Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose: Choline kinase (ChoKa) is an essential enzyme for phosphatidylcholine biosynthesis and is required for cell proliferation. The enzyme has also been implicated in cancer disease progression, metastasis, and invasiveness. The unique tumor-like behavior of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) led us evaluate whether this pathway could play a role in inflammation and joint damage due to synovitis. Therefore, we examined the role of ChoKa in RA FLS and in murine arthritis and performed a targeted metabolomics assessment of this pathway.
Methods: ChoKa expression was evaluated by immunohistochemistry (IHC) and Western blot (WB). The metabolic profile of the choline kinase was determined by 1H-MRS. FLS function using the ChoKa inhibitor MN58b (IC50=~5 nM) in medium and PDGF stimulated cells was evaluated by measuring 1) migration into a cleared area in cultured FLS monolayers; 2) proliferation using an MTT assay; and 3) protein expression by WB. Cell survival was determined in H2O2 treated cells by light microscopy. For arthritis experiments, mice were injected with K/BxN sera on day 0. MN58b (3mg/kg) was injected daily i.p. beginning on day 0 or day 4 after serum administration. Clinical arthritis scores were serially assessed. Joints were evaluated for inflammation and joint damage using histology and a semiquantitative scoring system.
Results: ChoKa mRNA and protein were highly expressed in RA synovial tissue and in cultured FLS. Its expression in FLS was increased 2-3-fold after TNF and PDGF stimulation, respectively with peak expression within 48 hours. Metabolomic studies of choline-containing compounds in cultured FLS extracts showed increased levels of phosphocholine in RA FLS compared to control FLS, confirming activation of this pathway. ChoKa regulated key FLS functions that might contribute to cartilage destruction in RA. For example, ChoKa inhibition with MN58b (5 uM) reduced proliferation by 79±3.2% and migration by 54±15% (p<0.05). ChoKa inhibition also markedly increased H2O2-induced apoptosis in FLS. Akt phosphorylation in response to PDGF as determined by WB was blocked by ChoKa inhibition. Finally, ChoKa inhibition signficantly decreased arthritis in pre-treatment protocols (day 0) as well as in established disease (day 4). For example, day 8 scores were 12±1 .6 and 7±2.6 (P<0.05) for vehicle and MN58b-treated mice, respectively, when initiated on day 0; and were 6.6±0.9 and 1.6±2.5 (P<0.05) for PBS and MN58b-treated mice when initiated on day 4. Joint histology scores for vehicle and MN58b-treated mice for inflammation were 3.2±0.5 and 1.25±1 (p<0.05), bone erosion scores were 2.7±0.5 and 0.25±0.5 (p<0.05), and cartilage damage scores were 1.5±1 and 1.6±0.5 (p<0.05) respectively.
Conclusion: Careful dissection of the metabolic profile in RA FLS indicate that choline metabolism is abnormal and is similar to transformed cells. Blocking this pathway with a selective ChoKa inhibitor suppressed inflammatory arthritis in mice as well as the aggressive behavior of cultured RA FLS, including cell migration and resistance to apoptosis. These data suggest that ChoKA inhibition could be an effective strategy for arthritis.
Disclosure:
M. Guma,
None;
E. Sanchez-Lopez,
None;
A. Lodi,
None;
S. Tiziani,
None;
J. C. Lacal,
Translational Cancer Drugs Pharma,
1;
M. Karin,
None;
G. S. Firestein,
None.
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