Transforming Growth Factor-B1 Induces Tissue Inhibitor of ...Transforming Growth Factor-B1 Induces...

Transforming Growth Factor-B1 Induces Tissue Inhibitorof Metalloproteinase-1 Expression via Activation ofExtracellular Signal-Regulated Kinase and Sp1 inHuman Fibrosarcoma Cells

Hee-Jin Kwak,1 Myung-Jin Park,1 Hyeyoung Cho,1 Chang-Min Park,1

Sang-Ik Moon,1 Hyung-Chan Lee,1 In-Chul Park,1 Mi-Suk Kim,4

Chang Hun Rhee,1,2 and Seok-Il Hong1,3

1Laboratory of Functional Genomics, 2Department of Neurosurgery, and 3Laboratory Medicine andClinical Pathology, Korea Institute of Radiological and Medical Sciences, Seoul, Korea and4Research Institute and hospital, National Cancer Center, Goyang, Korea

AbstractThe 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)-B1 on HT1080 human fibrosarcoma cells. In

in vitro Matrigel invasion and Transwell migration

assays, TGF-B1 dose-dependently inhibited the invasion

and migration of HT1080 cells, respectively. Gelatin

zymography, Western blot, and real-time PCR analysis

showed that TGF-B1 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-B1. Because augmentation of

TIMP-1 might be the major factor for the anti-invasive

and antimigrative activity of TGF-B1, we investigated

possible molecular mechanisms responsible for the

expression of TIMP-1 induced by TGF-B1. Treatment of

HT1080 cells with TGF-B1 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-B1. 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-B1-induced expression of TIMP-1. In addition,

electrophoretic mobility shift assay showed that TGF-B1

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-B1 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.

(Mol Cancer Res 2006;4(3):209–20)

IntroductionTumor cell invasion is a multistep process, involving

several interactions between tumor cells and extracellular

matrix (ECM), particularly 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 (2-5). Among the

ECM-degrading proteases, however, much attention have been

focused on the MMP family, especially MMP-2 and MMP-9,

and many evidences have shown that elevated levels of

MMP-2 and MMP-9 are well correlated with an invasive

phenotype of cancer cells (6). The extracellular activities of

these enzymes are inhibited, when they are complexed with

endogenous inhibitors, such as tissue inhibitor of metal-

loproteinase (TIMP)-1 and TIMP-2. Consequently, an imbal-

ance 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, thus maintaining ECM homeostasis

(9). Besides acting as proteinase inhibitors, TIMPs also modulate

cell growth, apoptosis, and angiogenesis (9). Currently, four

Received 8/19/05; revised 1/20/06; accepted 1/30/06.Grant support: National Nuclear R&D Program of Ministry of Science andTechnology (Seoul, Korea).The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).H-J. Kwak and M-J. Park contributed equally to this work.Requests for reprints: Seok-Il Hong, Laboratory of Functional Genomics, KoreaInstitute of Radiological and Medical Sciences, 215-4 Nowon-Ku, Gongneung-Dong, Seoul 139-706, Korea. Phone: 82-2-970-1260; Fax: 82-2-970-2402.E-mail: [email protected] D 2006 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-05-0140

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members of the TIMP family have been characterized: TIMP-1,

TIMP-2, TIMP-3, and TIMP-4 (10, 11). Among these, TIMP-1 is

a glycosylated protein with molecular mass ranging from 28.5 to

34 kDa, depending on the degree of glycosylation, and is secreted

in a soluble form by different cell types and forms a 1:1 complex

with MMP-9 (9). Tumor invasion and metastasis are negatively

regulated by TIMP-1 in different types of tumor cell lines

(12-16), and down-regulation of TIMP-1 expression or mutated

TIMP-1 is associated with increased invasiveness (17, 18).

Recent transgenic studies also showed that endogenous over-

expression or suppression of TIMP-1 inhibited or enhanced the

tumor growth in several cell types, respectively (19-21). In

addition, TIMP-1 has been shown to exert an antiangiogenic

activity both in vitro and in vivo (22, 23). TIMP-1 promoter

contains several 12-O -tetradecanoylphorbol-13-acetate –

responsive elements, such as activator protein-1 (AP-1), Sp1,

and Ets sites, and functional interaction of these transcription

factors has been shown to induce synergistic transcriptional

activation of the TIMP-1 promoter. Consequently, contrary to

TIMP-2, TIMP-1 expression is up-regulated by several factors,

including phorbol esters, interleukin-6, transforming growth

factor (TGF)-h1, epidermal growth factor, oncostatin M, or

erythropoietin (9, 24, 25).

TGF-h, 25-kDa homodimeric protein with autocrine

and paracrine activities, is a multifunctional protein that

has been implicated in various physiologic and pathologic

conditions, including cell cycle control, carcinogenesis in

human skin, and invasion and metastasis of carcinoma cells

(26). In tumor progression, TGF-h1 has been shown to have

dual function, either decreasing or promoting cancer

invasion, depending on the type and the progression stage

of tumor (27, 28). Consistent with proinvasive or anti-invasive

roles in cancer progression, TGF-h1 stimulates tumor invasion

by up-regulating integrin, MMP-2, MMP-9, and urokinase-

type plasminogen activator expression (29-33) while sup-

presses it by down-regulating plasminogen activator systems,

MMPs, and plasmin-dependent ECM degradation through

stimulating the expression of TIMP-1 and plasminogen

activator inhibitor-1 (34-37).

