Integrin-associated protein immunoglobulin domain is necessary for efficient vitronectin bead binding

Integrin-associated protein (IAP/CD47) is physically associated with the alpha v beta 3 vitronectin (Vn) receptor and a functionally and immunologically related integrin on neutrophils (PMN) and monocytes. Anti-IAP antibodies inhibit multiple phagocyte functions, including Arg- Gly-Asp (RGD)-initiated activation of phagocytosis, chemotaxis, and respiratory burst; PMN adhesion to entactin; and PMN transendothelial and transepithelial migration at a step subsequent to tight intercellular adhesion. Anti-IAP antibodies also inhibit binding of Vn- coated particles to many cells expressing alpha v beta 3. However, prior studies with anti-IAP did not directly address IAP function because they could not distinguish between IAP blockade and antibody- induced signaling effects on cells. To better determine the function of IAP, we have characterized and used an IAP-deficient human cell line. Despite expressing alpha v integrins, these cells do not bind Vn-coated particles unless transfected with IAP expression constructs. Increasing the level of alpha v beta 3 expression or increasing Vn density on the particle does not overcome the requirement for IAP. All known splice variants of IAP restore Vn particle binding equivalently. Indeed, the membrane-anchored IAP Ig variable domain suffices to mediate Vn particle binding in this system, while the multiply membrane-spanning and cytoplasmic domains are dispensable. In all cases, adhesion to a Vn- coated surface and fibronectin particle binding through alpha 5 beta 1 fibronectin receptors are independent of IAP expression. These data demonstrate that some alpha v integrin ligand-binding functions are IAP independent, whereas others require IAP, presumably through direct physical interaction between its Ig domain and the integrin.     

Expression of the 50-kDa integrin-associated protein on myeloid cells and erythrocytes.

Integrin-associated protein (IAP) is a 50-kDa intrinsic membrane protein that is involved in signal transduction during neutrophil activation by a variety of Arg-Gly-Asp-containing ligands. However, IAP does not itself directly bind these ligands, which are instead recognized by the leukocyte response integrin (LRI). In fact, IAP is more widely expressed than the LRI and is even on erythrocytes, which express no known integrins. This suggests that IAP may have additional functions besides signal transduction in association with the LRI. In this work we have quantitated IAP expression on several myeloid cells and cell lines, as well as erythrocytes, using a mAb which recognizes this protein. These data show that there are about 10,000 IgG-binding sites on each erythrocyte and 20 times that number on the myeloid cell lines U937 and HL60. Normal PMN and monocytes express about 25,000 IgG-binding sites. There are twice as many Fab'-binding sites on each cell, suggesting that each IgG binds via both Ag-combining sites. Binding data using several mAb to IAP suggest that IAP undergoes a temperature-dependent conformational change. Unlike some integrin receptors, IAP is not stored in a regulated secretory compartment in polymorphonuclear leukocytes (PMN). In addition, there is no evidence for internalization or shedding of IAP upon PMN activation. These data show that IAP is expressed at significant levels on myeloid cells and erythrocytes and that its expression is unaffected by the state of PMN activation.     

Adenocarcinoma cells exposed in vitro to Navelbine or Taxol increase Ep-CAM expression through a novel mechanism

