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J.N. Mckie, J. Mohamed, Z. Li, C.E. Arris, P. Hiscott, C. Sheridan, K.P. Langton, M.D. Barker, M.P. Clarke, D.J. Bevitt; Expression of ADAMTS Metalloproteinases in ARPE19 Cells: Transcriptional Regulation by TNF . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1703.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study the ARPE19 cell line for expression of members of the ADAMTS family of metalloproteinases and to investigate gene expression regulation for these enzymes under stimulation with the pro-inflammatory cytokine TNFα. Methods: ARPE19 cells were stimulated with TNFα at 5 and 50ng/ml and total RNA was harvested at 2,6,24 and 48hours post-stimulation. RT-PCR was used to detect expression of ADAMTS1-9, including short and long isoforms of ADMTS7 and ADAMTS9. The RT-PCR products were cloned and, after sequencing, were radiolabelled and used to probe Northern blots of total RNA harvested from the ARPE-19 cells. Results: RT-PCR: ADAMTS1, ADAMTS3, ADAMTS5, , ADAMTS7-S, ADAMTS7-L, ADAMTS9-S and ADAMTS9-L were all detected by RT-PCR in stimulated and unstimulated ARPE-19 cells. ADAMTS2 and ADAMTS6 were only detected by RT-PCR in cells which had been treated with TNFα. ADAMTS4 and ADAMTS8 were not detectable by RT-PCR. Northern blot: ADAMTS1, ADAMTS6, ADAMTS9-S and ADAMTS9-L were all upregulated by TNFα. ADAMTS5 and ADAMTS7-L expression was not regulated by TNFα. ADAMTS2 showed a moderate upregulation at 24hours, but not at other timepoints. ADAMTS3 and ADAMTS7-S were not detected by Northern blot even though they were detected by RT-PCR. This probably reflects the relatively low sensitivity of Northern blotting compared to RT-PCR. Conclusions: It has been shown by others that ADAMTS1 is involved in neovascularisation, ADAMTS2 and ADAMTS3 play a critical role in the early processing of collagen and ADAMTS5 is able to cleave the proteolglycans, aggrecan and versican. The substrate activity of ADAMTS6, ADAMTS7 and ADAMTS9, which are also expressed by the ARPE-19 cells, is not yet known, but they are likely to have similar roles in turnover of extracellular matrix. Expression of these ADAMTS genes in ARPE19 cells thus implies that they may be involved in turnover of the retinal ECM in both normal and pathological conditions. The upregulation of ADAMTS1, ADAMTS6 and both isoforms of ADAMTS9 by TNFα also suggests a possible role for these enzymes in inflammatory conditions of the retina.
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