Showing papers on "Protease-activated receptor 2 published in 2003"

PARticipation in inflammation.

Abstract: In this issue of the JCI, Ferrell and colleagues report that mice lacking the proteinase-activated receptor-2 (PAR-2) were protected against a form of adjuvant-induced arthritis (1). Herein we discuss protease-activated receptors, their mechanism of activation, and the contexts in which they are thought to function. A useful unifying hypothesis emerges: protease-activated receptors link tissue injury to appropriate cellular responses, and such responses, while normally homeostatic, may contribute to disease. Protease-activated receptor-1 (PAR-1), the prototypical PAR, was identified in a search for the receptors that mediate the activation of platelets and other cells by the coagulation protease thrombin. Known to be a seven transmembrane domain G-protein–coupled receptor, PAR-1 is activated by proteolysis (Figure ​(Figure1).1). Thrombin binds to PAR-1’s N-terminal exodomain and cleaves it after Arg41 to generate a new receptor N-terminus. The first six amino acids of this new N-terminus, SFLLRN, then serve as a tethered ligand that binds intramolecularly to the receptor’s heptahelical bundle to effect transmembrane signaling and G protein activation. Thus PARs are, in essence, peptide receptors that carry their own ligands, which lie hidden until revealed by receptor cleavage. The synthetic peptide SFLLRN, which mimics the PAR-1 tethered ligand, is a PAR-1 agonist and activates PAR-1 independent of thrombin and receptor cleavage. Such peptide agonists have provided a ready tool for probing the effects of PAR activation in vitro and in vivo. Figure 1 Mechanism of PAR-1 activation. The N-terminus of PAR-1, a seven transmembrane domain G protein–coupled receptor, contains a protease cleavage site that, once cleaved by thrombin, results in a new N-terminus. The new N-terminal sequence, SFLLRN, ... Four PARs are currently known (recent reviews include refs. 2–4). PAR-1, PAR-3, and PAR-4 can be activated by thrombin, and thrombin is almost certainly a physiological activator of these receptors in vivo. Several other proteases can cleave these receptors productively and may also contribute to their function in vivo. PAR-2 can be activated by multiple trypsin-like serine proteases including: trypsin itself, mast cell tryptase, neutrophil proteinase 3, tissue factor/factor VIIa/factor Xa, and membrane-tethered serine protease-1. However PAR-2 is not cleaved by thrombin.

Neutrophil Serine Proteinases Activate Human Nonepithelial Cells to Produce Inflammatory Cytokines Through Protease-Activated Receptor 2

Abstract: Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.

Protease-activated receptor 2: activation, signalling and function

Abstract: PARs (protease-activated receptors) are a family of four G-protein-coupled receptors for proteases from the circulation, inflammatory cells and epithelial tissues. This report focuses on PAR 2 , which plays an important role in inflammation and pain. Pancreatic (trypsin I and II) and extrapancreatic (trypsin IV) trypsins, mast cell tryptase and coagulation factors VIIa and Xa cleave and activate PAR 2 . Proteases cleave PAR 2 to expose a tethered ligand that binds to the cleaved receptor. Despite this irreversible activation, PAR 2 signalling is attenuated by β-arrestin-mediated desensitization and endocytosis, and by lysosomal targeting and degradation, which requires ubiquitination of PAR 2 . β-Arrestins also act as scaffolds for the assembly of multi-protein signalling complexes that determine the location and function of activated mitogen-activated protein kinases. Observations of PAR 2 -deficient mice support a role for PAR 2 in inflammation, and many of the effects of PAR 2 activators promote inflammation. Inflammation is mediated in part by activation of PAR 2 in the peripheral nervous system, which results in neurogenic inflammation and hyperalgesia.

