β1 Integrin/Focal Adhesion Kinase-mediated Signaling Induces Intercellular Adhesion Molecule 1 and Receptor Activator of Nuclear Factor κB Ligand on Osteoblasts and Osteoclast Maturation

Shingo Nakayamada,Yosuke Okada,Kazuyoshi Saito,Masahito Tamura,Yoshiya Tanaka

doi:10.1074/jbc.m308786200

Abstract

We have assessed characteristics of primary human osteoblasts, shedding light on signaling mediated by beta1 integrin. beta1 integrins are major receptors for these matrix glycoproteins. 1) Integrins beta1, alpha2, alpha3, alpha4, alpha5, alpha6, and alphav were highly expressed on primary osteoblasts. 2) Engagement of beta1 integrins on osteoblasts by cross-linking with specific antibody or ligand matrices, such as fibronectin or collagen, augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on the surface. 3) Up-regulation of ICAM-1 and RANKL on osteoblasts by beta1 stimulation was completely abrogated by pretreatment with herbimycin A and genistein, tyrosine kinase inhibitors, or transfection of dominant negative truncations of focal adhesion kinase (FAK). 4) Engagement of beta1 integrins on osteoblasts induced tartrate-resistant acid phosphatase-positive multinuclear cell formation in the coculture system of osteoblasts and peripheral monocytes. 5) Up-regulation of tartrate-resistant acid phosphatase-positive multinuclear cell formation by beta1 stimulation was completely abrogated by transfection of dominant negative truncations of FAK. Our results indicate that beta1 integrin-dependent adhesion of osteoblasts to bone matrices induces ICAM-1 and RANKL expression and osteoclast formation via tyrosine kinase, especially FAK. We here propose that beta1 integrin/FAK-mediated signaling on osteoblasts could be involved in ICAM-1- and RANKL-dependent osteoclast maturation.

Highlights

We have previously reported that human osteoblasts express intercellular adhesion molecule (ICAM)-1 and that interaction between ICAM-1, expressed on osteoblasts, and leukocyte function-associated antigen (LFA)-1, expressed on monocytes, is required for osteoclast maturation by RANKL on osteoblasts [12]
We have reported that ICAM-1 on rheumatoid synovial cells induced transcription of interleukin-1␤ by activation of a nuclear factor, AP-1, and that stimulation of ␤1 integrin up-regulated ICAM-1 and Fas, and Fas mediated apoptosis of rheumatoid synovial cells through focal adhesion kinase (FAK) [13, 14]
Our results demonstrate that engagement of ␤1 integrin by a specific antibody or ligand matrices up-regulated ICAM-1 and RANKL expression on osteoblasts and induced osteoclast formation via tyrosine kinase, especially FAK

Summary

EXPERIMENTAL PROCEDURES

The study protocol was approved by the Human Ethics Review Committee of the University of Occupational and Environmental Health, Japan, and a signed consent form was obtained from each subject prior to taking tissue samples used in the present study. Plates were washed three times with PBS, and the cells were added to each well as described above and were incubated in DMEM without FCS at 37 °C for the indicated duration. The plasmids and liposome complex were added to osteoblasts plated in a 6-well culture dish, incubated for 3 h in OPTI-MEM, and replaced with DMEM containing 10% FCS for 24 h. 2 ϫ 105 cells were incubated with negative control mAb thy1.2, integrin ␤1 mAb, ␤2 mAb, ␤3 mAb, ␣1 mAb, ␣2 mAb, ␣3 mAb, ␣4 mAb, ␣5 mAb, ␣6 mAb, ␣v mAb, CD54 (ICAM-1) mAb, RANKL mAb, or CD106 (VCAM-1) mAb in FACS medium consisting of Hanks’ balanced salt solution (Nissui, Tokyo, Japan), 0.5% human serum albumin (Yoshitomi, Osaka, Japan), and 0.2% NaN3 (Sigma) for 30 min at 4 °C. A p value less than 0.05 denoted the presence of a statistically significant difference

RESULTS

DISCUSSION

Intercellular κB Ligand on Osteoblasts and Osteoclast Maturation