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Article |
Address correspondence to Carl P. Blobel, Cell Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, Box 368, 1275 York Avenue, New York, NY 10021. Tel.: (212) 639-2915. Fax: (212) 717-3047. email: c-blobel{at}ski.mskcc.org
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Abstract |
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Key Words: EGF receptor; EGF receptor ligands; ADAMs; ectodomain shedding; growth factor signaling
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Introduction |
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Metalloproteases of the ADAM (a disintegrin and metalloprotease) family are thought to be responsible for shedding of certain EGFR ligands. ADAMs are membrane-anchored glycoproteins with diverse functions, including critical roles in fertilization, neurogenesis, angiogenesis, and in shedding of membrane-bound proteins from cells (Black and White, 1998; Schlöndorff and Blobel, 1999; Primakoff and Myles, 2000; Seals and Courtneidge, 2003). Mice lacking ADAM17 die perinatally and resemble mice lacking TGF (Mann et al., 1993), HB-EGF (Iwamoto et al., 2003; Jackson et al., 2003), and the EGFR (Miettinen et al., 1995; Sibilia and Wagner, 1995; Threadgill et al., 1995; Peschon et al., 1998). Consistent with these observations, ADAM17-deficient cells have been shown to be defective in shedding of TGF
, HB-EGF, and amphiregulin (Peschon et al., 1998; Merlos-Suarez et al., 2001; Sunnarborg et al., 2002). However, in addition to ADAM17, three other ADAMs have been linked to HB-EGF shedding. Overexpression of ADAM9 increases HB-EGF shedding in VeroH cells, whereas a mutant form of ADAM9 that is presumably unfolded and retained in the ER decreases HB-EGF shedding (Izumi et al., 1998); yet no defect in HB-EGF shedding was observed in cells lacking ADAM9 (Weskamp et al., 2002). Furthermore, ADAM12 reportedly has a role in HB-EGF shedding in the heart (Asakura et al., 2002) and in the down-regulation of cell-associated HB-EGF after stimulation with the phorbol ester PMA (Kurisaki et al., 2003). ADAM10 is the fourth ADAM to be implicated in HB-EGF shedding as part of the crosstalk between GPCRs and the EGFR (Lemjabbar and Basbaum, 2002; Yan et al., 2002). The remaining EGFR ligands, EGF, betacellulin, epiregulin, and epigen, are also known to be shed from cells, yet little information is available about the responsible enzyme(s) (Dempsey et al., 1997; Harris et al., 2003).
A crucial step toward understanding the mechanism underlying proteolytic cleavage of EGFR ligands (and its potential role in their activation) is to identify the responsible enzyme(s). In previous papers, different cell types and different approaches were used to analyze shedding of some EGFR ligands (see previous paragraphs), including antisense oligonucleotides and overexpression of both wild-type and putative dominant-negative ADAM constructs. Here, we chose a genetically defined system that is less prone to potential artifacts to evaluate the role of ADAMs in EGFR ligand shedding. To address potential compensatory or redundant functions between ADAMs 9, 12, 15, and 17, we generated adam9/12/15-/- and adam9/12/15/17-/- mice. Furthermore, we used cells isolated from wild-type, adam9/12/15-/-, adam10-/-, adam17-/-, adam19-/-, or adam9/12/15/17-/- mice to evaluate how loss of one or more widely expressed ADAMs affects the shedding of different EGFR ligands. This paper represents the first systematic characterization of EGFR ligand processing using mouse cells that lack one or more candidate sheddase of the ADAM family of metalloproteases.
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Results |
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Shedding of EGFR ligands in wild-type cells
Shedding of EGFR ligands was first evaluated in wild-type MEFs. As shown in Fig. 2 B, unstimulated mEFs shed basal amounts of TGF, amphiregulin, epiregulin, HB-EGF, betacellulin, and EGF. In the case of TGF
, amphiregulin, epiregulin, and HB-EGF, shedding was stimulated relatively strongly by PMA, whereas shedding of betacellulin and EGF was only weakly enhanced by PMA (see also Fig. 3 A). Treatment with the metalloprotease inhibitor batimastat strongly reduced both PMA-stimulated (unpublished data) and constitutive shedding of all EGF family members except HB-EGF. Although stimulated HB-EGF shedding was effectively inhibited by batimastat (unpublished data), constitutive release was only weakly affected (see also Fig. 4 A), suggesting that the predominant constitutive HB-EGF sheddase in primary mEFs is not a batimastat-sensitive metalloprotease, and is distinct from the sheddase(s) of other EGFR ligands.
