Regulation of in vitro glia-induced microvessel morphogenesis by urokinase

Plasminogen activators (PAs) regulate a variety of processes involved in tissue morphogenesis and differentiation. We used a coculture system in which microvascular endothelial cells are induced by glial cells to form capillary-like structures in order to examine the role of urokinase-type PA (uPA) during microvessel morphogenesis within the central nervous system (CNS). Endotheliaderived uPA activity decreased sevenfold within glial-endothelial cocultures when capillary-like structures were formed. Incubation of cocultures with concentrations of phorbol 12-myristate 13-acetate (0.1 and 1.0 nM) that induced endothelial uPA activity (45–210%) inhibited endothelial differentiation (25–70%). Furthermore, incubation of cocultures with proteolytically active low molecular weight uPA (5–500 IU/ml) inhibited endothelial differentiation (37–75%), whereas the amino terminal cell-binding fragment of uPA had minimal effect. Inhibition of plasminogen activation in cocultures with the serine protease/plasmin inhibitors aprotinin and soybean trypsin inhibitor increased glia-induced capillary-like structure formation (96–98%). These findings establish a paracrine/autocrine function for urokinase and its inhibitors in regulating endothelial responses to perivascular glia and provide insight into mechanisms of microvascular reactions to CNS pathology. © 1994 Wiley-Liss, Inc.     

Human 92- and 72-kilodalton type IV collagenases are elastases.

Elastin is critical to the structural integrity of a variety of connective tissues. Only a select group of enzymes has thus far been identified capable of cleaving insoluble elastin. Recently, we observed that human alveolar macrophages secrete elastase activity that is largely inhibited by the tissue inhibitor of metalloproteinases (TIMP). This finding suggested that one or more of the metalloproteinases released by alveolar macrophages has elastase activity. Accordingly, we tested pure human interstitial collagenase, stromelysin, 92-kDa type IV collagenase, and 72-kDa type IV collagenase for elastolytic activity using kappa-elastin zymography and insoluble 3H-labeled elastin. The 92- and 72-kDa type IV collagenases were found to be elastolytic in both assay systems. A recombinant preparation of 92-kDa type IV collagenase with gelatinolytic activity was also found to be elastolytic. Organomercurial activation was essential to detect elastolytic activity of the native 92- and 72-kDa type IV collagenases and enhanced the elastase activity of the recombinant 92-kDa enzyme. On a molar basis the recombinant 92-kDa type IV collagenase was approximately 30% as active as human leukocyte elastase in solubilizing 3H-labeled elastin. Exogenously added TIMP in significant molar excess abolished the elastase activity of the 92- and 72-kDa type IV collagenases. Stromelysin and interstitial collagenase showed no significant elastolytic activity, although both were catalytically active against susceptible substrates. Conditioned media from cultures of human mononuclear phagocytes containing the 92-kDa enzyme produced a distinct zone of lysis in the kappa-elastin zymograms at this molecular mass. These results definitively extend the spectrum of human proteinases with elastolytic activity to metalloproteinases and suggest the enzymatic basis for elastase activity observed with certain cell types such as human alveolar macrophages.     

An in vitro three-dimensional coculture model of cerebral microvascular angiogenesis and differentiation

Summary The microvasculature of the developing brain is plastic and responds differently to the many insults associated with preterm birth. We developed three-dimensional in vitro culture models for the study of the responses of the developing cerebral micro-vasculature. Beagle brain microvascular endothelial cells (BBMEC) were isolated by differential centrifugation from newborn beagle pups on postnatal Day 1 and placed in three-dimensional culture dispersed in a collagen gel. Alternatively, BBMEC were placed in a three-dimensional coculture with neonatal rat forebrain astrocytes. Cultures were analyzed for extracellular matrix components at 1 and 6 d, and total RNA was extracted for Northern analyses. Urokinase plasminogen activator activity was assayed in both mono- and cocultures of the two cell types. Studies of three-dimensional BBMEC/astrocyte cocultures demonstrated progressive tube formation with only low levels of endothelial proliferation. By 6 d in three-dimensional coculture, the BBMEC formed capillarylike tubes with a wrapping of glial processes, and basement membrane protein synthesis was noted. Urokinase plasminogen zymography suggested intercellular signaling by the two cell types. These data suggest that the three-dimensional beagle brain germinal matrix microvascular endothelial cell/neonatal rat astrocyte coculture provides a good model for the investigation of microvascular responses in the developing brain.     

Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro

Astrocytes maintain a unique association with the central nervous system microvasculature and are thought to play a role in neural microvessel formation and differentiation. We investigated the influence of astroglial cells on neural microvascular endothelial differentiation in vitro. Using an astroglial-endothelial coculture system, rat brain astrocytes and C6 cells of astroglial lineage are shown to induce bovine retinal microvascular endothelial (BRE) cells to form capillary-like structures. Light microscopic evidence for endothelial reorganization began within 48 hours and was complete 72-96 hours following the addition of BRE cells to 1-day-old astroglial cultures. The extent of BRE reorganization was quantitated by computer-assisted analysis and shown to be dependent upon the density of both the BRE and C6 cells within the cocultures. Coculture conditions in which BRE cells were separated from C6 cells by porous membranes failed to generate this endothelial cell change. Likewise, C6-conditioned media and C6-endothelial coculture conditioned media did not induce BRE cell reorganization. Extracellular laminin within the C6-endothelial cocultures, identified by indirect immunofluorescence, was concentrated at the endothelial-astroglial interface of capillary-like structures consistent with incipient basement membrane formation. Astroglial cells accumulated adjacent to capillary-like structures suggesting the presence of bidirectional influences between the reorganized endothelial cells and astroglia. This is the first demonstration of astroglial induction of angiogenesis in vitro and these findings support a functional role for perivascular astrocytes in the vascularization of neural tissue such as retina and brain.     

Identification of type IV collagenase in rat testicular cell culture: influence of peritubular-Sertoli cell interactions

In the testis, interactions between peritubular cells (mesenchyme) and Sertoli cells (epithelium), together with proteolytic remodeling of the extracellular matrix, may play a central role in testicular development, morphogenesis, and spermatogenesis. In this study we demonstrate that a metalloproteinase of 72 kDa present in rat Sertoli cell and Sertoli-peritubular cell coculture medium is activated by p-aminophenylmercuric acetate (p-APMA) to a lower molecular mass form, indicating that it is likely to be a latent collagenase. Immunoblots using antibodies to three different domains of type IV collagenase show that the 72-kDa protease and a 76-kDa protease are type IV pro-collagenases. Sertoli cells cultured alone produce basal levels of type IV collagenase that can be immunolocalized in the cytoplasm of cultured cells. Peritubular cells cultured alone produce much less type IV collagenase. However, Sertoli and peritubular cells in coculture do produce type IV pro-collagenase, and in cultures consisting predominantly of peritubular cells, the activated form of type IV collagenase was detected by both zymography and immunoblotting. Cells growing during the transitional phase (from cell attachment to confluence) secrete more metalloproteinases than during the confluent phase. In contrast, plasminogen activator levels are unaffected by time in culture. These results show that rat testicular cells in culture produce and secrete type IV collagenase, and that the secretion and activation of this enzyme and other metalloproteases is regulated by the ratio of mesenchymal cells to epithelial cells and time in culture.     

Differential Expression of Urokinase-Type Plasminogen Activator (uPA), Its Receptor (uPA-R), and Inhibitor Type-2 (PAI-2) during Differentiation of Keratinocytes in an Organotypic Coculture System

Cultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase-type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-I stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system.     

Interleukin 4 inhibition of prostaglandin E2 synthesis blocks interstitial collagenase and 92-kDa type IV collagenase/gelatinase production by human monocytes.

Activation of human monocytes results in the production of interstitial collagenase through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Inasmuch as interleukin 4 (IL-4) has been shown to inhibit PGE2 synthesis by monocytes, we examined the effect of IL-4 on the production of human monocyte interstitial collagenase. Additionally, we also assessed the effect of IL-4 on the production of 92-kDa type IV collagenase/gelatinase and tissue inhibitor of metalloproteinase-1 (TIMP-1) by monocytes. The inhibition of PGE2 synthesis by IL-4 resulted in decreased interstitial collagenase protein and activity that could be restored by exogenous PGE2 or dibutyryl cyclic AMP (Bt2cAMP). IL-4 also suppressed ConA-stimulated 92-kDa type IV collagenase/gelatinase protein and zymogram enzyme activity that could be reversed by exogenous PGE2 or Bt2cAMP. Moreover, indomethacin suppressed the ConA-induced production of 92-kDa type IV collagenase/gelatinase. These data demonstrate that, like monocyte interstitial collagenase, the conA-inducible monocyte 92-kDa type IV collagenase/gelatinase is regulated through a PGE2-mediated cAMP-dependent pathway. In contrast to ConA stimulation, unstimulated monocytes released low levels of 92-kDa type IV collagenase/gelatinase that were not affected by IL-4, PGE2, or Bt2cAMP, indicating that basal production of this enzyme is PGE2-cAMP independent. IL-4 inhibition of both collagenases was not a result of increased TIMP expression since Western analysis of 28.5-kDa TIMP-1 revealed that IL-4 did not alter the increased TIMP-1 protein in response to ConA. These data indicate that IL-4 may function in natural host regulation of connective tissue damage by monocytes.     