TGF-h1 signaling is initiated via binding of ligand to its

serine/threonine kinase receptor type II and then type I on the

cell surface (38). On activation, the type I and II receptors

interact to form hetero-oligomers, which then activate

receptor-associated Smads, Smad2 and Smad3. The receptor-

associated Smads then bind to Smad4, translocate to the

nucleus, and ultimately regulate transcription and expression

of target genes. Therefore, Smad proteins are the best known

intracellular substrates for signal transmitter of TGF-h1receptor activation (38). In addition to the Smad-mediated

signaling, TGF-h1 also can signal through mitogen-activated

protein kinase (MAPK) pathways, including extracellular

signal-regulated kinase 1/2 (ERK 1/2) and p38 (39-41).

Several studies have shown that MAPK pathways are

involved in the TGF-h1-mediated modulation of ECM-

degrading proteases and their inhibitors in the process of

cellular invasion (34, 42-44). However, there is little

information available about signaling mechanisms of TGF-

h1-mediated TIMP-1 expression and anti-invasion of tumor

cells.

In the present study, we investigated the role of TGF-h1 in

the regulation of MMPs and TIMPs expression in HT1080, a

cellular model of invasive fibrosarcoma cells, and observed that

treatment of HT1080 cells with TGF-h1 significantly up-

regulated TIMP-1 expression and blocked invasion and

migration of the cells and that ERK1/2 and Sp1 signaling

pathways were crucially involved in these events.

ResultsTGF-b1 Inhibited the Invasion and Migration of HT1080Cells In vitro

First, we examined the effect of TGF-h1 on invasiveness of

HT1080 cells using Matrigel invasion assay system. As shown

in Fig. 1A, invasion of HT1080 cells was significant and was

reduced dose-dependently by treatment with TGF-h1. Next, theeffects of TGF-h1 on the migration of HT1080 cells were

determined by Transwell migration assay. In line with the

results from the above invasion assays, TGF-h1 also inhibited

the migration of HT1080 cells in a dose-dependent manner

(Fig. 1B). No distinctive cellular morphologic changes, which

are typically associated with apoptosis, such as cell detachment,

rounding, or chromosomal fragmentation, were detected under

the experimental condition (data not shown). In addition,

significant cytotoxic effect was not detected by 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays

in the serum-free medium following up to 100 ng/mL TGF-h1treatment for 12 and 24 hours (Supplementary Fig. S1),

indicating that the anti-invasive activity of TGF-h1 was not due

to cytotoxic effect of this cytokine.

Augmentation of TIMP-1 Might Be Responsible for theAnti-Invasive and Antimigrative Activity of TGF-b1 inHT1080 Cells

Tumor cell invasiveness is strongly associated with the

balance between ECM-degrading proteases and their inhibitors,

especially MMPs and TIMPs. Therefore, to investigate the

effects of TGF-h1 on MMP-2, MMP-9, membrane type 1-MMP

(MT1-MMP), TIMP-1, and TIMP-2 induction in HT1080 cells,

we measured the expression of these molecules in the

conditioned medium or cell lysates by using gelatin zymography

analysis and Western blotting. As shown in Fig. 2A and B,

treatment of HT1080 cells with TGF-h1 significantly increased

the secretion of MMP-2 and TIMP-1, but not MT1-MMP and

TIMP-2. TGF-h1 also increased the secretion of MMP-9 but to a

lesser degree. Because TGF-h1 modulated the secretion of

MMP-2 and TIMP-1 significantly, we further investigated the

effect of TGF-h1 on the mRNA expression of these molecules

by using quantitative real-time PCR method. As shown in

Fig. 2C, TGF-h1 significantly augmented MMP-2 and TIMP-1

mRNA levels in HT1080 cells. Enhanced MMP-2 and TIMP-1

mRNA peaked at 3 and 6 hours, respectively, and declined

thereafter.

Because TGF-h1 significantly suppressed the invasion and

migration of HT1080 cells, we wondered whether TIMP-1

secretion by TGF-h1 might be critically involved in the

inhibition. Therefore, we did Matrigel invasion and Transwell

migration assays in the presence or absence of recombinant

TIMP-1 and found that TIMP-1 dose-dependently suppressed

Kwak et al.

Mol Cancer Res 2006;4(3). March 2006

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the invasion and migration of HT1080 cells (Fig. 2D and E).

However, the addition of TIMP-1 up to 100 ng/mL did not

completely inhibit invasion (Supplementary Fig. S2), indicating

that TIMP-1 is not the only invasion suppressor in this system.

The above data indicate that TGF-h1 regulates the

expression of MMP-2 and TIMP-1 at the transcriptional level

and that augmentation of TIMP-1 might be a key event in

modulating the balance of MMPs and TIMPs expression

against invasion and migration of HT1080 cells. To further

confirm that TIMP-1 was the direct mediator of the effects of

TGF-h1 on the inhibition of invasion, endogenous expression

of TIMP-1 genes in HT1080 cells was silenced by TIMP-1

small interfering RNA (siRNA), and efficient and specific

knockdown was observed by Western blot analysis of cell

lysates (Fig. 2F). Transfection of HT1080 cells with TIMP-1

siRNA reduced TGF-h1-mediated inhibition of invasion

compared with control siRNA-transfected cells, whereas

TIMP-1 siRNA transfection alone slightly up-regulated the

invasion of these cells (Fig. 2G). These results indicate that

TIMP-1 is the critical invasion and migration suppressor in

HT1080 cells, at least in part.

