Regulation of the synthesis and activity of urokinase plasminogen activator in A549 human lung carcinoma cells by transforming growth factor-beta

Transforming growth factor-beta (TGF beta) is a regulator of cellular proliferation which can alter the proteolytic activity of cultured cells by enhancing the secretion of endothelial type plasminogen activator inhibitor and affecting the secretion of plasminogen activators (PAs) in cultured fibroblastic cells. We used the TGF beta- responsive malignant human lung adenocarcinoma cell line A549 to study the relationships between the known TGF beta-induced growth inhibition and the effects of TGF beta on the secretion of PA activity by A549 cells. PA activity was quantitated by caseinolysis assays, and characterized by urokinase mRNA analysis, immunoprecipitation, and zymography assays. PA-inhibitor production was observed in autoradiograms of SDS-polyacrylamide gels and reverse zymography assays. It was found that TGF beta enhanced the production of PA activity by these cells, in accordance with an enhancement of urokinase mRNA levels. A concomitant stimulation of type 1 PA-inhibitor production was also observed in A549 cells in response to TGF beta. In contrast to the observations of A549 cells, TGF beta caused a decrease in the expression of both urokinase and the tissue-type PA mRNA in human embryonic WI-38 lung fibroblasts indicating opposite regulation of the expression of PAs in these cells. The results suggest that TGF beta may play a role in the regulation of the invasive, proteolytically active phenotype of certain lung carcinoma cells.     

Transforming growth factor-beta induction of type-1 plasminogen activator inhibitor. Pericellular deposition and sensitivity to exogenous urokinase

The human tumor cell line HT-1080 was used as a model system to study the effects of transforming growth factor-beta (TGF beta) on polypeptide synthesis and proteolytic activity of malignant cells. Confluent cultures were exposed to TGF beta under serum-free conditions, and alterations in the production of proteins were examined by metabolic labeling and polypeptide analysis. TGF beta induced the synthesis and secretion of the Mr 47,000 endothelial type plasminogen activator inhibitor (PAI-1) as shown by reverse zymography, immunblotting, and immunoprecipitation analyses. TGF beta-induced PAI-1 was rapidly deposited in the growth substratum of the cells as shown by metabolic labeling and extraction of the cultures with sodium deoxycholate. Using pulse-chase experiments, we found a relatively fast turnover of substratum-associated PAI-1. Exogenously added urokinase released PAI-1 from the substratum even in the presence of the plasmin inhibitor aprotinin, suggesting a direct effect of urokinase. Immunoreactive complexes of higher molecular weight were subsequently detected in the medium. Epidermal growth factor, transforming growth factor-alpha, platelet-derived growth factor, and insulin did not elicit similar effects on the amount of PAI-1. TGF beta also inhibited the anchorage-independent growth of HT-1080 cells at the same concentrations at which it induced PAI-1. These results indicate that TGF beta can modulate the extracellular proteolytic activity of cultured cells by enhancing the secretion and deposition of PAI-1 into their microenvironment. It remains to be established whether TGF beta inhibition of anchorage-independent growth of these cells is associated with the induction of PAI-1.     

Modulation of extracellular proteolytic activity and anchorage-independent growth of cultured cells by sarcoma cell-derived factors: relationships to transforming growth factor-beta

