TAT fusion proteins containing tyrosine 42-deleted IκBα arrest osteoclastogenesis

In most circumstances, NF-kappaB, which is essential for osteoclastogenesis, is activated following serine 32/36 phosphorylation of its cytosolic inhibitory protein, I kappaB alpha. In contrast to other cell types, I kappaB alpha, in bone marrow macrophages (BMMs), which are osteoclast precursors, is tyrosine-phosphorylated by c-Src kinase. To address the role of I kappaB alpha phosphorylation in osteoclastogenesis, we generated TAT fusion proteins containing wild-type I kappaB alpha (TAT-WT-I kappaB), I kappaB alpha lacking its NH2-terminal 45 amino acids (TAT-I kappaB(46-317)), and I kappaB alpha in which tyrosine residue 42, the c-Src target, is mutated into phenylalanine (TAT-I kappaB(Y42F)). TAT-I kappaB efficiently enters BMMs, and the NF-kappaB-inhibitory protein, once intracellular, is functional. While TAT-WT-I kappaB only Slightly inhibits osteoclastogenesis, osteoclast recruitment is diminished > 80% by TAT-I kappaB(46-317), an event mirrored by dentin resorption. The fact that TAT alone does not impact osteoclastogenesis, which also resumes following withdrawal of TAT-I kappaB(46-317), establishes that the mutant's anti-osteoclastogenic properties do not reflect toxicity. Affirming a functional role for I kappaB(Tyr(42)) in osteoclastogenesis, TAT-I kappaB(Y42F) is as efficient as TAT-I kappaB(46-317) in blocking osteoclast differentiation. Thus, dominant-negative I kappaB alpha constructs block osteoclastogenesis, and Tyr 42 is essential to the process, increasing the possibility that nonphosphorylatable forms Of I kappaB alpha may be a means of preventing pathological bone loss.