Characterization of Glycosylation Sites of the Epidermal Growth Factor Receptor

Abstract: The epidermal growth factor receptor is a transmembrane glycoprotein that mediates the cellular responses to epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). In this study of the human EGF receptor naturally expressed in A431 cells, the glycosylation sites of the full-length, membrane-bound receptor and of a secreted form of the receptor were characterized by mass spectrometry. Our data show that the naturally expressed human EGF receptor is fully glycosylated on eight of the 11 canonical sites; two of the sites are not glycosylated, and one is partially glycosylated, a pattern of site-usage similar but not identical to those reported for the recombinant human EGF receptor heterologously expressed in Chinese hamster ovary cells. We also confirm the partial glycosylation of an atypical NNC site first identified in the receptor expressed in Chinese hamster ovary cells. We show that an additional canonical site in the secreted form of the receptor is fully glycosylated. While the pattern of glycosylation is the same for the sites shared by the full-length and the secreted forms of the receptor, the oligosaccharides of the full-length receptor are more extensively processed. Finally, we provide evidence that in addition to the known secreted form of the receptor, a proteolytic cleavage product of the receptor corresponding to the full extracytoplasmic, ligand-binding domain is present in the conditioned medium.

Molecular regulation of the oxytocin receptor in peripheral organs.

Abstract: The oxytocin receptor belongs to the G-protein-coupled seven transmembrane receptor superfamily. Its main physiological role is regulating the contraction of uterine smooth muscle at parturition and the ejection of milk from the lactating breast. Oxytocin receptor expression is observed not only in the myometrium and mammary gland but also in the endometrium, decidua, ovary, testis, epididymis, vas deferens, thymus, heart and kidney, as well as in the brain. The expression profile shows a tissue-specific as well as a stage-specific pattern. The oxytocin receptor gene is a single-copy gene consisting of four exons and three introns, localized at 3p25-3p26.2 in the human chromosome. In transfection studies using a fusion construct containing the promoter region of the oxytocin receptor gene inserted in a reporter plasmid, neither proinflammatory cytokines nor oestrogen directly activate the gene. The nuclear fractions from up-regulated (term myometrium) and down-regulated (non-pregnant myometrium) tIssues show differential patterns of protein binding to the 5'-flanking region, and a human homologue of chicken MafF has been cloned as a term-myometrium-specific oxytocin receptor modulator. The oxytocin receptor gene appears to be highly methylated. Methylation around intron 1 and in intron 3 might contribute to tIssue-specific suppression of the gene. The oxytocin receptor is also regulated by desensitization, whose mechanism appears to involve loss of ligand-binding activity of the protein as well as suppression of the oxytocin receptor mRNA transcription. These findings taken together indicate that the oxytocin receptor is regulated in a very complicated manner, and the transcriptional regulatory elements critical for this regulation should be investigated further.

Expression of protease-activated receptor 2 in ulcerative colitis.

Abstract: Although tryptase released from mast cells might play a key role in the pathogenesis of ulcerative colitis (UC), the role of protease-activated receptor 2 (PAR2), tryptase receptor, remains unclear in the pathogenesis of this disease. The expressions of PAR2 and tumor necrosis factor (TNF) alpha in nine UC tissues and nine normal tissues were examined by immunohistochemistry. TNF-alpha levels secreted from human leukemic mast cell line (HMC-1) after the treatment of PAR2 agonists were also measured by enzyme-linked immunosorbent assay. The PAR2 and TNF-alpha proteins were more significantly detectable in UC tissues than in normal tissues. Furthermore, 65.2% of PAR2+ cells and 66.4% of TNF-alpha+ cells in UC tissues were tryptase-positive cells. In other words, 60.6% and 46.3% of tryptase-positive cells in UC tissues were PAR2+ cells and TNF-alpha+ cells, respectively. A chi2 analysis showed correlation (p < 0.007) between PAR2 and TNF-alpha in tryptase-positive mast cells. Moreover, PAR2 agonists significantly induced the TNF-alpha secretion from HMC-1. These results indicate that the activation of the mast cells through PAR2 may be involved in the pathogenesis of UC.

Ginsenoside-Rb1 acts as a weak phytoestrogen in MCF-7 human breast cancer cells.

