Paolo Mignatti

Control of type IV collagenase activity by components of the urokinase–plasmin system: a regulatory mechanism with cell‐bound reactants

The urokinase‐type plasminogen activator (uPA) and the matrix‐degrading metalloproteinases MMP‐2 and MMP‐9 (type IV collagenases/gelatinases) have been implicated in a variety of invasive processes, including tumor invasion, metastasis and angiogenesis. MMP‐2 and MMP‐9 are secreted in the form of inactive zymogens that are activated extracellularly, a fundamental process for the control of their activity. The physiological mechanism(s) of gelatinase activation are still poorly understood; their comprehension may provide tools to control cell invasion. The data reported in this paper show multiple roles of the uPA–plasmin system in the control of gelatinase activity: (i) both gelatinases are associated with the cell surface; binding of uPA and plasmin(ogen) to the cell surface results in gelatinase activation without the action of other metallo‐ or acid proteinases; (ii) inhibition of uPA or plasminogen binding to the cell surface blocks gelatinase activation; (iii) in soluble phase plasmin degrades both gelatinases; and (iv) gelatinase activation and degradation occur in a dose‐ and time‐dependent manner in the presence of physiological plasminogen and uPA concentrations. Thus, the uPA–plasmin system may represent a physiological mechanism for the control of gelatinase activity.

PLASMINOGEN ACTIVATORS AND MATRIX METALLOPROTEINASES IN ANGIOGENESIS

In the initial stages of capillary formation (angiogenesis) microvascular endothelial cells of preexisting blood vessels locally degrade the underlying basal lamina and invade into the stroma of the tissue to be vascularized. A consistent body of experimental evidence has shown that this process requires a wide array of dedradative enzymes. Components of the plasminogen activator (PA)-plasmin system and of the matrix metalloproteinase (MMP) family play important roles. PAs trigger a proteinase cascade that results is the generation of high local concentrations of plasmin and active MMPs. This increase in proteolytic activity has three major consequences: it permits endothelial cell degradation and invasion of the vessel basal lamina, generates extracellular matrix (ECM) degradation products that are chemotactic for endothelial cells, and activates and mobilizes growth factors localized in the ECM. In addition, urokinase-type PA modulates some endothelial cell functions, including proliferation and migration, with a mechanism independent of proteolytic activity. PA and MMP activities are modulated in endothelial cells by complex mechanisms, including transcriptional regulation by a variety of growth factors and cytokines with angiogenic activity, extracellular control of the proteolytic activities by tissue inhibitors, and interaction with binding sites on the cell membrane and ECM.

Proteinases and Tissue Remodeling

The term tissue remodeling describes transient or permanent changes in tissue architecture that involve breaching of histological barriers such as basement membranes, basal laminae, and interstitial stroma [extracellular matrix (ECM)]. Tissue remodeling is important to several stages of wound repair, such as inflammation and granulation tissue formation, and in a variety of other physiological or pathological states. These include ovulation, spermatogenesis, trophoblast implantation, mammary involution following lactation, uterine involution, nerve regeneration, rheumatoid arthritis, tumor invasion, and metastasis formation. A common feature of tissue remodeling involves the production of high levels of extracellular proteolytic activities by parenchymal and/or connective tissue cells. The ECM is organized into highly complex structures, each of which consists of different components including various collagen types, glycoproteins such as fibronectin and laminin, elastin, glycosaminoglycans (GAGs), and proteoglycans. Because these ECM components have distinct hydrolytic requirements for their degradation, remodeling of the ECM involves the action of an array of degradative enzymes.

Transformation-enhancing factor(s) released from chicken Rous sarcoma cells: effect on some transformation parameters.

Abstract The medium of chick embryo fibroblasts (CEF) transformed by Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) contains a factor(s) which complements the expression of some transformation parameters depending on the src gene. Notably, it reverses the block by puromycin of morphological transformation of cells infected with three ts -T mutants after shift-down from restrictive (41.5°) to permissive (37°) temperature. This reversal is not due to the release of inhibition of protein synthesis produced by puromycin, and is accompanied by the expression of two other src -dependent transformation parameters: disorganization of the cytoskeleton and loss of cell surface-associated fibronectin. The factor(s) able to overcome the puromycin block of morphological transformation was operationally called transformation-enhancing factor (TEF) like a previously reported factor favoring transformation by RSV ( Kryceve et al., Int. J. Cancer 17 , 370–379, 1976 ). It is lacking in media of untransformed cells, uninfected or infected with a nontransforming virus (RAV-1), and its production by RSV-infected cells seems to depend on the acquisition of the transformed phenotype, therefore on the expression of the src gene. Its effect was also shown to persist beyond the period of contact with the cells. It appears to be a glycoprotein which can be resolved by gel filtration into two peaks of 250K and 190K, apparently distinct from other known factors spontaneously released by transformed cells. A similar activity was also found in the medium of mammalian (rodent) cells transformed by SR-RSV and by other RNA and DNA oncogenic viruses, but not in the medium of untransformed controls.

Enhancement of plasminogen activator activity by the culture medium of Rous sarcoma virus-transformed cells.

We have tested the effect of the culture medium of chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV) on plasminogen activator (PA) activity of normal and RSV-infected cells. The results obtained showed that fibrin digestion by cultures of normal uninfected and RSV-infected cells directly attached to a fibrin substrate was increased when the cells were exposed to the supernatant of an 18 h-culture of CEF transformed by RSV. Addition of cycloheximide to the supernatant completely abolished this effect. Similarly, exposure of both normal and RSV-infected cells to the culture medium of RSV-transformed CEF resulted in a marked increase of the soluble PA activity present in the supernatant. This effect, however, was not abolished by inhibition of protein synthesis. For both the cell-bound and soluble PA activities of normal and RSV-infected cells the stimulatory effect was transient, being maximal between 8 and 12 h of exposure to the transformed cell-conditioned medium and disappearing after 24 h. A possible correlation with transformation-enhancing factor(s), previously reported to be present in the culture medium of transformed cells, is discussed.

Properties and Kinetics of Development of Rous Sarcoma Virus-Infected Cells Evidenced by Methylene Blue Staining

Three different kinds of areas of infected cells corresponding to different focus formation stages can be evidenced by methylene blue (MB) staining in cultures of chick embryo (CE) fibroblasts infected at low multiplicity with the temperature-sensitive (ts) mutant of Rous sarcoma virus (RSV), FU19, which transforms these fibroblasts at 37 degrees but not at 41 degrees. These are: (a) areas of MB-stainable cells with transformed phenotype (STP areas=foci); (b) areas of MB-stainable cells with normal phenotype (SNP areas), and (c) areas of MB-unstainable cells with normal phenotype (USNP areas). DNA and RNA synthesis and virus production were followed in these various stages at 37 degrees and at 41 degrees. The results show that when cultures are shifted from 37 degrees to 41 degrees, virus production in the SNP and USNP areas which arise by phenotypic reversion of STP foci remains comparable to that of the latter foci. On the contrary, DNA and RNA synthesis are markedly reduced in SNP and USNP areas, DNA synthesis falling down to the level of uninfected cells, and RNA synthesis remaining somewhat higher. The kinetics of development of SNP and STP areas in cultures infected with FU19 and with the parental virus SR4 were also compared. The results confirm that SNP areas are precursors of STP areas but that this passage occurs at a slower rate in cultures infected with FU19.