Faculty Bibliography

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 muM; 8-pCTP-2'-O-Me-cAMP-AM, 1 muM) or inhibitor brefeldin A (BFA), ESI-05, and ESI-09 (10 muM 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 this signaling intermediate may diminish bone destruction in inflammatory arthritis.-Mediero, A., Perez-Aso, M., Cronstein, B. N. Activation of EPAC1/2 is essential for osteoclast formation by modulating NFkappaB nuclear translocation and actin cytoskeleton rearrangements.

Senior housestaff and junior faculty are often expected to perform clinical research, yet may not always have the requisite knowledge and skills to do so successfully. Formal degree programs provide such knowledge, but require a significant commitment of time and money. Short-term training programs (days to weeks) provide alternative ways to accrue essential information and acquire fundamental methodological skills. Unfortunately, published information about short-term programs is sparse. To encourage discussion and exchange of ideas regarding such programs, we here share our experience developing and implementing INtensive Training in Research Statistics, Ethics, and Protocol Informatics and Design (INTREPID), a 24-day immersion training program in clinical research methodologies. Designing, planning, and offering INTREPID was feasible, and required significant faculty commitment, support personnel and infrastructure, as well as committed trainees. Clin Trans Sci 2014; Volume #: 1-7.

As patents expire on biological agents for the treatment of rheumatic diseases we have the opportunity to develop non-proprietary biologic agents, biosimilars. The development and approval of these agents present novel challenges to both pharma and regulatory agencies although there is great promise of high quality, less expensive biologic agents for the treatment of rheumatic diseases. Here we review the definitions of biosimilars, the regulatory challenges to approval of these agents and the record of approvals of biosimilars to date.

Activation of adenosine A2A receptor (A2AR) promotes fibrosis and collagen synthesis. However, the underlying mechanism is still unclear, not least because cAMP, its principal effector, has been found to inhibit TGFbeta1-induced collagen synthesis. Here, we show that in primary normal human dermal fibroblasts, A2AR stimulation with CGS21680 elicits a modest cAMP increase (150+/-12% of control; EC50 54.8 nM), which stimulates collagen1 (Col1) and collagen3 (Col3), but maximal cAMP resulting from direct activation of adenylyl cyclase by forskolin (15,689+/-7038% of control; EC50 360.7 nM) inhibits Col1 and increases Col3. Similar to Col1 expression, fibroblast proliferation increased following physiological cAMP increases by CGS21680 but was inhibited by cAMP increases beyond the physiological range by forskolin. The A2AR-mediated increase of Col1 and Col3 was mediated by AKT, while Col3, but not Col1, expression was dependent on p38 and repressed by ERK. TGFbeta1 induced phosphorylation of Smad2/3 and increased Col3 expression, which was prevented by Smad3 depletion. In contrast, CGS21680 did not activate Smad2/3, and Smad2/3 knockdown did not prevent CGS21680-induced Col1 or Col3 increases. Our results indicate that cAMP is a concentration-dependent switch for collagen production via noncanonical, AKT-dependent, Smad2/3-independent signaling. These observations explain the paradoxical effects of cAMP on collagen expression.-Perez-Aso, M., Fernandez, P., Mediero, A., Chan, E. S., and Cronstein, B. N. Adenosine 2A receptor promotes collagen production by human fibroblasts via pathways involving cyclic AMP and AKT but independent of Smad2/3.

Netrins have been extensively studied for their role in axonal guidance during neural development. In addition, netrins are chemopulsants for a variety of non-neuronal cell types via binding to their receptors Unc5b and DCC. Although thought to suppress inflammation in several settings, netrin1, acting via Unc5b, inhibits macrophage migration directed by chemokines CCL2 and CCL19 to promote macrophage retention in and exacerbation of atherosclerotic plaque. We asked whether Netrin1 was expressed during osteoclast (OC) differentiation and whether it plays a role in OC differentiation.DXAscan and MicroCT analysis were performed on Netrin1 deficient mice (radiation chimeras) and wildtype (WT, radiation chimeras) littermates. OC differentiation was studied as M-CSF/RANKL-stimulated differentiation of murine bone marrow precursors to TRAP+/multinucleated cells, in the presence/absence of recombinant Netrin1 and Unc5b antibody. Netrin1, Unc5b and DCC expression were studied by RT-PCR and Western Blot in primary bone marrow-derived osteoclasts. Netrin1 immunostaining was performed in human tissue obtained following primary prosthesis implantation or after prosthesis revision. During OC differentiation cell-associated Netrin1 and Unc5b (but not DCC) protein expression increased by 30+2% and 98+4% respectively (p<0.001,n=4) and Netrin1 secretion increased by 66+2% (p<0.001,n=4). Consistently, RANKL stimulates an increase in Netrin1 and Unc5b mRNA expression during OC differentiation (25+4 and 3+0.5 fold change respectively p<0.001,n=4). Moreover, in Netrin1-deficient marrow precursors OC differentiation was diminished by 65+2% as compared to control (p<0.001,n=6), an effect reversed by addition of recombinant netrin1 to cultures (121+5% increased, p<0.5,n=4). An antibody to the netrin1 receptor Unc5b reduces OC formation by 57+6% (p<0.001, =6) whereas an antibody to DCC had no effect on OC formation (5+4% reduction, p=NS vs. control,n=6). Finally, DXAscan and MicroCT analysis demonstrated an increase in!

Inflammasome pathways are important in chronic diseases; however, it is not known how the signalling is sustained after initiation. Inflammasome activation is dependent on stimuli such as lipopolysaccharide (LPS) and ATP that provide two distinct signals resulting in rapid production of interleukin (IL)-1beta, with the lack of response to repeat stimulation. Here we report that adenosine is a key regulator of inflammasome activity, increasing the duration of the inflammatory response via the A2A receptor. Adenosine does not replace signals provided by stimuli such as LPS or ATP but sustains inflammasome activity via a cAMP/PKA/CREB/HIF-1alpha pathway. In the setting of the lack of IL-1beta responses after previous exposure to LPS, adenosine can supersede this tolerogenic state and drive IL-1beta production. These data reveal that inflammasome activity is sustained, after initial activation, by A2A receptor-mediated signalling.

As one of the most utilized disease-modifying anti-rheumatic drugs, methotrexate (MTX) has revolutionized the treatment of rheumatoid arthritis as well as many other non-rheumatic chronic inflammatory diseases. Far from a simple anti- proliferative agent as was once thought, our understanding of how it exerts its anti-inflammatory effects has grown over the years. The mechanisms of action of MTX are reviewed here, and we look at how this knowledge helps to explain some of its most common side effects.