CD73 complexes with emmprin to regulate MMP-2 production from co-cultured sarcoma cells and fibroblasts.

BACKGROUND: Interaction between cancer cells and fibroblasts mediated by extracellular matrix metalloproteinase inducer (emmprin, CD147) is important in the invasion and proliferation of cancer cells. However, the exact mechanism of emmprin mediated stimulation of matrix metalloprotease-2 (MMP-2) production from fibroblasts has not been elucidated. Our previous studies using an inhibitory peptide against emmprin suggested the presence of a molecule on the cell membrane which forms a complex with emmprin. Here we show that CD73 expressed on fibroblasts interacts with emmprin and is a required factor for MMP-2 production in co-cultures of sarcoma cells with fibroblasts. METHODS: CD73 along with CD99 was identified by mass spectrometry analysis as an emmprin interacting molecule from a co-culture of cancer cells (epithelioid sarcoma cell line FU-EPS-1) and fibroblasts (immortalized fibroblasts cell line ST353i). MMP-2 production was measured by immunoblot and ELISA. The formation of complexes of CD73 with emmprin was confirmed by immunoprecipitation, and their co-localization in tumor cells and fibroblasts was shown by fluorescent immunostaining and proximity ligation assays. RESULTS: Stimulated MMP-2 production in co-culture of cancer cells and fibroblasts was completely suppressed by siRNA knockdown of CD73, but not by CD99 knockdown. MMP-2 production was not suppressed by CD73-specific enzyme inhibitor (APCP). However, MMP-2 production was decreased by CD73 neutralizing antibodies, suggesting that CD73-mediated suppression of MMP-2 production is non-enzymatic. In human epithelioid sarcoma tissues, emmprin was immunohistochemically detected to be mainly expressed in tumor cells, and CD73 was expressed in fibroblasts and tumor cells: emmprin and CD73 were co-localized predominantly on tumor cells. CONCLUSION: This study provides a novel insight into the role of CD73 in emmprin-mediated regulation of MMP-2 production.

Intergenerational transmission of post-traumatic stress disorder in Australian Vietnam veterans' families.

OBJECTIVE: To assess the association between parental post-traumatic stress disorder (PTSD) and offspring PTSD and its specificity for other disorders in a non-clinical epidemiological cohort of Australian Vietnam veterans, their partners and their sons and daughters. METHOD: Veterans were interviewed twice, in 1992-1994 and 2005-2006; partners were interviewed in 2006-2007, and their offspring in 2012-2014. A total of 125 sons and 168 daughters were interviewed from 197 families, 137 of which also included partners who were the mothers of the children. Statistical analysis used multi-level modelling to compute odds ratios and 95% confidence intervals while controlling for clustering effects within families. Parent PTSD diagnoses were examined for associations with offspring trauma exposure, PTSD and other psychiatric diagnoses. RESULTS: Veteran PTSD increased the risk of PTSD and no other disorder in both sons and daughters; partner PTSD did not. Veteran depression was also a risk factor for sons' PTSD, and alcohol disorder was linked to alcohol dependence in sons and PTSD in daughters, but not when controlling for veteran PTSD. CONCLUSION: We conclude that PTSD in a Vietnam veteran father increases the risk specifically for PTSD in his sons and daughters.

Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function.

To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro. Local release of soluble CD44 by the transfectants inhibited the ability of endogenous cell surface CD44 to bind and internalize hyaluronan and to mediate TA3 cell invasion of hyaluronan-producing cell monolayers. Mice intravenously injected with parental TA3/St cells developed massive pulmonary metastases within 21-28 d, whereas animals injected with TA3sCD44 cells developed few or no tumors. Tracing of labeled parental and transfectant tumor cells revealed that both cell types initially adhered to pulmonary endothelium and penetrated the interstitial stroma. However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis. Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells. These observations provide direct evidence that cell surface CD44 function promotes tumor cell survival in invaded tissue and that its suppression can induce apoptosis of the invading tumor cells, possibly as a result of impairing their ability to penetrate the host tissue hyaluronan barrier.

