Transforming growth factor-beta1 induces tissue inhibitor of metalloproteinase-1 expression via activation of extracellular signal-regulated kinase and Sp1 in human fibrosarcoma cells.
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.
Laboratory of Functional Genomics, Korea Institute of Radiological and Medical Sciences, Gongneung-Dong, Seoul 139-706, Korea.
Hee-Jin
HJ
Kwak
Myung-Jin
MJ
Park
Hyeyoung
H
Cho
Chang-Min
CM
Park
Sang-Ik
SI
Moon
Hyung-Chan
HC
Lee
In-Chul
IC
Park
Mi-Suk
MS
Kim
Chang Hun
CH
Rhee
Seok-Il
SI
Hong
D002465:Cell Movement / Q000187:drug effects; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005354:Fibrosarcoma / Q000378:metabolism* / Q000473:pathology*; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D009361:Neoplasm Invasiveness; D010766:Phosphorylation; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology / Q000502:physiology*; D053773:Transforming Growth Factor beta1
D005419:Flavonoids; D016329:Sp1 Transcription Factor; C508960:TGFB1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D020781:Matrix Metalloproteinase 1; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one