Increased release of the tumour necrosis factor receptor p75 by immortalized human keratinocytes results from an activated shedding mechanism and is not related to augmented steady-state levels of p75 mRNA.

Abstract

The soluble tumour necrosis factor receptor I (sTNFRI, p55) is produced at similar levels by both immortalized (A431, HaCaT, KB) and primary normal human keratinocytes (HNK), whereas the soluble TNFR II (sTNFR II, p75) appears to be specifically released only by immortalized human keratinocytes. The purpose of this study was to investigate whether the increase in p75 secretion by immortalized human keratinocytes is due to an increased shedding of the receptor from the cell membrane, or is related to increased steady-state levels of p75 mRNA. FACS analysis showed that levels of membranous p75 decreased in a time-dependent manner in immortalized cells cultured for 1, 3, 6, 12 and 24 h, while remaining unchanged in HNK throughout. Northern blot analysis showed that after 12 h of culture, when p75 expression was decreased on the cell membrane of all immortalized cells, there was no significant difference in steady state levels of p75 mRNA between immortalized keratinocytes and HNK. Supernatants of immortalized cells, cultured for 24 h contained distinct levels of p75, while levels of p75 in supernatants of HNK were under the detection limit, confirming that the p75 decrease on the cell membrane results from increased p75 shedding from the cell membrane of immortalized cells. In contrast to p75, p55 was continuously expressed on the cell membrane of normal and immortalized keratinocytes without significant variation throughout the entire 24-h culture period and was similarly shed by both cell types. These results suggest that immortalized keratinocytes are specifically activated for shedding of p75 from the cell membrane. Since p75 has a high affinity for TNF, the release of this receptor may imply a direct role in the escape of malignant/transformed keratinocytes from the TNF-mediated immune response.