Cell-substratum attachment and cell surface hyaluronate of Rous sarcoma virus-transformed chondrocytes.

Hyaluronate is associated with the cell surface of cultured Rous sarcoma virus-transformed chondrocytes. Detachment of these cells from their substratum by a variety of reagents is accompanied by release of 75-100% of this hyaluronate into solution. Treatment of the cells with 200 U/ml protease-free Streptomyces hyaluronidase at 37 degrees C cause release of greater than 90% of the cell surface hyaluronate and complete cell detachment. Treatment with a lower concentration of Streptomyces hyaluronidase (30 U/ml) at 25 degrees C or a corresponding activity of testicular hyaluronidase gives similar results, but only in the presence of mM EGTA. Treatment with the lower activities of either hyaluronidase or with 1 mM EGTA alone release only approximately 45% of the cell surface hyaluronate and does not cause significant cell detachment. It is concluded that there are two populations of cell surface hyaluronate differing in their accessibility or their resistance to dissociation from other components of the cell surface. It is proposed that the less readily released fraction is located between the transformed chondrocyte surface and substratum and is necessary for their interaction.

Epithelial collagens and glycosaminoglycans in the embryonic cornea. Macromolecular order and morphogenesis in the basement membrane.

The embryonic corneal epithelium synthesizes both collagen and chondroitin sulfate and excretes them across the basement membrane into the subepithelial space where they assemble into a spiraling orthogonal matrix of fibrils. The assembly of collagen into fibrils is first apparent at the outer face of the basement membrane in a region of ordered chondroitin sulfate molecules. Hyaluronate, another morphogenetically important corneal macromolecule, is produced at these early stages only by the inner endothelium. These correlated biosynthetic and ultrastructural data demonstrate discrete macromolecular products of the two corneal epithelia with differing morphogenetic properties and functions.

Hyaluronan-binding protein in endothelial cell morphogenesis.

Previous studies from several laboratories have provided evidence that interaction of hyaluronan (HA) with the surface of endothelial cells may be involved in endothelial cell behavior. We have recently characterized a mAb, mAb IVd4, that recognizes and neutralizes HA-binding protein (HABP) from a wide variety of cell types from several different species (Banerjee, S. D., and B. P. Toole. 1991. Dev. Biol. 146:186-197). In this study we have found that mAb IVd4 inhibits migration of endothelial cells from a confluent monolayer after "wounding" of the monolayer. HA hexasaccharide, a fragment of HA with the same disaccharide composition as polymeric HA, also inhibits migration. In addition, both reagents inhibit morphogenesis of capillary-like tubules formed in gels consisting of type I collagen and basement membrane components. Immunocytology revealed that the antigen recognized by mAb IVd4 becomes localized to the cell membrane of migrating cells, including many of their lamellipodia. Treatment with high concentrations of HA hexamer causes loss of immunoreactivity from these structures. We conclude that HABP recognized by mAb IVd4 is involved in endothelial cell migration and tubule formation.

Fluorescent morphological probe for hyaluronate.

Hyaluronate levels change dramatically during morphogenesis of various tissues and organs. Morphological detection of the exact temporal and spatial distribution patterns of hyaluronate may help to elucidate its role in morphogenesis. Since no specific direct method for visualizing hyaluronate with the light or electron microscope is currently available, we have developed a morphological probe by exploiting the high-affinity interaction of cartilage proteoglycan with hyaluronate. The core protein of this proteoglycan consists of a region that binds specifically to hyaluronate with a high association constant, and a region to which the majority of sulfated polysaccharide chains are covalently attached. The polysaccharide chains were removed by treatment with chondroitinase ABC, and the core protein, labeled with rhodamine, was used as the probe. This fluorescent probe binds reversibly and specifically to [3H]hyaluronate in a binding assay using ammonium sulfate precipitation of the core protein. The probe has been used to visualize the cell surface hyaluronate of rat fibrosarcoma cells, 3T3 cells, and SV-40 transformed 3T3 cells, three cell types with significantly different amounts of cell surface-associated hyaluronate.

Binding of hyaluronate to the surface of cultured cells.

