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Mol Biol Cell. Mar 15, 2009; 20(6): 1785–1794.
PMCID: PMC2655247

ADAMs 10 and 17 Represent Differentially Regulated Components of a General Shedding Machinery for Membrane Proteins Such as Transforming Growth Factor α, L-Selectin, and Tumor Necrosis Factor α

Benjamin Margolis, Monitoring Editor

Abstract

Protein ectodomain shedding is a critical regulator of many membrane proteins, including epidermal growth factor receptor-ligands and tumor necrosis factor (TNF)-α, providing a strong incentive to define the responsible sheddases. Previous studies identified ADAM17 as principal sheddase for transforming growth factor (TGF)-α and heparin-binding epidermal growth factor, but Ca++ influx activated an additional sheddase for these epidermal growth factor receptor ligands in Adam17−/− cells. Here, we show that Ca++ influx and stimulation of the P2X7R signaling pathway activate ADAM10 as sheddase of many ADAM17 substrates in Adam17−/− fibroblasts and primary B cells. Importantly, although ADAM10 can shed all substrates of ADAM17 tested here in Adam17−/− cells, acute treatment of wild-type cells with a highly selective ADAM17 inhibitor (SP26) showed that ADAM17 is nevertheless the principal sheddase when both ADAMs 10 and 17 are present. However, chronic treatment of wild-type cells with SP26 promoted processing of ADAM17 substrates by ADAM10, thus generating conditions such as in Adam17−/− cells. These results have general implications for understanding the substrate selectivity of two major cellular sheddases, ADAMs 10 and 17.

INTRODUCTION

All ligands of the epidermal growth factor receptor (EGFR) are made as membrane-anchored precursors, which must be released from their membrane tether to activate the EGFR in a paracrine manner (for reviews, see Harris et al., 2003 blue right-pointing triangle; Blobel, 2005 blue right-pointing triangle). The EGFR has critical roles in development and in diseases such as cancer (Yarden and Sliwkowski, 2001 blue right-pointing triangle; Gschwind et al., 2004 blue right-pointing triangle), so it is important to understand how ligand availability is regulated and which enzymes are responsible for releasing EGFR ligands under various conditions. Moreover, many other membrane proteins are proteolytically released or shed from cells, including other growth factors, cytokines, and receptors (Hooper et al., 1997 blue right-pointing triangle). Because the sheddases for other membrane proteins commonly have similar properties as EGFR ligand sheddases (e.g., Zheng et al., 2004 blue right-pointing triangle; Weskamp et al., 2006 blue right-pointing triangle; Kawaguchi et al., 2007 blue right-pointing triangle), insight about the identity and properties of sheddases gained from studying shedding of EGFR ligands usually has general relevance, because they can provide a framework for understanding which enzymes are responsible for shedding of other membrane proteins, and how these enzymes are regulated.

Two membrane-anchored metalloproteinases, ADAMs 10 and 17, have emerged as key molecules in most of the shedding events characterized to date (for recent examples, see Weskamp et al., 2006 blue right-pointing triangle; Chen et al., 2007 blue right-pointing triangle; Kawaguchi et al., 2007 blue right-pointing triangle; Li et al., 2007 blue right-pointing triangle), including in the release of EGFR ligands (Peschon et al., 1998 blue right-pointing triangle; Jackson et al., 2003 blue right-pointing triangle; Hinkle et al., 2004 blue right-pointing triangle; Sahin et al., 2004 blue right-pointing triangle; Horiuchi et al., 2007b blue right-pointing triangle; Sahin and Blobel, 2007 blue right-pointing triangle). Studies on the contribution of these two enzymes to constitutive and phorbol ester-stimulated EGFR ligand shedding have revealed a distinct substrate selectivity of ADAM10 and ADAM17 and also defined characteristic properties of these enzymes (Sahin et al., 2004 blue right-pointing triangle; Horiuchi et al., 2007b blue right-pointing triangle). “Loss of function” studies with cells lacking different ADAMs demonstrated that ADAM17 is the principal constitutive and phorbol ester-stimulated sheddase for transforming growth factor (TGF)-α, amphiregulin (AR), epiregulin (EPR), epigen, and heparin-binding epidermal growth factor (HB-EGF) (Peschon et al., 1998 blue right-pointing triangle; Jackson et al., 2003 blue right-pointing triangle; Sahin et al., 2004 blue right-pointing triangle; Sahin and Blobel, 2007 blue right-pointing triangle). The physiological relevance of this enzyme for activation of these EGFR ligands during development was corroborated through studies of mice lacking ADAM17, which resemble mice lacking TGF-α, HB-EGF, AR, or EGFR (Peschon et al., 1998 blue right-pointing triangle; Jackson et al., 2003 blue right-pointing triangle; Sternlicht et al., 2005 blue right-pointing triangle). ADAM10, in contrast, is responsible for the constitutive shedding of EGF and betacellulin (BTC), and its activity toward these substrates is not strongly enhanced by stimulation with phorbol 12-myristate 13-acetate (PMA) but instead can be stimulated by calcium influx (Sahin et al., 2004 blue right-pointing triangle; Horiuchi et al., 2007b blue right-pointing triangle). Mice lacking ADAM10 die very early during embryogenesis, most likely due to defects in Notch signaling (Hartmann et al., 2002 blue right-pointing triangle), so the contribution of ADAM10 to EGF and BTC signaling in development and adult animals remains to be determined. Nevertheless, inhibitors that are highly selective for ADAM10 over ADAM17 also block shedding of EGF in cell-based assays (Zhou et al., 2006 blue right-pointing triangle), further supporting the notion that ADAM10 is critical for this process. Evaluating the role of these ADAMs in EGFR ligand shedding has thus provided insight into their regulation and defined characteristic properties of these major sheddases.

