Abstract
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 alpha1(VI), collagen alpha2(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.
Highlights
Fibroblasts are the most ubiquitous cell types within our body
Identification of Proteins in Conditioned Media of human diploid fibroblasts (HDFs) by LC-MS/MS—Early passage HCA3 human skin fibroblasts were treated with H2O2 and were allowed to develop a stable senescence-like phenotype
The shotgun approach of ESI-LC-MS/MS-based proteomics has lead to the discovery that H2O2-induced senescentlike human diploid fibroblasts increase the production of Insulin-like growth factor-binding protein-6 (IGFBP-6) protein
Summary
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. During the process of aging, it is thought that such cells show changes in biochemistry and gene expression patterns Some of these changes, such as alterations in the secretion of degradative enzymes, inflammatory cytokines, and growth factors, are related to the senescent phenotype of normal diploid fibroblasts in culture [1,2,3,4,5]. These changes can alter a microenvironment, disrupt tissue structures, and cause growth of neighboring premalignant cells [1, 2, 5,6,7,8,9,10]. 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
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