TGF-b1 Induced Expression of MMP-2 and TIMP-1through p38 and c-Jun NH2-terminal kinase and ERK1/2Signaling Pathway, Respectively

To elucidate signaling mechanism possibly involved in anti-

invasive action of TGF-h1, we next examined whether TGF-h1influenced MAPKs and Akt signaling pathways in HT1080

cells. As shown in Fig. 3A, treatment of the cells with TGF-h1strongly up-regulated ERK1/2 and Akt phosphorylation at 15

minutes, lasting up to 180 minutes. TGF-h1 also increased p38

and c-Jun NH2-terminal kinase (JNK) phosphorylation, but the

effect was weak compared with ERK1/2 and Akt phosphory-

lation. To further ascertain the involvement of the activation

of these kinases in the expression of MMPs and TIMPs, the

cells were treated with TGF-h1 in the presence or absence of

highly specific inhibitors of MAPK/ERK kinase (MEK)-1

(PD98059), p38 (SB202190), JNK (SP600125), and phospha-

tidylinositol-3 kinase (PI3K; LY294002). Gelatin zymography,

Western blot, and real-time PCR analyses clearly showed that

pretreatment of the cells with SB202190 and SP600125, but not

PD98059 and LY294002, reduced the increase of MMP-2

induced by TGF-h1, whereas only PD98059 significantly

FIGURE 1. Effect of TGF-h1 on the invasion and migration of HT1080 cells. Chemotaxic cells were coated with Matrigel (100 Ag/cm2) on the upper side offilter for invasion assay or with type I collagen (100 Ag/cm2) on the underside of filter for migration assay, and 2 � 105 HT1080 cells cultured in the presence orabsence of TGF-h1 at indicated concentrations were placed in the upper well. Invasion and migration of the cells were determined by measuring the ability topass through a layer of Matrigel-coated and intact filters, respectively. Detailed procedures are described in Materials and Methods. Top, HT1080 cellsinvaded (A) and migrated (B) cells under the membrane after 12 hours (top ); bottom, invasion (A) and migration (B) were determined by counting cells infour microscopic fields per sample. Bars, SD. *, P < 0.05 versus control.

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blocked the increase of TIMP-1 by TGF-h1 (Fig. 3B-D).

Because the above data strongly implicated the involvement of

ERK1/2 signaling pathway in TGF-h1-induced TIMP-1

expression, we further investigated the role of ERK1/2 pathway

in TGF-h1-stimulated TIMP-1 expression by using dominant-

negative (DN) mutants of MAPKs. Thus, we transiently

FIGURE 2. Effect of TGF-h1 on MMP and TIMP expression and in vitro invasion and migration stimulated with TGF-h1 or TIMP-1 protein. HT1080 cellswere treated with various concentrations of TGF-h1. The conditioned serum-free medium was collected for analysis after 18 hours. A. Gelatin zymographyanalysis of conditioned medium from HT1080 cells to detect the secretion of MMP-2 and MMP-9. B. Western blot analysis of conditioned medium andmembrane extracts to detect MMP-2, MMP-9, TIMP-1, TIMP-2, and MT1-MMP. C. Quantitative real-time PCR analysis of total RNA isolated from HT1080cells treated with TGF-h1 (10 ng/mL) for indicated time points. Quantification of real-time PCR analysis. Columns, average for each sample normalized withthe amount of GAPDH; bars, SD. *, P < 0.05 versus control; TGF-h1. Matrigel invasion (D) and Transwell migration (E) of HT1080 cells cultured in theabsence or presence of TGF-h1 (10 ng/mL) or TIMP-1 (0.1 or 1 ng/mL). Left, HT1080 cells invaded and migrated under the membrane after 12 hours; right,invasion and migration rates were determined by counting cells in four microscopic fields per sample. Bars, SD. *, P < 0.05 versus control; TGF-h1; **, P <0.05 versus control; TIMP-1. F. Western blot analysis of HT1080 cells after transfection with TIMP-1 (200 nmol/L) or control siRNA for 24 hours. G. Matrigelinvasion assay of siRNA-transfected cells cultured in the absence or presence of TGF-h1 (10 ng/mL) for an additional 12 hours. Whole-cell lysates wereanalyzed for the detection of TIMP-1 by Western blot. Invasiveness was determined by counting cells in four microscopic fields per sample. Bars, SD. *, P <0.05 versus control; TGF-h1 in control siRNA-transfected cells; **, P < 0.05 versus TGF-h1; TGF-h1 in TIMP-1 siRNA-transfected cells.

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transfected DN mutant plasmids of MEK-1 (the immediate

upstream kinase of ERK1/2) and retroviruses for DN form of

p38 and JNK into HT1080 cells for 24 hours. After another 24

hours of incubation with TGF-h1 (10 ng/mL) in serum-free

medium, we measured the TIMP-1 in the conditioned medium

by using gelatin zymography and Western blot analyses.

Consistent with the results obtained with the pharmacologic

inhibitors, DN-MEK-1 abrogated TGF-h1-induced TIMP-1

expression (Fig. 3E). On the other hand, transduction of DN-

p38 and DN-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.

