©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Chondrocyte Matrix Metalloproteinase-8
HUMAN ARTICULAR CHONDROCYTES EXPRESS NEUTROPHIL COLLAGENASE (*)

(Received for publication, January 17, 1996; and in revised form, March 6, 1996)

Ada A. Cole (1)(§) Susan Chubinskaya (1) Barbara Schumacher (1) Klaus Huch (1) (2) Gabriella Cs-Szabo (1) Jianling Yao (1) Katalin Mikecz (1) (2) Karen A. Hasty (3) (4) Klaus E. Kuettner (1) (2)

From the  (1)Departments of Biochemistry and (2)Orthopedic Surgery, Rush Medical College, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, the (3)Department of Anatomy and Neurobiology, University of Tennessee, Memphis, Tennessee 38163, and (4)the Veterans Administration Medical Center, Memphis, Tennessee 38104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-1beta. 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.''


INTRODUCTION

Neutrophil collagenase (MMP-8) (^1)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.


MATERIALS AND METHODS

Human Blood Neutrophil Preparation

Polymorphonuclear leukocyte-enriched populations of cells were obtained from the blood of normal healthy volunteers by a modified Ficoll-Hypaque discontinuous gradient centrifugation method(18) . These cells were used as positive controls for MMP-8 mRNA for in situ hybridization.

Tissue Acquisition

Human cartilage was obtained from 13 donors (age range from 16 fetal weeks to 74 years) within 24 h of death through the Regional Organ Bank of Illinois. Two additional cartilages were obtained as surgical specimens from patients undergoing amputation. Full thickness non-calcified cartilage was removed from the articular surface of the knee cartilages with no macroscopically visible signs of fibrillation.

Organ Culture

Four intact human metatarsals (16 fetal weeks) including epiphyseal cartilages were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 25 µg/ml ascorbate for 48 h at 37 °C in 7% CO(2). Gelatinases A (MMP-2) and B (MMP-9) in the conditioned medium were identified based on their molecular masses on zymograms and by their ability to bind to a gelatin-Sepharose affinity column (Pharmacia Biotech Inc.)(19) .

Chondrocyte Cell Culture

Chondrocytes were released from the cartilages of three of the donors with a modification of the procedure of Aydelotte and Kuettner (20) using Pronase (0.4% (w/v), 60 min, 37 °C) followed by bacterial collagenase (0.025% (w/v), overnight, 37 °C). Chondrocytes (1.5 times 10^6/ml) were cultured for 24 h in Dulbecco's modified Eagle's medium, 10% fetal bovine serum (Life Technologies, Inc.), 25 µg/ml ascorbate, and 50 µg/ml gentamicin. Human recombinant IL-1beta (Genzyme Corp.) was added to the cultures at a concentration of 10 pg/ml. No IL-1beta was added to control chondrocytes.

Cartilage Extract

The articular cartilages were extracted in 1 M guanidine HCl for 2 h at 20 °C(21) . Aliquots of samples were analyzed by electrophoresis and immunoblotting without digestion. Other samples were digested with keratanase (0.01 unit/10 µg of proteoglycan), keratanase II (0.001 unit/10 µg of proteoglycan), and chondroitinase ABC (0.1 unit/10 µg of proteoglycan).

Western Blot Analyses

Samples were solubilized at an equal weight/volume in buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM CaCl(2), 0.25 M NaCl, 0.2% Brij 35. Samples (60 µg/40 µl/lane) were reduced with 5% dithiothreitol. Immunoblot analyses were performed following SDS-polyacrylamide gel electrophoresis with two antibodies, F2 and C44 (22) . F2 was prepared against recombinant MMP-8 and recognizes both MMP-1 and MMP-8. C44 is antiserum prepared against a synthetic peptide of MMP-8 and is specific for this enzyme. Protein was transferred from SDS-polyacrylamide slab gels (23) in buffer containing 192 mM glycine, 25 mM Tris, pH 8.3, and 20% (v/v) methanol. Additional binding sites were blocked in 5% non-fat powdered milk (Carnation). The blots were incubated with primary antibody (1:1000 dilution) and a goat anti-rabbit secondary antibody conjugated with alkaline phosphatase (1:5000; Promega). The blots were developed with Western Blue-stabilized substrate for alkaline phosphatase (Promega). Specificity of the antibody binding for C44 was determined by competitive binding of the antibody with the peptide antigen (22) prior to blot incubation(24) .

