Effect of UV irradiation on cell surface protease activity and amino acid uptake

The surface of most cells includes a coterie of resident proteins which act as receptors for a wide variety of ligands and other proteins which are potentially bioactive on cell-cell contact (juxtacrine effects), or else are released by enzyme activity to influence cell behaviour by autocrine or paracrine mechanisms. We previously found that UVC irradiation stimulates the release of TGFalpha from its membrane-bound preprocursor form whereby it acts as a stimulus to rapid, reparative cell multiplication; clearly this runs the risk hastening mitosis before UV-induced DNA damage is fully corrected, which in turn may increase the likelihood of residual lesions persisting and hence of new mutations being generated. We found that sublethal UVC irradiation (10 J m(-2)) of HeLa cell cultures also resulted in activation of ecto-aminopeptidase and ecto-endopeptidases which were maximal 16 and 20-24 h after irradiation, respectively. Both of these classes of protease were shown to be metalloproteases using a nonapeptide substrate (called P9) which is cognate to the N-terminal cleavage site of preproTGFalpha except for a reporter 125I-tyrosine [Piva et al., J. Cell. Biochem. 64 (1997) 353-368]. We now show that the N-terminal tyrosine cleaved from P9 by cell surface aminopeptidase activity, was found to be taken up by the cell resulting in its 10-25-fold concentration intracellularly, some two- to threefold higher than from a reservoir source, and may represent a novel salvage pathway for recovery of essential amino acids. Aminopeptidase activity was found to be both temperature- and FBS-dependent but was not reliant on ATP for its activity. Tyrosine transport across the cell membrane was also temperature and FBS-dependent but required ATP for maximal activity. UVC irradiation enhanced aminopeptidase activity but not tyrosine uptake by the cultures. The fraction of HeLa cells undergoing apoptosis increased in those cultures which were exposed to higher doses of UVC. The levels of ecto-aminopeptidase and ecto-endopeptidase activity in apoptotic cells were elevated compared to viable cells receiving the same dose of UVC. These results suggest that increased levels of cell surface protease activity in apoptotic cells would increase the amounts of free amino acids and growth factors in the extracellular medium and hence stimulate the proliferation of surrounding cells to replace those killed by UV irradiation.