Activation of EPAC1/2 is essential for osteoclast formation by modulating NFκB nuclear translocation and actin cytoskeleton rearrangements

Abstract

Bisphosphonates inhibit osteoclast differentiation/function via inhibition of Rap1A isoprenylation. As Rap1 is the effector of exchange protein directly activated by cAMP (EPAC) proteins, we determined the role of EPAC in osteoclast differentiation. We examined osteoclast differentiation as the number of primary murine/human bone-marrow precursors that differentiated into multinucleated TRAP-positive cells in the presence of EPAC-selective stimulus (8-pCTP-2=-O-Me-cAMP, 100 M; 8-pCTP-2=-O-Me-cAMP-AM, 1 M) or inhibitor brefeldin A (BFA), ESI-05, and ESI-09 (10 M each). Rap1 activity was assessed, and signaling events, as well as differentiation in EPAC1/2-knockdown RAW264.7 cells, were studied. Direct EPAC1/2 stimulation significantly increased osteoclast differentiation, whereas EPAC1/2 inhibition diminished differentiation (113؎6%, P<0.05, and 42؎10%, P<0.001, of basal, respectively). Rap1 activation was maximal 15 min after RANKL stimulation (147؎9% of basal, P<0.001), whereas silencing of EPAC1/2 diminished activated Rap1 (43؎13 and 20؎15% of control, P<0.001) and NFkB nuclear 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. RhoA, cdc42, Rac1, and FAK were activated in an EPAC1/2-dependent manner, and there was diminished cytoskeletal assembly in EPAC1/2-KO cells. In summary, EPAC1 and EPAC2 are critical signaling intermediates in osteoclast differentiation that permit RANKL-stimulated NFkB nuclear translocation and actin rearrangements. Targeting Osteoclasts, multinucleated giant cells derived from myeloid precursors belonging to the monocyte/ macrophage family (1, 2), resorb bone during normal bone remodeling and, by increasing in number and resorptive capacity, mediate pathological bone loss, as well. Osteoclasts adhere to bone and form a sealing zone into which they secrete HCl and proteases, such as collagenases, cathepsin K, and tartrate-resistant acid phosphatase (TRAP), which dissolve the mineral and matrix components of bone (3). Myeloid precursors differentiate into osteoclasts after stimulation by macrophage colony-stimulating factor 1 (M-CSF), which acts via its receptor CSF-1R. After interaction with M-CSF, differentiation and activation of osteoclasts is mediated by a complex network of regulatory factors (systemic hormones and locally produced cytokines) and cell-cell and cell-matrix interactions that are required for transition of the osteoclast precursor into a multinucleated and fully activated osteoclast (4, 5). Among these factors, receptor activator of nuclear factor -B ligand (RANKL) is a critical extracellular regulator of osteoclast differentiation and activation (6 -10). RANKL binds to its receptor, RANK, on the surface of osteoclast precursors (OCPs), resulting in the recruitment of TNF receptor associated factors (TRAFs), which activate nuclear factor B (NFB), c-Fos, phospholipase C␥ (PLC␥), and nuclear factor of

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…Contrasting the above results where PDE inhibitors decreased NFB and NFAT2 activity, EPAC1 or EPAC2 knockdown blocked NFB p50/p105 translocation, as well as abolished the three-to fivefold RANKLinduced increase in mRNA expression of NFAT2, cathepsin K, and osteopontin. Additionally, loss of EPAC1 versus EPAC2 exhibited differences in translocation and activation of NFB, suggesting potentially distinct roles for each isoform that require closer attention (682). In contrast to the above investigations, where transcriptional regulation was not modified by calcitonin, potential off-target effects of the pharmacological agents could partially explain some of the observed discrepancies.…”

“…"Clearly, the development of the first antagonists with (subtype) selectivity for EPAC represents another research milestone to ascribe biologic responses to EPAC" (915). Indeed, these selective EPAC2 inhibitors are becoming effective tools in probing isoform-specific EPAC functions (18,47,130,175,226,250,302,411,463,565,682,743,841,896,961,1114).…”

MicroRNA-22 negatively regulates poly(I:C)-triggered type I interferon and inflammatory cytokine production via targeting mitochondrial antiviral signaling protein (MAVS)

“…In contrast, inhibition of endogenous miR-22 expression enhanced the activities of both transcriptional factors (Figure 9C and 9D). It has been well established that translocation of NF-κB from the cytoplasm to the nucleus is a key determinant of NF-κB activation [37, 38]. Therefore, it was of interest to evaluate the effect of miR- 22 on NF-κB and IRF3 activation in poly(I:C)-treated cells.…”

Osteoclasts—Key Players in Skeletal Health and Disease

“…Consistent with the conventional view that bone marrow niches are the primary sites of postnatal hematopoiesis (1), medullary fractions from humans and animal models are excellent sources of OC precursors. Indeed, unfractionated marrow leukocytes (2–4) or macrophages expanded in vitro in the presence of macrophage colony-stimulating factor (M-CSF) (5, 6) are commonly used to induce in vitro osteoclastogenesis under the influence of appropriate cues, especially M-CSF and RANKL, as described below. OC can also be differentiated from peripheral blood (7–9), spleen and fetal liver (8, 10, 11).…”