TGF-b1-Induced Sp1 Activation Was Involved in TIMP-1Expression

Several transcriptional factors, including AP-1, Sp1, and 12-

O-tetradecanoylphorbol-13-acetate–responsive element, regu-

late the expressions of TIMP-1 (45, 46). Therefore, to identify

the possible transcription factor involved in TGF-h1-inducedTIMP-1 expression, we tested the effect of curcumin and

mithramycin, an inhibitor of AP-1 and Sp1, respectively, on

the event. As shown in Fig. 4A, pretreatment of the cells with

mithramycin, but not curcumin, significantly reduced the

secretion of TIMP-1 induced by TGF-h1. Mithramycin also

inhibited the induction of TIMP-1 mRNA by TGF-h1(Fig. 4B). Furthermore, transfection of Sp1 siRNA significantly

reduced the both Sp1 expression and TIMP-1 secretion induced

by TGF-h1 as well as basal levels (Fig. 4C). Next, we

investigated the involvement of Sp1 DNA-binding activity in

the TIMP-1 expression. Because TGF-h1-mediated TIMP-1

expression was blocked by ERK1/2 inhibition, as shown above,

it is likely that the inhibitory effect of PD98059 on TGF-h1-induced TIMP-1 expression might have been mediated, at least

in part, through down-regulation of Sp1 DNA-binding activity.

To examine this possibility, we did electrophoretic mobility

shift assay and found that TGF-h1 strongly increased Sp1

DNA-binding activities and that pretreatment of HT1080 cells

with PD98059 and mithramycin effectively inhibited the TGF-

h1-induced Sp1 DNA-binding activities in a dose-dependent

manner (Fig. 5A). Specificity of the Sp1 band was confirmed

by detecting supershift band, when the nuclear proteins were

preincubated with Sp1 antibodies (Fig. 5A). TGF-h1 also

induced the AP-1 DNA-binding activity and dose-dependently

blocked the event by pretreatment with PD98059 and curcumin

(Fig. 5B). These results together strongly suggest that TGF-h1-mediated TIMP-1 expression in HT1080 cells is regulated

dominantly by Sp1 transcriptional activity, which is mediated

by ERK1/2.

ERK1/2 Pathway and Sp1 Transcription Factor WereInvolved in TGF-b1-Induced Anti-Invasion and Antimigra-tion of HT1080 Cells

As shown above, because ERK1/2 and Sp1 signaling

pathways were critical for induction of TIMP-1 in HT1080

cells by TGF-h1, we next examined whether the inhibition

of these pathways restored the invasive and migrative activity

of these cells. As expected, treatment of HT1080 cells with

PD98059 and mithramycin, but not SB202190 and

SP600125, ameliorated the TGF-h1-mediated suppression

of HT1080 cell invasion (Fig. 6A and B) and migration

(Fig. 6D and E). However, treatment of the cells with

curcumin together with TGF-h1 more suppressed the

invasion (Fig. 6C) and migration (Fig. 6F) of HT1080 cells.

These additional inhibitory effects of HT1080 cell invasion

and migration by curcumin were not due to cytotoxicity,

because curcumin up to 10 Amol/L did not show any toxic

effect on HT1080 cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide assay (Supplementary Fig.

S3A). In addition, flow cytometric analysis revealed no

increase in the proportion of Annexin V–positive cells and

propidium iodide–positive cells with up to 10 Amol/L

curcumin (Supplementary Fig. S3B). However, significant

cytotoxicity was detected with >35 Amol/L concentration of

curcumin (Supplementary Fig. S3B). Therefore, these data

strongly indicate that ERK1/2 and Sp1 signaling pathways

are important for the induction of TIMP-1 and suppression of

invasion of HT1080 cells by TGF-h1.

DiscussionInvasion of tumor cells through ECM and basement

membrane barriers is a crucial step in tumor dissemination

and metastasis (47, 48). Proteolysis of ECM results from an

imbalance between activators and inhibitors of proteinases,

which are controlled by various cytokines or growth factors,

including TGF-h1. Tumor cell invasion and migration

activities are greatly increased by TGF-h1, leading to a

more malignant phenotype (31, 34). On the other hand,

several studies showed that TGF-h1 is a potent growth

suppressor of different normal and tumor cell types (27, 32).

Induction of TIMP-1 and inhibition of tumor cells invasion

by TGF-h1 have been described previously in an invasive

tumor cell line HT1080 (37); however, the regulation of

these events by TGF-h1 has not been investigated. In the

present study, therefore, we attempted to identify TGF-h1-induced signaling pathway that is involved in the regulation

of TIMP-1 expression and invasion and migration by

HT1080 cells and showed that TGF-h1 suppressed the

invasion and migration of HT1080 cells by modulating the

balance of ECM-degrading proteases and their inhibitor

proteins. Specifically, TGF-h1 up-regulated MMP-2, MMP-

9, and TIMP-1 expression, and augmentation of TIMP-1 by

TGF-h1 might be responsible for suppressing invasion and

migration of these cells. TGF-h1 induced TIMP-1 at both

mRNA and protein levels in a dose-dependent manner.

Furthermore, TGF-h1-induced TIMP-1 expression and anti-

invasion and antimigration were regulated through the ERK1/

2 signaling pathway and Sp1 transcription factor, suggesting

the possible mechanism of anti-invasive and antimigrative

action of TGF-h1 in HT1080 cells.

