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.     

Pregnancy-specific glycoprotein 1 induces endothelial tubulogenesis through interaction with cell surface proteoglycans

Pregnancy-specific β1 glycoproteins (PSGs) are the most abundant fetal proteins in the maternal bloodstream in late pregnancy. They are secreted by the syncytiotrophoblast and are detected around day 14 postfertilization. There are 11 human PSG genes, which encode a family of proteins exhibiting significant conservation at the amino acid level. We and others have proposed that PSGs have an immune modulatory function. In addition, we recently postulated that they are proangiogenic due to their ability to induce the secretion of VEGF-A and the formation of tubes by endothelial cells. The cellular receptor(s) for human PSGs remain unknown. Therefore, we conducted these studies to identify the receptor for PSG1, the highest expressed member of the family. We show that removal of cell surface glycosaminoglycans (GAGs) by enzymatic or chemical treatment of cells or competition with heparin completely inhibited binding of PSG1. In addition, PSG1 did not bind to cells lacking heparan or chondroitin sulfate on their surface, and binding was restored upon transfection with all four syndecans and glypican-1. Importantly, the presence of GAGs on the surface of endothelial cells was required for the ability of PSG1 to induce tube formation. This finding indicates that the PSG1-GAG interaction mediates at least some of the PSG1 proposed functions.     

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.     

Generation of hybrid transgenic silkworms that express Bombyx mori prolyl-hydroxylase alpha-subunits and human collagens in posterior silk glands: Production of cocoons that contained collagens with hydroxylated proline residues

Prolyl 4-hydroxylase (P4H) is a heterotetramer enzyme consisting of alpha-subunits (P4Halpha) and beta-subunits (P4Hbeta), and is required for collagen biosynthesis. Previously, we generated transgenic silkworms that produced human type III collagen fragments (mini-collagens) in the posterior silk gland (PSG). However, prolyl 4-hydroxylation did not occur on the mini-collagens, because in spite of an abundant expression of P4Hbeta in PSGs, P4Halpha expression was quite low there, thus resulting in an insufficient activity of P4H. In this study we aimed at generating hybrid transgenic silkworms whose PSGs are capable of producing mini-collagens and enough P4H for their prolyl 4-hydroxylation. Isolated PSGs were bombarded with fibroin L-chain gene promoter-driven vectors containing Bombyx mori P4Halpha (BmP4Halpha) cDNAs and were transplanted into the hemolymphatic cavity. The P4H activity in the PSG cells significantly increased, indicating that the expressed BmP4Halpha formed active tetramers with endogenous BmP4Hbeta. Using germ-line transgenesis technology, silkworms were generated that synthesized BmP4Halpha in PSG cells. The P4H activity in the transgenic silkworms was 130-fold higher than that of wild-type counterparts. Finally, we generated hybrid transgenic silkworms that expressed cDNAs of both BmP4Halpha and mini-collagen in PSG cells. They spun cocoons that contained mini-collagens whose appropriate proline residues had been adequately hydroxylated.     

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.     

Induction of scattering and cellular invasion by trefoil peptides in src- and RhoA-transformed kidney and colonic epithelial cells.

Trefoil factors (TFFs) are protease-resistant peptides that promote epithelial cell migration and mucosal restitution during inflammatory conditions and wound healing in the gastrointestinal tract. To date, the molecular mechanism of TFFs action and their possible role in tumor progression are unclear. In the present study, we observed that premalignant human colonic PC/AA/C1 and canine kidney MDCK epithelial cells are not competent to invade collagen gels in response to exogenously added TFFs (pS2, spasmolytic polypeptide, and intestinal trefoil factor). In contrast, activated src and RhoA exert permissive induction of invasion by the TFFs that produce similar parallel dose-response curves in src-transformed MDCKts.src and PCmsrc cells (EC50=20-40 nM). Cell scattering is also induced by TFFs in MDCKts.src cells. Stable expression of the pS2 cDNA promotes constitutive invasiveness in MDCKts.src-pS2 cells and human colonic HCT8/S11-pS2 cells established from a sporadic tumor. Furthermore, we found that TFF-mediated cellular invasion is dependent of several signaling pathways implicated in cell transformation and survival, including phosphoinositide PI3'-kinase, phospholipase C, protein kinase C, and the rapamycin target TOR. Constitutive and intense expression of pS2 was revealed by Western blot analyses and immunohistochemistry in human colorectal tumors and their adjacent control mucosa during the neoplastic progression, from the adenoma to the liver metastases. Our studies indicated that TFFs can be involved in cell scattering and tumor invasion via autocrine loops and may serve as potential targets in the control of colon cancer progression.     

