uPA mRNA Levels Research Articles

Elevations of tissue-type plasminogen activator and differential expression of urokinase-type plasminogen activator in diseased aorta.

Elevations of plasmin have been implicated in the pathogenesis of abdominal aortic aneurysms (AAA) because of its ability to digest extracellular matrix proteins. Plasminogen activators regulate the conversion of plasminogen to plasmin. Tissue-type plasminogen activator (tPA) is more important in modulation of fibrinolysis, and urokinase-type plasminogen activator (uPA) is predominant in tissue remodeling. The purpose of this study was to determine the levels of plasminogen activators in diseased aorta because they may be responsible for the increased plasmin levels previously described in AAA. Levels of tPA and uPA in AAA, occlusive, and normal (organ donor) aorta were studied in tissue explant supernatants. Supernatant tPA and uPA levels were measured with an enzyme-linked immunosorbent assay. Northern analysis was used to quantitate uPA messenger RNA (mRNA) levels in aortic tissue. Levels of tPA in the supernatants were similar in occlusive (20 +/- 4 ng/ml) and AAA (23 +/- 8) aorta, but threefold higher than in normal aorta (7 +/- 5; p < 0.005 for normal vs occlusive and p < 0.001 for normal vs AAA). In contrast, uPA supernatant levels were differentially expressed, with the highest level existing in AAA (9.7 +/- 2.7 ng/ml), followed by occlusive (4.9 +/- 3.5), and the lowest levels in normal aorta (1.2 +/- 0.7; p < 0.05 for normal vs occlusive, p < 0.001 for normal vs AAA, and p < 0.005 for occlusive vs AAA). Inhibition of protein or RNA synthesis by addition of cyclohexamide or actinomycin D, respectively, revealed no significant difference between treated and control supernatants, suggesting that the increases were caused by protein release rather than active synthesis. Levels of uPA mRNA followed the same trend as the supernatant uPA levels (AAA 1.07 +/- 0.54, occlusive 0.54 +/- 0.08, and normal aorta 0.01 +/- 0.01). Levels of tPA were similar in aneurysmal and occlusive aorta, but exhibited a threefold increase over normal aorta, suggesting that the elevations of tPA are associated with the arteriosclerosis present in both aneurysmal and occlusive disease. Differences in uPA levels were significant between all three groups, with the highest levels in AAA and the lowest levels in normal specimens. Northern analysis of uPA mRNA followed the same trend, suggesting that the increase in uPA may be regulated at the level of transcription. As uPA plays an important role in tissue remodeling, our findings may also reflect the relative tissue repair activities in these three types of specimens and may explain the previously reported increased levels of plasmin seen in AAA.

Inhibition of Urokinase-type Plasminogen Activator (uPA) Abrogates Myogenesis In Vitro

Urokinase-type plasminogen activator (uPA) is one of the components of blood's fibrinolytic cascade. uPA acts as a broad spectrum proteolytic enzyme involved in different physio-pathological processes including cellular fibrinolysis, adhesion, migration, invasion and remodeling. Here, we present evidence that uPA participates in myogenesis, a process which requires drastic cell membrane reorganization, leading to the plurinucleated myotube from the progenitor myoblast. We have dissected the expression of uPA throughout the different myogenic compartments and found an increase in uPA enzymatic activity associated with myotube formation in C2C12 myoblast cells, with uPA mRNA increasing prior the onset of fusion and differentiation. When both fusion and differentiation were blocked by specific inhibitors (DMSO, cytochalasin B) the levels of uPA were strongly downregulated. This process was reversible and specific: the removal of the inhibitors immediately restored the levels of uPA mRNA while the specific inhibition of uPA enzymatic activity by an anti-uPA antibody resulted in a 50% reduction of the extent of fusion and in the abrogation of muscle-specific gene products, such as alpha-actin and MyoD. Moreover, the conversion of fibroblasts to muscle-like cells upon acquisition of MyoD resulted in a dramatic increase of uPA mRNA, which was partially due to transcriptional activation of the uPA gene. These results indicate that the increase in uPA expression prior to fusion and differentiation occurs via a MyoD-mediated mechanism whereas the normal MyoD expression requires the plasminogen activation-dependent activity of this protease. Therefore, these studies extend the sphere of influence of myogenic factors to fibrinolysis, an intrinsic component of the hematological system. Taken together, one mechanism used by the myoblast cell to become a differentiated myotube, involving the inductive extracellular proteolysis of urokinase, is proposed.