Invasion of tumor cells is determined by the net activity

of ECM-degrading proteases and their inhibitors. Several

reports showed that TGF-h1 stimulates tumor invasion by



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up-regulating integrin, MMP-2, MMP-9, and urokinase-type

plasminogen activator expression (29-33), while suppresses it

by down-regulating plasminogen activator systems, MMPs, and

plasmin-dependent ECM degradation through stimulating the

expression of TIMP-1 and plasminogen activator inhibitor-1

(34-37). TGF-h1 suppression of invasion of HT1080 human

fibrosarcoma cells seems to be largely due to the induction of

TIMP-1 expression (9, 37). Consistent with the results of

Kubota et al. (37), our data also showed that TGF-h1 up-

regulated the expression and secretion of MMP-2, MMP-9, and

TIMP-1 but not MT1-MMP and TIMP-2. Although TGF-h1induced the expression of MMP-2 and MMP-9, the net invasive

and migrative activity of HT1080 cells was down-regulated due

to the induction of TIMP-1 by TGF-h1. Indeed, the addition of

recombinant TIMP-1 reduced the invasion and migration of

these cells. In other tumor cell systems, it has also been reported

that reconstitution of Smad3 restores the TGF-h1-inducedTIMP-1 expression and results in inhibiting the invasion of

FIGURE 3. Effect of various kinase inhibitors on the secretion of MMP-2 and TIMP-1 by TGF-h1 in HT1080 cells. A. Western blot analysis of HT1080cells stimulated with TGF-h1 (10 ng/mL) for indicated times. Cell extracts (50 Ag each) were resolved by SDS-PAGE and probed with phosphorylatedERK1/2 (p-ERK1/2), phosphorylated p38 (p-p38 ), phosphorylated JNK (p-JNK ), and phosphorylated Akt (p-Akt ) antibodies. To verify equal loading, theblots were probed with actin. B to D. HT1080 cells were pretreated with various kinase inhibitors, SB202190 (SB ; p38 inhibitor, 10 Amol/L), PD98059(PD ; MEK-1 inhibitor, 25 Amol/L), SP600125 (SP ; NK inhibitor, 10 Amol/L), and LY294002 (LY ; PI3K inhibitor, 5 Amol/L) for 1 hour before stimulation withTGF-h1 (10 ng/mL). B. Conditioned serum-free medium after 18 hours was collected for gelatin zymography to detect MMP-2. C. Western blot to detectMMP-2, TIMP-1, and TIMP-2. D. Quantitative real-time PCR analysis of total RNA isolated from HT1080 cells treated with various kinase inhibitors todetect the expression of MMP-2 and TIMP-1. Quantification by real-time PCR analysis. Columns, average for each sample normalized with the amount ofGAPDH; bars, SD. *, P < 0.05 versus control; TGF-h1; **, P < 0.05 versus TGF-h1. Western blot analysis of cell lysates and conditioned medium fromHT1080 cells transfected with DN mutant plasmids of MEK-1 (E) and retroviruses for DN form of p38 and JNK (F), respectively, in the presence orabsence of TGF-h1 (10 ng/mL). CTL, control.

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human choriocarcinoma cells (35). On the contrary, TGF-h1inhibited invasiveness of normal trophoblast by up-regulating

the TIMP-1 level, but not in JAR and JEG-2 choriocarcinoma

cells, which show very low level of TIMP-1 expression (49).

Therefore, TIMP-1 induction by TGF-h1 is an important

mechanism for inhibiting invasion of choriocarcinoma, at least

in part. In addition, human hepatoma cells (HepG2) also

produce TIMP-1 in response to TGF-h1 (50). Therefore, TGF-

h1-induced TIMP-1 production might be critical mechanism for

the suppression of invasion in some tumor cells, such as

fibrosarcoma, choriocarcinoma, and hepatoma, although further

studies are needed to uncover the critical role of TIMP-1 in

invasiveness of these tumors.

Several external stimuli, such as growth factors, phorbol

esters, and cytokines, induce TIMP-1 expression in various cell

types (9). However, the regulation mechanism of TIMP-1 is not

well understood. Tong et al. reported that ERK1/2 and p38

pathways are required for maximal regulation of TIMP-1 in

murine fibroblast by oncostatin M (24), and Kadri et al. showed

that erythropoietin-induced TIMP-1 expression and secretion

are mediated by ERK1/2 and PI3K/Akt signaling pathway (25).

In line with these studies, we also examined the involvement of

MAPK and PI3K/Akt pathways in the regulation of TIMP-1 by

TGF-h1 in HT1080 cells. TGF-h1 phosphorylated three

MAPKs (ERK1/2, p38, and JNK) and Akt, a PI3K downstream

effector molecule. By using specific kinase inhibitors of these

kinases, we found that the activation of ERK1/2 pathway was

involved in TGF-h1-induced TIMP-1 expression and secretion

in HT1080 cells. Actually, PD98059, a specific inhibitor of

MEK, which is upstream kinase of ERK1/2, but not SB202190,

SP600125, and LY294002, specific inhibitors of p38, JNK, and

PI3K, respectively, could effectively inhibit TIMP-1 expression

and secretion induced by TGF-h1 in these cells. Furthermore,

transient transfection of DN-MEK-1, but not DN-p38 and DN-

JNK, abrogated the induction of TIMP-1 by TGF-h1. To our

best knowledge, this is the first report to show that induction of

TIMP-1 by TGF-h1 is regulated by ERK1/2 signaling pathway.

