(Received for publication, January 30, 1997, and in revised form, May 27, 1997)
From the Departments of Pediatrics and
Pathology, Long Island Jewish Medical Center, Long
Island Campus for the Albert Einstein College of Medicine, New Hyde
Park, New York 11040, § Human Genome Sciences, Inc.,
Rockville, Maryland 20850-3338, the ¶ Department of Chemistry and
Institute of Molecular Biophysics, Florida State University,
Tallahassee, Florida 32306-3006, the
Division of Cancer
Genetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston,
Massachusetts 02115, and ** Triple Point Biologics, Forest
Grove, Oregon 97116
TIMP-4, a novel human tissue inhibitor of metalloproteinase, was identified and cloned (Greene, J., Wang, M., Raymond, L. A., Liu, Y. E., Rosen, C., and Shi, Y. E. (1996) J. Biol. Chem. 271, 30375-30380). In this report, the production and characterization of recombinant TIMP-4 (rTIMP4p) are described. rTIMP4p, expressed in baculovirus-infected insect cells, was purified to homogeneity by a combination of cation exchange, hydrophobic, and size-exclusion chromatographies. The purified protein migrated as a single 23-kDa band in SDS-polyacrylamide gel electrophoresis and in Western blot using a specific anti-TIMP-4 antibody. Inhibition of matrix metalloproteinase (MMP) activities by rTIMP4p was demonstrated in five MMPs. Enzymatic kinetic studies revealed IC50 values (concentration at 50% inhibition) of 19, 3, 45, 8, and 83 nM for MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, respectively. Purified rTIMP4p demonstrated a strong inhibitory effect on the invasion of human breast cancer cells across reconstituted basement membranes. Thus, TIMP-4 is a new enzymatic inhibitor in MMP-mediated extracellular matrix degradation and may have therapeutic potential in treating cancer malignant progression.
MMPs1 and their inhibitors TIMPs play a critical role in ECM homeostasis. Controlled remodeling of the ECM is an essential aspect of normal development, and deregulated remodeling has been indicated to have a role in the etiology of diseases such as arthritis, periodontal disease, and cancer metastasis (1-5). Four mammalian TIMPs have been identified so far: TIMP-1 (6), TIMP-2 (7), TIMP-3 (8-11), and the recently cloned TIMP-4 (12, 41). The proteins are classified based on structural similarity to each other, as well as their ability to inhibit metalloproteinases.
TIMPs are secreted multifunctional proteins that have anti-MMP activity as well as erythroid-potentiating and cell growth-promoting activities. The stimulating effect on cell growth was initially recognized when TIMP-1 and TIMP-2 were identified as having erythroid-potentiating activities (14, 15). It is now clear through several recent reports that TIMP-1 and TIMP-2 are mitogenic for non-erythroid cells, including normal keratinocytes (16), fibroblasts (17), lung adenocarcinoma cells (18), and melanoma cells (18). The involvement of TIMPs in the activation of pro-MMP has also been demonstrated (19). In addition, the recent evidence indicates that the TIMP family may be involved in steroidogenesis of rat testis and ovary indicating the potential role of TIMP in the reproduction (20).
The most widely appreciated biological function of the TIMPs is their role in the inhibition of cell invasions in vitro (21-24) and tumorigenesis (25-29) and metastasis in vivo (25-31). Since the net MMP activity is the result of the balance between activated enzyme levels and TIMP levels, an increase in the amount of TIMPs relative to MMPs could function to block tumor cell invasion and metastasis. The tumor-suppressing activity of TIMP on primary tumor growth may be in part due to its anti-angiogenic activity. In fact, both TIMP-1 (32) and TIMP-2 (33, 34) have been demonstrated to have an anti-angiogenic activity, and such inhibition of angiogenesis is mediated by inhibition of both endothelial cell proliferation (34) and migration (32). The underlying molecular mechanism for the tumor suppressing activities of TIMPs, nevertheless, is thought to depend on their anti-MMP activities.
We had recently cloned and characterized a human TIMP-4 (12). Transfection of TIMP-4 into human breast cancer cells inhibited the invasion potential of the cells in the in vitro invasion assay (13). When injected orthotopically into nude mice, TIMP-4 transfectants were significantly inhibited in their tumor growth and axillary lymph node and lung metastasis as compared with controls (13). These results suggest the therapeutic potential of TIMP-4 in treating cancer malignant progression. These results suggest an important role of TIMP-4 in inhibiting primary tumor growth and progression leading to invasion and metastasis. In the present study, we have produced and purified rTIMP4p from baculovirus infected cells. rTIMP4p was shown to inhibit MMP activity and tumor cell invasion across reconstituted basement membrane.
