Identification of cell markers of the nucleus and annulus of bovine intervertebral discs

Introduction  The intervertebral disc consists of three regions, the nucleus pulpous and the inner and outer annulus containing cells with individual phenotypes. However, no molecular markers are known to discriminate between the various cell types. Molecular markers would help identify the cell types through development of the disc, ageing of the disc, cell localization and in pathological states such as degenerative disc disease and scoliosis. Here, we present data revealing major difference between the cell types using SDS-PAGE and mass spectrometry. Such differences will help to develop molecular markers to identify the cell types using immunoblotting and immunohistochemistry.

Methods  Intervertebral discs were isolated from bovine tails and separated into three distinct regions of the nucleus, inner and outer annulus. The isolated regions were separately digested with collagenase and peptidase overnight. The remaining cell suspensions were sieved through a filter to remove large particles and then extensively washed. The cells were then separated into membrane and supernatant fractions by incubation with Triton followed by centrifugation. SDS-PAGE analysis using a variety of acylamide gels of the various fractions revealed bands of interest that where then cut from the gel, digested with trypsin and analysed by mass spectrometry. The peptide mass/charge data were collected and then compared with a databank of known peptide masses to give a composite protein mass.

Results  Analysis of the membrane and supernatant fractions of the cells from the three distinct regions in the disc by SDS-PAGE revealed unique protein patterns between the regions and fractions. A large broad band from the membrane of nucleus cells was analysed by mass spectrometry that revealed strong matches for myosin and clathrin and a match for basement membrane collagen-IV. A control band from the outer annulus also revealed a strong match for myosin and to a lesser extent basement membrane collagen type-IV. A strong SDS-PAGE band from the membrane fraction of the outer annulus revealed a mass spectrometry match to actin. Analysis of the corresponding supernatant fraction revealed a strong match to actin, whereas a band of similar molecular weight from the inner annulus revealed another myosin chain.

Conclusion  The differences revealed in the protein profile of cells from the three regions of disc and the identification of prominent proteins demonstrate that these differences can be used to identify molecular markers. SDS-PAGE and mass spectrometry analysis of significant bands showed strong matches in the membrane fraction for myosin and clathrin, and also basement membrane collagen-IV. By using immunoblotting and immunohistochemistry, it will be possible to follow the development, ageing and pathology of the three cell types and will hopefully be extended to finding markers that characterize the individual stages of the degenerative changes. This study will also include genechip technology to identify at the RNA level the difference between the three cell types.

This work was funded by the EU EURODISC project (QLK6-CT-2002–02582).