(Received for publication, November 18, 1994)
From the
The 72-kDa gelatinase/type IV collagenase (MMP-2) is a member of
the matrix metalloproteinase (MMP) family of enzymes. This enzyme is
known to cleave type IV collagen as well as degrade denatured
collagens. However, native interstitial collagens are reportedly
resistant to MMP-2 and are thought to be susceptible only to the
interstitial collagenases MMP-1 and MMP-8. In this study we report that
both human and chicken MMP-2, free of tissue inhibitors of
metalloproteinases (TIMPs) are capable of cleaving soluble, triple
helical type I collagen generating the ¾- and ¼-length
collagen fragments characteristic of vertebrate interstitial
collagenases. MMP-2 cleaves at the same Gly-Ile/Leu bond in the
collagen chains as interstitial collagenases with k
and K
values
similar to that of MMP-1. MMP-2 also is capable of degrading
reconstituted type I collagen fibrils. The closely related 92-kDa
gelatinase/type IV collagenase (MMP-9) is unable to cleave soluble or
fibrillar collagen under identical conditions indicating that the
specific collagenolytic activity of MMP-2 is not a general property of
gelatinases. That MMP-2, a potent gelatinase, also can cleave fibrillar
collagen provides an alternative to the proposal that two enzymes, an
interstitial collagenase and a gelatinase, are required for the
complete dissolution of stromal collagen during cellular invasion.
Extracellular matrix (ECM) ()degradation is mediated
by a battery of secreted enzymes produced by various cell
types(1) . The matrix metalloproteinases (MMPs) are a family of
at least 10 zinc-dependent endoproteinases that function at neutral pH
and cooperatively hydrolyze most of the proteins in the ECM (2) . Interstitial collagens (collagens I, II, and III), the
most abundant proteins of vertebrate connective tissue, are
particularly resistant to proteases including trypsin, plasmin, and
other members of the serine and sulfhydryl proteinase
families(1, 3) . The interstitial collagenases, MMP-1 (4) and MMP-8(5) , belong to an MMP subfamily that
specifically cleaves native triple helical collagens, yielding
¾- and ¼-length collagen fragments as a result of the
hydrolysis of a single Gly-Ile/Leu bond in each
chain of the
collagen molecule(2, 6, 7) .
The collagen fragments produced by the interstitial collagenases are susceptible to the gelatinases (MMP-2 and MMP-9), a second MMP subfamily, that rapidly degrade denatured collagens and collagen fragments(8, 9) . MMP-2 has been identified in a wide range of normal and malignant cells and in several species(2, 9, 10) . Cooperation between the interstitial collagenases and the gelatinases is thought to be essential for clearing interstitial collagens in connective tissue during inflammatory and invasive processes. While capable of cleaving Gly-Leu and Gly-Ile bonds (along with other peptide bonds) in denatured collagens(11, 12) , MMP-2 is thought to be unable to cleave the same bonds in native interstitial collagens(9, 13, 14) . However, MMP-2 is capable of cleaving native type IV and V collagens(2) . The expression of the gelatinases/type IV collagenases have been correlated with the metastatic state(15) , and these enzymes are believed to influence the ability of a cell to invade and metastasize due to their ability to degrade basement membrane type IV collagen(16) .
The study of the activation and substrate specificity of the gelatinases has been hampered by the co-isolation of the tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 and TIMP-2 usually co-purify in tight, noncovalent complexes with both the zymogen and active forms of the 92- and 72-kDa gelatinases, respectively(8, 17) . The ability to purify TIMP-free native enzyme (18, 19) and the production of recombinant progelatinases (20, 21) has allowed for more detailed and accurate assessments of the properties of these enzymes.
In the present study we show that both human and chicken
TIMP-free MMP-2 cleave collagen fibrils and soluble native type I
collagen specifically generating the ¾- and ¼-length
fragments characteristically produced by the interstitial collagenases.
