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CD30 Shedding from Karpas 299 Lymphoma Cells Is Mediated by TNF--Converting Enzyme¹

Hinrich P. Hansen^2,*, Sebastian Dietrich^*, Tatiana Kisseleva^3,*, Thilo Mokros^*, Rolf Mentlein, Hans H. Lange^*, Gillian Murphy and Hilmar Lemke^*

Departments of ^* Biochemistry and Anatomy, University of Kiel, Kiel, Germany; and School of Biological Science, University of East Anglia, Norwich, United Kingdom

	Abstract

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CD30 is a costimulatory receptor on activated lymphocytes and a number of human lymphoma cells. Specific ligation of membrane-bound CD30 or cellular stimulation by PMA results in a metalloproteinase-mediated down-regulation of CD30 and release of its soluble ectodomain (sCD30). In this report, it is demonstrated that PMA-induced CD30 cleavage from Karpas 299 cells was mediated by a membrane-anchored metalloproteinase which was active on intact cells following 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate extraction of membrane preparations. Moreover, CD30 shedding was blocked by the synthetic hydroxamic acid-based metalloproteinase inhibitor BB-2116 (IC₅₀, 230 nM) and the natural tissue inhibitor of metalloproteinases (TIMP)-3 (IC₅₀, 30 nM), but not by the matrix metalloproteinase inhibitors TIMP-1 and TIMP-2. This inhibition profile is similar to that of the TNF-- converting enzyme (TACE) and, indeed, mRNA transcripts of the membrane-bound metalloproteinase-disintegrin TACE could be detected in Karpas 299 cells. The ectodomain of TACE was expressed in bacteria as a GST fusion protein (GST-TACE) which cleaved CD30 from the surface of Karpas 299 cells and concomitantly increased the level of sCD30 in the cell supernatants. Hence, TACE does not only control the release of TNF-, but also that of sCD30.

	Introduction

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The CD30 is a receptor that has been detected on a subset of activated T and B cells and a variety of human lymphomas (1, 2). Cloning of its cDNA characterized CD30 as a member of the TNFR superfamily (3). The CD30 ligand (CD30L)⁴ is expressed on activated T cells, resting B cells, monocytes, and granulocytes and established as a member of the TNF superfamily (4, 5). The CD30-CD30L interaction induces pleiotropic biological effects on CD30⁺ cells, including activation, proliferation, differentiation, and cell death, depending on cell type and accessible other stimuli (6). Soluble CD30 (sCD30) is released from the cells by proteolytic cleavage of CD30. This CD30 shedding is enhanced by interaction with the CD30L⁺ cells and the resulting sCD30 is proposed to reduce the CD30L-dependent activation of CD30⁺ cells (7).

The ectodomains of many membrane proteins can be released proteolytically from the cell by receptor stimulation or in response to protein kinase C activators such as PMA. As demonstrated for the angiotensin-converting enzyme, the Alzheimer’s amyloid precursor protein, pro-TNF-, the Fas ligand, CD16, L-selectin, the TNFRs, CD30, CD43, CD44, and the IL-6R, the endoproteolytic conversion of membrane-anchored proteins is blocked by broad-spectrum hydroxamate inhibitors of matrix metalloproteinases (MMP). This indicates that MMPs or enzymes related to MMPs are responsible (8, 9, 10). The TNF--converting enzyme (TACE; ADAM 17), responsible for the release of TNF- by proteolytic cleavage of the membrane-associated precursor form (pro-TNF-), was recently characterized as a membrane-anchored metalloproteinase-disintegrin (ADAM, a metalloproteinase and disintegrin) (11, 12). Metalloproteinase-disintegrins belong to the metzincin superfamily of metalloproteinases encompassing the physiologically important families of MMPs, reprolysins and astacins. Due to their x-ray crystal structure, metalloproteinase-disintegrins are closely related to snake venom metalloproteinases (reprolysins) and are therefore regarded as members of the reprolysin family of metalloproteinases (13). Both snake venom proteinases and metalloproteinase-disintegrins exhibit membrane protein sheddase activity through their catalytic domain (11, 12, 14) and binding to cellular disintegrin receptors mediated by the disintegrin domain (15, 16, 17). Besides pro-TNF-, TACE cleaves the amyloid precursor protein (18) and mediates the shedding of L-selectin, the p75 TNFR, and TGF- (16). Other metalloproteinase-disintegrins such as the Drosophila ADAM 10 homologue KUZ and ADAM 9 have been found to cleave the Notch receptor or the membrane-anchored heparin-binding EGF-like growth factor, respectively (19, 20). Physiologically, the activity of metalloproteinases is regulated by tissue inhibitors of metalloproteinases (TIMPs). Although the activity of MMPs is blocked by TIMP-1, -2, and -3 (21), TACE is only inhibited by TIMP-3 (22). Hence, the shedding of TNF- and L-selectin is inhibited by TIMP-3 and not by TIMP-1 and -2 (23, 24, 25).