Restriction enzymes were obtained from Boehringer Mannhem. Chromatography supplies were purchased from PerSeptive. All the other reagents are listed as indicated below.
Preparation of rTIMP4p from BaculovirusThe coding sequence
of TIMP-4 is amplified using a standard polymerase chain reaction
approach with the primers corresponding to the 5 and 3
sequences of
the cDNA (5
primer: GCT AGT GGA TCC CTG CAG CTG CGC CCC GGC G; 3
primer: CGG CTT CTA GAA GGG CTG AAC GAT GTC AAC). The amplified
fragment was gel-purified and digested with BamHI and
XbaI. To construct the recombinant baculovirus expression
vector, the purified TIMP-4 polymerase chain reaction fragment was
ligated into pA2-GP vector, which was derived from pVL94 (35). The
resulting pA2-GP/TIMP4 vector was transfected into HB101 cells, and
positive clones were identified using polymerase chain reaction
screening and restriction enzyme analysis. The DNA sequence was
confirmed by automatic DNA sequencing of both strands. A recombinant
virus was produced and purified. Sf9 insect cells were infected with
recombinant baculovirus in EXCEL401 serum-free medium (JRH Scientific)
supplemented with 1% pencillin/streptomycin (Life Technologies, Inc.)
and 1% fetal bovine serum (Life Technologies, Inc.). A 5-L Bioreactor
was harvested 70 h post-infection, and cell viability was
estimated to be 80%. The bioreactor supernatant was clarified using a
continuous flow centrifuge (18,000 × g). The
harvesting and subsequent chromatography steps were carried out at
4-8 °C.
As we previously described (13, 36), an MMP-2-mediated gelatin degradation assay was used to monitor the anti-MMP activity of rTIMP4p during the purification. The clarified supernatant was directly loaded onto a strong cation-exchange column (POROS HS50 from PerSeptive Biosystems; column dimensions, 3 × 10 cm) at a linear flow rate of 800 cm/h. The column was previously equilibrated with 50 mM sodium acetate, 100 mM NaCl, pH 5.8, for 10 column volumes. The bound proteins were eluted using the step elution of 200 mM NaCl, 400 mM NaCl, 600 mM NaCl, 1 M NaCl, and 2 M NaCl (in the same equilibration buffer as before). The 600 mM eluted fractions were found containing anti-MMP-2 activity. The active fractions were pooled and diluted with 50 mM sodium acetate, pH 5.8, to a conductivity of six millisiemens.
A weak cation-exchange column (POROS CM20 from PerSeptive Biosystems; column dimensions, 2 × 7 cm) was equilibrated with 10 column volumes of 50 mM Tris-HCl, 100 mM NaCl, pH 7.5. The pooled active fractions from the strong cation-exchange column were loaded onto the column at a linear flow rate of 840 cm/h. The bound proteins were first washed with the equilibration buffer and then eluted by a gradient elution using the equilibration buffer and the elution buffer containing 50 mM Tris-HCl and 1 M NaCl, pH 7.5. The gradient elution was conducted from 0.1 M to 1 M NaCl within a 10-bed volume. The active fractions were pooled, and NaCl was added to raise the conductivity to 200 millisiemens.
A moderate hydrophobic interaction chromatography column (POROS PE50 from PerSeptive Biosystems; column dimensions, 2 × 10 cm) was equilibrated with 10 column volumes of buffer containing 50 mM sodium acetate and 4 M NaCl, pH 5.8. The pooled fractions from the weak cation-exchange column were loaded onto the column at the flow rate of 100 ml/45 min. The bound proteins were eluted using 50 mM sodium acetate and 100 mM NaCl, pH 5.8. The eluted active fractions from the column were pooled together.
The pooled fractions from the hydrophobic column were loaded onto a size-exclusion column (Superdex S-200 from Pharmacia Biotech Inc.; column dimensions, 2.5 × 90 cm) with a flow rate of 20 ml/30 min. The was previously equilibrated using 50 mM sodium acetate and 100 mM NaCl, pH 5.8. The sizing fractions were analyzed by SDS-PAGE and Coomassie Blue staining, and the relevant fractions corresponding to a size of 23 kDa were pooled and tested for purity and anti-MMP activity.