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ADAM10 has been implicated in shedding of HB-EGF as part of a pathway for crosstalk between a GPCR and the EGFR (Lemjabbar and Basbaum, 2002; Yan et al., 2002). As shown in Fig. 3, PMA-stimulated shedding of HB-EGF, TGF, amphiregulin, and epiregulin is not decreased in adam10-/- cells, suggesting that ADAM10 is not required for the PMA-stimulated shedding of these EGFR ligands. The enhanced stimulated shedding of these EGFR ligands in adam10-/- (Fig. 3 A) and adam10+/- cells (unpublished data) compared with the primary mEFs is presumably a consequence of immortalization.
Generation of adam9/12/15/17-/- quadruple knockout mice, and evaluation of PMA-stimulated EGFR ligand shedding in adam9/12/15/17-/- cells
Although ADAM17 is essential for the majority of stimulated shedding of HB-EGF in mEF cells, a residual amount of PMA-stimulated HB-EGF shedding is seen in the absence of ADAM17. Could this residual shedding depend on ADAMs 9 or 12, both of which have been implicated in HB-EGF shedding, or on the related ADAM15? To address this issue, we generated quadruple knockout mice lacking ADAMs 9, 12, 15, and 17 (see Materials and methods for details). Similar to adam17-/- mice, adam9/12/15/17-/- quadruple knockout mice that were born had open eyes and died in the first day after birth. The percentage of adam9/12/15/17-/- quadruple knockout embryos at E18.5 generated by mating adam9-/-12-/-15-/-17+/- parents was somewhat lower than the percentage of adam17-/- embryos at E17.518.5 produced by mating adam17+/- mice (Table III; Peschon et al., 1998).
To determine whether the loss of ADAMs 9, 12, and 15 exacerbates known defects in EGFR signaling in adam17-/- mice, we performed a histopathological examination of wild-type, adam9/12/15-/-, adam17-/-, and adam9/12/15/17-/- E18.5 embryos. As shown in Fig. 3 B (panels AD), the open-eye phenotype in adam17-/- mice that results from lack of TGF activation (Peschon et al., 1998) is similar in adam9/12/15/17-/- quadruple knockout mice, whereas it is not seen in adam9/12/15-/- triple knockout mice. Furthermore, Jackson et al. (2003) have described a defect in morphogenesis of the semilunar heart valves and the tricuspid and mitral valves in adam17-/- mice (Fig. 3 B, panels EP; mitral valve not depicted), which resembles the thickened and misshapen valves seen in hb-egf-/- mice and in mice with a knock-in mutation that abolishes HB-EGF shedding (Iwamoto et al., 2003; Yamazaki et al., 2003). As shown in Fig. 3 B, the heart valves in adam9/12/15-/- triple knockout mice are indistinguishable from those in wild-type mice, and again, the defects in heart valve morphogenesis in adam17-/- mice are comparable to the defects in adam9/12/15/17-/- quadruple knockout mice. A morphometric analysis of all heart valves of six adam9/12/15/17-/- E18.5 embryos also did not show an increased size compared with six adam17-/- E18.5 embryos (unpublished data). When we performed shedding experiments with adam9/12/15/17-/- quadruple knockout mEFs, the residual amount of PMA-stimulated HB-EGF shedding was comparable to what is observed in adam17-/- cells (percent increase in HB-EGF shedding after PMA stimulation in adam9/12/15/17-/- mEF: 33.3 ± 26.5%, n = 16; four embryos, 26 wells analyzed per embryo); compared with 33.7 ± 12.9% in adam17-/- mEF; Fig. 3 A). Together, these results argue against a significant contribution of ADAMs 9, 12, or 15 to the shedding of HB-EGF in these cells.
Constitutive shedding of EGFR ligands in adam-/- cells
Next, we evaluated the batimastat-sensitive component of constitutive shedding of EGFR ligands in the presence or absence of different ADAMs. No significant difference in the batimastat-sensitive constitutive shedding of all six ligands tested here was observed in adam9/12/15-/- or adam19-/- cells compared with wild-type controls (Fig. 4 A). Furthermore, constitutive shedding of TGF, amphiregulin, epiregulin, and HB-EGF was also not affected in adam10-/- cells.