In light of previous loss of function studies, which demonstrated that ADAM17 is required for the constitutive and PMA stimulated shedding of TGF-α, AR, EPR and HB-EGF (Peschon et al., 1998 blue right-pointing triangle; Jackson et al., 2003 blue right-pointing triangle; Sahin et al., 2004 blue right-pointing triangle), it was unexpected to find an activity in Adam17−/− cells, which was able to efficiently process these four EGFR-ligands upon stimulation with the calcium ionophore ionomycin (IM) (Horiuchi et al., 2007b blue right-pointing triangle). The critical role of shedding for activating EGFR-ligands prompted us to define the IM-stimulated sheddase for these proteins in Adam17−/− cells. Using several different activators and inhibitors of ectodomain shedding, we identified ADAM10 as the enzyme that can shed many substrates of ADAM17 in Adam17−/− cells. This observation raised the critical question about the physiological relevance of these two ADAMs in shedding various substrates in cells expressing both ADAM10 and 17. We therefore addressed whether both enzymes have largely equivalent or redundant functions, or whether one is the principal sheddase for substrates that can, in principle, be cleaved by both enzymes. Finally, we were interested in learning about the implications of our results for the development of ADAM17-selective pharmacological inhibitors, and whether ADAM10 can process substrates of ADAM17 in the presence of such inhibitors.

MATERIALS AND METHODS

Cell Lines and Reagents

Adam17−/− and Adam10−/− mouse embryonic fibroblasts (mEFs) were from embryonic day (E)13.5 and E9.5 embryos, respectively (Reddy et al., 2000 blue right-pointing triangle; Hartmann et al., 2002 blue right-pointing triangle). Adam10/17−/− mEFs were from Reiss and Saftig (unpublished data), and A431 and Chinese hamster ovary (CHO) cells were from American Type Culture Collection (Manassas, VA). CHO cells were grown in DMEM/F-12; all other cell lines were grown in DMEM, with antibiotics and 5% fetal calf serum (FCS), or with 10% FCS plus high glucose for A431 cells. All reagents were from Sigma-Aldrich unless otherwise indicated. Ionomycin was from Calbiochem (San Diego, CA). The hydroxamate inhibitor GI254023X (GI; 10-fold selective for ADAM10 over ADAM17; Hundhausen et al., 2003 blue right-pointing triangle; Weskamp et al., 2006 blue right-pointing triangle) was from David Becherer (GlaxoSmithKline, Research Triangle Park, NC), and marimastat was from Ouathek Ouerfelli (Sloan-Kettering Institute, New York, NY). The ADAM17-selective inhibitor SP26 (Mazzola et al., 2008 blue right-pointing triangle) was from Schering Plough (Kenilworth, NJ). Anti-Phospho-extracellular signal-regulated kinase (ERK) were from Cell Signaling Technology (Beverly, MA), anti-ERK2 antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-human CD23 (30X) was described previously (Weskamp et al., 2006 blue right-pointing triangle). Tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2 were from Dr. G. Murphy (University of Cambridge, Cambridge, United Kingdom), Calbiochem, or R&D Systems (Minneapolis, MN). TIMP-3 was from Dr. R. Black (Amgen, Seattle, WA). Antibodies for flow cytometry were from BD Biosciences Pharmingen, (San Diego, CA).

Expression Vectors

The expression vectors for ADAMs 10 and 17, for alkaline phosphatase (AP)-tagged proteins, and for human CD23 have been described previously (Sahin et al., 2004 blue right-pointing triangle; Weskamp et al., 2006 blue right-pointing triangle; Horiuchi et al., 2007b blue right-pointing triangle). The human P2X7R cDNA (ATCC no. 10658792) was subcloned into pcDNA3.1-Zeo+ (Invitrogen, Carlsbad, CA). The pEF expression plasmid for dominant-negative (DN) ADAM10 lacking its metalloprotease domain consists of the leader sequence and part of the prodomain linked to the disintegrin, transmembrane, and cytoplasmic domains, as described previously (Pan and Rubin, 1997 blue right-pointing triangle). An inactive ADAM9E>A was used as control for ADAM10-DN experiments. For expression of short hairpin RNA (shRNA), the mouse ADAM10 sequence (5′-GACAGUUCAACCUACGAAU-3′) followed by a nine nucleotide noncomplementary spacer (TCTCTTGAA) and the reverse complement sequence were inserted into the pSUPER vector (provided by Dr. T. Brummelkamp, Whitehead Institute, Cambridge, MA) after digestion with BglII and HindIII.

