Novel mimics of sialyl Lewis X: design, synthesis and biological activity of a series of 2- and 3-malonate substituted galactoconjugates.

A series of potent inhibitors of P-selectin as potential anti-inflammatory agents is reported. These compounds are derivatives of galactocerebrosides bearing a malonate side chain in positions 2 and 3 of the galactose moiety. Based on the binding mode of sialyl Lewis X, the two acidic groups of the malonate are designed to form ionic interactions with two important lysines in the active site of P-selectin, Lys113 and Lys111. On the other hand, the 4- and 6-hydroxy groups on the galactose ring are arranged to chelate the calcium ion in the P-selectin active site. The synthesis and the biological activity of this series of compounds are described. Lead compounds having a greater potency than sialyl Lewis X are identified.

Phosphorylation and calcium influx are not sufficient for the activation of cytosolic phospholipase A2 in U937 cells: requirement for a Gi alpha-type G-protein.

Differentiation with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U937 cell line toward a monocyte/granulocyte-like cell results in the cell acquiring an ability to release arachidonate upon stimulation. In contrast, the calcium ionophore ionomycin was able to stimulate phospholipase C, as measured by inositol 1,4,5-trisphosphate formation, to equal extents in both undifferentiated and dBcAMP-differentiated U937 cells. The role and regulation of cytosolic phospholipase A2 (cPLA2) in the production of arachidonate in these cells when either the chemotactic peptide fMLP or ionomycin are used as stimulus were investigated. The ionomycin- and fMLP-stimulated release of arachidonate were sensitive to the cPLA2 inhibitor arachidonyl trifluoromethylketone (IC50 values of 32 and 18 microM, respectively), but were not inhibited by E-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2 H-pyran-2-one, a bromoenol lactone inhibitor of the calcium-independent phospholipase A2. These results, coupled with the inhibition of ionomycin-induced arachidonate production by electroporation of differentiated cells to introduce an anti-cPLA2, demonstrate that the cPLA2 is the enzyme responsible for arachidonate release in differentiated cells. SDS-PAGE and immunoblot analysis of differentiated cells showed the cells to contain both phosphorylated and unphosphorylated forms of cPLA2 (ratio of about 2: 3). Surprisingly, undifferentiated cells contain 30% more enzyme than differentiated cells and contain a higher percentage (approximately 75%) of the phosphorylated in the absence of stimulation. The inability of undifferentiated cells to produce arachidonate is not due to insufficient intracellular calcium concentrations since ionomycin induces large (820-940 nM) influxes of intracellular calcium in both differentiated and undifferentiated cells. This demonstrates that phosphorylation of cPLA2 andan influx of intracellular calcium are not sufficient to activate the enzyme to produce arachidonate. Instead, activation of a pertussis toxin-sensitive Gi alpha-type G-protein is required as evidenced by the production of arachidonate in undifferentiated cells stimulated with mastoparan, an activator of Gi alpha subunits, in combination with ionomycin. This activation of a Gi alpha-type G-protein is independent of modulations of adenylyl cyclase activity since cellular cAMP levels were not modulated upon treatment with mastoparan and ionomycin.

Sulfated galactocerebrosides as potential antiinflammatory agents.

Native sulfatides, as well as many sulfated glycolipids, have been shown to avidly bind to the selectin receptors. In vivo, native sulfatides significantly block activity in selectin-dependent inflammatory responses. The fact that nonsulfated galactocerebrosides did not inhibit selectin-mediated adhesion identified a critical role for the anionic sulfate residue. We therefore initiated a program to evaluate the activity of position isomers. This study showed a binding selectivity for the positions 2 and 3 of the sulfate group on the carbohydrate ring as well as enhanced activity for the disulfated analogs. Furthermore, it was discovered that the attachment of lipophilic substituents on the carbohydrate ring was tolerated, consistent with the presence of a lipophilic pocket in the binding activity. This resulted in compounds with a 6-fold increased potency.

BMS-190394, a selectin inhibitor, prevents rat cutaneous inflammatory reactions.

