Murine pregnancy-specific glycoprotein 23 induces the proangiogenic factors transforming-growth factor beta 1 and vascular endothelial growth factor a in cell types involved in vascular remodeling in pregnancy

Haemochorial placentation is a unique physiological process in which the fetal trophoblast cells remodel the maternal decidual spiral arteries to establish the fetoplacental blood supply. Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen family. PSGs are produced by the placenta of rodents and primates and are secreted into the bloodstream. PSG23 is one of 17 members of the murine PSG family (designated PSG16 to PSG32). Previous studies determined that PSGs have immunoregulatory functions due to their ability to modulate macrophage cytokine secretion. Here we show that recombinant PSG23 induces transforming growth factor (TGF) beta1, TGFB1, and vascular endothelial growth factor A (VEGFA) in primary murine macrophages and the macrophage cell line RAW 264.7 cells. In addition, we identified new cell types that responded to PSG23 treatment. Dendritic cells, endothelial cells, and trophoblasts, which are involved in maternal vasculature remodeling during pregnancy, secreted TGFB1 and VEGFA in response to PSG23. PSG23 showed cross-reactivity with human cells, including human monocytes and the trophoblast cell line, HTR-8/SVneo cells. We analyzed the binding of PSG23 to the tetraspanin CD9, the receptor for PSG17, and found that CD9 is not essential for PSG23 binding and activity in macrophages. Overall these studies show that PSGs can modulate the secretion of important proangiogenic factors, TGFB1 and VEGFA, by different cell types involved in the development of the placenta.     

Early expression of pregnancy-specific glycoprotein 22 (PSG22) by trophoblast cells modulates angiogenesis in mice

Mouse and human pregnancy-specific glycoproteins (PSG) are known to exert immunomodulatory functions during pregnancy by inducing maternal leukocytes to secrete anti-inflammatory cytokines that promote a tolerogenic decidual microenvironment. Many such anti-inflammatory mediators also function as proangiogenic factors, which, along with the reported association of murine PSG with the uterine vasculature, suggest that PSG may contribute to the vascular adaptations necessary for successful implantation and placental development. We observed that PSG22 is strongly expressed around the embryonic crypt on Gestation Day 5.5, indicating that trophoblast giant cells are the main source of PSG22 during the early stages of pregnancy. PSG22 treatment up-regulated the secretion of transforming growth factor beta 1 and vascular endothelial growth factor A (VEGFA) in murine macrophages, uterine dendritic cells, and natural killer cells. A possible role of PSGs in uteroplacental angiogenesis is further supported by the finding that incubation of endothelial cells with PSG22 resulted in the formation of tubes in the presence and absence of VEGFA. We determined that PSG22, like human PSG1 and murine PSG17 and PSG23, binds to the heparan sulfate chains in syndecans. Therefore, our findings indicate that despite the independent evolution and expansion of human and rodent PSG, members in both families have conserved functions that include their ability to induce anti-inflammatory cytokines and proangiogenic factors as well as to induce the formation of capillary structures by endothelial cells. In summary, our results indicate that PSG22, the most abundant PSG expressed during mouse early pregnancy, is likely a major contributor to the establishment of a successful pregnancy.     

Identification of a new carcinoembryonic antigen (CEA) family member in human fetal liver--cloning and sequence determination of pregnancy-specific glycoprotein 7

The carcinoembryonic antigen gene family consists of the CEA- and the Pregnancy-Specific Glycoprotein- (PSG) subfamilies. Human fetal liver express several PSGs. Here we report cloning and sequencing of a new PSG subfamily member, PSG7. It is the fifth type of PSG found in fetal liver. PSG7 has the N-A1-A2-B2-C domain arrangement. Unlike other PSGs the N-terminal of PSG7 is unblocked. PSG7 has a cysteine in the C-terminal domain, which may allow dimerization. Variability analysis according to Wu and Kabat reveals that the region in the N-domain corresponding to complementarity determining region 3 of immunoglobulin is different between PSG subfamily members. Many members, including PSG7, contain the RGD sequence in this region. The CD2 region as well as two other short sequences (in N and A1 domains respectively) also show some variability. The function of PSGs is probably linked to the N-domain and the CDR2- and CD3-like regions are most likely responsible for ligand binding.     