In addition, Matrigel invasion and Transwell migration assays

showed that blockade of ERK1/2 pathway by PD98059

restored the invasion and migration of HT1080 cells from the

invasion and migration suppressive effect of TGF-h1. There-fore, in HT1080 cells, ERK1/2 pathway plays an important role

in TIMP-1 expression following the suppression of invasion

and migration by TGF-h1.Regulation of TIMP-1 gene expression is mainly mediated

at the transcription level and involves the activation of several

well-known transcription factors, including those belonging to

AP-1, Sp1, and Pea3/Ets families (45). A recent report shows

that the up-regulation of TIMP-1 promoter activity by TGF-

h1 requires the AP-1 response element (51). In other

stimulatory systems, such as oncostatin M and erythropoietin,

the dependence of TIMP-1 expression on AP-1 has also been

described (25, 49). In the present study, we examined which

transcription factor was related with the expression of TIMP-

1 and found that mithramycin, a specific Sp1 inhibitor, but

not curcumin, which is known as an AP-1 inhibitor,

significantly inhibited TGF-h1-induced TIMP-1 expression.

In addition, transfection of siRNA for Sp1 dramatically

diminished the TIMP-1 expression, and TGF-h1 increased the

Sp1 DNA-binding activity and PD98059 and mithramycin

effectively suppressed this increase. The inhibitory effect of

PD98059 on Sp1 DNA-binding activity implies that the

ERK1/2 signal pathway is critical for TGF-h1-induced Sp1

activation and TIMP-1 expression. We cannot, however, rule

out the possible involvement of AP-1, the well-known

transcription factor for TIMP-1 expression (25, 51, 52), and

other transcription factors. Nevertheless, it is certain that Sp1

transcription factor is dominantly involved in the induction of

TIMP-1 by TGF-h1 in these cell systems, partly at least.

Therefore, the above observations led us to conclude that

TGF-h1 increases TIMP-1 expression through ERK1/2 and

Sp1 signaling pathways.

FIGURE 4. Involvement of Sp1 transcription factor in TGF-h1-mediated TIMP-1 induction in HT1080 cells. A. Western blot analysis ofconditioned medium to detect the secretion of TIMP-1. Cells werepretreated with or without curcumin and mithramycin for 1 hour at theindicated concentrations and then stimulated with TGF-h1 (10 ng/mL) for18 hours in serum-free medium. B. Quantitative real-time PCR analysis oftotal RNA isolated from HT1080 cells treated with or without mithramycinin the presence or absence of TGF-h1 (10 ng/mL). Quantification by real-time PCR analysis. Columns, average for each sample normalized withthe amount of GAPDH; bars, SD. *, P < 0.05 versus control; TGF-h1; **,P < 0.05 versus TGF-h1; TGF-h1 + mithramycin. M, mithramycin. C.Western blot analysis of HT1080 cells to detect TIMP-1 and Sp1. Cellswere transfected with Sp1 siRNA (200 nmol/L) or control siRNA for 24hours and treated with TGF-h1 (10 ng/mL) for an additional 24 hours.Conditioned medium and whole-cell lysates were analyzed by Westernblot for the detection of TIMP-1 and Sp1 proteins, respectively.



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In summary, the data described above showed that TGF-h1inhibited pericellular proteolysis by inducing the expression of

TIMP-1 in HT1080 cells. Mechanistic analyses showed that

ERK1/2 activation of TGF-h1 regulated the levels of TIMP-1

expression and this regulation was largely mediated by the

regulation of transcription factor, Sp1. These results provide

important clues in the regulatory mechanisms underlying

TGF-h1-induced inhibition of tumor cell invasion and

migration.

Materials and MethodsReagents and Cell Culture

Recombinant human TGF-h1 was purchased from Bio-

source Co. (Camarillo, CA). Antibodies against MT1-MMP,

MMP-2, MMP-9, TIMP-1, and TIMP-2 were obtained from

Calbiochem (La Jolla, CA), and antibodies against phosphor-

ylated ERK1/2, p38, JNK, and Akt were from New England

Biolabs (Beverly, MA). The following kinase inhibitors were

purchased from Calbiochem and used in this study: PD98059

(ERK1/2), SB202190 (p38), SP600125 (JNK), and LY294002

(PI3K). Matrigel was purchased from Becton Dickinson

(Bedford, MA), and gelatin and type I collagen were purchased

from Sigma Chemical Co. (St. Louis, MO). HT1080 cells were

grown in DMEM (Life Technologies, Grand Island, NY)

supplemented with 2 mmol/L glutamine, 1 mmol/L sodium

pyruvate, 100 units/mL penicillin, 100 Ag/mL streptomycin

(Life Technologies), and 10% heat-inactivated fetal bovine

serum (Life Technologies) in a humidified incubator containing

5% CO2 at 37jC.

