Abstract
Intervertebral disc (IVD) degeneration is frequently characterized by increased cell proliferation, probably as a tissue regenerative response. Although many growth factors and their receptors have been shown to be expressed normally in the disc, and generally to be over-expressed during degeneration, not all of them have been thoroughly studied concerning their effects on IVD cell proliferation. In the present report, three potent mitogens, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) are examined regarding their capacity to induce proliferation in vitro of bovine coccygeal nucleus pulposus (NP) and annulus fibrosus (AF) cells, as well as to activate major intracellular signal transduction pathways. PDGF, bFGF and IGF-I were found to induce DNA synthesis in quiescent IVD cells in a dose-dependent manner. Maximum stimulation was induced by PDGF, while stimulation by all three factors simultaneously exceeded only slightly that caused by PDGF alone. All three growth factors were shown to phosphorylate immediately extracellular-signal regulated kinases (ERKs), while the stimulation by bFGF especially resulted in sustained ERK phosphorylation. Furthermore, all three growth factors induced phosphorylation of Akt in both Thr308 and Ser473 residues immediately after stimulation, although bFGF-induced phosphorylation was much weaker than that provoked by PDGF and IGF-I. In addition, the MEK inhibitor PD98059 and the PI 3-K inhibitor wortmannin were shown to block growth factor-induced ERK- and Akt-phosphorylation, respectively, in IVD cells. Inhibition of the MEK/ERK or the PI 3-K/Akt pathways provoked a significant decline of the proliferative effects of PDGF, bFGF or IGF-I on IVD cell cultures, while the simultaneous inhibition of both signaling pathways abolished completely the mitogenicity of these growth factors. The above effects of the three growth factors were reproduced similarly in both NP and AF cell cultures. Overall, the above results indicate that PDGF, bFGF and IGF-I stimulate the proliferation of IVD cells via the ERK and Akt signaling pathways.