HIF-2α regulates FLS proliferation, RANKL expression in FLS, osteoclastogenesis, and pannus formation.

Abstract

<p>(A) BrdU-incorporation assays in FLS infected with Ad-C or Ad-<i>Epas1</i> (MOI 800) (left), and FLS from WT or <i>Epas1</i>^+/− mice treated with 1 ng/ml of IL1β (right) (<i>n</i> = 6). (B) Ki67 staining in synovial sections from WT and <i>Epas1</i>^+/− mice without (NI) or with CIA, or from WT mice injected with Ad-C or Ad-<i>Epas1</i> (MOI 800) (<i>n</i> = 8). (C) Double-immunofluorescence staining for HIF-2α and Ki67 in mouse CIA synovium. Ki67-positive cells among HIF-2α–overexpressing cells were counted (<i>n</i> = 8). (D) RANKL mRNA levels were quantified in FLS infected with Ad-C or Ad-<i>Epas1</i> (MOI 800) or treated with 1 ng/ml of IL1β (<i>n</i> = 10). (E) Representative images of RANKL immunostaining in the knee synovium of WT or <i>Epas1</i>^+/− mice without (NI) or with CIA (Left). Typical immunofluorescence microscopy image of triple-stained CIA synovium (Right). (F) TRAP staining and counting of TRAP-positive multinucleated cells (<i>n</i> = 10) in the pannus of the bone–cartilage interface in WT and <i>Epas1</i>^+/− mice without (NI) or with CIA, or following injection with 1×10⁹ PFU of Ad-C or Ad-<i>Epas1</i>. (G) TRAP staining during <i>in vitro</i> osteoclastogenesis of precursor cells isolated from WT and <i>Epas1</i>^+/− mice or WT precursor cells infected with Ad-C or Ad-<i>Epas1</i> (800 MOI) (Left). Osteoclastogenesis was quantified by measuring the osteoclast area (<i>n</i> = 10) (Right). Values are means ± SEM (*<i>p</i><0.01, **<i>p</i><0.001). Scale bar, 50 µm.</p