Ectopic expression of the serine protease inhibitor PI9 modulates death receptor-mediated apoptosis

Abstract

Apoptosis is a highly controlled process, whose triggering is associated with the activation of caspases. Apoptosis can be induced via a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which recruit and activate pro-caspase-8 and -10. Regulation of apoptosis is achieved by several inhibitors, including c-FLICE-inhibitory protein, which prevents apoptosis by inhibiting the pro-apoptotic activation of upstream caspases. Here we show that the human intracellular serine protease inhibitor (serpin), protease inhibitor 9 (PI9), inhibits TNF-, TNF-related apoptosis-inducing ligand-and Fas ligand-mediated apoptosis in certain TNF-sensitive cell lines. The reactive center P1 residue of PI9 was required for this inhibition since PI9 harboring a GluAla mutation in its reactive center failed to impair death receptor-induced cell death. This suggests a classical serpin-protease interaction. Indeed, PI9 inhibited apoptotic death by directly interacting with the intermediate active forms of caspase-8 and -10. This indicates that PI9 can regulate pro-apoptotic apical caspases. Apoptosis is a highly controlled process, whose triggering is mainly associated with the activation of proteolytic enzymes.1,2 In the immune system, apoptotic death in target cells is mediated by cytotoxic lymphocytes (CL) including natural killer cells and cytotoxic T lymphocytes. These cells induce apoptosis via two different pathways: (a) the degranulation pathway and (b) a membrane receptor-ligand interaction involving members of the tumor necrosis family (TNF).2,3 In both pathways, activation of protease cascades plays an essential role in apoptotic death.Granules of CL contain several serine proteases called granzymes, of which granzyme B is the most important. Granzyme B possesses a unique Asp-ase activity and, upon entering the target cell, cleaves and activates pro-caspase-3 and other substrates to initiate apoptotic death. Target cell death can also be induced by members of the TNF family, such as membrane-bound Fas ligand (FasL) on the CL that interacts with its receptor Fas. Soluble-and membrane-bound TNF and TNF-related apoptosis-inducing ligand (TRAIL) also mediate death via their respective receptors, TNF receptor (TNF-R1) and TRAIL receptors (TRAIL-R1 and -R2). 4 Activation of these receptors lead to recruitment of the adaptor molecule, Fas-associated death domain (FADD). 5,6The death effector domain (DED) of FADD then recruits procaspase-8 and procaspase-10 via homotypic interaction with the DEDs in these caspases. Ligand, receptor, adaptor proteins and caspases form the death-inducing signaling complex (DISC). The high local concentration of procaspases promotes auto-activation and release into the cytosol triggering subsequent cleavage of downstream effector caspases (caspase-3, -6 and -7) leading to apoptotic death. Altogether, the induction and execution of apoptosis by CLs is mediated both by cysteine proteases, such as caspases, and by serine proteases, such as granzymes.Activation of apoptotic proteases within the...

Control of granzymes by serpins

“…Similarly, there has been one report of overexpressed ectopic SERPINB9 directly inhibiting intermediate active forms of caspases-8 and -10 during tumour necrosis factor and Fasinduced apoptosis. 61 However, the interaction could only be observed consistently in one of four tumour cell lines, which suggests that it is an overexpression artefact or it reflects an atypical characteristic of this particular cell line. On balance, coupled with the fact that no study has implicated rodent Serpinb9 in caspase regulation, evidence of a physiologically significant interaction between SERPINB9 and caspases is scant and unconvincing.…”

A Noncompetitive Small Molecule Inhibitor of Estrogen-regulated Gene Expression and Breast Cancer Cell Growth That Enhances Proteasome-dependent Degradation of Estrogen Receptor α

“…Induction of PI-9 may be a mechanism by which estrogens enable breast cancers to evade immune surveillance and apoptosis (21,23). PI-9 inhibits granzyme B and cytotoxic T lymphocyte and natural killer and cell-mediated apoptosis of target cancer cells (21,23,28) and caspase 8-dependent apoptosis induced by TNF-␣ family members (38,39). Expression of PI-9 is associated with a poor prognosis in some cancers (34 -36, 67).…”

“…The serpin PI-9 is a tumor lethality factor (34 -36) whose induction by estrogens enables breast cancer cells to evade apoptosis induced by the immune cells, cytotoxic T lymphocytes and natural killer cells (21,23,28). PI-9 also inhibits tumor necrosis factor-␣ (TNF-␣) Fas and TRAIL (TNF-related apoptosisinducing ligand)-mediated apoptosis (38,39). Induction of PI-9 results from direct binding of E 2 -ER␣ to EREs and ERE half-sites (19,40,41).…”

Granzyme B-induced apoptosis in cancer cells and its regulation (Review)

“…Recent findings indicate that PI-9 can inhibit not only the GrB/perforin-mediated death pathway but also the TNF-·-, TRAIL-and FasL-triggered death pathways (246,247). The inhibition of the death ligand-induced cell killing seems to reside in direct interaction of PI-9 with the intermediate active forms of caspase-8 and -10 (247).…”