Coordinated and cell-specific induction of both physiological plasminogen activators creates functionally redundant mechanisms for plasmin formation during ovulation.

Several lines of indirect evidence indicate that plasmin-mediated proteolysis plays a role in the breakdown of the follicle wall during ovulation. Consistent with this, the ovulation efficiency of mice lacking the two known physiological plasminogen activators (PAs), tissue-type PA (tPA) and urokinase-type PA (uPA), is reduced by 26%. Surprisingly, mice with a single deficiency of either tPA or uPA gene function were normal in their capacity to ovulate. In this study we used in situ hybridization and casein in situ zymography to localize the expression of messenger RNAs (mRNAs) encoding PAs and PA inhibitors and to examine the net PA activity in the mouse ovary at the time of ovulation. Although uPA mRNA expressed by granulosa cells is the most abundant and dramatically up-regulated PA before ovulation, a previously unnoticed coordinated induction oftPA mRNA was found in thecal-interstitial tissue. The existence of redundant mechanisms for plasmin production in the ovary may be the cause of the normal ovulation efficiency in single deficient mice lacking tPA or uPA. The expression of mRNAs for PA inhibitors, types 1 and 2, was low in the ovary, with minor inductions at restricted time points. In contrast, expression of protease nexin-1 (PN-1) by granulosa cells was high during the entire periovulatory period. Among subpopulations of granulosa cells, the expression of PN-1 and uPA was heterogeneous and complementary. Cumulus cells expressed high levels of PN-1 mRNA and low levels of uPA mRNA, thereby providing an inhibitory activity that may protect the mucified matrix of the cumulus oocyte complex from proteolytic degradation.

Coordinated and cell-specific induction of both physiological plasminogen activators creates functionally redundant mechanisms for plasmin formation during ovulation

Several lines of indirect evidence indicate that plasmin-mediated proteolysis plays a role in the breakdown of the follicle wall during ovulation. Consistent with this, the ovulation efficiency of mice lacking the two known physiological plasminogen activators (PAs), tissue-type PA (tPA) and urokinase-type PA (uPA), is reduced by 26%. Surprisingly, mice with a single deficiency of either tPA or uPA gene function were normal in their capacity to ovulate. In this study we used in situ hybridization and casein in situ zymography to localize the expression of messenger RNAs (mRNAs) encoding PAs and PA inhibitors and to examine the net PA activity in the mouse ovary at the time of ovulation. Although uPA mRNA expressed by granulosa cells is the most abundant and dramatically up-regulated PA before ovulation, a previously unnoticed coordinated induction oftPA mRNA was found in thecal-interstitial tissue. The existence of redundant mechanisms for plasmin production in the ovary may be the cause of the normal ovulation efficiency in single deficient mice lacking tPA or uPA. The expression of mRNAs for PA inhibitors, types 1 and 2, was low in the ovary, with minor inductions at restricted time points. In contrast, expression of protease nexin-1 (PN-1) by granulosa cells was high during the entire periovulatory period. Among subpopulations of granulosa cells, the expression of PN-1 and uPA was heterogeneous and complementary. Cumulus cells expressed high levels of PN-1 mRNA and low levels of uPA mRNA, thereby providing an inhibitory activity that may protect the mucified matrix of the cumulus oocyte complex from proteolytic degradation.

Embryo implantation in mouse: fetomaternal coordination in the pattern of expression of uPA, uPAR, PAI-1 and α 2MRLRP genes