In the present study, we characterized the CD30-cleaving enzyme found to be membrane-associated and inhibited by the TACE inhibitor TIMP-3. Since TACE-specific mRNA was detected in CD30⁺ Karpas 299 cells, we describe the bacterial expression of the TACE ectodomain. Its potency to cleave CD30 was compared with that of ADAM 10 and the related snake venom metalloproteinase hemorrhargin.

	Materials and Methods

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Materials

The mAbs Ki-2 (1,) and Ki-3 (2b,) were used for detection of cell-bound and sCD30 (26). The anti-2-phenyloxazolone (anti-phOx) Ab BH-1 (2b,) served as an isotype-matched control in flow cytometry (27). OKT-3 (anti-CD3 mAb) was obtained from American Type Culture Collection (Manassas, VA). The tissue inhibitors of metalloproteinases TIMP-1, TIMP-2, and TIMP-3 and the recombinant ADAM 10 were from G.M. (Norwich, U.K.). BB-2116 (N¹-(5-acetylamino-1S-methylcarbamoyl-pentyl)-2S-(2,2-dimethyl-propyl)-N⁴-hydroxy-3R-(4-hydroxy-phenylsulfanyl-methyl)-succinamide) was a kind gift from British Biotech Pharmaceuticals (Oxford, U.K.). Hemorrhargin from Echis pyramidum laekeyi was donated by Gavin Laing (Liverpool, U.K.). The large cell anaplastic lymphoma cell line Karpas 299 was a kind gift from Abraham Karpas (Cambridge, U.K.). The cells were cultured at 37°C and 7% CO₂ in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Stimulation of human lymphocytes

PBLs were isolated from buffy coat preparations of normal donors. The lymphocytes were purified by Ficoll density centrifugation (1.077 g/ml; Seromed, Berlin, Germany) and cultivated for 1 h in complete RPMI 1640 medium/10% FCS. Nonadherent lymphocytes were cultivated in complete RPMI 1640 medium/10% FCS + 100 U/ml IL-2 (10⁶/ml cells) for 72 h in the absence or presence of PHA (0.1% phytohemagglutinin P; Difco, Detroit, MI) and immobilized OKT3 (anti-CD3-mAb), respectively. For the OKT3-induced activation, dishes were coated with 10 µg/ml OKT3 for 1 h at room temperature and washed thoroughly before lymphocyte stimulation.

Membrane extraction

Karpas 299 cells were cultivated with or without PMA (30 ng/ml, 30 min) and lysed by ultrasonication (5 min) on ice. A crude membrane fraction was obtained by centrifugation (50,000 x g, 2 h, 4°C) which was resuspended by ultrasonication (2 min on ice) in 2 ml of 0.14 M NaCl and 20 mM HEPES (pH 7.4). Aliquots were then incubated under shaking with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Sigma, St. Louis, MO) for 2 h at 4°C. The mixture was then centrifuged (50,000 x g, 3 h, 4°C) and supernatants were intensively dialyzed against 140 mM NaCl and 20 mM HEPES (pH 7.4) and then collected as solubilizate.

