(Received for publication, December 29, 1994)
From the
The matrix metalloproteinase 92-kDa gelatinase is a major
product of inflammatory cells. Macrophages synthesize and secrete this
proteinase as a proenzyme in association with tissue inhibitor of
metalloproteinases (TIMP) (92), whereas neutrophils
store and release it from secondary granules as a TIMP-free proenzyme
(92
). Metalloproteinase proenzymes can be activated in vitro by a variety of agents, including organomercurials
and proteinases, resulting in loss of an 8-10-kDa
NH
-terminal domain which disrupts the interaction of a
conserved cysteine residue with the catalytic zinc molecule. We report
that the activation and processing of 92-kDa gelatinase differs
depending on its association with TIMP and the nature of the activating
agent. We observed that 92
undergoes classic activation
to 82 kDa by stromelysin, whereas exposure to 4-aminophenylmercuric
acetate (APMA) results in a final product of 83 kDa that still contains
the ``prodomain'' cysteine. Association with TIMP appears to
stabilize the COOH-terminal domain, whereas 92
is
converted by APMA to a final product of 67 kDa lacking the
COOH-terminal portion. In the continued presence of APMA, which
maintains cysteine-zinc disruption, the 67-kDa species is at least as
active as the classic 82 kDa. In contrast, activation of
92
by stromelysin initially generates the 82-kDa
form which is followed by final conversion to a 50-kDa species that
lacks the catalytic domain of the parent molecule. Therefore, although
stromelysin activation of 92
is initially
efficient, the active 82-kDa form is short-lived and is replaced by an
inactive 50-kDa product. This complex pattern of activation of the
92-kDa gelatinase may serve to restrict its proteolytic capacity
following exposure to stromelysin and may serve to regulate proteinase
activity in vivo.
The 92-kDa gelatinase (gelatinase B, MMP 9) ()is a
member of the metalloproteinase family of structurally related,
matrix-degrading enzymes. These proteinases play a major role in tissue
remodeling and repair associated with development and
inflammation(1, 2) . Abnormal expression of MMPs may
contribute to destructive processes, including tumor
invasion/metastasis(3, 4) , arthritis(5) ,
atherosclerosis(6) , aneurysm formation(7) , and
perhaps pulmonary emphysema related to cigarette smoking(8) .
The expression of metalloproteinases is subject to transcriptional
regulation by various cytokines, growth factors, and other agents (9, 10, 11, 12, 13, 35) .
Regulation of MMP activity is also tightly controlled
post-translationally in the extracellular space via coordinated
activation of secreted proenzymes as well as through interaction of
MMPs with the TIMPs(14, 15) . Proenzyme activation may
be achieved by various means in vitro that disrupt the
interaction between the active site zinc molecule, coordinated in the
catalytic domain, and a cysteine present within the proenzyme
domain(16, 17) . Activation results in loss of the
8-10-kDa NH-terminal prodomain, leaving the mature,
active proteinase. The mechanisms of MMP activation in vivo are currently unknown. Cleavage of the COOH-terminal domain has
also been described for selected MMPs during the activation
process(18, 19) . The effect of COOH-terminal
processing on enzyme function is unknown.
The 92-kDa gelatinase (gelatinase B), a major product of monocyte/macrophages, and the 72-kDa gelatinase (gelatinase A), produced by many cell types, especially fibroblasts, each efficiently degrade gelatins of all genetic types, native collagen types IV and V, fibronectin, entactin, and insoluble elastin(20, 21, 22, 23) . These proteinases are unique among MMPs in that they are secreted in physical association with specific TIMPs (72-kDa gelatinase-TIMP-2 complex and 92 kDa gelatinase-TIMP-1 complex)(24, 25) . TIMPs bind to the COOH-terminal domain of the progelatinases. The 92-kDa gelatinase is also produced by neutrophil precursors and stored in secondary granules. Because TIMP is not produced by neutrophils, the 92-kDa gelatinase in these cells is released TIMP-free(26) . The functional consequences of the unusual association of proenzyme and inhibitor are unknown. Furthermore, the mechanisms of zymogen activation of the 92-kDa progelatinase-TIMP complex secreted by macrophages versus the TIMP-free 92-kDa progelatinase released by neutrophils are incompletely understood.