Selectin binding is the first step in extravasation of leukocytes through the endothelium. Infiltration of leukocytes is a hallmark of an inflammatory response. Blockade of selectin-dependent adhesion, therefore, represents a specific mechanism-based anti-inflammatory strategy. We have used the natural product sulfatide, one of the selectin ligands, as a template to design a novel selectin antagonist. BMS-190394, a structural analog of sulfatide, is an inhibitor of cell binding to P-, E- and L-selectin-Ig fusion proteins. BMS-190394 also inhibits binding mediated by native P-selectin expressed on the surface of activated platelets. Pharmacokinetic analysis of BMS-190394 showed that the compound remained in circulation with a T1/2 of 7 hr, long enough to inhibit the development of an acute inflammatory response. The in vitro activity and pharmacokinetic profile of this selectin-blocking compound led to the determination of its in vivo anti-inflammatory activity. BMS-190394 was a potent inhibitor of the dermal immune complex-induced reverse passive Arthus reaction in rats when delivered by the i.v. or i.p. route. The ED50 of the compound in the reverse passive Arthus reaction compares favorably to that for dexamethasone. BMS-190394 was also an effective inhibitor of the delayed-type hypersensitivity reaction in the rat. Compared with previous reports of the use of antibodies and complex oligosaccharides to inhibit the activity of the selectins, this low-molecular-weight inhibitor of the selectins presents a novel class of anti-inflammatory agents.

Isolation and structure determination of sulfonoquinovosyl dipalmitoyl glyceride, a P-selectin receptor inhibitor from the alga Dictyochloris fragrans.

Bioassay-guided fractionation of the marine alga Dictyochloris fragrans led to the isolation and identification of sulfonoquinovosyl dipalmitoyl glyceride (1). The structure of 1 was determined by a combination of spectroscopic methods. On the basis of P-selectin inhibition assays (i.e., P-selectin-IgG ELISA, cell binding assay of receptor globulin, and platelet:HL60 adhesion, it was demonstrated that 1 selectively blocks the P-selectin-ligand interaction in vitro and could be considered a lead compound for synthetic modification in order to design more potent inhibitors of cell adhesion processes that play important roles in development of inflammatory-mediated disease states.

Phorbol ester-induced upregulation of polymorphonuclear leukocyte P-selectin ligand expression.

Proinflammatory stimuli cause the vascular endothelium to express P-selectin that tethers leukocytes by binding surface glycoprotein carbohydrate. While the activation of polymorphonuclear leukocytes (PMN) is associated with upregulation of the beta 2-integrins, there is little known about the regulated expression of the ligand for the endothelial P-selectin. We have used a soluble chimeric P-selectin protein as a probe for the expression of ligand on the surface of the PMN. Treatment with phorbol ester for more than 20 min stimulated P-selectin ligand expression. The upregulation of beta 2-integrin expression was affected in a similar manner. The mechanism of selectin ligand upregulation did not involve de novo protein synthesis, and may involve translocation of membranes containing performed intracellular ligand. C5a, which is generated in response to complement activation in vivo, also stimulated selectin ligand upregulation. Degranulation induced by nigericin increased ligand expression, and TNF-alpha treatment resulted in a modest upregulation.

Inhibition of Immune Complex-Induced Inflammation by A small Molecular Weight Selectin Antagonist.

The anti-inflammatory effect of a small molecular weight antagonist of P- and E-selectin-dependent cell adhesion was examined. The glycolipid sulphatide was shown to block the adherence of thrombin-activated rat platelets to HL-60 cells. This interaction is known to be dependent on P-selectin. The rat dermal reverse passive Arthus reaction was used to assess the effect of sulphatide on a neutrophil dependent inflammatory response. Sulphatide dosedependently blocked both the vascular permeability increase and cell infiltration after intraperitoneal administration. These results show that a small molecular weight compound which blocks P- and E-selectin dependent adhesion in vitro can effectively block the inflammation due to immune complex deposition. A compound with this type of profile may have therapeutic potential in the treatment of immune complex mediated diseases.

Cytosolic [Ca2+] homeostasis and tyrosine phosphorylation of phospholipase C gamma 2 in HL60 granulocytes.

Activated phagocytes produce large amounts of reactive oxygen intermediates, including peroxides. In addition to their microbicidal effect, it has recently been suggested that reactive oxygen species play a role as intracellular messengers. The mechanism of action remains unknown, but peroxides have been reported to increase tyrosine phosphorylation, an effect potentiated by vanadate. In this report we studied the effects of a combination of H2O2 and vanadate on Ca2+ homeostasis in granulocytic HL60 cells. The peroxides induced a transient elevation of cytosolic [Ca2+] associated with release from internal stores. Ca2+ mobilization was accompanied by increased generation of inositol 1,4,5-trisphosphate, implicating phospholipase C (PLC). A sizable increase in phosphotyrosine accumulation by several polypeptides in the M(r) 20,000 to 250,000 range preceded the [Ca2+] changes. We therefore considered the possibility that tyrosine phosphorylation of a phospholipase mediates the observed effects. Differentiated (granulocytic) HL60 cells did not have detectable levels of PLC gamma 1 but had substantial PLC gamma 2. Immunoprecipitation and immunoblotting experiments demonstrated that PLC gamma 2 becomes tyrosine-phosphorylated upon treatment of the cells with peroxides of vanadate. If associated with activation, such phosphorylation of PLC gamma 2 can account for the rise in [Ca2+]. Although capable of mobilizing internal Ca2+ stores, the peroxides failed to produce the sustained [Ca2+] increase predicted by the "capacitative" model. Mn2+ influx determinations indicated that this is due to impairment of divalent cation entry by the peroxides, uncoupling the plasma membrane from the internal stores. Changes in [Ca2+] homeostasis could mediate some of the messenger actions of reactive oxygen species.

