(Received for publication, January 17, 1996; and in revised form, March 6, 1996)
From the
This study confirms that normal human articular chondrocytes
express neutrophil collagenase or matrix metalloproteinase-8 (MMP-8), a
gene product previously thought to be expressed exclusively by
neutrophil leukocytes. Both MMP-8 protein and mRNA were present in
articular cartilages collected from normal human donors. Cartilage
extracts were assayed by immunoblotting and by analysis of enzymatic
activity on gelatin-substrate gels. Latent MMP-8 extracted from
cartilage has a molecular mass of 55 kDa; active MMP-8 was identified
at 46 and 42 kDa. In the absence of a reducing agent, MMP-8 migrated in
a high molecular mass complex above 200 kDa. Northern blotting results
demonstrated the expression of MMP-8 in chondrocytes, which could be
up-regulated by stimulation with interleukin-1. In addition,
reverse transcription-polymerase chain reaction using nested primers
and in situ hybridization revealed the presence of MMP-8 mRNA
in chondrocytes. The presence of both MMP-8 protein and message in
cartilage supports the concept that neutrophil collagenase could be the
enzyme described as ``aggrecanase.''
Neutrophil collagenase (MMP-8) ()is a
metalloproteinase gene product that is different from interstitial
collagenase (MMP-1). There is significant homology between MMP-8 and
MMP-1, with 57% identity to the deduced protein sequence for MMP-1 and
72% chemical similarity(1) . However, MMP-8 has been shown to
differ from MMP-1 in substrate specificity (2, 3) and
immunological cross-reactivity(4, 5) . Interstitial
collagenase is synthesized and released constitutively from cells into
the extracellular matrix, while MMP-8 has been shown to be synthesized
and stored in specific granules of the neutrophil
leukocyte(6) .
While MMP-8 was previously thought to be
expressed selectively by neutrophil precursors, recent evidence has
suggested that chondrocytes may also express this MMP and that its
function may not be limited to
collagenolysis(7, 8, 9, 10) . A
major component of articular cartilage matrix is a large aggregating
proteoglycan called aggrecan that consists of a core protein and
glycosaminoglycans. Analysis of the predominant cleavage site in the
core protein was identified by sequencing the peptides that had been
released into synovial fluid from human arthritic cartilage and from
interleukin-1-stimulated cartilage (11, 12, 13, 14) . The major
cleavage site was Glu-Ala
in the human
sequence. This site was different from the cleavage site
Asp
-Phe
produced by other
MMPs(15, 16) . The unidentified proteinase was called
``aggrecanase.'' Recently, MMP-8 was shown to be capable of
producing the aggrecanase cleavage product in
vitro(10, 17) .
The results from this study indicate that both MMP-8 protein and mRNA are produced by normal human chondrocytes and provide further supportive evidence that MMP-8 may be identical to aggrecanase.
Immunoblots of the cartilage extract showed no immunoreactive
bands without keratanase and chondroitinase digestion (data not shown)
when probed with either of the polyclonal antibodies C44 and F2.
Following digestion, immunoreactive bands were present (Fig. 1, A-D). MMP-8, recognized by both F2 and C44, was present
in the extracts of the five donor cartilages (Fig. 1, A and B, lanes 1-4 and 7). If the
extracts were reduced with dithiothreitol, the most prominent
immunoreactive band migrated at 55 kDa between the 57- and 52-kDa
bands of latent MMP-1 (Fig. 1B, lane 5).
Additional MMP-8 bands with molecular masses of 42, 40, 36, and 32 kDa
were also present (lanes 1 and 7). The extract from
only one donor contained MMP-1 (lane 3). The 68-kDa band (Fig. 1A) appears to be a nonspecific reactant for the
polyclonal C44 antibody since this band remained visible following
competitive binding with the MMP-8-specific peptide, while the other
bands disappeared (data not shown).
Figure 1: Identification of MMP-8 in cartilage extracts. A and C, Western blotting of cartilage extracts from five donors with the C44 antibody. B and D, Western blotting of cartilage extracts from the same donors in A, but with the F2 antibody. In A and B, samples were reduced; in C and D, samples were not reduced. E, gelatin zymogram. Lanes 1-4, 74 years, 62 years, 11 weeks, and 43 years, respectively; lane 5, purified MMP-1; lane 6, conditioned medium with MMP-2 and MMP-9; lane 7, 50 years. The positions of molecular mass standards at 200, 97, 69, 46, and 30 kDa are shown.
The extracts were subjected to
electrophoresis under nonreducing conditions (Fig. 1, C and D) so MMP-8 immunoreactive bands could be compared
with those on the gelatin zymograms. The extract from one donor (lane 7) contained one sharp band at 170 kDa and two
bands at 46 and 42 kDa. However, the majority of the immunoreactive
bands migrated above 200 kDa (lanes 1-4).
