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Increased expression of transforming growth factor (TGF)-β1 and tumor necrosis factor (TNF)-α are thought to play important roles in the development of pulmonary fibrosis. We recently reported that TNF-α upregulates TGF-β1 expression in primary mouse lung fibroblasts (MLFs), a key cell population in fibrogenesis. In the present study, we have investigated signal transduction pathways involved in TNF-α upregulation of TGF-β1 in both primary MLFs and the Swiss 3T3 fibroblast cell line. Treatment of fibroblasts with TNF-α resulted in a significant increase in TGF-β1 protein as measured by ELISA. The increase in protein was preceded by a 200–400% increase in TGF-β1 mRNA detected by quantitative, real-time, reverse transcriptase-polymerase chain reaction. Western blot analysis showed that TNF-α activated the extracellular signal-regulated kinase (ERK), and inhibitors of the ERK-specific mitogen-activated protein kinase pathway (PD98059 or U0126) blocked TNF-α induction of TGF-β1 mRNA and protein. mRNA stability experiments showed that TNF-α increased the half-life of TGF-β1 mRNA to more than 24 h compared with ˜ 15 h in unstimulated cells. Expression of constitutively active MEK1 that selectively phosphorylates ERK was sufficient for TGF-β1 mRNA stabilization in Swiss 3T3 fibroblasts. These results indicate that TNF-α activates the ERK-specific mitogen-activated protein kinase pathway leading to increased TGF-β1 production in fibroblasts, primarily via a post-transcriptional mechanism that involves stabilization of the TGF-β1 transcript.