Tissue inhibitor of metalloproteinase-3 expression is upregulated during human cytotrophoblast invasion in vitro.

Surprisingly, a successful human pregnancy requires cells from the fetal portion of the placenta (cytotrophoblasts) to adopt tumor-like properties. Cytotrophoblasts attach the conceptus to the endometrium by invading the uterus, and they initiate blood flow to the placenta by breaching maternal vessels. But unlike tumor metastasis, cytotrophoblast invasion is highly regulated both spatially and temporally. Our previous work showed that matrix metalloproteinase-9 (MMP-9) expression is upregulated during cytotrophoblast differentiation along the invasive pathway, and that activity of this proteinase specifies the cells' ability to degrade extracellular matrix (ECM) substrates in vitro. Here we tested the hypothesis that invading cytotrophoblasts express an unusual tissue inhibitor of metalloproteinase (TIMP) repertoire that allows them to regulate their MMP-9 proteolytic activity. By using protease-substrate gel electrophoresis, we found that human cytotrophoblasts express primarily TIMP-3. We showed that the cells' TIMP-3 expression is regulated in accord with that of MMP-9. The highest levels of protein and mRNA for both these molecules were detected after differentiation to a fully invasive phenotype and during early gestation, when invasion peaks, rather than at term, when invasion has stopped. Our results suggest that coexpression of MMP-9 and TIMP-3 by invading cytotrophoblasts plays an important role in regulating the depth of uterine invasion.

Murine tissue inhibitor of metalloproteinases-4 (Timp-4): cDNA isolation and expression in adult mouse tissues.

We have isolated cDNA clones corresponding to a new member of the murine tissue inhibitor of metalloproteinase (TIMP) family, designated Timp-4. The nucleotide sequence predicts a protein of 22,609 Da that contains the characteristic 12 cysteine TIMP signature. TIMP-4 is more closely related to TIMP-2 and TIMP-3 than to TIMP-1 (48%, 45% and 38% identity, respectively). Analysis of Timp-4 mRNA expression in adult mouse tissues indicated a 1.2 kb transcript in brain, heart, ovary and skeletal muscle. This pattern of expression distinguishes Timp-4 from other Timps, suggesting that the TIMP-4 protein may be an important tissue-specific regulator of extracellular matrix remodelling.

Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation.

Gelatinase B, a matrix metalloproteinase (MMP) of high specific activity, is highly expressed and activated by mouse blastocysts in culture, and inhibition of this enzyme activity inhibits lysis of extracellular matrix (Behrendtsen, O., Alexander, C. M. and Werb, Z. (1992) Development 114, 447-456). Because gelatinase B expression is linked to invasive potential, we studied the expression of gelatinase B mRNA and protein in vivo, in implanting trophoblast giant cells, and found that it was expressed and activated during colonization of the maternal decidua. mRNAs for several other MMPs (stromelysin-1, stromelysin-3 and gelatinase A) and MMP inhibitors (TIMP-1 and TIMP-2) were expressed in the undifferentiated stroma toward the outside of the decidua, and TIMP-3 mRNA was expressed in primary and some mature decidual cells during their differentiation. Both mRNA and TIMP-3 protein were present at high concentrations transiently, and declined from 6.5 days post coitum onward, as the cells underwent apoptosis during the main period of gelatinase B expression and ectoplacental growth and expansion. To assess the function of MMPs during implantation and decidual development, we either injected a peptide hydroxamate MMP inhibitor into normal mice or studied transgenic mice overexpressing TIMP-1. In both cases, decidual length and overall size were reduced, and the embryo was displaced mesometrially. Embryo orientation was less strictly regulated in inhibitor-treated deciduae than in control deciduae. Morphogenesis and development of oil-induced deciduomas were also slowed in the presence of the inhibitor. We conclude that administration of MMP inhibitors retards decidual remodeling and growth, and we suggest that the MMPs expressed in precursor stromal cells promote their differentiation and expansion.

Identification and characterization of human tissue inhibitor of metalloproteinase-3 and detection of three additional metalloproteinase inhibitor activities in extracellular matrix.

