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iRhom2 controls the substrate selectivity of stimulated ADAM17-dependent ectodomain shedding - PubMed

  • ️Tue Jan 01 2013

iRhom2 controls the substrate selectivity of stimulated ADAM17-dependent ectodomain shedding

Thorsten Maretzky et al. Proc Natl Acad Sci U S A. 2013.

Erratum in

Abstract

Protein ectodomain shedding by ADAM17 (a disintegrin and metalloprotease 17), a principal regulator of EGF-receptor signaling and TNFα release, is rapidly and posttranslationally activated by a variety of signaling pathways, and yet little is known about the underlying mechanism. Here, we report that inactive rhomboid protein 2 (iRhom2), recently identified as essential for the maturation of ADAM17 in hematopoietic cells, is crucial for the rapid activation of the shedding of some, but not all substrates of ADAM17. Mature ADAM17 is present in mouse embryonic fibroblasts (mEFs) lacking iRhom2, and yet ADAM17 is unable to support stimulated shedding of several of its substrates, including heparin-binding EGF and Kit ligand 2 in this context. Stimulated shedding of other ADAM17 substrates, such as TGFα, is not affected in iRhom2(-/-) mEFs but can be strongly reduced by treating iRhom2(-/-) mEFs with siRNA against iRhom1. Activation of heparin-binding EGF or Kit ligand 2 shedding by ADAM17 in iRhom2(-/-) mEFs can be rescued by wild-type iRhom2 but not by iRhom2 lacking its N-terminal cytoplasmic domain. The requirement for the cytoplasmic domain of iRhom2 for stimulated shedding by ADAM17 may help explain why the cytoplasmic domain of ADAM17 is not required for stimulated shedding. The functional relevance of iRhom2 in regulating shedding of EGF receptor (EGFR) ligands is established by a lack of lysophasphatidic acid/ADAM17/EGFR-dependent crosstalk with ERK1/2 in iRhom2(-/-) mEFs, and a significant reduction of FGF7/ADAM17/EGFR-stimulated migration of iRhom2(-/-) keratinocytes. Taken together, these findings uncover functions for iRhom2 in the regulation of EGFR signaling and in controlling the activation and substrate selectivity of ADAM17-dependent shedding events.

Keywords: ADAMs; Rhbdf1/2.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

iRhom2 is required for metalloproteinase-dependent crosstalk between the LPA receptor and ERK1/2 and for the substrate selectivity of stimulated ADAM17-dependent shedding events. (A) Western blot analysis shows similar levels of mature ADAM17 in mEFs from iRhom2−/− mice and littermate controls (Adam17−/− mEFs are shown as negative control; ADAM15 Western blot is shown as loading control; n = 3). (B) WT and iRhom2−/− mEFs, starved for 24 h, were treated for 15 min with 10 µM LPA, 20 ng/mL recombinant EGF (rEGF), or medium alone. Where indicated, cells were pretreated for 20 min with 20 µM the hydroxamate metalloproteinase inhibitor MM. The Western blot was probed for phospho-ERK1/2, with total ERK1/2 as loading control. (C) Densitometric quantification of ERK1/2 phosphorylation relative to total ERK (n = 3). (D–N) WT and iRhom2−/− mEFs were transfected with the AP-tagged EGFR ligands HB-EGF (D), amphiregulin (E), epiregulin (F), TGFα (G), BTC (H), or EGF (I) or the ADAM17 substrates EphB4 (J), KitL2 (K), Tie2 (L), CD62L (M), or ICAM-1 (N). Shedding of all ADAM17 substrates was activated by treatment with 25 ng/mL PMA for 30 min (D–G and J–N), whereas shedding of the ADAM10 substrates BTC and EGF was activated by 2.5 µM ionomycin (IO) (H and I). *P ≤ 0.05; ±SEM (n = 3).

Fig. 2.
Fig. 2.

iRhom2 is required for the stimulated shedding of HB-EGF and KitL2 in response to physiological stimuli but not of TGFα. (A–F) WT and iRhom2−/− mEFs were transfected with the AP-tagged ADAM17 substrates HB-EGF (A and B), KitL2 (C and D), or TGFα (E and F) and then activated with different stimuli for 30 min, as indicated [LPA (10 µM), PDGF (50 ng/mL), Thr (2 units/mL), or TNFα (10 ng/mL)]. All stimuli tested here activated ADAM17-mediated shedding, as evidenced by the significantly increased cleavage of HB-EGF (A), KitL2 (C), or TGFα (E) in WT mEFs. Identical experiments were performed with iRhom2−/− mEFs, in which stimulation for 30 min with LPA, Thr, PDGF, or TNFα did not increase the shedding of HB-EGF (B) or KitL2 (D) but activated the release of TGFα (F). *P ≤ 0.05; ±SEM (n = 3 for A–F). ctrl, control.