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Interestingly, when we measured the batimastat-sensitive constitutive shedding of EGF and betacellulin, we found that it was abrogated in two independent adam10-/- cell lines compared with control adam10+/- cells and the primary mEF cells (Fig. 4 A; unpublished data). This resulted in a strong decrease in overall unstimulated constitutive shedding of betacellulin (87.5%) and EGF (49.7%) from adam10-/- cells compared with adam10+/- cells (unpublished data). Constitutive shedding of betacellulin and EGF could be rescued with wild-type ADAM10, confirming that the defect in shedding in adam10-/- cells is indeed due to the lack of ADAM10 (Fig. 4 B). Next, we evaluated the role of ADAM10 in betacellulin-dependent EGFR signaling in adam10-/- cells. When either ADAM10 or betacellulin were introduced in adam10-/- cells, there was no increase in phosphorylation of ERK1/2, a commonly used indicator for activation of the EGFR (Fig. 4 C). However, when wild-type ADAM10 was cotransfected with betacellulin in adam10-/- cells, ERK1/2 phosphorylation was increased (Fig. 4 C). Thus, EGFR signaling via transfected betacellulin depends on the presence of functional ADAM10 in these cells. Together, these results are the first to identify the major sheddase for EGF and betacellulin in mouse embryonic cells, and thus also to uncover two novel substrates for ADAM10.
To address whether the results obtained in mEF cells could in principle also be relevant for other cells and tissues, Western blot analysis of the expression of ADAMs 10 and 17 in different mouse tissues was performed (Fig. 5). This confirmed that both ADAMs are widely expressed, even though their expression levels vary. Thus, it is likely that both ADAMs 10 and 17 are expressed in the cells and tissues in which the ligands analyzed in this paper exert their function as activators of EGFR signaling.
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Discussion |
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Ectodomain shedding experiments using these six major EGFR ligands in adam-/- MEFs corroborated previous reports that ADAM17 has a major role in the shedding of TGF, HB-EGF, and amphiregulin (Peschon et al., 1998; Merlos-Suarez et al., 2001; Sunnarborg et al., 2002). We found no evidence for a major contribution of other ADAMs besides ADAM17 to TGF
, HB-EGF, and amphiregulin shedding in these cells. Furthermore, epiregulin was identified as a novel ADAM17 substrate. Previous works have shown that adam17-/- mice resemble tgf
2/- mice (Peschon et al., 1998) in that they have open eyes at birth, as well as displaying similar vibrissae, hair, and skin defects (Mann et al., 1993). Furthermore, adam17-/- mice also resemble hb-egf-/- mice (Iwamoto et al., 2003) in that they have thickened aortic and pulmonic valves (Jackson et al., 2003). A similar phenotype is seen in mice with a mutation in the cleavage site of HB-EGF that abolishes its shedding (Yamazaki et al., 2003). Finally, the phenotype of adam17-/- mice resembles that of egfr-/- mice (Miettinen et al., 1995; Sibilia and Wagner, 1995; Threadgill et al., 1995; Peschon et al., 1998). Thus, genetic experiments have substantiated that ADAM17 is also essential for the activation of EGFR ligands in vivo. It remains to be determined whether the lack of processing of amphiregulin or epiregulin (or both) also contributes to the phenotype of adam17-/- mice.
ADAMs 9, 10, and 12 are also considered candidate HB-EGF sheddases (Izumi et al., 1998; Asakura et al., 2002; Kurisaki et al., 2003). However, although PMA-stimulated ectodomain shedding of HB-EGF was somewhat reduced in adam9/12/15-/- cells, this reduction was not statistically significant. In addition, the residual PMA stimulation of HB-EGF shedding in adam17-/- cells is most likely also not due to ADAMs 9, 12, or 15 because it remains unchanged in adam9/12/15/17-/- quadruple knockout cells. In a previous paper, Kurisaki et al. (2003) reported a significant reduction in the down-regulation of cell-associated HB-EGF in phorbol esterstimulated adam12-/- cells compared with wild-type controls. This apparent discrepancy may be due to differences in cell preparation or experimental design. Nevertheless, the main conclusion from the side-by-side comparison of different adam-/- cells isolated and cultured under identical conditions in this paper is that ADAM17 is the predominant PMA-stimulated HB-EGF sheddase in primary mEF cells.
The conclusion that ADAM17 has a critical role in shedding HB-EGF in vivo was further corroborated by an analysis of the role of these ADAMs during mouse development. As mentioned previously in this paper, adam17-/- mice resemble egfr-/-, tgf2/-, or hb-egf-/- mice, whereas no similar defects were seen in adam9/12/15-/- mice. Furthermore, the phenotype of adam17-/- mice does not appear to be considerably exacerbated when ADAMs 9, 12, and 15 are also deleted. Together, these findings argue against major compensatory or redundant roles for ADAMs 9, 12, and the related ADAM 15 in the activation of TGF
, HB-EGF, or the EGFR during development. However, it cannot be ruled out that ADAMs 9, 12, or 15 contribute to shedding of EGFR ligands in cells or tissues where these enzymes and potential substrates are highly expressed. Further analyses will address which ADAMs are capable of cleaving EGFR ligands when overexpressed, and in which tissues candidate EGFR ligand sheddases besides ADAMs 10 and 17 are highly expressed together with EGFR ligands that they can cleave.
