Inhibition of osteoclast function by adenovirus expressing antisense protein-tyrosine kinase 2.
Osteoclast activation is initiated by adhesion to bone, cytoskeletal rearrangement, formation of the sealing zone, and formation of the polarized ruffled membrane. Previous findings suggest that protein-tyrosine kinase 2 (PYK2), a cytoplasmic kinase related to focal adhesion kinase, participates in these events. This study examines the role of PYK2 in adhesion-mediated signaling and osteoclast function, using PYK2 antisense. We produced a recombinant adenovirus containing a 300-base pair reversed 5'-coding region of PYK2 and used full-length PYK2 as a control. Murine osteoclast-like cells or their mononuclear precursors were generated in a co-culture of bone marrow and osteoblasts. Infection with antisense adenovirus significantly reduced the expression of endogenous PYK2 protein relative to uninfected cells or to cells infected with sense PYK2 and caused: 1) a reduction in osteoclast formation in vitro; 2) inhibition of cell spreading and of actin ring formation in osteoclasts plated on glass or bone and of attachment and spreading of osteoclast precursors plated on vitronectin; 3) inhibition of bone resorption in vitro; 4) marked reduction in p130(Cas) tyrosine phosphorylation; and 5) no change in alpha(v)beta(3) integrin expression or c-Src tyrosine phosphorylation. Taken together, these findings support the hypothesis that PYK2 plays a central role in the adhesion-dependent cytoskeletal organization and sealing zone formation required for osteoclastic bone resorption.
Duong
L T
LT
Department of Bone Biology and Osteoporosis, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Nakamura
I
I
Lakkakorpi
P T
PT
Lipfert
L
L
Bett
A J
AJ
Rodan
G A
GA
D000199:Actins / Q000378:metabolism; D000256:Adenoviridae / Q000201:enzymology* / Q000235:genetics*; D000818:Animals; D017209:Apoptosis; D001862:Bone Resorption; D001842:Bone and Bones / Q000378:metabolism; D002448:Cell Adhesion; D002465:Cell Movement; D002478:Cells, Cultured; D018920:Coculture Techniques; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D051418:Focal Adhesion Kinase 2; D051379:Mice; D008856:Microscopy, Fluorescence; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D010010:Osteoclasts / Q000378:metabolism*; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics*; D011506:Proteins; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000096:biosynthesis; D019030:Receptors, Vitronectin / Q000096:biosynthesis; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D000199:Actins; C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins; D016376:Oligonucleotides, Antisense; D051419:Paxillin; D010750:Phosphoproteins; D011506:Proteins; C496062:Pxn protein, mouse; D019030:Receptors, Vitronectin; D050717:Retinoblastoma-Like Protein p130; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D016392:Proto-Oncogene Proteins pp60(c-src); C496053:Ptk2b protein, mouse