Importance of basic residues and quaternary structure in the function of MIP-1 beta: CCR5 binding and cell surface sugar interactions

Chemokines direct immune cells toward sites of infection by establishing a gradient across the extracellular matrix of the tissue. This gradient is thought to be stabilized by ligation of chemokines to sulfated polysaccharides known as glycosaminoglycans (GAGs) that are found on the surface of endothelial and other cells as well as in the tissue matrix. GAGs interact with chemokines and in some cases cause them to aggregate. The interaction between cell surface GAGs and chemokines has also been postulated to play a role in the anti-HIV activity of some chemokines, including MIP-1beta. Since many proteins interact with GAGs by utilizing basic residues, we mutated R18, K45, R46, and K48 in MIP-1beta to investigate the role of these residues in GAG binding and CCR5 function. We find that no single amino acid substitution alone has a dramatic effect on heparin binding, although change at R46 has a moderate effect. However, binding to heparin is completely abrogated in a mutant (K45A/R46A/K48A) in which the entire "40's loop" has been neutralized. A functional study of these mutants reveals that the charged residues in this 40's loop, particularly K48 and R46, are critical mediators of MIP-1beta binding to its receptor CCR5. However, despite the partially overlapping function of the residues in the 40's loop in binding to both CCR5 and heparin, the presence of cell surface sugars does not appear to be necessary for the ability of MIP-1beta to function on its receptor CCR5, as enzymatic removal of GAGs from cells results in little effect on MIP-1beta activity. Because the means by which the chemokine gradient transmits information to the recruited cells is not well defined, we also mutated the basic residues in MIP(9), a truncated form of MIP-1beta that is impaired in its ability to dimerize, to probe whether the quaternary structure of this chemokine influences its ability to bind heparin. None of the truncated variants bound as well as the full-length proteins containing the same mutation, suggesting that the MIP-1beta dimer participates in heparin binding.     

Contribution of the putative heparan sulfate-binding motif BBXB of RANTES to transendothelial migration

The chemokines are a family of small chemoattractant proteins that have a range of functions, including activation and promotion of vectorial migration of leukocytes. Regulation on activation, normal T cell expressed and secreted (RANTES; CCL5), a member of the CC-chemokine subfamily, has been implicated in a variety of immune responses. In addition to the interaction of CC-chemokines with their cognate cell-surface receptors, it is known that they also bind to glycosaminoglycans (GAGs), including heparan sulfate. This potential for binding to GAG components of proteoglycans on the cell surface or within the extracellular matrix might allow formation of the stable chemokine concentration gradients necessary for leukocyte chemotaxis. In this study, we created a panel of mutant RANTES molecules containing neutral amino acid substitutions within putative, basic GAG-binding domains. Despite showing reduced binding to GAGs, it was found that each mutant containing a single amino acid substitution induced a similar leukocyte chemotactic response within a concentration gradient generated by free solute diffusion. However, we found that the mutant K45A had a significantly reduced potential to stimulate chemotaxis across a monolayer of microvascular endothelial cells. Significantly, this mutant bound to the CCR5 receptor and showed a potential to mobilize Ca(2+) with an affinity similar to the wild-type protein. These results show that the interaction between RANTES and GAGs is not necessary for specific receptor engagement, signal transduction, or leukocyte migration. However, this interaction is required for the induction of efficient chemotaxis through the extracellular matrix between confluent endothelial cells.     