Temporal infiltration of leukocyte subsets into mouse skin inflamed with phorbol ester.

We have previously shown that multiple topical applications, over 11 days, of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a persistent inflammatory reaction characterized by edema, cell infiltration and epidermal hyperplasia. In order to characterize the cell infiltrate during the establishment of this inflammatory reaction, immunohistochemistry was performed using two monoclonal antibodies: MOMA-2, a macrophage antibody and Thy-1, a pan T-cell antibody. The level of polymorphonuclear leukocytes (PMNs) peaked by day 3 at 160-fold over nontreated controls and then subsided to a 30-fold elevation on days 7-10. By day 4, the number of macrophages increased 2.9-fold over the nontreated control and by day 10 were elevated 6.0-fold over the nontreated control. In comparison, the number of T-cells present by day 7 was significantly elevated 9.5-fold over the nontreated group and peaked at day 8 with a 19-fold elevation relative to nontreated controls. Topical treatment of animals with hydrocortisone valerate resulted in a dramatic (> 60%) reduction in the number of T-cells present in the tissue. In contrast, there was no effect of the steroid on the number of macrophages present in the tissue. The identification of specific cell types and their time course of infiltration is consistent with the development of a chronic inflammatory lesion.

Elevated membrane association of phospholipase C-gamma 1 in MDA-468 mammary tumor cells.

We have examined the distribution of phospholipase C-gamma 1 (PLC-gamma 1) between membrane and cytosolic fractions in several cell lines. In MDA-468 cells, which are derived from a human breast tumor, greater than one-half of the total PLC-gamma 1 is associated with the membrane fraction of the cell. Unlike the situation in A-431 cells [G. Todderud, M. I. Wahl, S. G. Ree, and G. Carpenter, Science, 248: 296-298, 1990], epidermal growth factor (EGF) stimulation of MDA-468 cells does not result in significantly increased PLC-gamma 1 association with membranes. Immunoblot analysis reveals low levels of phosphotyrosine in PLC-gamma 1 and EGF receptors in unstimulated MDA-468 cells and greatly increased phosphotyrosine levels in these proteins as a result of EGF stimulation of the cells. We conclude that autocrine activation of EGF receptors is not responsible for the elevated association of PLC-gamma 1 with membranes in these cells.

PMN binding to P-selectin is inhibited by sulfatide.

The endothelial adhesion protein P-selectin binds to a ligand present on the surface of leukocytes. We have characterized the binding interaction between P-selectin and polymorphonuclear leukocytes (PMNs) in an in vitro assay. These studies have utilized a soluble chimeric protein termed receptor globulin (Rg), which consists of the lectin-EGF-CR-CR extracellular domains of P-selectin fused to a human immunoglobulin G Fc domain. The PMNs bound to immobilized Rg in a saturable and concentration-dependent manner. The binding was specific for the Rg, as preincubation of the cells with soluble Rg inhibited binding to immobilized Rg, and binding was dependent on the presence of free divalent cations. The PMNs expressed a ligand for both P-selectin and E-selectin but not for L-selectin. Previously it was shown that sulfatide is a ligand for P-selectin binding in transformed cells. We have demonstrated that the presence of sulfatide in the P-selectin-PMN adhesion assay inhibits binding in a dose-dependent manner.

Altered distribution of phospholipase C-gamma 1 in benign hyperproliferative epidermal diseases.

Phospholipase C-gamma 1 (PLC-gamma 1) is a well characterized substrate for the epidermal growth factor receptor tyrosine kinase and has been implicated in the intracellular biochemical signaling cascade which occurs following stimulation of cells with epidermal growth factor. The in vivo localization of PLC-gamma 1 was examined by immunohistochemistry in sections of normal human skin and in skin sections from a diverse series of hyperproliferative epidermal conditions (psoriasis, seborrheic keratoses, acrochordons, and margins near second-degree burns). Immunoreactive PLC-gamma 1 was detected only in the basal compartment of normal skin but was readily detectable in both the basal and outer epidermal compartment in hyperproliferative skin conditions. In addition, immunoreactive PLC-gamma 1 colocalizes with immunoreactive epidermal growth factor receptor in both normal and hyperproliferative epidermis.

