The THP-1 cell line is a urokinase-secreting mononuclear phagocyte with a novel defect in the production of plasminogen activator inhibitor-2

Mononuclear phagocytes regulate the generation of plasmin by secreting urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2). We investigated the production of plasminogen activator (PA) and PA inhibitor by the human monocytic leukemia cell line, THP-1. Similar to U937 monoblast-like cells and peripheral blood monocytes (PBM), THP-1 cells produce a PA that is specifically neutralized by anti-uPA antibody and comigrates with human high molecular mass uPA (54 kDa) on casein-plasminogen zymogaphy. PA activity could be dissociated from intact THP-1 cells by brief treatment with a weak acid-glycine buffer, indicating that the uPA is secreted and bound to receptors on the plasma membrane. Regulation of uPA proceeds normally in THP-1 cells, with cell-associated PA activity increasing from 77 +/- 20 to 163 +/- 26 and 325 +/- 30 mPU/10(6) cells in response to PMA and LPS, respectively; parallel increases in steady state levels of uPA mRNA were observed. In contrast to normal expression of uPA activity, functional PAI-2 could not be demonstrated in either the conditioned media or cell lysates of THP-1 under basal or stimulated conditions. Both U937 and PBM secrete low levels of PA inhibitor activity that increase substantially in response to stimulation with PMA and LPS. Immunoreactive PAI-2, measured by ELISA, was undetectable in THP-1 lysates or conditioned medium, but was consistently present in U937 and PBM, paralleling the presence of PA inhibitor activity. THP-1 cells express low levels of an abnormally sized mRNA for PAI-2 and demonstrate a regulatory defect whereby steady state levels of PAI-2 mRNA are markedly reduced upon stimulation with PMA or LPS. By contrast, U937 and PBM respond to identical stimulation with increases in PAI-2 mRNA. We conclude that THP-1 cells express a structurally abnormal species of PAI-2 mRNA, with complete loss of inhibitory activity as well as altered function of PMA- and LPS-responsive regulatory elements.     

Evidence for intracellular cleavage of plasminogen activator inhibitor type 2 (PAI-2) in normal epidermal keratinocytes

Plasminogen activator inhibitor type 2 (PAI-2) is a serine proteinase inhibitor (serpin), present in high quantities in stratified squamous epithelia. Detergent extracts of human epidermis or cultured keratinocytes contain primarily active, nonglycosylated PAI-2. In keratinocytes, the vast majority of PAI-2 is retained within the cell, supporting the hypothesis that PAI-2 may serve specific intracellular function(s) through interaction with an unknown cytoplasmic proteinase. During interaction with the target proteinase, cleavage of PAI-2 within its reactive site loop leads to the formation of a more stable, "relaxed" conformation (PAI-2r). Using a monoclonal antibody specific for PAI-2r, we demonstrate here that PAI-2r is present in keratinocytes of the granular and basal layers of normal human epidermis. In addition, PAI-2r is detectable in cultured human epidermal keratinocytes, where it is concentrated in a detergent-insoluble fraction within differentiating cells. These data provide evidence for the presence of an endogenous, keratinocyte-derived proteinase that constitutively cleaves intracellular PAI-2 in normal human epidermal keratinocytes. Cleavage of PAI-2 by this proteinase may reflect specific intracellular action of PAI-2 in normal cells. Finally, we demonstrate that a commercially available anti-PAI-2 monoclonal antibody (#3750, American Diagnostica, Greenwich, CT), under native experimental conditions, preferentially recognizes the uncleaved, active form of PAI-2 and does not efficiently detect PAI-2r.     