Fig. 3.
Fig. 3.

Analysis of the requirement for the N-terminal cytoplasmic domain of iRhom2 in the rapid and posttranslational activation of ADAM17-dependent shedding of KitL2. (A–D) iRhom2−/− (A and C) or WT mEFs (B and D) were cotransfected with KitL2-AP (A and B) or TGFα-AP together with full-length WT-iRhom2, ΔCyto-iRhom2 (lacking the N-terminal cytoplasmic domain), or MAD2 as a control and either left untreated or stimulated with PMA (25 ng/mL; 30 min). (E) The membrane topology and domain organization of ΔCyto-iRhom2 and WT-iRhom2 is shown, with red asterisks indicating potential N-linked glycosylation sites in the large extracellular loop. C indicates C terminus; N indicates N terminus. The green cylinder at the C terminus marks the position of the T7 tag. (F) Representative Western blot of ΔCyto-iRhom2 or WT-iRhom2 expressed in iRhom2−/− mEFs and detected with antibodies against a C-terminal T7-tag. *P ≤ 0.05; ±SEM (n = 3 for A–F).

Fig. 4.
Fig. 4.

Analysis of the contribution of the extracellular or cytoplasmic domain of TGFα and HB-EGF to their stimulated shedding in iRhom2−/− mEFs. (A) Full-length TGFα-AP or HB-EGF-AP or chimeras of these EGFR ligands containing the extracellular EGF module and JM with the cleavage site of TGFα but the TM-CT of HB-EGF (TGFα/HB-EGF) or the EGF module of TGFα with the JM and TM-CT of HB-EGF (TGFα/HB-EGF JM) or the EGF module and JM of HB-EGF attached to the TM-CT of TGFα (HB-EGF/TGFα) or the EGF module of HB-EGF with the JM and TM-CT of TGFα (HB-EGF/TGFα-JM). The constructs depicted in A were transfected into iRhom2−/− (B) or WT (C) mEFs, and their constitutive and PMA-stimulated shedding into the supernatant was determined. *P ≤ 0.05, ±SEM (n = 3 for B and C).

Fig. 5.
Fig. 5.

Effect of inactivation of iRhom1 or iRhom2 or both iRhom1 and -2 on ADAM17- and ADAM10-dependent shedding. iRhom2−/− mEFs (A, E, G, H, I, and K) or WT controls (B, F, and J) were transfected with the AP-tagged ADAM17 substrates TGFα (A, B, and H), KitL2 (E–G), or the AP-tagged ADAM10 substrate BTC (I–K) and stimulated with 25 ng/mL PMA to activate ADAM17-dependent shedding (A, B, E, F, and H) or 2.5 µM ionomycin (IO) to activate ADAM10-dependent shedding (G and I–K) with or without siRNA against iRhom1 (siR1) (10 nM) (A, B, E, F, I, and J) or the ADAM17-specific inhibitor SP26 (G, H, and K). (C) ADAM17 Western blot shows a decrease of mature ADAM17 only in iRhom2−/− mEFs treated with iRhom1 siRNA but not in WT controls treated with iRhom1 siRNA. (D) Densitometric quantification of the percentage of mature ADAM17 relative to the proform in blots from three separate experiments like the one shown in C. *P ≤ 0.05; ±SEM (n = 3 for A–K).

Fig. 6.
Fig. 6.

iRhom2 controls ADAM17-dependent keratinocyte migration. (A and B) Primary WT (A) or iRhom2−/− (B) keratinocytes from 12-wk-old animals were cultured to confluence, and then a scratch wound was introduced, and the cultures treated with or without FGF7 (50 ng/mL) or HB-EGF (50 ng/mL), as indicated. Micrographs were taken at 0 and 48 h after scratch wounding. (Scale bar: 100 μm.) (C and D) Quantification of the results obtained with WT keratinocytes (C) or iRhom2−/− keratinocytes (D) (n = 3). (E) Western blot of ERK1/2 phosphorylation in primary WT or iRhom2−/− keratinocytes incubated with or without FGF7 (20 ng/mL) or HB-EGF (50 ng/mL) (ERK1/2 was loading control in E). (F) Densitometric quantification of the levels of pERK1/2 of three experiments like the one shown in E. *P ≤ 0.05; ±SEM.

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