Fibroblast growth factor-2 did not restore plasminogen system activity in endothelial cells on glycated collagen

People with diabetes experience morbidity and mortality from unregulated microvascular remodeling, which may be linked to hyperglycemia. Elevated glucose leads to extracellular matrix collagen glycation, which delays endothelial capillary-like tube formation in vitro. Glucose also increases endothelial cell fibroblast growth factor-2 (FGF-2) release and extracellular matrix storage, which should increase tube formation. In this study, we determined if FGF-2 could restore plasminogen system activity and angiogenic function in endothelial cells on glycated collagen. Human umbilical vein endothelial cells cultured on native or glycated collagen substrates were stimulated with FGF-2. Plasminogen system activity, cell migration, and capillary-like tube formation were measured, along with plasminogen system protein and mRNA levels. Glycated collagen decreased endothelial cell plasminogen system activity, cell migration, and tube length. FGF-2 did not restore plasminogen system activity or tube formation in cells on glycated collagen, despite decreasing plasminogen activator inhibitor-1 (PAI-1) protein level. We now show that PAI-1 binds to glycated collagen, which may localize PAI-1 to the extracellular matrix. These data suggest that FGF-2 may not restore angiogenic functions in endothelial cells on glycated collagen due to PAI-1 bound to glycated collagen.     

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.     

Differential susceptibility of type X collagen to cleavage by two mammalian interstitial collagenases and 72-kDa type IV collagenase

We have studied the degradation of type X collagen by human skin fibroblast and rat uterus interstitial collagenases and human 72-kDa type IV collagenase. The interstitial collagenases attacked the native type X helix at two loci, cleaving residues Gly92-Leu93 and Gly420-Ile421, both scissions involving Gly-X bonds of Gly-X-Y-Z-A sequences. However, the human and rat interstitial enzymes displayed an opposite and substantial selectivity for each of these potential sites, with the uterine enzyme catalyzing the Gly420-Ile421 cleavage almost 20-fold faster than the Gly92-Leu93 locus. Values for enzyme-substrate affinity were approximately 1 microM indistinguishable from the corresponding Km values against type I collagen. Interestingly, in attacking type X collagen, both enzymes manifested kinetic properties intermediate between those characterizing the degradation of native and denatured collagen substrates. Thus, energy dependence of reaction velocity revealed a value of EA of 45 kcal, typical of native interstitial collagen substrates. However, the substitution of D2O for H2O in solvent buffer failed to slow type X collagenolysis significantly (kH/kD = 1.1), in contrast to the 50-70% slowing (kH/kD = 2-3) observed with native interstitial collagens. Since this lack of deuterium isotope effect is characteristic of interstitial collagenase cleavage of denatured collagens, we investigated the capacity of another metalloproteinase with substantial gelatinolytic activity, 72-kDa type IV collagenase, to degrade type X collagen. The 72-kDa type IV collagenase cleaved type X collagen at both 25 and 37 degrees C, and at loci in close proximity to those attacked by the interstitial enzymes. No further cleavages were observed at either temperature with type IV collagenase, and although values for kcat were not determined (due to associated tissue inhibitor of metalloproteinases-2), catalytic rates appeared to be substantial in comparison to the interstitial enzymes. In contrast, type X collagen was completely resistant to proteolysis by stromelysin. Type X collagen thus appears to be highly unusual in its susceptibility to degradation by both interstitial collagenase and another member of the metalloproteinase gene family.     

Alkaline phosphatase dual‐binding sites for collagen dictate cell migration and microvessel assembly in vitro

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro‐ or microvasculature) play a role in the formation of microvessel‐like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor‐β1, and interleukin‐8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel‐like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins’ aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.     

Transforming growth factor-beta 1 up-regulates type IV collagenase expression in cultured human keratinocytes.

