Secreted metalloproteinases in testicular cell culture

It is well known that cultured Sertoli cells secrete plasminogen activators (Lacroix et al., Mol Cell Endocrinol 1977; 9:227-236; Hettle et al., Biol Reprod 1986; 34:895-904). We now show that testicular cells in culture also secrete gelatinolytic metalloproteinases. Gelatin zymographic analysis of concentrated culture medium proteins reveals that Sertoli cells secrete gelatinases of 185 kDa, 110 kDa, 83 kDa, 76 kDa, and 72 kDa in addition to plasminogen activators (PAs). Gelatinase 185 kDa is induced by FSH. Media from Sertoli (epithelial)/peritubular (mesenchymal) cell cocultures contain the Sertoli cell gelatinases and one FSH-stimulated gelatinase of 50 kDa, indicating that gelatinase 50 kDa is regulated by both FSH and cell-cell interactions. A 50-kDa fibronectinolytic activity is also present in the coculture medium from cells grown in the presence of FSH. Casein zymography demonstrates a prominent 30-kDa protease only in media from cocultures. Peritubular cells secrete urokinase-type plasminogen activator (u-PA) and exhibit slight degrading activity at 86 kDa and 74 kDa. The gelatinases are most active in the pH range 7.3-8.5 and are completely or partially inhibited by metal ion chelators indicating that they are metalloproteinases. Our data demonstrate that testicular cells in culture secrete several gelatinases in addition to PAs, and that FSH and coculture conditions regulate some of these secreted proteases. We suggest that the highly regulated secretion of these proteases may well be of physiological importance during testicular development and spermatogenesis.     

Tissue plasminogen activator is released into cultured medium by cultured human uveal melanocytes

Melanoma cells produce tissue plasminogen activator (t-PA) that plays an important role in tumor invasion and metastasis. The production of t-PA by normal human uveal melanocytes has not been reported previously. In order to explore this possibility, we studied the production of t-PA by cultured human uveal melanocytes and compared that with the production by cultured human uveal melanoma cells and epidermal melanocytes. Human adult uveal melanocytes were isolated and cultured from donor eyes. The cells were cultured in serum-free medium for 48 h and the conditioned medium then collected for the plasminogen activator (PA) activity assay. Free PA activity was tested in an amidolytic assay using a t-PA standard curve. PA type was identified by fibrinography and antihuman t-PA and urokinase plasminogen activator (u-PA) blocking antibodies. Free PA activity was found in the conditioned medium of normal melanocytes and melanoma cells. The predominant PA activity was t-PA. Normal uveal melanocytes produced more t-PA (3.23 +/- 0.73 IU/105 cells/24 h) than that of epidermal melanocytes (1.25 IU/105 cells/24 h) but much less than uveal melanoma cells (11.0 +/- 3.39 IU/105 cells/24 h). Western blot analysis revealed that most t-PA in conditioned media were one-chain t-PA with molecular weight of 69 kDa. Our study indicates that uveal melanocytes may contribute to the free t-PA activity previously found in aqueous humor and choroidal eye cup superfusions. Therefore, this function of uveal melanocytes may play a role in intraocular matrix remodeling, fibrinolysis and aqueous humor outflow.     

Modulation of plasminogen activator production by interleukin 1: differential responses of fibroblasts derived from human skin and rheumatoid and non-rheumatoid synovium