Cloning of the human TACE ectodomain and its expression as GST-TACE fusion protein

Total RNA from Karpas 299 cells was extracted with Trizol reagent (Life Technologies, Karlsruhe, Germany), digested with RNase-free DNase I (Roche Diagnostics, Mannheim, Germany), and cDNA synthesis was performed using a SuperScript Preamplification System and oligo(dT)_12–18 primers (Life Technologies). For an amplification of the cDNA fragment encoding the catalytic, disintegrin, and cysteine-rich domain of TACE, total cDNA was subjected to 30 cycles of PCR using proofreading polymerase (Elongase Enzyme mix; Life Technologies). As specific primers, which were 5' extended to allow restriction enzyme cleavage, we used the BamHI-TACE sense primer (5'-GTGAAAAGGATCCTTGCTGACCCAGATCCCATGAAGAACACG and the EcoRI-TACE antisense primer (5'-AGTGCTAGAATTCTACATCCTGTACTCGTTTCTCACATTTGCC. The sequences were derived from GenBank accession number U69611. The PCR product was purified by UV band elution (28) and restricted by BamHI and EcoRI enzymes (Roche Diagnostics) before the in-frame ligation with T4 DNA ligase (New England Biolabs, Beverly, MA) into the BamHI-EcoRI-restricted multiple cloning site of the pGEX-3X expression vector (Amersham Pharmacia Biotech, Freiburg, Germany). The insert of one clone was subsequently sequenced using an Abi Prism 310 Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany). Then the recombinant expression vector was transformed into competent Escherichia coli cell strain XL1-blue and grown on ampicillin-containing agar plates. PCR-tested colonies were used to inoculate 2-ml cultures of Luria-Bertani medium containing 100 µg/ml ampicillin and 1 ml of this culture was used to inoculate an ampicillin-containing 500-ml culture. When the culture reached an OD (A₆₀₀) of 0.5, the expression was induced by addition of isopropyl-1-thio-ß-D-galactopyranoside (1.5 mM final concentration) and the cells were incubated for 5 h more at 37°C. GST-TACE was obtained from the inclusion bodies and solubilized in 20 mM Tris-HCl (pH 8) containing 8 M urea and 100 mM 2-ME. The solubilized proteins were refolded by a 1:100 dilution in either 1) standard refolding buffer (20 mM Tris-HCl (pH 7.6) containing 200 mM NaCl, 5 mM CaCl₂, 100 µM ZnCl₂, 100 mM arginine, and 0.002% NaN₃) or 2) in this buffer which was further supplemented with 1 mM DTT and 3 mM oxidized glutathione (GSSG) (oxido-shuffling buffer). After incubation for 3 days at 4°C, the crude extract was diluted with the same volume of ice-cold water and the recombinant proteins were purified on glutathione-Sepharose (Amersham Pharmacia Biotech). The elution of GST-TACE was conducted with 10 mM of reduced glutathione in PBS.

Flow cytometry

Membrane-bound proteins were determined by direct immunofluorescence analysis. Cells (3 x 10⁵) were labeled with saturating amounts of FITC-conjugated Abs Ki-3-FITC (anti-CD30), anti-CD25-FITC (Dako, Hamburg, Germany), or BH1-FITC (anti-phOx) in PBS containing 0.1% BSA and 0.02% sodium azide for 20 min on ice. Cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany).