During the wound healing process lysis of basement membranes precedes keratinocyte migration into the wound bed. We studied, in vitro, whether this degradation of basement membranes could be regulated by transforming growth factor-beta 1 (TGF-beta 1), which is known to accelerate wound healing in vivo. Transforming growth factor-beta 1 was found to increase the expression of both 92- and 72-kDa type IV collagenases (gelatinases) in cultured human mucosal and dermal keratinocytes. The 92-kDa enzyme predominated in both unstimulated and stimulated cultures. The 92-kDa form was stimulated over 5-fold, and the other form by a factor of 2-3. This increase in the synthesis of type IV collagenases was associated with a marked increase in the mRNA levels of these enzymes as well. The induction of the 92-kDa enzyme was similar in culture medium containing either 0.15 or 1.2 mM calcium chloride. Rat mucosal keratinocytes secreted only 92-kDa type IV collagenase, the secretion of which was not regulated by TGF-beta 1. Also, TGF-beta 1 did not cause any significant induction (maximum about 1.2-fold) of either type IV collagenase in human gingival fibroblasts. The induction levels of both collagenases in human keratinocytes were independent of the type of the extracellular matrix the cells were grown on. However, the basement membrane matrix (Matrigel) activated about half of the 92-kDa type to its 84-kDa active form. The data suggest that TGF-beta 1 has a specific function in up-regulating the expression of type IV collagenases in human keratinocytes, offering a possible explanation of how keratinocytes detach from basement membranes prior to the migration over the wound bed.     

On the structure and chromosome location of the 72- and 92-kDa human type IV collagenase genes.

The 72- and 92-kDa type IV collagenases are members of a group of secreted zinc metalloproteases. Two members of this family, collagenase and stromelysin, have previously been localized to the long arm of chromosome 11. Here we assign both of the two type IV collagenase genes to human chromosome 16. By sequencing, the 72-kDa gene is shown to consist of 13 exons, 3 more than have been reported for the other members of this gene family. The extra exons encode the amino acids of the fibronectin-like domain which has so far been found in only the 72- and 92-kDa type IV collagenase. The evolutionary relationship among the members of this gene family is discussed.     

Synthesis of Urokinase-Type Plasminogen Activator and of Type- 1 Plasminogen Activator Inhibitor in Neuronal Cultures of Human Fetal Brain: Stimulation by Phorbol Ester

Human neuronal brain cultures established from 12- and 14-week-old fetuses synthesize and secrete urokinase-type plasminogen activator (uPA) and limited amounts of tissue-type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell-type PA inhibitor (PAI-1), which forms sodium dodecyl sulfate-stable tPA/PAI-1 complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI-1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates the synthesis of both uPA and PAI-1, resulting in a final increase in the plasmin-generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4 alpha-phorbol 12,13-didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin-generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI-1 synthesis.     

Coordinate induction of collagenase-1, stromelysin-1 and urokinase plasminogen activator (uPA) by the 120-kDa cell-binding fibronectin fragment in fibrocartilaginous cells: uPA contributes to activation of procollagenase-1.

Specific fibronectin (Fn) fragments found in synovial fluid of arthritic joints potentially contribute to the loss of cartilage proteoglycans by inducing matrix metalloproteinase (MMP) expression. However, whether or not the Fn fragment-modulated changes in expression of MMPs result in a net increase in matrix-degradative activity through alterations in the balance between MMP activation and inhibition has not been established. To understand the mechanisms by which proteolytic Fn fragments may contribute to joint degeneration, conditioned medium from fibrocartilaginous cells exposed to Fn, its 30-kDa fragment containing the collagen/gelatin-binding domain, its 120-kDa fragment containing the central cell-binding domain, and the RGD peptide were assayed for MMPs, and MMP activators and inhibitors. We found that the 120-kDa fragment of Fn (but not intact Fn), the 30-kDa fragment, and the RGD peptide, dose-dependently induced procollagenase-1 and prostromelysin-1 and decreased levels of the tissue inhibitor of metalloproteinases (TIMPs) -1 and -2. The alpha5beta1 integrin was implicated in the induction of collagenase by the 120-kDa Fn fragment, since collagenase induction was abrogated in the presence of blocking antibody to this integrin. Conditioned medium from cells exposed to the 120-kDa Fn fragment also demonstrated increased levels of the activated collagenase-1, which resulted in significantly elevated collagen degradative activity. That the urokinase plasminogen activator (uPA) was involved in the activation of procollagenase-1 was suggested by findings that the 120-kDa Fn fragment induced uPA coordinately with procollagenase-1, and the activation of procollagenase-1 was dose-dependently inhibited in the presence of plasminogen activator inhibitor-1. These data demonstrate that the 120-kDa cell-binding fragment of Fn induces a net increase in matrix-degradative activity in fibrocartilaginous cells by concomitantly inducing MMPs and their activator, uPA, while decreasing TIMPs.     

