Human chondrocyte expression of growth-arrest-specific gene 6 and the tyrosine kinase receptor axl: Potential role in autocrine signaling in cartilage

Abstract

Objective. To determine if human articular chondrocytes express the axl tyrosine kinase receptor and its ligand Gas-6, a protein product of growth- arrest-specific gene 6, and to determine if Gas-6 and axl function in the regulation: of chondrocyte growth and survival. Methods. The presence of Gas- 6 and axl was examined in situ in human articular cartilage by immunohistochemistry and in vitro in cell culture studies using primary human chondrocytes and immortalized human chondrocytes. The ability of recombinant Gas-6 to mediate adhesion of chondrocytes and to stimulate chondrocyte axl phosphorylation was determined. Studies of the role of Gas-6 and axl in cell proliferation and survival were also performed. Results. Both Gas-6 and axl were detected in cartilage by immunohistochemical staining. Gas-6 and axl messenger RNA (mRNA) and protein were also detected in cultures of primary and immortalized human chondrocytes. Compared with cells cultured in medium containing 10% serum, Gas-6 mRNA levels were increased in immortalized chondrocytes cultured in serum-free medium, while axl expression decreased. Chondrocytes attached to Gas-6-coated plastic, and the attachment was blocked by a soluble Ig fusion protein containing the axl extracellular domain. Recombinant human Gas-6 and serum-free conditioned medium from primary and immortalized human chondrocyte cultures stimulated chondrocyte axl tyrosine phosphorylation. A mitogenic effect was noted both when immortalized chondrocytes were stimulated with recombinant Gas-6 or when they were made to overexpress axl by transfection. Addition of recombinant Gas-6 to serum-free medium resulted in increased survival of primary chondrocytes cultured at low density in agarose. Conclusion. These findings present evidence for an autocrine signaling pathway in cartilage involving Gas-6 and the axl tyrosinekinase adhesion receptor. Stimulation of axl by Gas-6 may play an important role in the control of chondrocyte growth and survival.