Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose Bisphosphonates inhibit osteoclast differentiation/function via inhibition of Rap1A isoprenylation and cytoskeletal assembly. As Rap1 is the effector of EPAC proteins (exchange protein directly activated by cAMP), we determined the role of EPAC in osteoclast differentiation.
Methods
Osteoclast differentiation was studied as the number of TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine or human bone marrow precursors in the presence of the EPAC-selective cAMP analog 8-CPT-cAMP (100nM) and the EPAC inhibitor BFA (10µM). Rap1 activity assay was performed. Signaling events were studied by Western Blot in EPAC1/2 knockdown (lentiviral shRNA for EPAC1 or EPAC2 or scrambled shRNA) RAW264.7 cells. Osteoclast marker expression was studied by RT-PCR. Osteoclast morphological characterization was studied by phalloidin staining.
Results 8-CPT-cAMP significantly increased osteoclast differentiation whereas BFA inhibited differentiation (113±3% (p<0.05) and 42±2% (p<0.001) of control, respectively, n=6). Rap1 activation was maximal 15 min after RANKL stimulation (136±3% of basal, p<0.001, n=4) whereas silencing of EPAC1/2 diminished activated Rap1 (43±2% and 50±5% of control respectively, p<0.01, n=4) and NFkB translocation. TRAP staining revealed no osteoclast differentiation in EPAC1/2 KO cells. Cathepsin K, NFATc1 and Osteopontin mRNA expression decreased in EPAC1/2 KO cells when compared to control. Activation of RhoA, cdc42, Rac1 and FAK were observed in an EPAC1/2 dependent manner and there was diminished cytoskeletal assembly in EPAC1/2 KO cells.
Conclusion EPAC1/2 are critical signaling intermediates in osteoclast differentiation that permit RANKL-stimulated NFkB nuclear translocation and actin rearrangements. Targeting this signaling intermediate may diminish bone destruction in inflammatory arthritis.
Disclosure:
A. Mediero,
None;
M. Perez-Aso,
None;
B. N. Cronstein,
Canfite Pharma,
1,
AstraZeneca,
2,
Cellgene,
2,
Gilead,
2,
NIH,
2,
NYU School of Medicine,
3,
Bristol-Myers Squibb,
5,
Pfizer Inc,
5,
Eli Lilly and Company,
5,
Rheumatology Reseach Foundation,
6,
ACR,
6,
Arthritis Foundation,
6.
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