Involvement of heparin-like glycosaminoglycans and expression of a novel heparan sulfate binding protein (p24) during human placentation and in a model for human implantation

Previous studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. To investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were selected to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay showed that initial binding between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAG) with heparin-like properties, i.e., heparan sulfate and dermatan sulfate. Furthermore, a single class of highly specific, protease-sensitive heparin/heparan sulfate binding sites exist on the surface of RL95 cells. Three heparin binding, tryptic peptide fragments were isolated from RL95 cell surfaces and their amino termini partially sequenced. Reverse transcription-polymerase chain reaction (RT-PCR) generated 1 to 4 PCR products per tryptic peptide. Northern blot analysis of RNA from RL95 cells using one of these RT-PCR products identified a 1.2 Kb mRNA species (p24). The amino acid sequence predicted from the cDNA sequence contains a putative heparin-binding domain. A synthetic peptide representing this putative heparin binding domain was used to generate a rabbit polyclonal antibody (anti-p24). Indirect immunofluorescence studies on RL95 and JAR cells as well as binding studies of anti-p24 to intact RL95 cells demonstrate that p24 is distributed on the cell surface. Western blots of RL95 membrane preparations identify a 24 kDa protein (p24) highly enriched in the 100,000 g pellet plasma membrane-enriched fraction. p24 eluted from membranes with 0.8 M NaCl, but not 0.6 M NaCl, suggesting that it is a peripheral membrane component. Solubilized p24 binds heparin by heparin affinity chromatography and $\sp{125}$I-heparin binding assays. Furthermore, indirect immunofluorescence studies indicate that cytotrophoblast of floating and attached villi of the human fetal-maternal interface are recognized by anti-p24. The study also indicates that the HSPG, perlecan, accumulates where chorionic villi are attached to uterine stroma and where p24-expressing cytotrophoblast penetrate the stroma. Collectively, these data indicate that p24 is a cell surface membrane-associated heparin/heparan sulfate binding protein found in cytotrophoblast, but not many other cell types of the fetal-maternal interface. Furthermore, p24 colocalizes with HSPGs in regions of cytotrophoblast invasion. These observations are consistent with a role for HSPGs and HSPG binding proteins in human trophoblast-uterine cell interactions. ^

Cell surface galactosyltransferase structure and function during mesenchymal cell migration

In order to more fully understand the function of surface GalTase on mesenchymal cells, anti-GalTase IgG was used to (a) examine the role of surface GalTase during mouse mesenchymal cell migration on laminin and fibronectin; (b) define the plasma membrane distribution of GalTase by indirect immunofluorescence on migrating cells; (c) quantitate the level of surface GalTase on migrating cells; and (d) determine whether GalTase is associated with the cytoskeleton.^ Results show that anti-GalTase IgG was able to inhibit migration (48-80% as compared to basal rate) when cells were migrating on laminin-containing matrices. Monovalent Fab fragments inhibited migration on laminin by 90% after 4 hours. On the other hand, anti-GalTase IgG had no effect on cells migrating on fibronectin. This illustrates the substrate specificity of GalTase mediated-migration. When anti-GalTase IgG was used to localize surface GalTase on cells migratory on laminin, the enzyme was restricted to the leading and trailing edges of the cell. Assays indicate that GalTase is elevated approximately 3-fold when cells are migrating on laminin-containing matrices as compared to migratory cells on plastic or fibronectin, or as compared to stationary cells on any substrate. Laminin appears to recruit GalTase from preexisting intracellular pools to the growing lamellipodia.^ Double-label indirect immunofluorescence studies indicate that there is an apparent co-localization between some of the surface GalTase and some actin filaments. This relationship was explored by extracting cells prelabeled with anti-GalTase IgG and quantitated by radiolabeled second antibodies. Results show that 79% of the surface GalTase is associated with the cytoskeleton (as judged by detergent insolubility) when monovalent antibodies (Fab) are used. However virtually all (80-100%) of the surface GalTase can be induced to associate with the cytoskeleton when cross-linked with bivalent antibodies. Furthermore, when cells in suspension are incubated with divalent antibodies, an additional 66% of the surface GalTase can be induced to associate with the cytoskeleton. The elevated levels of surface GalTase detectable on cells migrating on laminin also appear to be associated with the cytoskeleton.^ Several lines of evidence suggest that GalTase is associated with F-actin. Data suggest that laminin induces the expression of surface GalTase to the growing lamellipodia where it becomes associated with the cytoskeleton leading to cell spreading and migration. (Abstract shortened with permission of author.) ^