Preparation of Anti-TIMP-4 AntibodyA peptide sequence corresponding to amino acids 207-225 of human TIMP-4 (12) was synthesized by an ABI 431A peptide synthesizer. Peptide synthesis reagents were from Advanced Chemtech, Louisville, KY. The purified peptide was conjugated to keyhole limpet hemocyanin (Sigma) via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Pierce). New Zealand rabbits were immunized with the carrier-hapten conjugate in Freund's complete adjuvant (Pierce) followed by Freund's incomplete adjuvant at the recommended intervals. Animals were anesthetized and exsanguinated, and antibodies were separated from serum by ion exchange. For final purification, a TIMP-4 peptide affinity column was made by conjugating 20 mg of TIMP-4 peptide to 5 ml of AminoLink resin (Pierce Chemical Co.) using sodium cyanoborohydride (Sigma).
Western BlotSamples were boiled in SDS/-mercaptoethanol
sample buffer and electrophoresed in 12% acrylamide-PAGE gels. Gels
were blotted onto polyvinylidene difluoride membrane in 25 mM Tris, 192 mM glycine buffer, pH 8.3, containing 20% (v/v) methanol. Blots were blocked in 5% bovine serum
albumin (Sigma) for 1 h. Primary antibodies were diluted 1:2000 in
TTBS (30 mM Tris, pH 7.4, 150 mM NaCl, 0.1%
Tween 20). After incubation with the primary antibodies overnight at
4 °C, the blots were washed 4 × 10 min in TTBS, then incubated for 1 h in goat anti-rabbit IgG-horseradish peroxidase (Sigma) diluted 1:6000 in TTBS. The blots were then washed 4 × 10 min in
TTBS, and the bands were visualized by chemiluminescence.
Inhibition of enzymatic activity by rTIMP4p was assayed by measuring degradation of [3H]gelatin as we previously described (13).
Kinetic StudiesActive human neutrophil gelatinase B/92-kDa type IV collagenase (MMP-9) was purified as described (37). Human fibroblast collagenase (MMP-1), gelatinase A/72-kDa type IV collagenase (MMP-2), and stromelysin (MMP-3) were obtained from Dr. L. Jack Windsor of the University of Alabama at Birmingham (38). MMP-1 was autoactivated and converted to active catalytic domain (cdMMP-1) during the storage. Pro-MMP-2 (0.2 µM) was partially activated by three-times-repeated freeze and thaw cycles. Recombinant human active matrilysin (MMP-7) was kindly provided by Dr. H. E. Van Wart (37, 38). The substrate used for determining the TIMP-4 inhibition parameters was a quenched fluorescent substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) (39), which was purchased from Bachem. The final substrate concentration in the assays was 1 µM. The final enzyme concentrations in the assays were 5, 5, 4.9, 6.7, and 1.4 nM for MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, respectively. The enzymes were incubated with various concentrations of TIMP-4 at 25 °C for 30 min before adding the substrate to start the kinetic assay. The assays were carried out at 25 °C using a Perkin Elmer LS-5 fluorescence spectrometer (40).
In Vitro Invasion AssayInhibition of breast cancer cell invasions by purified rTIMP4p was evaluated in the Matrigel invasion assay with reconstituted basement membrane as we previously described (13).
rTIMP4p was produced
in Sf9 insect cells using the baculovirus expression system. A
pVL94-based transfection vector PA2-GP/TIMP4 was constructed to
generate the recombinant virus which was subsequently used to infect
Sf9 cells. The optimal yield of rTIMP4p was obtained from the
conditioned medium of the infected cells at 70 h post-infection. The best purification of rTIMP4p was achieved by a 4-step
chromatography including a strong cation chromatography, a weak cation
chromatography, a hydrophobic interaction chromatography, and a
size-exclusion column. The rTIMP4p eluted from a size exclusion column
was stored in the buffer containing 50 mM sodium acetate
and 100 mM NaCl, pH 5.8. When analyzed by SDS-PAGE, this
preparation showed a single band at molecular mass of 23 kDa (Fig.
1A), which is consistent with
the predicted molecular mass based on the calculation from the protein
sequence (12, 41). The purified 23-kDa protein was confirmed as TIMP-4
by Western blot using a specific anti-TIMP-4 antibody (Fig.
1B). The consistency of calculated molecular mass and
the actual molecular mass of purified rTIMP4p suggests that there was
no post-translational glycosylation for rTIMP4p. This is in agreement
with the absence of the glycosylation site for TIMP-4 (41). In
addition, rTIMP4p gave a negative result in the glycosylation test
using modified periodic acid-Schiff method (42) (data not shown).