Determination of sCD30

Flexible 96-well microtiter plates (Integra Bioscience, Fernwald, Germany) were coated with 50 µl of Ki-2 mAb (50 µg/ml) by overnight incubation at 4°C. The plates were washed and subsequently blocked with PBS/10% FCS for 1 h at room temperature. After washing (three times with PBS/0.1% Tween 20), serial dilutions of both the CD30 standard (1–300 U/ml) (29) and the sCD30-containing samples were added and incubated for 1 h at room temperature. After washing,¹²⁵I-labeled Ki-3 mAb was added and incubated for 1 h and, after washing again, the radioactivity of the single wells was determined and compared with the internal standard.

Gelatin zymography

TACE was analyzed by SDS-polyacrylamide gel zymography. Samples were prepared in conventional nonreducing loading buffer without boiling and run on 10% polyacrylamide gels containing 0.1% gelatin. The gels were then washed twice for 15 min in 2.5% Triton X-100 to remove SDS, followed by a 20-min wash in metalloproteinase buffer (50 mM Tris-HCl (pH 7.6), 5 mM CaCl₂, 100 µM ZnCl₂, and 0.02% NaN₃). After a further incubation period for 20 h at 37°C in this buffer, the gels were stained with Coomassie brilliant blue. A clear zone indicated the presence of a proteinase with gelatinolytic activity.

	Results

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Hydroxamate inhibitor blocks PMA-induced CD30 shedding on normal and tumor cells

CD30 is expressed on CD30⁺ lymphoma cell lines, e.g., the Hodgkin’s disease derived cell line L540 and the large cell anaplastic lymphoma cell line Karpas 299 as well as on normal PBL after stimulation with PHA or anti-CD3 Ab (OKT-3). Constitutive shedding of CD30 was detected from all cells. This endoproteolytic release of the CD30 ectodomain (sCD30) was strongly activated by PMA, resulting in a loss of membrane-bound CD30 detected by flow cytometry and a corresponding increase of sCD30 in the supernatant detected by an CD30 Ab-based sandwich RIA (Fig. 1, A and C). The FITC-labeled 2-phOx Ab BH1 (BH1-FITC), which served as an isotype control in flow cytometry, showed no binding to any of the tested cells (Fig. 1B). PMA-induced shedding of CD30 was effectively blocked by the hydroxamic acid-based broad-spectrum MMP inhibitor BB-2116 (20 µM), whereas inhibition of constitutive shedding varied depending on the cell type. Although BB-2116 blocked the constitutive sCD30 release from tumor cells, the release from activated normal cells was hardly affected. These data showed that PMA-induced shedding was mediated by metalloproteinases; however other proteinases might be involved in constitutive shedding from normal cells.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Inhibition of CD30 shedding by hydroxamate inhibitor. Cells (106/ml) were incubated for 90 min in the absence or presence of PMA (30 ng/ml) with or without BB-2116 (20 µM). Cells used were: The CD30+ cell lines Karpas 299 and L540, CD30- normal PBL, PHA-stimulated PBL, and OKT3-stimulated PBL (anti-CD3 PBL). A, Membrane-bound CD30 was determined by FACS analysis using FITC-labeled Ki-3 mAb, B, FITC-labeled anti-phOx mAb was used as an isotype control. The results are given as means of the MFI for triplicate determinations. C, sCD30 was determined in the cell supernatants. The results show units per milliliter as means ± SD for triplicate determinations.

