Matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gliomas

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✓ The gene expression of five matrix metalloproteinases (MMPs) and two tissue inhibitors of metalloproteinases (TIMPs) was studied in human gliomas in vivo and in vitro to evaluate their roles in glioma invasion. Simultaneous expression of one to four MMP genes and two TIMP genes was found in 17 surgical glioma specimens, and one MMP (gelatinase A) gene and two TIMP genes were simultaneously expressed in tissue of three brains. The concomitant overexpression of gelatinase A, gelatinase B, and occasional matrilysin genes was associated with the malignancy of gliomas and accompanied by overexpression of the TIMP-1 gene. In five human glioma cell lines, gelatinase A, TIMP-1, and TIMP-2 genes were constitutively expressed in all cell lines: the matrilysin gene in three cell lines; the stromelysin gene in two cell lines; and the interstitial collagenase gene in one cell line. There was a clear difference in the expression of gelatinase B and stromelysin genes between surgical glioma specimens and glioma cell lines: the gelatinase B gene was not expressed constitutively in vitro but was overexpressed in vivo, whereas the stromelysin gene was not expressed in vivo but was expressed in some cell lines. To find the cause of that difference in vivo and in vitro, the transcriptional regulations of MMP and TIMP genes by tumor promoter, growth factors, or cytokines were studied in vitro. Interstitial collagenase, gelatinase B, stromelysin, and TIMP-1 genes were upregulated in many cell lines by phorbol-12-myristate-13-acetate (PMA) and in some cell lines by epidermal growth factor, tumor necrosis factor-α, or interleukin-1β. Transforming growth factor-β1 (TGFβ1) upregulated gelatinase A and matrilysin genes in some cell lines, and there were no clear responses from any MMP and TIMP genes to interleukin-6. Thus, the transcriptional modulation of MMP genes by these growth factors and cytokines seemed insufficient to explain the difference in gelatinase B and stromelysin gene expressionsin vivo and in vitro and was suggestive of the genetic alteration of glioma cells in vitro, the heterogeneous cell population in glioma tissues, or both. Furthermore, the in vitro invasion of glioma cells through Matrigel in response to PMA, TGFβ1, or TIMP-1 was assessed by chemoinvasion assay. In most cell lines, invasion was significantly stimulated by PMA or TGFβ1 but suppressed by TIMP-1. These in vivo and in vitro studies are strongly suggestive of the important roles of some MMPs, especially gelatinase A, gelatinase B, and matrilysin, in the glioma invasion.

Abstract

✓ The gene expression of five matrix metalloproteinases (MMPs) and two tissue inhibitors of metalloproteinases (TIMPs) was studied in human gliomas in vivo and in vitro to evaluate their roles in glioma invasion. Simultaneous expression of one to four MMP genes and two TIMP genes was found in 17 surgical glioma specimens, and one MMP (gelatinase A) gene and two TIMP genes were simultaneously expressed in tissue of three brains. The concomitant overexpression of gelatinase A, gelatinase B, and occasional matrilysin genes was associated with the malignancy of gliomas and accompanied by overexpression of the TIMP-1 gene. In five human glioma cell lines, gelatinase A, TIMP-1, and TIMP-2 genes were constitutively expressed in all cell lines: the matrilysin gene in three cell lines; the stromelysin gene in two cell lines; and the interstitial collagenase gene in one cell line. There was a clear difference in the expression of gelatinase B and stromelysin genes between surgical glioma specimens and glioma cell lines: the gelatinase B gene was not expressed constitutively in vitro but was overexpressed in vivo, whereas the stromelysin gene was not expressed in vivo but was expressed in some cell lines. To find the cause of that difference in vivo and in vitro, the transcriptional regulations of MMP and TIMP genes by tumor promoter, growth factors, or cytokines were studied in vitro. Interstitial collagenase, gelatinase B, stromelysin, and TIMP-1 genes were upregulated in many cell lines by phorbol-12-myristate-13-acetate (PMA) and in some cell lines by epidermal growth factor, tumor necrosis factor-α, or interleukin-1β. Transforming growth factor-β1 (TGFβ1) upregulated gelatinase A and matrilysin genes in some cell lines, and there were no clear responses from any MMP and TIMP genes to interleukin-6. Thus, the transcriptional modulation of MMP genes by these growth factors and cytokines seemed insufficient to explain the difference in gelatinase B and stromelysin gene expressionsin vivo and in vitro and was suggestive of the genetic alteration of glioma cells in vitro, the heterogeneous cell population in glioma tissues, or both. Furthermore, the in vitro invasion of glioma cells through Matrigel in response to PMA, TGFβ1, or TIMP-1 was assessed by chemoinvasion assay. In most cell lines, invasion was significantly stimulated by PMA or TGFβ1 but suppressed by TIMP-1. These in vivo and in vitro studies are strongly suggestive of the important roles of some MMPs, especially gelatinase A, gelatinase B, and matrilysin, in the glioma invasion.

Local invasive growth is one of the key features of gliomas, and leptomeningeal dissemination of malignant gliomas occurs in up to 20% of cases.39 Partial degradation of the extracellular matrix around tumor cells is an essential step in the process of tumor invasion and metastasis. Many recent studies have demonstrated that a family of structurally related metalloproteinases, namely “matrix metalloproteinases (MMPs),” plays an important role in matrix degradation by tumor cells. The MMPs are secreted as latent proenzymes that require the removal of propeptide domain to attain activity.54,60 At least 10 MMP members have been identified, and they can be divided into three main classes based on substrate specificity: interstitial collagenase, which degrades interstitial fibrillar collagen;59 gelatinase A and gelatinase B, which are specific for degradation of type IV collagen;28 and stromelysin, whose substrates include proteoglycans, glycoproteins such as fibronectin and laminin, and type IV and V collagens.5 Stromelysin is also an efficient activator of procollagenase, progelatinase B, and promatrilysin.16,32,35,38,55 Matrilysin is a subclass of stromelysin and has the broad stromelysin-like substrate specificity.31 The activities of MMPs are strictly regulated by natural MMP inhibitors called “tissue inhibitors of metalloproteinases (TIMPs).” There are two major members of this group: TIMP-161 and TIMP-2,53 both of which regulate the degradation of the extracellular matrix by selective inhibition of either autoactivation or substrate catalysis with differential specificity for different MMPs.

