The effect of UV radiation on furin activity in human keratinocyte cell lines
Creators: name: Ravi, Rethika
Publisher: name: RMIT University
Year: 2024

description: UV light is acknowledged to be the main carcinogen involved in the formation of skin cancer. It generates a range of biological responses in the skin, which includes adaptive, inflammatory and immunological reactions. In this thesis we exposed cultures of keratinocyte-derived cell lines (HEK cells-primary keratinocytes, HaCaT cells-immortalized keratinocytes and Colo 16 cells-squamous cell carcinoma cell line) to UVA and/or UVB radiation and observed the effect this had on a number of cellular processes. The UV dose used throughout the thesis was equivalent to the UV component observed in one medial erythemal dose which was 40 kJ/m2 UVA and 2 kJ/m2 UVB. In HEK cells, the viability of attached sham-irradiated cultures was 92%. UVA radiation had no effect on cell viability when compared to sham-irradiated controls. In HaCaT and Colo 16 cells, exposure to UVA reduced cell viability by 11% and 7%, respectively. In HEK cells, the attached viable cells were more sensitive to UVB and UVAB radiation as the cell viability was reduced by 46% and 33%, respectively. When HaCaT cells were exposed to UVAB radiation (cell viability was reduced by 32%), the result had characteristics similar to that seen for those cells irradiated by either UVA or UVB where the viability was reduced by 10% and 20%, respectively. In Colo 16 cells, the viability of attached cells in UVB- (49%) and UVAB-irradiated cells (49%) was reduced when compared to the sham-irradiated cultures (90%). The viability of the detached viable cells was low (&lt;5%) for all UV types and doses. The Colo 16 cells were more sensitive to UVR than were HEK or HaCaT cells. Furin levels in UV-irradiated HEK cells did not change 24 h post-irradiation unlike that seen for HaCaT or Colo 16 cells. Higher furin expression was seen in UVAB-irradiated Abstract XXIX HaCaT cells (12x) and UVB-irradiated Colo 16 cells (7.5x) when compared to un-irradiated controls. Furin mRNA levels were shown to be maximal after 24 h in HaCaT cells (91-fold) and 12 h for Colo 16 cells (1.5-fold) exposed to UVB-irradiation. There was a moderate correlation between furin mRNA and protein levels in irradiated HaCaT cells (r2 = 0.581) compared to that of Colo 16 cells (r2 = 0.399) 24 h post-irradiation. The turnover of furin protein was longer in Colo 16 cells compared to HaCaT cells (T½ = 17.0 h and 5.6 h, respectively). IL-1 had a slightly additive effect on furin expression and activity in HEK cells but a slightly suppressive effect on HaCaT and Colo 16 cells. The level of MMP-2 and -9 activity in HaCaT cells did not change in the 72 h period following exposure to UVR. Maximal MMP-2 and -9 activity was seen at 12 h (13489.9 and 15392.0 au/mg cell protein, respectively) in UVB-irradiated HEK cells. While high levels of MMP-2 activity was observed at 24 h (1998.1 au/mg cell protein) in UVB-irradiated Colo 16 cells, levels of MMP-9 were highest at 24 and 72 h (6512.8 and 2605.9 au/mg cell protein), respectively in these cells. In HaCaT cells, the expression of proMMP-2 in UVB- (63%) and UVAB-irradiated cells (64%) were similar to that of sham-irradiated controls. Active MMP-2 expression was higher in UVB-irradiated HaCaT cells (41%). In Colo 16 cells the highest induction of pro and active MMP-2 protein was seen in UVA-irradiated cells (139% and 112%, respectively). The level of active MMP-9 protein was only elevated in HaCaT cells exposed to either UVA (80%) or UVAB radiation (121%). This result was not seen in Colo 16 cells. Addition of IL-1 significantly increased MMP-9 activity, protein and mRNA expression in HaCaT and Colo16 cells. The mRNA levels for MMP-2 (73-fold) and -9 Abstract XXX (330-fold) were maximal 24 h post UVB-irradiation in HaCaT cells. In Colo 16 cells, maximal mRNA expression was seen at 0 h for MMP-2 and 12 h for MMP-9 (17-fold). There was a strong and positive correlation between furin and MMP mRNA levels in HaCaT cells but not in Colo 16 cells. 1, 10 phe and MMPI significantly decreased the activity of MMP-2 and -9 shed from UV-irradiated cells. When Dec RVKR cmk was added to HaCaT and Colo 16 cells, there was a loss of MMP-9 activity in the media of the UV-irradiated cells. In the presence of these inhibitors migration of the HaCaT and Colo 16 cells were also decreased. The effect of UVR on the release of TNF from human keratinocyte-derived cells was also examined. The addition of IL-1 increased the release of TNF in sham-irradiated HEK (2763.73 ng/mg cell protein), HaCaT cells (935.5 ng/mg cell protein) and Colo 16 cells (81.9 ng/mg cell protein) when compared to untreated controls. UVAB-irradiated cells treated with IL-1 induced the greatest increase in TNF release in all cell lines (32.9-, 4.2- and 7.7-fold in HEK, HaCaT and Colo 16 cells, respectively) compared to treated sham-irradiated cells. Addition of 1, 10 phe caused an inhibition of ~97% in IL-1 treated UVAB-irradiated HaCaT and Colo 16 cells. 95% in HaCaT cells and 87% in Colo 16 cells The level of TNF released from Dec RVKR cmk treated UVAB-irradiated cells was significantly reduced by 95% in HaCaT cells and 87% in Colo 16 cells. MMPI also inhibited TNF release from HaCaT and Colo 16 cells. The maximal mRNA levels post UVB-irradiation for TNF (59-fold) were at 16 h in HaCaT cells and 8 h (12.1-fold) in Colo 16 cells. The level of TACE mRNA was relatively constant in both these cells for up to 32 h post-irradiation. Abstract XXXI The results obtained suggest that UVR up-regulates furin mRNA and protein expression in these keratinocyte-derived cells. Colo 16 cells harbor furin protein for a much longer time which may contribute to increasing their metastatic nature as one of the proteases they process, MMP is known to cleave the ECM, allowing metastasis to occur. The results of this study suggest that (1) the changes in TACE activity are due to the conversion of pTACE to mTACE mediated by furin, and (2) TACE is the main enzyme involved in the release of TNF but MMPs may also be involved. The three keratinocyte cell lines respond differently when exposed to UVR. The response of HaCaT cells to UVR differs to that of HEK which suggest that these immortalized cell lines may not be a suitable model to study keratinocytes. Colo 16 exhibits a completely different profile to that of both HaCaT and HEK which most likely reflects its carcinogenic nature of these cells which may contribute to their metastatic potential, through changes in MMP and TACE activity.