Structure and function study of prostaglandin H synthase

Prostaglandin H synthase (PGHS) is a key enzyme in biosynthesis of prostaglandins, thromboxane, and prostacyclin. It has two activities, cyclooxygenase and peroxidase. "PGHS" means PGHS-1. A current hypothesis considers the cyclooxygenase reaction to be a free radical chain reaction, initiated by interaction of the synthase peroxidase with hydroperoxides leading to the production of a tyrosyl free radical. According to this hypothesis, tyrosyl residue(s) may play a key role in the cyclooxygenase reaction. Tetranitromethane (TNM) can relatively selectively nitrate tyrosines at pH 8.0. The effect of TNM on both cyclooxygenase activity and peroxidase activity has been examined: reaction of the synthase holoenzyme with TNM at pH 8.0 led to inactivation of both activities, with the cyclooxygenase activity being lost rapidly and completely, while the peroxidase activity was lost more slowly. Indomethacin, a non-steroidal anti-inflammatory agent, can protect the synthase from the inactivation of TNM. Amino acid analyses indicated that a loss of tyrosine and formation of nitrotyrosine residues occurred during reaction with TNM, and that TNM-reacted holoenzyme with $<$10% residual cyclooxygenase activity had about 2.0 nitrotyrosine/subunit.^ PGH synthase is known to be an endoplasmic reticulum membrane-associated protein. Antibodies directed at particular PGHS peptide segments and indirect immunofluorescence have been used to characterize the membrane topology of crucial portions of PGHS. PGHS was expressed in COS-1 cells transfected with the appropriate cDNA. Stably-transfected human endothelial cells were also used for the topology study. The cells were treated with streptolysin-O, which selectively permeabilizes the plasma membrane, or with saponin to achieve general membrane disruption, before incubation with the antipeptide antibodies. Bound antipeptide antibody was stained by FITC-labelled secondary antibody and visualized by fluorescence microscopy. With the antipeptide antibodies against residues 51-66, 156-170 or 377-390, there was a significant reticular and perinuclear pattern of staining in cells permeabilized with saponin but not in cells permeabilized with SLO alone. Antibodies directed against the endogenous C-terminal peptide or against residues 271-284 produced staining in cells permeabilized with saponin, and also in a lower, but significant fraction of cells permeabilized with SLO. Similar results were obtained when COS-1 cells expressing recombinant PGHS with a viral reporter peptide inserted at the C-terminus were stained with antibody against the reporter epitope.^ The PGHS C-terminal sequence is similar to that of the consensus KDEL ER retention signal. The potential function of the PGHS C-terminus segment in ER retention was examined by mutating this segment and analyzing the subcellular distribution of the mutants expressed in COS-1 cells. None of the mutants had an altered subcellular distribution, although some had greatly diminished the enzyme activities. (Abstract shortened by UMI.) ^

The seroprevalence of Rocky Mountain spotted fever in shelter dogs in Harris County

Rocky Mountain spotted fever (RMSF) is a tick-borne illness caused by the bacteria Rickettsia rickettsii, with infections occurring in humans and dogs. The prominent tick vector of RMSF, Dermacantor variabilis, and another potential vector, Rhipacephalus sanguineus, are prevalent in Texas. The goal of this study was to determine the prevalence of past infections by testing for IgG antibodies to R. rickettsii in dogs in an animal shelter in Harris County using an immunofluorescence assay (IFA) test. We found that 12.6% (24) of 191 dogs tested had a positive IFA test at 1:64 serum dilution, indicating infection at some time in the past. We also sampled the ticks present on dogs in the animal shelter to understand the prevalence of potential vector species. Of a total of 58 ticks, 86% were D. variabilis and the remaining 14% were R. sanguineus. The results of this study demonstrate that RMSF has the potential to be, and may already be, endemic to the Harris County area. Public health actions such as heightened surveillance and education that RMSF is present would be appropriate in the Harris County area.^

ANALYSIS OF ESTROGEN-INDUCED ANEUPLOIDY AND CHROMOSOME DISPLACEMENT

Diethylstilbestrol (DES) is a known human carcinogen and teratogen whose mechanism of action remains undetermined. As essentially diploid Chinese hamster cell line (Don) was used to test diethylstilbestrol (DES), dienestrol, hexestrol and the naturally occurring estrogens, estradiol and estriol for their ability to cause metaphase arrest and to induce aneuploidy. These compounds arrest mitosis within a narrow range of high concentrations and induce aneuploidy in recovering cell populations. DES was the most effective arrestant on a comparative molar basis. Estradiol and estriol were less potent as arrestants but were effective inducers of aneuploidy. Aneuploidy was induced in a non-random manner. The smallest chromosomes were most frequently recorded in aneuploid cells. Using anti-tubulin antibody and indirect immunofluorescence, it was found that DES inhibits bi-polar spindle assembly and disrupts the cytoplasmic microtubule complex (CMTC). Estradiol arrests mitosis in a manner that allows spindle assembly. Estradiol has no apparent effect on the CMTC. The naturally occurring estrogens caused chromosome displacement during mitotic arrest. Electron microscopy confirmed that the displaced chromosomes appeared at the polar regions of arrested cells. The arresting effect of estradiol, and to some extent DES, was reduced by the addition of dibutyryl cyclic adenosine monophosphate (db-cAMP). Aneuploidy induction by DES and similar compounds may be related to their carcinogenic and/or teratogenic potential. ^