Dexamethasone inhibition of tissue-type plasminogen activator (tPA) activity: paradoxical induction of both tPA antigen and plasminogen activator inhibitor

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits plasminogen activator (PA) activity and reveals the presence of a specific PA inhibitor (PAI-1). To determine whether the hormonal inhibition of PA activity reflects a decrease in the amount of PA or an increased amount of the inhibitor, or both, we have assayed PA and PAI-1 immunologically. HTC PA was determined to be entirely of the tissue type (tPA), and both free and complexed antigen was quantified by a RIA using rabbit antirat tPA, with rat insulinoma tPA as tracer and standard. PAI-1 was quantified by a Western blot assay using rabbit anti-HTC PAI-1 antibody and purified HTC PAI-1 as standard. Under conditions in which dexamethasone inhibited PA activity by 90%, there was no decrease in the cellular content of tPA antigen. Paradoxically, dexamethasone increased tPA antigen approximately 1.5-fold. Under these same conditions, dexamethasone increased PAI-1 antigen 4- to 5-fold. We conclude that the glucocorticoid inhibition of tPA activity in HTC cells is not secondary to a decrease in the amount of tPA but is secondary to the induction of a specific PA inhibitor.     

Plasminogen activator inhibitor type 2 (PAI-2) is present in normal human conjunctiva

The purpose was to characterize plasminogen activator inhibitor type 2 (PAI-2) expression in normal human conjunctiva in vivo and in vitro. PAI-2 antigen was assayed by immunostaining and immunoblotting of extracts from normal human conjunctival epithelial lysates and conditioned media (CM) of cultured human conjunctival keratinocytes. Immunostaining of normal human conjunctival epithelia revealed that PAI-2 was found consistently in the superficial keratinocytes and, in some biopsies, also in the lower keratinocyte layers. In all cases, PAI-2 was concentrated around the cell periphery. In extracts of conjunctival epithelia and cultured conjunctival keratinocytes, PAI-2 had an apparent molecular weight of 45 kDa, consistent with the non-glycosylated form. The majority of PAI-2, approximately 90%, was cell associated, however, a small percentage of PAI-2 was released into the CM in a linear manner with time. PAI-2 in the conditioned medium had a higher molecular weight, consistent with a glycosylated form. Conjunctival PAI-2 was active, as shown by its ability to complex with a target enzyme, urokinase plasminogen activator (uPA). Although PAI-2 was detectable both in monolayer (i.e., relatively undifferentiated) conjunctival keratinocyte cultures as well as in stratified (i.e., more differentiated) cultures, steady state levels of PAI-2 were greater in the latter. PAI-2 is constitutively expressed by normal human conjunctival epithelial cells. The expression of PAI-2 throughout all epithelial layers in some biopsies of conjunctiva in vivo contrasts with the previously established distribution of PAI-2 in corneal epithelia, where it is present exclusively in the most superficial (i.e. most highly differentiated) cells. The role of PAI-2 in either tissue is unclear. However, we speculate that its distinct distribution in conjunctival versus corneal epithelia underscores inherent differences between these tissues, and may reflect specific functions of this proteinase inhibitor in both conjunctival and corneal epithelial cells.     

Endotoxin induction of plasminogen activator and plasminogen activator inhibitor type 1 mRNA in rat tissues in vivo

The tissue-specific distribution of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA) and their inhibitor type 1 (PAI-1) was analyzed at mRNA level in five major rat organ tissues. t-PA mRNA was detected in lung, kidney, heart, and liver. u-PA mRNA was detected in kidney and lung. Presence of PA mRNA correlated with the detection of PA activity in extracts of these tissues. PAI-1 mRNA was detected predominantly in heart and lung. Although PAI activity could not be measured directly in tissue extracts, the presence of PAI-1 mRNA correlated with the occurrence of PA.PAI complex in fibrin autography of tissue extracts. Endotoxin injection caused a very large increase in plasma PAI activity. This increase correlated with a marked increase in PAI-1 mRNA in nearly all tissues studied. The increase in PAI-1 mRNA is most pronounced in lung and liver. Endotoxin injection also caused an increased level of t-PA mRNA in heart and kidney, and an increased u-PA mRNA level in kidney. mRNA analysis of freshly isolated and separated subfractionated liver cells showed that the marked increase in PAI-1 mRNA in the liver after endotoxin injection may be due mainly to a strong increase of PAI-1 mRNA in the liver endothelial cells.     

Transforming growth factor beta inhibits plasminogen activator (PA) activity and stimulates production of urokinase-type PA, PA inhibitor-1 mRNA, and protein in rat osteoblast-like cells.

