Cancer Investigation, 27:568–574, 2009ISSN: 0735-7907 print / 1532-4192 onlineCopyright c© Informa Healthcare USA, Inc.DOI: 10.1080/07357900802620810

ORIGINAL ARTICLEClinical Translational Therapeutics

The Effect of TGF-β2 on MMP-2 Production andActivity in Highly Metastatic Human Bladder

Carcinoma Cell Line 5637Ehsan Dehnavi,1,2 Zahra-Soheila Soheili,1 Shahram Samiei,3 Zahra Ataei,3 and Hajar Aryan4

Ministry of Science, Research and Technology, National Institute of Genetic Engineering and Biotechnology,1 Khatam University,2

Iranian Blood Transfusion Organization Research Center,3 and Payame-noor University of Tehran4

ABSTRACT

Transforming growth factor–beta (TGF-β) superfamily regulates matrix metalloproteinases(MMP), which intrinsically regulate various cell behaviors leading to metastasis. We investi-gated the effect of TGF-β2 on MMP-2 regulation in human bladder carcinoma cell line 5637.Zymography, ELISA, and real-time polymerase chain reaction revealed that TGF-β2 stimulatedMMP-2 production, but the transcription of its gene remained unchanged. Wortmannin could notinhibit MMP-2 secretion and activity and conversely the amount of the protein and its enzymaticactivity were increased. These data suggest that TGF-β2 increased MMP-2 at the posttranscrip-tional level and this upregulation was independent of phosphatidylinositol 3-kinase signalingpathway.

INTRODUCTION

TGF-β2 is a member of the multifunctional family oftransforming growth factor–beta. This family consists of a largenumber of structurally related secretory proteins. They act as alocal mediator (or hormone) to regulate a wide range of biolog-ical functions in animals (1). TGF-β has been reported to exertgrowth-inhibitory activity in normal epithelial cells whereas itinduces cell proliferation and invasive phenotypes in advancedcarcinomas (2, 3).

Invasion and metastasis are fundamental properties of ma-lignant cancer cells as well as bladder carcinoma cell line 5637.A number of proteolytic enzymes participate in these processes

Keywords: TGF-β2, MMP-2, Bladder CarcinomaCorrespondence to:Zahra-Soheila Soheili, PhDMinistry of Science Research and TechnologyNational Institute of Genetic Engineering and BiotechnologyP.O. Box 14965/161Pazhoohesh Boulevard17 Kilometers From Tehran-Karaj HighwayTehran, Iranemail: soheili@nigeb.ac.ir

to degrade environmental barriers such as extracellular matrix(ECM) and basement membrane. Among these enzymes, matrixmetalloproteinases (MMPs) play the major role. Furthermore,MMPs have an important role in normal physiology; they areinvolved in embryonic development, wound repair, bone remod-eling, and macrophage functions (4, 5).

Dysregulation of MMPs is involved in many diseases, suchas tumor metastasis. Metastasis is the major cause of deathamong cancer patients (6). The process of metastasis involves acascade of linked, sequential steps that involve multiple host–tumor interactions. Specifically, MMPs are involved in manysteps of tumor metastasis. These include tumor invasion, mi-gration, host immune escape, extravasations, angiogenesis, andtumor growth. Therefore, without MMPs, the tumor cells cannotperform successful metastasis (7–9).

Among the previously reported human MMPs, MMP-2(gelatinase A) and MMP-9(gelatinase B) are abundantly ex-pressed in various malignant tumors (10). They can degrade thecollagen type †V, which is the major structural component ofthe basement membrane and ECM (11).

The activities of MMPs are tightly regulated at the genetranscription levels, zymogen activation by proteolysis and in-hibition of active forms by endogenous inhibitors, tissue in-hibitor of metalloproteinase. MMP transcripts are generally ex-pressed at low levels, but these levels rise rapidly when tissues

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undergo cancer. Induction of their gene expression is mediatedby different signaling pathways depending on the type oftissue (12).