The binding of hyaluronate to SV-3T3 cells was measured by incubating a suspension of cells (released from the substratum with EDTA) with 3H-labeled hyaluronate and then applying the suspension to glass fiber filters which retained the cells and the bound hyaluronate. The extent of binding was a function of both the concentration of labeled hyaluronate and the cell number. Most of the binding took place within the first 2 min of the incubation and was not influenced by the presence or absence of divalent cations. The binding of labeled hyaluronate to SV-3T3 cells could be prevented by the addition of an excess of unlabeled hyaluronate. High molecular weight preparations of hyaluronate were more effective in preventing binding than low molecular weight preparations. The binding of [3H]hyaluronate was inhibited by high concentrations of oligosaccharide fragments of hyaluronate consisting of six sugars or more, and by chondroitin. The sulfated glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparin, and heparan sulfate) had little or no effect on the binding. The labeled hyaluronate bound to the cells could be totally removed by incubating the cells with testicular hyaluronidase, streptomyces hyaluronidase, or trypsin, indicating that the hyaluronate-binding sites are located on the cell surface.

Chick embryo fibroblasts produce two forms of hyaluronidase.

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.

Pericellular coat of chick embryo chondrocytes: structural role of hyaluronate.

Chondrocytes produce large pericellular coats in vitro that can be visualized by the exclusion of particles, e.g., fixed erythrocytes, and that are removed by treatment with Streptomyces hyaluronidase, which is specific for hyaluronate. In this study, we examined the kinetics of formation of these coats and the relationship of hyaluronate and proteoglycan to coat structure. Chondrocytes were isolated from chick tibia cartilage by collagenase-trypsin digestion and were characterized by their morphology and by their synthesis of both type II collagen and high molecular weight proteoglycans. The degree of spreading of the chondrocytes and the size of the coats were quantitated at various times subsequent to seeding by tracing phase-contrast photomicrographs of the cultures. After seeding, the chondrocytes attached themselves to the tissue culture dish and exhibited coats within 4 h. The coats reached a maximum size after 3-4 d and subsequently decreased over the next 2-3 d. Subcultured chondrocytes produced a large coat only if passaged before 4 d. Both primary and first passage cells, with or without coats, produced type II collagen but not type I collagen as determined by enzyme-linked immunosorbent assay. Treatment with Streptomyces hyaluronidase (1.0 mU/ml, 15 min), which completely removed the coat, released 58% of the chondroitin sulfate but only 9% of the proteins associated with the cell surface. The proteins released by hyaluronidase were not digestible by bacterial collagenase. Monensin and cycloheximide (0.01-10 microM, 48 h) caused a dose-dependent decrease in coat size that was linearly correlated to synthesis of cell surface hyaluronate (r = 0.98) but not chondroitin sulfate (r = 0.2). We conclude that the coat surrounding chondrocytes is dependent on hyaluronate for its structure and that hyaluronate retains a large proportion of the proteoglycan in the coat.

Hyaluronate inhibition of chondrogenesis: antagonism of thyroxine, growth hormone, and calcitonin.

The formation of cartilage-like aggregates in high-density stationary cultures of trypsinized chick embryo precartilage cells is blocked by low concentrations of hyaluronate. Thyroxine (and triiodothyronine), growth hormone, calcitonin, and adenosine 3',5'-monophosphate prevented this inhibition by hyaluronate, whereas other hormones tested did not.

Brain hyaluronidase: changes in activity during chick development.

Development of embryonic chick brain is characterized by high levels of hyaluronidase activity and of hyaluronate, both of which decrease rapidly soon after the chick hatches. By analogy to other systems, it is proposed that the sequence of hyaluronate production and its enzymatic removal may have a developmental role in brain formation.

Cancer incidence in patients with schizophrenia and their first-degree relatives - a meta-analysis.

