Genes for extracellular-matrix-degrading metalloproteinases and their inhibitor, TIMP, are expressed during early mammalian development.

Extracellular matrix (ECM) remodeling accompanies cell migration, cell-cell interactions, embryo expansion, uterine implantation, and tissue invasion during mammalian embryogenesis. We have found that mouse embryos secrete functional ECM-degrading metalloproteinases, including collagenase and stromelysin, that are inhibitable by the tissue inhibitor of metalloproteinases (TIMP) and that are regulated during peri-implantation development and endoderm differentiation. mRNA transcripts for collagenase, stromelysin, and TIMP were detected as maternal transcripts in the unfertilized egg, were present at the zygote and cleavage stages, and increased at the blastocyst stage and with endoderm differentiation. These data suggest that metalloproteinases function in cell-ECM interactions during growth, development, and implantation of mammalian embryos.

Hyperoxic exposure alters gene expression in the lung. Induction of the tissue inhibitor of metalloproteinases mRNA and other mRNAs.

Exposure to high concentrations of oxygen can result in tissue damage, particularly in the lung. Lung pathology induced by hyperoxia includes changes in lung cell populations and morphology. Presumably, alterations in gene expression underlie some of these cellular changes. In order to better understand the molecular basis of these events, a cDNA library was constructed from the mRNA of the lungs of a hyperoxia-exposed rabbit and differentially screened for clones corresponding to hyperoxia-induced messages. This approach has led to the isolation of four clones, three of which are presented in this communication. One clone corresponds to a message whose steady state levels were induced 6-fold and encodes the tissue inhibitor of metalloproteinases, a protein that plays a key role in the regulation of connective tissue turnover in some cells and potentiates erythroid development in others.

[Natural inhibitor of metalloproteinases: structural and functional study]. / Inhibiteur naturel des métalloprotéinases: étude structurale et fonctionnelle.

A potent collagenase inhibitor was purified from cells of calf aorta medial tissue maintained in culture. This molecule was characterized and identified as TIMP (Tissue Inhibitor of Metalloproteinases). Formation of a TIMP--collagenase complex was demonstrated chromatographically using pure TIMP and pure pig synovial cell collagenase. The N-terminal aminoacid sequence of TIMP was determined and, using appropriate oligonucleotide probes the human genes was cloned from a human cDNA bank. This gene was expressed in E. coli, and fully active TIMP was obtained after a denaturation renaturation process. The interest of TIMP as a model for the design of novel collagenase inhibitors is discussed.

Constitutive production of procollagenase and tissue inhibitor of metalloproteinases by human keratinocytes in culture.

Production of procollagenase and tissue inhibitor of metalloproteinases was demonstrated in human keratinocyte cultures. The two proteins were immunoprecipitated from keratinocyte-conditioned medium with antibodies to human dermal fibroblast collagenase and tissue inhibitor of metalloproteinases and quantitated with enzyme-linked immunosorbent assays. Treatment of the keratinocytes with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate, produced a six to 34-fold increase in procollagenase synthesis and secretion but only a threefold increase in the production of tissue inhibitor of metalloproteinases. Collagenase and tissue inhibitor of metalloproteinases mRNAs were present in normal keratinocytes, were the same size as their fibroblast counterparts, and both increased in response to treatment with 12-0-tetradecanoylphorbol-13-acetate. These data suggest that remodeling of type I collagen may be an important function of human keratinocytes in vivo.

Antisense RNA-induced reduction in murine TIMP levels confers oncogenicity on Swiss 3T3 cells.

Mouse 3T3 cell lines capable of constitutively synthesizing an RNA complementary to the messenger RNA encoding TIMP, tissue inhibitor of metalloproteinases, were constructed by transfection with appropriate plasmid constructs. Many of the lines were down-modulated for TIMP messenger RNA levels and secreted less TIMP into the culture medium. In comparison to noninvasive, nontumorigenic controls, these cells not only were invasive in a human amnion invasion assay, but also were tumorigenic and metastatic in athymic mice. These results indicate that TIMP suppresses oncogenicity, at least in immortal murine 3T3 cells.

Regional localization of the TIMP gene on the human X chromosome. Extension of a conserved synteny and linkage group on proximal Xp.

