Induction of Polyamine Oxidase 1 by Helicobacter pylori Causes Macrophage Apoptosis by Hydrogen Peroxide Release and Mitochondrial Membrane Depolarization

Abstract

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.

Helicobacter pylori Induces ERK-dependent Formation of a Phospho-c-Fos·c-Jun Activator Protein-1 Complex That Causes Apoptosis in Macrophages

“…A rabbit anti-mouse secondary antibody conjugated to FITC (Jackson ImmunoResearch Laboratories Inc., Westgrove, PA; 1:1000 dilution) was used. The nuclei were visualized with PI, and staining was imaged as described (8,37).…”

“…The cells were analyzed with a flow cytometer (FACSCalibur; BD Biosciences) using CellQuest software (BD Biosciences) for annexin V-FITC/ PI-stained cells (8,9) and FlowJo software (Tree Star, Inc., Ashland, OR) for the annexin V-FITC/7-AAD-stained cells (36).…”

“…For c-Fos and c-Jun, 1 g of mRNA was reverse-transcribed with the iScript TM cDNA synthesis kit (Bio-Rad). PCR was performed with thermal cycling conditions and methods to calculate the relative expression as described (2,8,9,35).…”

“…These findings suggest that the failure of the immune response could be directly related to the inability of effector cells, especially macrophages, to kill this bacterium. We have demonstrated that H. pylori induces apoptosis in macrophages in vitro by a polyamine-dependent mechanism (7)(8)(9). However, the signaling mechanisms involved in this process and their in vivo relevance remains to be elucidated.…”

“…This enhances expression of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis, which causes apoptosis by a mechanism that involves oxidation of spermine (8). We now investigated the particular MAPK pathways activated in macrophages, the components of the AP-1 complex, and the role of these responses in the induction of apoptosis.…”