ADAM17 is regulated by a rapid and reversible mechanism that controls access to its catalytic site

Abstract

Protein ectodomain shedding is crucial for cell-cell interactions because it controls the bioavailability of soluble tumor necrosis factor-α (TNFα) and ligands of the epidermal growth factor (EGF) receptor, and the release of many other membrane proteins. Various stimuli can rapidly trigger ectodomain shedding, yet much remains to be learned about the identity of the enzymes that respond to these stimuli and the mechanisms underlying their activation. Here, we demonstrate that the membrane-anchored metalloproteinase ADAM17, but not ADAM10, is the sheddase that rapidly responds to the physiological signaling pathways stimulated by thrombin, EGF, lysophosphatidic acid and TNFα. Stimulation of ADAM17 is swift and quickly reversible, and does not depend on removal of its inhibitory pro-domain by pro-protein convertases, or on dissociation of an endogenous inhibitor, TIMP3. Moreover, activation of ADAM17 by physiological stimuli requires its transmembrane domain, but not its cytoplasmic domain, arguing against inside-out signaling via cytoplasmic phosphorylation as the underlying mechanism. Finally, experiments with the tight binding hydroxamate inhibitor DPC333, used here to probe the accessibility of the active site of ADAM17, demonstrate that this inhibitor can quickly bind to ADAM17 in stimulated, but not quiescent cells. These findings support the concept that activation of ADAM17 involves a rapid and reversible exposure of its catalytic site.

Fig. 1.

Response of ADAM10 and ADAM17 to physiological stimuli of protein ectodomain shedding. (A) Wild-type (wt) mEF cells were transfected with TGFα-AP to monitor the activity of ADAM17, and stimulated for 30 minutes with LPA (10 μM), Thr (2 units/ml), TNFα (10 ng/ml), EGF (100 ng/ml) or BzATP (300 μM), as indicated. All stimuli tested here activated ADAM17, as evidenced by the significantly increased shedding of TGFα. (B) Identical experiments were performed with wild-type mEF cells transfected with the ADAM10 substrate BTC. Stimulation for 30 minutes with LPA, Thr, TNFα or EGF did not increase the shedding of BTC, whereas stimulation with BzATP did. Cells treated with BzATP in A and B were also co-transfected with its receptor, P2X7R. (C) In Adam17−/− mEFs transfected with TGFα and the inactive mutant ADAM17E>A (AD17E>A), shedding of TGFα was not stimulated above background levels by 30 minutes of treatment with LPA, Thr, TNF or EGF. (D,E) In Adam17−/− cells rescued with wild-type ADAM17 (AD17wt) (D) or ADAM17Δ-cyto (AD17Δ-cyto) (E) the stimulated shedding of TGFα by LPA, Thr, TNFα and EGF was restored to an equal extent. (F) To assess whether ADAM17 requires its cytoplasmic domain to respond to ionomycin or APMA, we performed similar rescue experiments in cells deficient in both ADAM10 and ADAM17 (Adam10/17−/−). Both ADAM17 and ADAM17Δ-cyto rescued IM- or APMA-stimulated shedding of ICAM-1 from these cells (30 minutes incubation) equally well. (G) To evaluate the role of the ADAM17 cytoplasmic domain in its response to stimulation with BzATP, Adam10/17−/− mEFs were transfected with P2X7R, ICAM-1 and wild-type ADAM17, ADAM17Δ-cyto or inactive ADAM17E>A There was no difference in the response of wild-type ADAM17 or ADAM17Δ-cyto to BzATP. (H–K) The role of the transmembrane domain of ADAM17 in its response to different stimuli was evaluated by generating chimera of the ADAM17 extracellular domain with the transmembrane domain of BTC (AD17-BTC) or CD62L (AD17-CD62L, see supplementary material Fig. S1A for details). Stimulated shedding of TGFα by LPA, Thr, TNFα or 25 ng/ml PMA was not restored by co-transfection of ADAM17-BTC (I) or ADAM17-CD62L (J) in Adam17−/− cells. ADAM17E>A (H) and ADAM17wt (K) served as negative and positive controls, respectively. Data are the average + s.e.m. of at least three separate experiments performed in duplicate. o indicates no statistical significance, *P<0.05, **P<0.01.

Fig. 2.

Downregulation of CD62L by ADAM10 or ADAM17 in primary B cells. (A,C) The cell surface expression of CD62L (A) or CD23 (C) was quantified by flow cytometry on freshly isolated control (Adam17flox/flox) and ADAM17-deficient (Adam17flox/flox/CD19-Cre) B cells. CD62L levels were higher in ADAM17-deficient cells, whereas the CD23 geomean showed no significant difference in the presence or absence of ADAM17 in unstimulated B cells. (B,D) The cell surface expression (geomean) of the ADAM17 substrate CD62L (B) and the ADAM10 substrate CD23 (D) was determined by flow cytometry in control and ADAM17-deficient B cells at different times after treatment with 300 μM BzATP compared with untreated controls. The change in CD62L geomean following BzATP treatment is expressed as percentage compared with untreated cells. The downregulation of CD62L on control B cells was significantly faster than in Adam17−/− cells (B). The shedding of the ADAM10 substrate CD23 was similar for both cell types (D). (E–G) To assess the relative contribution of ADAM10 to CD62L shedding in control versus Adam17−/− B cells, we tested how the ADAM10-selective inhibitor GI affected the decrease in CD62L levels following stimulation with BzATP. 1 μM GI did not affect the downregulation of CD62L on control cells (E), but reduced CD62L release by ~50% from Adam17−/− cells (F). 1 μM GI also reduced BzATP-stimulated shedding of CD23 by ~50% (G), providing a control for the effect of GI on ADAM10 under these conditions. Data are the average + s.e.m. of at least three separate experiments performed in duplicate. o signifies no statistical significance, *P<0.05, **P<0.01.

