Effects of hypoxia on protein and gene expression in fibroblast-like synoviocytes

Background

The synovial microenvironment in rheumatoid arthritis (RA) is often hypoxic with evidence of anaerobic metabolism. This results from an imbalance between high metabolic demands and impaired tissue perfusion due to microvascular damage. Cellular responses to hypoxia are mediated by the transcription factor HIF-1α, which is exquisitely and rapidly controlled by cellular oxygen tension. HIF-1α has been shown to play a key role in promoting tumor angiogenesis, and was recently shown to be critically important in inflammatory responses.

Methods

To better define the effects of hypoxia in the synovial microenvironment, we studied the response of fibroblast-like synoviocytes (FLS) to hypoxic stimulation using the gene expression microarray, quantitative RT-PCR, and matrix-assisted laser desorption-time of flight mass spectrometry. The expression of HIF-1α in fresh RA synovial tissue explants was evaluated under both normoxic and hypoxic conditions. In addition, FLS were infected by an adenoviral vector carrying a modified HIF-1α gene with the oxygen degradation domain removed. This vector has been shown to induce normoxic expression of genes with hypoxia responsive elements.

Results

Under normoxic conditions, HIF-1α expression in fresh samples of RA synovium was patchy and generally confined to the nuclei of cells in hyperplastic lining layers. Under conditions of 1% O₂ or CoCl₂, HIF-1α expression was markedly increased in FLS and in the lining cells of synovial tissue explants. Interestingly, while hypoxia induced the stabilization of HIF-1α protein by preventing its degradation, it also resulted in a significant downregulation of HIF-1α gene expression. Of the approximately 80 genes known to be directly regulated by HIF-1α, 75% were found to be upregulated by hypoxia in FLS. This included angiogenic mediators such as vascular endothelial growth factor, angiopoietins, and leptin, apoptosis mediators such as BNIP3, glycolysis-related enzymes such as G6PI, and the chemokine CXCL12 and its receptors CXCR4. Regulation of CXCL12 by HIF-1α was confirmed by its normoxic induction in adenoviral infected FLS lines. In addition, a spectrum of novel genes and proteins not known to be regulated by HIF-1α were shown to be induced in FLS by hypoxic stimulation.

Conclusions

Hypoxic conditions in RA synovium promote persistence by inducing angiogenesis, enhancing FLS survival, and enhancing lymphocyte recruitment. Hypoxic induction of G6PI may promote the development of anti-G6PI autoantibodies.