Caspase-7 mediates caspase-1-induced apoptosis independently of Bid.

Inflammasomes are innate immune mechanisms that activate caspase-1 in response to a variety of stimuli, including Salmonella infection. Active caspase-1 has a potential to induce two different types of cell death, depending on the expression of the pyroptosis mediator gasdermin D (GSDMD); following caspase-1 activation, GSDMD-sufficient and GSDMD-null/low cells undergo pyroptosis and apoptosis, respectively. Although Bid, a caspase-1 substrate, plays a critical role in caspase-1 induction of apoptosis in GSDMD-null/low cells, an additional mechanism that mediates this cell death independently of Bid has also been suggested. This study investigated the Bid-independent pathway of caspase-1-induced apoptosis. Caspase-1 has been reported to process caspase-6 and caspase-7. Silencing of caspase-7, but not caspase-6, significantly reduced the activation of caspase-3 induced by caspase-1, which was activated by chemical dimerization, in GSDMD/Bid-deficient cells. CRISPR/Cas9-mediated depletion of caspase-7 had the same effect on the caspase-3 activation. Moreover, in the absence of GSDMD and Bid, caspase-7 depletion reduced apoptosis induced by caspase-1 activation. Caspase-7 was activated following caspase-1 activation independently of caspase-3, suggesting that caspase-7 acts downstream of caspase-1 and upstream of caspase-3. Salmonella induced the activation of caspase-3 in GSDMD-deficient macrophages, which relied partly on Bid and largely on caspase-1. The caspase-3 activation and apoptotic morphological changes seen in Salmonella-infected GSDMD/Bid-deficient macrophages were attenuated by caspase-7 knockdown. These results suggest that in addition to Bid, caspase-7 can also mediate caspase-1-induced apoptosis and provide mechanistic insights into inflammasome-associated cell death that is one major effector mechanism of inflammasomes.

Visualization of Proliferative Vascular Endothelial Cells in Tumors in Vivo by Imaging Their Partner of Sld5-1 Promoter Activity.

Vascular endothelial cells (ECs) isolated from tumors characteristically express certain genes. It has recently been suggested that tumor vessel normalization facilitates effective drug delivery into tumors; however, how tumor vessel normalization can be recognized on the basis of the molecules expressed by tumor ECs is not clearly defined. The degree of cell proliferation is an important indicator to characterize the condition of the ECs. Herein, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) under the transcriptional control of the DNA replication factor partner of Sld5-1 (PSF1; official name GINS1) promoter to assess whether active ECs can be distinguished from dormant ECs. Predictably, ECs in the adult skin exhibited no EGFP signals. However, after s.c. injection of tumor cells, some ECs shifted to EGFP positivity, enabling distinction of EGFP-positive from EGFP-negative cells. We found that only a fraction of the EGFP-negative ECs strongly expressed the glycosylphosphatidylinositol-anchor protein CD109 associated with the phosphatidylinositol 3-kinase pathway. Taken together, these data indicate that areas of vascular normalization in tumors can be detected by CD109 expression, and this provides a window of opportunity for timing chemotherapy.

Gasdermin D mediates the maturation and release of IL-1α downstream of inflammasomes.

IL-1α serves as a pro-inflammatory cytokine. Although pro-IL-1α has cytokine activity, proteolytic maturation increases its potency and release from cells. IL-1α maturation occurs in a caspase-1-dependent manner following inflammasome activation. However, pro-IL-1α is not a substrate of caspase-1, and it remains unclear what mediates the maturation of this cytokine downstream of inflammasomes. Here, we show that gasdermin D (GSDMD), an executor of pyroptosis, is required for the rapid induction of IL-1α maturation by non-particulate inflammasome activators. Ablation of GSDMD abrogates the maturation of IL-1α, but not of IL-1ß. Inflammasome-induced maturation of IL-1α relies on extracellular Ca2+ and calpains. Ca2+ influx and calpain activation are induced in a GSDMD-dependent manner. Glycine, which inhibits cell lysis, but not GSDMD pore formation, does not affect IL-1α maturation. These results suggest that during inflammasome activation, GSDMD processed by caspase-1 forms plasma membrane pores that mediate Ca2+ influx, resulting in the calpain-dependent maturation of IL-1α.

Caspase-1 initiates apoptosis in the absence of gasdermin D.

Caspase-1 activated in inflammasomes triggers a programmed necrosis called pyroptosis, which is mediated by gasdermin D (GSDMD). However, GSDMD-deficient cells are still susceptible to caspase-1-mediated cell death. Therefore, here, we investigate the mechanism of caspase-1-initiated cell death in GSDMD-deficient cells. Inflammasome stimuli induce apoptosis accompanied by caspase-3 activation in GSDMD-deficient macrophages, which largely relies on caspase-1. Chemical dimerization of caspase-1 induces pyroptosis in GSDMD-sufficient cells, but apoptosis in GSDMD-deficient cells. Caspase-1-induced apoptosis involves the Bid-caspase-9-caspase-3 axis, which can be followed by GSDME-dependent secondary necrosis/pyroptosis. However, Bid ablation does not completely abolish the cell death, suggesting the existence of an additional mechanism. Furthermore, cortical neurons and mast cells exhibit little or low GSDMD expression and undergo apoptosis after oxygen glucose deprivation and nigericin stimulation, respectively, in a caspase-1- and Bid-dependent manner. This study clarifies the molecular mechanism and biological roles of caspase-1-induced apoptosis in GSDMD-low/null cell types.