Abstract
The net balance of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP) system has been known to be a key factor in tumor cell invasion. In the present study, we investigated the molecular mechanisms of anti-invasive and antimigrative activity of transforming growth factor (TGF)-beta1 on HT1080 human fibrosarcoma cells. In in vitro Matrigel invasion and Transwell migration assays, TGF-beta1 dose-dependently inhibited the invasion and migration of HT1080 cells, respectively. Gelatin zymography, Western blot, and real-time PCR analysis showed that TGF-beta1 enhanced the expression and secretion of MMP-2, TIMP-1, and, to a lesser degree, MMP-9 but not membrane type 1-MMP and TIMP-2. The addition of recombinant TIMP-1 protein reduced the Matrigel invasion and Transwell migration of HT1080 cells, similar to TGF-beta1. Because augmentation of TIMP-1 might be the major factor for the anti-invasive and antimigrative activity of TGF-beta1, we investigated possible molecular mechanisms responsible for the expression of TIMP-1 induced by TGF-beta1. Treatment of HT1080 cells with TGF-beta1 rapidly phosphorylated three mitogen-activated protein kinases [MAPK; extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH2-terminal kinase] and Akt. Among these kinases, the inhibition of only ERK1/2 pathway by PD98059, a specific inhibitor of MAPK/ERK kinase(MEK)-1, and transfection of dominant-negative MEK 1 effectively blocked the TIMP-1 induction by TGF-beta1. Mithramycin, a specific inhibitor of Sp1 transcription factor, but not curcumin, an inhibitor of activator protein-1, and transfection of Sp1 small interfering RNA significantly inhibited the TGF-beta1-induced expression of TIMP-1. In addition, electrophoretic mobility shift assay showed that TGF-beta1 up-regulated Sp1 DNA-binding activity, and PD98059 and mithramycin effectively inhibited these events. Finally, pretreatment of HT1080 cells with PD98059 and mithramycin, but not curcumin, restored the invasive activity of these cells. Taken together, these data suggest that TGF-beta1 modulates the net balance of the MMPs/TIMPs the systems in HT1080 cells for anti-invasion and antimigration by augmenting TIMP-1 through ERK1/2 pathway and Sp1 transcription factor.
Highlights
Tumor cell invasion is a multistep process, involving several interactions between tumor cells and extracellular matrix (ECM), a directed proteolysis of ECM, such as basement membranes required for intravasation and extravasation of tumor cells [1]
We investigated the role of transforming growth factor (TGF)-h1 in the regulation of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinase (TIMP) expression in HT1080, a cellular model of invasive fibrosarcoma cells, and observed that treatment of HT1080 cells with TGF-h1 significantly upregulated TIMP-1 expression and blocked invasion and migration of the cells and that extracellular signal-regulated kinase 1/2 (ERK1/2) and Sp1 signaling pathways were crucially involved in these events
Transduction of DNp38 and DN-Jun NH2-terminal kinase (JNK) retroviruses significantly suppressed MMP-2 but not TIMP-1 expression by TGF-h1 (Fig. 3F). These results suggest that ERK1/2 pathway is involved in the regulation of TGF-h1-induced TIMP-1 expression, whereas p38 and JNK pathway is involved in the regulation of MMP-2 expression in HT1080 cells, indicating that TGF-h1 regulates the expression of MMP-2 and TIMP-1 in HT1080 cells through different mechanisms
Summary
Tumor cell invasion is a multistep process, involving several interactions between tumor cells and extracellular matrix (ECM), a directed proteolysis of ECM, such as basement membranes required for intravasation and extravasation of tumor cells [1]. Proteolytic degradation of ECM components involves several proteases, such as matrix metalloproteinases (MMP), plasminogen activators, and serine proteases secreted by invading tumor cells [2,3,4,5]. An imbalance between MMP and TIMP activities results in an excessive ECM degradation necessary for tumor invasion and metastasis [7, 8]. TIMPs are multifunctional proteins that can inhibit the catalytic activity of MMPs, maintaining ECM homeostasis [9].
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