To decide whether PMA-induced CD30 cleavage was mediated by a secreted or a membrane-anchored metalloproteinase, the CD30-cleaving activities of supernatant fluids and membrane extracts of PMA-stimulated Karpas 299 cells were compared. Although a direct incubation of Karpas 299 cells with PMA (30 ng/ml, 30 min) induced a loss of membrane-bound CD30 to 65% of the untreated control, an incubation of the cells for 1 h with the supernatant of PMA-activated Karpas cells (30 ng/ml, 30 min) had no influence on the CD30 expression (Fig. 2). For the determination of CD30-cleaving activity in cell membranes, membrane fractions of PMA-activated Karpas 299 cells were extracted with the solubilizing reagent CHAPS. The extract induced a loss of CD30 to 57% of the dialysis buffer-incubated control (buffer). Interestingly, membrane extracts of nonstimulated cells exhibited CD30 sheddase activity as well. This activity was weaker but still capable to induce a reduction of CD30 to 77% of the untreated control. As with PMA, these membrane effects were dependent on metalloproteinases, since BB-2116 (20 µM) abolished the CD30-cleaving activity. The CHAPS-extracted membrane preparation of PMA-activated Karpas 299 cells also induced a loss of CD30 on PHA-activated normal PBLs, but had no influence on the expression of CD25 on both cells (data not shown). These data suggest that PMA-stimulated CD30 shedding was mediated by endogenous membrane-anchored metalloproteinases that could be activated or up-regulated by PMA. Moreover, membrane anchoring via the metalloproteinase transmembrane domain is not essential for activity in this system.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Localization of CD30 sheddase. Karpas 299 cells (1 x 106/ml) were incubated for 30 min with 30 ng/ml PMA (PMA Karpas) or not stimulated (Karpas). Cell supernatant and membranes were prepared as described in Materials and Methods. Membranes were extracted with 1% CHAPS. Supernatant (SN30) and membrane preparations of nonstimulated and PMA-activated cells were applied to untreated Karpas 299 cells with or without 20 µM BB-2116 and incubated for 1 h at 37°C. A, CD30 expression was determined by FACS analysis using FITC-labeled Ki-3 mAb. B, FITC-labeled anti-phOx mAb was used as an isotype control. The results are given as means of the MFI for triplicate determinations.

For a characterization of the responsible enzyme(s), the sensitivity of the PMA-induced CD30 shedding for a variety of metalloproteinase inhibitors was tested: 1) The efficacy of the broad-spectrum hydroxamate MMP inhibitor BB-2116 (20 µM) to prevent CD30 cleavage (10) ( Figs. 1–3) suggested the involvement of MMPs or related enzymes in the shedding of CD30. 2) In contrast, the natural inhibitors TIMP-1 (10 µg/ml) and TIMP-2 (10 µg/ml), which inhibit soluble and membrane-type MMPs (21, 30), could not prevent the PMA-induced CD30 shedding (Fig. 3A). This does not support an involvement of MMPs. 3) Phosphoramidon (10 µM) and captopril (10 µM), which are known inhibitors of thermolysin, neprilysin, and angiotensin-converting enzyme, respectively, also failed to influence CD30 cleavage. 4) Like BB-2116, TIMP-3 inhibited the PMA-induced CD30 shedding from Karpas 299 cells in a dose-dependent manner (Fig. 3B). The inhibitory potency of TIMP-3 (IC₅₀, 30 nM) was 8-fold higher than that of BB-2116 (IC₅₀, 230 nM). A similar inhibition by TIMP-3 was obtained when we analyzed CD30 shedding from CD30⁺ normal cells (data not shown). Since the MMP inhibitor TIMP-3 potently blocks the membrane-anchored metalloproteinase-disintegrin TACE (22), our data provide evidence for a role of TACE or a TACE-like proteinase in CD30 shedding.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. Inhibition of PMA-induced CD30 down-regulation. A, Karpas 299 cells (1 x 106/ml) were incubated for 1 h with 30 ng/ml PMA in the presence of the following metalloproteinase inhibitors: BB-2116 (20 µM), phosphoramidon (10 µM), captopril (10 µM), TIMP-1 (10 µg/ml), TIMP-2 (10 µg/ml), and TIMP-3 (800 nM). CD30 expression was determined by FACS analysis. sCD30 was determined in the cell supernatants. The results show units per milliliter as means ± SD for triplicate determinations. The sCD30 concentrations in the supernatants of untreated or only PMA-stimulated cells are indicated by dotted lines. B, The IC50 of TIMP-3 and BB-2116 was determined.

