Low fluences of ultraviolet irradiation stimulate hela cell surface aminopeptidase and candidate “TGFαase” activity

Abstract

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 TGF alpha 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 TGF alpha 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 proTGF alpha, 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 TGF alpha 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 TGF alpha 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".(ABSTRACT TRUNCATED AT 400 WORDS)

Increased ecto-metallopeptidase activity in cells undergoing apoptosis

“…Using the peptidase assay underpinning the present study, we showed that several agents capable of inducing ''genetic stress'' [Herrlich et al, 1986] caused an increase in an ectopeptidase activity that was a candidate ''TGF␣ase'' in HeLa cultures [Brown et al, 1993]. In addition, genetic stress was shown to be accompanied by activation of at least three other ectopeptidases (eAP, eDP, and eTP) in those experiments.…”

“…TGF␣ levels have been shown to be elevated in human skin after exposure to UV [Murphy et al, 1991], and this may be attributable to increased release from melanocytes and keratinocytes [Ellem et al, 1988]. Previous studies using cultured HeLa cells have shown that UV irradiation increased the level of ''TGF␣ase'' and aminopeptidase activity, which reached maximal levels (2-to 5-fold) at 16-20 h postirradiation, when assayed with either intact cells [Brown et al, 1993] or purified cell membranes [Piva et al, 1997]. Preliminary studies have shown that there was an increase in the activity of other ecto-metallopeptidases in UV-irradiated HeLa cells undergoing apoptosis [Piva et al, 1998].…”

Involvement of protein kinase C and an elastase‐like enzyme in the processing of transforming growth factor‐α in human colon carcinoma cell lines

“…Since many of these factors, including TGF-a, are expressed as membrane-bound precursors, control of the processing and release of these factors warrants investigation. Information concerning the processing of pro-TGF-a in some systems has been described (Mueller et al, 1990;Pandiella and MassaguC, 1991a, b;Brown et al, 1993), but very little is known about the processing of endogenous pro-TGF-a by endogenous cellular enzymes. Although incubation of HCT 116 cells with exogenous porcine pancreatic elastase was shown to release cellassociated TGF-a (Zorbas and Yeoman, 1993), comparisons of TGF-a processing in phenotypically different colon carcinoma cells have not been examined.…”

“…The fact that added elastase could release TGF-a from arterioles (Mueller et al, 1990) and HCT 116 colon carcinoma cells (Zorbas and Yeoman, 1993) confirms that the cleavage reaction occurs on the extracellular side of the membrane. A proteolytic enzyme, which could be responsible for the processing of the membrane inserted pro-TGF-a, has been described in transformed and nontransformed rat liver epithelial cells (Cappelluti et al, 1993) and HeLa cells (Brown et al, 1993). Therefore, Triton extracts of the colon carcinoma cells were examined and found to contain an elastase-like enzyme.…”

Erbb2 Regulates Inflammation and Proliferation in the Skin after Ultraviolet Irradiation

“…3 UV exposure also alters receptor degradation [3][4][5][6] and increases EGFR ligand expression. 7,8 Activation of EGFR by UV stimulates both MAPK and phosphatidyl inositol 3-kinase/Akt signaling. 9 EGFR activation also suppresses apoptosis, increases proliferation, delays hyperplasia, and increases skin carcinogenesis following UV exposure.…”