OBJECTIVE: Controversy concerning cancer incidence in schizophrenia exists because of heterogeneous study findings. METHOD: A meta-analysis was performed on standardized incidence ratios (SIR) of cancer in patients with schizophrenia and first-degree relatives and compared with general population samples. RESULTS: The pooled overall cancer incidence in patients was not significantly increased (SIR = 1.05, CI 0.95-1.15). Lung cancer incidence was slightly increased (SIR = 1.31, CI 1.01-1.71), but was reduced after adjusting for smoking prevalence. The incidence of several cancers unrelated to smoking was reduced in patients. Breast cancer rates were significantly increased in female patients. The pooled overall cancer incidence in siblings (SIR = 0.89, CI 0.84-0.94) and parents (SIR = 0.90, CI 0.88-0.93) was significantly reduced. A meta-regression detected a significant relationship between cancer risk in the general population and relative risk in patients. CONCLUSION: The meta-analysis aided exploration of inconsistent study findings. There is a discrepancy between cancer risk exposure and cancer incidence in schizophrenia consistent with a protective effect.

Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.

Basic fibroblast growth factor (FGF-2) is a pleiotropic growth factor detected in many different cells and tissues. Normally synthesized at low levels, FGF-2 is elevated in various pathologies, most notably in cancer and injury repair. To investigate the effects of elevated FGF-2, the human full-length cDNA was expressed in transgenic mice under control of a phosphoglycerate kinase promoter. Overexpression of FGF-2 caused a variety of skeletal malformations including shortening and flattening of long bones and moderate macrocephaly. Comparison by Western blot of FGF-2 transgenic mice to nontransgenic littermates showed expression of human FGF-2 protein in all major organs and tissues examined including brain, heart, lung, liver, kidney, spleen, and skeletal muscle; however, different molar ratios of FGF-2 protein isoforms were observed between different organs and tissues. Some tissues preferentially synthesize larger isoforms of FGF-2 while other tissues produce predominantly smaller 18-kDa FGF-2. Translation of the high molecular weight isoforms initiates from unconventional CUG codons and translation of the 18-kDa isoform initiates from an AUG codon in the FGF-2 mRNA. Thus the Western blot data from the FGF-2 transgenic mice suggest that tissue-specific expression of FGF-2 isoforms is regulated translationally.

Extracellular matrix metalloproteinase inducer regulates metalloproteinases in human uterine endometrium.

CONTEXT: Endometrial remodeling occurs during each menstrual cycle in women and also during the establishment of endometriosis. Both processes involve the production of metalloproteinases (MMPs) by uterine endometrial cells. OBJECTIVE: The objective of this study was to determine whether tissue remodeling and endometrial invasion involve activation of MMPs by extracellular matrix metalloproteinase inducer (EMMPRIN). MAIN OUTCOME MEASURES: EMMPRIN expression was examined by immunohistochemistry and real-time PCR in ectopic and eutopic endometria. For functional assays, human uterine fibroblasts were treated in the absence or presence of IL-1beta (10 ng/ml) or purified native EMMPRIN (0.5 or 1 microg/ml) for 24 h. Cellular RNA and conditioned medium were assayed by real-time PCR or immunoblotting. RESULTS: EMMPRIN protein localized to epithelial and fibroblast cells of eutopic and ectopic endometria. The pattern of localization was regulated by ovarian hormones. EMMPRIN mRNA levels varied throughout the menstrual cycle in parallel with the cyclic changes in estradiol. EMMPRIN treatment (0.5 microg/ml) of human uterine fibroblast cells stimulated MMP-1 (5.23-fold) and MMP-2 (8.55-fold), but not MMP-3, mRNA levels over levels in control cells (P < 0.05). EMMPRIN treatment (1 microg/ml) stimulated endogenous EMMPRIN (1.6-fold) mRNA levels (P > 0.05). IL-1beta stimulated MMP-1 (5.6-fold), MMP-2 (2.8-fold), and MMP-3 (75-fold) gene expression, but not EMMPRIN, over levels in control cells (P < 0.05). Both EMMPRIN and IL-1beta treatments stimulated MMP-1, -2, and -3, but not EMMPRIN protein secretion, with 0.5 microg/ml producing the greatest response. CONCLUSIONS: The ability of EMMPRIN to stimulate MMP secretion by endometrial fibroblasts indicates its potential role in uterine remodeling and the pathogenesis of endometriosis.