The gene encoding a tissue inhibitor of metallo-proteinases, TIMP, has previously been shown to be X-linked in both the human and mouse genomes. We have used a series of somatic cell hybrids segregating translocation and deletion X chromosomes to map the TIMP gene on the human X chromosome. In combination with previous data, the gene can be assigned to Xp11.23----Xp11.4. Genetic linkage analyses demonstrate that TIMP is linked to the more distal ornithine transcarbamylase (OTC) locus at a distance of about 22 centimorgans. The data are consistent with the conclusion that TIMP maps to a conserved synteny and linkage group on the proximal short arm of the human X chromosome and on the pericentric region of the mouse X chromosome, including loci for synapsin-1, a member of the raf oncogene family, OTC, and TIMP.

A protein kinase inhibitor gene reduces both basal and multihormone-stimulated prolactin gene transcription.

The possible role of the catalytic subunit of the cAMP-dependent protein kinase in mediating the regulation of prolactin gene transcription has been investigated through the use of a synthetic gene encoding the heat-stable inhibitor of the cAMP-dependent protein kinase. To assess the effects of protein kinase inhibitor expression on cAMP induction of prolactin gene transcription, a marker gene containing the rat prolactin promoter and adjacent 5'-flanking sequences linked to the bacterial chloramphenicol acetyltransferase gene was cotransfected with a protein kinase inhibitor-expression vector. The results demonstrate that the protein kinase inhibitor-expression vector reduced both basal and cAMP-stimulated expression of the cotransfected prolactin-chloramphenicol acetyltransferase gene. A mutant protein kinase inhibitor-expression vector, coding for an inactive inhibitor protein, did not inhibit basal or cAMP-stimulated prolactin gene transcription. Furthermore, the protein kinase inhibitor-expression vector did not inhibit zinc induction of the metallothionein promoter. Analysis of protein kinase activity in transfected cells demonstrated that the protein kinase inhibitor expression vector reduced cAMP-dependent protein kinase activity but did not reduce protein kinase C activity. Nuclease protection experiments confirmed that the effects of the inhibitor vector involved changes in correctly initiated transcripts produced from the prolactin promoter. Surprisingly, the protein kinase inhibitor-expression vector reduced the effects of several different agents including epidermal growth factor, thyrotropin-releasing hormone, phorbol esters, and estrogen on prolactin gene expression to the same extent as it altered cAMP effects.

Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leucine-rich repeats.

The primary structure of the ribonuclease inhibitor from pig liver has been determined by amino acid sequence analysis. The N alpha-acetylated polypeptide chain of 456 amino acids consists of 15 homologous leucine-rich repeats, characterized by leucyl residues at constant positions. Two types of alternating repeats occur, 29 (A) and 28 (B) residues long. The degree of identity between repeats of a given type ranged from 25 to 60%. Only one deletion in the B-repeat was necessary to perfectly align the leucyl residues between the two repeats. Leucine-rich repeats have previously been found in four membrane-bound proteins and one extracellular protein, and their amphiphilic character suggested that they could be involved in membrane binding. Ribonuclease inhibitor is the first example of a cytoplasmic protein containing this type of repeat. It seems likely, therefore, that leucine-rich repeats can have functions other than forming membrane binding structures.

Nucleotide sequence and expression of a Streptomyces griseosporeus proteinaceous alpha-amylase inhibitor (HaimII) gene.

The coding region of the alpha-amylase inhibitor (HaimII) gene from the producing strain Streptomyces griseosporeus YM-25 was localized on an 800-base-pair DNA segment. The nucleotide sequence of a 1,191-base-pair region including the HaimII gene was determined by the dideoxy-chain termination method. The nucleotide sequence data predicted an open reading frame of 363 base pairs starting with an ATG initiation codon and ending with a TGA translational stop codon. The amino acid sequence deduced from the nucleotide sequence indicated that the presumptive pre-HaimII protein extends 37 amino acids to the amino terminus and 6 amino acids to the carboxyl terminus of the mature HaimII protein. The pre-HaimII protein is believed to be processed both during and after secretion. Two forms of the inhibitor, which have a higher molecular weight than that of the HaimII protein isolated from S. griseosporeus, were partially purified from the culture filtrate of Streptomyces lividans containing the cloned HaimII gene.

Presence of transcription regulatory elements within an intron of the virus-inducible murine TIMP gene.