Cell Culture, Transfection, and Ectodomain Shedding Assays

Fibroblasts were transiently transfected with Lipofectamine 2000 and CHO cells with Lipofectamine and the indicated plasmids as described previously (Sahin et al., 2004 blue right-pointing triangle; Zheng et al., 2004 blue right-pointing triangle). Shedding assays were performed the day after transfection, except for shRNA and ADAM10-DN experiments, which were done 3 d after transfection. Chronic inactivation with 3 μM SP26 was for 2 d, with one change of medium, followed by a typical shedding experiment. For individual shedding experiments, cells were washed with DMEM, which was replaced after 1 h by fresh DMEM with or without activators or inhibitors of shedding, and then cells were incubated for 30 min to 4 h as indicated previously (Sahin et al., 2004 blue right-pointing triangle). AP activity in the supernatant and cell lysates was measured by colorimetry (Sahin et al., 2004 blue right-pointing triangle; Zheng et al., 2004 blue right-pointing triangle). The ratio between the AP activity in the supernatant and the total AP activity in the cell lysate plus supernatant was calculated from two identically prepared wells, and averaged. The ratio reflects the activity of a given sheddase toward a given AP-tagged protein. CD23 shedding assays were performed in Opti-MEM, and the same wells were used to collect supernatants after 1 h of constitutive shedding and 1 h of stimulated shedding, as described previously (Weskamp et al., 2006 blue right-pointing triangle).

Generation of CD19Cre/+Adam17flox/flox, Mmp7−/−Adam17−/− Mice and mEF Cells

Cd19(Cre/Cre) mice (C.Cg-Cd19tm1(cre)Cgn Ighb/J, The Jackson Laboratory, Bar Harbor, ME) were crossed with Adam17flox/flox mice (Horiuchi et al., 2007a blue right-pointing triangle) to generate Cd19Cre/+Adam17flox/flox mice. These were crossed with Adam17flox/flox mice to produce littermates that were Adam17flox/flox and either wild-type or Cd19Cre/+ at the Cd19 locus.

Mmp7−/− mice (B6.129-Mmp7tm1Lmm/J; Jackson labs, Bar Harbor, ME) were crossed with Adam17+/− mice (Horiuchi et al., 2007a blue right-pointing triangle) to produce Mmp7−/− Adam17+/− animals, which were viable and fertile. These were bred to obtain Mmp7−/−Adam17−/− double knock-out mice, which were born with the expected Mendelian ratio (Mmp7−/−Adam17−/−: 23.3%; Mmp7−/− Adam17+/−: 50.0%; Mmp7−/−Adam17+/+: 26.7%; n = 30). E13.5 mEFs were prepared as described previously (Sahin et al., 2004 blue right-pointing triangle). All mouse experiments were approved by the Institutional Animal Use and Care Committee of the Hospital for Special Surgery, New York, NY.

Shedding of CD23 and L-Selectin from Adam17+/+ or Adam17−/− B Cells

CD19Cre/+Adam17flox/flox mice (see above) were used to generate Adam17−/− B cells as follows. Spleen cell suspensions prepared from 4- to 5-mo-old mice were subjected to Ficoll-Hypaque density gradient centrifugation to remove erythrocytes and dead cells. Splenocytes (1 million cells/ml) were incubated for 40 min in RPMI 1640 medium with the indicated reagents and then washed in phosphate-buffered saline (PBS) and stained for 30 min on ice first with a monoclonal antibody to mouse Fcγ receptor to avoid nonspecific antibody binding, and then with phenotype-specific fluorescent antibodies (anti-CD90.1.2 Allophycocyanine for T cells and anti-B220 fluorescein isothiocyanate for B cells), and with phycoerythrin (PE)-conjugated anti-L-Selectin or anti-CD23. Cytometry was performed with a FACSCalibur (BD Biosciences, San Jose, CA) and data were analyzed with CellQuest software (BD Biosciences). B220+ CD90 cells were considered to represent B cells, and B220 CD90+ cells were considered to represent T cells. The relative amount of L-Selectin on the surface of the entire B or T cells population was calculated by multiplying the percentage of L-Selectin+ cells (L-Selectinlow to L-Selectinhigh) in B or T cells, with its corresponding mean of fluorescence in the L-Selectin+ subpopulation. CD23 cell surface levels were calculated similarly, but because its expression level varied greatly from one mouse to another, data were further normalized to the untreated control to allow comparisons of individual experiments.