Rheumatoid synovial fibroblasts were treated with purified porcine interleukin 1 alpha and recombinant human interleukin 1B, and the production of secreted and cell-associated plasminogen activator activity was measured. No stimulation of plasminogen activator activity was seen in response to either preparation of interleukin 1, and in more than half of the cell cultures interleukin 1 caused a significant decrease in the secreted levels of PA activity. Increased levels of prostaglandin E were produced in the same experiments, indicating that the cells were responsive to the interleukin 1 preparations. Both retinoic acid and unfractionated monocyte conditioned medium were able to stimulate the production of PA activity by the rheumatoid synovial fibroblast cultures. The rheumatoid synovial fibroblasts produced two species of plasminogen activator as indicated by SDS polyacrylamide gel electrophoresis, with apparent Mr of approx. 50,000 and 100,000. The Mr = 50,000 species co-migrates with urokinase-type plasminogen activator. No species is produced which co-migrates with tissue type plasminogen activator. Studies with antibodies also indicate that the activity produced is urokinase-type plasminogen activator. The Mr = 100,000 species may be an enzyme-inhibitor complex. Two non-rheumatoid synovial fibroblast cultures and two out of six human skin fibroblast cultures did produce elevated levels of plasminogen activator activity in response to recombinant human interleukin 1B. The results suggest that fibroblast populations may differ in their response to interleukin 1, in terms of production of plasminogen activator activity.     

Modulation of Proteolytic Activity During Neuritogenesis in the PC12 Nerve Cell: Differential Control of Plasminogen Activator and Plasminogen Activator Inhibitor Activities by Nerve Growth Factor and Dibutyryl-Cyclic AMP

Extracellular proteolysis is considered to be required during neuritic outgrowth to control the adhesiveness between the growing neurite membrane and extracellular matrix proteins. In this work, PC12 nerve cells were used to study the modulation of proteolytic activity during neuronal differentiation. PC12 cells were found to contain and release a 70-75-kDa tissue-type plasminogen activator (tPA) and a much less abundant 48-kDa urokinase-type plasminogen activator. A plasminogen activator inhibitor (PAI) activity with molecular sizes of 54 and 58 kDa was also detected in PC12 cell conditioned medium and formed high-molecular-mass complexes with released tPA. Release of PAI activity was dependent on treatment with nerve growth factor (NGF), whereas tPA synthesis and release were under control of a cyclic AMP-dependent mechanism and increased on treatment with dibutyryl-cyclic AMP [(But)2cAMP] or cholera toxin. Simultaneous treatment with NGF and (But)2cAMP resulted in increases of both tPA and PAI release and enhancement of tPA-PAI complex formation. The resulting plasminogen activator activity in conditioned medium was high in (But)2cAMP-treated cultures with short neuritic outgrowth but remained low in NGF- or NGF plus (But)2cAMP-treated cultures, where neurite extension was, respectively, large and very large. These results suggest that excess proteolytic activity may be detrimental to neuritic outgrowth and that not only PAI release but also tPA-PAI complex formation is associated with production of large and stable neuritic outgrowth. This can be understood as an involvement of PAI in the protection against neurite-destabilizing proteolytic activity.     

A stopped-flow calorimeter for biochemical applications.

In human skin fibroblast cultures a fraction of the procollagen that was processed to collagen and remained in the cell layer was further proteolytically modified by removal of both N- and C-terminal telopeptides. The proteolytic activity was associated with the cell layer, since secreted collagens were found always to contain intact telopeptides. The inclusion of neutral polymers, which caused the accumulation of the collagen in the cell layer [Bateman, Cole, Pillow & Ramshaw (1986) J. Biol. Chem. 261, 4198-4203], made the telopeptide cleavage more apparent in those cells which expressed the proteolytic activity. The extent of this cleavage was variable from cell culture to cell culture and between experiments with the same fibroblast line. The proteolytic activity was pH-dependent; cleavage was greatest at a culture-medium pH of 7.5 and 8.0 and was completely inhibited at a culture-medium pH of 7.0 and 6.5. The activity was significantly inhibited by soybean trypsin inhibitor, an elastase-specific inhibitor (N-acetylalanylalanylprolylvalylchloromethane) and the thrombin inhibitor hirudin. This cell-associated proteolytic activity may play a role in collagen degradation by removing the telopeptides, which are the primary sites of collagen cross-linking, thus destabilizing the collagen matrix sufficiently to render it susceptible to further proteolytic breakdown.     

Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli.