Values for the Michaelis-Menten constants K and k
were determined using soluble
collagen I as a substrate, and MMP-2 was found to be comparable with
MMP-1 in catalyzing this reaction. Implications of these findings to
the roles of MMP-2 in cell invasion are discussed.
Figure 1:
Specific cleavage of rat tendon and
bovine skin type I collagen by chicken and human MMP-2. Native type I
collagen was incubated in the presence of latent or active MMPs in 0.1
ml of CAB/Brij for 4 h at 25 °C, and the reactions were reduced,
electrophoresed on 5-15% polyacrylamide gels, and stained with
Coomassie Blue. A, rat collagen I (17.5 µg) was incubated
alone (NoEnzyme) or with chicken pro-MMP-2 (cproMMP-2), chicken MMP-2 (cMMP-2),
recombinant human MMP-2 (hMMP-2), recombinant human MMP-1 (hMMP-1), recombinant human MMP-9 (hMMP-9),
chicken or human MMP-2/TIMP-2 complexes (cMMP-2/TIMP-2 and hMMP-2/TIMP-2, respectively). The amount of enzyme used is
indicated in µg at the top of each lane. B, bovine collagen I (10 µg) was incubated alone (No
Enzyme) or with the indicated amounts of chicken MMP-2,
recombinant human MMP-2, recombinant human MMP-1, and recombinant human
MMP-9. C, rat collagen (17.5 µg, 1.35 µM) was
incubated with 0.1 µg of human MMP-2 or 0.025 µg of human MMP-1
for 0-4 h at 25 °C and analyzed by SDS-PAGE, and scanning
densitometry and the results were plotted as µg of collagen cleaved versus time. Inset shows the SDS-PAGE profile at the
indicated times used in the densitometric analysis. In A and B the positions of molecular mass standards are indicated on
the left ( 10
Da).
1 and
2 indicate the
1(I) and
2(I) peptide
chains of type I collagen, respectively.
indicates two cross-linked
1 chains and
an
1 chain cross-linked to an
2
chain. TC
and TC
indicate the ¾
NH
-terminal and COOH-terminal collagen cleavage products,
respectively.
In order to rule out that the collagenolytic activity exhibited by the TIMP-free preparation of chicken MMP-2 was due to a contaminating interstitial collagenase, a gel-slicing experiment was performed (Fig. 2). Chicken pro-MMP-2 was subjected to SDS-PAGE (Fig. 2A), and after electrophoresis the gel was washed and sliced into 2-mm slices, and each slice was incubated with APMA (to activate the zymogen) and rat type I collagen. Each reaction was analyzed by Coomassie Blue-stained SDS-PAGE (Fig. 2B) and gelatin zymography (Fig. 2C). The specific collagenolytic activity is present in the single fraction corresponding to 68-75 kDa (Fig. 2A and Fig. 2B, lane9) and coincides with the presence of the gelatinolytic activity of MMP-2 (Fig. 2C, lane9). No collagenolytic activity is found in the 30-50 kDa region of the gel where interstitial collagenases would migrate. When recombinant human MMP-1 is subjected to the same analysis, the collagenolytic activity is present only in the 40-45-kDa region of the gel (data not shown). These data support that the collagenolytic activity observed with the TIMP-free MMP-2 preparations is due to MMP-2 and not to the presence of a contaminating interstitial collagenase.