RESULTS

Sensitivity of the Calcium Influx-stimulated Sheddase for TGF-α in Adam17−/− Cells to Metalloproteinase Inhibitors

To characterize the ionomycin (IM)-stimulated sheddase for TGF-α in Adam17−/− cells, we compared its response to hydroxamate-type metalloproteinase inhibitors to that of ADAMs 10 and 17. As a selective assay for ADAM10, we used constitutive and IM-stimulated shedding of BTC, because this is abolished in Adam10−/− cells and can be rescued by transfection with ADAM10 (Sahin et al., 2004 blue right-pointing triangle; Horiuchi et al., 2007b blue right-pointing triangle). Moreover, constitutive and PMA-stimulated shedding of TGF-α served as selective readout for ADAM17, because both activities are strongly reduced in Adam17−/− cells and can be rescued by reintroduction of ADAM17 (Sahin et al., 2004 blue right-pointing triangle; Horiuchi et al., 2007b blue right-pointing triangle).

In Adam17−/− cells, IM-stimulated shedding of TGF-α was sensitive to the ADAM10-selective inhibitor GI at a concentration that blocks ADAM10 but not ADAM17 (0.2 μM; Figure 1A) (Weskamp et al., 2006 blue right-pointing triangle). Moreover, IM-stimulated shedding of TNF-α (Figure 1B) and of the ADAM10-substrate BTC (Figure 1C) in Adam17−/− cells was sensitive to 0.2 μM GI. In parallel experiments with Adam10−/− cells, PMA and IM-stimulated shedding of TGF-α (Figure 1, D and E) and TNF-α (Figure 1, F and G) were not inhibited by 1 μM GI, consistent with ADAM17 functioning as major sheddase for these substrates. Finally, we found that several other substrates, whose constitutive and PMA-dependent processing depends on ADAM17 (amphiregulin, epiregulin, and HB-EGF; Sahin et al., 2004 blue right-pointing triangle), neuregulin 1β1 and 1β2 (Horiuchi et al., 2005 blue right-pointing triangle), intercellular adhesion molecule (ICAM) (Tsakadze et al., 2006 blue right-pointing triangle), and L-Selectin (Li et al., 2006 blue right-pointing triangle) were shed from Adam17−/− cells after IM stimulation, and the increase in shedding of these substrates could also be blocked by 0.2 μM GI (Supplemental Figure 1).

Figure 1.
The ionomycin-stimulated sheddase of TGF-α and TNF-α in Adam17−/− cells is sensitive to the ADAM10 inhibitor GI254023X, to knockdown with ADAM10 shRNA, and to expression of a dominant-negative form of ADAM10. (A–G) ...

Calcium-stimulated TGF-α Shedding in Adam17−/− Cells Is Blocked by ADAM10-shRNA or Dominant-Negative ADAM10

To further assess whether the IM-stimulated sheddase of TGF-α in Adam17−/− cells could be ADAM10, we coexpressed single-hairpin RNA against mouse-ADAM10 (AD10-shRNA) with TGF-α, HB-EGF, or BTC in Adam17−/− cells. This resulted in a robust decrease (40–60%) in the IM-stimulated shedding of these substrates compared with a vector control (Figure 1, H–J). In addition, we found that a dominant-negative ADAM10 (AD10-DN) lacking its metalloproteinase domain inhibited IM-stimulated shedding of TGF-α, TNF-α, and BTC in Adam17−/− mEF cells compared with mock-transfected control cells (data not shown) or compared with control cells coexpressing an inactive ADAM (ADAM9E/A mutant, E/A; Figure 1, K–M). When ADAM10-DN was expressed in Adam10−/− cells as a control for nonspecific effects, it did not affect the constitutive, PMA-stimulated or IM-stimulated shedding of TGF-α (Figure 1, N and O), which depend on ADAM17 (see below and Figures 2, A–C, G, and H, and and33 B, D, and F).

Figure 2.
Contribution of ADAMs 10 and 17 to constitutive shedding of TGF-α. Wild-type mEF cells (A and D), Adam17−/− cells (B, E, G, and I), or Adam10−/− cells (C, F, H, and J) were transfected with TGF-α (A–C, ...
Figure 3.
In cells lacking both ADAM10 and -17, transfection of ADAM10 rescues the Ionomycin-stimulated shedding of proteins, for which ADAM17 is the major constitutive and PMA-stimulated sheddase. Adam10/17−/− double-knockout cells were transiently ...

Previous studies have shown that ADAM17 is the principal constitutive sheddase of TGF-α, HB-EGF, AR, and EPR in primary mouse embryonic fibroblasts (Sahin et al., 2004 blue right-pointing triangle). To determine whether ADAM10 also contributes to constitutive shedding of these molecules, we tested how ADAM10-DN affected TGF-α shedding in unstimulated cells. ADAM10-DN inhibited the constitutive shedding of TGF-α in Adam17−/− cells (Figure 2B), but not in wild-type (wt) controls (Figure 2A) or in Adam10−/− cells (Figure 2C), which both express ADAM17. Conversely, constitutive BTC shedding was decreased when ADAM10-DN was coexpressed in wt cells (Figure 2D) and Adam17−/− mEF cells (Figure 2E), thus in cells expressing ADAM10, whereas ADAM10-DN did not affect the residual BTC shedding seen in Adam10−/− cells (Figure 2F). In rescue experiments, expression of ADAM17 in Adam17−/− cells increased constitutive TGF-α shedding (Figure 2G), whereas expression of ADAM10 in Adam10−/− cells did not (Figure 2H), consistent with a dominant role of ADAM17 in TGF-α constitutive shedding. In similar rescue experiments with BTC, constitutive shedding could be increased by transfecting Adam10−/− cells with ADAM10 (Figure 2J) but not by expressing ADAM17 in Adam17−/− cells (Figure 2I).