The present study was initiated following the unexpected finding that activation of recombinant 92-kDa gelatinase proenzyme (TIMP-free) by stromelysin resulted in an initial burst in enzyme activity, but by 24-h proteolytic activity had markedly diminished. We now report that activation and processing of the 92-kDa gelatinase results in various processed forms with differing catalytic activities. The final product is dependent upon the association of the 92-kDa gelatinase with TIMP and the particular activating agent.
Recombinant 92-kDa
gelatinase (TIMP-free) was obtained from p2AHT2a cells transfected with
the adenovirus E1A gene, which inhibits TIMP and MMP transcription, and
the 92-kDa gelatinase gene driven by an SV40 promoter (kind gift of Dr.
Gregory Goldberg, Washington University, St. Louis, MO). Cells were
grown in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) containing 10% heat-inactivated fetal calf serum
and penicillin (10 units/ml) and streptomycin (10
µg/liter). Medium was changed to serum-free +
lactalbumin hydrolysate (0.2%) for 24 h, and the conditioned media were
collected and purified using the same protocol described above for
TIMP-associated 92-kDa gelatinase. Small amounts of 92
complex were separated from the major 92
species by the 0-10% Me
SO gradient gelatin
chromatography. SDS-PAGE Coomassie gels and Western blots using
separate polyclonal antibodies directed against the 92-kDa gelatinase
and TIMP-1 demonstrated that 92
consisted of two
equimolar bands on Coomassie stain which were identified by immunoblots
as the 92-kDa gelatinase and TIMP-1. Recombinant 92
consisted of pure 92-kDa gelatinase in the complete absence of
TIMP-1 (not shown).
Figure 1:
Stromelysin-mediated
activation and processing of 92 over time and
effect on catalytic activity. Purified 92
was
activated with stromelysin for 4 or 72 h at 37 °C. The reaction
products were applied to SDS-PAGE and Coomassie-stained. The activated
products were also incubated with insoluble
[
H]elastin and thiopeptolide. Note, that after 4
h of activation most of the proenzyme has been activated to the 82-kDa
form which has substantial catalytic activity. However, by 72 h, the
predominant band migrated at 50 kDa and had little catalytic
activity.
Figure 2:
Activation and processing of 92 and 92
by stromelysin and APMA. 92
and 92
were incubated with APMA or
stromelysin for 18 h at 37 °C. The reaction products were applied
to SDS-PAGE and Coomassie-stained. Note, the association with TIMP (lanes 1-3) largely inhibits processing past the
82-83-kDa forms. Note also the different 92
forms generated by APMA (67 kDa, lane 5) versus stromelysin (50 kDa, lane 6).
Figure 3:
Time course of APMA-mediated activation
and processing of 92 and 92
and
effect of exogenous TIMP. 92
purified from U937 cells,
recombinant 92
, and 92
+
exogenous TIMP (2-fold molar excess) were incubated with APMA for 0, 1,
4, and 18 h at 37 °C. The reaction products were applied to
SDS-PAGE and Coomassie-stained. Note, conversion of the 92-kDa
proenzyme to the 83-kDa form in the presence of endogenous (lanes
1-4) or exogenous (lanes 9-11) TIMP within 1
h. In the absence of TIMP (lanes 5-8) there is more
extensive processing to a 67-kDa final form over
time.
Figure 4:
Effect
of exogenous TIMP on APMA-activated 92.
92
proenzyme (lane 1) was activated by
APMA at 37 °C for 1 h (lane 2). After 1 h, APMA activation
continued for an additional 17 h either in the presence (+TIMP, lane 3) or the absence (-TIMP, lane
4) of TIMP. Reaction products were applied to SDS-PAGE and
Coomassie-stained. Note, conversion of the proenzyme to the 83-kDa form
and a smaller intermediate by 1 h. Exogenous recombinant TIMP added
(seen in lane 3 migrating at
21 kDa) inhibits further
processing. In the absence of TIMP, there is complete conversion to the
67-kDa species.