TACE-specific mRNA transcripts were detected by RT-PCR from PMA-activated Karpas 299 cells and CD30⁺ PBLs. Using TACE-specific primers, a 1299-bp cDNA fragment was amplified from both sources as shown for Karpas 299 (Fig. 4). The purified 1299-bp fragment from Karpas 299 cells, encoding the catalytic, disintegrin, and cysteine-rich domain of TACE, was cloned into the pGEX-3X GST gene fusion vector. Sequencing confirmed that it was identical with the published mRNA sequence of TACE derived from the GenBank accession number U69611 (data not shown).

View larger version (37K): [in this window] [in a new window]   FIGURE 4. RT-PCR of TACE mRNA. RT-PCR amplification of a cDNA fragment coding for the TACE ectodomain using mRNA from Karpas 299 cells. The arrow indicates the 1299-bp PCR-product.

The TACE ectodomain was expressed in bacteria as a GST fusion protein (GST-TACE) (Fig. 5A). Crude extracts from the insoluble fraction of the transfected bacteria were either refolded by 1) a standard dilution protocol that allowed the renaturation of the GST tag, but not of the cysteine-rich TACE ectodomain (GST-TACE(st)) or 2) dilution into the oxido-shuffling system which facilitates the formation of proper disulfide bonds after denaturing extraction (GST-TACE(ox)). After affinity purification on reduced glutathione-Sepharose, homogeneous GST-TACE was obtained from both preparations (Fig. 5B). From the oxido-shuffling buffer, a lower amount of material could be recovered, yielding about 20–30% of that from the standard refolded preparation. This might be due to the glutathione (GSSG) in the oxido-shuffling buffer which might interact with the subsequent purification procedure. However, the proteinase activity of GST-TACE(ox) recovered from the oxido-shuffling buffer was 100-fold higher, since 10 ng elicited a gelatinolytic activity equivalent to 1 µg of GST-TACE(st) in gelatin zymography (Fig. 5C). Hence, the two refolding procedures allowed the preparation of an active and inactive conformation of the TACE ectodomain.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Bacterial expression of TACE. A, Schematic representation of the GST-TACE fusion protein. B, SDS-PAGE analysis of the bacterial expressed GST-TACE fusion protein that was purified on glutathione-Sepharose, after refolding by dilution in the presence of GSSG/DTT (lane 1, GST-TACE(ox)) or in the absence of GSSG/DTT (lane 2, GST-TACE(st)). Control, Wild-type GST was refolded and purified (lane 3). C, Gelatinolytic activity of GST-TACE. Ten nanograms of affinity-purified GST-TACE(ox) (lane 1), 1 µg of GST-TACE(st), and 1 µg of wild-type GST (lane 3) were analyzed by gelatin zymography.

View larger version (42K): [in this window] [in a new window]   FIGURE 6. CD30 shedding by GST-TACE. Karpas 299 cells (106/ml) were incubated for 1 h with active GST-TACE(ox), inactive GST-TACE(st), ADAM 10 or hemorrhargin in concentrations, as indicated, with or without 20 µM BB-2116. A and B, Cell surface expression of CD30 was determined by flow cytometry using FITC-labeled Ki-3 mAb. C and D, FITC-labeled anti-phOx mAb was used as an isotype control. The results are given as means of the MFI ± SD for triplicate determinations. E and F, sCD30 was determined in the cell supernatants. The results show units per milliliter as means ± SD for triplicate determinations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Pro-TNF-, L-selectin, and the p75 TNFR are released by TACE which is a membrane-anchored metalloproteinase-disintegrin (ADAM) and belongs, along with soluble snake venom metalloproteinases, to the reprolysin family of metalloproteinases (11, 12, 16). TACE is strongly inhibited by TIMP-3, but not affected by the MMP inhibitors TIMP-1 and -2 (22). The strong inhibition of CD30 shedding from Karpas 299 cells by TIMP-3 and the inability of TIMP-1 and -2 suggested that TACE might be responsible for the cleavage of CD30 as well. This assumption was further substantiated: 1) It could be demonstrated that CD30 shedding was mediated by a membrane-associated metalloproteinase since solubilized membrane extracts of Karpas 299 cells were able to cleave CD30 at the cell surface of intact cells. 2) TACE-specific mRNA transcripts were detected in Karpas 299 cells by means of RT-PCR. 3) It could be demonstrated that the recombinant fusion protein containing GST and the TACE ectodomain (GST-TACE) elicited CD30 sheddase activity in a dose-dependent manner. These findings demonstrated that TACE is expressed by Karpas 299 cells and is able to cleave membrane-bound CD30. The CD30-cleaving activity of the construct was comparable to that of the snake venom metalloproteinase hemorrhargin, both belong to the reprolysin family of metalloproteinases. Since hemorrhargin is a TNF--releasing enzyme (14), the cleaving of CD30 serves to corroborate the common releasing mechanism.