Widespread Divergence of the CEACAM/PSG Genes in Vertebrates and Humans Suggests Sensitivity to Selection

In mammals, carcinoembryonic antigen cell adhesion molecules (CEACAMs) and pregnancy-specific glycoproteins (PSGs) play important roles in the regulation of pathogen transmission, tumorigenesis, insulin signaling turnover, and fetal–maternal interactions. However, how these genes evolved and to what extent they diverged in humans remain to be investigated specifically. Based on syntenic mapping of chordate genomes, we reveal that diverging homologs with a prototypic CEACAM architecture–including an extracellular domain with immunoglobulin variable and constant domain-like regions, and an intracellular domain containing ITAM motif–are present from cartilaginous fish to humans, but are absent in sea lamprey, cephalochordate or urochordate. Interestingly, the CEACAM/PSG gene inventory underwent radical divergence in various vertebrate lineages: from zero in avian species to dozens in therian mammals. In addition, analyses of genetic variations in human populations showed the presence of various types of copy number variations (CNVs) at the CEACAM/PSG locus. These copy number polymorphisms have 3–80% frequency in select populations, and encompass single to more than six PSG genes. Furthermore, we found that CEACAM/PSG genes contain a significantly higher density of nonsynonymous single nucleotide polymorphism (SNP) compared to the chromosome average, and many CEACAM/PSG SNPs exhibit high population differentiation. Taken together, our study suggested that CEACAM/PSG genes have had a more dynamic evolutionary history in vertebrates than previously thought. Given that CEACAM/PSGs play important roles in maternal–fetal interaction and pathogen recognition, these data have laid the groundwork for future analysis of adaptive CEACAM/PSG genotype-phenotypic relationships in normal and complicated pregnancies as well as other etiologies.     

Conservation of pregnancy-specific glycoprotein (PSG) N domains following independent expansions of the gene families in rodents and primates

Background  

Rodent and primate pregnancy-specific glycoprotein (PSG) gene families have expanded independently from a common ancestor and are expressed virtually exclusively in placental trophoblasts. However, within each species, it is unknown whether multiple paralogs have been selected for diversification of function, or for increased dosage of monofunctional PSG. We analysed the evolution of the mouse PSG sequences, and compared them to rat, human and baboon PSGs to attempt to understand the evolution of this complex gene family.     

Characterization of a monoclonal antibody, OVB1, which binds to a unique determinant in human ovarian carcinomas and myeloid cells

A monoclonal antibody, OVB1, was generated against a human ovarian carcinoma cell line, OVCAR-3. The antigen reacting with this antibody was strongly expressed on the external surface of the plasma membrane of OVCAR-3 cells and cells of 4/4 other ovarian carcinoma lines. Variable density and homogeneity of expression was found on cells from 5/5 breast carcinoma lines. Various ovarian tumor specimens and normal human tissues were frozen, cryostat-sectioned, and examined for OVB1 reactivity using immunoperoxidase methods. A strong, uniform, homogeneous reaction on 10/10 ovarian carcinoma specimens and variable, non-homogeneous reactions on breast tumors were seen. Normal tissues reacting with the antibody include thyroid, pituitary pars intermedia, breast ductal epithelium, Auerbach's plexus and neuronal processes in the GI tract, colonic mucosal epithelium, and salivary gland ductal epithelium. Polymorphonuclear leukocytes, eosinophils, and approximately 13% of peripheral lymphocytes, as well as cells around germinal centers in lymph nodes and spleen, showed strong reactivity by immunofluorescence and/or immunoperoxidase. Expression of the OVB1 antigen in the myeloid cells of normal human bone marrow occurred from the promyelocyte stage through to more mature cells in a subpopulation of myeloblasts. Indirect immunofluorescence of live peripheral blood cells showed localization to the surface of PMNs, eosinophils, and certain lymphocytes. Double-immunofluorescence studies (with a direct fluorescein-anti-lactoferrin antibody conjugate) showed co-localization of OVB1 and OKM1 (anti-C3bi receptor) antibodies to specific granules of PMNs. Localization of OVB1 and OKM1 antibodies to granular structures in the PMN was confirmed by electron microscopy using the ferritin bridge technique. The antigen reacting with the OVB1 antibody was shown to be neuraminidase sensitive, but protease insensitive. The OVB1 monoclonal antibody may be useful in identification of ovarian tumors and subclassification of myeloid leukemias.     