Ep-CAM antigen expression was shown to vary by phase across the cell cycle. Following pretreatment of various adenocarcinoma cells in culture with clinically relevant concentrations of vinorelbine tartrate (Navelbine) or paclitaxel (Taxol), cell surface expression of Ep-CAM antigen increased by two- to ten-fold compared to that of untreated control cells and was associated with arrest of cell cycle progression and accumulation of cells in the S and G2/M phases. We demonstrated that increases in cell surface antigen expression resulted in improved biological effectiveness of the targeting antibody as measured in vitro by antibody-dependent cellular cytotoxicity and in vivo by enhanced antibody targeting to Ep-CAM-expressing xenografts in mice pretreated with Navelbine. No effect on cell cycle progression or Ep-CAM antigen expression was seen with human interferon-alpha and interferon-gamma, agents that increase gene expression of various tumor and normal antigens and may upregulate some antigens. Thus, the upregulation of cell surface Ep-CAM expression following pretreatment with G2/M blockers is through a novel mechanism involving residence time of the antigen on the cell surface. This significant increase in Ep-CAM expression appears to be tumor-specific since we saw no increase in antigen expression on normal epithelial cells. Studies to reveal relative internalization rates suggest that the increase in cell surface expression of Ep-CAM following pretreatment with G2/M blockers is a consequence of an inhibition of normal cycles of antigen endocytosis and expression on the cell surface. The present work provides a mechanism for the improved clinical efficacy of therapeutic antibodies used in combination with traditional cell cycle-specific chemotherapeutic drugs.     

A role of the B-oligomer moiety of islet-activating protein, pertussis toxin, in development of the biological effects on intact cells

Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein (Tamura, M., Nogimori, K., Murai, S., Yajima, M., Ito, K., Katada, T., Ui, M., and Ishii, S. (1982) Biochemistry 21, 5516-5522), the biggest subunit (Mr = 28,000, referred to as the A-protomer) of which catalyzes transfer of the ADP-ribose moiety of NAD to the membrane Mr = 41,000 protein. The pentamer, termed the B-oligomer, consisting of the residual subunits was the moiety of IAP that was responsible for binding to the cell surface, as revealed by competitive inhibition of the development of the IAP actions on intact rat C6 glioma cells and rat adipocytes. The binding of the B-oligomer to its receptor proteins was divalent via the constituent two dimers; it stimulated mitosis of lymphocytes and caused an insulin-like action to enhance glucose oxidation in adipocytes, just as did concanavalin A, presumably as a result of cross-linking or aggregation of the membrane proteins. The A-promoter displayed its biological action on adipocytes only when the B-oligomer had been bound to the cells. Thus, IAP is a typical A-B toxin in which the B-oligomer is first bound to the cell surface proteins to enable the A-protomer to reach to the site of its action within the cell. Diverse biological actions of pertussis toxin may be accounted for by the mitogenic action of the B-oligomer as well as ADP-ribosyltransferase activity of the A-promoter.     

HD39 (B3), a B lineage-restricted antigen whose cell surface expression is limited to resting and activated human B lymphocytes

The B cell-restricted antigen HD39, whose cell surface expression is limited to resting and activated human B lymphocytes, is described in this report. The monoclonal antibody HD39 detects a two-chain glycoprotein with apparent molecular weights of 130,000 and 140,000. During B cell ontogeny, HD39 is first expressed in the cytoplasm of bone marrow derived pre-B cells, then appears on the cell surface of sIgM+ B cells, and finally on the majority of sIgM+ sIgD+ resting B cells. After activation in vitro, the expression of HD39 on the cell surface first increases, and then the antigen is lost as cells begin to differentiate. HD39 is weakly expressed on very few non-T cell ALL and B cell CLL, on approximately 50% of B cell lymphomas, and not on Waldenstr?m's macroglobulinemias and myelomas. In contrast, it is strongly expressed on all hairy cell leukemias. Its limited cell surface expression in B cell ontogeny suggests that HD39 may be important in the events that regulate the activation of the human resting B lymphocytes.     