FIGURE 5. Effect of MEK-1 and Sp1 inhibitors on TGF-h1-induced DNA-binding activity of Sp1 and AP-1 in HT1080 cells. Cells were grown to 70%confluence in DMEM containing 10% fetal bovine serum, and medium was changed to serum-free medium. Cells were stimulated with TGF-h1 (10 ng/mL) inthe presence or absence of MEK inhibitor, PD98059, mithramycin, or curcumin for 4 hours. Nuclear extracts from the cells were subjected to gel shift assaywith Sp1 (A) and AP-1 (B) consensus oligonucleotides as described in Materials and Methods. For the detection of Sp1 supershift band, nuclear extractsfrom TGF-h1-treated cells were incubated with anti-Sp1 antibody for 30 minutes on ice and analyzed by electrophoretic mobility shift assay. Negative control,32P-labeled Sp1 (A) or AP-1 (B) oligonucleotides without HeLa nuclear extract; Positive control, nuclear extract of HeLa cells incubated with 32P-labeled Sp1(A) and AP-1 (B) oligonucleotides; SS, supershift.

Kwak et al.


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Transfection and TransductionDN-MEK-1 plasmids (generous gift from Dr. Su-Jae Lee,

Korea Institute of Radiological and Medical Sciences, Seoul,

Korea) were transfected in HT1080 cells, using Effectene

transfection reagent (Qiagen, Valencia, CA), by following the

supplier’s instructions. For retrovirus transduction, cells were

infected with retrovirus harboring DN-p38 and DN-JNK-1

(generous gift from Dr. Su-Jae Lee) for 90 minutes. After 24

hours, transfected and transduced cells were washed twice

with serum-free medium and incubated with or without TGF-

h1 (10 ng/mL) in the same medium. The infection and

selection of the target cells by puromycin were done as

described previously (51). The siRNA against Sp1 and TIMP-

1 were purchased from Santa Cruz Biotechnology (Santa

Cruz, CA), and transfections of cells with siRNA molecules

were done using RNAiFect transfection reagent (Qiagen) as

described by the manufacturer.

ZymographyProduction of MMPs by HT1080 cells was analyzed by

gelatin zymography. HT1080 cells in subconfluent culture

condition (f70-80% confluent) were washed and replenished

with serum-free DMEM and incubated with or without

indicated concentrations of TGF-h1 in the presence or absence

of various kinase inhibitors for 18 hours. Serum-free condi-

tioned medium (20 AL) was mixed with SDS sample buffer

without heating or reduction and applied to 10% polyacryl-

amide gels copolymerized with 1 mg/mL gelatin. After

electrophoresis, gels were washed in 2.5% (v/v) Triton X-100

for 2 hours at room temperature to remove SDS, rinsed twice

with water, and then incubated in developing buffer [50 mmol/

L Tris-HCl (pH 7.5), 5 mmol/L CaCl2, 1 Amol/L ZnCl2, and 0.1

NaN3] for 18 hours at 37jC.Subsequently, gels were fixed and stained with 10% 2-

propanol and 10% acetic acid containing 0.5% Coomassie blue

R250. The protease activity was visualized as clear bands

within the stained gel.

Invasion and Migration AssaysMatrigel invasion assays were done using modified Boyden

chambers with polycarbonate Nucleopore membrane (Corning,

Corning, NY). Precoated filters (6.5 mm in diameter, 8-Am pore

size, Matrigel 100 Ag/cm2) were rehydrated with 100 ALmedium. Then, 2 � 105 cells in 200 AL serum-free DMEM

FIGURE 6. Effect of PD98059, mithramycin, and curcumin on the anti-invasion of HT1080 cells induced by TGF-h1. Cells were pretreated for 1 hour withvarious kinase inhibitors (A and D), mithramycin (B and E), or curcumin (C and F) in the presence or absence of TGF-h1 (10 ng/mL). Left, HT1080 cellsinvaded (A-C) and migrated (D-F) under the membrane after 12 hours; right, invasion and migration rates were determined by counting cells in fourmicroscopic fields per sample. T, TGF-h1; M, mithramycin; C, curcumin. Bars, SD. *, P < 0.05 versus control; **, P < 0.05 versus TGF-h1.



217



supplemented with 0.1% bovine serum albumin were seeded

into the upper part of each chamber, whereas the lower

compartments were filled with 1 mL of the same medium

mentioned above in the presence or absence of TGF-h1(10 ng/mL) with or without specific kinase inhibitors or

TIMP-1. Following incubation for 18 hours at 37jC, non-

invaded cells on the upper surface of the filter were wiped out

with a cotton swab, and the invaded cells on the lower surface

of the filter were fixed and stained with Diff-Quick kit

(Fisher Scientific, Pittsburgh, PA). Invasiveness was determined

by counting cells in five microscopic fields per well, and the

extent of invasion was expressed as an average number of cells

per microscopic field. Transwell migration assays were done

using the same procedure as for invasion assay, except coating

the underside of filters with type I collagen (100 Ag/cm2).

Western Blot AnalysisHT1080 cells were lysed in lysis buffer [20 mmol/L Tris-

HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L

EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate,

1 mmol/L h-glycerophosphate, 1 mmol/L Na3VO4, 1 Ag/mL

leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride]. After

a brief sonication, the lysates were clarified by centrifugation at

12,000 � g for 10 minutes at 4jC, and protein content in the

supernatant was measured by the Bradford method. An aliquot

(30-50 Ag protein/lane) of the total protein was separated by

10% or 12% SDS-PAGE and blotted to nitrocellulose transfer

membrane (0.2 Am; Amersham, Arlington Heights, IL). The

membrane was blocked with 5% nonfat skim milk in TBST

[20 mmol/L Tris-HCl (pH 7.6), 137 mmol/L NaCl, and 0.01%

Tween 20] for 1 hour at room temperature followed by

incubation with the primary antibodies. After extensive

washing with TBST, the membrane was reprobed with

horseradish peroxidase–linked anti-rabbit immunoglobulin, at

1:3,000 diluted in 5% nonfat skim milk in TBST, for 40

minutes at room temperature. Immunoblots were visualized by

enhanced chemiluminescence (Amersham) according to the

manufacturer’s protocol.

