HIF-2α is up-regulated by pro-inflammatory cytokines in FLS of RA synovium.

<p>(A and B) Typical immunofluorescence microscopy images of DAPI, HIF-2α, and FLS markers (vimentin and CD55) or the macrophage marker CD68 in human RA synovium (A) and mouse CIA synovium (B). (C, Left) Primary culture of total synovial cells isolated from DBA/1J mice. DC, dendritic cells; MΦ, macrophages. (Center) Typical immunofluorescence microscopy images of HIF-2α, DAPI, and vimentin or CD68. (Right) The percentage of FLS and macrophages positive for HIF-2α staining was determined from six microscopic fields (<i>n</i> = 4). (D) Raw264.7 cells were treated with LPS (50 ng/ml) or TNFα (50 ng/ml) for 24 h. mRNA levels were detected by RT-PCR analysis (<i>n</i> = 6). (E) Primary cultured FLS were treated with IL1β (1 ng/ml), IL6 (100 ng/ml), IL17 (10 ng/ml), or TNFα (100 ng/ml) for 24 h. mRNA levels of HIF-1α and HIF-2α were quantified by qRT-PCR (<i>n</i> = 6). (F) FLS were treated with PD98059 (PD; 20 µM) to inhibit ERK, SB203580 (SB; 20 µM) to inhibit p38 MAP kinase, SP600125 (SP; 20 µM) to inhibit JNK, or the indicated concentration (µM) of BAY 11-7085 (BAY) to inhibit NF-κB. The cells were exposed to IL1β or TNFα for 24 h, and HIF-2α mRNA levels were quantified (<i>n</i> = 6). (G) Mouse CIA synovium was stained for the hypoxia marker pimonidazole (upper). Primary cultured FLS were maintained under hypoxic conditions or were infected with Ad-<i>Epas1</i> at an MOI of 800 for 24 h. HIF-1α and HIF-2α proteins were detected by Western blotting (lower). Values are presented as means ± SEM (*<i>p</i><0.01, **<i>p</i><0.005, ***<i>p</i><0.001). Scale bar, 50 µm.