The pro-inflammatory cytokines, IL-1beta and TNF-alpha, inhibit intestinal alkaline phosphatase gene expression

High levels of the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), are present in the gut mucosa of patients suffering form various diseases, most notably inflammatory bowel diseases (IBD). Since the inflammatory milieu can cause important alterations in epithelial cell function, we examined the cytokine effects on the expression of the enterocyte differentiation marker, intestinal alkaline phosphatase (IAP), a protein that detoxifies bacterial lipopolysaccharides (LPS) and limits fat absorption. Sodium butyrate (NaBu), a short-chain fatty acid and histone deacetylase (HDAC) inhibitor, was used to induce IAP expression in HT-29 cells and the cells were also treated +/- the cytokines. Northern blots confirmed IAP induction by NaBu, however, pretreatment (6 h) with either cytokine showed a dose-dependent inhibition of IAP expression. IAP Western analyses and alkaline phosphatase enzyme assays corroborated the Northern data and confirmed that the cytokines inhibit IAP induction. Transient transfections with a reporter plasmid carrying the human IAP promoter showed significant inhibition of NaBu-induced IAP gene activation by the cytokines (100 and 60% inhibition with IL-1beta and TNF-alpha, respectively). Western analyses showed that NaBu induced H4 and H3 histone acetylation, and pretreatment with IL-1beta or TNF-alpha did not change this global acetylation pattern. In contrast, chromatin immunoprecipitation showed that local histone acetylation of the IAP promoter region was specifically inhibited by either cytokine. We conclude that IL-1beta and TNF-alpha inhibit NaBu-induced IAP gene expression, likely by blocking the histone acetylation within its promoter. Cytokine-mediated IAP gene silencing may have important implications for gut epithelial function in the setting of intestinal inflammatory conditions.     

Phenotyping human leukemic T-Cell lines: Enzyme markers; surface antigens,and cytogenetics

We have compared phenotypic markers for a series of established human leukemic T-cell lines collected from different laboratories. Cell lines were tested first for genetic markers using polymorphic enzymes and then for expression of T lymphoid cell surface differentiation antigens using monoclonal antibodies. Chromosomal analysis was used as an additional method for identification of selected cell lines. On the basis of enzyme markers, it was possible to assign each of the cell lines examined to one of nine different groups. With two exceptions, surface antigen phenotypes for each of 12 cell lines were clearly distinctive. Thus, some groups of cell lines indistinguishable by enzyme markers could be further subdivided by surface antigen phenotyping. However, significant quantitative variation in expression of individual antigens was observed. In addition, surface antigen expression was not uniform in different subcultures of one cell line studied in detail. These results indicate that leukemic T-cell lines cannot be used generally as simple models of surface antigen expression in normal T-cell differentiation.     

Expression of CD47/integrin-associated protein induces death of cultured cerebral cortical neurons

The death and survival of neuronal cells are regulated by various signaling pathways during development of the brain and in neuronal diseases. Previously, we demonstrated that the neuronal adhesion molecule brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1 (BIT/SHPS-1) is involved in brain-derived neurotrophic factor (BDNF)-promoted neuronal cell survival. Here, we report the apoptosis-inducing effect of CD47/integrin-associated protein (IAP), the heterophilic binding partner of BIT/SHPS-1, on neuronal cells. We generated a recombinant adenovirus vector expressing a neuronal form of CD47/IAP, and found that the expression of CD47/IAP by infection with CD47/IAP adenovirus induced the death of cultured cerebral cortical neurons. The numbers of TdT-mediated biotin-dUTP nick-end labelling (TUNEL)-positive neurons and of cells displaying apoptotic nuclei increased by expression of CD47/IAP. Neuronal cell death was prevented by the addition of the broad-spectrum caspase inhibitor Z-VAD-fmk. Furthermore, we observed that co-expression of CD47/IAP with BIT/SHPS-1 enhanced neuronal cell death, and that BDNF prevented it. These results suggest that CD47/IAP is involved in a novel pathway which regulates caspase-dependent apoptosis of cultured cerebral cortical neurons. CD47/IAP-induced death of cultured cortical neurons may be regulated by the interaction of CD47/IAP with BIT/SHPS-1 and by BDNF.     