Steroid Inhibition of Neural Micro vessel Morphogenesis In Vitro: Receptor Mediation and Astroglial Dependence

Steroid hormones alter several aspects of microvascular function within the CNS. Both microvessel formation and blood-brain barrier expression appear to be influenced by interactions between astrocytes and endothelial cells. To determine if steroids alter astrocyte-endothelial interactions, we studied their effects on astroglial-induced microvessel morphogenesis in vitro. C6 astroglial cells induce bovine retinal microvascular endothelial cells to differentiate into capillary-like structures. Dexamethasone, hydrocortisone, and progesterone at 10 nM inhibited C6-induced microvessel morphogenesis by 75, 35, and 30%, respectively. Inhibition by dexamethasone was both time and concentration dependent, reaching 80-100% at 1 microM. Tetrahydrocortisone and 17 alpha-hydroxyprogesterone had only marginal inhibitory effects. Cortexolone, a glucocorticoid receptor antagonist, blocked inhibition by dexamethasone. Progesterone receptors were expressed in C6 but not bovine retinal microvascular endothelial cells, identifying the astroglial cell as the likely effector of progesterone-mediated inhibition. Astroglial cells were further implicated as the effectors of steroid-mediated inhibition because none of the steroids inhibited astroglial-independent capillary-like structure formation in response to a reconstituted extracellular matrix, Matrigel. These findings are evidence that steroids modulate neural microvascular endothelial cell functions indirectly through perivascular astrocytes via a receptor-mediated mechanism.     

Angiostrongylus cantonensis: induction of urokinase-type PA and degradation of type IV collagen in rat lung granulomatous fibrosis

Granuloma formation and subsequent fibrosis around Angiostrongylus cantonensis larvae in the lungs were induced experimentally in Sprague-Dawley strain rats. Casein zymogram analysis demonstrated that urokinase-type plasminogen activator (uPA) activity was increased during lung inflammation and fibrosis. Granulomatous fibrosis, type IV collagen degradation and activation of uPA occur simultaneously. Furthermore, the present study demonstrated that collagen avidly binds uPA. Immunohistochemical observations showed localization of uPA within the infiltrating leucocytes. We propose that uPA may participate in A. cantonensis-induced granulomatous fibrosis.     

Thrombospondin forms complexes with single-chain and two-chain forms of urokinase

Thrombospondin (TSP), an adhesive glycoprotein found in platelets and extracellular matrix, has been shown previously to interact with plasminogen and tissue plasminogen activator, resulting in efficient plasmin generation. We now demonstrate specific complex formation of TSP with both the single-chain and two-chain forms of urokinase (scuPA and uPA). Binding of uPA and scuPA to immobilized TSP was detected and quantified using colorimetric immunoassays and a functional amidolytic assay. Binding was time and concentration dependent with apparent affinity constants of 40-50 nM. Binding was not affected by serine protease inhibitors, EDTA, or epsilon-aminocaproic acid. scUPA and uPA bound to TSP retained functional activity. Using a sensitive amidolytic assay we found that TSP. scuPA complexes were efficiently converted to TSP. uPA by catalytic plasmin concentrations. Additionally, TSP.uPA complexes were found to have plasminogen-activating activity equivalent to fluid-phase uPA and to be protected from inhibition by plasminogen activator inhibitor type 1, the major plasma and matrix plasminogen activator inhibitor. Using immunohistochemical techniques, we also demonstrated co-distribution of TSP and uPA in normal and malignant breast tissue. Complex formation of TSP with uPA may serve to localize, concentrate, and protect these enzymes on cell surfaces and within the extracellular matrix, thereby providing a reservoir of plasminogen activator activity.     

Collagen-binding domain of human plasma fibronectin contains a latent type-IV collagenase.

Cleavage of the 45-kDa gelatin-binding fragment of human plasma fibronectin with fibronectinase resulted in the activation of two forms of metalloproteinase with different substrate specificities. The 40-kDa FN-type-IV collagenase A degrades heat-denatured type-I collagen, laminin and also native collagen type IV. The 27-kDa FN-type-IV collagenase B degrades native collagen type IV, but it does not cleave laminin and only poorly degrades gelatin. Both enzymes begin with the same N-terminal sequence VYQPQPH- (residues 262-268 of fibronectin) but, contrary to the FN-type-IV collagenase A, the FN-type-IV collagenase B has lost the C-terminal region of type I repeats, where the major gelatin-binding determinants of fibronectin are located. The FN-type-IV collagenases A and B are sequentially similar to the middle domain (domain II) of collagenase type IV, secreted by H-ras-transformed human bronchial epithelial cells. Substrate and inhibition specificity of FN-type-IV collagenase A and B are different from those of FN-gelatinase and FN-laminase, isolated previously from the central and C-terminal fibronectin domains, respectively. The substrate specificity of both enzymes, characterized in this study, is also different from that of already known matrix-degrading metalloproteinases.