Figure 5:
Gelatinolytic activity of the activated
and processed forms of the 92-kDa gelatinase. 92 was activated by APMA and truncated stromelysin (
22 kDa) at
37 °C for 1, 4, and 18 h. A, equal amounts (5 µg) from
each reaction mixture were applied to SDS-PAGE and Coomassie-stained.
The 92-kDa proenzyme(-) was activated by APMA to the 83-kDa form
by 1 h and further processed after 4 and 18 h. Stromelysin activation
resulted in complete conversion to 82 kDa after 1 h. Further processing
was evident at 4 h with more extensive processing after 18 h. B, 0.05 µg of each mixture was also subjected to gelatin
zymography. Following Coomassie staining, white bands represent zones of lysis of the gelatin substrate. Note, activity
of the 92-kDa proenzyme, activated in situ(-).
Regardless of activating agent, the predominant active forms migrate at
82-83 and 67 kDa. There is minimal activity of the 50-kDa
stromelysin activation product.
To more definitively
quantify the gelatin-degrading capacity of the activated and processed
forms of 92, without introducing the variable of
partial denaturation/refolding encountered in zymography, each species
was isolated by HPLC gel-filtration, and the amount of enzyme required
to degrade 50% of a known amount of gelatin was determined. As shown in Table 1, the APMA-activated 83-and 67-kDa forms are nearly
equivalent gelatinases. However, their activity is dependent on the
continued presence of APMA. When APMA is removed from the buffer
following APMA activation, there is no detectable catalytic activity.
Stromelysin-activated 82-kDa enzyme had similar gelatinolytic
activity to the APMA-induced forms. However, the 50-kDa species showed
only 20-25% of this activity. Nevertheless, this assay revealed
greater activity of the 50-kDa form than might have been expected from
the zymography data (Fig. 5) and the results of
NH-terminal sequencing (see below). This activity could be
related to small amounts of the 65-kDa intermediate, transient species
which may have co-purified with the 50-kDa form. Thus, much of the
activity observed may have come from the active, larger species.
Alternatively, this 50-kDa preparation might also retain a small amount
of the NH
-terminal, catalytic site-containing portion of
92-kDa gelatinase (see below).
Stromelysin activation (Table 2) yields the 82-kDa
species which is the classic NH-terminal active form of
MMPs described previously(32) . NH
-terminal
sequence begins with FQT (amino acid 88), which is the start of the
catalytic domain just downstream from the conserved cysteine. The
50-kDa form has the NH
-terminal sequence EPE (amino acid
429). Accordingly, this 50-kDa fragment starts past the catalytic site,
within the proline-rich hinge region. We were unable to identify a
fragment containing the catalytic zinc-binding domain, but we assume
that a minor fragment of
50 kDa remains which has this motif based
on the small but detectable activity of the HPLC-generated 50-kDa form (Table 1) and the barely detectable zymographic activity present
at this M
(Fig. 5). Presumably, much of
this NH
-terminal ``half'' of the 92-kDa
gelatinase has been degraded to lower M
forms
during stromelysin activation.
In this report we demonstrate that activation and processing
of the 92-kDa gelatinase can result in several final products,
depending upon association of the proteinase with TIMP and the
activating agent applied. Native 92-kDa gelatinase produced by
mononuclear phagocytes is secreted in physical association with TIMP
(92). The presence of TIMP results in classic
NH
-terminal activation and prevents further processing.
TIMP-free 92-kDa gelatinase (92
), as secreted from
neutrophil secondary granules, undergoes further processing to a
COOH-terminally truncated, active proteinase following exposure to
either organomercurial agents or MMPs (stromelysin). This 67-kDa form
is the final active product when activation is achieved with APMA.
However, this is only a transient product when activation is mediated
by stromelysin. In the latter case, over time, the enzyme continues to
undergo cleavage, resulting in its diminished catalytic activity.
