Expression OF membrane type 1 matrix metalloproteinase in human articular cartilage.
код для вставкиСкачатьARTHRITIS & RHEUMATISM Vol. 40, No. 4, April 1997, pp 704-709 0 1997, American College of Rheumatology 704 EXPRESSION OF MEMBRANE TYPE 1 MATRIX METALLOPROTEINASE IN HUMAN ARTICULAR CARTILAGE FRANK H. BUTTNER, SUSAN CHUBINSKAYA, DANIEL MARGERIE, KLAUS HUCH, JOHANNES FLECHTENMACHER, ADA A. COLE, KLAUS E. K U E n N E R , and ECKART BARTNIK Objective. To detect the message for membrane type 1 (MT1) matrix metalloproteinase (MMP) in articular cartilage and chondrosarcoma cells, to study its expression in osteoarthritis (OA), and to determine whether i nt e r l eu k in -lp (IL-1p) influences its expression. Methods. Reverse transcription-polymerase chain reaction methods were used to detect message. Cloning and sequencing were applied to confirm the sequence. Northern blotting was used to quantify the message for MT1-MMP and to compare its expression in OA cartilage with or without IL-1p treatment. In situ hybridization was utilized to show MT1-MMP transcripts in cartilage and to study the influence of IL-1p. Results. The results show that MT1-MMP messenger RNA (mRNA) is expressed in chondrosarcoma cells and OA chondrocytes. Results of the in situ hybridization confirmed the expression in OA cartilage as well as in normal cartilage. The level of mRNA was not modulated following IL-1p stimulation. Conclusion. This study shows that MT1-MMP is expressed by chondrocytes. The similarities in mRNA levels in OA and normal chondrocytes suggest that regulation of MT1-MMP mRNA may not be a primary factor in OA. Proteolysis of the extracellular matrix of articular cartilage is a hallmark of inflammatory and degenerative joint diseases like rheumatoid arthritis and osteoarthritis Frank H. Buttner, Diplom Biologe, Daniel Margerie, Diplom Biologe, Eckart Bartnik, PhD: Hoechst AG, Kalle-Albert, Wiesbaden, Germany; Susan Chubinskaya, PhD, Ada A. Cole, PhD, Klaus E. Kuettner, PhD: Rush Medical College at Rush-Presbyterian-St. Luke’s Medical Center, Chicago, Illinois; Klaus Huch, MD, Johannes Flechtenmacher, MD: University of Ulm, Ulm, Germany. Address reprint requests to Eckart Bartnik, PhD, Hoechst AG, Kalle-Albert, Biomedical Research, H 528, Rheingaustrasse 190, 65174 Wiesbaden, Germany. Submitted for publication August 1, 1996; accepted in revised form October 25, 1996. (OA). The process of matrix degradation is thought to be mediated through the activity of matrix metalloproteinases (MMPs) (1). The most recent additions to this expanding family include the membrane-type MMPs, the first of which was identified by Sat0 et a1 (2) and designated MT1-MMP. This MMP is bound to cellular membranes. Strongin et a1 ( 3 ) were the first to describe one possible function of MT1-MMP. Once activated, MT1-MMP acts as a receptor for tissue inhibitor of metalloproteinases 2 (TIMP-2), and the resultant dimer then binds latent gelatinase A (MMP-2), activating the gelatinase. Cao et a1 (4) showed that the transmembrane domain of MT1-MMP plays an essential role in the activation of progelatinase A. That study reported that truncated forms of MT1-MMP containing no transmembrane domain were unable to activate gelatinase A, whereas mutants of MT1-MMP with a transmembrane domain from an unrelated protein were able to activate gelatinase A. More recent reports show that transmembrane-deficient mutants can still process progelatinase A; however, different methods were used for the two studies. Pei and Weiss ( 5 ) demonstrated intrinsic matrix-degrading activity of transmembranedeletion mutants of MT1-MMP and suggested that there may be other functions of MT1-MMP than activation of gelatinase A at the cellular membrane ( 5 ) . The current study concentrates on the expression of MT1-MMP in articular cartilage, since it has been previously reported that gelatinase A is present in that tissue. In fact, activated gelatinase A as well as activated collagenase