Duong L T, Nakamura I, Lakkakorpi P T, Lipfert L, Bett A J, Rodan G A
Department of Bone Biology and Osteoporosis, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
J Biol Chem. 2001 Mar 9;276(10):7484-92. doi: 10.1074/jbc.M008368200. Epub 2000 Dec 1.
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.
破骨细胞的激活始于对骨的黏附、细胞骨架重排、封闭区的形成以及极化皱褶膜的形成。先前的研究结果表明，蛋白酪氨酸激酶2（PYK2），一种与黏着斑激酶相关的胞质激酶，参与了这些过程。本研究使用PYK2反义核酸来研究PYK2在黏附介导的信号传导和破骨细胞功能中的作用。我们构建了一种重组腺病毒，其包含PYK2的300个碱基对的反向5'-编码区，并使用全长PYK2作为对照。在骨髓和成骨细胞的共培养物中生成小鼠破骨细胞样细胞或其单核前体。与未感染的细胞或感染正义PYK2的细胞相比，反义腺病毒感染显著降低了内源性PYK2蛋白的表达，并导致：1）体外破骨细胞形成减少；2）抑制铺在玻璃或骨上的破骨细胞的细胞铺展和肌动蛋白环形成，以及铺在玻连蛋白上的破骨细胞前体的黏附和铺展；3）体外骨吸收受到抑制；4）p130（Cas）酪氨酸磷酸化显著降低；5）α（v）β（3）整合素表达或c-Src酪氨酸磷酸化无变化。综上所述，这些发现支持了以下假设，即PYK2在破骨细胞骨吸收所需的黏附依赖性细胞骨架组织和封闭区形成中起核心作用。