Abstract: Ginseng has been recommended to alleviate the menopausal symptoms, which indicates that components of ginseng very likely contain estrogenic activity. We have examined the possibility that a component ofPanax ginseng, ginsenoside-Rb1, acts by binding to estrogen receptor. We have investigated the estrogenic activity of ginsenoside-Rb1, in a transient transfection system using estrogen-responsive luciferase plasmids in MCF-7 cells. Ginsenoside-Rbb1 activated the transcription of the estrogen-responsive luciferase reporter gene in MCF-7 breast cancer cells at a concentration of 50 μM. Activation was inhibited by the specific estrogen receptor antagonist ICI 182,780, indicating that the estrogenic effect of ginsenoside-Rbb1 is estrogen receptor dependent. Next, we evaluated the ability of ginsenoside-Rbb1, to induce the estrogen-responsive gene c-fos by semi-quantitative RT-PCR assays and Western analyses. Ginsenoside-Rbb1 increased c-fos both at mRNA and protein levels. However, ginsenoside-Rbb1 failed to activate the glucocorticoid receptor, the retinoic acid receptor, or the androgen receptor in CV-1 cells transiently transfected with the corresponding steroid hormone receptors and hormone responsive reporter plasmids. These data support our hypothesis that ginsenoside-Rbb1 acts a weak phytoestrogen, presumably by binding and activating the estrogen receptor.

Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2

Abstract: We evaluated the contribution of rab5a and rab11a to trafficking and signaling of protease-activated receptor 2 (PAR2), a receptor for trypsin and tryptase. Agonists stimulated internalization of PAR2 into early endosomes containing rab5a. Dominant negative rab5aS34N disrupted early endosomes and inhibited agonist-stimulated endocytosis of PAR2. Internalized PAR2 was sorted to lysosomes, and rab5a remained in early endosomes. Rab5a promoted and rab5aS34N impeded resensitization of trypsin-induced calcium mobilization. Rab11a was detected in the Golgi apparatus with PAR2, and PAR2 agonists stimulated redistribution of rab11a into vesicles containing PAR2 that migrated to the cell surface. Dominant negative rab11aS25N was mostly confined to the Golgi apparatus. Although expression of rab11aS25N caused retention of PAR2 in the Golgi apparatus, it did not abolish trafficking of PAR2 to the cell surface. However, expression of wild-type rab11a accelerated both recovery of PAR2 at the cell surface and resensitization of PAR2 signaling. Thus rab5a is required for PAR2 endocytosis and resensitization, whereas rab11a contributes to trafficking of PAR2 from the Golgi apparatus to the plasma membrane.

Protease-Activated Receptor-2 Expression in IgA Nephropathy: A Potential Role in the Pathogenesis of Interstitial Fibrosis

Abstract: An increasing body of evidence suggests that pro- teases may play a key role in the pathogenesis of tissue fibrosis. Protease-activated receptor-2 (PAR-2) is cleaved and activated by trypsin-like proteolytic enzymes, including tryptase and activated coagulation factor X (FXa). Both these soluble mediators have been demonstrated, directly or indi- rectly, at the interstitial level in progressive renal diseases, including IgA nephropathy (IgAN). PAR-2 mRNA and protein levels were investigated by RT-PCR and immunohistochemis- try, respectively, in 17 biopsies from IgAN patients and 10 normal kidneys. PAR-2 expression was also evaluated, by RT-PCR and western blotting, in cultured human mesangial and proximal tubular cells. Finally, gene expression of plas- minogen activator inhibitor-1 (PAI-1) and TGF-, two power- ful fibrogenic factors, was evaluated in FXa-, trypsin-, and PAR-2 activating peptide-stimulated human proximal tubular cells by Northern blot. In normal kidneys, PAR-2 gene expres- sion was barely detectable, whereas in IgAN biopsies the mRNA levels for this protease receptor were strikingly in- creased and directly correlated with the extent of interstitial fibrosis. Immunohistochemical staining demonstrated that PAR-2 protein expression in IgAN biopsies was mainly local- ized in the proximal tubuli and within the interstitial infiltrate. Proximal tubular cells in culture expressed PAR-2. Activation of this receptor by FXa in tubular cells induced a striking increase in intracellular calcium concentration. In addition, incubation of both cell lines with trypsin, FXa, or PAR-2 activating peptide caused a marked upregulation of PAI-1 gene expression that was not counterbalanced by an increased ex- pression of plasminogen activators. Finally, PAR-2 activation induced a significant upregulation of TGF- gene and protein expression in both mesangial and tubular cells. On the basis of our data, we can suggest that PAR-2 expressed by renal resi- dent cells and activated by either mast cell tryptase or FXa may induce extracellular matrix deposition modifying the PAI-1/PA balance and inducing TGF- expression. These molecular mechanisms may underlie interstitial fibrosis in IgAN.