EMMPRIN (CD147), an inducer of matrix metalloproteinase synthesis, also binds interstitial collagenase to the tumor cell surface.

Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as basigin or CD147, is a glycoprotein that is enriched on the surface of tumor cells and stimulates production of several matrix metalloproteinases by adjacent stromal cells. In this study, we have found that EMMPRIN not only stimulates the production of interstitial collagenase (MMP-1) but also forms a complex with MMP-1 at the tumor cell surface. Complex formation was demonstrated by phage display, affinity chromatography, and immunocytochemistry. Presentation of MMP-1 complexed to EMMPRIN at the tumor cell surface may be important in modifying the tumor cell pericellular matrix to promote invasion.

Divalent cation-independent aggregation of rat-1 fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus.

Rat-1 fibroblasts infected with the temperature-sensitive transformation mutant LA 24 of Rous sarcoma virus have a high rate of divalent cation-independent homotypic cell aggregation when grown at the permissive temperature, 34 degrees. Cells grown at the nonpermissive temperature, 39 degrees, have a low rate of homotypic cell aggregation. Hyaluronic acid is involved in the homotypic aggregation of permissively grown cells since aggregation is blocked by either treatment of the cells with hyaluronidase or the presence of exogenously added hyaluronic acid. Despite their low rate of homotypic aggregation, nonpermissively grown cell are capable of heterotypically adhering to premissively grown cells. The formation of heterotypic aggregates is blocked by treatment of the nonpermissively grown cells with hyaluronidase but not by treatment of permissively grown cells with hyaluronidase. These results provide evidence that the divalent cation-independent aggregation of Rat-1 LA 24 cells is mediated by interactions between hyaluronic acid and other adhesive components on the cell surface.

Human keratinocytes express EMMPRIN, an extracellular matrix metalloproteinase inducer.

Increased levels of matrix metalloproteinases are associated with tissue degradation and remodeling during tumor invasion and wound healing. In both processes, there is evidence that cell interactions between fibroblasts and tumor cells or keratinocytes lead to increases in metalloproteinase production. We have previously isolated and purified a tumor cell surface protein, EMMPRIN (extracellular matrix metalloproteinase inducer), which stimulates production of interstitial collagenase, gelatinase A, and stromelysin-1 by fibroblasts, and we have obtained cDNA clones that encode the EMMPRIN protein from LX-1 human lung carcinoma cells. In this study we report immunolocalization of EMMPRIN around the surface of human keratinocytes in vitro and in vivo, and isolation of cDNAs that encode the entire open reading frame for EMMPRIN from a human keratinocyte library. Comparison of the EMMPRIN cDNAs from normal human keratinocytes and LX-1 human tumor cells by nucleotide sequence analysis, expression of the recombinant proteins, and in vitro translation using the cDNAs from the two sources indicate that they express very similar forms of EMMPRIN. Native EMMPRIN isolated directly from extracts of keratinocytes, however, is slightly smaller in size and is present at a lower concentration compared with that from LX-1 tumor cells. These results establish the presence of EMMPRIN in the normal epidermis and raise the possibility of its involvement in regulation of matrix remodeling at the epidermal-dermal interface.

Characterization of the gene for human EMMPRIN, a tumor cell surface inducer of matrix metalloproteinases.

EMMPRIN (extracellular matrix metalloproteinase inducer) also known as CD147 and basigin, is a member of the immunoglobulin family that is present on the surface of tumor cells and stimulates nearby fibroblasts to synthesize matrix metalloproteinases. Using our EMMPRIN cDNA, we have isolated a cosmid clone that contains the human EMMPRIN gene. S1 analysis with a fragment of the gene clone and primer extension of the mRNA was performed to determine the transcription start site. PCR and sequence analysis have defined the exon/intron organization of the gene and show that it is highly conserved with the mouse EMMPRIN/basigin gene. About 950 bases of the 5'-flanking region were examined for transcription factor consensus binding sites, locating three SP1 sites and two AP2 sites. The transcription start site was found to be located in a CpG island. Elements in the proximal promoter region were conserved in the human and mouse genes.