The specific activity of the recovery of rTIMP4p is summarized in Table
I. The yield of purified rTIMP4p was
approximately 1.7 mg/2 × 107 cells.
|
The
inhibitory activity of rTIMP4p on MMPs was analyzed by a soluble
gelatin degradation assay. As shown in Fig.
2, when MMP-2 and MMP-9 were incubated
with purified rTIMP4p at the mole ratio of 1 to 2, the gelatinolytic
activities were inhibited 88% for MMP-2 and 66% for MMP-9,
respectively. A similar pattern with higher magnitude of inhibition was
also observed for TIMP-2, suggesting that TIMP-4 may be more specific
for MMP-2 in a manner similar to TIMP-2.
Kinetic analysis of the inhibition of MMPs by rTIMP4p was performed in
a continuous fluorometric assay with a quenched fluorescent peptide
substrate. The inhibition kinetics of rTIMP4p were analyzed against
human MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9. The MMPs were incubated
with different concentrations of rTIMP4p. As shown in Fig.
3, the inhibitor concentrations that
reached to 50% inhibition of MMP activities (IC50) were
determined to be 19, 3, 45, 8, and 83 nM for MMP-1, MMP-2,
MMP-3, MMP-7, and MMP-9, respectively. Therefore, TIMP-4 is a potent
inhibitor of all five tested MMPs, and it has preference for MMP-2 and
MMP-7.
Inhibition of Invasion Potential of Human Breast Cancer Cells
Previously, we demonstrated that transfection of TIMP-4
cDNA into human breast cancer cells inhibited tumor cell invasion cross-reconstituted basement membrane (Matrigel) (13). The effect of
purified rTIMP4p on the invasion of MDA-MB-435 human breast cancer
cells was investigated. MDA-MB-435 cells were moderately invasive. At
the end of a 24-h incubation, about 10% of MDA-MB-435 cells had
crossed the Matrigel barrier. A significant reduction in invasive
potential was noted when rTIMP4p was added at two different
concentrations. The percentages of invaded cells were 1.5% for the
cells treated with 10 nM rTIMP4p and 0.6% for the cells
treated with 100 nM rTIMP4p, respectively. To facilitate the comparison of the relative invasiveness between controls and rTIMP4p-treated cells in this study, all values were normalized to the
percent invasion of control MDA-MB-435 cells which were taken as 100%
(Fig. 4).
To rule out the possibility that the different invasion potentials between the control cells and rTIMP4p-treated cells are due to the potential inhibitory effect of TIMP-4 on cell growth, we conducted growth rate experiments to determine whether addition of rTIMP4p affects the growth of MDA-MB-435 cells. When the cells were treated with 20, 50, and 100 rTIMP4p in the Dulbecco's modified Eagle's medium containing 5% fetal calf serum (changing the fresh medium and rTIMP4p every 2 days) for 7 days, no significant differences in growth rate were observed between the control and rTIMP4p-treated cells (data not shown). These results are consistent with our previous report on the similar growth rates of the control MDA-MB-435 cells and TIMP-4-transfected cells (13).
Active recombinant TIMP-4 protein is required for characterization of its biochemical activity against MMPs and biological functions in inhibiting tumor growth and metastasis. Proteins of eukaryotic cells expressed in Escherichia coli are often generated as inactive, insoluble aggregates known as inclusion bodies and therefore require in vitro complicated refolding. In the present study, we expressed, purified, and characterized recombinant TIMP-4 protein prepared from baculovirusinfected insect Sf9 cells. The identity of rTIMP4p was confirmed by several criteria. First, as expected, the purified protein had a molecular mass of 23 kDa in SDS-PAGE, which is in close agreement with the calculated molecular mass of the 22.5-kDa protein, based on the mature protein of 195 amino acids after removal of the signal sequence (12). Second, the purified protein possessed a metalloproteinase inhibitory activity against MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9. Third, the purified protein can be recognized immunochemically by an affinity-purified specific anti-TIMP-4 polyclonal antibody. Fourth, the purified protein inhibited tumor cell invasion in the Matrigel invasion assay, and a similar effect was also reported for other TIMPs (21, 22).