Transforming growth factor beta (TGF beta) treatment of rat osteoblast-rich calvarial cells or of the clonal osteogenic sarcoma cells, UMR 106-01, resulted in dose-dependent inhibition of plasminogen activator (PA) activity, and increased production of 3.2 kb mRNA and protein for PA inhibitor -1 (PAI-1). Although tissue-type PA (tPA) protein was not measured, TGF beta did not influence production of mRNA for tPA. Production of 2.3 kb mRNA for urokinase-type PA (uPA) was also increased by TGF beta in a dose-dependent manner. The effects of TGF beta on synthesis of mRNA for PAI-1 and uPA were maintained when protein synthesis was inhibited, and were abolished by inhibition of RNA synthesis. Although uPA had not been detected previously as a product of rat osteoblasts, treatment of lysates of osteoblast-like cells with plasmin yielded a band of PA activity on reverse fibrin autography, corresponding to a low Mr form of uPA. Untreated conditioned media from normal osteoblasts or UMR 106-01 cells contained no significant TGF beta activity, but activity could be detected in acidified medium. Treatment of conditioned media with plasmin resulted in activation of approximately 50% of the TGF beta detectable in acidified media. The results identify several effects of TGF beta on the PA-PA inhibitor system in osteoblasts. Net regulation of tPA activity through the stimulatory actions of several calciotropic hormones and the promotion of PAI-1 formation by TGF beta could determine the amount of osteoblast-derived TGF beta activated locally in bone. Stimulation of osteoblast production of mRNA for uPA could reflect effects on the synthesis of sc-uPA, a precursor for the active form of the enzyme.     

Different mechanisms contribute to simultaneous inhibition of urokinase and tissue-type plasminogen activators by glucocorticoids in human ovarian carcinoma cells

Previous studies from our laboratory have demonstrated that OVCA 433 human ovarian carcinoma cells are glucocorticoid responsive by several criteria and contain high affinity, saturable, steroid-specific glucocorticoid receptors. These cells secrete both mammalian plasminogen activators (PAs), urokinase (uPA) and tissue-type PA (tPA). Treatment of OVCA 433 cells with 1 x 10(-7) M dexamethasone (Dex) for 4 days led to 77% and 83% reductions in the extracellular activities of uPA and tPA, respectively, released into serum-free conditioned medium during a 1-h period. Dex treatment led to a 71% decrease in the rate of extracellular uPA antigen accumulation, as determined by enzyme-linked immunosorbent assay, as well as a 73% reduction in steady state uPA mRNA levels. In contrast, Dex treatment led to only a 42% decrease in the rate of extracellular tPA antigen accumulation and a 48% decrease in tPA mRNA levels; such decreases were insufficient to account for the 83% reduction in tPA activity. Thus, while Dex-induced decreases in uPA antigen and mRNA levels accounted for all but 6% of the decrease in uPA activity, a large discrepancy existed between the magnitudes of decreased tPA activity and decreased tPA antigen and mRNA levels. OVCA 433 cells produce both PAI-1 and PAI-2, two specific PA inhibitors. Treatment of cells with 1 x 10(-7) M Dex for 4 days led to a 3.3-fold increase in the rate of extracellular PAI-1 accumulation, with little or no effect on PAI-2 accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin E2 regulates production of plasminogen activator isoenzymes,urokinase receptor,and plasminogen activator inhibitor-1 in primary cultures of rat calvarial osteoblasts

