An Inhibitor of the F1 subunit of ATP synthase (IF1) modulates the activity of angiostatin on the endothelial cell surface

Abstract

Angiostatin binds to endothelial cell (EC) surface F(1)-F(0) ATP synthase, leading to inhibition of EC migration and proliferation during tumor angiogenesis. This has led to a search for angiostatin mimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F(1) ATP synthase and inhibited F(1)-dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. Although exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared with angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis.

FIG. 1

Inhibition of purified F₁ ATP synthase activity by IF1. Purified F₁ ATP synthase activity was measured as a change in fluorescence (emission λ =355 nm, excitation λ =460 nm) by coupling the production of ADP to the oxidation of NADH via pyruvate kinase and lactate dehydrogenase. Inhibition of F₁ activity is represented by an increase in relative fluorescence units (RFU). IF1 (0–4 μM) was added to a constant amount of F₁ ATP synthase at either pH 7.5 (open bars) or pH 6.5 (hatched bars). Azide, a known inhibitor of F₁F₀ ATP synthase completely inhibited F₁ activity, similar to IF1 at pH 6.5.

FIG. 2

Binding of IF1 to purified bovine F₁ ATP synthase. ELISA was employed to demonstrate concentration-dependent binding of IF1 to F₁ ATP synthase. Each well was coated with 10 μg/ml of F₁ ATP synthase before addition of increasing amounts of IF1. Control lane (-) shows binding of secondary antibody only. n=3.

FIG. 3

Inhibition of HUVEC proliferation at low pH (pH <7.0) in the presence of IF1 as measured by BrdU incorporation. HUVEC proliferation at 48 h was inhibited 20% by IF1 at concentrations of 1 μg/ml (closed squares) and 10 μg/ml (open triangles) compared to media only (open squares) and PBS controls (closed circles). Cycloheximide, an inhibitor of protein synthesis, inhibited cell proliferation by 65%; n=3 (open circles).

FIG. 4

HUVEC tube differentiation in the presence of IF1. EC, pre-incubated at pH 6.5 or pH 7.5, were plated on Matrigel-coated wells in the presence of PBS only or 1 μM IF1. At pH 6.5, PBS only positive control (a) was comparable to 1 μM IF1 (b). At pH 7.5, PBS only control (c) was also identical to 1 μM IF1 (d). Cycloheximide, a known inhibitor of protein synthesis, completely inhibited tube formation (e).

FIG. 5

Endogenous IF1 on the surface of HUVEC. a) HUVEC were incubated at pH 7.4 overnight before incubating with anti-IF1 and anti-CD31 antibodies. IF1 was shown to be endogenously present on the surface of EC (light gray peak) compared to secondary only control (black peak). CD31, a known marker on the surface of EC was used as a positive control (medium gray peak). b) HUVEC were incubated at pH 7.4 before treatment with exogenous IF1 (IF1 ex) (gray peak) or HBSS+ buffer only to demonstrate endogenous IF1 (IF1 en) (black peak). Exogenous IF1 increased the signal (median intensity) of IF1 on the surface of EC by 41%.

FIG. 6

IF1 binding to bovine F₁ ATP synthase in the presence of angiostatin by ELISA. Wells were coated with F₁ ATP synthase (10 μg/ml) before incubation with angiostatin 100 μg/ml “+angiostatin” or PBS only “−angiostatin”. IF1 was then incubated at increasing concentrations (0–10 μg/ml). Pre-incubation with angiostatin inhibited IF1 (10 μg/ml) binding to ATP synthase approximately 70%. A K_d of 5 nM was calculated from binding data in the binding isotherm using statistics software called Systat for Windows, version 5 (Systat Inc. Evanston, IL).

FIG. 7

The effects of IF1 and angiostatin on EC tube differentiation. Competition experiments with sequential addition of angiostatin and IF1 are shown. The first bar (white) represents the control where cells were plated in the absence of either angiostatin or IF1. The second bar (light gray) represents the effect of 0.50 μM angiostatin. The third bar (hatched) shows the effect of sequential addition of IF1 (1 μM) followed by angiostatin (0.50 μM). The fourth bar (black) represents sequential addition of angiostatin (0.50 μM) and IF1 (5 μM). The fifth bar (dark gray) represents angiostatin (0.50 μM) followed by IF1 (1 μM).