We demonstrated here that human recombinant TIMP-4 can effectively
inhibit human MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, with the
IC50 values of 19, 3, 45, 8, and 83 nM,
respectively. This relatively higher potency of TIMP-4 against MMP-2
than other MMP suggests that TIMP-4, like TIMP-2, is more specific for
MMP-2. In fact, the predicted structure of the TIMP-4 shares 37%
sequence identity with TIMP-1 and 51% identity with TIMP-2 (12). In
addition, we also demonstrated a high affinity interaction between
TIMP-4 and the C domain of MMP-2 and showed that TIMP-4 bound both
full-length MMP-2 and the C domain of MMP-2 in a manner similar to
TIMP-2 (43). Binding of MMP-2 to TIMP-4 was of high affinity with an apparent Kd of 1.7 × 107
M but sightly weaker than that to TIMP-2 (apparent
Kd of 6.6 × 10
8 M)
(43). These Kd differences are in agreement with the
relatively more potent inhibitory effect of TIMP-2 on MMP-2 than that
of TIMP-4 (Fig. 2). The overall sequence identity between TIMP-4 and
other TIMPs suggests that TIMP-4 may inhibit MMPs through a similar
mechanism by forming a strong noncovalent complex with a 1:1
stoichiometry (44). Although the inhibitory activity of TIMP is
distributed throughout the molecule, the N-terminal regions of the TIMP
family are highly conserved and thus may contribute to the inhibitory
activities, and the C-terminal regions are divergent and may enhance
the selectivity to the target enzymes (41, 45). A more detailed
structural comparison indicated that TIMP-4 shares a relatively high
identity with TIMP-2 particularly in the loops of 4 and 5 within the
C-terminal domain (41). Thus, it is possible that TIMP-4 and TIMP-2 may
share similar mechanistic and functional properties based on the
sequence identity, similar enzymatic kinetics, and the high affinity
binding to MMP-2 (43).
Augmented MMP activities are associated with the metastatic phenotype of carcinomas, especially breast cancer (46-49). The down-regulation of MMPs may occur at the levels of transcriptional regulation of the genes, activation of secreted proenzymes, and through interaction with TIMPs. The clinical importance of MMPs during the tumor progression emphasizes the need to effectively block MMPs and the subsequent tumor cell invasion. The inhibitory effect of TIMPs on MMP activity leads one to expect that an increase in the amount of TIMPs relative to MMPs could function to block tumor cell invasion and metastasis. Indeed, tumor cell invasion and metastasis can be inhibited by up-regulation of TIMP expression or by an exogenous supply of TIMPs (23, 24, 28-31). Alternatively, down-regulation of TIMP-1 and TIMP-2 have also been reported to contribute significantly to the tumorigenic and invasive potentials of the cells (25-27). These results suggest that an inhibitory activity of TIMPs play an important role in inhibiting tumor cell malignant progression leading to invasion and metastasis.
In this study, we demonstrated an inhibitory effect of the purified rTIMP4p on the invasion of human breast cancer cells, which is consistent with our previous studies on the inhibition of cell invasion on the TIMP-4-transfected cells compared with the TIMP-4 negative control cells (13). In the experimental Matrigel invasion assay, approximately 95% inhibition of invasion potential was achieved when the breast cancer cells were treated with 100 nM rTIMP4p. Similar inhibitory effects with much less magnitude were also reported for TIMP-1 (21) and TIMP-2 (22) on different tumor cells. The almost complete suppression of invasion potential of breast cancer cells by rTIMP4p suggests that the major matrix degradation proteinases required for the invasion of breast cancer cells in the Matrigel invasion assay are MMPs, and their enzymatic activities can be inhibited effectively by TIMP-4. The inhibition of breast cancer cell invasion by both an exogenous supply of rTIMP4p and the endogenous expressed TIMP-4 suggest that the TIMP-4-mediated anti-invasion activity could be physiologically or pathologically relevant in the tumor microenvironment.
Using in situ hybridization analysis, we have demonstrated a stromal expression of TIMP-4 mRNA in the fibroblasts surrounding the breast carcinomas.2 The expression of TIMP-4 in the stroma adjunct to the breast carcinomas may indicate one of the host responses to try to balance the local tissue degradation due to the tumor cell invasion. Therefore, availability of the excess TIMP-4 relative to MMP (either by exogenous supply or endogenous expression) would create a microenvironment in the tumoral-stromal interface where the MMP-mediated ECM degradation and the subsequent tumor cell invasion can be inhibited by TIMP-4. While we are aware that the Matrigel in vitro invasion assay may not be an accurate predictor of breast cancer cell invasion as it occurs in vivo, we have recently demonstrated the TIMP-4-mediated anti-tumor and anti-metastasis activities of TIMP-4 transfected breast cancer cells in the animal model (13). These results support a role for MMPs and the inhibitor TIMP-4 in breast cancer cell invasion. Therefore, the potential therapeutic value of TIMP-4 for controlling cancer progression warrants further investigation.