The phosphatidyl inositol 3–kinase pathway regulates a num-ber of cellular processes including cell cycle progression, glu-cose metabolism, angiogenesis, cell motility, and apoptosis (13).Activation of this pathway in response to TGF-β1has beendemonstrated in several normal cells like epithelial cells andcancer cells like HT-1080 (14, 15). There is limited informa-tion, however, on the role and regulation of this pathway byTGF-β2 in 5637 cells. Since 5637 cells constitutively secretplentiful amounts of MMP-2, we aimed to determine the role ofTGF-β2 on MMP-2 regulation and the role of PI3K as a probabledownstream signaling pathway for TGF-β2 effects.

MATERIALS AND METHODS

Cell culture

Recombinant human TGF-β2 was purchased from Chemicon(Brussel, Belgium); wortmannin was from Calbiochem (Brus-sel, Belgium). The 5637 cells (American Type Culture Collec-tion, Tehran, Iran) were grown in RPMI 1640 (Gibco, Brussel,Belgium) supplemented with streptomycin (Jabberebn Hayyan,Tehran, Iran), penicillin (Sigma, Brussel, Belgium), and 10%heat-inactivated fetal bovine serum (Sigma) in a humidified in-cubator under 5% CO2 at 37◦C.

Cells in subconfluent culture were washed with PBS (Merck),fed with serum-free RPMI 1640 and incubated with or without5 ng/mL of TGF-β2 in the presence or absence of wortmannin(100 nM). At the end of 24 hr, conditioned medium was collectedand centrifuged and the supernatants were stored at −80◦C untiluse and the adherent cells were lysed in Tripure RNA-isolatingreagent (Roche, Tehran, Iran). Experiments were performed atleast three times using separate sets of cultures.

Zymography

Enzymatic activity of MMP-2 in culture conditioned mediawas analyzed by gelatin zymography. The media supernatantswere treated with SDS-PAGE sample buffer without boiling orreduction and were fractionated on 8% polyacrylamide gel con-taining gelatin (2 mg/mL) by electrophoresis at 10◦C. HT-1080cell culture conditioned media was used as marker for MMPactivity along with protein size marker (Invitrogen, Brussel,Belgium). After electrophoresis, gels were washed in 2.5%Triton X-100 (Sigma) for 60 min at room temperature to re-move SDS. Then they were incubated in zymography buffer[50 mL Tris–HCl (pH 7.2 0), 0.15 M NaCl, 5 mM CaCl2, and0.05% NaN3] (Merck, Tehran, Iran) for 18 hr at 37◦C. Subse-quently, gels were fixed and stained with 10% acetic acid and25% ethanol (Merck) containing 0.5% Coomassie Brilliant BlueR250 (Merck). Gelatinolytic activities appeared as clear bandsof digested gelatin against a dark-blue background of stainedgelatin.

ELISA

Collected supernatants were diluted to appropriate concen-tration. The amount of MMP-2 was determined using ELISAKit (Calbiochem) according to the instruction provided by themanufacturer.

Real-time polymerase chain reaction (PCR)

Real-time PCR was performed in 25 µL of a reaction consist-ing of 12.5 µL Superscript III Platinum

©R SYBR Green one-stepRT supermix (Invitrogen), 1 µM concentration of each primer(MMP2, MMP9, and 18S rRNA) and 4µL RNA as template. ThePCR thermal profile consists of an initial incubation of 30 min at50◦C and 10 min at 95◦C followed by 30 cycles of 95◦C for 30 s,54◦C for 30 s, 72◦C for 30 s, and 80◦C for 10 s (data acquisitionpoint). Amplification, detection, and data analysis were per-formed with MYiQ real-time detection system (Bio-rad, Milan,Italy). Each sample was run in triplicate. Relative quantificationhas been estimated according to the 2−��CT method.

RNA isolation

Total RNA from near confluent cells was extracted withTripure

©R Isolation Reagent (Roche). All samples were extractedaccording to manufacturer instruction resulting purified intactRNA which was stored at −80◦C.