We have identified and characterized a novel human tissue inhibitor of metalloproteinase (TIMP). It is found exclusively in the extracellular matrix of a large number of cultured human cells, including: primary embryonal kidney (293), neuroblastoma (SK-N-SH), normal whole embryo (FHs 173We), cervical carcinoma (HeLa S3), colon adenocarcinoma (Caco-2), ileocecal adenocarcinoma (HCT-8), fibrosarcomas (SW 684 and Hs 913T) and normal gingival fibroblasts (GF11 and 1292). It was not detected in the conditioned media from any of these cell lines. Its apparent molecular mass of 24-25 kDa, as determined by its migration on protease-substrate gels, is intermediate between TIMP-1 (28.5 kDa) and TIMP-2 (21 kDa). Like the latter two proteins, human TIMP-3 contains intrachain disulfide bonds and displays altered electrophoretic mobility in the presence of beta-mercaptoethanol. The N-terminal, amino acid sequence of the protein is identical to that of chicken TIMP-3 (ChIMP-3), and its amino acid composition is similar. The protein is not N-glycosylated, as determined by treatment with N-glycosidase-F. Finally, it is recognized by antisera raised against pure ChIMP-3 but not by anti-human TIMP-1 or anti-human TIMP-2 antibodies. Based on these properties, we propose that this protein is TIMP-3 and is the human counterpart of ChIMP-3 (Pavloff et al., J. Biol. Chem. 267: 17321-17326, 1992). Two additional inhibitors detected in the matrix of human cell lines, designated inhibitor of metalloproteinase (IMP)-a and IMP-b, migrate with apparent masses of 29 kDa and 30 kDa. Both are N-glycosylated. A fourth inhibitor activity, which is smaller in mass than TIMP-3 and is also pecifically located in the matrix, is detectable in some cell lines.

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues.

We have isolated cDNA clones corresponding to a novel mouse metalloproteinase inhibitor. Five overlapping cDNA clones contain most of the information for a prominent 4.5-kilobase transcript that was detected in RNA from mouse fibroblasts and adult tissues. Sequence analysis revealed an open reading frame (ORF) for a protein of 212 amino acids that is 80% identical to chicken inhibitor of metalloproteinases-3 (ChIMP-3). The 3'-untranslated sequence also showed remarkable conservation with the chicken gene. The ORF directed the expression of a 24-kDa protein in COS-1 cells that localized to the extracellular matrix (ECM). On the basis of these similarities we propose to identify the new gene as murine tissue inhibitor of metalloproteinases-3 (TIMP-3). Mouse C3H 10T1/2 fibroblasts produced a 24-kDa metalloproteinase inhibitor that also localized to the ECM and was recognized by a polyclonal antibody to ChIMP-3. Like TIMP-1, TIMP-3 was highly inducible in mouse C3H 10T1/2 fibroblasts by phorbol ester (PMA), epidermal growth factor (EGF), and transforming growth factor-beta 1, but nuclear run-on assays showed that the on/off transcription kinetics were faster for TIMP-3 than TIMP-1. A major difference in vitro was the stimulation of expression of TIMP-3 by dexamethasone which inhibits EGF- and PMA-induced TIMP-1 transcription. Also, TIMP-3 showed a distinctive pattern of expression in adult tissues with abundant transcripts detected in kidney, lung, and brain but only low levels detected in bone, a prominent location of TIMP-1 transcripts. We propose that TIMP-3 functions in a tissue-specific fashion as part of an acute response to remodeling stimuli.

Role of the 21-kDa protein TIMP-3 in oncogenic transformation of cultured chicken embryo fibroblasts.

The 21-kDa protein is an extracellular matrix (ECM) component whose synthesis is stimulated transiently during oncogenic transformation of chicken embryo fibroblasts (CEF) or after treatment of normal cells with the tumor promoter phorbol 12-myristate 13-acetate. Biochemical characterization indicates that the protein is related, but not identical, to two members of the family of tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2. The cDNA of the 21-kDa protein was recently cloned, and based upon its deduced amino acid sequence and other supporting data we propose that it is another member of this family, a TIMP-3. We now report electrophoretic purification of sufficient quantities of this protein to determine its function. The protein promotes the detachment of transforming cells from the ECM. Although its presence in the matrix may be necessary for cell release it is not the only factor involved because it does not influence the adhesive properties of nontransformed cells. It also appears to accelerate the morphological changes associated with cell transformation and stimulates the proliferation of growth-retarded, nontransformed cells maintained under low serum conditions. Based on these data we hypothesize that the 21-kDa protein promotes the development of the transformed phenotype in cultured cells.

A new inhibitor of metalloproteinases from chicken: ChIMP-3. A third member of the TIMP family.