The expression of the gene for the murine tissue inhibitor of metalloproteinases (TIMP) is induced in response to viruses, growth factors, and phorbol esters. In this report we show that the accumulation of TIMP mRNA after Newcastle disease virus induction is caused by transcriptional activation of the gene. Comparison of the sequences of cDNA and genomic clones along with RNase protection and primer extension analyses revealed that the murine TIMP gene possesses multiple cap sites and that the exon 1 consists exclusively of 5'-noncoding sequences. We observed that DNA regions analogous to those found upstream of the virus-inducible interferon genes are present within intron 1 of the TIMP gene. To investigate the possible role of TIMP intron 1 in gene expression, we used a functional assay based on the transfection of plasmids in which the DNA segment to be tested is placed in proximity to a marker gene driven by the heterologous herpes simplex virus thymidine kinase promoter. Our results indicate that TIMP intron 1 contains DNA sequence elements capable of modulating the activity of a heterologous promoter in two different ways: (i) by enhancing constitutive expression and (ii) by conferring virus inducibility. These results suggest that intron 1 may be involved in the transcriptional regulation of TIMP gene expression.

In vitro synthesis of the active tissue inhibitor of metalloproteinases encoded by a complementary DNA from virus-infected murine fibroblasts.

We have recently described the characterization and expression of a murine gene highly homologous to the human tissue inhibitor of metalloproteinases/erythroid potentiating activity (TIMP/EPA) gene. We have also reported that expression of this gene is regulated in response to virus infection. In the present report we describe the use of a cDNA clone derived from mRNA isolated from Newcastle disease virus-induced murine cells to direct in vitro synthesis of proteins encoded by this murine TIMP/EPA gene. This approach was used to analyze structural and functional parameters of the TIMP/EPA protein. Translation experiments using microsomes revealed a murine protein similar in size to that of human TIMP: Mr of approximately 22,000 for the core protein and 28,000 for the processed protein. Processing in microsomes involved N-glycosylation and cleavage of the signal peptide. Both the processed and unprocessed proteins were able to inhibit degradation of collagen by collagenase but unable to inhibit virus replication. Synthesis of truncated TIMP proteins showed that the collagenase-inhibiting activity was not encoded within a delimited portion of the molecule. This result suggests that conformation is probably essential for TIMP activity.

Sequence of the bacteriophage P22 anti-recBCD (abc) genes and properties of P22 abc region deletion mutants.

The nucleotide sequence of a segment of the bacteriophage P22 chromosome to the left (downstream in the PL operon) of the erf gene was determined. Previous studies (A. C. Fenton and A. R. Poteete, 1984, Virology 134, 148-160) have shown that this region encodes a function that is required for efficient growth of P22 in wild-type, but not in recB- Salmonella. The gene or genes encoding this function were designated abc (anti-recBCD). The DNA sequence reveals three open reading frames that potentially encode polypeptides with molecular weights of 10,900, 11,600, and 6600 (in order of transcription). P22 deletion mutants lacking each of the open reading frames were constructed. In addition, plasmids were constructed placing each of the open reading frames under control of the lac UV5 promoter. The phenotypes of the deletion mutants, and the results of plasmid-phage complementation tests, indicate that Abc activity depends primarily on sequences that encode the 11.6-kDa polypeptide; the 10.9-kDa polypeptide-encoding sequence makes a minor contribution to Abc activity as well. These sequences have been designated abc2 and abc1, respectively. The 6.6-kDa polypeptide is apparently uninvolved.

On the use of anti-sense RNA: down-regulation of mRNA encoding a metalloproteinase inhibitor.

In this article we describe methods used for producing anti-sense RNA and the mechanisms by which specific eukaryotic gene functions are inhibited. We also describe some of our studies with plasmid constructs producing anti-sense RNA targeted to a murine metalloproteinase inhibitor and review the evidence for naturally occuring anti-sense transcripts in eukaryotes.

Characterization and expression of a murine gene homologous to human EPA/TIMP: a virus-induced gene in the mouse.

A genomic clone encompassing the entire coding region of a murine gene homologous to human erythroid potentiating activity/tissue inhibitor of metalloproteinase (EPA/TIMP) was isolated and sequenced. Based on alignment with human EPA/TIMP cDNAs we deduce a structure comprising five exons and four introns extending over 4.3 kb of DNA. In mouse and hamster cell lines transcription from this gene and interferon genes is induced by Newcastle Disease virus (NDV). Examination of the 5'-flanking sequences of the gene reveals a set of repeated elements with structural similarity to those previously described as inducer-responsive elements in the human IFN-beta 1 gene. The 4.3-kb DNA fragment encompassing the homologous murine EPA/TIMP gene was transfected into human T98G cells and transfectants tested for NDV inducibility. In contrast to the endogenous human gene, the integrated murine EPA/TIMP gene was NDV-inducible and TIMP activity was detectable in the cell culture fluid.

