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Molecular & Cellular Proteomics 4:1273-1283, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Proteomic Identification of Insulin-like Growth Factor-binding Protein-6 Induced by Sublethal H₂O₂ Stress from Human Diploid Fibroblasts^*

Lifang Xie, George Tsaprailis and Qin M. Chen^,¶

From the Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona 85724 and the Southwest Environmental Health Sciences Center, College of Pharmacy, Tucson, Arizona 85721

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 1(VI), collagen 2(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.

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