have been described in extracts of articular cartilage from O A patients in which tissue destruction is evident (6). The expression of an activator of gelatinase A in tissue in which the enzyme has been shown to be active prompted the examination of whether MT1-MMP is also expressed in articular cartilage. The results of studies using amplification by reverse transcriptionpolymerase chain reaction (RT-PCR), Northern blot analyses, and in situ hybridization show that MT1-MMP MT1-MMP IN HUMAN ARTICULAR CARTILAGE is expressed in both normal a n d OA articular cartilage, as well as in human chondrosarcoma cells. While levels of expression in the chondrosarcoma cells were relatively high, levels of expression in the OA cartilage were much lower. N o detectable differences were evident between levels of expression in t h e normal or the OA chondrocytes beyond those observed between individual cartilage samples. In addition, there was no detectable change in MT1-MMP expression following stimulation by the catabolic cytokine, interleukin 1p (IL-lp), which is known to up-regulate t h e expression of other MMPs (1). These data suggest that MT1-MMP expression in chondrocytes may be constitutive. A n up-regulation of the gelatinase A activator messenger RNA (mRNA) may not be necessary for increased activity of the enzyme protein during the destructive proteolytic events associated with OA. It is also possible that the primary function of MT1-MMP in cartilage is involvement in some process other than the activation of gelatinase A. PATIENTS AND METHODS Chondrosarcoma cell culture. Human chondrosarcoma cells (SM-0219/JJ-012; a gift from Dr. J. Block, Rush Medical College, Chicago, IL) were grown as monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM) with 25 Fl,g/mlof ascorbic acid (Sigma, Munich, Germany), 0.1 ngiml of insulin (Hoechst, Marburg, Germany), 100 mM hydrocortisone (Sigma), 50 wg/ml of gentamicin (Gibco, Grand Island, NY), 2 mM glutamine (Gibco), and 10% fetal bovine serum (FBS; Sigma) in a humidified atmosphere of 5% CO,, at 37°C. Acquisition of human articular cartilage. Human hyaline articular cartilage was obtained from OA patients undergoing total joint replacement. These cartilage samples were used for the RT-PCR and Northern blot analyses. For comparison with normal cartilage, samples were obtained from donors with no joint disease through the Regional Organ Bank of Illinois (ROBI). These normal cartilage samples were used with the in situ hybridizations. For stimulation with IL-lp, cartilage samples used in the explant cultures were also obtained from normal donors and OA patients. Tissue culture. To determine whether MT1-MMP expression could be up-regulated in the presence of IL-lp, cartilage explants (3 X 3 mm’) both from normal donors and OA patients were cultured with recombinant human IL-lP (Genzyme, Boston, MA) at concentrations of 0.05-50 pg/ml. The media consisted of DMEM supplemented with 10% FBS, 25 &ml of ascorbate, and 50 pl,g/ml of gentamicin. Control explants were cultured in identical media, except that the IL-1p was omitted. Each explant was cultured in 1 ml of medium in 24-well plates in a humidified atmosphere of 5% CO, at 37°C (7,8). Cell culture of human chondrocytes. Osteoarthritic chondrocytes were isolated from the remaining articular cartilage from the OA patients and cultured in alginate beads for 705 72 hours as described by Hauselmann et a1 (9). The alginate beads containing the chondrocytes were maintained in Ham’s F-12DMEM (50/50) medium (Gibco), supplemented with 10% FBS, 25 pg/ml of ascorbic acid, and 50 &ml gentamicin in a humidified atmosphere of 5% CO, at 37°C. For stimulation with IL-lp, cells were subdivided into 2 populations for a further 3-day cultivation in the presence or absence of 50 pg/ml of IL-lP. RNA isolation. Total RNA was prepared according to a modification of the protocol of Chomczynski and Sacchi (lo), from chondrosarcoma cells, cultured 0.4 