Organomercurial agents activate MMPs by chelating the highly
conserved prodomain cysteine residue, interrupting its interaction with
the active site
zinc(14, 15, 16, 17) . Freeing of
the zinc leads to autolytic activity whereby the enzyme first cleaves a
portion of the proenzyme domain yielding an 86-kDa intermediate species
which has been described previously(32) . We consistently
observed this product, however, only when the reaction was stopped
within 1 h of activation. This intermediate alters the structure of the
protein, resulting in autolytic cleavage of nearly the entire proenzyme
domain. However, the next cleavage is not COOH-terminal to the
conserved cysteine, as one might expect, but just upstream or
NH-terminal yielding an 83-kDa product, as recently
described (33) and shown here. The retention of the cysteine
explains why the continued presence of APMA is required for catalytic
activity. In the presence of TIMP, this is the final product of APMA
activation; however, the enzyme has limited catalytic potential due to
inhibition caused by the associated TIMP molecule.
In the absence of
TIMP, APMA allows further autolytic processing to a 67-kDa form. Given
the same NH-terminal sequence of both the 83- and 67-kDa
forms, this processing event must come from the COOH terminus (Fig. 6). This COOH-terminal truncated product has equal to or
greater catalytic activity than the 83-kDa form. Increased catalytic
activity after loss of a portion or all of the COOH-terminal domain has
been observed for other MMPs(27) , with the exception of
interstitial collagenase degradation of interstitial collagens, where
the presence of the COOH-terminal domain is required, probably for
substrate binding(34) .
Figure 6:
Domain structure of activated and
processed forms of the 92-kDa gelatinase. The domain structure of the
92-kDa gelatinase is shown (top), including the proenzyme,
catalytic domain that coordinates the active site zinc molecule, the
fibronectin-like domain (Fn), the type V collagen-like domain (coll V), and the COOH-terminal domain. The APMA- and
stromelysin-activated and processed products are aligned based on
NH-terminal cleavage sites (Table 2).
It is unlikely that activation with
APMA mimics processes that occur in vivo. Interaction of MMPs
with other proteinases may more closely reflect activation in
vivo. Stromelysin has been reported to activate the 92-kDa
gelatinase to an 82-kDa product (NH-terminal sequence
F
QT) with loss of the entire NH
-terminal
proenzyme domain, including the Cys
(32) . However,
this 82-kDa form is the final product only in the presence of TIMP.
92
, or neutrophil gelatinase, undergoes further
processing. A 50-kDa protein was isolated that contained the
COOH-terminal collagen-like and hemopexin-like domains. This fragment
represented cleavage in the Pro-rich hinge region, past the catalytic
domain. Although this fragment cannot have catalytic activity, we did
observe greatly reduced, but detectable, enzymatic function in the
HPLC-purified 50-kDa form and at 50-kDa by gelatin zymography. Most
likely, this was caused by the NH
-terminal, catalytic
domain-containing fragment also produced by this cleavage in the hinge
region. We were unable to isolate this fragment, however, and therefore
the reason it is likely to have been largely degraded to smaller
nonfunctional fragments. Nevertheless, it is clear that stromelysin
causes extensive processing of 92
, ultimately
leading to diminished catalytic activity.
Although this study was
conducted in vitro, it may provide important clues to
post-translational mechanisms of regulating proteinase activity in
vivo. Neutrophils and macrophages recruited during an inflammatory
response have the capacity to release the 92-kDa gelatinase. Within
this inflammatory mileu, the enzyme may come in contact with other
proteinases and inhibitors that regulate the proteolytic activity of
the gelatinase. The current study demonstrates that proteinases may not
only activate the 92-kDa gelatinase, but upon continued exposure may
inactivate the enzyme, thereby limiting further tissue damage. Such
inactivation may rather rapidly follow activation, as for stromelysin
processing of 92-kDa gelatinase. Finally, these data demonstrate that
the catalytic function, processing, and fate of neutrophil-released
92-kDa progelatinase free of associated TIMP may be very different from
macrophage-secreted 92-kDa progelatinase-TIMP complex. Interestingly,
92-kDa gelatinase released from neutrophils appears to be more labile
than the 92 produced by macrophages. Perhaps neutrophil
gelatinase is ``turned over'' more quickly in vivo,
consistent with a role in acute inflammatory processes which typically
involve neutrophils.