LRegulation of cytokine-induced HW-1 alpha expression in rheumatoid synovial fibroblasts

The transcription factor hypoxia-inducible factor (HIF)-1 plays a central physiological role in oxygen and energy homeostasis, and is activated during hypoxia by stabilization of the subunit HIF-1 alpha. Activation can also occur by proinflammatory cytokines during inflammation. Hypoxia, as well as proinflammatory cytokines, plays an important role in the synovia in rheumatoid arthritis (RA) patients. Expression of HIF-1 alpha has been demonstrated in RA synovial lining layer. The aim of the study was to investigate the regulation of the intracellular signal transduction pathways, involved in the expression of HIF-1 alpha, and in the expression of genes regulated by HIF-1 alpha in rheumatoid synovial fibroblasts (RSF). RSF were cultured under proinflammatory conditions (IL-1 beta and TNF-alpha stimulation) and under chemical hypoxia (CoCl2 treatment). Expression of HIF-1 alpha was analyzed in nuclear extracts by Western blotting. The effect of inhibitors of the PI3K and the ERK pathway on HIF-1 alpha protein expression was measured. mRNA expression of HIF-1 alpha, COX-2, vascular endothelial growth factor (VEGF), and stromal cell-derived factor (SDF)-1 was determined by real-time RT-PCR, and protein production of VEGF and SDF-1 by ELISA. Treatment of the synovial fibroblasts with 150 MM CoCl2 as well as stimulation with 10 ng/mL IL-1 beta or TNF-alpha resulted in strong protein expression of HIF-1 alpha, measured with Western blotting. Pretreatment with the MEK1/2 inhibitor PD98059 as well as the PI3K inhibitor LY294002 resulted in inhibition of the cytokine-induced HIF-1 alpha expression. Furthermore, it was shown that cytokine-induced mRNA expression of HIF-1 alpha was inhibited by the PI3K inhibitor. We found that cytokine stimulation induced VEGF mRNA and protein production, but no significant effect of kinase inhibition was found on VEGF production in cytokine-stimulated RSF, Both the ERK pathway and the PI3K pathway are involved in the cytokine-induced HIF-1 alpha expression in RSF and in the expression of proangiogenic factors