Figure 2:
Collagenolytic activity of chicken
TIMP-free MMP-2 preparation fractionated by SDS-PAGE. A preparation of
chicken pro-MMP-2 (0.2 µg), isolated by gelatin-Sepharose and Mono
Q chromatography, was electrophoresed on a 5.5 10-cm SDS-12%
polyacrylamide gel and washed with 2.5% Triton X-100 and water. The gel
was then sliced into 2-mm fragments. Each slice was incubated with 10
µg of rat collagen I and 2 mM APMA in 150 µl of
CAB/Brij at 25 °C for 72 h. Following incubation the samples were
analyzed for the presence of both the collagen cleavage products and
gelatinase activity as described under ``Experimental
Procedures.'' A, silver stain of chicken TIMP-free
pro-MMP-2 run in a parallel lane of the gel that was sliced. Marks on the right of the gel indicate where the slices were
made and numbered. B, Coomassie Blue-stained 5-15%
gradient polyacrylamide gel of the collagen cleavage products generated
after 72 h. C, a 10-µl aliquot from each reaction was
analyzed by gelatin zymography to indicate the slice that contained
MMP-2 gelatinolytic activity. Positions of molecular mass standards are
indicated on the left (
10
Da), and
slice numbers are indicated at the top. Slice number 9, which
contained the 62-kDa active gelatinase, also generated the specific
TC
cleavage fragment.
Figure 3:
Degradation of fibrillar collagen by MMP-1
and MMP-2 and lack of degradation by MMP-9. Fifty micrograms of H-labeled rat tail tendon type I collagen (1.6 mg/ml) was
allowed to gel at 37 °C in microtiter plate wells as described
under ``Experimental Procedures.'' After the collagen fibrils
were washed and equilibrated with CAB/Brij, proteases were added to the
wells in triplicate and allowed to incubate at 37 °C for 18 h.
Following incubation, the supernatants were removed and analyzed by
liquid scintillation spectroscopy. Buffer alone was used as a control
for background and was subtracted from the mean counts/min released by
each enzyme. Data are represented as percent of total collagen released
above background. Errorbars indicate the standard
errors of the mean. Enzymes were used at the indicated concentrations
and are labeled as follows: TPCK-treated trypsin (Trypsin),
recombinant human MMP-1 (MMP-1), recombinant human
MMP-2 (MMP-2), and recombinant human MMP-9 (MMP-9).
This study demonstrates that MMP-2 is capable of cleaving native type I collagens under conditions that preclude any helix denaturation (27) . This activity is not common to gelatinases as equal amounts of MMP-9 failed to cleave collagen under identical conditions. That the collagenolytic activity of the MMP-2 preparations was not due to a contaminating protease was shown by a gel-slicing analysis (Fig. 2) in which the single slice containing the 62-kDa MMP-2 gelatinolytic activity also exhibited the specific cleavage of collagen. The use of purified recombinant human MMP-2 further supports that MMP-2 alone is responsible for the observed collagenolysis.
MMP-2 cleaves the same peptide bonds in the collagen
chains as the interstitial collagenases. The TC
fragments produced by MMP-2 (Table 1) have the same
amino-terminal sequence as the TC
produced by other
vertebrate collagenases(30) . These data, in conjunction with
the apparent identical electrophoretic mobility of the TC
fragments, suggest that the MMP-2 behaves as an interstitial
collagenase cleaving the
(P
)Gly
-(P
`)Ile/Leu
bond in the
chains of native triple helical collagen. MMP-2
is known to cleave after glycine with hydrophobic residues, glutamine,
asparagine, or serine in the P
site(11, 12) . However, it has long been thought
that the gelatinases/type IV collagenases were unable to cleave these
bonds in native interstitial
collagens(2, 8, 9) . Why the ability of MMP-2
to cleave native collagen I has not been detected previously is
unclear. When we initially isolated and characterized chicken MMP-2
this collagenolytic activity was not observed (22) as was the
case for a number of studies using human MMP-2 isolated from cultured
cells(9, 13, 14) . One explanation is the
presence of TIMP-2, which co-purifies with MMP-2 from many sources. The
presence of TIMP-2 in MMP-2 preparations markedly decreases the rate
and extent of activation of pro-MMP-2 and also decreases the specific
activity of the enzyme
preparations(19, 20, 32) . TIMP-2 also may
interfere with the ability of MMP-2 to bind to collagen in a productive
fashion. We have shown that an equal molar amount of TIMP-complexed
MMP-2, activated in the same manner as TIMP-free MMP-2, generates
markedly reduced amounts of specific collagen cleavage products
compared with TIMP-free MMP-2 (Fig. 1A). The ability of
avian MMP-2 to cleave native type I collagen albeit at reduced levels
has been previously reported(33) . However, this activity was
thought to be a peculiarity of the avian enzyme, and the effect of the
associated TIMP-2 on the reduced activity was not explored. It would
appear that any TIMP-2, free or in a complex with the zymogen, in MMP-2
preparations would markedly diminish the specific interstitial
collagenolytic activity of MMP-2.
