PDGF, bFGF and IGF-I stimulate the proliferation of intervertebral disc cells in vitro via the activation of the ERK and Akt signaling pathways

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 overexpressed 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.

Human lung fibroblasts prematurely senescent after exposure to ionizing radiation enhance the growth of malignant lung epithelial cells in vitro and in vivo

“…DNA synthesis in young and senescent fibroblasts was estimated after dual labeling with 5-bromo-2'-deoxyuridine (BrdU) and 4',6-diamino-2-phenylindole (DAPI) dihydrochoride, as previously described (23). Briefly, cells were plated sparsely on glass coverslips in DMEM containing 10% FBS.…”

“…For measuring the effect of conditioned media on cancer cell growth, A549 or H1299 cells were plated at a density of 8x10 3 cells/cm 2 , in DMEM containing 10% FBS. After 6 h the medium was changed to DMEM containing 1% FBS for another 24 h. Then, the conditioned media to be tested were added to the sparse cultures, diluted 1:1 with fresh DMEM containing 2% FBS along with [methyl-3 H]-thymidine (0.2 µCi/ml, 25 Ci/mmol) and after 24 h of thymidine incorporation was measured by scintillation counting, as previously described (23).…”

Biological impact of the fibroblast growth factor family on articular cartilage and intervertebral disc homeostasis

“…It has been extensively studied in the literature and is found to be involved in numerous cellular functions in various cell types, including angiogenesis, tumorigenesis, cell proliferation, differentiation, wound healing, limb formation, and tissue remodeling (Bodo et al, 2002;Bobick et al, 2007;Douwes Dekker et al, 2007;Kakudo et al, 2007;Kanayama et al, 2007;Pratsinis and Kletsas, 2007;Schmal et al, 2007;Choi et al, 2008). In chondrocytes, the role of bFGF as an important cell growth regulatorin the growth plate is already well established (Kilkenny and Hill, 1996).…”

Identification of the potential molecular targets for human intervertebral disc degeneration based on bioinformatic methods

“…Additionally, Takahashi et al (14) revealed that the polymorphism 5A allele, which often occurs in the promoter region of the gene that regulates MMP-3 expression, was a possible risk factor for accelerated IDD in the elderly. In addition, Pratsinis et al (15) reported that platelet-derived growth factor, insulin-like growth factor-I and basic fibroblast growth factor could stimulate the proliferation of intervertebral disc cells via the activation of the extracellular-signal regulated kinase and Akt signaling pathways. By contrast, microRNAs (miRNAs) that can regulate RNA degradation or repression of translation have been identified as key regulators in numerous biological processes (16).…”