Identification and cDNA cloning of a novel RNA-binding protein that interacts with the cyclic nucleotide-responsive sequence in the Type-1 plasminogen activator inhibitor mRNA.

Incubation of HTC rat hepatoma cells with 8-bromo-cAMP results in a 3-fold increase in the rate of degradation of type-1 plasminogen activator inhibitor (PAI-1) mRNA. We have reported previously that the 3'-most 134 nt of the PAI-1 mRNA is able to confer cyclic nucleotide regulation of message stability onto a heterologous transcript. R-EMSA and UV cross-linking experiments have shown that this 134 nt cyclic nucleotide-responsive sequence (CRS) binds HTC cell cytoplasmic proteins ranging in size from 38 to 76 kDa. Mutations in the A-rich region of the CRS both eliminate cyclic nucleotide regulation of mRNA decay and abolish RN-protein complex formation, suggesting that these RNA-binding proteins may be important regulators of mRNA stability. By sequential R-EMSA and SDS-PAGE we have purified a protein from HTC cell polysomes that binds to the PAI-1 CRS. N-terminal sequence analysis and a search of protein data bases revealed identity with two human sequences of unknown function. We have expressed one of these sequences in E. coli and confirmed that the recombinant protein interacts specifically with the PAI-1 CRS. Mutation of the A-rich portion of the PAI-1 CRS reduces binding by the recombinant PAI-1 RNA-binding protein. The amino acid sequence of this protein includes an RGG box and two arginine-rich regions, but does not include other recognizable RNA binding motifs. Detailed analyses of nucleic acid and protein data bases demonstrate that blocks of this sequence are highly conserved in a number of metazoans, including Arabidopsis, Drosophila, birds, and mammals. Thus, we have described a novel RNA-binding protein that identifies a family of proteins with a previously undefined sequence motif. Our results suggest that this protein, PAI-RBP1, may play a role in regulation of mRNA stability.

Growth state-dependent binding of USF-1 to a proximal promoter E box element in the rat plasminogen activator inhibitor type 1 gene.

Induced PAI-1 gene expression in renal epithelial (NRK-52E, clone EC-1) cells occurs as part of the immediate-early response to serum. PAI-1 transcripts are maximally expressed early in G(1) (within 4 h of serum addition to quiescent EC-1 cells) and then subsequently decline to basal levels prior to entry into DNA synthetic phase. Comparative analysis of PAI-1 mRNA abundance and de novo-synthesized thiolated RNA in quiescent cells, as well as at 4 h (early G(1)) and 20 h (late G(2)) postserum addition, in conjunction with RNA decay measurements indicated that PAI-1 gene regulation upon growth activation was predominantly transcriptional. An E box motif (CACGTG), important in the induced expression of some growth state-dependent genes, mapped to nucleotides -160 to -165 upstream of the transcription start site in the PAI-1 proximal promoter. Mobility-shift assessments, using a 18-bp deoxyoligonucleotide construct containing the E box within the context of PAI-1-specific flanking sequences, confirmed binding of EC-1 nuclear protein(s) to this probe and, specifically, to the E box hexanucleotide site. The specificity of this protein-probe interaction was verified by competition analyses with double-stranded DNA constructs that included E box deoxyoligonucleotides with non-PAI-1 flanking bases, mutant E box sequences incapable of binding NRK nuclear proteins, and unrelated (i.e., AP-1) target motifs. Extract immunodepletion and supershift/complex-blocking experiments identified one PAI-1 E box-binding protein to be upstream stimulatory factor-1 (USF-1), a member of the HLH family of transcription factors. Mutation of the CACGTG site to TCCGTG in an 18-bp PAI-1 probe inhibited the formation of USF-1-containing complexes confirming that an intact E box motif at -160 to -165 bp in the PAI-1 promoter and, in particular, the CA residues at -165 and -164 are essential for USF-1 binding. Incorporation of this 2 bp change into a reporter construct containing 764 bp of the proximal PAI-1 "promoter" ligated to a CAT gene effectively reduced (by 74%) CAT activity in cycling cells. An intact E box motif at nucleotides -160 to -165 in the PAI-1 promoter, thus, is an important functional element in the regulation of PAI-1 transcriptional activity in renal cells.