During the process of embryo implantation, trophoblast cells invade deep into uterine stroma and play a key role in establishing fetomaternal exchange of molecules. We have studied the in vivo expression patterns of the molecules of the urokinase system, during the process of mouse embryo implantation and early placentation. The sites of synthesis of urokinase-type plasminogen activator (uPA), uPA-receptor (uPAR), plasminogen activator inhibitor type 1 (PAI-1) and α 2-macroglobulin receptor/low density lipoprotein receptor-related protein (α 2MRLRP) transcripts were determined by in situ hybridization. These genes were found to be expressed in a finely regulated pattern. High levels of uPA mRNA were found in invasive trophoblast cells, while the same cells did not appear to synthesize PAI-1. Starting from day 6.5, endothelial cells of newly forming vessels also transcribed uPA gene. uPAR and α 2MRLRP were in all stages expressed by decidual tissue, and their expression domains overlapped in large areas. Immunohistochemistry with uPA and PAI-1 antibodies revealed areas of co-localization of these secreted proteins with the expression domains of uPAR and α 2MRLRP, which is of great interest in view of the role of these two receptors in clearing uPA-PAI-1 complexes. In situ zymography demonstrated the pressence of active uPA in the ectoplacental cone region at 7.5 and 8.5 days. Our studies outline the expression of a set of functionally related genes that is well coordinated between fetal and maternal tissues. This coordination may model other physiological and pathological invasive processes.

Effects of thrombin on steroid-modulated cultured endometrial stromal cell fibrinolytic potential.

By virtue of their unique chronic expression of tissue factor, the primary initiator of hemostasis, decidualized endometrial stromal cells are capable of significant thrombin generation after vascular disruption. In addition to its potent procoagulant effects, thrombin modifies endothelial and glomerular cell fibrinolytic activity. Therefore, we evaluated whether thrombin affected the expression of endometrial stromal cell urokinase-type (uPA) and tissue-type (tPA) plasminogen activators and their primary inhibitor, type 1 plasminogen activator inhibitor (PAI-1), and whether ovarian steroids modulated putative thrombin effects. Confluent stromal cell cultures were incubated in a defined medium containing vehicle control, 10(-8) mol/L estradiol (E2), 10(-7) mol/L medroxyprogesterone acetate (MPA), or E2 plus MPA for 4 days. The medium was then collected and exchanged for medium containing the corresponding steroids with or without thrombin and the specific thrombin inhibitor, D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, for an additional 24 h. The conditioned medium was then collected and analyzed for immunoreactive (ir) uPA, tPA, and PAI-1 by enzyme-linked immunosorbent assay and for PA activity by chromogenic assay, whereas Northern analysis of the cells was employed to evaluate the expression of thrombin receptor, uPA, tPA, and PAI-1 messenger ribonucleic acid (mRNA) species. The latter studies revealed that confluent cultures incubated in defined medium expressed the 3.45-kilobase thrombin receptor message. Steady state levels of thrombin receptor mRNA were unaffected by exogenous steroids. Thrombin added in the absence of exogenous steroids elevated concentrations of ir tPA, uPA, and PAI-1 compared with control cultures. Conversely, in the absence of added thrombin, MPA added alone or together with E2 inhibited levels of ir tPA and uPA while stimulating PAI-1 levels despite the lack of a response to E2 alone. Interestingly, thrombin counteracted this progestin inhibition of tPA and uPA expression and augmented the progestin-enhanced expression of PAI-1. Northern analysis revealed that steady state levels of tPA and uPA mRNA were also enhanced by thrombin in both control and steroid-containing cultures. Net PA activity reflects the balance between PA and PAI-1. In the absence of thrombin, there is virtually no detectable tPA activity and minimal uPA activity in progestin-exposed cultures. However, thrombin elicited significant increases in tPA and uPA activity in control and E2-treated cultures. Despite the molar excess of PAI-1 in MPA-treated and E2- plus MPA-treated cultures, thrombin reversed progestin inhibition of PA activity. Predictably, the addition of D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, blocked the effects of thrombin on PAI-1, tPA, and uPA protein and mRNA expression and PA activity. In summary, thrombin enhances endometrial stromal cell fibrinolytic and extracellular matrix-degrading protease activity in vitro. Such processes occurring in vivo would probably play a role in menstruation and abnormal uterine bleeding.