An early event in malignant transformation is the increased expression of proteases, such as plasminogen activator, which can degrade surrounding extracellular matrices, thereby conferring an advantage for tumour cell invasion and metastasis. The present studies provide evidence that plasma fibronectin (Fn), which is a component of the extracellular matrix, is a direct substrate for the plasminogen activator urokinase (UK). Human plasma Fn was incubated with human UK under plasminogen-free conditions. Fn cleavage was both time- and dose-dependent and was evident within 30 min. The proteolytic digestion was limited and complete within 12 h at an enzyme/substrate ratio of 1:20. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native dimeric 440 kDa structure of Fn with the concomitant appearance of three proteolytic fragments of 210, 200 and 25 kDa. Since two large fragments of similar size to the 220 kDa monomeric chains of Fn were obtained following proteolysis, it is proposed that UK cleaves Fn at two sites, one towards the N-terminal and one close to the C-terminal, but N-terminal to its interchain disulphide bonds. These studies suggest that the local proteolytic digestion and release of Fn from the extracellular matrix by tumour cells possessing high levels of UK may involve the direct proteolytic breakdown of Fn by UK.     

Plasminogen activator inhibitor-type I is a major biosynthetic product of retinal microvascular endothelial cells and pericytes in culture.

Previous studies have shown that a glycoprotein of Mr 47,000 (designated Gp47) is a major biosynthetic product of retinal endothelial cells in vitro (Canfield, Schor, West, Schor & Grant (1987) Biochem. J. 246, 121-129). We now present data indicating that (a) an identical protein is secreted by bovine retinal pericytes, (b) this protein is plasminogen activator inhibitor-type I (PAI-1), as revealed by immunoprecipitation with specific antibodies and reverse fibrin zymography, and (c) retinal endothelial cells and pericytes synthesize different species of matrix macromolecules, that is: type IV collagen is the major collagen secreted by endothelial cells, whereas pericytes produce predominantly type I collagen; fibronectin and thrombospondin are synthesized by both cell types. Our studies also indicate that PAI-1 is produced, albeit at considerably lower levels, by large vessel vascular cells (aortic endothelial and smooth muscle cells) and human skin fibroblasts. PAI-1 produced by human skin fibroblasts appears to be a distinct molecular species compared to its bovine counterpart as assessed by its slower mobility on SDS/polyacrylamide-gel electrophoresis. The potential significance of elevated PAI-1 production by retinal endothelial cells and pericytes, as well as their distinctive patterns of matrix biosynthesis, is discussed in terms of the involvement of these cells in the maintenance and remodelling of microvessel basement membrane.     

Recombinant interferon-γ secreted by chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium

Summary Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-γ (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and-2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.     

Activation of gelatinases by fibrin is PA/plasmin system-dependent in human glomerular endothelial cells

Evidence suggests that fibrin deposit is related to severity of glomerulonephropathy. Fibrin is considered to play an active role beyond a haemostatic plug or temporary matrix in response to injury. We have reported that fibrin induced specific morphological changes and up-regulated intercellular adhesion molecule-1 expression of glomerular endothelial cells (GECs). Changes of gelatinases activity have been implicated playing a prominent role in glomerular diseases involving matrix turnover. This study examined whether overlying fibrin influences the expression of gelatinase A and B in cultured human GECs and mechanism underlying the activation. No gelatinase activity was detectable in supernatant of cultured GECs; however, physiological concentration of fibrin (0.5–2.0 mg/ml) induced a dramatic expression of activated MMP-2 and MMP-9 at both mRNA and protein level in a dose and time dependent manner. Increased mRNA level of membrane-type 1 matrix metalloproteinases (MT1-MMPs) was also found. Interestingly, we observed that fibrin also induced the expression of tissue type plasminogen activator (tPA), urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 by casein zymographic and reverse zymographic analysis. Fibrin plate assay revealed the net activity was PA predominant. Serine protease inhibitor aprotinin blocked the conversion of pro-gelatinase A and B to their active forms. The results demonstrate that overlying fibrin increased the secretion of gelatinase A and B from GECs. PA/plasmin proteolytic pathways contributed to the activation of gelatinases.     