In the central nervous system, the secretion of interstitial collagenase and 65-kD type IV collagenase (gelatinase A) was observed in cultured fetal astrocytes and in glioma cell lines.4 The production of matrilysin and stromelysin is also reported in human glioma cell lines,37 and the production of gelatinase B is elevated in human malignant gliomas.42 In addition to TIMPs, human glioma cell lines secrete smaller inhibitors of metalloproteinases named IMP-1, IMP-2, and IMP-3,4 the first two of which are also detected in the conditioned medium of rabbit brain capillary endothelial cells.18 The inhibitor IMP-2 has been considered to be identical to TIMP-2, but the other two small inhibitors have not yet been characterized. Two myelin-derived proteins with molecular weights of 35 kD and 250 kD are also found to contain the MMP blocking sequence and are thought to be responsible for inhibiting the spread of TB16 melanoma cells and 3T3 fibroblasts in the central nervous system.9,40 In addition, the expressions of MMP and TIMP genes in tumors are known to be regulated not only by tumor cells themselves but also by tumor cell—host cell interaction.10,17,34,36 The plasminogen activator43,46 and a variety of biologically active agents such as growth factors, cytokines, and extracellular matrix components,2,7,8,24,33,58 have been shown to regulate MMP and TIMP gene expression.

A systematic study of MMP and TIMP gene expression has not been reported in human gliomas. The present study was designed to examine the expression of five MMP (interstitial collagenase, gelatinase A, gelatinase B, stromelysin, and matrilysin) genes as well as two TIMP (TIMP-1 and TIMP-2) genes in human gliomas to evaluate the relationship between the expression levels of MMP or TIMP genes and the malignancy of gliomas. In addition, the transcriptional modulation of MMP and TIMP genes has been examined in human glioma cell line by means of treatment with phorbol-12-myristate-13-acetate (PMA), transforming growth factor-β1 (TGFβ1), epidermal growth factor (EGF), tumor necrosis factor-α (TNFα), interleukin (IL)-1β, or IL-6 (the latter five of which are detected in malignant gliomas in vivo6,12,26,47–49,57) to study their roles in glioma invasion.

Materials and Methods

Clinical Source of Tissues

Surgical specimens of brain tissues and gliomas were obtained at surgery, particularly from the edge of the contrast-enhancing border in gliomas, and immediately snap-frozen in liquid nitrogen; they were then stored at −70°C until analysis. Age and gender of the patients as well as location and histology of their gliomas are shown in Table 1. All recurrent gliomas had received irradiation and chemotherapy prior to the sampling. The brain specimens were obtained from the temporal lobes of three patients with partial complex seizure.

TABLE 1

Clinical features of three brain and 17 glioma samples*

Case No.Age (yrs), SexLocationHistology
130, Mrt temporal lobeB
221, Frt temporal lobeB
329, Mrt temporal lobeB
447, Flt frontal lobeA
544, Mlt frontal lobeA
637, Flt temporal lobeAA
741, Mrt frontal lobeAA
841, Flt temporal lobeAA
944, Frt frontal lobeAA, R
1030, Mlt parietal lobeG
1173, Mrt temporal lobeG
1266, Mrt temporal lobeG
1367, Mrt temporal lobeG
1458, Mlt parietal lobeG
1575, Mrt temporal lobeG
1661, Mrt frontal lobeG
1769, Mrt temporal lobeG
1862, Flt temporal lobeG
1944, Mrt temporal lobeG, R
2062, Mrt temporal lobeG, R

Abbreviations: B = brain tissue; A = astrocytoma; AA = anaplastic astrocytoma; G = glioblastoma; R = recurrent.

Glioma Cell Lines

Five human glioma cell lines (GB-1, U-373MG, A-172, T98G, and U-87MG) were used: GB-1 is a human glioblastoma-derived cell line established in our laboratory30 and the others were obtained from a human tumor cell bank (See Sources of Supplies and Equipment). All glioma cell lines were maintained in tissue culture with Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal calf serum and antibiotic agents (penicillin G, 100 U/ml; and streptomycin, 100 µg/ml) in a humidified atmosphere of 5% carbon dioxide and 95% air at 37°C.

Treatment of Glioma Cell Lines

After the cultured cells reached approximately 80% confluence, the medium was replaced by chemically defined serum-free MCDB 105. The cells were incubated in MCDB 105 supplemented with 0.2% bovine serum albumin (BSA) for 24 hours at 37°C to prevent protein deprivation of the cells under the serum-free condition, and then treated with 10 ng/ml of PMA or 5 ng/ml of TGFβ1 for 24 hours for analyses of transcriptional regulation of MMP and TIMP genes and chemoinvasiveness of gliomas. In addition, the cells were treated with 10 ng/ml of TNFα for 24 hours, 20 ng/ml of EGF for 16 hours, 2.5 ng/ml of IL-1β for 10 hours, or 50 ng/ml of IL-6 for 4 or 24 hours, for study of transcriptional regulation of MMP and TIMP genes. The treatment with each agent was performed three times for analysis of transcriptional regulation of MMP and TIMP genes or chemoinvasiveness.