Primer for MMP2 and 18SrRNA

Using criteria required for designing syber green assay,primer were selected for human MMP-2 (F: 5′-TGAAGAGCGTGAAGTTTGGAAG-3′, R: 5′-GAAGGCAGTGGAGAGGAAGG-3′) and 18SrRNA (F: 5′-CGGCGACGACCCATTCGAAC-3′, R: 5′-GAATCGAACCCTGATTCCCCGTC-3′). Theprimers for MMP-2 were designed using beacon designer soft-ware 4.01 (www.primerbiosoft.com) and mfold zuker program(www.bioinfo.rpi.edu/application/mfold) to ensure the absenceof secondary structure. Additional criteria for a good SYBRGreen real-time assay included a relatively short amplicon(<200 bp) for both housekeeping gene (99-bp) and MMP-2gene (73-bp) specific primers. A blast search was performed tocheck the specificity of DNA sequences of primers. To amplify18SrRNA, the offered primers by Bio-rad were used.

RESULTS

TGF-β2-induced MMP-2 productionand activity

The protein levels and activity of MMP-2 in serum-free con-ditioned medium were determined by ELISA and gelatin zy-mography. Since 5637 cells produce large amount of MMP-2,supernatants were diluted before performing tests. By zymogra-phy, it was shown that TGF-β2 at 5 ng/mL markedly stimulatedproteolytic activity of MMP-2 (Fig. 1(A)). ELISA results re-vealed that TGF-β2 increased the amount of MMP-2 as well asits protein activity (Fig. 1(B)).

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Figure 1. TGF-β2 upregulated MMP-2 production and activity. (A)induction of MMP-2 gelatinolytic activity in human bladder carci-noma cell line 5637. Cells were starved in serum-free medium for24 hr and then treated with 5 ng/mL human recombinant TGF-β2. Cultures were harvested 24 hr later and conditioned mediumsubjected to zymography. Supernatant from HT-1080 was used aspositive control of MMP-2. (B) ELISA test was performed at thesame conditions for three independent experiments.

PI3K inhibition increased MMP-2 proteinproduction and enzymatic activity

To elucidate the signaling mechanism that is possibly in-volved in the upregulation of MMP-2, we examined if the TGF-β2 executes its role through PI3K pathway. Cells were treatedwith 100 nM wortmannin in the absence or presence of 5 ng/mLTGF-β2. Zymography and ELISA analyses showed that treat-ment of the cultures with wortmannin clearly stimulated both theproduction and activity of MMP-2 (Fig. 2(A)). Data showed thatusing TGF-β2 with wortmannin synergistically induced MMP-2gelatinolytic activity (Fig. 2(B)).

MMP-2 induction by TGF-β2 was not at thegene expression level

We examined if the amount of MMP-2 was increased atthe transcriptional level. Samples of total RNA were collectedfrom control and treated cultures. Real-time PCR was per-formed on the corresponding samples. The 2−��CT method(2−(Ct,Target/treated−Ct,18S/treated)−(Ct,Target/control−Ct,18S/control)) wasused to calculate relative changes in gene expression. Usingthe obtained Ct values for relative quantitation, it was shown

that the TGF-β2 had no effect on MMP-2 transcription intreated samples. Real-time PCR analysis showed that wort-mannin had no effect on MMP-2 at the gene expression level(Fig. 3(A)). The amplification product of MMP-2 amplicon wassubjected to electrophoresis to confirm specific amplification(Fig. 3(B)).

DISCUSSION

In mammals there are three isoforms of TGF-β (1, 2, and3) that are functionally similar and interchangeable under manyconditions. Although they may possess unique functions (16–18) all three isoforms are expressed by fibroblasts and epithelialcells (19, 20). Control of ECM production as one of the essen-tial functions of the TGF-β family is accomplished in variousways, for example, the regulation of MMPs. Previous studieshave shown upregulation of MMP-2 (21, 22) and MMP-9 (23,24) by TGF-β1 in a great number of cell lines. Increased un-derstanding of the mechanisms through which growth factorsimpress MMP activity can provide insights into a wide varietyof both physiological and diseased states.

We used TGF-β2 to investigate its effects on regulation ofMMP-2, the most important collagenase which expresses byhuman bladder carcinoma cell line 5637. The role of TGF-β2 inregulating MMP-2 has not been studied in bladder cancer cellsyet. But it has been found that TGF-β1 stimulated productionand activity of MMP-9 in many cell types. TGF-β1upregulatedMMP-9 in keratinocytes (25), human corneal epithelial cell(26), human fibroblast cells (27), lens epithelial cells (28), hu-man HT-1080 colon carcinoma cells, MCF10A breast epithelialcells (29), and human prostate epithelial cells (30). Furthermore,Wolfram Eichler and his colleagues have reported that TGF-β2

stimulated MMP-2 in human RPE cells (31).In the current study, we demonstrated that TGF-β2 increased

MMP-2 protein production and activity in 5637 cell culturemedia but it did not have any substantial effects on its geneexpression by the cells.