Plasminogen detection in oocytes and plasminogen activator activities in the porcine oviduct during the estrous cycle

Plasminogen activators (PAs) are highly specific serine proteases that convert the extracellular zymogen plasminogen into the active proteinase plasmin. Plasminogen-dependent proteolytic activity was detected by zymography both in the tissue membrane fraction of oviducts and in the oviductal flushing obtained at the preovulatory (Pre-Ov), postovulatory (Post-Ov) and mid-luteal (Mid-L) stages of the estrous cycle. A main proteolytic band, with a relative mobility similar to a human melanoma cell tissue-type plasminogen activator (t-PA), was found in all samples. Two additional components were observed in Pre-Ov and Post-Ov oviductal flushing but not in the tissue membrane fraction. In the oviductal flushing the PA activity was significantly higher in the Post-Ov stage than in the Pre-Ov one. Both urokinase-type plasminogen activator (u-PA, 50 kDa) and t-PA (72 kDa) were detected by Western blot; they showed differences in their relative concentration between Post-Ov and Pre-Ov oviductal flushing. The main PA substrate, plasminogen, was detected by indirect immunofluorescence in the cumulus cell extracellular matrix (ECM) and oocyte zona pellucida (ZP). In denuded oocytes, plasminogen was also detected on the surface of the plasma membrane. It is possible that oviductal PAs may act on the plasminogen present in the cumulus cell ECM and ZP; consequently, the generated plasmin could be involved in the rebuilding or degradation of these oocyte structures during fertilization or early development.     

The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants

Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus-transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus- transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and -independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator.     

Vectorial secretion of extracellular matrix proteins, matrix-degrading proteinases, and tissue inhibitor of metalloproteinases by endothelial cells

In a study of the vectorial secretion of proteins by bovine aortic arch endothelial cells, we found that the extracellular matrix macromolecules collagen and fibronectin as well as several matrix-degrading metalloproteinases were secreted selectively in the basal direction. In contrast, the tissue inhibitor of metalloproteinases showed only a weak preference for the basal direction. Three proteins at 18-35 kDa were secreted with preference apically, counter to the basal secretion of approximately 70% of the total secreted protein. As expected, rabbit synovial fibroblasts, which were used as a control, secreted proteins, including collagen, gelatin-degrading proteinases, and casein-degrading proteinases, equally in apical and basal directions. The basal secretion of collagen, fibronectin, gelatinases, and tissue inhibitor of metalloproteinases by bovine aortic arch endothelial cells suggests that the structural and functional polarity of these cells is manifested, in part, at the level of polarized secretion of matrix-related proteins.     

Purification and characterization of exudate gelatinases in the chronic-phase of carrageenin-induced inflammation in rats

Gelatinases have been purified from the exudate in the chronic-phase (day 7) of carrageenin-induced inflammation in rats. The day-7 exudate gelatinases gave two peaks on Sephadex G-150 gel filtration, the initial step of the purification. The molecular weights of the gelatinases corresponding to the two peaks were about 300 kDa (HMW fraction) and about 110 kDa (LMW fraction), respectively. The gelatinase in the HMW fraction has been purified to homogeneity; the purified gelatinase gave a single band corresponding to a molecular weight of 57 kDa on both SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-gelatin PAGE. On the other hand, the gelatinase purified from the LMW fraction was found to consist of three species, with molecular weights of 66, 64, and 57 kDa, as judged on SDS-gelatin PAGE. Granulation tissue-derived fibroblasts in culture mainly produced the 64-kDa species, which was converted to a 57-kDa species on treatment with 4-amino-phenylmercuric acetate, while rat macrophages and polymorphonuclear leukocytes mainly secreted the 96-kDa species. These results suggest that exudate gelatinases are largely produced by fibroblasts in granulation tissue and that they bind to exudate proteins, resulting in the formation of complexes with molecular weights of about 300 kDa and about 110 kDa. The gelatinases purified from the HMW and LMW fractions are metalloproteinases, as judged from the results of inhibitor experiments. Both the gelatinases degraded gelatin, but showed to proteolytic activity toward alpha-casein or type I collagen. Type IV collagen was degraded at 35 degrees C by the gelatinases purified from the LMW fraction but not by that from the HMW fraction.     