chondrocytes, and intact OA cartilage. The RNA concentration was determined spectrophotometrically. PCR, cloning, and sequencing. Two sets of primers (each 21 basepairs in length) were employed: set A for nucleotides 112-1860 of MT1-MMP and set B for nucleotides 964-671 (2). RT-PCR was performed according to standard procedures using Tuq polymerase. For generating the fulllength message for MT1-MMP, the PCR conditions were modified by using 10% DMSO and increasing the annealing temperature. Fragments generated by PCR were first analyzed by restriction endonuclease digests. A full-length PCR message for MT1-MMP was cloned into the pCDM8 vector (Invitrogen, San Diego, CA) and sequenced. Northern blot analyses. Total RNA was isolated either directly from the OA cartilage (5 different samples; age range 64-80 years) or from cultured OA chondrocytes. The RNA was resolved on agarose/formaldehyde gels, transferred to Nylon membranes, and ultraviolet light-crosslinked. A probe was generated from human chondrocyte RNA using RT-PCR with primer set B, and was radiolabeled for hybridization with w3’P-dCTP using random nonamer primers (Amersham, Braunschweig, Germany). Hybridized blots were washed under conditions of high stringency. The same blots were rehybridized with either p-actin or glyceraldehyde-3-phosphatedehydrogenase (GAPDH) probes to confirm that equal amounts of RNA were loaded per lane. Quantitation was performed using a phosphor image analyzer. In situ hybridization. Articular cartilage from 7 normal donors (age range newborn to 69 years) and 3 OA patients (ages 59,69, and 74) as well as cultured cartilage explants were fixed in 4% paraformaldehyde and paraffin-embedded. In situ hybridization was also performed on sections of chondrosarcoma tissue from which the human chondrosarcoma cells (JJ-012) were derived (a gift from Dr. J. Block). The probe used for in situ hybridization was the reverse primer of set B. The probe was 3’4abeled with 5’-(~t-thioI-~~S)-dCTP using terminal deoxynucleotidyl transferase (New England Nuclear, Boston, MA). The radiolabeled probe was hybridized to cartilage sections as previously described by Chubinskaya et a1 (11). Competitive inhibition controls were performed by mixing the radiolabeled primers with the same unlabeled probe in ratios of 1:1, 1:2, 1:4, and 1%. RESULTS PCR identification of MT1-MMP. Two primer sets were designed for the amplification of m R N A by PCR. Primer set A corresponds to codons in the amino BUTTNER ET AL 706 M 1749 bp - 707 bp - 1 2 3 4 M Figure 1. Reverse transcription-polymerase chain reaction (RTPCR) products of RNA samples extracted from human chondrosarcoma cells (SM0219) or from human osteoarthritic articular cartilage, employing primer sets A and B (see Patients and Methods for details). Lanes 1 and 2, PCR products from human chondrosarcoma cells; Lanes 3 and 4, PCR products from human articular cartilage; lanes 1 and 3, full-length message for membrane type 1 matrix metalloproteinase (MT1-MMP; 1,749 bp); lanes 2 and 4, amplified PCR fragment of MT1-MMP (707 bp); lane M, molecular size markers (Boehringer Mannheim nos. I11 and VI). and the carboxyl terminals of MTl-MMP (nucleotides 112-1860) whereas set B corresponds to nucleotides 964-1671 (hinge to hemopexin domain). With RNA from the chondrosarcoma cells, the amplification by RT-PCR using both primer sets A and B resulted in PCR fragments with the expected sizes of 1,749 bp and 707 bp, respectively (Figure 1, lanes 1 and 2). Fragments of the same size were identified following amplification of RNA originating from 4 different patients and 1 normal donor. RNA was either extracted directly from cartilage (Figure 1, lanes 3 and 4, showing amplification products from RNA directly isolated from the cartilage of 1 patient) or from OA chondrocytes cultured in alginate beads (data not shown). Restriction enzyme analysis confirmed that the fragments indeed corresponded to MT1-MMP complementary DNA