To further explore the requirement for ADAMs 10 and 17 for constitutive shedding, we used Adam10/17−/− double-deficient cells (Reiss and Saftig, unpublished data). Introducing ADAM10 into Adam10/17−/− cells restored constitutive BTC shedding, whereas ADAM17 did not (Figure 3A). In contrast, expression of either ADAM10 or 17 increased constitutive shedding of TGF-α and L-Selectin, with higher levels of both shed proteins observed in the presence of ADAM17 (Figure 3, B and C). With respect to stimulated shedding, PMA-dependent release of TGF-α (Figure 3D) and L-Selectin (data not shown) in Adam10/17−/− cells was only rescued by ADAM17 but not ADAM10. Shedding of BTC was not sensitive to PMA stimulation, regardless of whether ADAM10 or ADAM17 were coexpressed (Figure 3E). Finally, IM stimulation of Adam10/17−/− cells rescued with ADAM10 strongly enhanced shedding of TGF-α (Figure 3F), L-Selectin (data not shown) and of BTC (Figure 3G), demonstrating that ADAM10 can shed TGF-α in the absence of ADAM17. In contrast, IM stimulation of Adam10/17−/− cells expressing ADAM17 strongly increased shedding of TGF-α (Figure 3F) and L-Selectin (data not shown) but not of BTC (Figure 3G), corroborating that calcium influx also activates ADAM17. In Adam10/17−/− cells, ADAM10 rescued the IM-stimulated shedding of other proteins whose principal PMA-stimulated sheddase is ADAM17 (TNF-α, ICAM, and L-Selectin; Figure 3, H–J; HB-EGF, data not shown). Together, these experiments confirm that IM-stimulated shedding of TGF-α and several other proteins is mediated by ADAM10 when ADAM17 is absent.

Stimulation of ADAM10-dependent Shedding by a Physiologically Relevant Signaling Pathway

The stimuli described above, IM and PMA, are useful to define the characteristic properties (“fingerprint”) of protease activities in cell-based assays (Overall and Blobel, 2007 blue right-pointing triangle), but they are not physiological stimuli. We therefore tested whether a ligand-activated receptor-signaling pathway can also stimulate ADAM10 to serve as an alternative protease for ADAM17 substrates. Previous studies showed that shedding of CD23, a substrate for ADAM10 (Weskamp et al., 2006 blue right-pointing triangle), is stimulated by activation of the P2X7 receptor in B cells (Gu et al., 1998 blue right-pointing triangle). CHO cells express endogenous P2X7R (Michel et al., 1998 blue right-pointing triangle), whereas mEF cells do not respond to activators of this receptor (Figures 4, D–F, and and5,5, A–E). Shedding of CD23 from CHO cells was stimulated by IM or dibenzoyl ATP (BzATP) (Michel et al., 1998 blue right-pointing triangle), an agonist of P2X7R but not by the ADAM17 activator PMA. Moreover, the BzATP-stimulated CD23 shedding was sensitive to 0.75 μM GI, consistent with an ADAM10-mediated processing (Figure 4A). Similarly, BTC shedding from CHO cells was also stimulated by BzATP and was sensitive to 0.75 μM GI and to marimastat (Figure 4B) and to coexpression of ADAM10-DN (Figure 4C). When P2X7R was coexpressed with BTC in Adam17−/− cells, addition of BzATP enhanced BTC shedding, and this was sensitive to 0.75 μM GI (Figure 4D). When these experiments were repeated in Adam10−/− cells (Figure 4E) and Adam10/17−/− cells (Figure 4F), no stimulation of BTC shedding by BzATP was observed, strongly suggesting that ADAM10 is responsible for this P2X7R-stimulated shedding.

Figure 4.
Stimulation of the P2X7R by BzATP activates ADAM10-dependent shedding. (A) CHO cells, which endogenously express P2X7R, were transfected with the ADAM10 substrate CD23, or left untransfected (NT), and stimulated with IM (2.5 μM), PMA (25 ng/ml), ...
Figure 5.
BzATP/P2X7R stimulates both ADAM10 and ADAM17 as sheddases of proteins that require ADAM17 as their major constitutive and PMA stimulated sheddase (A–E). Adam17−/− cells (A and D), Adam10−/− cells (B and E), and ...