We report cDNA cloning and primary structure of a new metalloproteinase inhibitor (ChIMP-3) produced by chicken embryo fibroblasts. ChIMP-3, formerly called the 21-kDa protein, is one of five ChIMPs (Chicken Inhibitor of MetalloProteinases). In this paper, we report that of the three most abundant ChIMPs, ChIMP-3 and ChIMP-a are extracellular matrix components, whereas ChIMP-2 is found in the media conditioned by the cells. Treatment of ChIMP-3 and ChIMP-a with N-glycosidase-F indicates that ChIMP-a is N-glycosylated whereas ChIMP-3 is not. The deduced amino acid sequence of ChIMP-3 predicts a protein whose properties are consistent with experimental measurements. Analysis of sequence alignments with the two previously described members of the TIMP (tissue inhibitor of metalloproteinases) family, TIMP-1 and TIMP-2, from various species indicates that ChIMP-3 is a related but distinct protein. This conclusion is supported by lack of significant binding with anti-TIMP-1 and anti-TIMP-2 antibodies. Based on these data, its unusual solubility properties, and its exclusive location in the matrix, we propose that ChIMP-3 is a new member of this family of metalloproteinase inhibitors, a TIMP-3.

The 21-kDa protein is a transformation-sensitive metalloproteinase inhibitor of chicken fibroblasts.

We report the electrophoretic purification and characterization of the 21-kDa protein, an extracellular matrix component synthesized during the early stages of transformation of chicken embryo fibroblasts infected with Rous sarcoma virus (Blenis, J., and Hawkes, S. P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 770-774; Blenis, J., and Hawkes, S. P. (1984) J. Biol. Chem. 259, 11563-11570). The NH2-terminal amino acid sequence of the protein is greater than 60% identical to a consensus sequence of mammalian tissue inhibitor of metalloproteinases (TIMP). It shares several biochemical properties with other metalloproteinase inhibitors, including evidence of intrachain disulfide bonds and resistance to cleavage by trypsin. An electrophoretic assay employing a metal ion-dependent gelatinase from conditioned cell culture medium demonstrates inhibitor activity for purified 21-kDa protein. The 21-kDa protein is the major inhibitor in the extracellular matrix and appears unique in solubility properties among inhibitors with a TIMP-like sequence. Statistical analysis of amino acid composition data for these inhibitors defines two distinct groups (TIMP and TIMP-2) and supports a close relationship for the 21-kDa protein with the TIMP group. However, the apparent size and lack of glycosylation align it more closely with the TIMP-2 group of proteins. Therefore, it is possible that the 21-kDa protein is a variant of TIMP or, alternatively, represents a third protein within the metalloproteinase inhibitor family. This report provides the first evidence that avian metalloproteinase inhibitors are similar in sequence to their mammalian counterparts.

Molecular weight dependence of hyaluronic acid produced during oncogenic transformation.

Cultured chicken embryo fibroblasts synthesize two distinct molecular size classes of hyaluronic acid. The high molecular weight material (form I, 2.98 x 10(6) is the predominant species synthesized by transformed cells, whereas form II (1.42 x 10(5)) is the major product of non-transformed cells. A shift to synthesis of predominantly form I hyaluronic acid is an early transformation event in cells infected with LA24 Rous sarcoma virus and maintained at the permissive temperature for transformation (35 degrees C). Form I hyaluronic acid exhibits greater binding to preparations of cellular fibronectin and to both normal and transformed cells than does form II. Both forms bind more to transformed cells than to normal, uninfected cells. Hyaluronic acid (predominantly form I) isolated from transforming cells stimulates proliferation in growth-retarded, non-transformed cells.

Indirect monitoring of carbon-13 metabolism with NMR: analysis of perfusate with a closed-loop flow system.

NMR studies of living cells and of perfused organs almost invariably require a major effort in the design of either the probe or the support system. For enriched 13C studies of metabolism, it is sometimes possible to use a simpler approach, one amenable to use in narrow-bore high-resolution NMR spectrometers. Since some of the metabolic changes are reflected by changes in the chemical composition of the media, it may be necessary to provide only for NMR measurements of the perfusate. A simple apparatus is described which allows NMR measurement of the perfusate. A commercially available concentric NMR tube, modified to incorporate flow, is inserted in the flow loop and placed in the spectrometer. An example is given of the metabolism of 13C-enriched glucose to lactate by chick embryo fibroblasts grown in a hollow fiber bundle assembly.