fragments (data not shown). The full-length PCR fragment of 1,749 bp derived with the use of primer pair A was subcloned. Subsequent sequencing revealed a match to the sequence reported by Sat0 et al (2) (with the corrections provided by Okada et a1 [12]), except for 10 nucleotides. These differences may reflect true differences in the sequence of cartilage MT1-MMP rnRNA; however, they may also have been introduced by Taq polymerase mistakes generated under the altered PCR conditions needed for the successful amplification of the full-length MT1-MMP mRNA. These data show that MT1-MMP is expressed by cbondrosarcoma cells and chondrocytes from OA cartilage. Northern blot analyses. As an additional control and in order to quantify MT1-MMP transcripts, Northern blot analyses were performed using total RNA isolated directly from the cartilage of an additional 5 OA patients. The Northern blots were incubated with a radiolabeled probe that was generated from the same primer pair B used for the PCR (Figure 2). The probe hybridized to a 4.5-kilobase message, the same size as the mRNA reported by Sat0 et a1 (2) and Takino et a1 (13) for MT1-MMP. Levels of mRNA were quantitated using a phosphor imager and expressed as a percentage of the detected p-actin or GAPDH signal after reprobing with 0-actin-specific or GAPDH-specific probes. The results showed variations between 47% and 110% (Figure 2, lanes 3 and 5 ) among the 5 OA patients evaluated. There were also no substantial differences between the mRNA levels in the OA chondrocytes cultured with and without IL-1p (Figure 2, lanes 6 and 7). The quantification showed only a 15% difference. Expression of MT1-MMP in cartilage as detected by in situ hybridization. The results from the in situ hybridization confirmed the Northern blot data, in that all OA cartilage tested contained chondrocytes with detectable levels of MT1-MMP mRNA (Figure 3A). There was, however, variation in the levels of expression, from weak to strong, within the chondrocytes from different OA donors. In addition, no up-regulation of MT1-MMP message was evident compared with controls following stimulation with IL-1p over the concentration range tested. Within the cartilage sections following in situ hybridization, mRNA was detectable in all zones, from superficial to deep. In the OA cartilage where the superficial layer was disrupted or absent, mRNA levels for MT1-MMP did not appear to be elevated in the middle and deep layers. When the levels of expression in OA chondrocytes (from 3 patients) were compared with the levels in chondrocytes from 7 normal donors, no substantial differences could be found. As with the expression in OA chondrocytes, levels of expression varied among the normal chondrocytes, and upregulation by IL-1p could not be detected. MT1-MMP IN HUMAN ARTICULAR CARTILAGE 1 2 3 4 4.5 kb - 5 707 6 7 - 4.5 kb p-actin GAPDH Figure 2. Northern blot analysis of membrane type 1 matrix metalloproteinase (MT1-MMP) messenger RNA derived from 5 different osteoarthritis (OA) patients or from OA chondrocytes cultivated in alginate beads with or without interleukin-lp (IL-lp). Each lane contains 15 pg of total RNA. Lanes 1-5, RNA extracted directly from 5 different OA patients; lanes 6 and 7, RNA extracted from cultivated articular OA chondrocytes, without (lane 6) and with (lane 7) 50 pg/ml of IL-16. Hybridization with a labeled 707-bp probe specific for MT1-MMP (lanes 1-7) resulted in a detection of a 4.5-kb MT1-MMP transcript. p-actin or GAPDH was used as a control for loading equal amounts of RNA. For all tissues, the variation in expression did not appear to be related to heterogeneity of chondrocyte density. While some of the OA cartilage had reduced A numbers of chondrocytes compared with the normal cartilage, the observed variation related to the number of grains over the chondrocytes. Within any 1 field, not R Figure 3. In situ hybridization. Dark field photomicrographs of A, osteoarthritic