Substrate Zymography

Gelatinolytic activity of the cartilage extracts was assayed by gelatin-substrate zymography(25) . To activate the proenzyme, aliquots of cartilage extracts, recombinant MMP-8, and purified human MMP-1 (gift from Dr. H. G. Welgus, Washington University Medical Center) were incubated with 0.5 mM aminophenylmercuric acid. Phenanthroline (2 mM) was added to inhibit MMP activity in some samples.

Northern Blotting

Total RNAs were obtained from freshly isolated chondrocytes, cultured in the presence or absence of IL-1beta (26) from monolayer cultures. The cells were lysed in 4 M guanidine isothiocyanate; Tris-equilibrated phenol, pH 7.4, was added. RNA in the aqueous phase was purified by precipitation with 70% ethanol. Total RNAs (10 µg/sample) were denatured, electrophoresed in 1% agarose gel, and transferred to membranes. The membranes were prehybridized with unlabeled DNA and hybridized with the radioactively labeled MMP-8-specific cDNA probe (1) at a concentration of 3 times 10^6 cpm/ml. The cDNA probes were labeled with [P]dCTP (specific activity of 3000 Ci/mM; Amersham Corp.) by the random primer extension method (27) using the Amersham Multiprime DNA labeling system. The specific activity of the labeled probe was 2 times 10^9 cpm/µg of cDNA. The membranes were exposed to film for 9 days.

PCR Amplification

Total RNA was isolated directly from cartilage tissue by a modified method of Chomczynski and Sacchi(28) . Approximately 0.1 µg of total RNA was transcribed using reverse transcriptase with a MMP-8-specific antisense primer. The resulting cDNA was amplified by PCR (RT-PCR kit, Perkin-Elmer; sense for bp 439-462 and antisense for bp 774-797)(29) . 10 µl of the first amplification product and a second sense primer (bp 594-613; nested primer) were used for a subsequent amplification step. PCR products were separated in 1% agarose gel and stained with ethidium bromide. PCR products were purified with the Geneclean system (Qiagen, Inc.) and sequenced by the dideoxy chain termination method (30) using the fmol DNA sequencing system (Promega).

In Situ Hybridization

In situ hybridization was performed (31) with two of the probes used as primers for RT-PCR (antisense for bp 774-797 and sense for bp 439-462). The probes were 3`-end-labeled with 5`-alpha-thiol-S-dCTP (DuPont NEN) using terminal deoxynucleotidyltransferase (DuPont NEN) under conditions of high stringency.


RESULTS

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-1beta 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-1beta; lane 2 contains RNA from chondrocytes cultured with 10 pg/ml IL-1beta. 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 times 45.




DISCUSSION

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-1beta. 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants 2-P50-AR-39239 and AI-22603 (to K. A. H.) and by a Deutscher Akademischer Austausch Dienst fellowship (to K. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Rush Medical College, Rush-Presbyterian-St. Luke's Medical Center, 1653 W. Congress Pkwy., Chicago, IL 60612. Tel.: 312-942-4879; Fax: 312-942-3053.

(^1)
The abbreviations used are: MMP, matrix metalloproteinase; IL-1beta, interleukin-1beta; PCR, polymerase chain reaction; RT, reverse transcription; bp, base pairs.


ACKNOWLEDGEMENTS

We thank Dr. Allan Valdellon and the staff of the Regional Organ Bank of Illinois for help in providing human cartilage as well as Dr. Adam I. Harris and Susie Ro, Jie Yang, Doug Johnson, and Lincoln Michal for technical assistance.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.