Glycosaminoglycans (GAGs) suppress the toxicity of HypF-N prefibrillar aggregates

A group of diverse human pathologies is associated with proteins unable to retain their native state and convert into prefibrillar and fibrillar amyloid aggregates that are then deposited in the extracellular space. Glycosaminoglycans (GAGs) have been found to physically associate with these deposits and also to promote their formation in vitro. However, the effect of GAGs on the toxicity of these aggregates has been investigated in only one protein system, the amyloid β peptide associated with Alzheimer's disease. In this study, we investigate whether GAGs affect the toxicity of the N-terminal domain of Escherichia coli HypF (HypF-N) oligomers on Chinese hamster ovarian cells and the mechanism by which such suppression is mediated. The results show that heparin and other GAGs inhibit the toxicity observed by HypF-N oligomers in a dose-dependent manner. GAGs were not found to bind preformed HypF-N oligomers, change their morphological and structural characteristics or disaggregate them. Nevertheless, they were found to bind to the cells' surface and prevent the interaction of the oligomers with the cells. Overall, the results indicate that GAGs have a generic ability to inhibit the toxicity of aberrant protein oligomers and that such toxicity suppression can occur through different mechanisms, such as through binding to the oligomers with consequent loss of interaction of the oligomers to the GAGs present on the cell surface, as proposed previously for amyloid β aggregates, or through mechanisms independent of direct GAG-oligomer binding, as shown here for HypF-N aggregates.     

Pregnancy-specific glycoprotein expression in normal gastrointestinal tract and in tumors detected with novel monoclonal antibodies

Pregnancy-specific glycoproteins (PSGs) are immunoglobulin superfamily members related to the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family and are encoded by 10 genes in the human. They are secreted at high levels by placental syncytiotrophoblast into maternal blood during pregnancy, and are implicated in immunoregulation, thromboregulation, and angiogenesis. To determine whether PSGs are expressed in tumors, we characterized 16 novel monoclonal antibodies to human PSG1 and used 2 that do not cross-react with CEACAMs to study PSG expression in tumors and in the gastrointestinal (GI) tract using tissue arrays and immunohistochemistry. Staining was frequently observed in primary squamous cell carcinomas and colonic adenocarcinomas and was correlated with the degree of tumor differentiation, being largely absent from metastatic samples. Staining was also observed in normal oesophageal and colonic epithelium. PSG expression in the human and mouse GI tract was confirmed using quantitative RT-PCR. However, mRNA expression was several orders of magnitude lower in the GI tract compared to placenta. Our results identify a non-placental site of PSG expression in the gut and associated tumors, with implications for determining whether PSGs have a role in tumor progression, and utility as tumor biomarkers.     

Binding of interferon gamma by glycosaminoglycans: a strategy for localization and/or inhibition of its activity.

Glycosaminoglycans (GAGs) are a group of negatively charged molecules present in many tissues as components of the extracellular matrix, basement and cellular membranes. This work analysed the ability of this group of substances to interact with human interferon gamma and the effect of those interactions on its biologic activity. A variety of GAGs (heparin, heparan sulfate, chondroitin sulfate and hyaluronic acid), and a related sulfated polysaccharide (dextran sulfate), were found to interact with IFN-gamma as determined by inhibition of the binding of [125I]IFN-gamma to COLO-205 cells and binding to wells coated with GAGs. These interactions were inhibited by synthetic peptides mimicking the sequences of the basic amino acid cluster located at the C-terminal end of mouse and human IFN-gamma, or by poly-L-lysine, suggesting that ionic interactions between the positively-charged C-terminus and negatively charged groups in GAGs were involved. IFN-gamma molecules bound to plate-immobilized or endothelial cell surface GAGs retained biological activity, since they could induce major histocompatibility complex (MHC) class II expression on COLO-205 cells, suggesting that cell surface GAGs might be able to present IFN-gamma to its receptors. These results suggest important regulatory roles for GAGs on the activity of IFN-gamma in vivo.     

