Protein kinase C inhibitors, such as H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine), staurosporine, and sphingosine, reduce TNF alpha secretion stimulated by PMA. The effect of PMA has been investigated at each stage of TNF alpha biogenesis. Treatment of Mono Mac 6 cells with LPS alone results in rapid, transient, and full expression of TNF alpha mRNA. Concomitant addition of PMA does not increase TNF alpha mRNA synthesis any further, but it prolongs the half-life of TNF alpha mRNA about 3-fold. However, mRNA stabilization does not account for the striking effect of PMA on TNF alpha secretion. Analysis of TNF alpha synthesis and secretion by immunoprecipitation indicates that LPS alone is fully effective in stimulating the formation of the intracellular 26-kDa TNF alpha precursor. LPS alone is not sufficient to allow processing of the precursor and secretion of mature 17-kDa TNF alpha. The rate of TNF alpha secretion observed immediately after the addition of PMA to LPS-pretreated cells is similar to the maximum rate from LPS/PMA-treated cells, but without the lag observed in cells after being exposed to LPS and PMA simultaneously. In summary, PMA is required for the completion of TNF alpha precursor processing and secretion in LPS-treated human Mono Mac 6 cells, whereas murine RAW cells are able to complete the terminal steps of TNF alpha processing in the absence of PMA.     

Differential response of myofibrillar and cytoskeletal proteins in cells treated with phorbol myristate acetate

Muscle-specific and nonmuscle contractile protein isoforms responded in opposite ways to 12-o-tetradecanoyl phorbol-13-acetate (TPA). Loss of Z band density was observed in day-4-5 cultured chick myotubes after 2 h in the phorbol ester, TPA. By 5-10 h, most I-Z-I complexes were selectively deleted from the myofibril, although the A bands remained intact and longitudinally aligned. The deletion of I-Z-I complexes was inversely related to the appearance of numerous cortical, alpha-actinin containing bodies (CABs), transitory structures approximately 3.0 microns in diameter. Each CAB consisted of a filamentous core that costained with antibodies to alpha-actin and sarcomeric alpha-actinin. In turn each CAB was encaged by a discontinuous rim that costained with antibodies to vinculin and talin. Vimentin and desmin intermediate filaments and most cell organelles were excluded from the membrane-free CABs. These curious bodies disappeared over the next 10 h so that in 30-h myosacs all alpha-actin and sarcomeric alpha-actinin structures had been eliminated. On the other hand vinculin and talin adhesion plaques remained prominent even in 72-h myosacs. Disruption of the A bands was first initiated after 15-20 h in TPA (e.g., 15-20-h myosacs). Thick filaments of apparently normal length and structure were progressively released from A segments, and by 40 h all A bands had been broken down into enormous numbers of randomly dispersed, but still intact single thick filaments. This breakdown correlated with the formation of amorphous cytoplasmic aggregates which invariably colocalized antibodies to myosin heavy chain, MLC 1-3, myomesin, and C protein. Complete elimination of all immunoreactive thick filament proteins required 60-72 h of TPA exposure. The elimination of the thick filament-associated proteins did not involve the participation of vinculin or talin. In contrast to its effects on myofibrils, TPA did not induce the disassembly of the contractile proteins in stress fibers and microfilaments either in myosacs or in fibroblastic cells. Similarly, TPA, which rapidly induces the translocation of vinculin and talin to ectopic sites in many types of immortalized cells, had no gross effect on the adhesion plaques of myosacs, primary fibroblastic cells, or presumptive myoblasts. Clearly, the response to TPA of contractile protein and some cytoskeletal isoforms not only varies among phenotypes, but even within the domains of a given myotube the myofibrils respond one way, the stress fibers/microfilaments another.     

Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate.

Ectodomain shedding of cell surface membrane-anchoring proteins is an important process in a wide variety of physiological events(1, 2). Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is the first discovered mammalian sheddase responsible for cleavage of several important surface proteins, including TNF-alpha, TNF p75 receptor, L-selectin, and transforming growth factor-a. Phorbol myristate acetate (PMA) has long been known as a potent agent to enhance ectodomain shedding. However, it is not fully understood how PMA activates TACE and induces ectodomain shedding. Here, we demonstrate that PMA induces both reactive oxygen species (ROS) generation and TNF p75 receptor shedding in Mono Mac 6 cells, a human monocytic cell line, and l-selectin shedding in Jurkat T-cells. ROS scavengers significantly attenuated PMA-induced TNF p75 receptor shedding. Exogenous H2O2 mimicked PMA-induced enhancement of ectodomain shedding, and H2O2-induced shedding was blocked by TAPI, a TACE inhibitor. Furthermore, both PMA and H2O2 failed to cause ectodomain shedding in a cell line that lacks TACE activity. By use of an in vitro TACE cleavage assay, H2O2 activated TACE that had been rendered inactive by the addition of the TACE inhibitory pro-domain sequence. We presume that the mechanism of TACE activation by H2O2 is due to an oxidative attack of the pro-domain thiol group and disruption of its inhibitory coordination with the Zn++ in the catalytic domain of TACE. These results demonstrate that ROS production is involved in PMA-induced ectodomain shedding and implicate a role for ROS in other shedding processes.     

Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells.

Recent evidence has implicated cytokines and growth factors in the initiation of parturition in women. In the present study, the amnion-derived cell line WISH was used to determine whether proinflammatory cytokines (interleukins 1 beta, 6, and 8, tumor necrosis factor-alpha, and granulocyte/macrophage colony stimulating factor) could amplify epidermal growth factor-induced prostaglandin E2 production. WISH cells were preincubated with cytokines (0.0001-10 ng/ml) for 60 min and then challenged with EGF (10 ng/ml) for 4 hrs after which PGE2 production was measured by radioimmunoassay. EGF, IL-1 beta and TNF-alpha alone caused a dose-dependent increase in PGE2 production, while IL-6, IL-8 and GM-CSF were ineffective over the dose range tested. When cells were preincubated with IL-1 beta or TNF-alpha, there was a dose-dependent potentiation of EGF-induced PGE2 production that was greater than the sum of EGF alone and IL-1 beta or TNF-alpha alone. In each case, the minimum dose of IL-1 beta or TNF-alpha which amplified EGF-induced PGE2 production was 0.1 ng/ml (p less than 0.05, Student's t-test). These data show that low concentrations of IL-1 beta or TNF-alpha may serve to amplify EGF-mediated PGE2 biosynthesis in amnion-derived cells and suggest that cytokines may modulate EGF function in responsive cells.     

Regulation of protein kinase C by nerve growth factor, epidermal growth factor, and phorbol esters in PC12 pheochromocytoma cells

We have used a permeabilized cell assay and a synthetic peptide substrate (KRTLRR) to specifically monitor the activity of protein kinase C in PC12 cells preincubated with nerve growth factor (NGF), epidermal growth factor (EGF), or phorbol esters. Pretreatment of PC12 cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate or 1 microM phorbol dibutyrate stimulated the rate of KRTLRR peptide phosphorylation 4.8- and 2.6-fold, respectively. Furthermore, pretreatment of cells with NGF or EGF transiently increased the KRTLRR peptide kinase activity. Peak stimulations of KRTLRR peptide kinase (1.3-2-fold) were observed after 1-5 min of growth factor treatment and returned to control levels within 15-20 min. The KRTLRR peptide kinase activity fulfilled two criteria of protein kinase C. A synthetic peptide inhibitor of protein kinase C inhibited both growth factor- and phorbol ester-stimulated KRTLRR peptide kinase activity. In addition, growth factors and phorbol esters failed to stimulate KRTLRR peptide kinase activity in cells rendered protein kinase C-deficient by long-term treatment with 1 microM 12-O-tetradecanoylphorbol 13-acetate. In contrast to the transient activation of protein kinase C, ribosomal S6 kinase, assayed with the synthetic peptide RRLSSLRA, was persistently activated by NGF and EGF. The findings indicate that protein kinase C serves an early and transient role in the molecular actions of NGF and EGF in PC12 cells.     

Dexamethasone inhibition and phorbol myristate acetate stimulation of plasminogen activator in human embryonic lung cells

Treatment of human embryonic lung cells with dexamethasone resulted in a decrease in plasminogen activator activity measured in the fibrinolytic assay. The decrease in activity could at least partially be explained by the presence of an inhibitory substance(s) based on the following observations of lysates of dexamethasone-treated vs. control cells: a) an increase in specific activity following subcellular fractionation; b) an increase in fibrinolytic activity following separation by gel electrophoresis; c) an increase in fibrinolytic activity following mild acid-treatment; and d) a decrease in urokinase-directed fibrinolytic activity in mixing experiments. Phorbol myristate acetate increased plasminogen activator activity without affecting the level of inhibitory substance.     