Glucocorticoid receptor inhibits transforming growth factor-beta signaling by directly targeting the transcriptional activation function of Smad3.

The transforming growth factor-beta (TGF-beta) family of cytokines and glucocorticoids regulate diverse biological processes through modulating the expression of target genes. Here we report that glucocorticoid receptor (GR) represses TGF-beta transcriptional activation of the type-1 plasminogen activator inhibitor (PAI-1) gene in a ligand-dependent manner. Similarly, GR represses TGF-beta activation of the TGF-beta responsive sequence containing Smad3/4-binding sites. Using mammalian two-hybrid assays, we demonstrate that GR inhibits transcriptional activation by both Smad3 and Smad4 C-terminal activation domains. Finally, we show that GR interacts with Smad3 both in vitro and in vivo. These results suggest a molecular basis for the cross-regulation between glucocorticoid and TGF-beta signaling pathways.

IL-1beta mediates induction of hepatic type 1 plasminogen activator inhibitor in response to local tissue injury.

Type 1 plasminogen activator inhibitor (PAI-1), a major physiological inhibitor of plasminogen activation, is an important component of the hepatic acute phase response. We studied the acute phase regulation of murine hepatic PAI-1 in response to systemic toxicity and local tissue injury in both wild-type mice and in mice in which the interleukin (IL)-1beta gene had been inactivated by gene targeting. Endotoxin induced plasma PAI-1 antigen levels and PAI-1 mRNA accumulation in liver to the same extent in both wild-type and IL-1beta-deficient mice. In contrast, turpentine increased plasma PAI-1 and hepatic PAI-1 mRNA accumulation in wild-type mice but not in IL-1beta-deficient mice. Intraperitoneal injection of murine IL-1beta rapidly increased plasma PAI-1 and hepatic PAI-1 mRNA in both wild-type and IL-1beta-deficient mice. These results suggest that IL-1beta is a critical inducer of hepatic PAI-1 gene expression during the acute phase response to local tissue injury. In situ hybridization studies revealed that hepatocytes are the cells primarily responsible for the hepatic expression of the PAI-1 gene induced by lipopolysaccharide and turpentine.

Cyclic nucleotide regulation of PAI-1 mRNA stability. Identification of cytosolic proteins that interact with an a-rich sequence.

Incubation of HTC rat hepatoma cells with the cyclic nucleotide analogue 8-bromo-cAMP results in a 3-fold increase in the rate of degradation of type-1 plasminogen activator-inhibitor (PAI-1) mRNA. Previous studies utilizing HTC cells stably transfected with beta-globin:PAI-1 chimeric constructs demonstrated that at least two regions within the PAI-1 3'-untranslated region mediate the cyclic nucleotide-induced destabilization of PAI-1 mRNA; one of these regions is the 3'-most 134 nucleotides (nt) of the PAI-1 mRNA (Heaton, J. H., Tillmann-Bogush, M., Leff, N. S., and Gelehrter, T. D. (1998) J. Biol. Chem. 273, 14261-14268). In the present study, ultraviolet cross-linking analyses of this region demonstrate HTC cell cytosolic mRNA-binding proteins ranging from 38 to 76 kDa, with a major complex migrating at approximately 50 kDa. RNA electrophoretic mobility shift analyses demonstrate high molecular weight multiprotein complexes that specifically interact with the 134-nt cyclic nucleotide-responsive sequence. The 50, 61, and 76 kDa and multiprotein complexes form with an A-rich sequence at the 3' end of the cyclic nucleotide-responsive region; a 38-kDa complex forms with a U-rich region at the 5' end of the 134 nt sequence. Mutation of the A-rich region prevents both the binding of the 50-, 61-, and 76-kDa proteins and formation of the multiprotein complexes, as well as cyclic nucleotide-regulated degradation of chimeric globin:PAI-1 transcripts in HTC cells. These data suggest that the proteins identified in this report play an important role in the cyclic nucleotide regulation of PAI-1 mRNA stability.