Modulation of carbohydrates and carbohydrate binding proteins (lectins) during retinoic acid -induced differentiation of murine embryonal carcinoma cells

The current studies were undertaken to examine the effect of retinoic acid (RA)-induced differentiation of the murine embryonal carcinoma cell line, F-9, on the glycosylation of specific cellular glycoproteins and on the expression of two members of the family of endogenous lactoside-binding lectins. It was found that RA-induced differentiation of these cells into cells with the properties of primitive endoderm results in the increased fucosylation of 3 glycoproteins with molecular weights of 175 (gp175), 250 (gp250), and 400 (pg400) kDa. These three fucose-containing glycoproteins can be considered as new markers of differentiation in this system. The increased fucosylation of these glycoproteins preceded the 3-fold increase in fucosyltransferase (FT) activity that was seen upon RA-induced differentiation of these cells, indicating that an increase in fucosyltransferase activity alone cannot explain the increased fucosylation of these glycoproteins.^ The effect of RA and Ch55, a chalcone carboxylic acid with retinoid-like properties, induced differentiation of a variety of murine embryonal carcinoma cell lines on the activities of both FT and sialyltransferase (ST) was examined. The effect of differentiation on the activities of both glycosyltransferases was modulated and most probably is dependent upon the differentiation pathway that is triggered by the retinoids for each of the embryonal carcinoma cell lines.^ Two glycoproteins, Lysosomal Associated Membrane Glycoproteins 1 and 2 (LAMP-1 and LAMP-2) were examined in more detail during the course of RA-induced differentiation of F-9 cells. Both the levels and glycosylation of both glycoproteins are increased following differentiation of these cells. Differentiation results in the increased binding of $\sp{125}$l-labelled L-phytohemagglutinin to bind to LAMP-1 which indicates increased GlcNAc $\beta$1,6 branching of the oligosaccharide side chains.^ We found that RA-induced differentiation of F-9 cells results in the decreased expression of the 34 kDa lectin 24 h after addition of the retinoid to the medium. Additionally, 48 h of RA-treatment results in the increased expression of the 14.5 kDa lectin. By indirect immunofluorescence we were able to colocalize the 14.5 kDa lectin and laminin which suggests that laminin may be a ligand for the lectin in the F-9 cells. (Abstract shortened with permission of author.) ^

The role of the conserved N -terminal domain of STAT3 in STAT3 dephosphorylation and nuclear export

Rapid redistribution of STAT subcellular localization is an essential feature of cytokine signaling. To elucidate the molecular basis of STAT3 function, which plays a critical role in controlling innate immune responses in vivo, we initiated studies to determine the mechanisms controlling STAT3 nuclear trafficking. We found that STAT3 is transported to the nucleus in the absence of cytokine treatment, as judged by indirect immunofluorescence studies in the presence of leptomycin B, an inhibitor of CRM1-dependent nuclear export, suggesting that the non-phosphorylated STAT3 protein contains a functional nuclear import signal. An isoform lacking the STAT3 N-terminal domain (Δ133STAT3) retains the ability to undergo constitutive nuclear localization, indicating that this region is not essential for cytokine-independent nuclear import. Δ133STAT3 is also transported to the nucleus following stimulation with interleukin-6 (IL-6). Interestingly, IL-6-dependent tyrosine phosphorylation of Δ133STAT3 appears to be prolonged and the nuclear export of the protein delayed in cells expressing endogenous STAT3, consistent with defective Δ133STAT3 dephosphorylation. Endogenous STAT3 does not promote the nuclear export of Δ133STAT3, although dimerization between endogenous Stat3 and Δ133STAT3 is detected readily. Thus, the STAT3 N-terminal domain is not required for dimerization with full-length STAT3, yet appears to play a role in proper export of Stat3 from the nucleus following cytokine stimulation. STAT3-deficient cells reconstituted with Δ133STAT3 show enhanced and prolonged Stat1 signaling in response to IL-6, suggesting that induction of the STAT3-dependent negative regulator SOCS3 is impaired. In fact, Δ133STAT3 fails to induce SOCS3 mRNA efficiently. These studies collectively indicate that the STAT3 N-terminal region may be important for IL-6-dependent target gene activation and nuclear dephosphorylation, while dispensable for nuclear import. STAT3 is an oncogene. STAT3 is constitutively activated in primary tumors of many types. Thus far, research in the design of STAT3 protein inhibitors has focused on the SH2 and DNA-binding domains of STAT3. Interference with these domains eliminates all signaling through STAT3. If the N-terminal domain is involved in tetramerization on a subset of target genes, inhibition of this region may lead to a more selective inhibition of some STAT3 functions while leaving others intact. ^