HIF-1α is a negative regulator of interferon regulatory factors: Implications for interferon production by hypoxic monocytes.

Patients with severe COVID-19 infection exhibit a low level of oxygen in affected tissue and blood. To understand the pathophysiology of COVID-19 infection, it is therefore necessary to understand cell function during hypoxia. We investigated aspects of human monocyte activation under hypoxic conditions. HMGB1 is an alarmin released by stressed cells. Under normoxic conditions, HMGB1 activates interferon regulatory factor (IRF)5 and nuclear factor-κB in monocytes, leading to expression of type I interferon (IFN) and inflammatory cytokines including tumor necrosis factor α, and interleukin 1ß, respectively. When hypoxic monocytes are activated by HMGB1, they produce proinflammatory cytokines but fail to produce type I IFN. Hypoxia-inducible factor-1α, induced by hypoxia, functions as a direct transcriptional repressor of IRF5 and IRF3. As hypoxia is a stressor that induces secretion of HMGB1 by epithelial cells, hypoxia establishes a microenvironment that favors monocyte production of inflammatory cytokines but not IFN. These findings have implications for the pathogenesis of COVID-19.

Cholesterol depletion reduces Helicobacter pylori CagA translocation and CagA-induced responses in AGS cells.

Infection with Helicobacter pylori cagA-positive strains is associated with gastritis, ulcerations, and gastric cancer. CagA is translocated into infected epithelial cells by a type IV secretion system and can be tyrosine phosphorylated, inducing signal transduction and motogenic responses in epithelial cells. Cellular cholesterol, a vital component of the membrane, contributes to membrane dynamics and functions and is important in VacA intoxication and phagocyte evasion during H. pylori infection. In this investigation, we showed that cholesterol extraction by methyl-beta-cyclodextrin reduced the level of CagA translocation and phosphorylation. Confocal microscope visualization revealed that a significant portion of translocated CagA was colocalized with the raft marker GM1 and c-Src during infection. Moreover, GM1 was rapidly recruited into sites of bacterial attachment by live-cell imaging analysis. CagA and VacA were cofractionated with detergent-resistant membranes (DRMs), suggesting that the distribution of CagA and VacA is associated with rafts in infected cells. Upon cholesterol depletion, the distribution shifted to non-DRMs. Accordingly, the CagA-induced hummingbird phenotype and interleukin-8 induction were blocked by cholesterol depletion. Raft-disrupting agents did not influence bacterial adherence but did significantly reduce internalization activity in AGS cells. Together, these results suggest that delivery of CagA into epithelial cells by the bacterial type IV secretion system is mediated in a cholesterol-dependent manner.

Helicobacter pylori neutrophil-activating protein promotes myeloperoxidase release from human neutrophils.

Helicobacter pylori infection induces acute and chronic inflammation and plays a key role in gastric mucosal diseases. H. pylori neutrophil-activating protein (HP-NAP), one of its virulence factors, induces not only chemotactic but also oxidative burst responses of neutrophils. Activated neutrophils use myeloperoxidase (MPO) to generate many cytotoxic oxidants, which might result in gastric mucosal injury. In this study, we evaluated whether HP-NAP could promote MPO release from human neutrophils. Recombinant HP-NAP expressed in Escherichia coli was purified by two sequential gel filtration chromatographies and then subjected to syringe filtration for endotoxin removal. The purified recombinant HP-NAP assembles into a multimer with a secondary structure of the typical alpha-helix. In addition to stimulating the production of reactive oxygen species, HP-NAP is able to induce the secretion of MPO in human neutrophils. The increased MPO release from neutrophils induced by HP-NAP may be related to the pathogenesis of H. pylori-associated gastritis.

Expression of lncRNA MIR222HG co-transcribed from the miR-221/222 gene promoter facilitates the development of castration-resistant prostate cancer.

Mechanisms by which non-coding RNAs contribute to the progression of hormone-sensitive prostate cancer (PCa) (HSPC) to castration-resistant PCa (CRPC) remain largely unknown. We previously showed that microRNA-221/222 is up-regulated in CRPC and plays a critical role in modulating androgen receptor function during CRPC development. With further investigation, we characterized a putative promoter region located 23.3 kb upstream of the miR-221/222 gene, and this promoter is differentially activated in CRPC LNCaP-Abl cells, leading to the up-regulation of miR-221/222. Upon promoter activation, a set of polyadenylated long non-coding RNA (lncRNA) MIR222HGs was transcribed from this promoter region. Over-expression of these MIR222HGs increased androgen-independent cell growth and repressed the expression of androgen receptor-regulated dihydrotestosterone (DHT)-induced KLK3, TMPRSS2, and FKBP5 in HSPC LNCaP cells, hallmarks of the CRPC phenotype. Clinically, increased expression of MIR222HG is associated with PCa progression to CRPC. In primary tumors, expression levels of MIR222HG and miR-221/222 inversely correlate with Gleason score and androgen receptor (AR) pathway activity. Interestingly, MIR222HG is Argonaute 2-bound and its expression is Dicer 1-dependent, suggesting its functional association with the RNA-induced silencing complex. Further studies led to the hypothesis that MIR222HG may potentially affect miR-mediated expression silencing, subsequently leading to AR reprogramming. Our study highlights an essential role of a non-coding RNA in CRPC development and that differential activation of a single promoter can up-regulate two different types of non-coding RNAs, miR-221/222 and lncRNA MIR222HG, in CRPC. Additionally, this study reveals a novel function of lncRNAs as a modulator of Argonaute-mediated RNA-induced silencing complex.