Cell surface complex of cathepsin B/annexin II tetramer in malignant progression

The cysteine protease cathepsin B is upregulated in a variety of tumors, particularly at the invasive edges. Cathepsin B can degrade extracellular matrix proteins, such as collagen IV and laminin, and can activate the precursor form of urokinase plasminogen activator (uPA), perhaps thereby initiating an extracellular proteolytic cascade. Recently, we demonstrated that procathepsin B interacts with the annexin II heterotetramer (AIIt) on the surface of tumor cells. AIIt had previously been shown to interact with the serine proteases: plasminogen/plasmin and tissue-type plasminogen activator (tPA). The AIIt binding site for cathepsin B differs from that for either plasminogen/plasmin or tPA. AIIt also interacts with extracellular matrix proteins, e.g., collagen I and tenascin-C, forming a structural link between the tumor cell surface and the extracellular matrix. Interestingly, cathepsin B, plasminogen/plasmin, t-PA and tenascin-C have all been linked to tumor development. We speculate that colocalization through AIIt of proteases and their substrates on the tumor cell surface may facilitate: (1) activation of precursor forms of proteases and initiation of proteolytic cascades; and (2) selective degradation of extracellular matrix proteins. The recruitment of proteases to specific regions on the cell surface, regions where potential substrates are also bound, could well function as a 'proteolytic center' to enhance tumor cell detachment, invasion and motility.     

Differential effects of transforming growth factor-beta on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations

To determine the effects of transforming growth factor-beta (TGF-beta) on the different cell types that exist in bone, cell populations (I-IV), progressively enriched in osteoblastic cells relative to fibroblastic cells, were prepared from fetal rat calvaria using timed collagenase digestions. TGF-beta did not induce anchorage-independent growth of these cells, nor was anchorage-dependent growth stimulated in most populations studied, despite a two- to threefold increase in the synthesis of cellular proteins. In all populations the synthesis of secreted proteins increased 2-3.5-fold. In particular, collagen, fibronectin, and plasminogen activator inhibitor synthesis was stimulated. However, different degrees of stimulation of individual proteins were observed both within and between cell populations. A marked preferential stimulation of plasminogen activator inhibitor was observed in each population, together with a slight preferential stimulation of collagen; the effect on collagen expression being directed primarily at type I collagen. In contrast, the synthesis of SPARC (secreted protein acidic rich in cysteine/osteonectin was stimulated approximately two-fold by TGF-beta, but only in fibroblastic populations. Collectively, these results demonstrate that TGF-beta stimulates matrix production by bone cells and, through differential effects on individual matrix components, may also influence the nature of the matrix formed by different bone cell populations. In the presence of TGF-beta, osteoblastic cells lost their polygonal morphology and alkaline phosphatase activity was decreased, reflecting a suppression of osteoblastic features. The differential effects of TGF-beta on bone cell populations are likely to be important in bone remodeling and fracture repair.     

Formation of Intercellular Collagen Matrix by Cultured Liver Epithelial Cells and Loss of its Ability in Hepatocarcinogenesis in vitro

Epithelial cells of normal rat liver origin (strain RL34) synthesized the α1 peptide of type I collagen. In nononcogenic cultures (RL34 and RL34EC) and a marginally oncogenic culture (RL34HII), the peptide was continuously secreted from the proliferating cells. Part of the soluble peptide was incorporated into the intercellular matrix of contact-inhibited cells after confluency, while the remainder was degraded. The intercellular matrix contained characteristic collagen fibrils which were argyrophilic and revealed a 64 nm axial periodicity. Epithelial cells of an oncogenic culture (RL34HT) secreted procollagen into the medium continuously throughout their proliferative phases and were unable to accumulate collagen fibrils in the intercellular matrix. The depletion of collagen accumulation in the hepatocarcinoma cell culture was ascribed to lack of the binding of native collagen molecules to the cell membrane and the persistence of high proteolytic activity on the cell surface.     