Northern Blot Analysis

The surgical specimens were pulverized and the glioma cell lines were washed with phosphate-buffered saline. Thereafter, both samples were homogenized in 4 M guanidinium isothiocyanate buffer. The homogenates were layered on CsCl cushions and centrifuged at 150,000 G for 18 hours. The pellets were suspended in Tris-HCl/EDTA buffer (10 mM Tris-HCl buffer, pH 7.5/1 mM ethylenediaminetetraacetic acid), extracted with phenol and chloroform, then precipitated with ethanol twice, and dissolved in Tris-HCl/EDTA buffer. The total RNA sample (15 µg) was electrophoresed on 1% agarose gel containing 0.66 M formaldehyde, and transferred onto a Hybond-N membrane after three rinses of the gel in 10 × standard saline citrate buffer (SSC) at pH 7.0. The membrane was baked in a vacuum oven for 2 hours at 80°C. Complementary DNA probes of interstitial collagenase, gelatinase A, gelatinase B, stromelysin, matrilysin, TIMP-1, or TIMP-2 were labeled by random priming with [32P]deoxycytidine triphosphate. The baked membrane was hybridized with the 32P-labeled cDNA probe in 50% formamide, 5 × SSC, 5 × Denhardt's solution, 0.5% sodium dodecylsulfate (SDS), 50 µg/ml of denatured salmon sperm DNA and 10% dextran sulfate, for 16 hours at 42°C. Thereafter, the membrane was washed in 2 × SSC and 0.5% SDS for 10 minutes, 2 ′ SSC and 0.1% SDS for 30 minutes at room temperature, and literature, 0.2 × SSC and 0.1% SDS for 30 minutes at 60°C, and exposed to hyperfilm-MP with intensifying screen for 24 to 72 hours at −80°C. Transcript levels of MMP and TIMP genes were quantitated with a radioanalytic imaging system. The loading of equal amounts of total cellular RNA was confirmed by staining the gels with ethidium bromide and examining the ribosomal band under ultraviolet light. The amounts of agents used for the analysis of transcriptional regulation of MMP and TIMP genes were determined on the basis of related7,11,24,29,33 and the optimum times of treatment were determined by examination of MMP and TIMP gene expression on Northern blots at regular intervals.

Chemoinvasion Assay

The chemoinvasion assay was performed in a chemotaxis chamber with polyvinylpyrrolidone-free polycarbonate porous filters (pore size 8 µm, diameter 8 mm) according to the modified method of Albini and colleagues.1 The polycarbonate filters were coated with a uniform layer of 100 µg protein/cm2 of Engelbreth-Holm-Swarm tumor—derived basement membrane, Matrigel. Glioma cells grown to approximately 80% confluence were treated with or without PMA or TGFβ1 for 24 hours and added to the chemotaxis chamber, which had been placed in a 24-well culture plate containing fibroblast-conditioned medium (FCM) and incubated in a CO2 incubator for 24 hours at 37°C with or without 10 ng/ml of PMA or 5 ng/ml of TGFβ1. The effect of TIMP-1 was examined by adding that agent (final concentrations, 3 or 10 µg/ml) to the chamber for 24 hours at 37°C. Fibroblast-conditioned medium was obtained from confluent BALB/c3T3 fibroblasts incubated in serum-free MCDB 105 containing 0.2% BSA for 48 hours and used as a chemoattractant. After the incubation, the nonmigratory cells on the upper surface of the filter were mechanically removed. The migratory cells on the lower surface were fixed, stained with Giemsa, and counted under a microscope. Five to ten random fields per filter were examined using × 200 magnification. The cells used for the chemoinvasion assay were counted with a Coulter counter, and their viability was confirmed by using the trypan blue dye exclusion method. Each assay was performed in triplicate and repeated on at least two separate occasions.

Statistical Analysis

Statistical significance of the radioactivities of Northern blots of MMP and TIMP genes in surgical specimens of human brain and glioma was assessed by means of the Mann—Whitney U test. The significance of the chemoinvasiveness of five human glioma cell lines was estimated with the Student t-test.

Sources of Supplies and Equipment

Human glioma cell lines (U-373MG, A-172, T98G, and U-87MG) were obtained from American Type Culture Collection, Rockville, Maryland. Complementary DNA probes of interstitial collagenase, stromelysin, and TIMP-1 were kindly supplied by Dr. N. I. Hutchinson, Merck Research Laboratory, Rahway, New Jersey; those of gelatinase A and gelatinase B by Drs. B. L. Marmer and G. I. Goldberg, Washington University School of Medicine, St. Louis, Missouri; that of matrilysin by Dr. R. Breathnach, INSERUM, Nantes, France; and that of TIMP-2 by Dr. W. G. Stetler-Stevenson, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

Transforming growth factor-β1 and TNFα were supplied by Wako Pure Chemical Industries, Osaka, Japan; EGF by Higeta Shoyu Co., Ltd., Choshi, Chiba, Japan; IL-6 by R&D Systems, Inc., Minneapolis, Minnesota; and IL-1β and Matrigel were supplied by Collaborative Biochemical Products, Bedford, Massachusetts. MCDB 105 was obtained from Sigma Chemical Company, St. Louis, Missouri. Hybond-N membrane and [32P]deoxycytidine triphosphate were supplied by Amersham International plc., Buckinghamshire, England.

The radioanalytic imaging system was manufactured by AMBIS Systems, Inc., San Diego, California; the chemotaxis chamber was supplied by KURABO, Osaka, Japan.