ATR inhibition controls aggressive prostate tumors deficient in Y-linked histone demethylase KDM5D.

Epigenetic modifications control cancer development and clonal evolution in various cancer types. Here, we show that loss of the male-specific histone demethylase lysine-specific demethylase 5D (KDM5D) encoded on the Y chromosome epigenetically modifies histone methylation marks and alters gene expression, resulting in aggressive prostate cancer. Fluorescent in situ hybridization demonstrated that segmental or total deletion of the Y chromosome in prostate cancer cells is one of the causes of decreased KDM5D mRNA expression. The result of ChIP-sequencing analysis revealed that KDM5D preferably binds to promoter regions with coenrichment of the motifs of crucial transcription factors that regulate the cell cycle. Loss of KDM5D expression with dysregulated H3K4me3 transcriptional marks was associated with acceleration of the cell cycle and mitotic entry, leading to increased DNA-replication stress. Analysis of multiple clinical data sets reproducibly showed that loss of expression of KDM5D confers a poorer prognosis. Notably, we also found stress-induced DNA damage on the serine/threonine protein kinase ATR with loss of KDM5D. In KDM5D-deficient cells, blocking ATR activity with an ATR inhibitor enhanced DNA damage, which led to subsequent apoptosis. These data start to elucidate the biological characteristics resulting from loss of KDM5D and also provide clues for a potential novel therapeutic approach for this subset of aggressive prostate cancer.

Cholesterol glucosylation by Helicobacter pylori delays internalization and arrests phagosome maturation in macrophages.

BACKGROUND/PURPOSE: Helicobacter pylori colonizes the human stomach and contributes to chronic inflammation of the gastric mucosa. H. pylori persistence occurs because of insufficient eradication by phagocytic cells. A key factor of H. pylori, cholesterol-α-glucosyltransferase encoded by capJ that extracts host cholesterol and converts it to cholesteryl glucosides, is important to evade host immunity. Here, we examined whether phagocytic trafficking in macrophages was perturbed by capJ-carrying H. pylori. METHODS: J774A.1 cells were infected with H. pylori at a multiplicity of infection of 50. Live-cell imaging and confocal microscopic analysis were applied to monitor the phagocytic trafficking events. The viability of H. pylori inside macrophages was determined by using gentamicin colony-forming unit assay. The phagocytic routes were characterized by using trafficking-intervention compounds. RESULTS: Wild type (WT) H. pylori exhibited more delayed entry into macrophages and also arrested phagosome maturation more than did capJ knockout mutant. Pretreatment of genistein and LY294002 prior to H. pylori infection reduced the internalization of WT but not capJ-knockout H. pylori in macrophages. CONCLUSION: Cholesterol glucosylation by H. pylori interferes with phagosome trafficking via a lipid-raft and PI3K-dependent manner, which retards engulfment of bacteria for prolonged intracellular survival of H. pylori.

Helicobacter pylori neutrophil-activating protein induces release of histamine and interleukin-6 through G protein-mediated MAPKs and PI3K/Akt pathways in HMC-1 cells.

Helicobacter pylori neutrophil-activating protein (HP-NAP) activates several innate leukocytes including neutrophils, monocytes, and mast cells. It has been reported that HP-NAP induces degranulation and interleukin-6 (IL-6) secretion of rat peritoneal mast cells. However, the molecular mechanism is not very clear. Here, we show that HP-NAP activates human mast cell line-1 (HMC-1) cells to secrete histamine and IL-6. The secretion depends on pertussis toxin (PTX)-sensitive heterotrimeric G proteins but not on Toll-like receptor 2. Moreover, HP-NAP induces PTX-sensitive G protein-mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38-mitogen-activated protein kinase (p38 MAPK), and Akt in HMC-1 cells. Inhibition of ERK1/2, p38 MAPK, or phosphatidylinositol 3-kinase (PI3K) suppresses HP-NAP-induced release of histamine and IL-6 from HMC-1 cells. Thus, the activation of HMC-1 cells by HP-NAP is through Gi-linked G protein-coupled receptor-mediated MAPKs and PI3K/Akt pathways.

CSE1L/CAS, a microtubule-associated protein, inhibits taxol (paclitaxel)-induced apoptosis but enhances cancer cell apoptosis induced by various chemotherapeutic drugs.

CSE1L/CAS, a microtubule-associated, cellular apoptosis susceptibility protein, is highly expressed in various cancers. Microtubules are the target of paclitaxel-induced apoptosis. We studied the effects of increased or reduced CAS expression on cancer cell apoptosis induced by chemotherapeutic drugs including paclitaxel. Our results showed that CAS overexpression enhanced apoptosis induced by doxorubicin, 5-fluorouracil, cisplatin, and tamoxifen, but inhibited paclitaxel-induced apoptosis of cancer cells. Reductions in CAS produced opposite results. CAS overexpression enhanced p53 accumulation induced by doxorubicin, 5-fluorouracil, cisplatin, tamoxifen, and etoposide. CAS was associated with alpha-tubulin and beta-tubulin and enhanced the association between alpha-tubulin and beta-tubulin. Paclitaxel can induce G2/M phase cell cycle arrest and microtubule aster formation during apoptosis induction, but CAS overexpression reduced paclitaxel-induced G2/M phase cell cycle arrest and microtubule aster formation. Our results indicate that CAS may play an important role in regulating the cytotoxicities of chemotherapeutic drugs used in cancer chemotherapy against cancer cells.