MMP-2 has been reported to be induced by TGF-β1 throughincreasing both the transcription rate and mRNA stability inhuman gingival fibroblasts (32), human fibrosarcomas (33), andhuman prostate cancer cell lines (30). In addition, TGF-β1 wasfound to increase the half-life of the MMP-2 mRNA from 46to 150 hr in human fibroblasts (27). Since TGF-β2and TGF-β1

are functionally similar under many conditions (34), it seemsthat TGF-β2upregulates MMP-2 activity and production in 5637cells through posttranscriptional mechanisms.

To investigate the assumed role of the PI3K signaling path-way in MMP-2 production by 5637 cells, wortmannin was usedas a universal inhibitor of PI3K isoforms. Previous studies haveshowed that wortmannin decreased MMP-2 and MMP-9 pro-duction by LNCAP, PC-3, and DU-145 prostate cancer celllines (35), Lewis lung carcinoma subline H-59 cells (36), andovarian carcinoma cell line OVCA429 (37). Furthermore, treat-ment of extravillous trophoblast cell line (HTR8/Svneo) withLY294002 (another inhibitor of PI3K) decreased MMP-9 pro-duction and activity in conditioned media (38). Moreover, it

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Figure 2. Wortmannin increased MMP-2 protein activity and production in 5637 cells. (A) the effects of 5 ng/mL TGF-β2 and 100 nM wortmanninon MMP-2 gelatinolytic activity. Conditioned medium of HT-1080 cells used as MMP-2 marker. (B) MMP-2 ELISA for the same culture treatedwith 100 nM wortmannin or with 5 ng/mL TGF-β2 and with both 100 nM wortmannin and 5 ng/mL TGF-β2.

has been reported that the PI3K pathway promotes activation ofMMP-2 and invasion in mouse mammary epithelial cells (39).In this study we showed that wortmannin caused a substantialincrease in both the protein amount and enzymatic activity forMMP-2 through the 5637 cell line. These data are in accord withregulation of collagenase gene by cytokines and growth factorsthrough complex signaling pathways. It seems that mechanismsinvolved in gelatinase expression by the bladder cells are differ-ent, at least in part, from known pathways mediating gelatinaseproduction by other cells. While in most of the cell lines, PI3Kpathway is one of the principal ways involved in MMP up-regulation, our data indicate that probably signals transduced

through PI3K inhibit MMP-2 production by 5637 cells. JordyEsparza and his colleagues in 1999 had shown that wortman-nin strongly increased fibronectin-induced MMP-2 and MMP-9production by T lymphocytes (40). The mechanisms involvedin this pathway were not known yet. Our finding showed thatPI3K has an inhibitory role in MMP-2 production and activ-ity by 5637 bladder cells. In other words, some growth factorsand cytokines would impress their inhibitory roles on mediat-ing MMP production (activity) via PI3K signaling pathways in5637 bladder cells. Furthermore, these data suggested that us-ing PI3K inhibitors and TGF-βas antiinvasive therapy shouldbe done more carefully.

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Figure 3. (A) Relative quantitation analysis of MMP-2 mRNA expression for 5637 cells treated with 100 nM wortmanin, 5 ng/mL TGF-β2, and5 ng/mL TGF-β2+ 100 nM wortmannin. (B and C) PCR products generated from the MMP-2 and 18Sr RNA-specific messages in total cellularRNA.

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ACKNOWLEDGMENTS

This research was supported by the National Institute ofGenetic Engineering and Biotechnology through grant no. 218.We would like to acknowledge and extend our gratitude to AliTalebian, Ahmad Gharabaghian, Mahnaz Kavari, Maryam Ab-dollahi, Semiramis Tootian, and Farzaneh Tavassoli for theircontribution to this study.

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