Results
Expression of MMP and TIMP Genes in Gliomas In Vivo

Representative Northern blots of MMP and TIMP genes in surgical specimens of tissue from three brains and 17 gliomas are shown in Fig. 1A and their mean radioactivities in brain tissues and gliomas (excluding recurrent gliomas) in Fig. 2. The transcripts were revealed as 2.5 kb in interstitial collagenase, 3.1 kb in gelatinase A, 2.8 kb in gelatinase B, 2.0 kb in stromelysin, 1.2 kb in matrilysin, and 0.9 kb in TIMP-1. The TIMP-2 transcripts were detected as a major 3.5 kb52,56 with additional minor 2.3, 1.5, and 1.0 kb in brain tissues and gliomas. Only gelatinase A gene was slightly-to-moderately expressed in the brain tissue; expression of the other MMP genes was not found. The level of major 3.5-kb TIMP-2 transcript was rather high and the TIMP-1 gene was slightly expressed in the brain tissue. Surgical glioma specimens generally showed simultaneous expression of one to four MMP genes and two TIMP genes, but no identical patterns. The interstitial collagenase transcript was barely detectable in only two of the nine primary glioblastomas and both of the two recurrent glioblastomas. The gelatinase A transcript was detected in all gliomas, and the mean expression level of the gelatinase A gene was approximately threefold higher in both astrocytomas and anaplastic astrocytomas and approximately sixfold higher in glioblastomas than in the brain tissues. The gelatinase B gene was scarcely expressed in the astrocytomas and in two of the four anaplastic astrocytomas but was markedly expressed together with the TIMP-1 gene in glioblastomas. Characteristically, the stromelysin transcript was not detected in any gliomas in vivo. The matrilysin transcript was detected in only five of nine primary and in both of the two recurrent glioblastomas. In addition, the minor 1.0-kb TIMP-2 transcript was significantly elevated in anaplastic astrocytomas and glioblastomas.

Fig. 1.
Fig. 1.

Representative Northern blot analysis. Total cellular RNA was isolated from surgical specimens of three human brain tissues and 17 human gliomas (A) as well as from five human glioma cell lines (GB-1, U-373MG, A-172, T98G, and U-87MG) (B) and hybridized with 32P-labeled complementary DNA probes of interstitial collagenase (MMP-1), gelatinase A (MMP-2), gelatinase B (MMP-9), stromelysin (MMP-3), matrilysin (MMP-7), tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2. Representative ethidium bromide-stained gels (ribosomal (r)RNA) are shown to allow comparison of the total amount of RNA per sample. Ribosomal RNA is used as the size standard. Arrowheads on TIMP-2 Northern blots show 3.5-, 2.3-, 1.5-, and 1.0-kb transcripts. A: Lanes 1 to 3 = brain tissues; Lanes 4 and 5 = primary astrocytomas; Lanes 6 to 8 = primary anaplastic astrocytomas; Lane 9 = recurrent anaplastic astrocytoma; Lanes 10 to 18 = primary glioblastomas; and Lanes 19 and 20 = recurrent glioblastomas. B: Lane 1 = GB-1; Lane 2 = U-373MG; Lane 3 = A-172; Lane 4 = T98G; and Lane 5 = U-87MG. MMP = matrix metalloproteinase.

Fig. 2.
Fig. 2.

Bar graph displaying the mean radioactivities of Northern blots of interstitial collagenase (MMP-1), gelatinase A (MMP-2), gelatinase B (MMP-9), stromelysin (MMP-3), matrilysin (MMP-7), tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 in three human brain tissues and 14 nonrecurrent human gliomas. Each bar shows mean ± standard deviation. MMP = matrix metalloproteinase. In each MMP and TIMP section, 1 = brain tissue; 2 = astrocytoma; 3 = anaplastic astrocytoma; 4 = glioblastoma. *p < 0.05 between brain tissue and anaplastic astrocytoma or glioblastoma; p < 0.05 between glioblastoma and astrocytoma or anaplastic astrocytoma.

Responses of MMP and TIMP Gene Expression to Agents in Gliomas In Vitro

To understand the regulatory mechanism of expression of MMP and TIMP genes in gliomas, their expression was analyzed in five glioma cell lines as shown representatively in Fig. 1B. The constitutive expression of the gelatinase B gene was not detected in any cell lines, although the gelatinase A gene was constitutively expressed in all cell lines, the matrilysin gene in three cell lines, the stromelysin gene in two cell lines, and the interstitial collagenase gene in one cell line. The TIMP-1 and TIMP-2 genes were constitutively expressed in all cell lines, and the TIMP-2 transcript in vitro was detected as major 3.5 kb and minor 1.0 kb.

Responses of MMP and TIMP gene expressions to PMA, TNFα, EGF, IL-1β, IL-6, or TGFβ1 in five human glioma cell lines are shown in Fig. 3A to G, and the mean radioactivities of responding Northern blots are divided into five grades as shown in Table 2. The induction of more than twofold changes in radioactivity was defined as clear responses (“+” or “−” in Table 2). Characteristically, there was no clear upresponse of the gelatinase B gene or downregulation of MMP and TIMP genes, particularly the stromelysin gene, in any cell lines to agents other than PMA. Treatment with PMA often upregulated the expression of many MMP genes and the TIMP-1 gene in all or some cell lines, except U-87MG in which gelatinase A and TIMP-2 genes were downregulated. Treatment with TNFα, EGF, or IL-1β induced the upregulation of MMP genes other than the gelatinase B gene only in one to three cell lines, and the upregulation of the TIMP-1 gene in T98G and the TIMP-2 gene in GB-1 was shown after treatment with EGF. No clear responses of MMP and TIMP genes were shown in any cell lines after treatment with IL-6. Treatment with TGFβ1 upregulated the gelatinase A gene in GB-1 and U-373MG and the matrilysin gene in T98G and U-87MG.

Fig. 3.
Fig. 3.