The bone resorbing agent, prostaglandin E₂ (PGE₂), was found to alter several components of the plasminogen activator (PA)/plasmin pathway in primary cultures of rat neonatal osteoblast-like cells. The mRNA and activities of both urokinase-type PA (uPA) and tissue-type PA (tPA) were enhanced by PGE₂ treatment. The presence of mRNA for the uPA receptor (uPAR) has been demonstrated in these cells and steady-state levels shown to be greatly enhanced, the response being rapid and sustained for at least 24 hours. mRNA for plasminogen activator inhibitor 1 (PAI-1) was modulated in a biphasic manner, with inhibition of the constitutive level apparent at 4 hours of treatment and stimulation apparent at 12 hours and longer, while PAI-1 protein, measured by an ELISA assay for rat PAI-1, was diminished over this period. Neither PAI-2 mRNA nor mRNA for the broad spectrum protease inhibitor, protease nexin-1 (PN-1), was found to be modulated by PGE₂. Therefore, PGE₂ is likely to stimulate cell surface proteolytic activity, since uPA mRNA and cell-associated activity were elevated, as was mRNA for the cellular receptor for uPA. Although it was not possible to measure uPAR number and affinity it seems likely that elevated uPAR mRNA would translate into increased uPARs which would localize the increased uPA activity to the pericellular region. tPA mRNA and activity were also increased transiently with the activity inhibited with prolonged incubations, apparently by PAI-1. Elevation of tPA mRNA and activity may result in elevated activity within the extracellular matrix as tPA has been reported to associate with several matrix proteins. Thus the early effect of PGE₂ would be to promote proteolysis, both pericellularly and in the extracellular matrix. The inhibition of PAI-1 mRNA and protein, which would contribute to the elevation of activity, is due to PGE₂, but the later stimulatory effect on PAI-1 mRNA may be due to feedback regulation by transforming growth factor beta (TGFβ), secreted by osteoblasts and activated by elevated levels of PA. © 1995 Wiley-Liss Inc.     

Dolichos biflorus agglutinin (DBA) reveals a similar basal cell differentiation in normal and psoriatic epidermis

Binding of N-acetyl galactosamine (GalNAc)-specific Dolichos biflorus agglutinin (DBA) conjugates to frozen sections of normal epidermis and of psoriatic uninvolved and lesional skin was studied in fluorescence microscopy. The DBA conjugates bound only to single basal cell layer in normal and uninvolved psoriatic epidermis from patients with different blood group status. In the lesional area of psoriatic skin a similar reaction with a single basal cell layer was revealed. Other lectin-conjugates applied, presenting also GalNAc specificity, reacted with most cell layers of normal and both uninvolved and lesional psoriatic epidermis and gave an attenuated reaction with the middle epidermal layers. The results show that the basal cell characteristics are confined only to the cells along the basal membrane also in psoriatic epidermis, although cells in three lowest layers may be able to proliferate.     

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.     

Effects of cellular transformation on expression of plasminogen activator inhibitors 1 and 2. Evidence for independent regulation

Expression of plasminogen activators (PA) has been reported to be associated with invasive tumor growth and increased metastatic ability. In order to delineate changes in PA and PA inhibitor (PAI) expression that accompany cellular transformation, we studied oncogene-containing variants of the Rat-1 cell line. We report here that transfection of the oncogenes v-src, erbB, c-myc, v-myc, N-myc, and EJras into these cells does not result in detectable PA activity in conditioned media or cell extracts. In addition, Northern blot analysis fails to demonstrate urokinase mRNA in Rat-1 cells or transfectants. Moreover, cells transformed by EJras and v-src but not other oncogenes secrete an active placental-type PAI, PAI-2. Using inducible EJras constructs, we find that increased PAI-2 gene expression is detectable within 6-12 h after treatment with the inducing agent. Peak expression of PAI-2 mRNA is increased 10-15-fold over base line, and high levels are maintained for at least 72 h. In contrast to the results with PAI-2, secretion of endothelial-type PAI-1 into conditioned media is sharply down-regulated by several oncogenes. Thus, we have found that PAI-1 and PAI-2 are independently regulated in transformed variants of Rat-1 cells. The specific induction of PAI-2 in cells transformed by oncogenic ras and src suggests that this protease inhibitor may have a previously unsuspected role in malignancy.     

Dolichos biflorus agglutinin (DBA) reveals a similar basal cell differentiation in normal and psoriatic epidermis

Summary Binding of N-acetyl galactosamine (GalNAc)-specific Dolichos biflorus agglutinin (DBA) conjugates to frozen sections of normal epidermis and of psoriatic uninvolved and lesional skin was studied in fluorescence microscopy. The DBA conjugates bound only to single basal cell layer in normal and uninvolved psoriatic epidermis from patients with different blood group status. In the lesional area of psoriatic skin a similar reaction with a single basal cell layer was revealed. Other lectin-conjugates applied, presenting also GalNAc specificity, reacted with most cell layers of normal and both uninvolved and lesional psoriatic epidermis and gave an attenuated reaction with the middle epidermal layers. The results show that the basal cell characteristics are confined only to the cells along the basal membrane also in psoriatic epidermis, although cells in three lowest layers may be able to proliferate.     