Smad4/DPC4 and Smad3 mediate transforming growth factor-beta (TGF-beta) signaling through direct binding to a novel TGF-beta-responsive element in the human plasminogen activator inhibitor-1 promoter.

The transforming growth factor-beta (TGF-beta) family of cytokines mediates multiple biological effects by regulating the expression of target genes. The Smad family proteins function as intracellular signal transducers downstream of the receptors to transmit the TGF-beta signal from cell membrane to nucleus. The mechanisms by which Smads mediate transcriptional activation of target genes is largely unknown. Here we report the identification of a novel TGF-beta-responsive element in the human type 1 plasminogen activator inhibitor promoter that is required for mediating strong transcriptional activation of this gene by TGF-beta. Smad3 and Smad4 are incorporated into a TGF-beta-inducible complex formed on this element following TGF-beta stimulation of human hepatoma cells. Both Smad3 and Smad4 bind directly to this TGF-beta-responsive element through their conserved MH1 domains. These results indicate that Smad3 and Smad4 mediate TGF-beta signaling by directly interacting with a specific response element in a physiological target gene.

Cyclic nucleotide regulation of type-1 plasminogen activator-inhibitor mRNA stability in rat hepatoma cells. Identification of cis-acting sequences.

Type-1 plasminogen activator-inhibitor (PAI-1) is a major physiologic inhibitor of plasminogen activation. Incubation of HTC rat hepatoma cells with the cyclic nucleotide analogue, 8-bromo-cAMP, causes a dramatic increase in tissue-type plasminogen activator activity secondary to a 90% decrease in PAI-1 mRNA. Although 8-bromo-cAMP causes a modest decrease in PAI-1 transcription, regulation is primarily the result of a 3-fold increase in the rate of PAI-1 mRNA degradation. To determine the cis-acting sequences required for cyclic nucleotide regulation, we have stably transfected HTC cells with chimeric genes containing sequences from the rat PAI-1 cDNA and the mouse beta-globin gene and examined the effect of cyclic nucleotides on the decay rate of these transcripts. The mRNA transcribed from the beta-globin gene is stable and not cyclic nucleotide-regulated, whereas the transcript from a construct containing the beta-globin coding region and the PAI-1 3'-untranslated region (UTR) is destabilized in the presence of 8-bromo-cAMP, suggesting that this response is mediated by sequences in the PAI-1 3'-UTR. Analyses by deletion of sequences from this chimeric construct indicate that, whereas more than one region of the PAI-1 3'-UTR can confer cyclic nucleotide responsiveness, the 3'-most 134-nucleotide sequence alone is sufficient to do so. Insertion of PAI-1 sequences within the beta-globin 3'-UTR confirms that the 3'-most 134 nucleotides of PAI-1 mRNA can confer cyclic nucleotide regulation of stability on a heterologous transcript, suggesting that this sequence may play a major role in hormonal regulation of PAI-1 mRNA stability.

Interleukin-1 induction of type-1 plasminogen activator inhibitor (PAI-1) gene expression in the mouse hepatocyte line, AML 12.

Type-1 plasminogen activator inhibitor (PAI-1), the major regulator of fibrinolysis, is an important component of the acute phase (AP) response, the coordinated systemic reaction of an organism to tissue injury. As part of a combined in vivo and in vitro study of AP regulation of PAI-1 gene expression in murine hepatocytes, we have characterized the cytokine regulation of PAI-1 gene expression in AML 12 cells, an established line of normal hepatocytes derived from an adult transgenic mouse overexpressing transforming growth factor alpha. Interleukin (IL)-1 caused a rapid and transient 4-fold increase in PAI-1 mRNA that was maximal at 1 h. Half-maximal induction by IL-1 was obtained at 50 U/ml and maximal effects were seen at approximately 500 U/ml. Tumor necrosis factor alpha induced PAI-1 mRNA accumulation with the same magnitude and time course as IL-1, and was not additive with IL-1. IL-6 and dexamethasone alone had no effect on PAI-1 mRNA accumulation and did not enhance the effect of IL-1. Transforming growth factor beta caused a sustained 5- to 7-fold increase in the accumulation of PAI-1 mRNA that was maximal after 2 to 4 h. The IL-1 induction of PAI-1 was inhibited by actinomycin D, but not by cycloheximide. Nuclear run-on studies demonstrated that IL-1 induced a rapid and transient increase in PAI-1 gene transcription that was maximal at 30 min. IL-1 did not stabilize PAI-1 mRNA, and might, in fact, accelerate its rate of decay. These data demonstrate that IL-1, a potent mediator of AP response, induces the accumulation of PAI-1 mRNA in murine hepatocytes, at least in part, by rapidly and transiently increasing the rate of transcription of the PAI-1 gene.

