Effect of nerve growth factor and its transforming tyrosine kinase protein and low-affinity nerve growth factor receptors on apoptosis of notochordal cells

Abstract

Purpose: The disappearance of notochordal cells by apoptosis is thought to be the starting point of intervertebral disc degeneration. The aim of this study was to determine the apoptotic pathway of notochordal cells as well as the anti-apoptotic potential of caspase inhibitors.

Methods: Rat notochordal cells were isolated, cultured, and placed in either 0 % (apoptosis-promoting condition) or 10 % (normal control) foetal bovine serum (FBS). We identified and quantified apoptotic cell deaths and caspase activities. In addition, we examined the cells for expression of nerve growth factor (NGF) and its two receptors--TrkA (survival signal) and p75 (apoptotic signal)--and downstream pathways. Finally, we analysed the degree of anti-apoptotic effects of caspase inhibitors on the cells.

Results: The apoptotic rate and expressions of caspase-8 (extrinsic pathway), -9 (intrinsic pathway), and -3 (common executioner) of notochordal cells were increased in 0 % FBS compared with those in 10 % FBS. Expressions of NGF, p75 receptor and JNK downstream pathways were also increased in 0 % FBS. In contrast, expressions of the TrkA receptor and Akt and MAPK downstream pathways were decreased in 0 % FBS. Pancaspase, capase-9 and capase-8 inhibitors significantly reduced apoptotic cell death.

Conclusions: Our results suggest that notochordal cells undergo apoptosis through both the intrinsic and extrinsic pathways by activation of NGF, p75 receptor, and the JNK downstream pathway. We also found that apoptosis of notochordal cells can be attenuated by caspase inhibitors. Caspase inhibitors may play a therapeutic role in delaying the starting point of disc degeneration that is due to inappropriate or premature excessive apoptosis of notochordal cells.

Fig. 1

Notochordal cells were treated with 10 % or 0 % fetal bovine serum (FBS) for 48 hours. Cell death was assayed with flow cytometry after double staining with annexin V-FITC and propidium iodide and APOPercentage

Fig. 2

The percentage of apoptotic cell death of notochordal cells, expressed as the mean and standard deviation of three independent experiments. ^***p < 0.001

Fig. 3

Western blot analysis showing increased expression of NGF, p75 receptor, and JNK downstream pathway but decreased expression of TrkA receptor and Akt and MAPK downstream pathway in notochordal cells treated with 0 % fetal bovine serum (FBS) for 48 hours compared with 10 % FBS

Fig. 4

Western blot analysis shows that notochordal cells treated with 0 % fetal bovine serum (FBS) of serum deprivation for 48 hours caused activations of initiators of the intrinsic (caspase-9) and extrinsic (caspase-8) pathways as well as their common executioner (caspase-3) compared with 10 % FBS

Fig. 5

Assay of the antiapoptotic effects of caspase inhibitors. Notochordal cells were incubated with 0 % fetal bovine serum (FBS) with and without Z-IETD-FMK (caspase-8 inhibitor, 100 μM), Z-LEHD-FMK (caspase-9 inhibitor, 100 μM), and Boc-D-FMK (pancaspase inhibitor, 100 μM) for 48 hours (a). Apoptotic cell death was determined by double staining with annexin V-FITC and propidium iodide followed by analysis with flow cytometry. The results are presented as the percentage of cell death (b). *p < 0.05; **p < 0.01