ADAM10 has also been implicated in HB-EGF shedding as part of a pathway that involves crosstalk between GPCRs and the EGFR (Lemjabbar and Basbaum, 2002; Yan et al., 2002; Lemjabbar et al., 2003). On the other hand, our results indicate that ADAM10 does not make a major contribution to PMA-stimulated or constitutive shedding of HB-EGF in the cells tested here. This is consistent with the notion that different stimuli may activate different ADAMs, such that HB-EGF shedding depends mainly on ADAM17 under the conditions used here, and mainly on ADAM10 when the appropriate GPCR is stimulated.
Little was previously known about the sheddases responsible for the release of EGF and betacellulin from cells. Here, we show that constitutive shedding of both EGF and betacellulin was strongly reduced in adam10-/- cells compared with heterozygous controls, and could be rescued by reintroduction of wild-type ADAM10. Furthermore, stimulation of the EGFR by transfected betacellulin in adam10-/- cells is only seen when these cells are rescued by cotransfection with wild-type ADAM10. These results are the first to identify ADAM10 as the major sheddase for these two crucial EGFR ligands in mouse cells. Because ADAM10 is widely expressed, it is tempting to speculate that it may participate in the functional regulation of these two EGFR ligands in development and in diseases such as cancer.
In light of the genetic evidence for a key role of ADAM17 in activation of EGFR ligands in mice (Peschon et al., 1998; Sunnarborg et al., 2002; Jackson et al., 2003), it is surprising that no ADAM has been identified as an essential part of the EGFR pathway in Drosophila (Lee et al., 2001; Urban et al., 2001; Ghiglione et al., 2002; Tsruya et al., 2002; Shilo, 2003). Instead, Rhomboids (integral membrane proteins with seven membrane-spanning domains) have been implicated in cleaving EGFR ligands (Urban et al., 2002), whereas reducing the expression of a putative ADAM17 orthologue in Drosophila via small interfering RNA did not block development of EGFR-dependent structures (Lee et al., 2001).
These results suggest that there are critical differences in the mechanism underlying proteolytic activation of EGFR ligands between flies and mice. However, the finding that all EGFR ligands tested here are processed by ADAM10 or ADAM17 in mEFs suggests a possible alternative explanation for these findings. Drosophila carry orthologues of ADAM10 (KUZ) and ADAM17 (AAF56986, the ADAM targeted by RNA interference in Lee et al. [2001]), as well as a third ADAM related to ADAM17 and KUZ with no evident orthologue in mammals (AAF56926). It is conceivable that two or three of these ADAMs fulfill redundant or compensatory roles in activation of EGFR ligands during development in Drosophila. This may only become apparent once two or three of these putative EGFR ligand sheddases are simultaneously inactivated. Conversely, the results in Drosophila suggest that it will be worthwhile to further investigate the potential role of Rhomboids and intramembrane proteolysis in EGFR ligand activation in mammals.
In summary, we report the first systematic analysis of the shedding of EGFR ligands in cells lacking one or more widely expressed and catalytically active ADAM. Our results uncover critical roles for both ADAM10 and ADAM17 in shedding of EGFR ligands in mEF cells. ADAM17 emerged as the major PMA-stimulated and constitutive sheddase of TGF, amphiregulin, HB-EGF, and epiregulin, which is consistent with the essential role for ADAM17 in activation of the EGFR during development. Furthermore, ADAM10 was found to be the major batimastat-sensitive sheddase for betacellulin and EGF in mEF. Further experiments, including the generation of conditional adam10-/- knockout mice, as well as knock-in mutations that abolish shedding of EGF and betacellulin, will be necessary to address the biological relevance of ADAM10 in shedding the endogenous forms of these EGFR ligands in vivo. The identification of different EGFR ligands as substrates for ADAM10 and ADAM17 sets the stage for the further analysis of how these ADAMs are regulated and how their substrate specificity is achieved. Because proteolysis of EGFR ligands may be critical for their functional activation, and signaling via the EGFR has been implicated in diseases such as cancer, ADAM10 and ADAM17 may be attractive targets for the design of drugs that modulate the action of these ligands.
