LOW FLUENCES OF ULTRAVIOLET-IRRADIATION STIMULATE HELA-CELL SURFACE AMINOPEPTIDASE AND CANDIDATE TGF-ALPHA-ASE ACTIVITY

Several forms of perturbation result in the release of bioactive molecules into the microenvironment of injured cells to mediate the inflammatory or reparative reactions which restore normal tissue structure and function. Amongst other products, ultraviolet irradiation (UV) causes the release of the growth factor TGFalpha from a variety of epithelial cell sources, apparently by a post-translational mechanism. Here we have explored the hypothesis that UV results in the activation of cell surface proteases which may then be capable of excising mature TGFalpha from its plasma membrane-bound precursor. Using a recently described, sensitive assay of peptidase activity tailored to the substrate requirements for cleavage of the scissile bonds in proTGFalpha, we have found that nonlethal fluences of UV (< 12 Jm-2) to HeLa cell cultures are followed by large increases in cell surface proteolytic activities. Amongst these, endopeptidase activity produces a similar product profile from the nonapeptide substrate to that of human leukocyte elastase, an enzyme previously shown to be capable of releasing a bioactive, mature form of TGFalpha from its cell-bound precursor. However, in addition to this candidate ''TGFase'' activity, cell surface aminopeptidase activity was also very significantly increased. The increase in the two classes of peptidase function differed in the timing of their responses. Aminopeptidase activation occurred immediately following UV, peaking after some 15-20 h, whereas the increase in endopeptidase activity lagged 6 h behind, cresting after 20-24 h. No evidence for a role for aminopeptidase in the activation of the endopeptidase could be found. Also, there was no increase in the total proteolytic activity demonstratable in cell extracts following UV. Attempts to interrupt the UV peptidase activation by inhibiting protein synthesis with cycloheximide were unsuccessful; rather, the inhibitor itself caused an increase in both classes of peptidase activity during the first 20 h. Unlike the UV response, both the aminopeptidase and endopeptidase ectoactivities increased simultaneously within a few hours of introducing cycloheximide into the medium of unirradiated cultures. The cycloheximide induced activity peaked after 20 h. Interestingly, cycloheximide alone has previously been shown to potentiate TGFalpha release from a cell line producing its precursor constitutively. These data suggest that both UV and cycloheximide can initiate reactions in HeLa cells which result in ectopeptidase activation of a global nature. Since both agents result in rapid interruption of DNA synthesis, it is possible that this cell surface proteolytic response may be analogous to, or part of, the ''mammalian genetic stress response.'' The mechanism of the activation of the cell surface proteases appears to be post-translational, perhaps part of a proteolytic cascade originating from perturbed macromolecular synthesis.