In the zymogram, bands of activity were present at molecular masses of 46 and 42 kDa in the extract from one donor (Fig. 1E, lane 7), identical to those present on the Western blot (Fig. 1C, lane 7). The 46- and 42-kDa species appear to be MMP-8 since their molecular masses were unchanged with aminophenylmercuric acid activation. All activity on the zymogram was inhibited by 2 mM phenanthroline (data not shown), indicating that the clearing was due to metalloproteinases.
On Northern blot
hybridization, barely detectable levels of mRNA for MMP-8 were present
in freshly isolated chondrocytes from normal cartilage (Fig. 2A, lane 1). However, in response to 10
pg/ml IL-1 for 24 h, the MMP-8 mRNA levels were up-regulated, and
a single band of mRNA transcript was detected with an approximate size
of 3.3 kilobases (lane 2).
Figure 2:
Northern blot analysis of total RNA
extracted from isolated normal chondrocytes cultured for 24 h showing
one representative sample of three independent experiments. A,
membrane was hybridized with P-labeled cDNA probe specific
to MMP-8. Lane 1 contains RNA from chondrocytes cultured
without IL-1
; lane 2 contains RNA from chondrocytes
cultured with 10 pg/ml IL-1
. B, shown is an ethidium
bromide-stained gel demonstrating RNA
loading.
RT-PCR products using a nested primer set specific for neutrophil collagenase gave the same molecular size products as was expected/calculated (Fig. 3). The message level was low since only a nested primer PCR could detect the signal. The final PCR product was sequenced after purification and had 100% identity to the published cDNA sequence(1) .
Figure 3:
Nested primer RT-PCR products of RNA
samples extracted from cartilage tissue of normal human donors. Lanes 1-5, 1 day, 3.5 months, 4 years, 22 years, and 68
years, respectively; lane 6, molecular size markers in base
pairs from a X174 replicative form digest of HindI
DNA.
In situ hybridization was performed with a polymorphonuclear leukocyte-enriched population of blood cells and with cartilage sections using antisense and sense primers. These were positive with the antisense probe (Fig. 4, A and B). The antisense probe hybridized to chondrocytes in cartilage (Fig. 4C), while the sense probe did not show a cell-specific signal above background (Fig. 4D).
Figure 4:
In
situ hybridization. A, polymorphonuclear
leukocyte-enriched population of cells hybridized with the antisense
primer identical to bp 774-797 in the MMP-8 gene
sequence and visualized using dark-field microscopy. B,
bright-field photomicrograph of A. C, cartilage (34
years) hybridized with the same antisense primer used in A. D, cartilage (34 years) hybridized with the sense primer
identical to bp 439-462 in MMP. Magnification
45.
We have shown that both MMP-8 protein and message are present in articular cartilage from normal human donors over the age range of 1 day to 74 years. These data demonstrate that this MMP is not expressed exclusively by neutrophils. Latent MMP-8 extracted from cartilage has a molecular mass of 55 kDa, while active MMP-8 is 46 and 42 kDa. These are comparable to the molecular masses of latent and active recombinant neutrophil MMP-8 constitutively released from transfected COS cells (32) . We have observed that recombinant MMP-8 is not glycosylated to the same degree as native MMP-8 and is not stored in specific granules within the transfected cells(1) .
Chondrocyte mRNA for MMP-8 was demonstrated using 1) Northern
blotting of RNA isolated from chondrocytes, 2) RT-PCR using nested
primers, and 3) in situ hybridization. While the presence of
MMP-8 mRNA was shown with all three procedures, the amount of mRNA
expressed by either isolated chondrocytes or chondrocytes within
cartilage was relatively low. Expression of this mRNA in normal
chondrocytes was barely detectable by Northern blotting, although it
was significantly up-regulated after stimulation with IL-1. In
cartilage, RT-PCR amplification using nested primers was required to
show the presence of mRNA. The most sensitive method for detecting
MMP-8 mRNA was in situ hybridization. The increased
sensitivity of the in situ hybridization technique allowed the
detection of MMP-8 mRNA in circulating neutrophil leukocytes.
Neutrophils were previously thought to synthesize and store all MMP-8
protein in secretion granules during their development in bone marrow
prior to entering circulation; our evidence shows that the mRNA is
still present in these cells and suggests that they may continue to
synthesize the protein while in circulation.
The results of this
study show that normal human chondrocytes express an inducible MMP-8 gene with an mRNA of 3.3 kilobases. The gene
product is similar, if not identical, to neutrophil MMP-8 expressed by
COS cells. The fact that chondrocytes express the MMP-8 gene
provides supporting evidence that MMP-8 could be the proteinase in
cartilage responsible for degrading the aggrecan core protein, leading
to the loss of aggrecan from the extracellular matrix of cartilage and
the development of osteoarthritis.