Production of tissue-type plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1) in mildly cirrhotic rat liver.

We have studied the production of tissue-type plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1) in liver of normal rats and in rats with mild cirrhosis induced by carbon tetrachloride inhalation, to demonstrate the production of these fibrinolytic components and their pathophysiologic role in the liver in vivo. Immunohistochemical study of paraffin-embedded liver sections and fibrin autography of frozen sections showed that the normal rat liver produces very little t-PA or PAI-1. On the contrary, striking t-PA activity and both t-PA and PAI-1 antigens were observed in the cirrhotic liver. Both t-PA and PAI-1 in plasma were also markedly increased in the cirrhotic rats. Because the hepatocyte can internalize t-PA or PA/PAI-1 complexes from circulation, Northern blot analysis of the total liver RNA was performed to demonstrate the endogenous synthesis of t-PA and PAI in the liver. Although the normal liver hardly expresses either t-PA or PAI-1 mRNA, striking t-PA and PAI-1 mRNA expression was observed in the liver of rats with mild cirrhosis. These data demonstrate that t-PA and PAI-1 production is strongly upregulated in the liver in rats with mild cirrhosis. These fibrinolytic components, whose production is closely associated with liver failure, may play important roles in the regulation of hepatocyte proliferation and liver regeneration in vivo.

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.

Induction of plasminogen activator inhibitor-1 in HepG2 human hepatoma cells by mediators of the acute phase response.

The liver plays a central role in the systemic acute phase response of an organism to injury. Plasminogen activator inhibitor-1 (PAI-1), a major regulator of fibrinolysis, is an important component of the acute phase response in humans. The source of plasma PAI-1 has been a matter of controversy, but recent in situ hybridization experiments have demonstrated that human hepatocytes express the PAI-1 gene in vivo. However, little is known about regulation of human hepatic PAI-1 gene expression by mediators of the acute phase response. We have analyzed the regulation of PAI-1 mRNA accumulation by interleukin (IL)-1, IL-6, and dexamethasone, known mediators of the acute phase response, in HepG2 cells, a highly differentiated human hepatoma cell line that produces a broad spectrum of acute phase proteins including PAI-1. Incubation of HepG2 cells with IL-1 resulted in a rapid and transient 40-fold induction of the 3.2-kilobase PAI-1 mRNA and a 30-fold induction of the 2.2-kilobase PAI-1 mRNA. Although IL-6 alone had only a modest effect on PAI-1 expression, in combination with IL-1, it caused a synergistic induction of PAI-1 mRNA accumulation. Dexamethasone alone did not increase PAI-1 mRNA accumulation but enhanced it in combination with IL-1. Using nuclear run-on experiments, we determined that the mechanism by which IL-1 alone, or in combination with IL-6, induced PAI-1 mRNA accumulation was to cause a 10-15-fold, transient stimulation of PAI-1 gene transcription. We found no evidence of an effect of these cytokines on PAI-1 mRNA stability. These data demonstrate that mediators of the acute phase response induce the accumulation of PAI-1 mRNA in human hepatoma cells by rapidly and transiently increasing the transcription of the PAI-1 gene.

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.

Type 1 plasminogen activator inhibitor is not an acute phase reactant in rats. Lack of IL-6- and hepatocyte-dependent synthesis.

