Protease Inhibitor 10 Inhibits Tumor Necrosis Factor α-induced Cell Death

Abstract

Protease inhibitor 10 (PI10) is a member of the ovalbumin family of serine protease inhibitors (ov-serpin) that is expressed at elevated levels in patients with acute myeloid leukemia and chronic myelomonocytic leukemia. Based upon the ability of the related serpin plasminogen activator inhibitor 2 (PAI-2) to protect cells against tumor necrosis factor ␣ (TNF␣)-induced cell death, this study was initiated to investigate the potential cytoprotective activity of PI10. Two different expression systems (i.e. plasmids encoding either PI10 alone or PI10 fused to the tag: enhanced green fluorescent protein, EGFP) were utilized to stably transfect an eukaryotic model cell system (i.e. HeLa cells) that neither expresses PAI-2 nor PI10. The level of PI10 expression in the stable transfectants was found to correlate with their resistance to TNF␣-induced cell death. Immunoprecipitation/immunoblotting experiments demonstrated that PI10 is able to form SDS-stable complexes (i.e. M r >100,000) with a cytosolic protein(s). Increased levels of the PI10-containing complexes can be detected by TNF␣ treatment by preventing intracellular degradative activities with the proteasome inhibitor N-carbobenzyloxy-leucine-leucine-norvalinal. PI10-containing complexes are dissociated with conditions known to separate classical protease-serpin complexes (i.e., 1.5 M ammonium hydroxide in the presence of SDS). These data support a role for the regulation of intracellular protease activities by ov-serpins. Protease inhibitor 10 (PI10)1 (1) is a member of the ovalbumin family of serine protease inhibitors (ov-serpin) that exhibits an expression pattern restricted to cells within the monocytic lineage (2). The absence of a signal leader sequence in PI10's coding sequence (1, 2) coupled with a nuclear localization signal in its C-D interhelical loop (3) results in this inhibitor being present both as a cytoplasmic and a nuclear molecule. The unique tissue and subcellular distribution for this serpin raises the possibility that PI10 may play a key role in normal hematopoietic development, a process that requires the regulation of cell viability, growth, and differentiation.The ability of diverse types of serine protease inhibitors to prevent cell death suggests that intracellular proteases other than cysteine aspartic-specific proteases can also participate in this process (for reviews, see Refs. 4 and 5). This concept is supported by the observation that the ov-serpin PAI-2 is able to confer resistance from the cytotoxic effects of tumor necrosis factor ␣ (TNF␣) in two cell lines (i.e. HT1080 (6) and HeLa (7)). Unfortunately, high M r SDS-stable complexes between PAI-2 and another molecule have not been observed during these cell death experiments, thus impeding further research on the pathway. Based upon (i) the high amino acid homology (48%) between PAI-2 and PI110 (1) and (ii) our observations that patients with acute myeloid leukemia and chronic myelomonocytic leukemia express high levels of PI10 (2), we hypothesized that this ov-serpin might ...

Maspin sensitizes breast carcinoma cells to induced apoptosis

“…On the other hand, since several other serpins implicated in apoptosis primarily regulate inducedapoptosis (Dickinson et al, 1995;Kanamori et al, 2000;Kwaan et al, 2000;Schleef and Chuang, 2000), the negligible e ect of maspin on the growth and survival of MDA-MB-435 cells in maintenance culture may be a manifestation in an environment that lacks appropriate apoptotic stimuli.…”

Bomapin is a redox-sensitive nuclear serpin that affects responsiveness of myeloid progenitor cells to growth environment

“…Bomapin is assumed to have P1-Arg at its reactive centre which is consistent with in vitro inhibition of thrombin and trypsin [14]. Bomapin complex with a possible intracellular protease was detected in HeLa cells over-expressing the serpin, but the protease was not identified [17]. It was proposed that bomapin possibly regulates a protease in the early stages of hematopoietic differentiation [15].…”

“…It is expressed only in bone marrow, leukocytes of patients with myeloid leukaemia that correspond to myeloid progenitors [14], and promyelocytic leukaemia cell lines (HL60, THP1, and AML-193), but it is not present in terminally differentiated leukocytes [15]. This protein has a 22 amino acid-long CD-loop that contains a nuclear localization signal [16,17]. Bomapin is assumed to have P1-Arg at its reactive centre which is consistent with in vitro inhibition of thrombin and trypsin [14].…”

Identification and Characterization of A Novel Rat Ov-Serpin Family Member, Trespin

“…6, A and B). Also, the only physiological role of bomapin described is resistance to tumor necrosis factor ␣-induced apoptosis (22) in HeLa cells, a cell line that does not express endogenous bomapin. Even so, we investigated a similar role for trespin and found no protection from tumor necrosis factor ␣-induced apoptosis in HeLa cells by both transient transfection and stable clone assays (data not shown), further substantiating that bomapin and trespin have distinct physiological roles.…”

“…Other ov-serpins such as plasminogen inhibitor-9 (PI-9) can inhibit cells from undergoing granzyme B-mediated apoptosis and may regulate host defense against microbial or viral proteinases (20,21). Bomapin (proteinase inhibitor-10) as well as plasminogen activator inhibitor-2 (PAI-2) confers apoptotic resistance to tumor necrosis factor ␣ in several cell lines (22)(23)(24). Maspin (protease inhibitor-5) has been identified as an inhibitor of cell motility and metastasis along with demonstrating anti-angiogenesis properties (25-28).…”