Representative Northern blot responses of MMP and TIMP genes to agents. Total cellular RNA was isolated from five human glioma cell lines (GB-1, U-373MG, A-172, T98G, and U-87MG) and hybridized with 32P-labeled complementary DNA probe of interstitial collagenase (MMP-1) (A), gelatinase A (MMP-2) (B), gelatinase B (MMP-9) (C), stromelysin (MMP-3) (D), matrilysin (MMP-7) (E), tissue inhibitor of metalloproteinases (TIMP)-1 (F), or TIMP-2 (G). Northern blot analyses after treatment without and with phorbol-12-myristate-13-acetate (PMA) or transforming growth factor (TGF)-β1 were conducted together, whereas analyses after treatment without and with tumor necrosis factor (TNF)-α, epidermal growth factor (EGF), interleukin (IL)-1β or IL-6 were done separately from each other. Ribosomal RNA was used as the size standard. The upper thick bands (arrows) in B in Lanes TNFα, EGF, IL-1β, and IL-6 in the GB-1 and U-373MG cells show 28S ribosomal RNA. Representative ethidium bromide—stained gels (H) are shown to allow comparison of the total amount of RNA per sample. MMP = matrix metalloproteinase; Lane PMA • TGFβ1 (−) = no treatment with PMA or TGFβ1 for 24 hours; Lane PMA(+) = treatment with 10 ng/ml PMA for 24 hours; Lane TGFβ1 (+) = treatment with 5 ng/ml TGFβ1 for 24 hours; Lanes TNFα(−) and (+) = treatment without and with 10 ng/ml TNFα, respectively, for 24 hours; Lanes EGF(−) and (+) = treatment without and with 20 ng/ml EGF, respectively, for 16 hours; Lanes IL-1β(−) and (+) = treatment without and with 2.5 ng/ml IL-1β, respectively, for 10 hours; Lanes IL-6(−) and (+) in A, C, D, and E = treatment without and with 50 ng/ml IL-6, respectively, for 4 hours; Lanes IL-6(−) and (+) in B, F, and G = treatment without and with 50 ng/ml IL-6, respectively, for 24 hours; Lanes IL-6(−) 4h and IL-6(+) 4h in H = treatment without and with 50 ng/ml IL-6, respectively, for 4 hours; Lanes IL-6(−) 24h and IL-6(+) 24h in H = treatment without and with 50 ng/ml IL-6, respectively, for 24 hours.

TABLE 2

Responses of MMP and TIMP genes in five human glioma cell lines to various agents as shown in Northern blot analyses*

Agents Used
MMPs and TIMPsCell LinesPMA (10 ng/ml 24 hrs)TNFα (10 ng/ml 24 hrs)EGF (20 ng/ml 16 hrs)IL-1β (2.5 ng/ml 10 hrs)IL-6 (50 ng/ml 4 or 24 hrs)TGFβ1 (5 ng/ml 24 hrs)
interstitial collagenaseGB-1+nononono no
 (MMP-1)U-373MG+nononono no
A-172+nononono no
T98G+ (7.31)+ (11.29)++(23.31)± ±
U-87MG++no+no no
gelatinaseA GB-1++± + (6.39)
 (MMP-2)U-373MG±+± +
A-172+±±± ±
T98G±±±± ±
U-87MG±± ±
gelatinase BGB-1+nononono no
 (MMP-9)U-373MG+nononono no
A-172+nonononono 
T98Gnonononono no
U-87MGnonononono no
stromelysinGB-1nonononono no
 (MMP-3)U-373MG+nono+no no
A-172++nonono no
T98G+ (10.98)+ (17.04)±+(25.98)± 
U-87MG+ (11.62)+ (68.67)no+ (152.00) ±
matrilysinGB-1++(7.98)±+(19.48)± 
 (MMP-7)U-373MG++(12.00)no+ (16.48)no ±
A-172nonononono no
T98Gnonononono +
U-87MG±±±± + (18.00)
TIMP-1GB-1+±± 
U-373MG+±±±± 
A-172+±±± ±
T98G+±+±± ±
U-87MG+ (12.27)±±± ±
TIMP-2GB-1±+± ±
U-373MG± ±
A-172±±± ±
T98G±±± ±
U-87MG 

The mean radioactivities of Northern blots of MMP and TIMP genes are classified into five grades: + = induced or increased more than twofold; ± = increased less than twofold; − = decreased two- to threefold; ∓ = decreased less than twofold; no = no response. Particular numbers in parentheses indicate increased multiple values more than fivefold. Abbreviations: MMP = matrix metalloproteinase; TIMP = tissue inhibitor of metalloproteinases; PMA = phorbol-12-myristate-13-acetate; TNFα = tumor necrosis factor-α; EGF = epidermal growth factor; IL = interleukin; TGFβ1 = transforming growth factor-β1.

Cell lines were treated with 50 ng/ml IL-6 for 4 hours for analysis of responses of interstitial collagenase, gelatinase B, stromelysin, and matrilysin genes; or for 24 hours for analysis of responses of gelatinase A, TIMP-1, and TIMP-2 genes.

If more than a fivefold increase in radioactivity of Northern blots by agents other than PMA is defined as a marked transcriptional upregulation, the marked upregulation by TNFα and IL-1β was observed in the interstitial collagenase gene in T98G, in the stromelysin gene in T98G and U-87MG, and in the matrilysin gene in GB-1 and U-373MG. In addition, the marked upregulation was induced by TGFβ1 in the gelatinase A gene in GB-1 and in the matrilysin gene in U-87MG.

Chemoinvasion Assay

The invasive potential of tumor cells can be determined in vitro using chemotaxis chambers with a porous filter coated with Matrigel. To evaluate the roles of MMPs and TIMPs in the invasion of glioma cells, the effects of PMA, TGFβ1, or TIMP-1 on the in vitro invasion of five human glioma cell lines were examined using the chemotaxis chamber. Cells that migrated through the Matrigel were shown to protrude from the porous filter as demonstrated representatively in Figs. 4A to C. The effects of PMA or TGFβ1 on chemoinvasiveness are shown in Fig. 5A. The migrated cell numbers per field were significantly increased in GB-1, U-373MG and T98G cells and decreased in U-87MG cells by treatment with PMA. They were also significantly increased in the GB-1, U-373MG, T98G, and U-87MG cell lines by treatment with TGFβ1 and not changed in A-172 by treatment with PMA or TGFβ1. There was no significant change in cell growth after treatment with PMA or TGFβ1 (data not shown). The marked stimulation of the invasion of GB-1 and U-373MG cells by PMA or TGFβ1 may be explained, at least partially, by their stimulatory effects on the expression of gelatinase A, gelatinase B, or matrilysin, which can degrade the type IV basement membrane collagenase, whereas the suppression of the invasion of U-87MG by PMA may be due to its suppressive effect on gelatinase A expression (see Table 2). Those observations indicated the essential role of MMPs in the in vitro glioma invasion, but the unresponsiveness of A-172 to these agents cannot be explained by modulation in the expression of MMP and TIMP genes. Treatment with TIMP-1 significantly decreased, in a dose-dependent manner, the migrated cell number in all cell lines, except in A-172 cells, which were treated with 3 µg/ml TIMP-1 (Fig. 5B).

