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Tumor cells express NKG2D ligands on their cell surface, which are the ligandsof the activating receptor, NKG2D, that is expressed on the surface of NK cells.The binding of NK cells to tumor cells through the interaction of NKG2D and itsligands induces the cytolysis of the tumor cells. In the present study, we investigatedthe effects of hypoxia on the expression of NKG2D ligands on the surface of humanosteosarcoma cells using three cell lines. To produce hypoxic and normoxic conditions,the osteosarcoma cell lines were cultured under 1 and 20% O2 conditions, respectively.The osteosarcoma cells expressed NKG2D ligands such as MHC class I-related chainmolecules A and B (MICA and MICB) and the UL16-binding proteins 1, 2 and 3 (ULBP1, 2 and 3). MICA was the most frequently expressed NKG2D ligand in the osteosarcomacells. Hypoxia decreased the expression of cell surface MICA only without increasingthe secretion of soluble MICA, which is produced by proteolytic cleavage of cellsurface MICA. Hypoxia consistently decreased the susceptibility of the osteosarcomacells to the cytotoxicity of the NK cells. Hypoxia induced the expression of hypoxia-induciblefactor-1α (HIF-1α), and knockdown of the expression of HIF-1α using small interferingRNA increased the expression of cell surface MICA and concomitantly increasedthe level of soluble MICA. Hypoxia decreased the production of nitric oxide (NO)metabolites (nitrite and nitrate), thus, indicating a decreasing effect on NOproduction. However, a NO donor, NOC18, decreased the expression of cell surfaceMICA without any apparent effects on the expression of HIF-1α under both hypoxicand normoxic conditions. The present results indicate that hypoxia downregulatesthe expression of cell surface MICA without increasing the level of soluble MICAin a HIF-1α-dependent manner and suggest that the effects of hypoxia are not linkedto the hypoxia-induced reduction of NO production.

Hypoxia downregulates the expression of cell surface MICA without increasing soluble MICA in osteosarcoma cells in a HIF-1α-dependent manner