Synergistic induction of tissue-type plasminogen activator gene expression by glucocorticoids and cyclic nucleotides in rat HTC hepatoma cells.

We have reported previously that tissue-type plasminogen activator (tPA) gene expression is regulated by glucocorticoids and cyclic nucleotides in HTC rat hepatoma cells. Incubation of HTC cells with the synthetic glucocorticoid dexamethasone (Dex) transiently increases tPA messenger RNA accumulation 2-fold, whereas incubation with 8-bromo-cAMP (cAMP) alone results in a sustained 2-fold increase. Nuclear run-on studies indicate that these effects occur at the level of gene transcription. In combination, however, Dex and cAMP act synergistically to induce tPA messenger RNA levels 10- to 15-fold; this synergistic induction is at least in part transcriptional. We now report that this synergistic induction of tPA gene transcription requires concomitant protein synthesis. Furthermore, the action of Dex must precede that of cAMP, and the action of Dex requires ongoing protein synthesis, whereas the action of cAMP has no such requirement. To further investigate the mechanism of the synergistic induction of tPA gene transcription, we cloned the tPA promoter from an HTC genomic library. We established the start site of transcription in HTC cells by primer extension and determined the nucleotide sequence of 2.3 kilobase-pairs (kb) of the 5'-flanking region, including 1.7 kb of sequence not previously reported. A 2.3-kb segment of the rat tPA promoter has been ligated to a chloramphenicol acetyltransferase reporter gene and its hormonal regulation evaluated in transient and stable transfection studies in HTC cells. Although this promoter length is sufficient to mediate the 2-fold induction in gene expression seen with cAMP alone, it is not sufficient to recapitulate the synergistic induction of endogenous tPA gene transcription seen with Dex plus cAMP in combination. We have ruled out relief of transcriptional arrest as the mechanism of the synergistic induction. Therefore, we suggest that sequences lying outside the most proximal 2.3 kb of tPA promoter mediate the synergistic interaction of Dex and cAMP.

Plasminogen activator inhibitor-1 is an immediate early response gene in regenerating rat liver.

Plasminogen activator inhibitor-1 (PAI-1), a M(r) 50,000 serine protease inhibitor, is the major physiological inhibitor of plasminogen activation. Quiescent rat hepatocytes do not express the PAI-1 gene in vivo; however, PAI-1 is synthesized both by primary cultures of rat hepatocytes and by hepatoma cells in vitro. Furthermore, PAI-1 is expressed by fibroblastic cells in vitro, in response to mitogen stimulation, suggesting a possible connection between hepatocyte PAI-1 expression and cell proliferation. To determine whether PAI-1 is an early growth response gene in hepatocytes in vivo, we analyzed its expression in regenerating rat liver. Male rats underwent partial (70%) hepatectomy (PH) or sham operation (SO), and liver samples were analyzed by Northern blot analysis and in situ hybridization. PAI-1 mRNA was not present at time 0 h, nor at any other time in SO rats but was induced rapidly in regenerating livers, peaking at 2 h and declining to negligible levels by 8 h posthepatectomy. This induction was not inhibited by cycloheximide. In situ hybridization analysis localized PAI-1 transcripts to hepatocytes. Immunohistochemical analysis demonstrated PAI-1-specific staining in hepatocytes in the livers of both PH and SO rats, but the temporal and spatial distribution profiles differed between PH and SO rats. Our studies demonstrate that PAI-1 is an immediate early response gene, transiently expressed in regenerating liver, expression of which may be important in hepatocyte growth and proliferation in vivo.

Mechanism of glucocorticoid induction of the rat plasminogen activator inhibitor-1 gene in HTC rat hepatoma cells: identification of cis-acting regulatory elements.

Type 1 plasminogen activator inhibitor (PAI-1) is the major physiological inhibitor of plasminogen activation, inhibiting both tissue- and urokinase-type plasminogen activators. In HTC rat hepatoma cells, glucocorticoids increase PAI-1 activity, antigen and mRNA accumulation 3- to 5-fold; this increase is due solely to an increase in the rate of PAI-1 gene transcription. We have identified the cis-acting sequences in the 5'-flanking sequence of the HTC PAI-1 gene that mediate this induction. Analysis of a series of hybrid genes containing various portions of the PAI-1 5'-flanking region fused to the chloramphenicol acetyltransferase reporter gene transfected into HTC cells localized the region involved in the transcriptional regulation by glucocorticoids to between -1237 and -764. Electrophoretic mobility shift assays and DNase-I protection assays showed that a glucocorticoid response element (GRE) 15-mer located at -1212 bound the glucocorticoid receptor DNA-binding domain protein in a concentration-dependent manner. Mutations created within this GRE eliminated its ability both to confer a glucocorticoid response and to bind the glucocorticoid receptor. When placed upstream of a heterologous promoter in either orientation, this GRE conferred glucocorticoid inducibility. We, therefore, conclude that the sole cis-acting sequence required for the glucocorticoid response of the PAI-1 gene in rat HTC hepatoma cells is the GRE at -1212.

