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.

Curcumin inhibits cell cycle progression of immortalized human umbilical vein endothelial (ECV304) cells by up-regulating cyclin-dependent kinase inhibitor, p21WAF1/CIP1, p27KIP1 and p53.

To elucidate possible mechanisms of anti-angiogenic activity by curcumin, we performed cDNA microarray and found that curcumin modulated cell cycle related gene expression. For further confirmation, DNA contents and expression levels of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CDKIs) were examined by FACS analysis and Western blotting, respectively. Curcumin was found to induce G0/G1 and/or G2/M phase cell cycle arrest, up-regulate CDKIs, p21WAF1/CIP1, p27KIP1, and p53, and slightly down-regulate cyclin B1 and cdc2 in ECV304 cells. However, expression level of other cyclins and CDKs were not changed by curcumin. We, therefore, conclude that the up-regulation of CDKIs by curcumin plays a critical role in the regulation of cell cycle distribution in these cells, which may have a major role in anti-angiogenic activity of curcumin.

Protein kinase C-alpha activation by phorbol ester induces secretion of gelatinase B/MMP-9 through ERK 1/2 pathway in capillary endothelial cells.

In the present study, phorbol 12-myristate 13-acetate (PMA) was found to increase secretion of matrix metalloproteinase (MMP)-9 and in vitro invasion in bovine capillary endothelial (BCE) cells, which were blocked by specific inhibitors of protein kinase C (PKC). To elucidate molecular mechanisms involved, we studied the effect of PMA on the activation of mitogen activated protein kinases (MAPKs), and found that PMA activated extracellular signal-regulated kinase (ERK)1/2 and PD98059, a specific inhibitor of MAPK kinase, significantly reduced PMA-induced MMP-9 secretion as well as in vitro invasion of BCE cells. Treatment of safingol, a specific PKC-alpha inhibitor, and introduction of antisense PKC-alpha into these cells reduced the secretion of MMP-9 and activation of ERK1/2 by PMA. Furthermore, we employed adenoviral PKC-alpha and found that weak PMA stimulation (5 ng/ml) enhanced ERK1/2 activation and MMP-9 secretion in these cells. Therefore, we strongly suggest that PKC-alpha, partly at least, have a crucial role in MMP-9 secretion and invasion of BCE cells which are mediated via ERK1/2 signaling pathway.

Modulation of phorbol ester-induced regulation of matrix metalloproteinases and tissue inhibitors of metalloproteinases by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase.

OBJECT: Expression of matrix metalloproteinases (MMPs) has been postulated to play a central role in brain tumor invasion; however, its underlying mechanism is not yet fully understood. In the present study, by assessing the effect of a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, on the secretion of MMPs and in vitro invasion of various glioma cells, the authors attempt to define the role of the p38 MAPK pathway in the regulation of MMPs and tissue inhibitors of metalloproteinases (TIMPs) activated by phorbol ester (phorbol-12-myristate-13-acetate [PMA]) in the D54 human glioblastoma cell line. METHODS: The activation of MAPKs was determined using Western blot analysis after addition of phospho-specific antibodies against these kinases, the status of MMPs and TIMPs was analyzed using gelatin zymography and Western blot analysis, and the invasion rate of D54 cells and other glioma cells was analyzed using a modified Boyden chamber assay. Treatment of D54 cells with PMA activated two distinct MAPKs, extracellular signal-regulated kinase (ERK) 1/2 and p38 MAPK, but not c-Jun N-terminal kinase/stress-activated protein kinase. Induction of MMP-9 production and MMP-2 activation by PMA were blocked by SB203580, a specific inhibitor of p38 MAPK, but not by PD98059, a specific inhibitor of ERK 1/2. In addition, PMA-induced downregulation of TIMP-1 and TIMP-2 secretion and upregulation of the membrane type I MMP, a major activator of MMP-2 on the cell surface, were reversed by SB203580 in these cells; the PMA-induced increase of invasion in vitro decreased when SB203580 was added to the top compartment of a modified Boyden chamber; and the inhibitor also reduced the MMP secretion and PMA-induced in vitro invasion in various glioma cell lines. CONCLUSIONS: These results indicate that activation of p38 MAPK by PMA plays a central role in the regulation of MMPs and TIMPs in D54 cells, which has a major influence in tumor invasion and metastasis. Furthermore, inhibition of p38 MAPK by SB203580 blocked the secretion of MMPs and in vitro invasion of various glioma cells, underscoring a possible role of p38 MAPK inhibitors as antiinvasive and/or antimetastatic agents of malignant gliomas.