Adenovirus-Mediated Gene Transfer of Inhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons

Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 m M potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 m M potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N -acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, not at later time points suggesting that IAPS delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 μ M or 1 m M glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

Comparative analysis of CEA and SCC serum markers with IAP in human lung cancer

Serum levels of carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC) and immunosuppressive acidic protein (IAP) were measured in 37 patients with lung cancers, in 24 with non-cancer pulmonary diseases and in 24 normal controls We evaluated the sensitivity, specificity and accuracy of these three markers alone and combined. The highest specificity was observed for SCC (83.3%) and the highest sensitivity for IAP (94.6%). The best accuracy was obtained with the combined determination of CEA and SCC. In cancer and non-cancer pulmonary diseases the best correlation was observed between CEA and SCC (r = 0.30 in cancer and r = 0.45 in non-cancer pulmonary diseases). Although the IAP test is not specific in the detection of lung cancer, its use may be helpful in monitoring the acute phase reactions that occur very frequently in this malignancy.     

OTUB1 modulates c‐IAP1 stability to regulate signalling pathways

The cellular inhibitor of apoptosis (c‐IAP) proteins are E3 ubiquitin ligases that are critical regulators of tumour necrosis factor (TNF) receptor (TNFR)‐mediated signalling. Through their E3 ligase activity c‐IAP proteins promote ubiquitination of receptor‐interaction protein 1 (RIP1), NF‐κB‐inducing kinase (NIK) and themselves, and regulate the assembly of TNFR signalling complexes. Consequently, in the absence of c‐IAP proteins, TNFR‐mediated activation of NF‐κB and MAPK pathways and the induction of gene expression are severely reduced. Here, we describe the identification of OTUB1 as a c‐IAP‐associated deubiquitinating enzyme that regulates c‐IAP1 stability. OTUB1 disassembles K48‐linked polyubiquitin chains from c‐IAP1 in vitro and in vivo within the TWEAK receptor‐signalling complex. Downregulation of OTUB1 promotes TWEAK‐ and IAP antagonist‐stimulated caspase activation and cell death, and enhances c‐IAP1 degradation. Furthermore, knockdown of OTUB1 reduces TWEAK‐induced activation of canonical NF‐κB and MAPK signalling pathways and modulates TWEAK‐induced gene expression. Finally, suppression of OTUB1 expression in zebrafish destabilizes c‐IAP (Birc2) protein levels and disrupts fish vasculature. These results suggest that OTUB1 regulates NF‐κB and MAPK signalling pathways and TNF‐dependent cell death by modulating c‐IAP1 stability.     

Regulation of intracisternal A particles in mouse teratocarcinoma cells: involvement of DNA methylation in transcriptional control

The methylation state of Intracisternal A Particle (IAP) genes in mouse teratocarcinoma cell lines has been investigated as an approach to study the regulation of the expression of these particles. Treatment of the cells with the methylation inhibitor 5-azacytidine induces the production of the particles in all the cells; the induction is particularly striking in an embryonal carcinoma cell line which is normally devoid of IAPs. The induction is accompanied by decrease in DNA methylation as demonstrated by using the methylation sensitive isoschizomer enzymes MspI and HpaII. Hypomethylation of the IAP genes correlates with accumulation of IAP, specific polyadenylated RNA reinforcing the hypothesis that methylation plays an important role in the control of IAP expression.     

Regulation of intracisternal A particles in mouse teratocarcinoma cells: involvement of DNA methylation in transcriptional control

The methylation state of Intracisternal A Particle (IAP) genes in mouse teratocarcinoma cell lines has been investigated as an approach to study the regulation of the expression of these particles. Treatment of the cells with the methylation inhibitor 5-azacytidine induces the production of the particles in all the cells; the induction is particularly striking in an embryonal carcinoma cell line which is normally devoid of IAPs. The induction is accompanied by decrease in DNA methylation as demonstrated by using the methylation sensitive isoschizomer enzymes MspI and HpaII. Hypomethylation of the IAP genes correlates with accumulation of IAP, specific polyadenylated RNA reinforcing the hypothesis that methylation plays an important role in the control of IAP expression.