Background/Purpose: Fibroblast-like synoviocytes (FLS) in the intimal lining are major effectors of cartilage damage in rheumatoid arthritis (RA). PI3Kdelta, a member of the Class IA phosphoinositide 3-kinases (PI3K), was recently identified as a regulator of aggressive behavior by RA FLS. Invasive migration requires dynamic interactions between the extracellular matrix (ECM), the actin cytoskeleton and the capacity for matrix degradation. The specialized ECM-degrading membrane protrusions like invadopodia in cancer cells or podosomes in macrophages are known to facilitate invasive migration. Therefore, we investigated the role of PI3Kdelta in formation of invadosomes and matrix degradation.

Methods:

To visualize invadosomes, RA FLS were cultured in fluorescent  labeled gelatin coated dishes for 16 h. Invadosomes were identified with anti-cortactin, anti-TKS5, anti-MT1-MMP and phalloidin staining and observed with confocal microscopy.  To quantify the gelatin degradation, we measured the degradation area and normalized it to total cell surface area using Image J software. Co-localization analysis was performed for PI3Kdelta and Akt  with cortactin, TKS5, MT1-MMP or F-actin. MMP expression was analyzed with qPCR following TNF stimulation for 24 h in the presence or absence PI3Kdelta inhibitors (INK007, CAL101) or a PI3Kdelta/gamma inhibitor (IPI145).

Results:

The collective term invadosomes includes podosomes that form in monocytic cells and invadopodia that are associated with cancer cells. First, we examined whether RA synoviocytes spontaneously form extracellular matrix degrading invadosomes. 25-40% of the cells spontaneously formed invadosomes, which was increased by 2.5 fold in presence of TNF or IL-1 (n=5, p<0.04). Because TNF regulates expression and activity of PI3Kdelta in FLS, we next examined cellular distribution of p110delta using confocal microscopy. Immunofluorescence staining revealed colocalization of p110delta with invadopodia markers cortactin, TKS5 and MT1-MMP in TNF treated and control cells. To determine whether PI3Kdelta activity is required for invadosome formation and matrix degradation, cells were plated onto fluorescent gelatin coated dishes in the presence or absence of PI3Kdelta inhibitors INK007, CAL101, IPI145 (0.1-3 uM) or vehicle control. PI3Kdelta inhibition significantly decreased gelatin degradation in a concentration dependent manner, with 65±6% inhibition at 1 uM for INK007 (p< 0.035; similar results for other inhibitors). Furthermore, the percentage of cells with invadosomes was reduced by PI3Kdelta inhibition. Consistent with these findings, phosphorylation and accumulation of Akt, a downstream target of PI3Kdelta, was decreased by PI3Kdelta inhibition at the invadopodia-mediated gelatin degradation site. PI3Kdelta inhibition had no effect on MMP1, 2, 3 and MT1-MMP gene expression in TNF-treated RA FLS.

Conclusion:

PI3Kdelta plays a critical role in invasive matrix degradation by RA FLS by regulating spontaneous and TNF-induced invadosome formation. These observations, together with previous findings that PI3Kdelta regulates FLS migration, growth and survival, suggest that PI3Kdelta inhibition could protect cartilage in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/pi3kdelta-regulates-invadosome-formation-and-invasiveness-of-fibroblast-like-synoviocyte-in-rheumatoid-arthritis/