Structural and functional analysis of the RANTES-glycosaminoglycans interactions

Chemokines mediate their biological activity through activation of G protein coupled receptors, but most chemokines, including RANTES, are also able to bind glycosaminoglycans (GAGs). Here, we have investigated, by site-directed mutagenesis and chemical acetylation, the role of RANTES basic residues in the interaction with GAGs using surface plasmon resonance kinetic analysis. Our results indicate that (i) RANTES exhibited selectivity in GAGs binding with highest affinity (K(d) = 32.1 nM) for heparin, (ii) RANTES uses the side chains of residues R44, K45, and R47 for heparin binding, and blocking these residues in combination abolished heparin binding. The biological relevance of RANTES-GAGs interaction was investigated in CHO-K1 cells expressing CCR5, CCR1, or CCR3 and the various GAGs that bind RANTES. Our results indicate that the heparin binding site, defined as the 40s loop, is only marginally involved in CCR5 binding and activation, but largely overlaps the CCR1 and CCR3 binding and activation domain in RANTES. In addition, enzymatic removal of cell surface GAGs by glycosidases did not affect CCR5 binding and Ca(2+) response. Furthermore, addition of soluble GAGs inhibited both CCR5 binding and functional response, with a rank of potency similar to that found in surface plasmon resonance experiments. Thus, cell surface GAGs is not a prerequisite for receptor binding or signaling, but soluble GAGs can inhibit the binding and the functional response of RANTES to CCR5 expressing cells. However, the marked selectivity of RANTES for different GAGs may serve, in vivo, to control the concentration of specific chemokines in inflammatory situations and locations.     

Formation and dissociation of proteasome storage granules are regulated by cytosolic pH

The 26S proteasome is the major protein degradation machinery of the cell and is regulated at many levels. One mode of regulation involves accumulation of proteasomes in proteasome storage granules (PSGs) upon glucose depletion. Using a systematic robotic screening approach in yeast, we identify trans-acting proteins that regulate the accumulation of proteasomes in PSGs. Our dataset was enriched for subunits of the vacuolar adenosine triphosphatase (V-ATPase) complex, a proton pump required for vacuole acidification. We show that the impaired ability of V-ATPase mutants to properly govern intracellular pH affects the kinetics of PSG formation. We further show that formation of other protein aggregates upon carbon depletion also is triggered in mutants with impaired activity of the plasma membrane proton pump and the V-ATPase complex. We thus identify cytosolic pH as a specific cellular signal involved both in the glucose sensing that mediates PSG formation and in a more general mechanism for signaling carbon source exhaustion.     

Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses.

Chemokines selectively recruit and activate a variety of cells during inflammation. Interactions between cell surface glycosaminoglycans (GAGs) and chemokines drive the formation of haptotactic or immobilized gradients of chemokines at the site of inflammation, directing this recruitment. Chemokines bind to glycosaminoglycans on human umbilical vein endothelial cells (HUVECs) with affinities in the micromolar range: RANTES > MCP-1 > IL-8 > MIP-1alpha. This binding can be competed with by soluble glycosaminoglycans: heparin, heparin sulfate, chondroitin sulfate, and dermatan sulfate. RANTES binding showed the widest discrimination between glycosaminoglycans (700-fold), whereas MIP-1alpha was the least selective. Almost identical results were obtained in an assay using heparin sulfate beads as the source of immobilized glycosaminoglycan. The binding of chemokines to glycosaminoglycan fragments has a strong length dependence, and optimally requires both N- and O-sulfation. Isothermal titration calorimetry data confirm these results; IL-8 binds heparin fragments with a K(d) of 0.39-2.63 microM, and requires five saccharide units to bind each monomer of chemokine. In membranes from cells expressing the G-protein-coupled chemokine receptors CXCR1, CXCR2, and CCR1, soluble GAGs inhibit the binding of chemokine ligands to their receptors. Consistent with this, heparin and heparin sulfate could inhibit IL-8-induced neutrophil calcium flux. Chemokines can therefore form complexes with both cell surface and soluble GAGs; these interactions have different functions. Soluble GAG chemokines complexes are unable to bind the receptor, resulting in a block of the biological activity. Previously, we have shown that cell surface GAGs present chemokines to the G-protein-coupled receptors, by increasing the local concentration of protein. A model is presented which brings together all of these data. The selectivity in the chemokine-GAG interaction suggests selective disruption of the haptotactic gradient may be an achievable therapeutic approach in inflammatory disease.     