To measure the MMP-2, MMP-9, TIMP-1, and TIMP-2

proteins secreted, the conditioned medium from each sample

was also subjected to protein analysis. For this purpose, culture

medium in each tissue culture dish was collected and

concentrated 10-fold using a Centricon 10 microconcentrator

(Amicon, Beverly, MA). The concentrates were subjected to

SDS-PAGE analysis.

Real-time PCRReal-time PCR assays were done using QuantiTect SYBER

Green PCR kit (Qiagen) with SYBR Green I as the fluorescent

dye enabling real-time detection of PCR products according to

the manufacturer’s protocol. Gene-specific primers were used,

and the specificity was tested under normal PCR conditions.

Oligonucleotide primer sequences were as follows: human

TIMP-1 sense (5V-ACCAGACCACCTTATACCAGCG-3V) andantisense (5V-GGACTGGAAGCCCTTTTCAGAG-3V), human

MMP-2 sense (5V-GAGAACCAAAGTCTGAAGAG-3V) and

antisense (5V-GGAGTGAGAATGCTGATTAG-3V), and human

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense

(5V-TGAACGGGAAGCTCACTGG-3V) and antisense (5V-TCCACCACCCTGTTGCTGTA-3V). Cycling conditions were

95jC for 15 minutes followed by 35 cycles of 94jC for 15

seconds, 60jC for 30 seconds, and 72jC for 30 seconds. For

quantification, the target gene was normalized to the internal

standard GAPDH gene. Results of the real-time PCR data were

represented as Ct values, where Ct is defined as the threshold

cycle of PCR at which amplified product was first detected. To

compare the different RNA samples in an experiment, we used

the comparative Ct method (53) and compared the RNA

expression in samples with that of the control in each

experiment. The primers were constructed so that the dynamic

range of both the targets and the GAPDH reference were similar

over a wide range of dilutions (1:1-10,000). PCR was done in

triplicates for each sample. The results were expressed as mean

F SD for the relative expression levels compared with the

control, and minimum values of three independent experiments

were taken.

3-(4,5-Dimethylthiazol-2-yl)-2,5-DiphenyltetrazoliumBromide Assays

A tetrazolium-based assay was used to determine the

cytotoxicity. Briefly, 2,000 cells were seeded in 96-well plates

and cultured for 24 hours, and seven concentrations of TGF-h1or curcumin were added, respectively. Cell viability was

examined at 12 and 24 hours after treatment. Before testing,

10 AL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide labeling reagent (5 mg/mL 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide in PBS) was added and the

cells were incubated for 4 hours at 37jC. Then, 100 ALdissolving reagent (DMSO) was added and the plate was further

incubated for 15 minutes at 37jC to dissolve the formazan

crystals. The absorbance was measured at a wavelength of 570

nm on a Labsystem multiscan microplate reader (Merck

Eurolab, Dietikon, Switzerland). Results represented the

absorbance ratio between treated and untreated cells at

indicated time points. Results are expressed as mean F SD.

Evaluation of ApoptosisApoptosis was evaluated by staining the cells with Annexin

V-FITC (PharMingen, San Diego, CA) and propidium iodide

according to the manufacturer’s protocol. Cells were then

analyzed by FACScan flow cytometer (Becton Dickinson,

Franklin Lakes, NJ).

Electrophoretic Mobility Gel Shift AssayElectrophoretic mobility shift assay was done as described

elsewhere (54) with some modifications. Nuclear extracts were

prepared from HT1080 cells treated with or without TGF-h1(10 ng/mL) and specific kinase inhibitors. Synthetic oligonu-

cleotides consisting of consensus sequences of Sp1 (5V-ATTCG-ATCGGGGCGGGGCGAGC-3V) and AP-1 (5V-CGCTTGA-TGACTCAGCCGGAA-3V; Santa Cruz Biotechnology) were

used as probes after annealing sense and antisense fragments.

The double-stranded oligonucleotides were then labeled with

[g-32P]ATP. Nuclear extracts (10 Ag) were incubated with

5 � 104 counts/min labeled probes for 30 minutes at 22jC and

analyzed in 6% PAGE followed by autoradiography. For

Kwak et al.


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supershift analysis, nuclear extracts were preincubated with Sp1

antibody (2 or 4 Ag) on ice for 30 minutes before the addition of

labeled double-stranded oligonucleotides followed by incuba-

tion at room temperature for an additional 20 minutes.

Statistical AnalysisAll results are meanF SD or SE. Statistical significance was

determined using Student’s t test.

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2006;4:209-220. Mol Cancer Res Hee-Jin Kwak, Myung-Jin Park, Hyeyoung Cho, et al. Fibrosarcoma CellsExtracellular Signal-Regulated Kinase and Sp1 in HumanMetalloproteinase-1 Expression via Activation of

1 Induces Tissue Inhibitor ofβTransforming Growth Factor-

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