Inhibitory Effect of a Synthetic Prostacyclin Analogue, Beraprost, on Urokinase-type Plasminogen Activator Expression in RC-K8 Human Lymphoma Cells

Plasminogen activation by urokinase-type plasminogen activator (uPA) is implicated in tumor invasion and metastasis by the breakdown of extracellular matrix. We have recently demonstrated the inhibitory effect of cAMP on uPA gene transcription in RC-K8 human lymphoma cells (Biochim Biophys Acta 1268: 293-9, 1995). Prostacyclin produced by endothelial cells is shown to increase cellular cAMP levels by activating adenylate cyclase. We, therefore, examined the effect of a stable analogue of prostacyclin, Beraprost, on uPA production in RC-K8 cells. uPA activity gradually increased in the conditioned medium with time. Beraprost (0.1 nM-1.0 microM) inhibited uPA accumulation in a dose-dependent manner without affecting cell viability. Fibrinzymography demonstrated that high and low molecular forms of uPA were present in the conditioned medium and that after Beraprost-treatment all forms of uPA decreased and no PA/PA inhibitor complex was present. Northern blot analysis revealed that after exposure to Beraprost, uPA mRNA levels increased transiently and then rapidly decreased to below control levels. Treatment with Beraprost resulted in a rapid activation of cellular cyclic AMP-dependent protein kinase (PKA). Beraprost completely negated uPA gene expression induced by phorbol myristate acetate, an activator of protein kinase C (PKC). These results suggest that Beraprost inhibits uPA production by suppressing uPA gene expression through the PKA pathway and that PKA-mediated signals are dominant in uPA gene expression as compared to those medicated by PKC. This inhibition of uPA expression by a prostacyclin analogue may be an important fact to explain the mechanism of anti-metastatic effects of prostacyclin.

Induction of plasminogen activator inhibitor type 1 in murine lupus-like glomerulonephritis

Three major components of the plasminogen activators (PA)/plasmin system are synthesized physiologically in glomeruli, and can be involved in glomerular proteolysis and extracellular matrix metabolism: tissue-type PA (tPA), urokinase (uPA) and PA inhibitor type 1 (PAI-1). To explore the possible role of a dysregulation of the plasmin protease system in the development and progression of lupus-like glomerulonephritis, we studied the expression of the renal plasmin protease components during the course of the disease, either acute, induced by IgG3 monoclonal cryoglobulins, or chronic, occurring spontaneously in three different lupus-prone mice: (NZBxNZW)F1, BXSB and MRL-lpr/lpr. RNase protection assays and in situ hybridizations revealed a marked glomerular induction of PAI-1 mRNA abundance without any significant changes in renal tPA and uPA mRNA levels in the two different types of lupus-like glomerulonephritis. The overexpression of PAI-1 mRNA occurred in parallel with a significant decrease in glomerular tPA-catalyzed enzymatic activity as determined by zymographic analysis. In addition, a concomitant increase in glomerular expression of transforming growth factor beta 1 (TGF-beta 1) mRNA was observed. The demonstration of a close correlation between the PAI-1 and TGF-beta 1 mRNA levels and the severity of lupus-like glomerular lesions suggests that a pertubation of the glomerular PA/PAI balance, resulting from a marked TGF-beta 1-mediated induction of PAI-1 gene expression, plays an important role in the progression of lupus-like glomerular lesions, leading to glomerulosclerosis.

Amiloride modulates urokinase gene expression at both transcription and post-transcription levels in human colon cancer cells

Activity of receptor-bound urokinase plasminogen activator (uPA) on the surface of colon cancer cells appears to be a function of the number of uPA receptors. The regulation of uPA therefore may determine the invasive phenotype. The effects of amiloride on the modulation of uPA mRNA and protein induced by phorbol ester (PMA) and cycloheximide (CHX) were studied in four colon cancer cell lines, HCT116, KM12SM, LIM1215 and LS123. Northern blot analyses showed that PMA induced uPA mRNA that peaked at 2-48 h in HCT116 cells. In all colon cancer cell lines tested, the expression of uPA mRNA by PMA was super-induced after the addition of the protein synthesis inhibitor CHX, suggesting that stimulation of uPA gene expression does not require de novo protein synthesis. uPA mRNA was also induced by CHX alone, indicating that there may be a labile protein which inhibits uPA mRNA processing. Amiloride profoundly inhibited uPA mRNA production at concentrations between 0.1-1 mM in the presence or absence of PMA or CHX. uPA protein levels on the colon cancer cell surface reflected PMA induction and amiloride inhibition of uPA mRNA levels. Transcriptional elongation experiments using isolated nuclei indicated that while the induction effects of PMA or CHX on uPA gene expression were mediated at the post-transcriptional level, amiloride acted at both transcription and post-transcription levels. The inhibitory effects of amiloride on uPA gene expression reported in this paper may offer the prospect of developing new therapeutic approaches to the prevention of invasion and metastasis by adenocarcinomas.