Characterization of a transformation-sensitive protein in the extracellular matrix of chicken embryo fibroblasts.

The extracellular matrix of cultured chicken embryo fibroblasts undergoes a number of modifications during the early stages of oncogenic transformation. One alteration is increased production of a small protein (Mr approximately 21,000) which is transiently deposited in the matrix by transforming cells infected with LA24, a temperature-sensitive mutant of Rous sarcoma virus (RSV) (Blenis, J., and Hawkes, S.P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 770-774). This protein is a major component of substratum-associated material (material which remains attached to culture dishes after removal of cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid). Its synthesis is stimulated by transformation of cells with NY68, another ts mutant of RSV, and also by treatment of normal, uninfected cells with the tumor promoter, phorbol myristate acetate. Accessibility of the 21-kDa protein to lactoperoxidase-catalyzed iodination indicates an exposed location within the matrix. The protein binds strongly to the culture dish and/or other matrix components. This interaction can be disrupted by sodium dodecyl sulfate but not by several nonionic detergents, unless beta-mercaptoethanol or KCl (0.5 M) are also present. High concentrations of urea or guanidine hydrochloride also remove the protein from the matrix. The 21-kDa protein is resistant to trypsin, collagenase, and the hydrolytic enzymes associated with cells transformed by the wild-type Prague A RSV but not to Pronase or chymotrypsin. A 21-kDa protein with properties similar to those described above is also detected in the medium and binds to the matrix, suggesting that a potential route of deposition of the 21-kDa protein in the matrix may be via shedding and subsequent interaction with other matrix components.

The effect of the tumor promoter, phorbol myristate acetate (PMA), on hyaluronic acid (HA) synthesis by chicken embryo fibroblasts.

Treatment of chicken embryo fibroblasts (CEF) with the tumor promoter, phorbol myristate acetate (PMA), resulted in a rapid increase in their ability to synthesize the glycosaminoglycan, hyaluronic acid (HA), whereas the parent compound, phorbol, had no effect. CEF cultures incubated with PMA (100 ng/ml) for 6 h resulted in a 15-fold increase in HA synthetase activity compared with phorbol-treated control cultures. The incorporation of [3H]acetate into HA and chemical determination of this polymer also demonstrated increased synthesis of HA by cells treated with PMA. We have previously shown that CEF infected with a temperature-sensitive mutant of Rous sarcoma virus, LA24, exhibit increased synthesis of HA upon transformation. PMA treatment of cells transformed with RSV-LA24 results in a further 1.5-fold stimulation of HA synthesis. These data indicate that PMA causes an increased synthesis of HA in CEF which is analogous to the increased synthesis of HA found in virally transformed CEF.

Transformation-sensitive protein associated with the cell substratum of chicken embryo fibroblasts.

The interaction of cultured cells with their growth substrata has been studied as a function of oncogenic transformation by using chicken embryo fibroblasts infected with the temperature-sensitive mutant of Rous sarcoma virus, LA24, and grown in plastic culture dishes. In comparison to total cell fractions, substratum-associated material (SAM), prepared by EGTA release of transforming cells from culture dishes, is enriched in a 21-kilodalton (kDal) protein. Synthesis and deposition of this protein in SAM are stimulated within hours of transfer of cells to the permissive temperature (35 degrees C), peak around 8 hr, and decline to levels 1.3-fold higher than those of controls at 41 degrees C by 20 hr after the temperature shift. In contrast, incorporation of (3)H-labeled amino acids into newly synthesized fibronectin in SAM is not significantly influenced by the transformation process during this time. Furthermore, although the presence of fibronectin in SAM is influenced by cell density, the 21-kDal protein is increased in SAM of transforming cells at all densities examined. The 21-kDal protein is not present in increased amounts in SAM from normal, uninfected chicken embryo fibroblasts grown at 41 degrees C and 35 degrees C or from cells infected with the wild-type Rous sarcoma virus (Prague A), which are fully transformed. It is not a mannose-containing glycoprotein and does not appear to be phosphorylated. Furthermore, it is not a product of normal cell protein degradation induced by transformation but results from de novo protein synthesis after shift of LA24-infected cells to the permissive temperature. Finally, turnover of the 21-kDal protein is slower at 35 degrees C than at 41 degrees C. This amplifies the effect of increased synthesis and results in a net accumulation in SAM during the early stages of transformation.