A growth-responsive gene (16C8) in normal mouse fibroblasts homologous to a human collagenase inhibitor with erythroid-potentiating activity: evidence for inducible and constitutive transcripts.

We present the DNA sequence of an essentially full-length cDNA clone of 16C8, a growth factor-inducible gene isolated from a mouse embryo fibroblast cDNA library. The 0.9-kb mRNA encodes an Mr 22,500 protein that has substantial homology to a human protein with the reported abilities to potentiate erythroid differentiation and to inhibit collagenases and other tissue metalloproteinases. The N-terminus of the predicted protein has a hydrophobic nature characteristic of secreted proteins, and two potential sites for N-linked glycosylation are present. The cytoplasmic concentration of 16C8 mRNA is maximal in mid G1 at about 6 h after serum stimulation of quiescent fibroblasts. Northern blot analysis showed a progressive reduction in the size of the induced 16C8 transcripts with increasing time after serum stimulation. This was shown to be due to the reduction in length of the poly(A) tails. S1 analysis of the 5' portion of the mRNA revealed the presence of three different species of transcript, only one of which was inducible.

Primary structure and cDNA cloning of human fibroblast collagenase inhibitor.

We report the primary structure and cDNA cloning of human fibroblast collagenase inhibitor, a glycoprotein that appears to play a central role in modulating the activity of a number of metalloendoproteases of connective tissue origin including collagenase, gelatinase, and proteoglycanase. Secreted human fibroblast collagenase inhibitor was purified and subjected to automated Edman degradation. The secreted protein consists of 184 amino acid residues; it contains two sites of N-linked oligosaccharide linkage and six disulfide bonds. Synthetic oligonucleotide probes based on selected amino acid sequences of the inhibitor were used to screen a lambda gt10 cDNA library from a human fibroblast line. Two overlapping cDNA clones were characterized to determine the complete coding and noncoding sequences of the specific mRNA. The amino acid sequence deduced from the nucleotide sequence agrees with that determined by protein sequencing. One clone appears to contain the complete 5' end and, in addition, the cDNA sequence predicts a 23-amino acid leader peptide. The other clone represents the 3' end of the mature message and includes a short poly(A)+ tract. This 3' sequence is remarkably similar to a reported cDNA encoding part of the protein derived from mouse fibroblast poly(A)+ RNA. However, this inhibitor has no substantial homology with previously sequenced protease inhibitors.

Differentiation of a human leukemia cell line and expression of collagenase inhibitor.

A human collagenase inhibitor (CI) of Mr 28,500 has been extensively characterized in skin fibroblasts and identified in a variety of connective tissues. Because human alveolar macrophages synthesize and secrete both a collagenase and CI that are immunologically and functionally identical to their counterparts in fibroblasts, we studied the production of such proteins by an immature human cell line (HL60) that can be induced to differentiate along monocytic or granulocytic pathways. The cells failed to synthesize collagenase under any culture condition tested. However, upon exposure to 1,25-dihydroxyvitamin D3 or phorbol esters (PMA), both of which promote monocytic differentiation of HL60, these cells synthesized and released CI in a dose-dependent manner. Furthermore, the extent of CI expression was paralleled by the acquisition by such cells of the monocytic marker 63D3, indicating that inhibitor production and differentiation are closely correlated. This CI was immunologically and functionally identical to that produced by human macrophages and human skin fibroblasts. The quantity of CI synthesized by PMA-stimulated cells was 3- to 5-fold greater than produced by human alveolar macrophages, approximately equal to 1 microgram per 10(6) cells per day. In contrast, undifferentiated HL60 cells produced little or no detectable CI (less than or equal to 10-20 ng per 10(6) cells per day). Interestingly, when HL60 cells were stimulated to undergo granulocytic differentiation by dimethyl sulfoxide or retinoic acid, they also produced the "monocytic" CI.

New applications of microbial products.

Microbial secondary metabolites are now being used for applications other than as antibacterial, antifungal, and antitumor agents. These applications include use against parasites (coccidia, helminths) and insects as well as for animal and plant growth stimulation, immunosuppression, uterocontraction, and other pharmacological activities. Further applications are possible in various areas of pharmacology and agriculture, a development catalyzed by the use of simple enzyme assays for screening prior to testing in intact animals or in the field.