When the ADAM17-substrates ICAM or TNF-α were transfected together with P2X7R in mEF cells, BzATP stimulated their shedding from both Adam17−/− (Figure 5, A and D) and Adam10−/− cells (Figure 5, B and E). The BzATP-stimulated shedding was sensitive to 0.75 μM GI in Adam17−/− cells, which express endogenous ADAM10, but not in Adam10−/− cells, which express endogenous ADAM17. The BzATP-stimulation did not increase ICAM shedding in Adam10/17−/− double-knockout cells (Figure 5C). These results are similar to those obtained with IM stimulation (Figure 3) and are consistent with a P2X7R stimulated processing of the substrates by both ADAM10 and ADAM17, in Adam17−/− and Adam10−/− cells, respectively. However, in CHO cells, which express P2X7R and both proteases, the ADAM10 inhibitor GI did not detectably affect shedding of TGF-α, TNF-α, or ICAM when stimulated with BzATP (Figure 5, F–H), corroborating that a contribution of ADAM10 to the shedding of these substrates is most evident in the absence of ADAM17.

ATP and Calcium-stimulated Shedding of the ADAM17-Substrate L-Selectin from Primary Adam17−/− B Cells

To assess whether activators of ADAM10 can lead to the release of endogenous substrates of ADAM17 from primary cells ex vivo, we analyzed shedding of L-Selectin (Li et al., 2006 blue right-pointing triangle) from ADAM17-deficient B cells that were stimulated with ionomycin or by activation of the P2X7R. For this purpose, we generated mice that carried a floxed Adam17 and expressed the B cell specific CD19-Cre to obtain animals that lack ADAM17 in B cells (CD19Cre/+Adam17flox/flox mice; see Materials and Methods for details). FACS analysis was used to evaluate the expression of L-Selectin on the surface of T cells or B cells from CD19+/+Adam17flox/flox controls (Figure 6, A and B) or from CD19Cre/+Adam17flox/flox mice (Figure 6, A and D). The level of cell surface L-Selectin as determined by the total fluorescence dramatically increased in the B cells from CD19Cre/+Adam17flox/flox mice compared with the controls CD19+/+Adam17flox/flox (Figure 6, B and D), in agreement with the deletion of ADAM17 by the Cre-recombinase, and as reported for Adam17ΔZn/ΔZn-chimeric mice (Li et al., 2006 blue right-pointing triangle). After treatment with PMA, IM, or ATP, we observed an almost complete decrease in L-Selectin on the surface of T cells from CD19+/+Adam17flox/flox mice and from CD19Cre/+Adam17flox/flox mice (Figure 6, A and C). This decrease was not seen in B cells from CD19Cre/+Adam17flox/flox mice that were stimulated with PMA, but a partial reduction in L-Selectin was achieved after treatment with IM or ATP (Figure 6D). Moreover, a partial shedding of CD23 from B cells isolated from CD19+/+Adam17flox/flox (Figure 6E) and CD19Cre/+ Adam17flox/flox mice (Figure 6F) was detected upon treatment with IM or ATP, but not with PMA, so this was not affected by the presence of CD19-Cre. These results corroborate that ADAM10 can be activated by IM and ATP in B cells, just as in mEFs. Moreover, they suggest that only ADAM17 can completely deplete the cell surface L-Selectin pool, depending on the activating signal, which might be a prerequisite for an adequate immune response.

Figure 6.
Shedding of the ADAM17 substrate L-Selectin from primary Adam17−/− B cells (A–D). Primary T cells (A and C) or B cells (B and D) were isolated from CD19+/+ Adam17flox/flox mice (A and B) or CD19Cre/+ Adam17flox/flox mice (C and ...

Selective Inhibitors for ADAM17 Demonstrate That the Ability of ADAM10 to Compensate for ADAM17 Develops over Time

The results presented above, which demonstrate that ADAM10 can efficiently process many ADAM17 substrates when ADAM17 is inactive, raised important questions about the relative contribution of ADAM10 to shedding of ADAM17 substrates in wild-type cells. To address this question, we analyzed shedding from wild-type cells treated with a highly selective inhibitor of ADAM17. When wt mEFs expressing TGF-α or L-Selectin were stimulated with PMA or IM, we found complete inhibition of the PMA- and IM-stimulated shedding by the highly ADAM17-selective inhibitor SP26 (Mazzola et al., 2008 blue right-pointing triangle), whereas the ADAM10-selective GI had no detectable effect (Figure 7, A–D, see Supplemental Figure 2 for controls regarding the selectivity of these inhibitors toward ADAM10 and -17). Identical experiments performed with BTC confirmed that GI completely blocked IM-stimulated shedding of this ADAM10 substrate, whereas SP26 did not (Figure 7E). Similar profiles of inhibition were obtained for TGF-α when we repeated these experiments in CHO cells (Supplemental Figure 2). Together with our previous results (see above and Figures 2, A–C, G, and H; H;5,5, F–H, and and6,6, B and D), these data demonstrate that ADAM10 does not significantly contribute to the shedding of substrates of ADAM17 when both ADAM10 and -17 are present, even though ADAM10 can efficiently shed ADAM17 substrates in Adam17−/− cells. Thus, ADAM17 is indeed the principal sheddase for ADAM17 substrates such as TGF-α, L-Selectin, or TNF-α, and ADAM10 only cleaves these substrates when ADAM17 is inactivated.

