Tendon healing in vitro: activation of NIK, IKKalpha, IKKbeta, and NF- kappaB genes in signal pathway and proliferation of tenocytes.

Abstract

Initiation of DNA transcription and proliferation of tendon cells are critical to tendon healing and require pivotal signals to the nucleus. Exploring intracellular signaling pathways pertinent to the healing process may reveal new approaches to accelerating the healing rate of the tendon. The authors investigated expression of NIK, IKKalpha, IKKbeta, and NF- kappaB genes in the signal pathway and tenocyte proliferation in an in vitro model in which cultured tenocytes were exposed to basic fibroblast growth factor (bFGF). Tenocytes were obtained from explant culture of rabbit intrasynovial tendons and were treated with bFGF at concentrations of 0, 2, or 10 ng/ml. Levels of expression of a series of genes for key factors along the signaling route--nuclear factor (NF)-kappaB-inducing kinase, inhibitor of kappa B kinase alpha and beta, and the NF-kappaB--were examined by quantitative analysis of products of reverse transcription and multiplex polymerase chain reactions. Proliferation of the cells was assessed with evaluation of growth curves and immunochemical labeling of the DNA of the cells. Expression levels of NIK, IKKalpha, IKKbeta, and NF-kappaB genes were significantly increased by bFGF at concentrations of 2 and 10 ng/ml. Western blot confirmed the increase of NF-kappaB in the tenocytes. The proliferation rate of the cells was significantly promoted by bFGF. Expression of these genes increased proportionately to the amounts of bFGF stimulating the cells and was correlated with increases in the proliferation rate. This study showed that expression of a series of genes along the NF-kappaB pathway was remarkably promoted by bFGF. The effects were proportionate to in vitro cell proliferation rate. Results of the study suggest that activation of a series of genes along the NF-kappaB pathway may play a pivotal role in initiating cell proliferation during the healing process of intrasynovial tendons. As activation of genes in signal transduction pathways is a new field in the biology of growth factor action with tremendous potential in promoting tissue repairs, manipulation of expression of a series of genes along the NF-kappaB pathway can be a new target of enhancing tendon healing through molecular mechanisms.