Down-regulation of osteopontin mediates a novel mechanism underlying the cytostatic activity of TGF-β.

Abstract

Loss of a cytostatic response to TGF-β has been implicated in multiple hyper-proliferative disorders, including cancer. Although several key genes involved in the cytostatic activity of TGF-β have in the past been identified, its exact mode of action is yet to be elucidated. A comprehensive understanding of the mechanisms underlying the cytostatic activity of TGF-β may open up new avenues for the development of therapeutic strategies. Quantitative real-time RT-PCR was used to assess osteopontin (OPN) gene expression in human hepatoma-derived Huh-7 and lung adenocarcinoma-derived A549 cells. Reporter assays using an OPN promoter-luciferase construct and its mutated counterparts were performed to assess its transcriptional activity. Binding of Smad4 to the OPN gene promoter was investigated using chromatin immunoprecipitation (CHIP). The putative role of Smad4 in OPN gene expression down-regulation was also assessed using a shRNA-mediated knockdown strategy. The anti-proliferative effect of TGF-β on different cancer-derived cell lines was determined using the cell proliferation reagent WST-1. We found that the OPN expression levels dose-dependently decreased in TGF-β-treated Huh-7 and A549 cells. Our reporter assays indicated that this TGF-β-induced repression occurred at the transcriptional level, and could largely be abrogated by disruption of an element (TIE2) similar to the TGF-β inhibitory element found in other TGF-β-repressed genes. Our CHIP assay revealed that the Smad protein complex specifically binds to the OPN gene promoter, and that the TGF-β-mediated inhibition of OPN was lost upon shRNA-mediated knockdown of Smad4. Moreover, we found that the deregulation of OPN gene expression by TGF-β occurred concomitantly with loss of the TGF-β anti-proliferative response, whereas a neutralizing anti-OPN antibody partially restored this response. Our results indicate that the OPN gene is a direct target of Smad-mediated TGF-β signaling, implying that OPN expression inhibition serves as a novel mechanism underlying the cytostatic activity of TGF-β.