Growth factor phosphorylation of PLC-gamma 1.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate has a central role in many signalling pathways. One of the phospholipase C (PLC) isozymes that mediates this reaction is a direct substrate for the tyrosine kinase activity of several growth factor receptors. Growth factors elicit increases in both the phosphoserine and the phosphotyrosine content of the PLC-gamma 1 isozyme. PLC-gamma 1 contains three tyrosine phosphorylation sites, which have been identified as residues 771, 783 and 1254. Phosphorylation of tyrosine residues is sufficient to increase the catalytic activity of PLC-gamma 1, though other proteins may modulate this activation. However, the role of growth factor-enhanced phosphorylation of serine residues on PLC-gamma 1 remains obscure. In vitro studies of PLC-gamma 1, recovered from growth factor-treated cells, indicate that activation by tyrosine phosphorylation is not due to increased sensitivity to Ca2+, a required co-factor, but is reflected in altered kinetic constants, i.e. V(max) and, to a lesser extent, Km.

Elevated content of the tyrosine kinase substrate phospholipase C-gamma 1 in primary human breast carcinomas.

Phospholipase C-gamma 1 (PLC-gamma 1) is a substrate for several receptor tyrosine kinases and its catalytic activity is increased by tyrosine phosphorylation. However, the biological significance of this molecule in normal or malignant human epithelial cell proliferation is unknown. We determined the relative content of PLC-gamma 1 in primary human mammary carcinomas and in nonmalignant mammary tissues. By Western blot and immunohistochemistry, considerably higher levels of PLC-gamma 1 protein were detectable in the majority of carcinomas and in one of two benign fibroadenomas compared to normal breast tissues. In 18 of 21 carcinomas that contained high levels of PLC-gamma 1, the presence of phosphotyrosine on PLC-gamma 1 could also be detected. All carcinomas in which tyrosine phosphorylated PLC-gamma 1 was present also expressed detectable levels of the epidermal growth factor receptor or erbB-2, two tyrosine kinases known to phosphorylate this enzyme. Thus, a high percentage of mammary carcinomas concomitantly display increased levels of receptor tyrosine kinases and a direct tyrosine phosphorylation substrate, thereby potentially amplifying two successive steps in a signal transduction pathway.

Stimulation of phospholipase C-gamma 1 membrane association by epidermal growth factor.

Epidermal growth factor (EGF) treatment of A-431 epidermoid carcinoma cells elicited a redistribution of phospholipase C-gamma 1 (PLC-gamma 1) from a predominantly cytosolic localization to membrane fractions. The temporal coincidence of this redistribution with EGF stimulation of inositol phosphate formation and EGF increased phosphorylation of PLC-gamma 1 suggests that the membrane association of PLC-gamma 1 is a significant event in second messenger transduction.

Epidermal growth factor: the receptor and its function.

Epidermal growth factor (EGF) is a small polypeptide hormone with mitogenic properties in vivo and in vitro. EGF elicits biologic responses by binding to a cell surface receptor which is a transmembrane glycoprotein containing a cytoplasmic protein tyrosine kinase. EGF responses are mediated by ligand binding and activation of this intrinsic protein kinase. The receptor can be phosphorylated by other protein kinases, and this may regulate receptor function. Stimulation of the receptor tyrosine kinase activity by ligand binding must regulate the activity of an as yet undefined molecule(s) responsible for transmitting a mitogenic signal to the nucleus.

Presence of mannose phosphate on the epidermal growth factor receptor in A-431 cells.

In this report, we demonstrate a novel post-translational modification of the epidermal growth factor (EGF) receptor. This modification involves the presence of phosphate, previously thought to exist only on amino acid residues in the EGF receptor, on oligosaccharides of the receptor. We have utilized several independent approaches to determine that mannose phosphate is present on the EGF receptor in A-431 cells. Following metabolic labeling with 32P, immunoisolation of the EGF receptor, and digestion with Pronase radioactivity was determined to be present on high mannose type oligosaccharides by concanavalin A chromatography. Also, after acid hydrolysis of in vivo 32P-labeled EGF receptor, radioactivity was detected that co-migrated with mannose 6-phosphate on two-dimensional thin layer electrophoresis. This radiolabeled material co-eluted with a mannose 6-phosphate standard from a high pressure liquid chromatography anion exchange column. Last, an acid hydrolysate of [3H]mannose-labeled EGF receptor contained two radiolabeled fractions, as analyzed by thin layer electrophoresis, and the radioactivity in one of these fractions was substantially reduced by alkaline phosphatase treatment prior to electrophoresis. These experiments indicate that the mature EGF receptor in A-431 cells contains mannose phosphate. This is a novel modification for membrane receptors and has only been reported previously for lysosomal enzymes and a few secreted proteins.