Faculty Bibliography

A diffusible factor produced and secreted by malignant murine cells was capable of inducing plasminogen activator production by normal diploid human fibroblasts. The factor's ability to induce plasminogen activator was insensitive to treatment with nucleases, but its activity was destroyed by digestion with proteases. It is proposed that such a factor would play a role in malignancy if it would recruit normal cells that were adjacent to transformed cells to produce plasminogen activator which could result in tumor-promoted proteolysis

Bovine capillary endothelial (BCE) cells produce increased amounts of the proteases plasminogen activator (PA) and latent collagenase when cultured in the presence of the following preparations which are known to contain angiogenic activities: bovine retinal extract, mouse adipocyte conditioned medium, and human hepatoma cell lysate. These preparations stimulated both BCE cell PA and collagenase activities in a dose-dependent manner. Both activities were increased to about the same level by these preparations as by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. Mitogens that are not angiogenic, such as insulin, epidermal and fibroblast growth factors, and endothelial cell growth supplement, had no effect on BCE cell PA and collagenase activities. Two of the angiogenic preparations (retinal extract and mouse adipocyte-conditioned medium) had no effect on PA activity in endothelial cells derived from bovine aortae (BAE cells). The angiogenic preparations had little (human hepatoma cell lysate, mouse adipocyte-conditioned medium) or no (bovine retinal extract) effect on BAE cell collagenase activities. In the bovine system, the induction of high levels of both PA and collagenase activities by angiogenic preparations is limited to capillary endothelial cells

Human A431 epidermoid carcinoma cells in culture exhibit epidermal growth factor (EGF)-induced 'down-regulation' of cell-surface and total cellular (Triton X-100 extractable) EGF receptors caused entirely by an enhanced rate (4-fold) of receptor inactivation [Krupp, M. N., Connolly, D. T. & Lane, M. D. (1982) J. Biol. Chem. 257, 11489-11496]. The following observations show that this enhanced rate of EGF receptor inactivation is closely correlated with an increased cellular activity of plasminogen activator (PA), a serine protease. First, EGF-induced down-regulation of cell-surface and total cellular EGF receptors and the concomitant increase in cellular PA activity occur with identical kinetics, the t 1/2 for both processes being 3-3.5 hr. Second, the EGF dose-response curves for down-regulation of total cellular EGF receptor and increased PA activity are similar. The EGF concentrations for half-maximal responses of both processes are 10-15 nM and 20 nM, respectively. Third, the removal of EGF from previously down-regulated cells results in the recovery of total cellular EGF binding activity with a concurrent loss of cellular PA activity. Fourth, blocking PA synthesis or activity with cycloheximide or dexamethasone prevents down-regulation of the EGF receptor. Fifth, the addition of leupeptin, an inhibitor of PA and plasmin action, blocks EGF-induced receptor down-regulation as well as the increase of PA activity. That EGF receptor down-regulation is independent of plasminogen per se in the culture medium suggests that PA-mediated events may initiate the rapid inactivation of the EGF receptor that occurs during down-regulation

This study was undertaken to determine whether plasminogen was present within the epidermis. Cryostat sections of normal human skin were incubated with either whole rabbit antihuman plasminogen serum, the IgG fraction thereof, or the immune-specific IgG fraction thereof, purified by affinity chromatography on plasminogen-Sepharose. Standard indirect immunofluorescent techniques using rhodamine-conjugated goat antirabbit IgG were subsequently employed. Fluorescence was localized to the basal layer of the epidermis. Nonimmune serum and immune serum depleted of its specific anti-plasminogen IgG by prior affinity chromatography yielded negative results, demonstrating the specificity of staining for plasminogen. Our findings indicate that plasminogen is present within the epidermis, localized to the peripheral intracytoplasmic area of basal keratinocytes. This finding supports the hypothesis that a proteolytic event mediated by plasminogen activator with the subsequent generation of plasmin may be involved in normal keratinocyte physiology

The activity of the serine protease plasminogen activator (PA) was measured in cell lysates from primary mouse keratinocyte cultures as well as from a number of established mouse keratinocyte lines. Enzyme activity was generally higher in the transformed lines than in the primary cultures; however, among the lines tested, those that expressed the highest degree of morphologic differentiation had the highest levels of cell-associated PA. In both the normal (primary) and transformed (established) keratinocyte cultures, PA activity increased when cultures reached confluence and morphologic evidence of differentiation was noted. The highest specific activity of the enzyme was found in cells shed from differentiating cultures, which consisted predominantly of detergent-resistant cornified envelopes. As the cultures differentiated and these cells were shed from the culture surface, the total cell-associated PA activity of the culture decreased accordingly. In both the normal and transformed keratinocyte cultures, peak PA activity occurred at a time when DNA synthesis was declining. These findings indicate that as keratinocytes differentiate, their intracellular levels of PA increase. The modulation of this endogenous keratinocyte enzyme may play an important, although as yet undefined, role in the normal maturation and terminal differentiation of these cells

The role of the matrix glycoprotein fibronectin in the formation of the external granular layer of the developing mouse cerebellum was investigated by in vitro studies of the binding of cerebellar cells to a fibronectin-coated culture substratum and by in vivo immunocytochemical localization of antiplasma fibronectin antiserum in cerebellar tissue. The adhesion of cells dissociated from embryonic and early postnatal mouse cerebellum is developmental stage-specific when the cells are plated on tissue culture substrata derivatized with human plasma fibronectin. Cells dissociated from mouse cerebellum at embryonic day 13 form cellular aggregates on insoluble plasma fibronectin. In contrast, cells dissociated from embryonic day 16 through postnatal day 7 cerebellum form a monolayer. Time-lapse video recordings reveal extensive cell movement of late embryonic and early postnatal cerebellar cells on insoluble plasma fibronectin. Late embryonic and early postnatal cerebellar cells bind to fibronectin but do not degrade the fibronectin substratum. Immunocytochemical studies of the binding of antiplasma fibronectin antisera to cryostat sections of intact embryonic and early postnatal cerebellar tissue reveal a brightly stained region of endogenous fibronectin along the route of granule cell migration from the lateral caudal part of the neuroepithelium lining the fourth ventricle up onto the external surface of the cerebellar anlage. When the formation of the external granular layer is completed, the intense region of fibronectin is no longer visible