Protease-activated receptor-2 signaling triggers dendritic cell development.

Abstract: Dendritic cells (DC) are potent antigen-presenting cells that govern the effector cell responses of the immune system. DC are thought to continuously develop from circulating progenitors in a process that is accelerated by inflammatory stimuli. However, the physiological signals that regulate the development of DC from precursor cells have not been well defined. Here we show that a serine protease acting via protease-activated receptor-2 (PAR-2) stimulates the development of DC from bone marrow progenitor cells cultured in granulocyte-macrophage colony-stimulating factor and IL-4. DC fail to develop in bone marrow cultures treated with soy bean trypsin inhibitor, a serine protease inhibitor, but this inhibition is overcome by a PAR-2 agonist peptide. DC do not spontaneously develop from the bone marrow of PAR-2-deficient mice, but can be stimulated to do so by inflammatory mediators. These results suggest that endogenous serine proteases stimulate DC development in vitro. Thus, serine proteases may help trigger adaptive immune responses in vivo.

G PROTEIN-COUPLED RECEPTOR SIGNALLING IN NEUROENDOCRINE SYSTEMS Molecular regulation of the oxytocin receptor in peripheral organs

Abstract: The oxytocin receptor belongs to the G-protein-coupled seven transmembrane receptor superfamily. Its main physiological role is regulating the contraction of uterine smooth muscle at parturition and the ejection of milk from the lactating breast. Oxytocin receptor expression is observed not only in the myometrium and mammary gland but also in the endometrium, decidua, ovary, testis, epididymis, vas deferens, thymus, heart and kidney, as well as in the brain. The expression profile shows a tissue-specific as well as a stage-specific pattern. The oxytocin receptor gene is a single-copy gene consisting of four exons and three introns, localized at 3p25–3p26·2 in the human chromosome. In transfection studies using a fusion construct containing the promoter region of the oxytocin receptor gene inserted in a reporter plasmid, neither proinflammatory cytokines nor oestrogen directly activate the gene. The nuclear fractions from up-regulated (term myometrium) and down-regulated (non-pregnant myometrium) tissues show differential patterns of protein binding to the 5′-flanking region, and a human homologue of chicken MafF has been cloned as a term-myometrium-specific oxytocin receptor modulator. The oxytocin receptor gene appears to be highly methylated. Methylation around intron 1 and in intron 3 might contribute to tissue-specific suppression of the gene. The oxytocin receptor is also regulated by desensitization, whose mechanism appears to involve loss of ligand-binding activity of the protein as well as suppression of the oxytocin receptor mRNA transcription. These findings taken together indicate that the oxytocin receptor is regulated in a very complicated manner, and the transcriptional regulatory elements critical for this regulation should be investigated further.

Protease-Activated Receptor 2–Mediated Vasodilatation in Humans In Vivo Role of Nitric Oxide and Prostanoids

Abstract: Background— Systemic hypotension as a consequence of vascular dysfunction is a well-recognized and important feature of critical illness. Although serine protease activation has been implicated as a cause of vascular dysfunction in systemic inflammation, the mechanism is unknown. Recently, a class of receptors with an entirely novel mechanism of action, protease-activated receptors (PARs), has been identified that would explain the link between protease activation and systemic hypotension. Our aim was to test the hypothesis that in vivo activation of protease-activated receptor 2 (PAR-2) in humans would mediate vasodilatation. Methods and Results— For these first-in-human studies, an activating peptide for the human PAR-2 receptor was synthesized and administered to healthy volunteers. Using both the dorsal hand vein technique and forearm plethysmography, we studied the effects of PAR-2 activation in human blood vessels and investigated the mechanism of vasodilation. Activation of PAR-2 receptors in vivo ...

Protease-activated receptor (PAR)-independent growth and pro-inflammatory actions of thrombin on human cultured airway smooth muscle.