Glucocorticoid induction of plasminogen activator and plasminogen activator-inhibitor messenger RNA in rat hepatoma cells

HTC rat hepatoma cells synthesize and secrete both tissue-type plasminogen activator (tPA) and type 1 plasminogen activator-inhibitor (PAI-1). Incubation with the synthetic glucocorticoid dexamethasone causes a rapid decrease in tPA activity which is secondary to a 5-fold increase in PAI-1 antigen and activity. Paradoxically, dexamethasone increases tPA antigen by 50%. We have analyzed HTC cell RNA by Northern and slot blot analysis, using as probes radiolabeled human PAI-1 and rat tPA cDNAs. HTC cells have a single species of PAI-1 mRNA of approximately 3.2 kilobases, which is increased 4-fold upon incubation with dexamethasone. Maximal induction occurs after 8-10 h of incubation. Half-maximal induction occurs at 5 nM dexamethasone. Dexamethasone also transiently increases the 2.8 kilobase tPA mRNA. The protein synthesis inhibitor cycloheximide does not affect accumulation of PAI-1 mRNA and does not block its induction by dexamethasone. In contrast, cycloheximide alone causes an increase in tPA mRNA, and in combination with dexamethasone, no further increase is observed. Induction of both mRNAs is prevented by actinomycin D. We conclude that the dexamethasone-induced increase in HTC cell PAI-1 activity and antigen is the result of a direct effect on accumulation of PAI-1 mRNA.     

Increased matriptase zymogen activation in inflammatory skin disorders

Matriptase, a type 2 transmembrane serine protease, and its inhibitor hepatocyte growth factor activator inhibitor (HAI)-1 are required for normal epidermal barrier function, and matriptase activity is tightly regulated during this process. We therefore hypothesized that this protease system might be deregulated in skin disease. To test this, we examined the level and activation state of matriptase in examples of 23 human skin disorders. We first examined matriptase and HAI-1 protein distribution in normal epidermis. Matriptase was detected at high levels at cell-cell junctions in the basal layer and spinous layers but was present at minimal levels in the granular layer. HAI-1 was distributed in a similar pattern, except that high-level expression was retained in the granular layer. This pattern of expression was retained in most skin disorders. We next examined the distribution of activated matriptase. Although activated matriptase is not detected in normal epidermis, a dramatic increase is seen in keratinocytes at the site of inflammation in 16 different skin diseases. To gain further evidence that activation is associated with inflammatory stimuli, we challenged HaCaT cells with acidic pH or H(2)O(2) and observed matriptase activation. These findings suggest that inflammation-associated reactive oxygen species and tissue acidity may enhance matriptase activation in some skin diseases.     

Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells.

Extracellular proteolysis is believed to be an essential component of the angiogenic process. The effects of VEGF, a recently described angiogenic factor, were assessed on PA activity and PA and PAI-1 mRNA levels in microvascular endothelial cells. u-PA and t-PA activity were increased by VEGF in a dose-dependent manner, with maximal induction at 30 ng/ml. u-PA and t-PA mRNAs were increased 7.5- and 8-fold respectively after 15 hours, and PAI-1 mRNA 4.5-fold after 4 hours exposure to VEGF. At equimolar concentrations (0.5 nM), VEGF was a more potent inducer of t-PA mRNA than bFGF, while bFGF was a more potent inducer of u-PA and PAI-1 mRNAs. In addition, VEGF induced u-PA and PAI-1 mRNAs with kinetics similar to those previously demonstrated for bFGF. These results demonstrate the regulation of PA and PAI-1 production by VEGF in microvascular endothelial cells and are in accord with the hypothesis that extracellular proteolysis, appropriately balanced by protease inhibitors, is required for normal capillary morphogenesis.