Detection of transformed cells using a fluorescent probe: the molecular basis for the differential reaction of fluorescamine with normal and transformed cells.

Normal and transformed fibroblasts can be discriminated by a flow cytometry assay on the basis of their differential reaction with fluorescamine. The cause of altered reactivity of transformed cells with this fluorescent probe has been investigated by a detailed analysis of its reaction with chicken embryo fibroblasts transformed by a temperature sensitive mutant of Rous sarcoma virus. The subcellular distribution of fluorescent adducts characterized by cell fractionation and gel electrophoresis procedures supports the hypothesis that transformed cells possess a surface barrier which decreases the accessibility of fluorescamine to reactive macromolecules. The barrier has been identified as being composed at least partly of hyaluronic acid, because of the ability of purified and specific hyaluronidase (from Streptomyces hyalurolyticus) to modulate the response of transformed cells to fluorescamine. Enzyme treatment of transformed cells prior to reaction with fluorescamine causes them to resemble nontransformed cells both in the nature of components labeled and in their fluorescence intensity. It is suggested that fluorescamine monitors an altered surface hyaluronic acid composition which occurs upon transformation. Its significance is discussed in terms of the known physical properties of the molecule and the finding that it is an early event in the process of transformation.

Detection of an early surface change during oncogenic transformation.

Fluorescamine, which can label surface components of cells grown as monolayers in culture, has been used to probe alterations in chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus, Prague A, LA24. The fluorescence of bound fluorescamine on cells at the permissive temperature (35 degrees) was found to be about 1/3 that of cells cultured at the nonpermissive temperature (41 degrees). During development of the transformed phenotype, i.e., after transfer of the cells from 41 degrees to 35 degrees, the decrease in surface fluorescence was observed to be an early event occurring within the first 4-8 hr after temperature shift. This alteration took place on a time scale similar to that of changes in 2-deoxyglucose transport and an increased rate of DNA synthesis, but before any major morphological changes. The change was related to cell transformation rather than to growth differences of the cells at the two temperatures. Further, it was found that fluorescamine was not monitoring the loss of LETS glycoprotein from the surface or the loss of any other surface components that could be detected by lactoperoxidase-catalyzed iodination of surface proteins. When fluorescamine-labeled components were resolved by polyacrylamide gel electrophoresis, significant differences were seen between components from cells cultured at 35 degrees compared with those from cells cultured at 41 degrees. Based on these results, possible mechanisms accounting for the fluorescence differences are suggested.

Neurotoxins of Bungarus multicinctus vernom. Purification and partial characterization.

The purification to homogeneity of nine neurotoxic components of the venom of Bungarus multicinctus is described. The purified components include alpha-bungarotoxin and two other alpha-type synaptic toxins and beta-bungarotoxin and five other beta-type synaptic toxins. The purified toxins have been characterized by electrophoresis, isoelectric focusing, amino acid analysis, and N-terminal amino acid determination. The alpha-type synaptic neurotoxins constitute a discrete class with molecular weights of 7000-8500, isoelectric points (pI) of 9.0-9.2, and N-terminal isoleucine or methionine. The beta-type synaptic neurotoxins constitute a second group with molecular weights of 20 000-22 000 and pI = 8.8-9.7. Fractions 10 through 13 exhibit a chain structure consisting of a 6000-7000 light chain and a 11 000-15 000 heavy chain apparently covalently stabilized by interchain disulfides. Fractions 9A and 14 were single chains of 11 000-14 000 which resemble the sequenced beta-type synaptic neurotoxin notexin (Halpert, J., and Eaker, D. (1975), J. Biol. Chem. 250, 6990). All of the beta-type synaptic toxins have a single tryptophan and N-terminal aspartic acid or asparagine.

Quantitative determination of transformed cells in a mixed population by stimultaneous fluorescence analysis of cell surface and DNA an individual cells.

Cell-surface labeling with fluorescamine indicates that the fluorescence of Balb 3T3 A31 cells in considerably decreased after both viral and chemical transformation. This phenomenon, coupled with the technique of flow microfluorometry, enabled nontransformed and transformed cells to be distinguished. A second fluroescent probe, propidium iodide, which intercalates into DNA, was used in combination with fluorescamine in order to obtain a ratio of cell-surface labeling to DNA content. This manipulation allowed enhanced resolution of two populations and the detection of small numbers of transformants in a predominantly normal population.