Background/Purpose: Hexokinases (HKs) catalyze the first step in glucose metabolism. HK2 constitutes the principal inducible isoform with a restricted distribution in normal adult tissues. Fibroblast-like synoviocytes (FLS) are a key component of rheumatoid arthritis (RA) invasive synovium, and display unique aggressive features, including increased migration and invasion. We have recently showed a critical role of glucose metabolism and specifically of HK2 in RA FLS phenotype. Of interest, HK2 localizes not only in the cytosol but also at mitochondria and protects mitochondria against apoptosis. We hypothesize that mitochondrial-bound HK2 is key regulator of RA FLS phenotype.

Methods: HK2 localization at baseline and after RA FLS activation with platelet derived growth factor (PDGF, 10ng/ml) was evaluated by cell fractionation and western blot (WB), and confocal microscopy. RA FLS were infected with GFP, full-length (FL)-HK2 or HK2 lacking its mitochondrial binding motif (HK2ΔN) expressing adenovirus (ad). RA FLS were also incubated with metiljasmonate (MJ, 2.5mM), which dissociates HK2 from mitochondria. FLS function in medium and PDGF stimulated cells was evaluated by measuring 1) migration of cultured FLS monolayers (scratch assay); 2) in vitro invasion assay using matrigel. For arthritis experiments, mice were injected with K/BxN sera on day 0. MJ (25mg/kg) was injected daily i.p. beginning on day 0 after serum administration or starting at the peak of arthritis (from day 5). Clinical arthritis scores were serially assessed. Joint histology was evaluated using a semiquantitative scoring system.

Results: 30 minutes after PDGF stimulation, cell fractionation and confocal microscopy revealed that PDGF induced the translocation of HK2 to the mitochondrial fraction. Overexpression of the HK2 mutant reversed the invasive phenotype induced by full-length HK2 after PDGF stimualtion, and also FLS migration rate, GFP-ad: 101.2 ±12.69; FL-HK2-ad: 135.7±38.88; HK2ΔN-ad 112.6±35.72 (FL-HK2-ad vs. HK2ΔN-ad: p<0.01). MJ treatment also significantly reduced RA FLS invasion from 46.79±8,961 to 22.45±7.48 (p<0.001) and migration rate from 253±14 to 152±6 (p<0.001) after PDGF stimulation. Finally, MJ treatment significantly decreased arthritis severity. Day 10 scores were 4.8±0.9 and 1.2±0.58 (P<0.01) for vehicle and MJ-treated mice respectively, when mice were treated from day 0 after serum administration, and 12.5±0.5 and 9.2±0.663 (p<0.05) for vehicle and MJ-treated mice when mice were treated from day 5. Joint histology scores for vehicle and MJ-treated mice from day 0 were: for inflammation 0.9±0.821 and 0.4±0.42 (p<0.05), bone erosion scores were 1.5±0.61 and 0.3±0.45 (p<0.01), and cartilage damage scores were 1.9±0.42 and 0.3±0.45 (p<0.01), respectively.

Conclusion: Our results suggest that mitochondrial HK2 is key regulator of aggressive FLS phenotype, which contributes to joint destruction in RA. Targeting HK2, as an isoform-specific contributor to RA FLS phenotype, offers a safer approach than global glycolysis inhibition. Other possible strategy to improve selectivity would be to target HK2 binding to the mitochondria.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-mitochondrial-bound-hk2-in-rheumatoid-arthritis-fibroblast-like-synoviocytes/