Proteomic identification of insulin-like growth factor-binding protein-6 induced by sublethal H₂O₂ stress from human diploid fibroblasts

Fibroblasts are the most ubiquitous cell types within our body. They produce various factors to maintain the texture and structure of a particular organ or tissue. To identify protein factors secreted by fibroblasts and alteration of these protein factors upon oxidative stress, HCA₃ human skin diploid fibroblasts were exposed to a sublethal dose of H₂O₂, which induces a prematurely senescent phenotype. Conditioned media from prematurely senescent cells versus control cells were analyzed for proteins using an LC-MS/MS-based proteomic technique. Collagen α1(VI), collagen α2(I), fibronectin, lumican, and matrix metalloproteinase 2 were among the proteins consistently detected from control and H₂O₂-treated cells. Insulin-like growth factor-binding protein-6 (IGFBP-6) consistently showed up in the conditioned medium of H₂O₂-treated cells but not from untreated cells. Increased IGFBP-6 production due to H₂O₂ treatment was confirmed by RT-PCR and Western blot analyses. While H₂O₂ induced a dose-dependent elevation of IGFBP-6 mRNA, Western blot analyses detected elevated levels of IGFBP-6 protein in the conditioned medium of H₂O₂-treated cells. In comparison, fibronectin or matrix metalloproteinase 2 did not show changes at the mRNA level in cell lysates or at the protein level in the conditioned medium by H₂O₂ treatment. Using several types of toxins at sublethal doses, including cis-platin, hydroxyurea, colchicine, L-mimosine, rhodamine, dithiothreitol, or N-ethylmaleimide, we found that these agents induced increases of IGFBP-6 at mRNA and protein levels. An increased level of IGFBP-6 protein was detected in the plasma of aging mice and of young mice treated with doxorubicin. These data suggest that IGFBP-6 may serve as a sensitive biomarker of cell degeneration or injury in vitro and in vivo.