Abstract: (1) Thrombin, a mitogen for human cultured airway smooth muscle (HASM), has many actions that have been attributed to activation of protease-activated receptor (PARs). However, the role of PARs in the proliferative action has not been clearly identified. Moreover, thrombin elicits cytokine production in a number of cell types, but these effects have not been characterized in human ASM. (2) Thrombin (0.03-3 U ml(-1))-stimulated increases in the levels of the pro-inflammatory and fibrogenic cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed over the same concentration range observed for thrombin-stimulated mitogenesis. (3) Inhibition of thrombin proteolytic activity, with either D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK)- or hirudin-treated thrombin (0.3 U ml(-1)) or in the presence of the thrombin serine protease-selective inhibitor, SDZ 217-766 (0.15 micro M), reduced the thrombin-stimulated GM-CSF levels by 91+/-3, 65+/-12 and 83+/-9% (n=8, P<0.05), respectively. PPACK treatment, hirudin and SDZ 217-766 inhibited thrombin-stimulated increase in cell number by 70+/-8, 63+/-11 and 69+/-8%, respectively. (4) PAR-selective peptides SFLLRN (PAR1; 10 micro M), SLIGKV (PAR2; 10 micro M), GYPGQV (PAR4; 100 micro M) or the combination of SFLLRN and GYPGQV elicited mitogenic responses of only 15% of that to thrombin and surprisingly, had no effect on GM-CSF levels (n=8). Nevertheless, inhibition of thrombin responses by pertussis toxin (50 ng ml(-1)) suggests that the PAR-independent actions also involve a G-protein-coupled receptor. (5) PAR1 receptor expression was evident by immunohistochemistry and these receptors were coupled to increases in intracellular calcium, but not to the phosphorylation of ERK or the increases in cyclin D1 protein levels that are essential for cell proliferation. Cross-desensitization of intracellular calcium increases by thrombin and the PAR1-selective peptide provides evidence that the PAR1 receptor responds to both ligands. (6) The failure of PAR-selective peptides to mimic thrombin responses together with the inhibition of thrombin responses by serine protease inhibitors suggest the involvement of novel proteolytic receptor targets for thrombin-induced mitogenesis and cytokine production.

Molecular characterization of the rat Kupffer cell glycoprotein receptor.

Abstract: The Kupffer cell receptor for glycoproteins has been reported to have a role in clearance of galactose- and fucose-terminated glycoproteins from circulation. Although the gene and a cDNA encoding the receptor have been described, there has been little study of the receptor protein. To address some questions about possible ligands and functions for this receptor, fragments representing portions of the extracellular domain have been expressed and characterized. The extracellular domain consists of a trimer stabilized by an extended coiled-coil of alpha-helices. The receptor displays monosaccharide-binding characteristics similar to the hepatic asialoglycoprotein receptor, but with somewhat less selectivity. The two best monosaccharide ligands are GalNAc and galactose. alpha-Methyl fucoside is a particularly poor ligand. Analysis of Kupffer cell receptor binding to glycoproteins and oligosaccharides released from them reveals highest affinity for desialylated, complex N-linked glycans. The best glycoprotein ligands contain multiple highly branched oligosaccharides. A human ortholog of the rat receptor gene does not encode a full-length protein and is not expressed in liver. These characteristics suggest that the receptor may have functions parallel to those of the hepatocyte asialoglycoprotein receptor in some (but not all) mammalian species.

Protease-activated receptor-2 expression and the role of trypsin in cell proliferation in human pancreatic cancers.

Abstract: Protease-activated receptor (PAR)-2 is a G protein-coupled receptor that is activated by trypsin. The purpose of this study was to examine PAR-2 expression and the role of trypsin in cell proliferation in human pancreatic cancer cells. All four pancreatic cancer cell lines studied, from well to undifferentiated types, AsPC-1, BxPC-3, Panc-1, and MIAPaCa-2, had significant levels of PAR-2 mRNA detected by reverse transcription-polymerase chain reaction, and showed a band of about 55 kDa corresponding to the known molecular weight of PAR-2: AsPC-1, BxPC-3 and Panc-1 showed a strong band, and MIAPaCa-2 showed a weak one. Immunocytochemically, AsPC-1, BxPC-3, and Panc-1 showed intense immunostaining for PAR-2, predominantly in the plasma membrane, while in MIAPaCa-2, immunostaining was weak. Proliferative activity of AsPC-1 cells was increased by concentrations of trypsin as low as 10 nM, and activity peaked at a concentration of 100 nM, representing almost 60% of that induced by 10% fetal bovine serum. In contrast, trypsin had no significant effect on proliferation of MIAPaCa-2 cells. These findings suggest that trypsin plays a role in the growth of PAR-2-positive pancreatic cancer cells and serves as a potent mitogen in vitro, functioning as a growth factor.