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Materials and methods |
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The histopathological analysis of adam9/15-/- mice was performed as described previously for adam9-/- or adam15-/- mice (Weskamp et al., 2002; Horiuchi et al., 2003). Histopathological analysis of adam9/12/15-/- mice was performed by the Memorial Sloan-Kettering Cancer Center mouse phenotyping core. No abnormalities or pathological phenotypes were observed in adam9/15-/- and adam9/12/15-/- mice. Serial sections of tissues affected in egfr-/-, hb-egf-/-, and tgf2/- mice did not uncover any evident defects in adam9/12/15-/- mice. Specifically, there were no defects in the development of the heart or its valves (Fig. 3 B, panels EP), and also no defects in epithelia, intestine, lung, or in hair development. Finally, adam9/12-/- double knockout and adam9/12/15-/- triple knockout mice were indistinguishable from wild-type controls in their appearance and behavior during routine handling.
To generate adam9/12/15/17-/- quadruple knockout mice, adam9/12/15-/- triple knockout mice were mated with adam17+/- animals. Offspring from this mating that were heterozygous for the targeted allele of all four ADAMs were identified by Southern blotting, and were backcrossed several times with adam9/12/15-/- triple knockout mice to obtain adam9-/- 12-/-15-/-17+/- mice. Crosses of adam9-/-12-/-15-/-17+/- mice produced litters with a similar distribution of the targeted ADAM17 allele at E18.5 to what has been reported from crosses of adam17+/- mice (Table III; Peschon et al., 1998). Histopathological evaluation of newborn adam9/12/15/17-/- quadruple knockout mice did not uncover any significant worsening of the developmental defects described for adam17-/- mice. The cause for the slightly increased embryonic lethality of adam9/12/15/17-/- mice compared with adam17-/- mice (Table III) remains to be determined. Images of fixed and hematoxylin and eosinstained heart sections mounted in Permount/Histoclear were acquired with Axiovision software via an Axiocam HRC camera mounted on an Axioplan2 microscope (software, camera, and microscope all from Carl Zeiss MicroImaging, Inc.). The objective was a Plan-Neofluar 10x/0,30 (44 03 30; Carl Zeiss MicroImaging, Inc.) lens. Images were processed with Adobe Photoshop® 7.0, and the surface area of heart valves in serial sections was measured using NIH Image 1.63 software.
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Generation of primary MEFs
Primary MEFs were generated from wild-type or adam-/- E13.5 embryos and were cultured as described previously (Weskamp et al., 2002). In addition to adam9/12/15-/- triple knockout and adam9/12/15/17-/- quadruple knockout mice, we also used adam17-/- (Peschon et al., 1998) and adam19-/- (Zhou et al., 2004) mice as well as wild-type controls of mixed genetic background (129Sv/C57Bl6) to generate the corresponding primary mEF cells. All genotyping was performed by Southern blot analysis. adam10-/- fibroblast cell lines derived from E9.5 embryos have been described previously (Hartmann et al., 2002).
Northern blot analyses
Procedures for isolation of mRNA, gel electrophoresis, transfer to membranes, and generation of 32P-labeled cDNA probes of the indicated ADAMs under high stringency were described previously (Weskamp and Blobel, 1994).
Transfections and shedding assays
cDNA constructs encoding AP-EGFR ligand fusion proteins were transfected with LipofectAMINETM (Invitrogen). Fresh Opti-MEM (Invitrogen) medium was added the next day, incubated for 1 h, and then replaced with fresh medium containing either 20 ng/ml PMA or 1 µM batimastat (provided by D. Becherer, GlaxoSmithKline, Research Triangle Park, NC), which was also collected after 1 h. Evaluation of AP activity by SDS-PAGE or by colorimetric assays was performed as described previously (Zheng et al., 2002). No AP activity was present in conditioned media of nontransfected cells.
Western blot analysis
Western blot analysis of the expression of ADAM10 and ADAM17 in MEFs and in different mouse tissues was performed as described previously (Weskamp et al., 1996). The blots were probed with a polyclonal antiserum against ADAM10 (CHEMICON International) and against ADAM17 (Schlöndorff et al., 2000).
Statistical analyses
t tests for two samples assuming equal variances were used to calculate the P values. P values <0.05 were considered statistically significant.
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Acknowledgments |
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This work was supported by National Institutes of Health grant RO1 GM65740 (to C.P. Blobel), by the Memorial Sloan-Kettering Cancer Center support grant NCI-P30-CA-08748, and by the Samuel and May Rudin Foundation, the DeWitt Wallace Fund, the Fonds der Chemischen Industrie, and the Deutsche Forschungsgemeinschaft.
Submitted: 21 July 2003
Accepted: 13 January 2004
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