Fig. 4.
Fig. 4.

Representative photomicrographs showing invading GB-1 glioma cells that were untreated (A) or treated with 10 ng/ml of phorbol-12-myristate-13-acetate (B) or 5 ng/ml of transforming growth factor-β1 (C) for 24 hours. The cells migrated through Matrigel were stained with Giemsa and shown to protrude from a porous filter(arrows). Original magnification × 100.

Fig. 5.
Fig. 5.

Bar graphs showing the effects of treatment with 10 ng/ml phorbol-12-myristate-13-acetate (PMA), 5 ng/ml of transforming growth factor (TGF)-β1, or 3 to 10 µg/ml tissue inhibitor of metalloproteinases (TIMP)-1, for 24 hours on the chemoinvasiveness of human glioma cell lines (GB-1, U-373MG, A-172, T98G and U-87MG). The migrated cells through the porous filter were stained with Giemsa and counted under a microscope. Each bar shows mean ± standard deviation cell numbers. A: Treatment with PMA or TGFβ1. B: Treatment with TIMP-1. *p < 0.001, **p < 0.005; p < 0.01; and ¶ ¶p < 0.05 compared to the no treatment group.

Discussion

Expression of MMP and TIMP Genes in Gliomas In Vivo

The present systematic study shows high expression of the TIMP-2 gene, low or moderate expression of the gelatinase A and TIMP-1 genes, and undetectable expression of interstitial collagenase, gelatinase B, stromelysin, and matrilysin genes in human brain tissues. There is no evidence of stromelysin gene expression in glioma tissues. Interstitial collagenase gene expression is found in some glioblastomas, but the concomitant overexpression of gelatinase A, gelatinase B, and occasional matrilysin genes is closely associated with malignancy of gliomas in vivo and accompanied by the overexpression of the TIMP-1 gene. These results strongly suggest that gelatinase A, gelatinase B, and matrilysin play important roles in the expression of the malignant phenotype in human gliomas. The overexpression of the TIMP-1 gene may prevent the excess action of MMPs. Rao, et al.,42 also reported an overexpression of gelatinase B gene in human malignant gliomas in vivo. The 2.3- and 1.5-kb TIMP-2 transcripts were not detected in gliomas in vitro but in human brain tissues and gliomas in vivo and may be derived from a novel TIMP gene with high homology to the TIMP-2 gene or produced by an alternative splicing of the TIMP-2 gene.

Chemoinvasiveness of Gliomas

Paulus and Tonn41 reported that human malignant glioma cells derived from primary cultures, fifth passages, and established cell lines were able to migrate through Matrigel. The present chemoinvasion assay demonstrates that TIMP-1 inhibits chemoinvasiveness in a dose-dependent manner. The TIMP-2 inhibits gelatinase A 10-fold more effectively than does TIMP-1, and the TIMP-1 inhibits interstitial collagenase twofold more effectively than TIMP-2.21 Because the gelatinase A gene is constitutively more expressed than the interstitial collagenase gene in human glioma cell lines, TIMP-2 might reduce the chemoinvasiveness of gliomas more efficiently than TIMP-1. The changes in the invasiveness of human glioma cells by treatment with PMA or TGFβ1 are probably dependent on the complicated concerted action of various MMPs and the inhibitory action of TIMP-1 and TIMP-2 in response to PMA or TGFβ1, but it is difficult to estimate a strict correlation between the chemoinvasiveness and the expression levels of MMP and TIMP genes, particularly in response to PMA or TGFβ1 in glioma cell lines, mainly because of the difference in responses and substrate specificities of individual MMPs and TIMPs and to the presence of other factors involved in glioma invasion, such as adhesiveness and motility. It is also noted that in vitro migration through Matrigel does not necessarily represent a general measure of in vivo invasiveness, but reflects the ability to penetrate the basement membrane.50

Responses of MMP and TIMP Gene Expression to PMA, TNFα, EGF, IL-1β, IL-6, or TGFβ1

Although the present study is limited in number, there is a clear difference between the expression of gelatinase B and stromelysin genes in gliomas in vitro and in vivo: the gelatinase B gene is not expressed in vitro but is over-expressed in vivo, whereas the stromelysin gene is not expressed in vivo but is expressed in some cell lines. That difference may be induced by host tissue environmental factors, such as plasminogen activator43,46 and a variety of biologically active agents such as growth factors and cytokines,2,7,8,24,33 in addition to the genetic changes of glioma cells in vitro and the heterogeneity of glioma cells in vivo. In the present study we examined what transcriptional modulation of MMP and TIMP genes is induced in gliomas in vitro by treatment with PMA, TNFα, EGF, IL-1β, IL-6, or TGFβ1, the latter five of which are detected in malignant gliomas in vivo.6,12,26,47–49,57