Tissue factor-dependent coagulation protease signaling in acute lung injury

Abstract: Objectives: To review the role of tissue factor-dependent coagulation in acute lung injury. To interpret preclinical and clinical data on therapeutic intervention of the coagulation cascade, focusing on the principles of proteolytic cell signaling of the coagulant and anticoagulant pathways. Data Extraction and Synthesis: This review is based on published original research and relevant review articles on cell signaling by coagulation proteases and on experimental models that implicate the tissue factor-initiated coagulation cascade in acute lung injury and systemic inflammation. Conclusions: The coagulation cascade signals via protease activated receptors in the tissue factor-initiation phase and downstream via the effector protease, thrombin. Bleomycin-induced acute lung injury is an example of thrombin signaling-dependent pathology. Frequently, thrombin signaling is a major contributor to inflammation in the extravascular space but intravascular thrombin signaling is a threshold-regulated event. At low concentrations, intravascular thrombin activates the protein C pathway by converting protein C (bound to endothelial cell protein C receptor) to activated protein C and this generates antiinflammatory signals along the activated protein C-endothelial cell protein C receptor-protease activated receptor 1 pathway on endothelial cells. Direct thrombin signaling only occurs when intravascular thrombin concentrations exceed a coagulant threshold. In systemic bacterial toxin-mediated inflammation, inhibition of thrombin is not sufficient to limit inflammation, whereas tissue factor inhibition interrupts a self-sustaining inflammatory escalation in acute lung injury. Therefore, in the vasculature, inflammatory signaling by the tissue factor initiation complex is favored over thrombin signaling.

Marked enhancement in gene expression by targeting the human insulin receptor

Abstract: Background Exogenous genes can be delivered to cells without viral vectors using an “artificial virus” comprised of nonviral plasmid DNA encapsulated in the interior of 85 nm pegylated immunoliposomes (PIL). The liposomes are targeted to cells with receptor-specific targeting ligands such as receptor-specific peptidomimetic monoclonal antibodies. Methods The levels of luciferase gene expression in human or rat glioma cells are measured after targeting the PIL-encapsulated plasmid DNA via the human insulin receptor, the human epidermal growth factor receptor, or the rat transferrin receptor. The luciferase expression plasmids were either derived from pCEP4, which contains the Epstein-Barr nuclear antigen-1/oriP replication system, or from pGL2, which lacks this system for episomal replication of plasmid DNA. Results Depending on the plasmid construct used and the receptor targeted, the peak luciferase gene expression varied more than 200-fold from 1.8 ± 0.1 to 419 ± 31 pg luciferase per mg cell protein. With the same plasmid, the peak level of gene expression following delivery to the cell via the human insulin receptor was 100–200-fold higher than gene expression following delivery via either the epidermal growth factor receptor or the transferrin receptor. There was no gene expression if the targeting ligand on the PIL was replaced with a nonspecific isotype control antibody. Conclusions The extent to which an exogenous gene is expressed within a cell via a nonviral, receptor-mediated gene transfer technology is determined by the receptor specificity of the targeting ligand. The highest levels of gene expression are obtained after targeting the insulin receptor, and this may derive from the nuclear targeting properties of this receptor system. Copyright © 2002 John Wiley & Sons, Ltd.

IL-10 Mediates Sigma 1 Receptor-Dependent Suppression of Antitumor Immunity

Abstract: Sigma receptors are unique endoplasmic reticulum proteins that mediate signaling for a variety of drugs. We determined the effect of sigma(1) receptor agonists on immune responses in a syngeneic lung cancer model. Sigma(1) receptor agonists, including cocaine, up-regulated splenocyte IL-10 mRNA and protein production in vitro in a sigma receptor-dependent, pertussis toxin-sensitive manner. In vivo, sigma(1) receptor agonists promoted tumor growth and induced IL-10 at the tumor site. Increased tumor growth was prevented by administration of specific Abs to IL-10 or by administration of specific sigma(1) receptor antagonists. We report that sigma(1) receptor ligands, including cocaine, augment tumor growth through an IL-10 dependent mechanism.