Transcriptional and posttranscriptional regulation of urokinase‐type plasminogen activator expression in endothelial cells by basic fibroblast growth factor

The mechanism of induction of urokinase-type plasminogen activator (uPA) by basic fibroblast growth factor (bFGF) was explored in fetal bovine aortic endothelial GM 7373 cells. A three- to four-fold increase in the steady-state levels of uPA mRNA was observed after 6 h of incubation of the cell cultures with bFGF. Accordingly, nuclear run-on experiments showed a 2-2.4-fold increase in the rate of uPA gene transcription during the first 4 h of treatment with the growth factor. bFGF did not affect uPA mRNA stability, as evaluated by chase experiments with the mRNA synthesis inhibitor actinomycin D. Upregulation of uPA mRNA was followed by a delayed increase in uPA protein synthesis paralleled by an increase in secreted and cell-associated uPA activity. Twelve h were required before accumulated uPA mRNA was translated into the corresponding protein. During this time interval, the continuous presence of biologically active bFGF in the extracellular environment represented an absolute requirement for uPA mRNA translation. Substitution of residues Lys-27, Lys-30, and Arg-31 to glutamine residues in the bFGF molecule resulted in a mutant (M1Q-bFGF) that caused uPA mRNA accumulation in the absence of a significant increase in cell-associated uPA activity. M1Q-bFGF also induced an increase in cell-associated uPA activity only when added to the cell cultures in the presence of soluble heparin. These results provide evidence that bFGF can affect uPA expression in endothelial GM 7373 cells both at transcriptional and posttranscriptional translational levels. They also show the possibility to dissociate upregulation of uPA mRNA from upregulation of uPA activity by mutagenesis of the bFGF molecule.

A NOVEL MODEL FOR METASTASIS SUPPRESSION INVOLVING REGULATION OF NUCLEAR PRECURSOR MESSENGER-RNA STABILITY

Our previous data correlated urokinase plasminogen activator (uPA) gene activation with metastasis in different rat mammary carcinomas, However, inconsistent with this was the suppression of the uPA gene in the metastatic rat mammary carcinoma cell line, BC1. We have now re-examined this cell line, selecting in vivo specifically for those BC1 cells capable of lung metastasis. We demonstrate that within the polyclonal BC1 cell line, it is possible to enrich for a metastatic sub-population which synthesizes 4 to 5-fold higher levels of uPA mRNA and enzyme activity. Therefore, the ability of a BC1 cell to metastasize appears to correlate with its escape from suppression of the uPA gene in this tumour. The results are consistent with the mechanism of uPA gene activation in metastatic DMBA-8 rat mammary tumour cells, and suggest a novel model of tumour progression based on regulation of pre-mRNA stability.

Inhibition of NF-KB-Rel A expression by antisense oligodeoxynucleotides suppresses synthesis of urokinase-type plasminogen activator (uPA) but not its inhibitor PAl-1

The essential role of urokinase-type plasminogen activator (uPA) in tumor invasion and metastasis stresses the necessity of a fine-tuned cellular control over its expression. It has been shown that changes in uPA directly correlate with changes in cell invasiveness. We examined the role of Rel-related proteins in uPA synthesis by human ovarian cancer cells by inhibiting their expression using the antisense (AS) oligodeoxynucleotide (ODN) technology. Exposure of OV-MZ-6 cells to 10 microM phosphorothioate (PS)-derivatized AS-ODN directed to Rel A led to a maximal 50% decrease of uPA antigen in cell lysates and a 70% reduction in cell cultures supernatants accompanied by a significant transient decline in uPA mRNA levels. Antisense-PS-ODN directed to NF-kappa B1 (p50) or c-rel had no effect on uPA protein expression. AS-PS-ODN directed to Rel A also affected the proteolytic capacity of OV-MZ-6 cells reflected by an approximately 70% decrease in the fibrinolytic capacity of the cells within 24 h compared to untreated controls. AS-PS-ODN directed to I kappa B alpha expression increased uPA in cell culture supernatants up to 50%. uPA receptor (uPAR) production and synthesis of plasminogen activator inhibitor type-1 (PAI-1) were not altered by either AS-PS-ODN applied. Western blot and gel retardation analyses revealed constitutive expression of Rel-related proteins in nuclear protein extracts of OV-MZ-6 cells. Thus these proteins seem to be implicated in uPA regulation and may thereby contribute to tumor spread and metastasis.