We have previously described a factor(s) produced by 8387 fibrosarcoma cells, which can affect plasminogen activator (PA) activity of cultured cells. Since then, transforming growth factor-beta (TGF beta) has been established as a major growth factor/growth inhibitor that regulates both the expression and activity of PAs and their endothelial-type inhibitor (PAI-1). The present study was undertaken to characterize the 8387 fibrosarcoma cell-derived factor(s) and to investigate its relationships to TGF beta by analysis of modulation of PA activity and cell growth. The fibrosarcoma cell-derived proteins were partially purified from serum-free conditioned culture medium using Bio-Gel P-10 chromatography. Two separate fractions with apparent molecular weights of 16,000 and 12,000 contained activities that both decreased the secretion of PA activity by human lung fibroblasts and inhibited the soft agar growth of A549 lung adenocarcinoma cells. Both factors affected similarly the production of urokinase-type PA and PAI-1 in various cell lines and enhanced anchorage-independent growth of murine AKR-2B fibroblasts. The effects of these factors thus resembled those of TGF beta. The immunological relationships between the Mr 16,000 and Mr 12,000 factors and TGF beta were therefore studied using neutralizing anti-TGF beta antibodies. The TGF beta antibodies efficiently inhibited the effects of the Mr 16,000 factor but not those of the Mr 12,000 factor in cell culture assays. The results suggest that 8387 fibrosarcoma cells produce two major growth inhibitors, one of which is closely related to TGF beta.     

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.     

The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells

Basic fibroblast growth factor (bFGF), a potent inducer of angiogenesis in vivo, stimulates the production of both urokinase- and tissue-type plasminogen activators (PAs) in cultured bovine capillary endothelial cells. The observed increase in proteolytic activity induced by bFGF was effectively diminished by picogram amounts of transforming growth factor beta (TGF beta), but could not be abolished by increasing the amount of TGF beta. However, the inhibition by TGF beta was greatly enhanced if the cells were pretreated with TGF beta before addition of bFGF. After prolonged incubation of cultures treated simultaneously with bFGF and TGF beta, the inhibitory effect of TGF beta diminished and the stimulatory effect of the added bFGF dominated as assayed by PA levels. TGF beta did not alter the receptor binding of labeled bFGF, nor did a 6-h pretreatment with TGF beta reduce the amount of bFGF bound. The major difference between the effects of bFGF and TGF beta was that while bFGF effectively enhanced PA activity expressed by the cells, TGF beta decreased the amounts of both cell-associated and secreted PA activity by decreasing enzyme production. Both bFGF and TGF beta increased the secretion of the endothelial-type plasminogen activator inhibitor.     

Treatment of mouse L-cells with phorbol myristate acetate induces the secretion of a plasminogen activator inhibitor which binds to human and mouse urokinase and human tissue plasminogen activator

1. Serum-free conditioned medium from L-cells or L-cells treated with the tumor-promotor phorbol myristate acetate (PMA) was analyzed for plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activity. Conditioned medium from control or PMA-treated cells did not contain detectable PA activity when assayed by SDS-PAGE and zymography. 2. Conditioned medium from PMA-treated cells, but not control cells, contained a PAI of Mr = 40,000 da when assayed by reverse zymography. 3. The L-cell PAI formed SDS-stable complexes with purified human (homo sapiens) urokinase and tissue plasminogen activator, as well as, mouse (Mus musculus) urinary PA. 4. These results indicate that biochemical and immunological differences between human and mouse urokinase and human urokinase and human tissue plasminogen activator do not influence the interaction of the L-cell PAI with these enzymes.     

Transforming growth factor beta alters plasminogen activator activity in human skin fibroblasts

Adult human skin fibroblasts were used as a model to study the effects of transforming growth factor beta (TGF beta) on the secreted plasminogen activator (PA) activity of cultured cells. TGF beta, at nanogram concentrations, enhanced the secretion of pro-PA from two fibroblast strains in a time- and dose-dependent manner. The induced enzymatic activity was inhibited by anti-urokinase antibodies and it co-migrated with purified urokinase in polyacrylamide gels. The secretion of PA activity was abolished when cycloheximide (0.1 microgram/ml) was added to the cultures. The activity was thus dependent on protein synthesis rather than just on direct activation of a plasminogen proactivator. TGF beta had only a slight mitogenic effect on the test cells. Epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin were ineffective alone in inducing PA. Insulin, on the contrary, had an inhibitory effect on the TGF beta-induced PA activity. In addition to its effects on the secretion of PA, TGF beta enhanced the production of a proteinase inhibitor by these cells. The results suggest a role for TGF beta in the regulation of PA activity and pericellular proteolysis in fibroblastic cells.     

