Session Type: Abstract Submissions (ACR)
Background/Purpose: 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.
Methods: DEXAscan 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, n=6) whereas an antibody to DCC had no effect on OC formation (5±4% reduction, p=NS vs. control, n=6). Finally, DEXAscan and MicroCT analysis demonstrated an increase in bone mineral density (BMD), total volume (TV), bone volume (BV) and TV/BV in both cortical and trabecular bone in Netrin1 deficient mice when compared to WT (p<0.01 for all, n=5). Netrin1 immunostaining in human tissue biopsies reflect enhanced expression in tissue from implant revision when compared to primary implants.
Conclusion: The chemorepulsant Netrin1 is required for osteoclast differentiation and stimulates OC differentiation by an autocrine mechanism. This finding suggests that Netrin1 may be a novel target to reduce OC-mediated bone resorption and to prevent joint prosthesis loosening.
Filed a patent on use of adenosine A2AR agonists to prevent prosthesis loosening (pending). ,
P. E. Purdue,
S. R. Goldring,
Pfizer Inc, Bone Therapeutics, Fidia Pharma, Inc, Abbott Laboratories,
B. N. Cronstein,
NIH, Gilead, Takeda, AstraZeneca,
NYU School of Medicine,
Merck-SeronoBristol-Myers Squibb, Novartis, CanFite Biopharmaceuticals, Cypress Laboratories, Regeneron (Westat, DSMB), Endocyte, Protalex, Allos, Inc., Savient, Gismo Therapeutics, Antares Pharmaceutical, Medivector,
Multiple patents on adenosine receptors and bone metabolism, pharmacology,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/netrin1-is-a-critical-autocrine-factor-for-osteoclast-differentiation/