The contributing role of hepatocytes and IL-6 in the acute phase-like elevation of plasma type 1 plasminogen activator inhibitor (PAI-1) in vivo is not known. We addressed this question by comparing the effects of two inflammatory stimuli known to increase plasma concentrations of IL-6 on the plasma concentrations and site of synthesis of PAI-1 and cysteine proteinase inhibitor (CPI) in rats. Subcutaneous injection of turpentine results in a sustained increase in plasma IL-6 and CPI Ag levels over a 24-h period. In contrast, plasma PAI-1 activity was not altered by turpentine treatment. Northern blot analysis of poly(A)+ mRNA extracted from freshly isolated hepatocytes of saline- or turpentine-treated animals demonstrated induction of CPI mRNA expression but failed to detect basal or induced PAI-1 or IL-6 mRNA expression. However, PAI-1 mRNA was detected in rat hepatocytes in primary culture for 24 h and was induced by dexamethasone. Intravenous infusion of bacterial LPS (4 mg/kg) induced a sustained increase in plasma CPI Ag and transient increases in plasma IL-6 and PAI-1. Northern blot analysis of freshly isolated, fractionated liver cells indicated that LPS treatment (3 h) induced PAI-1 mRNA expression most significantly in the endothelial and Kupffer cell fractions. IL-6 mRNA expression was induced in Kupffer cells and CPI mRNA was induced in hepatocytes. Immunocytochemical analysis revealed LPS-induced accumulation of PAI-1 Ag associated with the vascular endothelium, subendothelial matrix, and sinusoidal lining cells. Our results indicate that PAI-1 mRNA is not significantly expressed by rat hepatocytes in vivo and that plasma PAI-1 levels are not influenced by increased IL-6 expression in Kupffer cells or in plasma.

Thrombin induction of plasminogen activator inhibitor mRNA in human umbilical vein endothelial cells in culture.

We have previously reported that incubation of human umbilical vein endothelial cells (HUVECs) with human alpha-thrombin causes a time- and concentration-dependent increase in secreted plasminogen activator inhibitor type 1 (PAI-1) activity (Gelehrter TD, Sznycer-Laszuk R. J Clin Invest 1986;77:165-9). Here we report that the regulation of PAI-1 activity by thrombin is secondary to the thrombin-induced increase in PAI-1 mRNA accumulation. Incubation of HUVECs for 6 to 24 hours with 0.3 to 1.0 U/ml thrombin causes a 1.8-fold to 10-fold increase in PAI-1 activity and a 1.5-fold to threefold increase in accumulation of both the 3.2 and the 2.2 kilobase PAI-1 mRNAs. These effects are prevented by the prior addition of hirudin, a specific thrombin inhibitor. Inhibition of RNA synthesis with actinomycin D blocks the thrombin induction of PAI-1 mRNA accumulation. The protein synthesis inhibitor, cycloheximide, which itself markedly stimulates the accumulation of PAI-1, appears to prevent the induction by thrombin, suggesting that thrombin may act by inducing another effector such as interleukin-1. Consistent with this hypothesis is our observation that simultaneous addition of antibodies to interleukin-1-alpha prevents the thrombin induction of PAI-1 activity and mRNA.

Plasminogen activator regulation in osteoblasts: parathyroid hormone inhibition of type-1 plasminogen activator inhibitor and its mRNA.

In order to determine the mechanism by which parathyroid hormone (PTH) stimulates plasminogen activator (PA) activity in rat osteoblasts, we investigated the effect of human PTH(1-34) [hPTH(1-34)] on the synthesis of mRNAs for tissue-type PA (tPA), urokinase-type PA (uPA), and PA inhibitor-1 (PAI-1), and on release of PA activity and PAI-1 protein in both normal rat calvarial osteoblasts and UMR 106-01 osteogenic sarcoma cells. hPTH(1-34) (0.25-25 nM) decreased PAI-1 mRNA and protein, and increased PA activity in both cell types in a dose-dependent manner with ED50 of about 1 nM for both responses. Forskolin and isobutylmethylxanthine also stimulated PA activity and decreased PAI-1 protein and mRNA in both cell types. hPTH(1-34) did not show any consistent effect on tPA and uPA mRNA in calvarial osteoblasts, but a modest (two-fold) increase of both mRNAs was observed in UMR 106-01 cells treated with 25 nM hPTH(1-34). However, when protein synthesis was inhibited with 100 microM cycloheximide, the increase of tPA and uPA mRNA by hPTH(1-34) was enhanced in UMR 106-01 cells and became evident in calvarial osteoblasts. Fibrin autography also revealed that hPTH(1-34) increases tPA and uPA activity, especially after cycloheximide treatment in UMR 106-01 cells. These results strongly suggest that PTH increases PA activity predominantly by decreasing PAI-1 protein production through a cyclic adenosine monophosphate (cAMP)-dependent mechanism in rat osteoblasts. The reduction of PAI-1 protein by PTH results in enhanced action of both tPA and uPA, and would contribute to the specific roles of these PAs in bone.