cartilage or B, chondrosarcoma tissue, showing hybridization with radiolabeled antisense primer of primer set B (original magnification X 112.5). BUTTNER ET AL 708 all chondrocytes were positive: of the positive chondrocytes in the same field, there appeared to be the same approximate number of grains. But the number of grains over the positive cells varied among patients and donors. In situ hybridization with the chondrosarcoma tissue showed strong expression of mRNA for MT1-MMP (Figure 3B). DISCUSSION The results of this study clearly show that MT1MMP is expressed in human articular cartilage. Message was detected by RT-PCR and Northern blot analyses in RNA extracted from chondrosarcoma cells and from OA chondrocytes. The fragments generated by the RTPCR corresponded to the size expected for the amplified products of MT1-MMP. The full-length message for MTl-MMP was cloned and sequenced. The sequence was identical to that reported by Sat0 and Okada and colleagues (2,12). Using Northern blot analyses, an mRNA of 4.5 kb was detected-the same size for the MT1-MMP mRNA reported by Sat0 and Takino et a1 (2,13). Finally, the technique of in situ hybridization was employed to confirm the results obtained from Northern blot analyses showing that mRNA for MTl-MMP was present in OA chondrocytes. This technique also provided information that mRNA levels comparable to those in OA chondrocytes were detectable in chondrocytes from normal donors. In addition, the mRNA levels in cartilage both from OA patients and from normal donors appeared similar in superficial-, middle-, and deep-layer chondrocytes. In the OA cartilage where the superficial layer was disrupted or absent, mRNA levels for MT1-MMP did not appear to be elevated in the middle and deep layers; an elevation has been reported for other MMPs (11). In the chondrosarcoma tissue, mRNA expression was stronger than those found in the cartilage. Stimulation with the catabolic cytokine, IL-lp, did not appear to modulate the expression levels of mRNA for MT1-MMP over the concentration range tested (0.05-50 pg/ml) for the explant cultures using the technique of in situ hybridization or at 50 pg/ml for the Northern blot analyses. These concentrations have previously been shown to be effective in up-regulating mRNA for MMP-8 (neutrophil collagenase) (11,14). There are several possible explanations for the lack of modulation by IL-1P. First, the concentrations may not have been appropriate for gene stimulation. Second, it is not clear whether there is an IL-1P-responsive element in the regulatory sequences of this gene. Third, the influence of IL-lP may be at the protein level, control- ling the stability of the mRNA. Finally, the IL-1P influence may be on the translation efficiency for the MT1-MMP mRNA. The actual pathway site at which IL-1p exerts its control is a subject for future studies. There appeared to be no difference between the level expression of mRNA for MT1-MMP in cartilage from normal donors or from patients with end-stage OA. However, if MT1-MMP is constitutivelyproduced by the chondrocytes, the regulation of degradative enzymes may reside at a point other than the regulation of the MT1-MMP message, for example, at the level of the activation of the proenzyme. Studies involving the trimolecular complex of MT1-MMP, TIMP-2, and gelatinase A ( 3 ) may be necessary before a full understanding of the regulation of the proteolytic activity of MT1MMP is possible. The finding that MT1-MMP transmembrane domain-deficient mutants degrade extracellular matrix proteins such as gelatin, fibronectin, laminin, vitronectin, and dermatan sulfate proteoglycan ( 5 ) further stresses the potential importance of MT1MMP in cartilage-degrading processes common to degenerative joint diseases like osteoarthritis. REFERENCES 1. Woessner J F Jr: Matrix metalloproteinase and their inhibitor in connective tissue remodeling. FASEB J 5:2145-2154, 1991 2. Sat0 H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seiki