Heme oxygenase-1 (HO-1) is a heme-degrading enzyme anchored in the endoplasmic reticulum by a carboxyl-terminal transmembrane segment (TMS). HO-1 is highly expressed in various cancers and its nuclear localization is associated with the progression of some cancers. Nevertheless, the mechanism underlying HO-1 nuclear translocation and its pathological significance remain elusive. Here we show that the signal peptide peptidase (SPP) catalyzes the intramembrane cleavage of HO-1. Coexpression of HO-1 with wild-type SPP, but not a dominant-negative SPP, promoted the nuclear localization of HO-1 in cells. Mass spectrometry analysis of cytosolic HO-1 isolated from HeLa cells overexpressing HO-1 and SPP revealed two adjacent intramembrane cleavage sites located after S275 and F276 within the TMS. Mutations of S275F276 to A275L276 significantly hindered SPP-mediated HO-1 cleavage and nuclear localization. Nuclear HO-1 was detected in A549 and DU145 cancer cell lines expressing high levels of endogenous HO-1 and SPP. SPP knockdown or inhibition significantly reduced nuclear HO-1 localization in A549 and DU145 cells. The positive nuclear HO-1 stain was also evident in lung cancer tissues expressing high levels of HO-1 and SPP. Overexpression of a truncated HO-1 (t-HO-1) lacking the TMS in HeLa and H1299 cells promoted cell proliferation and migration/invasion. The effect of t-HO-1 was not affected by a mutation in the catalytic site. However, blockade of t-HO-1 nuclear localization abolished t-HO-1-mediated effect. The tumorigenic effect of t-HO-1 was also demonstrated in the mouse model. These findings disclose that SPP-mediated intramembrane cleavage of HO-1 promotes HO-1 nuclear localization and cancer progression independent of HO-1 enzymatic activity.

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Recent evidence indicates that indigenous Clostridium species induce colonic regulatory T cells (Tregs), and gut lymphocytes are able to migrate to pancreatic islets in an inflammatory environment. Thus, we speculate that supplementation with the well-characterized probiotics Clostridium butyricum CGMCC0313.1 (CB0313.1) may induce pancreatic Tregs and consequently inhibit the diabetes incidence in non-obese diabetic (NOD) mice. CB0313.1 was administered daily to female NOD mice from 3 to 45 weeks of age. The control group received an equal volume of sterile water. Fasting glucose was measured twice a week. Pyrosequencing of the gut microbiota and flow cytometry of mesenteric lymph node (MLN), pancreatic lymph node (PLN), pancreatic and splenic immune cells were performed to investigate the effect of CB0313.1 treatment. Early oral administration of CB0313.1 mitigated insulitis, delayed the onset of diabetes, and improved energy metabolic dysfunction. Protection may involve increased Tregs, rebalanced Th1/Th2/Th17 cells and changes to a less proinflammatory immunological milieu in the gut, PLN, and pancreas. An increase of α4β7+ (the gut homing receptor) Tregs in the PLN suggests that the mechanism may involve increased migration of gut-primed Tregs to the pancreas. Furthermore, 16S rRNA gene sequencing revealed that CB0313.1 enhanced the Firmicutes/Bacteroidetes ratio, enriched Clostridium-subgroups and butyrate-producing bacteria subgroups. Our results provide the basis for future clinical investigations in preventing type 1 diabetes by oral CB0313.1 administration.

Chemotherapy resistance is one of the most common causes of death among patients with ovarian cancer, and identifying novel antitumor agents is a priority. Here, we report that the novel molecule 2‐(anaphthoyl)ethyl‐trimethylammonium iodide (α‐NETA) induces epithelial ovarian cancer (EOC) cell pyroptosis through the gesdermin‐d (GSDMD)/caspase‐4 pathway. Furthermore, Cell Counting Kit‐8 fluorescence‐activated cell sorting analysis showed that α‐NETA treatment led to cell death in different ovarian cancer cell lines, including Ho8910, Ho8910PM, and A2780. Morphologic examination by electron microscopy indicated that cells treated with α‐NETA produced multiple microbubbles, typical of cells undergoing pyroptosis. α‐NETA also significantly increased expression of pyroptosis‐associated molecules including caspase‐4 and GSDMD in EOC cells. Knockdown of either caspase‐4 or GSDMD in ovarian cancer cells strongly interfered with α‐NETA cell‐killing activity, indicating that α‐NETA acts through the pyroptosis pathway. In vivo, α‐NETA treatment dramatically decreased the size of EOC tumors in mice. Our findings suggest that α‐NETA represents a potential new antitumor molecule or lead compound for EOC chemotherapy.—Qiao, L., Wu, X., Zhang, J., Liu, L., Sui, X., Zhang, R., Liu, W., Shen, F., Sun, Y., Xi, X. α‐NETA induces pyroptosis of epithelial ovarian cancer cells through the GSDMD/caspase‐4 pathway. FASEB J. 33, 12760–12767 (2019). http://www.fasebj.org