Figure 7.
Effect of acute and chronic treatment of wild-type mEF cells with the ADAM17-selective inhibitor SP26 on the ionomycin-stimulated shedding of TGF-α and BTC. (A–E) Wild-type mEF cells were transiently transfected with TGF-α (A and ...

These findings raised questions about whether chronic treatment with selective inhibitors of ADAM17, which are being developed for treatment of cancer and rheumatoid arthritis, could generate conditions that mimic those in Adam17−/− mice. To directly address the possibility of compensatory changes in the cellular shedding mechanism, we performed essentially identical shedding experiments after a chronic incubation of wt mEF cells with SP26 for 2 d. Unlike the results shown in Figure 7, B and D, in which SP26 was used for acute inhibition, IM could stimulate shedding of TGF-α and L-Selectin despite the presence of SP26 after chronic treatment with this inhibitor, and the responsible sheddase was completely blocked by GI, consistent with a role for ADAM10 in this process (Figure 7, F and G; BTC shedding is shown as a control for ADAM10 activity under these conditions in Figure 7H, dimethyl sulfoxide as a control had no effect; data not shown). Evidently, ADAM10 does not make a detectable contribution to the shedding of ADAM17 substrates in wild-type cells treated with an ADAM17-selective inhibitor for 30 min to 2 h, but after prolonged treatment with this inhibitor, the contribution of ADAM10 to shedding of ADAM17 substrates is similar to what is seen in Adam17−/− cells. These experiments demonstrate that chronic inhibition of ADAM17 creates conditions under which ADAM10 can make a significant contribution to the IM-stimulated shedding of ADAM17 substrates in wt cells.

DISCUSSION

Ectodomain shedding is critical for regulating the function of membrane proteins such as TNF-α and EGFR ligands. Because dysregulation of EGFR signaling occurs in diseases such as cancer, and TNF-α is a causative factor in rheumatoid arthritis, it is important to understand the underlying proteolytic machinery. Here, we used the ectodomain shedding of TNF-α, TGF-α, and other membrane proteins such as L-Selectin to identify ADAM10 as a sheddase that can, in principle, release these proteins almost as efficiently as their primary sheddase, ADAM17, but only in Adam17−/− cells stimulated with ionomycin. Nevertheless, despite the ability of ADAM10 to efficiently shed many substrates of ADAM17 in Adam17−/− cells, ADAM17 nevertheless clearly emerged as the functionally dominant enzyme, or “principal sheddase” for these substrates when both enzymes were present.

An important criterion for identifying ADAM10 as an efficient sheddase of ADAM17-substrates in Adam17−/− cells was its “fingerprint,” defined as its characteristic response to activators and inhibitors of ectodomain shedding (Overall and Blobel, 2007 blue right-pointing triangle). ADAM10 emerged as the relevant IM-stimulated sheddase for TGF-α and other membrane proteins in Adam17−/− cells by all criteria we could apply, including its response to an ADAM10-selective metalloprotease inhibitor (GI), ADAM10-shRNA, and dominant-negative ADAM10. In Adam10/17−/− double knockout cells, IM-stimulated shedding of TGF-α could be rescued by both ADAM10 and ADAM17, demonstrating that calcium influx activates both ADAMs. However, PMA-dependent shedding of TGF-α could only be rescued by ADAM17, corroborating that only ADAM17 responds to short-term stimulation with PMA. Interestingly, IM-stimulated ADAM10 is sensitive to low nanomolar concentrations of TIMPs 1, 2, and 3 in cell-based assays, whereas purified soluble ADAM10 is inhibited by TIMPs 1 and 3, but not TIMP2, in vitro (Amour et al., 2000 blue right-pointing triangle) (Supplemental Figure 3). Evidently, ADAM10 has a different inhibitor profile in cell-based assays compared with biochemical assays. Because MMP7 is a known sheddase of TNF-α and other membrane proteins (Powell et al., 1999 blue right-pointing triangle; Haro et al., 2000 blue right-pointing triangle; Li et al., 2002 blue right-pointing triangle; Lynch et al., 2005 blue right-pointing triangle), we also ruled out its involvement in IM-stimulated shedding by using Mmp7−/−/Adam17−/− double-knockout cells (Supplemental Figure 4). Moreover, following up on a previous report of an aminophenylmercuric acetate (APMA)-activated TGF-α sheddase in CHO cells lacking functional ADAM17 (Merlos-Suarez et al., 2001 blue right-pointing triangle), we showed that ADAM10-dependent TGF-α shedding can be activated by APMA in Adam17−/− cells (Supplemental Figure 5). Finally, we confirmed that TGF-α released by ADAM10 retains its biological activity (Supplemental Figure 6).

