Signaling in osteoclastogenesis: use of affinity purified chicken antibodies

Objective: RANKL is known to induce osteoclast differentiation and bone resorption by binding to its receptor RANK in osteoclasts. A number of well-known signaling pathways have been shown to be involved in this process, but recent studies suggest a less known signaling pathway comprising the vertebrate serine-threonine kinases MST1/2, Lats1/2 and a scaffolding protein, WW45, may also play a role in regulating proliferation and differentiation of osteoclast precursor cells. Very little is known about the function and endogenous expression of WW 45. Our goal in this study was to generate anti-WW45 antibodies in chickens, taking advantage of differences in the amino acid sequence of chicken vs mammalian WW45, and to use these antibodies to elucidate the role of this protein in vertebrates.

Methods: Peptide antigens derived from mouse WW45 were constructed as multiple antigenic peptides (MAPs) and injected into wing muscles of laying NH Red chickens with Freund's adjuvant. A second injection followed ten days later. Eggs were collected prior to injections and 14 days post-injection forward. IgY was purified from the egg yolks by ammonium sulfate precipitation and then affinity purified and used for western blot and immunoprecipitation studies of cultured osteoclasts and osteoclast precursor cells.

Results: Affinity purified anti-WW45 IgY's were able to detect high levels of endogenous mouse WW45 in RANKL-induced differentiated osteoclasts and low levels in proliferating osteoclast precursor cells. Results of our western blot and RT-PCR analyses demonstrated that endogenous

WW45 was up-regulated at the mRNA and protein levels upon RANKL-induced osteoclast differentiation. Immunoprecipitation studies demonstrate the interaction of WW45 with MST1/2 and Lats1/2 in differentiated osteoclasts.

Conclusions: WW45 is expressed in differentiating osteoclast precursor cells and interacts with its partners MSTI/2 and Latsl/2 upon RANKL-induced osteoclast differentiation. Disruptions in this signaling complex may underly diseases characterized by excessive osteoclastic activity and bone resorption.