Cloning and characterization of the bovine plasminogen activators uPA and tPA

Abstract A bovine cDNA library was screened with cDNA probes coding for the two human plasminogen activators, uPA and tPA. An almost full-length uPA cDNA and a fulllength tPA cDNA were isolated and sequenced. The almost full-length uPA cDNA of 2228 bp codes for nine amino acids of the signal peptide and the 413 amino acids of the bovine pro uPA. Bovine and human uPA had 81% identical nucleotides and 75% identical amino acids. The tPA cDNA of 2402 bp codes for a prepro protein of 562–566 amino acids. The exact length of the protein depends on which of three putative translational start sites is used. The similarity to human tPA was 78% at the nucleotide level and 82% at the amino acid level. Northern blots could not detect the expression of either uPA or tPA in normal lactating udder. In RNA from a mastitic udder, low levels of both uPA mRNA and tPA mRNA were detected.

Modulation of Urokinase-type Plasminogen Activator Gene Expression by Inflammatory Cytokines in Human pre-B Lymphoma Cell Line RC-K8

We examined the effects of inflammatory cytokines, such as interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF alpha), transforming growth factor-beta (TGF beta) and lipopolysaccharide (LPS), on the urokinase-type plasminogen activator (uPA) gene expression in RC-K8 human pre-B lymphoma cells. Recombinant IL-1 alpha, recombinant IL-1 beta and LPS but not recombinant IL-6, recombinant TNF alpha and TGF beta dose-dependently increased uPA accumulation in the conditioned medium. Northern blot analysis revealed that uPA mRNA levels rapidly increased with a peak induction at 2 h after stimulation with IL-1 alpha and IL- 1 beta, but uPA mRNA increase by LPS began at 9 h after stimulation and the increase was maintained until the experiment ended at 24 h. These responses were independent of de novo synthesis, rather amplified in the presence of a protein synthesis inhibitor. The effects by IL-1 alpha and Il-1 beta were prevented by addition of anti-IL-1 alpha and anti-IL-1 beta neutralizing antibodies, respectively. In contrast, both antibodies did not prevent LPS-induced uPA gene expression. Therefore, it is unlikely that the effect by LPS is through induction of IL-1. Both IL-1 alpha and IL- 1 beta rapidly activated uPA gene transcription, but not increased stability of uPA mRNA. These results suggest that both IL-1 alpha and IL-1 beta cause a rapid activation of uPA gene transcription in which de novo protein synthesis is not required and that LPS induces uPA gene expression independently of the IL-1 pathway. These modulations of uPA production by inflammatory mediators may be implicated in tumor growth and metastasis.

Regulation of plasminogen activator inhibitor-1 (PAI-1) expression by 1,25-dihydroxyvitamin D-3 in normal and malignant rat osteoblasts

In rat calvarial osteoblast-like cells and in clonal osteogenic sarcoma cells (UMR 106-01), 1,25-dihydroxyvitamin D-3 (1,25(OH) 2D 3) enhanced plasminogen activator (PA) activity and decreased PA inhibitor-1 (PAI-1) production over the same concentration range. Steady-state levels of mRNA for PAI-1 were also decreased by 1,25(OH) 2D 3 in a dose-dependent manner, without significant effects on mRNA for either tissue-type PA (tPA) or urokinase-type PA (uPA). When protein synthesis was inhibited by cycloheximide treatment in UMR 106-01 cells, the action of 1,25(OH) 2D 3 on PAI-1 mRNA was abolished, as was observed previously with parathyroid hormone (PTH) treatment. In osteoblast-like cells however, 1,25(OH) 2D 3 and PTH actions differred, in that 1,25(OH) 2D 3 had no effect on either PAI-1 or uPA mRNA levels under conditions of protein synthesis inhibition, whereas PTH decreased PAI_1, and increased uPA mRNA. Identification of proteins involved in these actions may help to explain differences in molecular regulation by PTH and 1,25(OH) 2D 3, two agents which have similar actions on osteoblasts, but employ different signal transduction pathways.