The stimuli described above, such as PMA, IM, and APMA, are pleiotropic and not physiological, so we also evaluated shedding activated by the P2X7 nucleotide receptor, which is involved in many physiological aspects of the immune response (Chen and Brosnan, 2006 blue right-pointing triangle; Moore and MacKenzie, 2007 blue right-pointing triangle). Experiments in Adam−/− mEFs as well as ADAM17-deficient primary B cells clearly established that both ADAMs 10 and 17 can be stimulated by the P2X7R in adherent fibroblasts and nonadherent primary B cells. Moreover, ADAM10 stimulated via P2X7R was able to shed substrates such as TNF-α, ICAM, and L-Selectin. Nevertheless, selective ADAM inhibitors confirmed that ADAM17 is the major sheddase for TNF-α, TGF-α, HB-EGF, and ICAM in P2X7R-stimulated CHO cells, where both ADAMs 10 and 17 are present. So, the ability of ADAM10 to shed substrates such as TNF-α or TGF-α after activation of P2X7R is also only evident in the absence of ADAM17.

Interestingly, acute inhibition of ADAM17 with an ADAM17-selective inhibitor blocked stimulated shedding of ADAM17 substrates from wt mEFs or CHO cells, whereas chronic inhibition of ADAM17 generated conditions that mimicked those in Adam17−/− cells, i.e., ADAM10 could take over shedding of ADAM17 substrates. These observations could be relevant for chronic and specific inhibition of ADAM17 in patients. A compensatory up-regulation of ADAM10 activity during chronic treatment with SP26 is unlikely, because there was no detectably increase in shedding of the ADAM10-substrate BTC. Perhaps chronic inactivation of ADAM17 leads to an accumulation of its substrates, which then become more accessible to ADAM10, possibly by “spilling over” into a compartment where ADAM10 is most active. The results obtained in primary B cells with endogenously expressed L-Selectin are consistent with this interpretation, because higher levels of L-Selectin are seen in the absence of ADAM17. Moreover, activation of these cells with IM or ATP does not lead to complete consumption of L-Selectin in Adam17−/− cells, suggesting that a subpopulation of L-Selectin is not accessible to ADAM10, even in the absence of ADAM17.

We predict that the results obtained with TGF-α, TNF-α, and several other membrane proteins (Supplemental Figure 1) are likely representative for many, if not most or all, proteins whose constitutive and PMA-stimulated shedding depends on ADAM17. However, although ADAM10 can, in principle, process substrates of ADAM17, it nevertheless normally does not when both enzymes are present, and a role for ADAM10 as a secondary sheddase has yet to be demonstrated in vivo. For example, ADAM10 cannot efficiently compensate for the loss of ADAM17 with respect to activating the EGFR during mouse development (Peschon et al., 1998 blue right-pointing triangle; Jackson et al., 2003 blue right-pointing triangle; Sternlicht et al., 2005 blue right-pointing triangle), or in terms of generating soluble TNF-α in a mouse model for endotoxin shock (Bell et al., 2007 blue right-pointing triangle; Horiuchi et al., 2007a blue right-pointing triangle). Therefore, we predict that ADAM17 will also emerge as the physiologically or pathologically more relevant sheddase of other membrane proteins that can be shed by both ADAM10 and 17, such as the amyloid precursor protein (Buxbaum et al., 1990 blue right-pointing triangle; Lammich et al., 1999 blue right-pointing triangle) and Klotho (Chen et al., 2007 blue right-pointing triangle). In contrast, we found no evidence that ADAM17 can substitute as a sheddase for substrates of ADAM10 in Adam10−/− cells, at least in the presence of the stimuli used here. Finally, it should be noted that other ADAMs or non-ADAM metalloproteinases may also play significant roles as sheddases in other cell types or under different conditions than those tested here.

In summary, loss of function studies with cells lacking ADAM10 or ADAM17 or both have provided new insight into the principal components of a general, yet differentially regulated cellular shedding machinery for TGF-α, HB-EGF, TNF-α, L-Selectin, and several other membrane proteins. Because ectodomain shedding is increasingly being recognized as a critical signaling switch that affects the function of a large number of membrane proteins, these results are likely to provide a framework for understanding the regulation of processing of other membrane proteins by ADAMs 10 and 17. Based on our findings, we hypothesize that the substrate repertoire of ADAM10 can, in principle, overlap with that of ADAM17 in cells activated with IM, APMA or via the P2X7R. Nevertheless, identifying ADAM10 as an alternative sheddase for ADAM17 substrates is only relevant in the case of the deletion or the chronic inhibition of ADAM17, and for stimuli that will normally activate ADAM10. Clearly, defining the individual fingerprints of these two major sheddases under various conditions is a prerequisite for probing the mechanism underlying their regulation under specific physiological and pathological conditions. Moreover, it will be important to consider the implications of these results for the use of selective ADAM inhibitors to treat human diseases.

Supplementary Material

[Supplemental Materials]

ACKNOWLEDGMENTS

We thank Dr. Gillian Murphy for kindly providing TIMP1 and TIMP2 and Dr. Roy Black for providing TIMP3. This work was supported by National Institutes of Health grant GM-64750 (to C.P.B.), the Deutsche Forschungsgemeinschaft SFB/B9, the Belgian Interuniversity Attraction Poles Program, and the Center of Excellence “Inflammation at Interfaces” (to P. S. and K. R.).

Footnotes

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-11-1135) on January 21, 2009.

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