Regulation of protein secretion in Chinese hamster ovary cells by cell cycle position and cell density : Plasminogen activator, procollagen and fibronectin

To study the relationship between cell growth control, cell contact, and protein secretion, we examined the production of plasminogen activator, procollagen, and fibronectin by Chinese hamster ovary (CHO) fibroblasts, both as a function of position in the cell cycle and as a function of cell density. CHO fibroblasts that were synchronized at hourly intervals throughout the cell cycle by mitotic selection in an automated roller bottle apparatus secreted plasminogen activator only during the G2 and M phases of the cell cycle (10–14 h after mitotic selection). Cell-associated plasminogen activator activity was variable during G1 and S, but was greatly reduced during G2 and M. In contrast, secretion of the connective tissue matrix proteins, procollagen and fibronectin, was controlled by cell density rather than by cell cycle position. Type III procollagen and fibronectin were secreted throughout the cell cycle with no pronounced variations. Type I procollagen was not secreted by cycling cells and was observed in confluent cultures only after 24–48 h. To correlate these changes in protein secretion patterns with cell shape and contact, we used scanning electron microscopy (SEM) to study the appearance of CHO cells after mitotic selection. Actively dividing cells retained a high proportion of rounded, ruffled, and blebbed cells during all phases of the cell cycle. Only with increased cell density in contact-inhibited confluent cultures did most cells begin to flatten and spread. Thus, secretion of and attachment to extracellular matrix did not occur in rapidly dividing cells, but appeared to require the increased cell-cell contact and spreading that accompanies contact inhibition of growth. On the other hand, increased secretion of plasminogen activator was directly related to cell division and may be part of a sequence of events that allows cells growing in culture to loosen extracellular attachments in preparation for rounding and cytokinesis.     

Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan

Basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow stromal cells and stimulates haematopoiesis in vitro. We report here that primary human bone marrow cultures contain bFGF and express heparin-like bFGF binding sites on the cell surface and in the extracellular matrix (ECM). bFGF bound predominantly to a 200-kD cell surface heparan sulfate proteoglycan (HSPG), which was also found in conditioned medium. bFGF was released from bone marrow cultures by incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) and, less efficiently, by plasmin. Solubilized bFGF was found as a complex with the 200-kD HSPG. The complex was biologically active as shown by its ability to stimulate plasminogen activator production in bovine aortic endothelial cells. bFGF-HSPG complexes of bovine endothelial cells, however, were not released by PI-PLC. While only trace amounts of the bFGF-binding 200-kD HSPG were released spontaneously from bone marrow cultures, incubation with PI-PLC solubilized almost all of the 200-kD HSPG. The HSPG could be metabolically labeled with ethanolamine or palmitate, which was partially removed by treatment with PI-PLC. These findings indicate linkage of the HSPG to the cell surface via a phosphatidylinositol anchor. Plasmin released the 200-kD HSPG less efficiently than PI-PLC. We conclude that HSPGs of human bone marrow serve as a reservoir for bFGF, from which it can be released in a biologically active form via a dual mechanism; one involving a putative endogenous phospholipase, the other involving the proteolytic cascade of plasminogen activation.     

Identification of cells responsible for urokinase-type plasminogen activator synthesis and secretion in human diploid kidney cell cultures

Human urokinase-type plasminogen activator (uPA) is a serine protease that converts plasminogen to plasmin. It is produced and secreted by a variety of different human cells in vivo and in vitro. We have studied human diploid kidney cell (HKC) cultures prepared from neonatal kidney tissue and cultures of purified populations of HKC to determine which cells synthesize and secrete uPA into the culture medium. Antibodies against cell specific antigens and uPA were used to correlate specific kidney cell types with uPA synthesis. In addition, secretion of uPA activity into growth and uPA production media was determined for each cell type and cultures containing a mixture of cell types. The results of these studies demonstrated that glomerular visceral epithelial and kidney tubular epithelial cells synthesize and secrete uPA into the culture medium.