Cleavage of native type I collagen
required two to four times more MMP-2 than MMP-1 to generate similar
amounts of the specific cleavage fragments (Fig. 1). This might
indicate that MMP-2 is less efficient than MMP-1 in bringing about
collagen dissolution. However, determination of the kinetic constants
for this reaction revealed that the k values of
MMP-2 and MMP-1 were equal (Table 2), although the K
value for MMP-2 (8.5 µM) was higher
than that of MMP-1 (1 µM). Given these data and the
concentration of collagen I used in this study (1.4 µM)
the Michaelis-Menten equation indicates that the rate of cleavage of
type I collagen by MMP-1 should be 4.1 times greater than that of
MMP-2, a value consistent with our observations. Interestingly, the 8.5
µMK
value for MMP-2 indicates that
assays carried out with collagen concentrations of 100 µg/ml (0.4
µM), 20-fold below the K
, would yield
little specific collagen cleavage products, and this also may explain
why this activity has not been observed in other studies. That the k
values for MMP-2 and MMP-1 are similar
suggests that at suitably high collagen concentrations (i.e. >8 µM) MMP-2 and MMP-1 would cleave native
collagen at similar rates. Such high concentrations of collagen might
only be encountered in the interstitial stroma.
Most significantly,
MMP-2 is capable of extensively degrading reconstituted fibrillar
collagen. Since soluble, monomeric collagen exists at negligible levels
in the extracellular stroma, fibrillar collagen is a more relevant
biological substrate. That MMP-2 degrades fibrillar collagen to an
equal or greater extent as MMP-1 (Fig. 3) may indicate that in
the fibril assay the K for MMP-2 is exceeded and
MMP-2 functions as efficiently as MMP-1. When an equal gelatinolytic
amount of MMP-9 was assayed, the level of fibrillar collagen
degradation did not exceed that of trypsin (Fig. 3). These data
suggest that MMP-2 may have a role as an interstitial collagenase in vivo and be sufficient for the degradation of both the
basement membrane collagen IV and the connective tissue collagens.
Degradation of interstitial collagens has long been believed to be dependent on the presence of two enzymes: interstitial collagenases and gelatinases. The data presented here suggest that MMP-2 may be capable of effecting the removal of interstitial collagens in the absence of collagenases. The role of MMP-2 in connective tissue remodeling in normal and pathological processes might, therefore, be re-evaluated. Expression of the gelatinases/type IV collagenases has been linked to the invasive potential of cells(15) . The functional role of the gelatinases/type IV collagenases is thought to be the degradation of type IV collagens, thereby allowing cells to traverse the basement membrane. While this may be true, MMP-2 may also facilitate the invasion of the underlying connective tissue ECM in which type I collagen predominates.
While several malignant cells, including A2058 and MDA-231, express MMP-1 in vitro(34) , others such as HT1080, HT-144, MDA-435, and MCF-7 do not express interstitial collagenase in vitro, yet these cells are invasive and appear to be capable of invading tissues that contain interstitial collagens(34, 35) . MMP-2, which is produced by these cells, could catalyze the degradation of such collagen. Furthermore, there are malignant cells that do not express interstitial collagenase in vivo as determined by in situ hybridization(36, 37) . In the latter case, it is possible that MMP-2 supplies the collagenolytic potential necessary to move across the basement membrane and through the interstitium. Tumor cells have been demonstrated to induce adjacent normal stromal cells to produce MMP-1(38) ; yet, the constitutive expression of MMP-2 may alone suffice for local collagenolysis.