Fig. 3.

Stimulation of ADAM17 does not require removal of TIMP3 or pro-domain processing. (A) Primary mEF cells isolated from Timp3−/− and Timp3+/− embryos were transfected with the ADAM17 substrates TGFα or CD62L and tested for their ability to respond to stimulation with 25 ng/ml PMA for 30 minutes. No significant difference was observed between Timp3−/− mEFs and Timp3+/− controls. (B) Cos7 cells transfected with the ADAM17 substrate AR were preincubated for different times (2–24 hours) with 50 μM RVKR, and then stimulated with 25 ng/ml PMA for 30 minutes with or without RVKR. No significant effect of RVKR on shedding of AR was observed. (C) As a positive control for inhibition of pro-protein convertases by 50 μM RVKR, Cos7 cells expressing AD17-EC-Fc were preincubated for 4 hours with 50 μM RVKR, and then fresh medium with or without 50 μM RVKR was added for 1hour. Processing of AD17-EC-Fc to the mature form was blocked by 50 μM RVKR. (D) Western blot of endogenous ADAM17 in Cos7 cells treated with 50 μM RVKR for 2–24 hours (as in B) and probed with anti-ADAM17 cytoplasmic domain antibodies. There was no major decrease in the ratio of mature to pro-ADAM17 in treated versus untreated cells up to 8 hours, whereas an accumulation of pro-ADAM17 accompanied by a decrease of mature ADAM17 became apparent after treatment for 24 hours. Data represent the average + s.e.m. of at least three separate experiments performed in duplicate, **P<0.01.

Fig. 4.

PMA stimulation of ADAM17 is rapidly reversible. (A) Cos7 cells transfected with AR were stimulated with 25 ng/ml PMA continuously for 110 minutes, or for 10 minutes followed by washing out of excess PMA. Removal of PMA had no significant effect on the shedding of AR compared with continuously stimulated cells. (B) Cos7 cells transfected with AR were sequentially incubated with or without MM (4 μM), or 25 ng/ml PMA, or MM and PMA for 4 hours. Then the cells were washed, and incubated in conditioned medium (CM) for 1 hour. MM blocked both constitutive and PMA-stimulated shedding in the first 4 hours, and could be readily washed out, resulting in increased AR shedding from unstimulated cells treated with MM compared with cells not incubated with MM. Moreover, PMA increased shedding over constitutive levels even after being washed out, and shedding from cells treated with MM + PMA was further increased after removing MM. (C) To test whether the activation of ADAM17 is reversible, we performed similar experiments as in B; however, following stimulation of cells with MM + PMA for 5 minutes, the cells were washed and then treated with MM only, or with MM and BimI (4 μM) for an additional 5 minutes, before these compounds were washed out. The final treatment consisted of incubation in normal medium for 20 minutes with no additions or addition of PV (50 μM), as indicated. The increase in shedding from cells treated with MM + PMA following washout was blocked by treatment with BimI for 5 minutes, demonstrating that the activation of ADAM17 is reversible. When cells that were pretreated with MM + PMA and then with BimI were incubated with 50 μM PV, this strongly stimulated AR shedding. Data are representative of experiments performed at least three times, and are shown as average + s.e.m. of duplicates. Statistical significance was determined relative to the control, *P<0.05, **P<0.01.

Fig. 5.

Use of a tight-binding active site inhibitor of ADAM17, DPC333, as a probe for the accessibility of the catalytic site of ADAM17 in unstimulated and stimulated cells. (A) Cos7 cells transfected with AR were treated with conditioned medium (CM), or medium containing MM (4 μM) + PMA (25 ng/ml), DPC333 (0.25 μM) + PMA, or MM + PV (50 μM) or DPC333 + PV. After 40 minutes, all preincubated cells were washed, and then incubated in control medium without further additions, or in medium with PV or PMA, as indicated, for an additional 20 minutes. The results show that DPC333 cannot be washed out after PMA treatment, as the rebounding activity seen after washing out the reversible inhibitor MM in PMA treated samples is not observed in samples treated with DPC333 + PMA. Moreover, in samples treated with DPC333 + PMA for 40 minutes, then washed and treated with PV, no activation of ADAM17 was observed. A similar absence of stimulated shedding was found for samples preincubated with DPC333 + PV and then treated with PMA as a second stimulus. (B) AR-transfected Cos7 cells were incubated for 40 minutes in the presence or absence of DPC333, then washed and incubated for 20 minutes in the presence or absence of PMA or DPC333 or DPC333 + PMA, as indicated. DPC333 reduced constitutive shedding of AR in the first 40 minutes of treatment. However, after washing out of DPC333, the constitutive activity recovered to almost normal levels. PMA stimulation of cells that had been preincubated with DPC333 for 40 minutes under non-stimulated conditions resulted in almost identical stimulation as in cells that were not preincubated with DPC333. (C,D) RAW cells were cultured in six-well plates and preincubated for 90 minutes with or without 0.25 μM DPC333 together with 25 ng/ml PMA (C) or without PMA (D). Then, all compounds were washed out and the cells treated for 1 hour with fresh medium with or without 100 ng/ml LPS in the presence or absence of 0.25 μM DPC333. The conditioned culture supernatants were then used to quantify the amount of shed TNF using ELISA. Pretreatment of RAW cells with PMA and DPC333 significantly decreased the subsequent stimulation of TNF shedding by LPS, whereas pretreatment with DPC333 alone had no effect on LPS-stimulated TNF shedding compared with controls that were not pretreated. The results in A and B are representative of experiments performed at least three times, and the data in C and D represent the average + s.e.m. of three separate experiments performed in duplicates. o indicates not statistically significant, *P<0.05, **P<0.01.