Gene expression profiles of TNF-alpha, TACE, furin, IL-1beta and matrilysin in UVA- and UVB-irradiated HaCat cells.
OBJECTIVE
It is known that solar ultraviolet (UV) irradiation exerts multiple effects on mammalian skin tissues, one of which is the induction of local and systemic immunosuppression as well as inflammation. Tumor necrosis factor-alpha (TNF-alpha) and other cytokines are suggested to play a role in these responses. Quantitative real-time polymerase chain reaction (TaqMan RTPCR) was used to elucidate the effect of UVA and UVB irradiation on the expression of genes coding for TNF-alpha, IL-1beta, IL-10, FasL, matrilysin, TACE and furin in HaCaT cells over a 48 h period (IL-1beta, interleukin-1beta; FasL, Fas ligand).


METHODS
Cultured HaCaT cells were either sham irradiated (control) or exposed to UVA (2000 and 8000 J/m2) or UVB (200 and 2000 J/m2) radiation. RNA was extracted from cells at 0, 4, 8, 12, 16, 24, 48 h post-irradiation and reverse transcribed to generate cDNA for subsequent real-time PCR amplification.


RESULTS
Significant increases in the mRNA levels for all genes tested were detected in both UVA- and UVB-irradiated HaCaT cells compared with control (sham-irradiated) cells. TNF-alpha mRNA levels were immediately up-regulated (0 h) after irradiation, with maximal induction at 8 h post 2000 J/m2 UVA and 200 J/m2 UVB irradiation, at 4 h post 8000 J UVA irradiation and at 48 h post 2000 J/m2 UVB irradiation. No correlation was observed between TNF-alpha, TACE and furin mRNA induction in the different irradiated cohorts.


CONCLUSIONS
Results suggest that time-distinct gene induction of TNF-alpha, furin, IL-1beta and matrilysin may be involved in UV-induced cellular responses, but not for TACE. In general, mRNA induction was dose dependent at some time points post-irradiation, but not throughout the whole time course tested. Our results show that quantitative real-time PCR is a useful tool in the analysis of quantitative changes of mRNA levels in cultured HaCaT cells after UV exposure.
School of Medical Sciences, RMIT University, Bundoora, Vic., Australia. beata.skiba@rmit.edu.au
Beata
B
Skiba
Bronwyn
B
Neill
Terrence J
TJ
Piva
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D002460:Cell Line; D045683:Furin / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics*; D016753:Interleukin-10 / Q000235:genetics; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism / Q000528:radiation effects; D020783:Matrix Metalloproteinase 7 / Q000235:genetics*; D008666:Metalloendopeptidases / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D014466:Ultraviolet Rays; D015854:Up-Regulation
D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D045683:Furin; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D020783:Matrix Metalloproteinase 7; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human