Plasminogen activators and their inhibitors in the neuromuscular system: I. Developmental regulation of plasminogen activator isoforms during in vitro myogenesis in two cell lines

Plasminogen activators (PAs), were estimated qualitatively and quantitatively in two different clonal murine skeletal muscle cell lines. Both cell lines produced the two major types of PAs found in mammalian cells, urokinase-type (uPA) and tissue type (tPA). These two lines are models for the study of myogenesis in vitro, but differ in several growth and differentiation characteristics. Because of their possible involvement in these characteristics we assayed the expression of PAs in both cell systems during development in culture. Utilizing fibrin zymography two isoforms of tPA were detected. One co-migrated with human tPA at 75 kd and another may represent a tPA:inhibitor complex at 105 Kd. Several isoenzymes of uPA were detected and these changed depending on whether cell homogenates or conditioned medium was analyzed and whether myogenic cells were at single-cell myoblast or multi-nucleated myotube stage. Species-specific antisera to mouse uPA identified 4 uPA bands in muscle cell medium and 5 in cell layers. Antigenic uPA bands also varied depending on stage of myogenesis. Quantitative amidolytic studies using chromogenic substrates showed that maximal PA activity, both uPA and tPA, occurred at the time of myoblast fusion. Furthermore, uPA activity in membranes increased during myogenesis, while both uPA and tPA in medium decreased after fusion. These studies indicate that muscle PA expression is developmentally regulated and may correlate with growth and differentiation in skeletal muscle.     

Urokinase-type and tissue-type plasminogen activators have different distributions in cultured bovine capillary endothelial cells

The cell extracts and conditioned medium from cultured bovine capillary endothelial (BCE) cells were examined to determine the types of plasminogen activator (PA) present in each of these two fractions. The fractions were first analyzed by fibrin autography after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The cell extracts contained two species of PA of Mr 48,000 and 28,000. Multiple forms of PA were detected in the conditioned medium: variable amounts of the Mr 48,000 and 28,000 forms and a broad band of activity with Mr in the range of 67,000-93,000. The major fraction of the Mr 48,000 form was in the cell extract. Treatment of the cells with 12-0-tetradecanoyl phorbol-13-acetate or with a preparation containing angiogenic activity resulted in a proportionate increase in the levels of all forms. The Mr 48,000 form was demonstrated to be a urokinase-like PA, since it was immunoprecipitated with antibodies to urokinase. When conditioned medium or cell extracts from biosynthetically labelled BCE cells were incubated with antiserum to urokinase, the Mr 48,000 form was immunoprecipitated only from the cell extract. The Mr 67,000-93,000 forms were demonstrated to be tissue-type PAs, since they were immunoprecipitated with antibodies to tissue PA. When the same conditioned medium or cell extracts were incubated with antiserum to tissue-type PA, the Mr 67,000-93,000 forms were immunoprecipitated only from the conditioned medium. Therefore, BCE cells are able to produce both tissue-type PA, which is primarily secreted, and urokinase-type PA, which remains primarily cell associated.     

A beta-type transforming growth factor, present in conditioned cell culture medium independent of cell transformation, may derive from serum

An alpha-type transforming growth factor (TGF alpha) is produced at high levels by rat embryo cells transformed by the Snyder-Theilen strain of feline sarcoma virus (FeSV). Addition of 2 ng mouse epidermal growth factor (mEGF) during purification identified the presence of a second, EGF-dependent growth factor of the TGF beta type (TGF beta) in this conditioned medium. This factor had an approximate Mr of 12,000 and eluted at 37% acetonitrile during high performance liquid chromatography. This extracellular type of TGF beta activity also was present in conditioned medium of rat cells after infection with a transformation defective strain of Abelson leukemia virus, and hence expression of this growth factor activity was independent of cell transformation. Moreover, the presence of an EGF-dependent, 12,000 Mr clonogenic activity in extracts of bovine serum alone suggests serum as an origin for the B-type transforming growth factor initially observed in conditioned medium of Snyder-Theilen FeSV transformed cells. This does not, however, preclude the possibility that TGF beta is also secreted by the transformed rat embryo cells themselves.     