Recent analysis of the promoter sequences of several MMP genes at the transcriptional level has shed light on the possible molecular basis for their inducibility by a variety of biologically active agents. The promoter regions of human interstitial collagenase, gelatinase B, stromelysin, matrilysin, and TIMP-1 genes have certain common features important for their transcriptional regulation: the TATA box and the activator protein 1 (AP-1) binding site.2,3,8,14,15,27,51 In addition, polyoma virus enhancer activator 3 (PEA-3) upstream of the AP-1 binding site in interstitial collagenase, stromelysin, and matrilysin genes acts synergistically with AP-1,2,3,14,15,27,51,58 but upstream regulatory sequences in the stromelysin promoter contain additional EGF- and tumor promoter—responsive elements, which are lacking in the corresponding matrilysin sequences.14 In the gelatinase B gene, three motifs homologous to the binding sites for AP-1, nuclear factor—κβ (NF-κβ), and Sp-1 proteins are detected, and the AP-1 binding site is indispensable but not sufficient for the induction and requires synergistic cooperation with either the κβ or Sp-1 site.45 Accordingly, treatment with PMA, TNFα, EGF, or IL-1β theoretically upregulates through the AP-1 binding site the expressions of interstitial collagenase, gelatinase B, stromelysin, matrilysin, and TIMP-1 genes,2,7,8,14,24,33,58 but such upregulation is not consistent in human glioma cell lines. However, TNFα and IL-1β each occasionally induce the marked upregulation of interstitial collagenase, stromelysin, and matrilysin genes in the same population of glioma cell lines, which suggests the action of both cytokines on the promoter regions of the particular MMP genes. The promoter region of the gelatinase A gene is very different from that of other MMP genes, most notably by the absence of the obvious TATA box and the AP-1 binding site,13,22 and the response of the gelatinase A gene to PMA, TNFα, EGF, or IL-1β is correspondingly variable in glioma cell lines.

Interleukin-6 is reported to use the signaling molecule gp130,20 and to upregulate the TIMP-1 gene but not MMP genes;29,44 however, no clear responses are shown in glioma cell lines. The transcriptional regulation in the TIMP-2 gene is not yet characterized in detail, and the responses of the TIMP-2 gene to PMA, TNFα, EGF, or IL-1β are rather variable in glioma cell lines. Transforming growth factor-β1 inhibitory element (TIE) is present in the promoter region of interstitial collagenase, stromelysin, and matrilysin genes to repress their transcriptional expression,14,25 but the clear transcriptional inhibition of interstitial collagenase, stromelysin, and matrilysin genes by TGFβ1 is not found in any glioma cell lines; rather, TGFβ1 is an important positive regulator of the matrilysin gene expression in two glioma cell lines. The TIE is not observed in the gelatinase A gene23 and that gene's expression is either unaffected or moderately upregulated by TGFβ1, as shown in glioma cell lines. The inhibitory effect of TGFβ1 on gelatinase B gene expression is not particularly clear,24 and cannot be estimated in glioma cell lines, because there is no detectable gelatinase B gene expression in vitro both constitutively and after treatment with TGFβ1. The region containing the AP-1 binding site in the TIMP-1 gene confers a positive response to TGFβ1 in the presence of serum8 and TGFβ1 upregulates TIMP-1 gene expression.11 However, there is no definite upregulation of TIMP-1 gene expression in response to TGFβ1 in any glioma cell lines. In addition, there is no clear response of the TIMP-2 gene to TGFβ1 in any glioma cell lines.

Thus, the transcriptional modulation of MMP and TIMP genes in response to TNFα, EGF, IL-1β, IL-6, or TGFβ1 is not consistent and is limited to some glioma cell lines, suggesting the genetic changes of the promoter region in glioma cells in vitro. In addition, the agents listed above do not induce either elevation of gelatinase B gene expression or absence of stromelysin gene expression in human glioma cell lines, suggesting the impossibility of changing the expressions of gelatinase B and stromelysin genes from the in vitro to the in vivo phenotype. Gelatinase B activity is detected in macrophages;19 thus macrophages in malignant gliomas may participate in gelatinase B gene expression in vivo. However, it is still unknown why the stromelysin gene expression observed in two of five glioma cell lines is undetected in 17 gliomas in vivo. Further studies of MMPs and TIMPs under various conditions are needed for the full understanding of local invasion and the characteristic absence of metastasis of malignant gliomas outside the central nervous system.

In conclusion, we can infer from the present study that gelatinase A, gelatinase B, and matrilysin are at least partially responsible for the invasive potential of human malignant gliomas. In addition, TIMP-1 and TIMP-2, as well as some growth factors and cytokines such as EGF, TGFβ1, TNFα, and IL-1β, may modulate the activities or gene expressions of MMPs in glioma tissues and the invasion of the glioma cells.

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This work was supported by Grant-in-Aid No. 06771112 from the Ministry of Education, Science, and Culture of Japan.

Article Information

Address reprint requests to: Eiichi Tani, M.D., Department of Neurosurgery, Hyogo College of Medicine, 1–1 Mukogawa-cho, Nishinomiya, Hyogo 663, Japan.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Representative Northern blot analysis. Total cellular RNA was isolated from surgical specimens of three human brain tissues and 17 human gliomas (A) as well as from five human glioma cell lines (GB-1, U-373MG, A-172, T98G, and U-87MG) (B) and hybridized with 32P-labeled complementary DNA probes of interstitial collagenase (MMP-1), gelatinase A (MMP-2), gelatinase B (MMP-9), stromelysin (MMP-3), matrilysin (MMP-7), tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2. Representative ethidium bromide-stained gels (ribosomal (r)RNA) are shown to allow comparison of the total amount of RNA per sample. Ribosomal RNA is used as the size standard. Arrowheads on TIMP-2 Northern blots show 3.5-, 2.3-, 1.5-, and 1.0-kb transcripts. A: Lanes 1 to 3 = brain tissues; Lanes 4 and 5 = primary astrocytomas; Lanes 6 to 8 = primary anaplastic astrocytomas; Lane 9 = recurrent anaplastic astrocytoma; Lanes 10 to 18 = primary glioblastomas; and Lanes 19 and 20 = recurrent glioblastomas. B: Lane 1 = GB-1; Lane 2 = U-373MG; Lane 3 = A-172; Lane 4 = T98G; and Lane 5 = U-87MG. MMP = matrix metalloproteinase.