Opsonic properties of C-reactive protein. Stimulation by phorbol myristate acetate enables human neutrophils to phagocytize C-reactive protein-coated cells

We examined phagocytosis of sheep erythrocytes passively sensitized with pneumococcal C-polysaccharide (E-PnC) and of E-PnC coated with C-reactive protein (E-PnC-CRP) by human polymorphonuclear leukocytes (PMN). PMN isolated from blood of normal individuals failed to ingest either E-PnC or E-PnC-CRP; however, after stimulation with 12-O-tetradecanoylphorbol-13-acetate (PMA; 2 ng/ml), PMN ingested E-PnC-CRP efficiently with a mean phagocytic index (PI) of 99.5 +/- 4.8 (mean +/- SD, n = 11), and E-PnC to a lesser extent with a mean PI of 33.2 +/- 11.7 (mean +/- SD, n = 11). PMN that had adhered to PnC-coated glass and that were stimulated with PMA attached but did not ingest E-PnC-CRP. In contrast, PMN plated on E-PnC-CRP-coated glass and stimulated with PMA did not attach or ingest E-PnC-CRP. These data indicate that PMN can be induced to phagocytize PnC-CRP and that both PnC and CRP are required for ingestion. They also suggest that specific receptors for these ligands are expressed by stimulated PMN. Neither attachment nor phagocytosis of E coated with rabbit anti-E IgG (E-IgG) was affected by plating PMN on PnC or PnC-CRP. On the other hand, both phagocytosis and ingestion of E-PnC-CRP as well as E-IgG was blocked by plating PMA-stimulated PMN on immune complexes containing rabbit IgG. Inhibition experiments with the use of 3G8, a monoclonal antibody to the Fc gamma receptor of PMN, and human monomeric IgG1 demonstrated that attachment of E-PnC-CRP is mediated by receptors other than the Fc gamma receptors. These combined results indicated a nonreciprocal association between the putative CRP receptors and the Fc gamma receptors of stimulated PMN, resulting in the clearance of both types of receptors from the apical surface of PMN by antigen-immobilized rabbit IgG.     

Insulin and epidermal growth factor stimulate glycolysis in quiescent 3T3 fibroblasts with no changes in key glycolytic enzyme activities

In order to study the effect of insulin and epidermal growth factor (EGF) on glycolysis in quiescent 3T3 fibroblasts and their mechanisms of action, lactic acid produced by cells and activities of key glycolytic enzymes in cell extracts were determined. Insulin increased lactic acid production; the maximal stimulation occurred at the concentrations above 250 ng/ml and the half-maximal dose was 50 ng/ml. This effect of insulin appeared as early as one hour, and lactic acid production in the presence of insulin linearly increased up to 4 h. The 24-h pretreatment with insulin exhibited no significant effect on the production by cells afterward incubated either with or without insulin. Lactic acid production decreased as the concentration of phloridzin increased. However, insulin stimulation of the production still remained in the presence of phloridzin. Parahydroxymercuribenzoate reduced production only by the equivalent of the increase due to insulin. EGF also increased lactic acid production; this effect occurred at 1 ng/ml and was maximal at 100 ng/ml. The activities of hexokinase, phosphofructokinase and pyruvate kinase in quiescent cells were not increased by insulin, and the affinities for substrates of these enzymes remained unaltered. These findings suggest that glucose uptake is a rate-limiting step in glycolysis in quiescent 3T3 fibroblasts and that the stimulatory effect of insulin on glycolysis is mediated by enhanced glucose entry.     

Regulation by ceramide of epidermal growth factor signal transduction and mitogenesis in cell lines overexpressing the growth factor receptor.

Ceramide has emerged as a pleiotropic signal mediator of cellular responses including differentiation, proliferation, cell cycle arrest and apoptosis. In the present study we evaluated the effect of cell permeant ceramide analogues on ligand-induced tyrosine phosphorylation of the EGF receptor (EGFR), phospholipase Cy (PLCgamma) activity and cell proliferation. Treatment with N-acetylsphingosine (C2-cer) and N-hexanoylceramide (C6-cer) prevented EGF-induced tyrosine trans-phosphorylation of the receptor in two different cell lines overexpressing the human EGFR (A431 and EGF-T17 cells). In contrast, treatment of A431 and EGFR-T17 cells with C2-cer or C6-cer did not affect the ligand binding capacity of the receptor, an effect that was however observed after TPA-induced activation of PKC. In addition EGF-stimulated PLCgamma activity was transiently decreased in A431 cells treated with C6-cer and only a modest, albeit significant reduction on ligand-induced 3H-InsP3 generation was observed in EGFR-T17 cells pretreated with ceramide. We also examined the effect of C2-cer on serum (A431)- or EGF (EGFR-T 17)-induced cell proliferation. Treatment of EGFR-TI7 cells with C2-cer (0.1-10 microM) did not affect cell viability, but prevented EGF-induced 3H-thymidine incorporation in a dose-dependent manner. In contrast, 3H-thymidine incorporation in serum-stimulated A431 cells decreased only at the higher doses of C2-cer used (1-10 microM), being this effect accompanied by a slight, albeit significant (20-25%), reduction in cell viability.