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.

Glucocorticoid regulation of plasminogen activator inhibitor-1 messenger ribonucleic acid and protein in normal and malignant rat osteoblasts.

Glucocorticoids exert potent inhibitory effects on bone formation. We have previously shown that glucocorticoids suppress plasminogen activator (PA) activity in normal and malignant rat osteoblasts. To clarify the mechanism of this suppression, we investigated the effects of dexamethasone on PA inhibitor-1 (PAI-1), tissue-type PA (tPA), and urokinase-type PA (uPA) expression and also on PAI-1 protein and PA activity in both normal rat calvarial osteoblasts and a clonal osteogenic sarcoma cell line, UMR 106-01. Dexamethasone increased PAI-1 mRNA and protein in both cell types. The increase in PAI-1 protein and the decrease in PA activity were obtained over the same concentration range, with a half-maximally effective concentration of dexamethasone of about 10(-9) M. The increase in PAI-1 mRNA caused by dexamethasone was retained with cycloheximide treatment, but abolished with actinomycin-D. Dexamethasone had no effect on tPA or uPA mRNA in either cell type. The glucocorticoid antagonist RU 486 prevented the effects of dexamethasone on PA activity and PAI-1 protein. Dihydrotestosterone, progesterone, and 17 beta-estradiol did not influence PA activity or PAI-1 formation. Although tPA and uPA protein could not be measured, these results suggest that glucocorticoids suppress PA activity predominantly by increasing PAI-1 synthesis in rat osteoblasts. Suppression of PA activity through actions on PAI-1 formation by glucocorticoids could contribute to the mechanisms by which glucocorticoids inhibit bone formation.

Transcriptional and posttranscriptional regulation of type 1 plasminogen activator inhibitor and tissue-type plasminogen activator gene expression in HTC rat hepatoma cells by glucocorticoids and cyclic nucleotides.

We have reported previously that incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone causes a 90% decrease in tissue-type plasminogen activator (tPA) activity secondary to a 4-fold increase in plasminogen activator inhibitor-1 (PAI-1) mRNA accumulation. Dexamethasone also induces a modest and transient increase in tPA mRNA. The cyclic nucleotide analog 8-bromo-cAMP (cA) causes a greater than 50-fold increase in PA activity, the result of a 90% decrease in PAI-1 and a sustained 2-fold increase in tPA mRNA accumulation. Dexamethasone and cA in combination cause a 150-fold increase in PA activity, the result of an 80% decrease in PAI-1 and a synergistic 15-fold increase in tPA mRNA. To determine the mechanism of this complex hormonal regulation, we have examined rates of synthesis and decay of PAI-1 and tPA mRNAs. Here we report that dexamethasone induces a 5-fold increase in PAI-1 gene transcription and does not significantly alter PAI-1 message decay; PAI-1 mRNA has a half-life of about 4 h in both untreated and dexamethasone-treated cells. In contrast, cA regulates PAI-1 mRNA by both decreasing the rate of PAI-1 gene transcription by 60% and accelerating the rate of PAI-1 message decay. Regulation of tPA by cA, both alone and in combination with dexamethasone, occurs primarily at the level of transcription. Dexamethasone and cA-induced tPA mRNA has a half-life of 2.75 h; tPA mRNA degradation is significantly inhibited by either cycloheximide or actinomycin-D.(ABSTRACT TRUNCATED AT 250 WORDS)