Ontogeny of apelin and its receptor in the rodent gastrointestinal tract

Apelin is the endogenous ligand for the APJ receptor and both apelin and APJ are expressed in the gastrointestinal (GI) tract. The aim of this study was to define ontogeny of apelin and APJ in the developing rodent GI tract by measuring expression levels and characterizing abundance and cellular localization at an embryonic stage (E18.5 or E21), two postnatal stages (P4, P16) and in the adult. Apelin and APJ mRNA levels were measured by real time RT-PCR, apelin and APJ-containing cells were identified by immunohistochemical (IHC) staining. Gastric, duodenal and colonic apelin and APJ mRNA levels were highest at birth and declined postnatally. In the postnatal rat stomach, few apelin peptide-containing cells were identified, the density of gastric apelin-containing cells increased progressively after weaning and into adulthood. A robust APJ immunostaining was observed postnatally in the epithelium, intestinal goblet cells and in smooth muscle cells. In the adult rat, APJ immunostaining in the surface epithelium and goblet cells decreased markedly. During the early postnatal period, in an apelin-deficient mouse, APJ expression and immunostaining in the gut were reduced suggesting that apelin regulates APJ. Together, our data support a role for the apelin–APJ system in the regulation of smooth muscle, epithelial and goblet cell function in the GI tract.     

Human kallikrein 3 (prostate specific antigen) and human kallikrein 5 expression in salivary gland tumors

The human kallikrein 5 protein (hK5) is expressed in many normal tissues, most notably in skin, breast, salivary gland and esophagus. It has also been shown to be a potential biomarker for breast, ovarian and testicular cancer. Human kallikrein 3 (hK3; prostate-specific antigen) is the most useful marker for adenocarcinoma of the prostate gland. The aim of this study was to determine whether hK3 and hK5 are expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), in order to compare normal with tumor tissues. Pleomorphic adenomas, adenoid cystic carcinomas, polymorphous low-grade adenocarcinomas, acinic cell carcinomas, mucoepidermoid carcinomas and adenocarcinomas not otherwise specified of both minor and major salivary glands were examined. The results of this study indicate that most salivary gland tumors do not show high levels of expression of hK5. Staining was most prominent in keratinizing epithelia in pleomorphic adenomas. hK3 is not expressed in salivary gland tumors.     

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.     

Systematic Analysis of Pericarp Starch Accumulation and Degradation during Wheat Caryopsis Development

Although wheat (Triticum aestivum L.) pericarp starch granule (PSG) has been well-studied, our knowledge of its features and mechanism of accumulation and degradation during pericarp growth is poor. In the present study, developing wheat caryopses were collected and starch granules were extracted from their pericarp to investigate the morphological and structural characteristics of PSGs using microscopy, X-ray diffraction and Fourier transform infrared spectroscopy techniques. Relative gene expression levels of ADP-glucose pyrophosphorylase (APGase), granule-bound starch synthase II (GBSS II), and α-amylase (AMY) were quantified by quantitative real-time polymerase chain reaction. PSGs presented as single or multiple starch granules and were synthesized both in the amyloplast and chloroplast in the pericarp. PSG degradation occurred in the mesocarp, beginning at 6 days after anthesis. Amylose contents in PSGs were lower and relative degrees of crystallinity were higher at later stages of development than at earlier stages. Short-range ordered structures in the external regions of PSGs showed no differences in the developing pericarp. When hydrolyzed by α-amylase, PSGs at various developmental stages showed high degrees of enzymolysis. Expression levels of AGPase, GBSS II, and AMY were closely related to starch synthesis and degradation. These results help elucidate the mechanisms of accumulation and degradation as well as the functions of PSG during wheat caryopsis development.     

The use of tissue microarrays for semiquantitative evaluation of ATPaseC1 expression is ineffective

Abstract

We described earlier the possible role of ATPaseC1 expression as a diagnostic and prognostic marker for oral cancer; others have reported its use for tumors of the lung and breast. We assessed ATPaseC1 expression in a sample of oral squamous cell carcinoma (OSCC) using tissue microarrays (TMAs) to analyze the relation between ATPaseC1 expression and clinical, histopathological and prognostic parameters. We performed a retrospective study of 48 cases of OSCC. We constructed TMAs using two different regions of each tumor. V-ATPaseC1 immunohistochemistry was performed and assessed semiquantitatively. ATPaseC1 staining was observed in most of the neoplastic cells in all tumors. Staining was diffusely cytoplasmic and, to a lesser extent, nuclear. The degree of concordance between the measurements performed in tissue microarray 1 (TMA1) and tissue microarray 2 (TMA2), as evaluated using the intra-class correlation coefficient (ICC), was low. We found great variability in the immunohistochemical staining of the different regions of each tumor. We found 16 cases with mild expression (33.3%), 20 with moderate expression (41.7%) and 12 with intense expression (25%). Differences in the clinical-pathological variables studied were not statistically significant. The difficulty of immunohistochemical evaluation, the heterogeneity of the carcinomas and the fact that evaluation of expression requires semiquantitative analysis render the reliability of the results obtained from TMA-based techniques questionable.