Background/Objectives Cartilage destruction mediated by invasive fibroblast-like synoviocytes (FLS) plays a central role in pathogenesis of RA. Increased cell migration and degradation of extracellular matrix are fundamental to these processes. The Class I phosphoinositide 3-kinases (PI3K) control cell survival, proliferation and migration, which might be involved with cartilage damage in RA. PI3Kdelta isoform expression was recently identified as a key regulator of FLS growth and survival, suggesting that it could contribute to synoviocyte aggressive behaviour. Therefore, we assessed the role of PI3Kdelta in synoviocyte invasion and matrix degradation using isoform selective PI3K inhibitors.
Methods FLS were cultured in Matrigel coated transwells. PI3K inhibitors or vehicle were added to the upper chamber and PDGF was used as a chemoattractant in the lower chamber. The invading cells were quantified by staining the philtres with 1% crystal violet. F-actin was visualised with Rhodamin phalloidin and analysed with fluorescent microscopy. Rac1 activation was measured using PAK1/PBD GST pull down and quantified by Western blot analysis. PI3K inhibitors: pan (GDC-0941), PI3Kalpha (A66), PI3Kbeta (TGX-221), PI3Kgamma (AS-252424), PI3Kdelta (INK007 and CAL-101) and PI3K-delta/gamma (IPI-145).
Results PDGF-directed invasion was completely inhibited by the pan PI3K inhibitor (1 uM). To define the role of the individual isoforms, we tested the effect of the isoform selective PI3K inhibitors. PI3Kdelta inhibition (INK007) significantly decreased the number of invading cells, with 60 ± 5% inhibition at 1 uM (p < 0.04). Similar results were observed with two other inhibitors with distinct chemical structures (CAL-101 and INK055). The PI3Kalpha inhibitor decreased invasion by 40 ± 5% while PI3Kbeta and PI3Kgamma inhibitors had no effect. Phalloidin staining was then used to visualise FLS actin rearrangement in response to PDGF with or without PI3K inhibitors. PI3Kdelta inhibition by INK007, CAL-101 and IPI-145 decreased lamellipodia formation by 50 ± 6% (p < 0.05). Similar inhibition was seen with the pan PI3K inhibitor, while the selective inhibitors of PI3Kalpha, PI3Kbeta or PI3Kgamma had no effect. We then hypothesised that PI3Kdelta might modulate activation of Rho GTPases in synoviocytes, which regulate actin organisation. PI3Kdelta inhibition with INK007 had no effect on baseline Rac1 activation but blocked activation in response to PDGF by 95 ± 6% (p < 0.03). Similar findings were observed with the pan PI3K inhibitor, while PI3Kalpha inhibition had no significant effect.
Conclusions PI3Kdelta is a major regulator of FLS migration and invasion and functions by inhibiting Rac1 activation and modulating F actin cytoskeleton rearrangement. These observations, together with previous findings that PI3Kdelta regulates FLS growth and survival, suggest that PI3Kdelta inhibition could be chondroprotective in RA by modulating synoviocyte growth, migration and invasion.