Regulatory sequences and protein-binding sites involved in the expression of the rat plasminogen activator inhibitor-1 gene.

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor that inhibits both tissue-type and urokinase-type plasminogen activators. Expression of PAI-1 is regulated by growth factors, cytokines, and hormones. To determine the molecular mechanisms involved in the basal expression of the rat PAI-1 gene, we have analyzed the cis-acting sequences and the trans-acting factors involved in the transcription of this gene in the HTC rat hepatoma cell line. DNase I protection analyses revealed eight regions within the first 764 base pairs of 5'-flanking sequence that interact specifically with HTC cell nuclear proteins. The proteins that bind to five of the eight footprinted sites were identified as PEA3-, Sp1-, and CTF/NF-1-like proteins using competition electrophoretic mobility shift assays. The expression of fusion genes containing progressive 5' deletions of the rat PAI-1 promoter linked to the chloramphenicol acetyltransferase reporter gene were analyzed in transient transfection experiments in HTC cells. These studies demonstrated the Sp1 and CTF/NF-1 sites to be important for transcriptional activation. Two of the footprinted sites contain the sequence 5'-TTTGn(n)TCAAT-3' and were shown in competition electrophoretic mobility shift assays to bind the same or related protein(s). Sequences containing these sites, from -764 to -628 base pairs, and from -266 to -188 base pairs, were identified in functional studies as repressor elements of transcription.

Isolation and characterization of the rat plasminogen activator inhibitor-1 gene.

We have reported that glucocorticoids and cyclic nucleotides regulate tissue-type plasminogen activator activity in HTC rat hepatoma cells primarily by modulating plasminogen activator inhibitor (PAI-1) gene expression. To investigate the molecular mechanisms underlying this hormonal regulation, we have cloned the rat PAI-1 gene from an HTC genomic library. The gene is approximately 10.5 kilobases (kb) in size and is comprised of nine exons and eight introns. Comparison of this gene with the human PAI-1 gene (Bosma, P. J., van den Berg, E., Kooistra, T., Siemieniak, D. R., and Slightom, J. L. (1988) J. Biol. Chem. 263, 9129-9141) revealed strict conservation of the exon-intron structure. The start site of transcription is identical to that in the human PAI-1 gene. The complete nucleotide sequence of the rat PAI-1 gene, including 2.4 kb of 5'- and 1 kb of 3'-flanking DNA, has been determined. Two regions in the 5'-flanking sequence of the rat PAI-1 gene show a high degree of similarity to sequences in the upstream region of the human PAI-1 gene: a 60-base pair region from -90 to the TATA box (90% identical), and a more distal sequence located at -753 to -510 (greater than 80% identical). Both of these regions have been shown to be involved in the glucocorticoid regulation of expression of the human PAI-1 gene (van Zonneveld, A.-J., Curriden, S. A., and Loskutoff, D. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5525-5529), although neither region contains the hexamer consensus sequence for glucocorticoid response elements. Five putative glucocofticoid response elements, 100% identical to the hexamer consensus sequence, were identified in the 5'-flanking region of the rat gene, none of which is present in the regions of sequence similarity. Seven putative cyclic AMP response elements that are 75% identical to the octamer consensus sequence are located upstream of the rat gene. There is only one such sequence within the 5'-flanking region of the human gene; however, that sequence, at -61, is 100% conserved in the rat gene.

Determination of exon-intron structure: a novel application of the polymerase chain reaction technique.

We describe a novel application of the polymerase chain reaction (PCR) technique of DNA amplification to study the exon-intron structure of the rat plasminogen activator inhibitor (PAI-1) gene. This technique is relatively simple and also allows the isolation of introns for sequencing. Primers were selected based on a knowledge of the cDNA sequences of human and rat PAI-1 and of the gene structure of human PAI-1. However, knowledge of a cDNA sequence and/or the structure of a gene in another species is not a prerequisite. Sequences selected from positions along the cDNA of interest could be used to amplify the DNA either from an isolated but uncharacterized gene or directly from genomic DNA, making this technique generally applicable. Thus, this method is a useful advance in the study of gene structure and evolution.

The occurrence of the alpha G-Philadelphia-globin allele on a double-locus chromosome.

Hb G-Philadelphia, an alpha-globin allele, is expressed as either 20%, 30%, or 40% of the total hemoglobin. Restriction analyses published thus far have shown that among persons with 30% and 40% hemoglobin (Hb) G the alpha G allele is seen only in a single-locus haplotype. We now report the identification of a second haplotype in which the alpha G allele is found in tandem with an alpha A allele. This haplotype has been found present in DNA from the members of one family in which Hb G is expressed as 20% of the total hemoglobin, determined by both cellulose acetate electrophoresis and high-performance liquid chromatography (HPLC). Synthesis was balanced in all individuals. The identification of a variant alpha-globin allele in two distinct haplotypes presents the possibility of independent mutation. However, an alternative explanation cannot be ruled out; namely, that the original allele may have become distributed among the two haplotypes by unequal crossing-over.