Characterization of the catalytic properties of the membrane-anchored metalloproteinase ADAM9 in cell-based assays

Abstract

ADAM9 (A Disintegrin And Metalloprotease 9) is a membrane-anchored metalloproteinase that has been implicated in pathological retinal neovascularization and in tumor progression. ADAM9 has constitutive catalytic activity in both biochemical and cell-based assays and can cleave several membrane proteins, including epidermal growth factor and Ephrin receptor B4; yet little is currently known about the catalytic properties of ADAM9 and its post-translational regulation and inhibitor profile in cell-based assays. To address this question, we monitored processing of the membrane-anchored Ephrin receptor B4 (EphB4) by co-expressing ADAM9, with the catalytically inactive ADAM9 E > A mutant serving as a negative control. We found that ADAM9-dependent shedding of EphB4 was not stimulated by three commonly employed activators of ADAM-dependent ectodomain shedding: phorbol esters, pervanadate or calcium ionophores. With respect to the inhibitor profile, we found that ADAM9 was inhibited by the hydroxamate-based metalloprotease inhibitors marimastat, TAPI-2, BB94, GM6001 and GW280264X, and by 10 nM of the tissue inhibitor of metalloproteinases (TIMP)-3, but not by up to 20 nM of TIMP-1 or -2. Additionally, we screened a non-hydroxamate small-molecule library for novel ADAM9 inhibitors and identified four compounds that selectively inhibited ADAM9-dependent proteolysis over ADAM10- or ADAM17-dependent processing. Taken together, the present study provides new information about the molecular fingerprint of ADAM9 in cell-based assays by showing that it is not stimulated by strong activators of ectodomain shedding and by defining a characteristic inhibitor profile. The identification of novel non-hydroxamate inhibitors of ADAM9 could provide the basis for designing more selective compounds that block the contribution of ADAM9 to pathological neovascularization and cancer.

Keywords: ADAM9 (A Disintegrin And Metalloproteinase 9); EphB4; ectodomain shedding; molecular fingerprint.

Figure 1.. Overexpression of ADAM9 results in increased release of EphB4-AP.

COS7 cells (A, E), Adam9−/− (B), Adam10/17−/− (C) and wild type (WT)-mEFs (D) were co-transfected with EphB4-AP and WT ADAM9 (A9) or the inactive ADAM9 E>A control (EA9), in which the catalytic site consensus sequence HEXXH was mutated to HAXXH. EphB4 was tagged with an alkaline phosphatase (AP) moiety to allow detection of the cleaved ectodomain in the cell supernatant. Then the cells were washed and incubated in conditioned medium for 2 hours. (A-D) In all cases, there was a significant increase in EphB4-AP shedding from cells overexpressing A9 compared to cells overexpressing EA9. Western blot analysis confirmed similar levels of overexpressed A9 or the EA9 control in the different cell lines shown here. Please note that the anti-mouse ADAM9 cyto antibodies used here do not cross-react with endogenous ADAM9 in COS7 cells (derived from monkey kidney), but recognize a minor non-specific band in Adam9−/− mEFs, and detect endogenous ADAM9 in wild type and Adam10/17−/− mEFs. Student’s t-test, *P ≤ 0.05; ±SEM. (E) Increasing amounts of transfected A9 cDNA increased the shedding of co-transfected EphB4-AP from COS7 cells. Dunnett’s test, *P ≤ 0.05; ±SEM. Increased levels of transfected A9 or EA9 cDNA correlated with increased levels of A9 or EA9 protein, as confirmed by Western blot analysis. All Western blots and graphs are representative of at least 3 separate experiments.

Figure 2.. TIMP-inhibitor profile of ADAM9 in cell-based assays.

The effect of 5, 10 or 20 nM TIMP-1 (A), TIMP-2 (B), or TIMP-3 (C) was tested on Adam10/17−/− mEFs expressing EphB4-AP together with ADAM9 (A9) or catalytically inactive ADAM9 E>A (EA9). The numbers indicate the fold increase in shedding after 2 hours of incubation under different conditions, using untreated cells expressing EA9 as a reference for background shedding, which is set to 1. TIMP-1 had no significant effect on background shedding in the EA9 expressing cells. In cells expressing A9, there was an increase in EphB4 shedding of approximately 250%, and this was not significantly affected by up to 20 nM TIMP-1. Concentrations of up to 20 nM TIMP-2 also did not affect EphB4 background shedding in the presence of EA9. TIMP-3 partially blocked EphB4 shedding at concentrations of 10 nM and 20 nM. These results are representative of at least 3 separate experiments. Dunnett’s test, *P ≤ 0.05; ±SEM

Figure 3.. The catalytic activity of ADAM9 in cell-based assays is sensitive to the hydroxamates TAPI-2, GM6001, marimastat, BB94 and GW280264X but not to the ADAM10 inhibitor GI254023X.

Panels A – F show the effect of different concentrations of the hydroxamates TAPI-2 (A), GM6001 (B), marimastat (C), BB94 (D), GW280264X (E) and GI254023X (F) on the increase in EphB4-AP shedding after 2 hours of incubation in Adam10/17−/− mEFs overexpressing ADAM9 compared to cells expressing ADAM9 E>A. Each panel is representative of at least 3 separate experiments. Dunnett’s test, *P ≤ 0.05; ±SEM

Figure 4.. Evaluation of the response of ADAM9 to stimuli of ectodomain shedding in cell-based assays.

The effect of 20 ng/ml PMA, 100 μM of the phosphatase inhibitor pervanadate (PV), or 2.5 μM Ionomycin (IO) on ADAM9 (A9)-dependent shedding of EphB4-AP from Adam10/17−/− mEFs is shown after 30 minutes of stimulation. The numbers in each bar represent the percent shedding compared to cells expressing ADAM9 E>A (EA9), where shedding in the absence of treatment was used as a reference, and set to 1. Treatment with these compounds had no effect on EphB4-AP shedding in the presence or absence of active A9. These results are representative of at least 3 separate experiments. Dunnett’s test, ns = not significant; ±SEM

Figure 5.. Identification of four ADAM9 inhibitors based on a screen of a small molecule library.

The structures of compounds 28, 41, 50, and 67 are shown in A-D. The effect of different compound concentrations on the increase in EphB4 shedding in Adam10/17−/− mEFs overexpressing ADAM9 compared to cells expressing ADAM9 E>A after 2 hours is shown for compound 28 (E), compound 41 (F), compound 50 (G), and compound 67 (H). In wild type mEFs, none of the inhibitors had an effect on the IO-stimulated shedding of BTC-AP (2.5 μM, 30 minutes), which depends on ADAM10 (I-L) or the PMA-stimulated shedding of TGFα–AP (20 ng/ml, 30 minutes), which depends on ADAM17 (M-P) respectively. MM, marimastat (5 μM). Dunnett’s test, *P ≤ 0.05; ±SEM