Proteolytic activation of latent transforming growth factor-beta from fibroblast-conditioned medium

Transforming growth factor-beta (TGF beta) is produced by most cultured cells in an inactive form. Potential activation mechanisms of latent TGF beta were studied using fibroblastic (NRK-49F and AKR-MCA) cell-conditioned medium as a model. Active TGF beta was monitored by radioreceptor and soft agar assays as well as by antibody inhibition and immunoprecipitation. Little or no TGF beta was detected in untreated conditioned medium. Treatment of the medium with extremes of pH (1.5 or 12) resulted in significant activation of TGF beta as shown by radioreceptor assays, while mild acid treatment (pH 4.5) yielded only 20-30% of the competition achieved by pH 1.5. In an effort to define more physiological means of TGF beta activation, the effects of some proteases were tested. Plasmin and cathepsin D were found to generate 25-kD bands corresponding to the active form of TGF beta as shown by immunoprecipitation analysis of radiolabeled cell-conditioned medium. Plasmin treatment of the medium resulted in activity that was quantitatively similar to that of mild acid treatment as measured by radioreceptor and soft agar assays. In addition, the plasmin-generated activity was inhibited by anti-TGF beta antibodies. Sequential treatments of AKR-MCA cell-conditioned medium with mild acid followed by plasmin or plasmin followed by mild acid gave activation comparable to either treatment alone. The data suggest that conditioned medium may contain at least two different pools of latent TGF beta. One pool is resistant to mild acid and/or plasmin and requires strong acid or alkali treatment for activation. A second pool is activated by mild pH change and/or plasmin. Activation of this form of latent TGF beta may take place by dissociation or proteolytic digestion from a precursor molecule or hypothetical TGF beta-binding protein complex.     

Type beta transforming growth factor from feline sarcoma virus-transformed rat cells. Isolation and biological properties

We have isolated a strongly mitogenic, type beta transforming growth factor (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal growth factor (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of 21%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (20 pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on growth-arrested NRK, human lung, and Swiss mouse 3T3 fibroblast monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting factors responsible for the transforming action of culture fluids from FeSV-Fre cells.     

Urokinase-type plasminogen activator and its inhibitor secreted by cultured human monocyte-macrophages

Human blood monocytes in culture differentiate to macrophagelike cells within 1 week. Coinciding with this morphological transition the cells started releasing increasing amounts of the serine proteinase plasminogen activator (PA; Mr 56,000) of the urokinase (u-PA) type and the proteinase inhibitor alpha-2-macroglobulin (alpha 2M). Unlike the cell-associated PA activity, which was also readily detected in fresh monocytes, the activity secreted into the serum-free culture medium could be measured only after treatment of the samples with sodium dodecyl sulphate. Heat or acid treatment of the medium was not sufficient to reveal the PA activity, suggesting that, apart from alpha 2M, another PA-inhibiting substance was present in the culture medium. The inhibitor (Mr 65,000) was found to be synthesized by macrophages and specifically inhibited u-PA activity but not tissue-type PA (t-PA) or plasmin activity. Dexamethasone decreased the secretion of PA by differentiated macrophages without affecting the production of alpha 2M or the PA inhibitor. Dexamethasone also inhibited the morphological differentiation of the cells when added to the monocyte-phase cells.     