Regarding the related shedding of TNF-, it has not been clarified whether pro-TNF- is exclusively cleaved by TACE on all cell types since inactivation of the TACE gene in mouse T cells caused a marked decrease but not complete inhibition of TNF- release (11). ADAM 10, another membrane-anchored metalloproteinase-disintegrin closely related to TACE, was also able to cleave pro-TNF- (40, 41) from the cell surface, indicating that other metalloproteinases might play a role. Similarly, sCD30 was not only released by TACE but also by ADAM 10, although less effectively. Since we detected mRNA of both TACE and ADAM 10 (data not shown) in CD30⁺ Karpas 299 cells, the question arises whether TACE represents the only physiologically relevant CD30-cleaving enzyme. The data from direct application of the recombinant ADAMs do not clearly answer this question. The evaluation of their inhibition profile appears to be more promising. Although inhibition data on ADAMs are incomplete, TIMPs play a role in distinguishing different ADAMs. TACE is inhibited by TIMP-3 and not by TIMP-1 and 2, whereas both TIMP-1 and -3 inhibit ADAM 10 (22, 42). Thus, the inhibition of CD30 shedding by TIMP-3 and not by TIMP-1 and -2 argues in favor of TACE as the physiologically relevant CD30-cleaving metalloproteinase. TACE and ADAM 10 are structurally very similar and differ from other known ADAMs (13, 40, 41). Nonetheless, catalytic activity has also been described for two other metalloproteinase-disintegrins, i.e., ADAM 12 and ADAM 9 (MDC 9) (43, 44). The inhibition profile of ADAM 12 appears to be completely different from that of TACE since neither the hydroxamate inhibitor BB-94 nor TIMP-1, -2, and -3 were effective (45). Inhibition data on MDC 9 are not yet available. Hence, although inhibition data suggest a role of TACE in CD30 shedding, the involvement of so far uncharacterized TACE-related enzymes cannot be excluded.

	Acknowledgments

 
We thank M. Burmester for excellent assistance with membrane preparations.

	Footnotes

 
¹ This research was supported by a grant from Haensel Stiftung (to H.H.) and a grant from the Bundesministerium für Bildung und Forschung (to H.L.).

² Address correspondence and reprint requests to Dr. Hinrich Hansen, Department of Biochemistry, University of Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany.

³ Current address: Department of Microbiology, Columbia University, New York, NY.

⁴ Abbreviations used in this paper: CD30L, CD30 ligand; ADAM, a metalloproteinase and disintegrin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; GSSG, oxidized glutathione; MFI, mean fluorescence intensity; MMP, matrix metalloproteinase; phOx, phenyloxazolone; sCD30, soluble CD30; TACE, TNF--converting enzyme; TIMP, tissue inhibitor of metalloproteinases.

Received for publication July 12, 1999. Accepted for publication September 7, 2000.

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