Tumor necrosis factor-alpha and interleukin-1 induce activation of MAP kinase and SAP kinase in human neuroma fibroblasts

Two cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), which are released by macrophages during the early inflammatory phase of nerve injury, are known to induce activation of mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK), which locate at different signal transduction pathways and are involved in cell cycle G0/G1 transition and cellular proliferation in human fibroblasts. Activation of these two protein kinases by the cytokines may stimulate fibroblast proliferation in damaged nerves and thereby play a role in the formation of a neuroma, a disorganized mass of tissue that interferes with neural regeneration and repair. To investigate the possibility that this mechanism is operative in neuroma formation, we used cultured, serum-starved fibroblasts from surgically removed human neuromas stimulated with TNF-alpha and/or IL-1 alpha and IL-1 beta, and measured the activation of MAPK and SAPK using myelin basic protein (MBP) and human c-Jun (1-169) glutathione S-agarose transferase (GST) fusion protein as substrates. For comparison, neuroma fibroblast cultures were also stimulated with phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor-AB (PDGF-AB), a potent activator for MAPK. TNF-alpha and both forms of IL-1 produced a rapid activation of MAPK, with a peak at 15 min for TNF-alpha stimulation, and a peak at 30 min for IL-1 stimulation. TNF-alpha combined with either IL-1 alpha or IL-1 beta produced a synergistic effect on the activation of MAPK. The increases in MAPK induced by TNF-alpha and IL-1 were similar to the increases induced by PMA and PDGF-AB. To confirm the presence of MAPK, immunoprecipitation and immunoblotting were carried out on experimental and control lysates. TNF-alpha and IL-1 also increased activation of SAPK, but to a lesser extent than MAPK. PMA and PDGF-AB were also much less effective in stimulating activation of SAPK. Our findings indicate that TNF-alpha and IL-1 activate parallel signal transduction pathways in human neuroma fibroblasts, and that they are relatively stronger activators of MAPK than of SAPK. Previous studies have convincingly demonstrated that MAPK and SAPK are involved in human fibroblast proliferation. The results of our study suggest that TNF-alpha and IL-1 may play a role in frustrating functional nerve regeneration after injury by stimulating these two kinases, which, in turn, leads to fibroblast proliferation and formation of neuromas.