Background/Purpose: Previous work demonstrated that one mechanism by which bisphosphonates inhibit osteoclast differentiation and function is via inhibition of Rap1A isoprenylation and, as a result, function. As Rap1 is the effector of the cAMP-binding EPAC protein (exchange protein directly activated by cAMP), we determined the role of EPAC1 in osteoclast differentiation.

Methods: Osteoclast differentiation was studied as the M-CSF/RANKL stimulated formation of multinucleated TRAP-positive cells from primary murine (C57Bl/6) bone marrow-derived precursors or EPAC1 knockdown (lentiviral shRNA for EPAC1) RAW264.7 cells in the presence/absence of the EPAC-selective cAMP analog 8-CPT-cAMP 100nM and EPAC inhibitor BFA (10µM). Rap1 activity assay was carried out according to the manufacturers’ protocol. Signaling events (EPAC, NFkB and MAPK) were studied by Western Blot in EPAC1 KO RAW264,7 cells (scrambled shRNA transfection is control for these experiments). Osteoclast marker expression was studied by RT-PCR.

Results: The EPAC-selective cAMP analogue 8-CPT-cAMP did not significantly affect osteoclast maturation (106±4% of control, p=NS, n=5) but the EPAC inhibitor BFA inhibited differentiation (59±9% inhibition, p<0.001 vs control, n=5). Activation of Rap1 was maximal 10 minutes after RANKL stimulation (161±4% of control, p<0.001, n=4). NFkB nuclear translocation induced by RANKL was diminished 15 minutes after stimulation in EPAC1 KO cells (63±3% of scrambled shRNA infected cells, p<0.001, n=4) and TRAP staining revealed no osteoclast differentiation when EPAC1 was knocked down. Moreover, pERK1/2 and pJNK are marginally activated in control cells after RANKL stimulation (117±3% and 107±2% of control, respectively, p=0.5, n=4) and deletion of EPAC1 slightly decreased this activation (93±2% and 95±1% of control, respectively, p=NS, n=4). Interestingly, the MEK inhibitor U0126 restores RANKL-stimulated osteoclast formation in the EPAC1 KO cells (109±2% of control, p=NS, n=6). Finally, in EPAC1 knockdown cells inhibition of osteoclast differentiation is correlated with decreased expression of osteoclast differentiation markers Cathepsin K, NFATc1 and Osteopontin when compared to control (4±0.7, 2.3±0.5 and 3±0.4 fold decreased respectively, p<0.001, n=3).

Conclusion: EPAC1 is a critical intermediate in osteoclast differentiation which permits increased ERK1/2 activation and NFkB nuclear translocation. Targeting this signaling intermediate may diminish bone destruction in inflammatory arthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/epac1-activation-is-required-for-nfkb-nuclear-translocation-and-osteoclast-differentiation/