Phenotypic and functional characteristics of porcine peritoneal mesothelial cells

The objective of this study was to establish a technique to isolate porcine mesothelial cells (PMCs) from omental tissue and to compare them to human mesothelial cells (HMCs). The PMCs were dispersed by collagenase digestion and isolated on a Ficoll layer. Their morphologic and ultrastructural features were assessed at confluence by light and electronic microscopy, and they were characterized by immunohistochemistry using specific HMC markers. PMC proliferation was studied in the presence of growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or transforming growth factors beta1, beta2, or beta3 (TGF). Fibrinolytic PMC activity was detected by zymography for tissue plasminogen activator (tPA) and by reverse zymography for plasminogen activator inhibitor-1 (PAI-1). The recalcification time of cell lysates was used to define PMC procoagulant activity, and gelatinase zymography was used to detect metalloproteinase production. At confluence, PMCs formed typical cobblestone monolayers and exhibited structural features characteristic of HMCs. Weibel Palade bodies were never seen. Specific HMC markers (HBME1, ME1, WT1) cross-reacted with PMCs. As HMCs and PMCs coexpressed cytokeratin and vimentin, and also expressed vinculin and alpha-actin. Addition of PDGF or EGF to the culture medium stimulated PMC proliferation. PMCs constitutively expressed fibrinolytic and procoagulant activity and secreted MMP9 and MMP2. The technique described in this study allows isolation of mesothelial cells from porcine omental tissue. These porcine cells exhibit a mesothelial phenotype and functional properties similar to those of HMCs. Our data warrant an evaluation of mesothelial cells as targets in several therapeutic strategies with porcine models.     

A plasminogen activator is induced during goldfish optic nerve regeneration.

The use of purified piscine plasminogen in a chromogenic solution assay enabled us to detect plasminogen activator (PA) activity in crude homogenates of goldfish optic nerve following nerve injury. In contrast, no activity was detected in the homogenates of uninjured nerve. Under conditions allowing regeneration of the optic axons (optic nerve crush), PA activity peaked 8 days after crush, and decreased to undetectable levels by 60 days. Under conditions allowing only degeneration of the axons (enucleation), the activity peaked at 8 days but decreased more rapidly. Casein zymography of samples after fractionation in SDS-PAGE showed that PA activity migrated as a doublet at Mr = 60-65 kd. Using this assay, activity was also observed in uninjured control nerves. This plasminogen-dependent activity migrated as three bands of higher molecular weight (Mr = 75, 95 and 120 kd) and was undetectable in solution assays of unfractionated extracts, suggesting complex formation with an inhibitor(s). Fibrin overlay assay of retinal explants and isolated primary cells in culture suggest that the goldfish PA is associated with the glial cells of the goldfish visual pathway.     

Inhibition of endothelial cell proliferation by type beta-transforming growth factor: interactions with acidic and basic fibroblast growth factors

TGF beta is a potent (ED50 approximately 10(-11) M) inhibitor of the proliferative activities of both acidic and basic FGF on vascular and capillary endothelial cells in vitro. The inhibition of cell growth is dose-dependent and characteristic of a non-competitive interaction. The results demonstrate that TGF beta and FGF can interact at the cellular level to modulate growth and suggest that many of the biological activities of FGF observed in vitro and in vivo (ie angiogenesis, cell growth, cell differentiation) may be regulated by the presence of TGF beta and related proteins (ie inhibin) in the local cellular milieu. The possible identity of TGF beta with the inhibitors of endothelial cell growth detected in in vitro assays of crude extracts is discussed.     

Phosphorylation of nuclear protein is an early event in TGF beta 1 action.

Transforming growth factor beta (TGF beta) is a family of polypeptides that modulate growth and differentiation. TGF beta exerts its effects on target cells through interaction with specific cell surface receptors, but the signal transduction pathways are as yet largely unresolved. In this study we report that the growth inhibitory action of TGF beta on mink lung CCl 64 cells is associated with a rapid and transient phosphorylation of a number of nuclear proteins. In parallel, a transient expression of the immediate early gene jun B is observed. The expression of jun B can be inhibited by the protein kinase inhibitor H7 and can be augmented by the phosphatase inhibitor okadaic acid. Thus, protein phosphorylation can be a possible mechanism through which TGF beta 1 initiates early genomic responses.