Pregnancy-Specific Glycoproteins Bind Integrin αIIbβ3 and Inhibit the Platelet—Fibrinogen Interaction

Pregnancy-specific glycoproteins (PSGs) are immunoglobulin superfamily members encoded by multigene families in rodents and primates. In human pregnancy, PSGs are secreted by the syncytiotrophoblast, a fetal tissue, and reach a concentration of up to 400 ug/ml in the maternal bloodstream at term. Human and mouse PSGs induce release of anti-inflammatory cytokines such as IL-10 and TGFβ1 from monocytes, macrophages, and other cell types, suggesting an immunoregulatory function. RGD tri-peptide motifs in the majority of human PSGs suggest that they may function like snake venom disintegrins, which bind integrins and inhibit interactions with ligands. We noted that human PSG1 has a KGD, rather than an RGD motif. The presence of a KGD in barbourin, a platelet integrin αIIbβ3 antagonist found in snake venom, suggested that PSG1 may be a selective αIIbβ3 ligand. Here we show that human PSG1 binds αIIbβ3 and inhibits the platelet – fibrinogen interaction. Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit αIIbβ3 function. Human PSG9 and mouse Psg23 are also inhibitory suggesting conservation of this function across primate and rodent PSG families. Our results suggest that in species with haemochorial placentation, in which maternal blood is in direct contact with fetal trophoblast, the high expression level of PSGs reflects a requirement to antagonise abundant (3 mg/ml) fibrinogen in the maternal circulation, which may be necessary to prevent platelet aggregation and thrombosis in the prothrombotic maternal environment of pregnancy.     

Role of scavenger receptors in the binding and internalization of heat shock protein 70

Extracellular heat shock protein 70 (Hsp70) exerts profound effects both in mediating tumor rejection by Hsp70-based vaccines and in autoimmunity. Further progress in this area, however, awaits the identification of the cell surface receptors for extracellular Hsp70 that mediate its immune functions. We have examined a wide range of candidate Hsp70 receptors and find significant binding through two main families of cell surface proteins, including 1) the scavenger receptor (SR) family and 2) C-type lectins of the NK family. In addition, given that the anticancer effects of Hsp70 vaccines have been shown to involve uptake of Ags by APC exposed to Hsp70-tumor Ag complexes, we have examined the ability of the receptors identified here to internalize Hsp70-peptide complexes. Our findings indicate that three members of the SR family (lectin-like oxidized low density lipoprotein receptor 1; fasciclin, epidermal growth factor-like, laminin-type epidermal growth factor-like, and link domain-containing scavenger receptor-1; and SR expressed by endothelial cells-1) are able to bind Hsp70-peptide complexes and mediate its efficient internalization. Indeed, each of the SR was able to mediate efficient uptake of Hsp70 when transfected into Chinese hamster ovary cells previously null for uptake. Curiously, Hsp70 internalization occurs independently of the intracellular domains of the SR, and Hsp70 uptake could be detected when the entire intracellular domain of lectin-like oxidized low density lipoprotein receptor 1 or SR expressed by endothelial cells-1 was truncated. The existence of a wide repertoire of cell surface Hsp70-binding structures may permit intracellular responses to extracellular Hsp70 that are cell specific and discriminate between Hsp70 family members.     

Glypican-1 is a VEGF165 binding proteoglycan that acts as an extracellular chaperone for VEGF165

Glypican-1 is a member of a family of glycosylphosphatidylinositol anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for endothelial cells and a potent angiogenic factor in vivo. Heparin binds to VEGF165 and enhances its binding to VEGF receptors. However, native HSPGs that bind VEGF165 and modulate its receptor binding have not been identified. Among the glypicans, glypican-1 is the only member that is expressed in the vascular system. We have therefore examined whether glypican-1 can interact with VEGF165. Glypican-1 from rat myoblasts binds specifically to VEGF165 but not to VEGF121. The binding has an apparent dissociation constant of 3 x 10(-10) M. The binding of glypican-1 to VEGF165 is mediated by the heparan sulfate chains of glypican-1, because heparinase treatment abolishes this interaction. Only an excess of heparin or heparan sulfates but not other types of glycosaminoglycans inhibited this interaction. VEGF165 interacts specifically not only with rat myoblast glypican-1 but also with human endothelial cell-derived glypican-1. The binding of 125I-VEGF165 to heparinase-treated human vascular endothelial cells is reduced following heparinase treatment, and addition of glypican-1 restores the binding. Glypican-1 also potentiates the binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1. Furthermore, we show that glypican-1 acts as an extracellular chaperone that can restore the receptor binding ability of VEGF165, which has been damaged by oxidation. Taken together, these results suggest that glypican-1 may play an important role in the control of angiogenesis by regulating the activity of VEGF165, a regulation that may be critical under conditions such as wound repair, in which oxidizing agents that can impair the activity of VEGF are produced, and in situations were the concentrations of active VEGF are limiting.     

