TAT fusion proteins containing tyrosine 42-deleted IkappaBalpha arrest osteoclastogenesis.
In most circumstances, NF-kappaB, which is essential for osteoclastogenesis, is activated following serine 32/36 phosphorylation of its cytosolic inhibitory protein, IkappaBalpha. In contrast to other cell types, IkappaBalpha, in bone marrow macrophages (BMMs), which are osteoclast precursors, is tyrosine-phosphorylated by c-Src kinase. To address the role of IkappaBalpha phosphorylation in osteoclastogenesis, we generated TAT fusion proteins containing wild-type IkappaBalpha (TAT-WT-IkappaB), IkappaBalpha lacking its NH(2)-terminal 45 amino acids (TAT-IkappaB(46-317)), and IkappaBalpha in which tyrosine residue 42, the c-Src target, is mutated into phenylalanine (TAT-IkappaB(Y42F)). TAT-IkappaB efficiently enters BMMs, and the NF-kappaB-inhibitory protein, once intracellular, is functional. While TAT-WT-IkappaB only slightly inhibits osteoclastogenesis, osteoclast recruitment is diminished >80% by TAT-IkappaB(46-317), an event mirrored by dentin resorption. The fact that TAT alone does not impact osteoclastogenesis, which also resumes following withdrawal of TAT-IkappaB(46-317), establishes that the mutant's anti-osteoclastogenic properties do not reflect toxicity. Affirming a functional role for IkappaB(Tyr(42)) in osteoclastogenesis, TAT-IkappaB(Y42F) is as efficient as TAT-IkappaB(46-317) in blocking osteoclast differentiation. Thus, dominant-negative IkappaBalpha constructs block osteoclastogenesis, and Tyr(42) is essential to the process, increasing the possibility that nonphosphorylatable forms of IkappaBalpha may be a means of preventing pathological bone loss.
Abu-Amer
Y
Y
Department of Orthopedic Research, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. abuamery@msnotes.wustl.edu
Dowdy
S F
SF
Ross
F P
FP
Clohisy
J C
JC
Teitelbaum
S L
SL
D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D001862:Bone Resorption; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003804:Dentin / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D015696:Gene Products, tat / Q000737:chemistry*; D020672:I-kappa B Proteins; D015151:Immunoblotting; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D010010:Osteoclasts / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry*
D004268:DNA-Binding Proteins; D015696:Gene Products, tat; D020672:I-kappa B Proteins; C000606205:Nfkbia protein, mouse; D011993:Recombinant Fusion Proteins; D000072000:NF-KappaB Inhibitor alpha; D014443:Tyrosine; D012694:Serine