Tissue-type plasminogen activator is a target of the tumor suppressor gene maspin

Abstract

The maspin protein has tumor suppressor activity in breast and prostate cancers. It inhibits cell motility and invasion in vitro and tumor growth and metastasis in nude mice. Maspin is structurally a member of the serpin (serine protease inhibitors) superfamily but deviates somewhat from classical serpins. We find that single-chain tissue plasminogen activator (sctPA) specifically interacts with the maspin reactive site loop peptide and forms a stable complex with recombinant maspin [rMaspin(i)]. Major effects of rMaspin(i) are observed on plasminogen activation by sctPA. First, rMaspin(i) activates free sctPA. Second, it inhibits sctPA preactivated by poly-D-lysine. Third, rMaspin(i) exerts a biphasic effect on the activity of sctPA preactivated by fibrinogen/gelatin, acting as a competitive inhibitor at low concentrations (< 0.5 microM) and as a stimulator at higher concentrations. Fourth, 38-kDa C-terminal truncated rMaspin(i) further stimulates fibrinogen/gelatin-associated sctPA. rMaspin(i) acts specifically; it does not inhibit urokinase-type plasminogen activator, plasmin, chymotrypsin, trypsin, or elastase. Our kinetic data are quantitatively consistent with a model in which two segregated domains of maspin interact with the catalytic and activating domains of sctPA. These complex interactions between maspin and sctPA in vitro suggest a mechanism by which maspin regulates plasminogen activation by sctPA bound to the epithelial cell surface.

Figure 1

(A) Purification of sctPA from MDA-MB-435-conditioned medium. Zymographic analysis of the plasminogen-dependent activity is shown. Lanes 1–5 are 15 μg of protein sample from MDA-MB-435-conditioned medium, 15 μg of protein of elution fraction 5, 0.45 NIH unit of high molecular weight uPA standard, 0.43 NIH unit of commercial sctPA standard, and 6.15 NIH units of elastase. (B) Dose-dependent inhibition of plasminogen activator activity by tPA-blocking monoclonal antibody. A coupled assay for plasminogen activation by free enzyme was performed. The rates of plasminogen activation by 0.4 NIH unit of pure sctPA (solid line) and by 15 ml of fraction 5 (dashed line) are normalized and presented as percentages of the corresponding positive controls in the absence of antibody. The error bars represent standard deviations from two parallel experiments.

Figure 2

Formation of a detergent-resistant complex between rMaspin(i) and sctPA. (A) Western blotting of sctPA–rMaspin(i) complex. The monomer form of rMaspin(i), dimer form of rMaspin(i), and commercial sctPA–rMaspin(i) complex had molecular masses of 42, 84, and 110 kDa, respectively. Lanes 1–6 are the mixtures of pure sctPA and rMaspin(i) at indicated molar ratios. Lane 7 is polymerized rMaspin(i) standard. (B) Western blotting of the sctPA–ovalbumin complex. Lanes 1–5 are the mixtures of sctPA and ovalbumin at indicated molar ratios.

Figure 3

The dose-dependent effect of rMaspin(i) on plasminogen activation by free sctPA activity (A) and by poly-d-lysine-coimmobilized sctPA (B). The initial velocities are presented as the rate of plasmin production in units of pM/min (⧫). The error bars represent the standard deviations of two parallel experiments. The dashed line represents calculated initial velocities by using Eq. 2 with α = 0.01 pM/min, β = 8.3 × 10⁻⁷/min, ξ = 0.425, ψ = 1.7 × 10⁻⁷ pM⁻¹, and ζ = 1.0 × 10⁻¹⁶ pM⁻² for A and α = 0.435 pM/min, ξ = 0.1, ψ = 4.0 × 10⁻⁷ pM⁻¹, and ζ = 1.0 × 10⁻¹⁶ pM⁻² for B.

Figure 4

(A) The dose-dependent effect of rMaspin(i) (○) and trypsin-cleaved maspin (•) on the plasminogen activation by fibrinogen/gelatin-activated sctPA. Dashed lines represent calculated initial velocities by using Eq. 2 with α = 0.21, β = 1.45 × 10⁻⁷/min, ξ = 0.1, ψ = 6.0 × 10⁻⁸ pM⁻¹, and ζ = 1.0 × 10⁻¹⁶ pM⁻² for rMaspin(i), and α = 1.01, β = 1.727 × 10⁻⁶/min, ξ = 0.425, ψ = 1.7 × 10⁻⁷ pM⁻¹, and ζ = 1.0 × 10⁻¹⁶ pM⁻² for trypsin-cleaved maspin. (B) Double-reciprocal plot of 1/v vs. 1/s for the plasminogen activation by fibrinogen/gelatin-activated sctPA at low concentrations of rMaspin(i). The substrate, S, is plasminogen. (C) Replot of the slopes of the double-reciprocal plot vs. the concentration of rMaspin(i). The intercept on the horizontal axis indicates the K_i value as 0.13 μM. The initial velocities are presented as rate of the production of plasmin in units of pM/min. The error bars represent the standard deviations of two parallel experiments.

Figure 5

Models for interactions between rMaspin(i) and sctPA. Positions left and right of sctPA represent bindings to regulatory and catalytic sites, respectively. Ks are equilibrium constants and ks are rate constants. v and M in Eq. 2 represent the initial velocity of plasmin production in pM/min and the concentration of rMaspin(i) (or 38-kDa C-terminal truncated rMaspin(i)) in μM, respectively. α, β, χ, ξ, ψ, and ζ are kinetic constants.