Homeostatic modulation of cell surface KDR and Flt1 expression and expression of the vascular endothelial cell growth factor (VEGF) receptor mRNAs by VEGF

Vascular endothelial cell growth factor (VEGF) is a potent angiogenic factor expressed during embryonic development, during wound healing, and in pathologies dependent on neovascularization, including cancer. Regulation of the receptor tyrosine kinases, KDR and Flt-1, to which VEGF binds on endothelial cells is incompletely understood. Chronic incubation with tumor-conditioned medium or VEGF diminished (125)I-VEGF binding to human umbilical vein endothelial cells, incorporation of (125)I-VEGF into covalent complexes with KDR and Flt1, and immunoreactive KDR in cell lysates. Receptor down-regulation desensitized VEGF activation of mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) and p38 mitogen-activated protein kinase. Preincubation with VEGF or tumor-conditioned medium down-regulated cell surface receptor expression but up-regulated KDR and Flt-1 mRNAs, an effect abrogated by a neutralizing VEGF antibody. Removal of VEGF from the medium led to recovery of (125)I-VEGF binding and resensitization of human umbilical vein endothelial cells. Recovery of receptor expression was inhibited by cycloheximide, indicating that augmented VEGF receptor mRNAs, and not receptor recycling from a cytoplasmic pool, restored responsiveness. As the VEGF receptors promote endothelial cell survival, proliferation, and other events necessary for angiogenesis, the noncoordinate regulation of VEGF receptor proteins and mRNAs suggests that human umbilical vein endothelial cells are protected against inappropriate or prolonged loss of VEGF receptors by a homeostatic mechanism important to endothelial cell function.     

The WASP-binding protein WIRE has a role in the regulation of the actin filament system downstream of the platelet-derived growth factor receptor

To facilitate the study of human endothelial cells we have used a replication defective retrovirus encoding the catalytic subunit of telomerase (hTERT) to derive populations of telomerase-immortalized human microvascular endothelial (TIME) cells. Whereas parental HMVECs became senescent on average within 35-45 population doublings (PDs), TIME cells have continued to proliferate for at least 200 PDs. TIME cells express readily detectable telomerase activity but display only a modest increase in telomere length. Karyotypic analysis reveals the cells to have a normal complement of human chromosomes with no evidence of gross genetic abnormalities. Furthermore, TIME cells retain many of the characteristics of the primary endothelial cells from which they were derived. For example, they express a panel of characteristic endothelial cell surface marker proteins such as CD31/PECAM-1 and alpha(v)beta3-integrin. In addition, TIME cells express receptors for low-density lipoprotein (LDL) receptor as they are competent for receptor-mediated endocytosis of fluorescent acetylated LDL. Importantly, when plated on matrigel, TIME cells undergo tubule formation. Moreover, when cocultured in the presence of human glioma cells, but not primary human astrocytes, TIME cells are induced to form stable tubules. Detachment of TIME cells from extracellular matrix leads to a form of programmed cell death known as anoikis. Conditional activation of the protein kinase Akt (Akt:ER*) significantly inhibited the onset of TIME cell anoikis under these conditions. We believe that the ability of hTERT to immortalize primary human endothelial cells, and the fact that such cells retain the endothelial characteristics of the cells from which they were derived, will greatly facilitate the analysis of human endothelial cell biology in vitro.