  • View in gallery

    Bar graph displaying the mean radioactivities of Northern blots of interstitial collagenase (MMP-1), gelatinase A (MMP-2), gelatinase B (MMP-9), stromelysin (MMP-3), matrilysin (MMP-7), tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 in three human brain tissues and 14 nonrecurrent human gliomas. Each bar shows mean ± standard deviation. MMP = matrix metalloproteinase. In each MMP and TIMP section, 1 = brain tissue; 2 = astrocytoma; 3 = anaplastic astrocytoma; 4 = glioblastoma. *p < 0.05 between brain tissue and anaplastic astrocytoma or glioblastoma; p < 0.05 between glioblastoma and astrocytoma or anaplastic astrocytoma.

  • View in gallery

    Representative Northern blot responses of MMP and TIMP genes to agents. Total cellular RNA was isolated from five human glioma cell lines (GB-1, U-373MG, A-172, T98G, and U-87MG) and hybridized with 32P-labeled complementary DNA probe of interstitial collagenase (MMP-1) (A), gelatinase A (MMP-2) (B), gelatinase B (MMP-9) (C), stromelysin (MMP-3) (D), matrilysin (MMP-7) (E), tissue inhibitor of metalloproteinases (TIMP)-1 (F), or TIMP-2 (G). Northern blot analyses after treatment without and with phorbol-12-myristate-13-acetate (PMA) or transforming growth factor (TGF)-β1 were conducted together, whereas analyses after treatment without and with tumor necrosis factor (TNF)-α, epidermal growth factor (EGF), interleukin (IL)-1β or IL-6 were done separately from each other. Ribosomal RNA was used as the size standard. The upper thick bands (arrows) in B in Lanes TNFα, EGF, IL-1β, and IL-6 in the GB-1 and U-373MG cells show 28S ribosomal RNA. Representative ethidium bromide—stained gels (H) are shown to allow comparison of the total amount of RNA per sample. MMP = matrix metalloproteinase; Lane PMA • TGFβ1 (−) = no treatment with PMA or TGFβ1 for 24 hours; Lane PMA(+) = treatment with 10 ng/ml PMA for 24 hours; Lane TGFβ1 (+) = treatment with 5 ng/ml TGFβ1 for 24 hours; Lanes TNFα(−) and (+) = treatment without and with 10 ng/ml TNFα, respectively, for 24 hours; Lanes EGF(−) and (+) = treatment without and with 20 ng/ml EGF, respectively, for 16 hours; Lanes IL-1β(−) and (+) = treatment without and with 2.5 ng/ml IL-1β, respectively, for 10 hours; Lanes IL-6(−) and (+) in A, C, D, and E = treatment without and with 50 ng/ml IL-6, respectively, for 4 hours; Lanes IL-6(−) and (+) in B, F, and G = treatment without and with 50 ng/ml IL-6, respectively, for 24 hours; Lanes IL-6(−) 4h and IL-6(+) 4h in H = treatment without and with 50 ng/ml IL-6, respectively, for 4 hours; Lanes IL-6(−) 24h and IL-6(+) 24h in H = treatment without and with 50 ng/ml IL-6, respectively, for 24 hours.

  • View in gallery

    Representative photomicrographs showing invading GB-1 glioma cells that were untreated (A) or treated with 10 ng/ml of phorbol-12-myristate-13-acetate (B) or 5 ng/ml of transforming growth factor-β1 (C) for 24 hours. The cells migrated through Matrigel were stained with Giemsa and shown to protrude from a porous filter(arrows). Original magnification × 100.

  • View in gallery

    Bar graphs showing the effects of treatment with 10 ng/ml phorbol-12-myristate-13-acetate (PMA), 5 ng/ml of transforming growth factor (TGF)-β1, or 3 to 10 µg/ml tissue inhibitor of metalloproteinases (TIMP)-1, for 24 hours on the chemoinvasiveness of human glioma cell lines (GB-1, U-373MG, A-172, T98G and U-87MG). The migrated cells through the porous filter were stained with Giemsa and counted under a microscope. Each bar shows mean ± standard deviation cell numbers. A: Treatment with PMA or TGFβ1. B: Treatment with TIMP-1. *p < 0.001, **p < 0.005; p < 0.01; and ¶ ¶p < 0.05 compared to the no treatment group.

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Angel PBaumann IStein Bet al: 12-O-tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5′-flanking region. Mol Cell Biol 7:225622661987Mol Cell Biol 7:

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Caroni PSchwab ME: Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading. J Cell Biol 106:128112881988J Cell Biol 106:

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Dabbous MKNorth SMHaney Let al: Macrophage and lymphocyte potentiation of syngeneic tumor cell and host fibroblast collagenolytic activity in rats. Cancer Res 48:683268361988Cancer Res 48:

11.

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Gaire MMagbanua ZMcDonnell Set al: Structure and expression of the human gene for the matrix metalloproteinase matrilysin. J Biol Chem 269:203220401994J Biol Chem 269:

15.

Gutman AWasylyk B: The collagenase gene promoter contains a TPA and oncogene-responsive unit encompassing the PEA3 and AP-1 binding sites. EMBO J 9:224122461990EMBO J 9:

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He CWilhelm SMPentland APet al: Tissue cooperation in a proteolytic cascade activating human interstitial collagenase. Proc Natl Acad Sci USA 86:263226361989Proc Natl Acad Sci USA 86:

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Henry Nvan Lamsweerde ALVaes G: Collagen degradation by metastatic variants of Lewis lung carcinoma: cooperation between tumor cells and macrophages. Cancer Res 43:532153271983Cancer Res 43:

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