LPS induces major changes in the extracellular proteolytic balance in the murine kidney

Recent investigations have shown that in the murine kidney urokinase (uPA) and tissue-type plasminogen activator (tPA) are synthesized and released in urine by tubular epithelial cells, raising the possibility that plasminogen activators (PAs) may be involved in the maintenance of patency and fluidity in renal tubules. To further investigate the contribution of the PA system in renal pathology, we have determined the effects of LPS on the renal production of PAs: we localized PA-catalyzed proteolysis by zymographic analysis of tissue sections and studied the accumulation of mRNAs for PAs and their inhibitors (PAI-1 and PAI-2) by in situ hybridization. Both a single and two injections of LPS induced a dramatic reduction in urinary and renal uPA enzymatic activity; this decrease in catalytic activity was attributable to a reduction in uPA mRNA levels in both proximal and distal tubules. By contrast, we noticed a marked increase of tPA mRNA content in glomerular cells which was not accompanied by a concomitant increase in tPA-mediated proteolytic activity. In addition, a major up-regulation in PAI-1 mRNA levels was observed throughout the kidney, while PAI-2 mRNA was not detectable in the kidneys of control or LPS-injected animals. Our investigations document the profound alterations of the PA/PAI balance in renal tissue following in vivo LPS administration. They suggest that imbalanced extracellular proteolysis might participate in the alterations of kidney function observed in septic shock.

Okadaic acid-dependent induction of the urokinase-type plasminogen activator gene associated with stabilization and autoregulation of c-Jun.

We have previously shown that the tumor promoter okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, transcriptionally induces the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells. This induction occurs independently of the protein kinase C- and cAMP-dependent signaling pathways. Here we show that a sequence located 2.0 kilobases upstream of the uPA gene, which resembles an AP-1-recognition sequence, mediates the action of OA. DNA-protein interaction studies, together with mRNA and protein analyses, indicate that c-Jun, but not c-Fos, is involved in OA-dependent uPA gene induction. The appearance of high levels of uPA mRNA and DNA binding activity of c-Jun to the AP-1-like site correspond to the appearance of c-Jun accumulation, suggesting that c-Jun accumulation is a critical event in OA-dependent uPA gene induction. c-Jun protein levels increase significantly between 100 and 160 min following OA treatment, whereas c-Jun translation increases only slightly in this time frame, suggesting that post-translation mechanisms are also involved in c-Jun accumulation. Pulse-chase analyses shows that OA specifically stabilizes c-Jun. We discuss our results with respect to the possibility that protein phosphatase 2A maintains c-Jun in its down-regulated state in LLC-PK1 cells.

Differential Modulation of Plasminogen Activator Gene Expression by Oncogene-Encoded Protein Tyrosine Kinases

Urokinase-type plasminogen activator (uPA) gene transcription is increased &gt; or = 50-fold in chicken embryo fibroblasts (CEF) following transformation by the protein tyrosine kinase pp60v-src. Protein phosphorylation appears to play a critical role in uPA gene expression in these cells; protein kinase C-activating phorbol esters cooperate with pp60v-src to synergistically increase uPA mRNA, whereas cyclic AMP (cAMP)-dependent protein kinase-activating agents (e.g., 8-bromo cAMP) repress uPA mRNA levels. To explore the relationship between transforming oncogenes and uPA gene expression, uPA mRNA levels were measured in CEF infected with selected avian retroviruses. We report that v-ras and the transforming protein tyrosine kinases v-src, v-yes, and v-ros all increase cellular uPA mRNAs. However, transformation with the protein tyrosine kinase encoded by v-erbB, or the nuclear proteins encoded by v-jun, v-ski, or v-myc, did not increase uPA mRNA detectably. Ras and all of the protein tyrosine kinases analyzed, including the v-erbB product, but none of the nuclear oncoproteins sensitized cells to phorbol ester induction of uPA gene expression. Thus, increased uPA gene expression is not simply a secondary consequence of cell transformation but, rather, is regulated or comodulated by only a subset of oncogene products. Analysis of cells expressing site-directed mutants of pp60v-src showed that the induction of the uPA gene is dependent on protein tyrosine kinase catalytic activity, myristylation, and plasma membrane localization. However, these properties together are not sufficient; an additional feature in the src homology 2 domain is also required. The major sites of serine phosphorylation, serines 12 and 17, and the autophosphorylation site, tyrosine 416, are not essential for uPA gene induction. However, the reduction of uPA mRNA in pp60v-src-transformed cells by 8-bromo cAMP is dependent on tyrosine 416.