M: A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370:61-65, 1994 3. Strongin AY, Collier I, Bannikov G, Marmer BL, Grants GA, Goldberg GI: Mechanism of cell surface activation of 72-kDa type 1%' collagenase. J Biol Chem 270:5331-5338, 1995 4. Cao J, Sato H, Takino T, Seiki M: The C-terminal region of membrane type matrix metalloproteinase is a functional transmembrane domain required for pro-gelatinase A activation. J Biol Chem 2705301-805, 1995 5. Pei D, Weiss SJ: Transmembrane-deletion mutants of the membrane-type matrix metalloproteinase-1 process progelatinase A and express intrinsic matrix-degrading activity. J Biol Chem 271:9135-9140, 1996 6. Yu LP Jr, Smith GN Jr, Brandt KD, Capello W: Type XI collagen-degrading activity in human osteoarthritic cartilage. Arthritis Rheum 33:1626-1633, 1990 7. Hauselmann HJ, Opplinger L, Michel BA, Stefanovic-Racic M, Evans CH: Nitric oxide and proteoglycan biosynthesis by human articular chondrocytes in alginate culture. FEBS Lett 352:361-364, 1994 8. Campbell IK, Piccoli DS, Hamilton JA: Stimulation of human chondrocyte prostaglandin E2 production by recombinant human interleukin-1 and tumor necrosis factor. Biochem Biophys Acta 1051:310-318, 1990 9. Hauselmann HJ, Aydelotte MB, Schumacher BL, Kuettner KE, Gitelis SH, Thonar EJ: Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads. Matrix 12:116-129, 1992 10. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem 162:156-159, 1987 MT1-MMP IN HUMAN ARTICULAR CARTILAGE 11. Chubinskaya S, Huch K, Mikecz K, Cs-Szabo G, Hasty KA, Kuettner KE, Cole AA: Chondrocyte matrix metalloproteinase-8: up-regulation of neutrophil collagenase by interleukin-l/3 in human cartilage from knee and ankle joints. Lab Invest 74:232-240, 1996 12. Okada A, Bellocq JP, Rouyer N, Chenard MP, Rio MC, Chambon P, Basset P: Membrane-type matrix metalloproteinase (MTMMP) gene is expressed in stromal cells of human colon, breast, and head and neck carcinomas. Proc Natl Acad Sci U S A 92~2130-2734,1995 13. Takino T, Sato H, Yamamoto E, Seiki M: Cloning of a human gene potentially encoding a novel matrix metalloproteinase having a C-terminal transmembrane domain. Gene 155:293-298, 1995 14. Cole AA, Chubinskaya S, Schumacher B, Huch K, Cs-Szabo G , Yao J, Mikecz K, Hasty KG Kuettner KE: Chondrocyte matrix metalloproteinase-8. J Biol Chem 271:11023-11026, 1996 Clinical Images: Severe destructive P,-microglobulin arthropathy after 28 years of hemodialysis The patient, a 58-year-old man with a 28-year history of hemodialysis, was admitted to our hospital because of recurrent hemarthrosis of the right shoulder (A). The joint had had to be aspirated with increasing frequency over the preceding months to relieve pain, despite joint immobilization and antiphlogistic therapy. Blood loss from a 150-250-m1 hemorrhagic effusion at every arthrocentesis had to be replaced. Radiography and magnetic resonance imaging (B) revealed erosions, hypertrophic pannus-like material in synovium, and massive joint effusion (H = humerus; arrow 1 = erosion; arrow 2 = hypertrophic pannus). Synovectomy was performed. Histologic evaluation of the synovium with hematoxylin and eosin staining showed marked hypervascularization, cellular infiltrates, and hemorrhagic and hyaline structures (C). Congo red staining was diagnostic of amyloidosis. The green birefringence seen on Congo red staining with polarizing microscopy (D) documented &-microglobulin, which was confirmed by immunohistologic staining (not shown). After synovectomy, the patient was discharged and has had no recurrence of hemarthrosis to date. We are indebted to Dr. B. Kreklau and Prot R. Ramanzadeh for per$orming the surgery, Dr. N. Prosenc for immunohistologic analyses, and Dr. 0. Liangos for help with the clinical records. H. Appel, MD J. Sieper, MD A. Distler, MD J. Braun, MD Klinikum Benjamin Franklin Free University Berlin, Germany
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