Differential modulation of plasminogen activator gene expression by oncogene-encoded protein tyrosine kinases.

Urokinase-type plasminogen activator (uPA) gene transcription is increased > or = 50-fold in chicken embryo fibroblasts (CEF) following transformation by the protein tyrosine kinase pp60v-src. Protein phosphorylation appears to play a critical role in uPA gene expression in these cells; protein kinase C-activating phorbol esters cooperate with pp60v-src to synergistically increase uPA mRNA, whereas cyclic AMP (cAMP)-dependent protein kinase-activating agents (e.g., 8-bromo cAMP) repress uPA mRNA levels. To explore the relationship between transforming oncogenes and uPA gene expression, uPA mRNA levels were measured in CEF infected with selected avian retroviruses. We report that v-ras and the transforming protein tyrosine kinases v-src, v-yes, and v-ros all increase cellular uPA mRNAs. However, transformation with the protein tyrosine kinase encoded by v-erbB, or the nuclear proteins encoded by v-jun, v-ski, or v-myc, did not increase uPA mRNA detectably. Ras and all of the protein tyrosine kinases analyzed, including the v-erbB product, but none of the nuclear oncoproteins sensitized cells to phorbol ester induction of uPA gene expression. Thus, increased uPA gene expression is not simply a secondary consequence of cell transformation but, rather, is regulated or comodulated by only a subset of oncogene products. Analysis of cells expressing site-directed mutants of pp60v-src showed that the induction of the uPA gene is dependent on protein tyrosine kinase catalytic activity, myristylation, and plasma membrane localization. However, these properties together are not sufficient; an additional feature in the src homology 2 domain is also required. The major sites of serine phosphorylation, serines 12 and 17, and the autophosphorylation site, tyrosine 416, are not essential for uPA gene induction. However, the reduction of uPA mRNA in pp60v-src-transformed cells by 8-bromo cAMP is dependent on tyrosine 416.

Transforming growth factor-beta 1 stimulates macrophage urokinase expression and release of matrix-bound basic fibroblast growth factor.

Macrophage expression of urokinase-type plasminogen activator (uPA) appears to play a role in their release of matrix-bound basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta). In experiments reported here, we have examined the potential regulatory effects of bFGF and TGF-beta 1 on macrophage uPA expression. TGF-beta 1 stimulated in a dose- and time-dependent manner the expression of secreted membrane and intracellular uPA activities by a macrophage cell line (RAW264.7). When examined at similar concentrations, bFGF had little effect, and interleukin-1 alpha, tumor necrosis factor-alpha, and monocyte colony stimulating factor had no effect on macrophage uPA expression. Exposure of macrophages to TGF-beta 1 led to a rapid and sustained increase in the steady-state levels of uPA mRNA that was independent of de novo protein synthesis and was completely inhibited by actinomycin D. However, the TGF-beta 1-induced increase in uPA mRNA was largely unaffected by subsequent incubation of cells with actinomycin D. The protein kinase C inhibitor H7 markedly reduced the ability of TGF-beta 1 to stimulate expression of uPA activity. Likewise, okadaic acid and microcystin, inhibitors of serine/threonine phosphatases, potentiated the ability of TGF-beta 1 to upregulate macrophage uPA expression. TGF-beta 1 primed cells converted nearly all added plasminogen to plasmin and expressed sixfold more membrane-bound plasmin than control cells. Preincubation of TGF-beta 1 with either serum or methylamine-modified alpha 2-macroglobulin did not affect its ability to induce macrophage uPA expression. When control and TGF-beta 1-primed macrophages were cultured on matrices containing bound 125I-bFGF, their release of 125I-bFGF was increased five and tenfold, respectively, in the presence of plasminogen. The ability of TGF-beta to induce macrophage uPA expression and the plasmin-dependent release of matrix-bound bFGF may provide an indirect mechanism by which TGF-beta stimulates angiogenesis.