Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

Tumor suppressor p53 plays a central role in response to DNA damage. DNA-damaging agents modulate nuclear actin dynamics, influencing cell behaviors; however, whether p53 affects the formation of nuclear actin filaments remains unclear. In this study, we found that p53 depletion promoted the formation of nuclear actin filaments in response to DNA-damaging agents, such as doxorubicin (DOXO) and etoposide (VP16). Even though the genetic probes used for the detection of nuclear actin filaments exerted a promotive effect on actin polymerization, the detected formation of nuclear actin filaments was highly dependent on both p53 depletion and DNA damage. Whilst active p53 is known to promote caspase-1 expression, the overexpression of caspase-1 reduced DNA damage-induced formation of nuclear actin filaments in p53-depleted cells. In contrast, co-treatment with DOXO and the pan-caspase inhibitor Q-VD-OPh or the caspase-1 inhibitor Z-YVAD-FMK induced the formation of nuclear actin filament formation even in cells bearing wild-type p53. These results suggest that the p53-caspase-1 axis suppresses DNA damage-induced formation of nuclear actin filaments. In addition, we found that the expression of nLifeact-GFP, the filamentous-actin-binding peptide Lifeact fused with the nuclear localization signal (NLS) and GFP, modulated the structure of nuclear actin filaments to be phalloidin-stainable in p53-depleted cells treated with the DNA-damaging agent, altering the chromatin structure and reducing the transcriptional activity. The level of phosphorylated H2AX (γH2AX), a marker of DNA damage, in these cells also reduced upon nLifeact-GFP expression, whilst details of the functional relationship between the formation of nLifeact-GFP-decorated nuclear actin filaments and DNA repair remained to be elucidated. Considering that the loss of p53 is associated with cancer progression, the results of this study raise a possibility that the artificial reinforcement of nuclear actin filaments by nLifeact-GFP may enhance the cytotoxic effect of DNA-damaging agents in aggressive cancer cells through a reduction in gene transcription.

Statins Show Anti-Atherosclerotic Effects by Improving Endothelial Cell Function in a Kawasaki Disease-like Vasculitis Mouse Model.

Kawasaki disease (KD) is an acute inflammatory syndrome of unknown etiology that is complicated by cardiovascular sequelae. Chronic inflammation (vasculitis) due to KD might cause vascular cellular senescence and vascular endothelial cell damage, and is a potential cause of atherosclerosis in young adults. This study examined the effect of KD and HMG-CoA inhibitors (statins) on vascular cellular senescence and vascular endothelial cells. Candida albicans water-soluble fraction (CAWS) was administered intraperitoneally to 5-week-old male apolipoprotein E-deficient (ApoE-) mice to induce KD-like vasculitis. The mice were then divided into three groups: control, CAWS, and CAWS+statin groups. Ten weeks after injection, the mice were sacrificed and whole aortic tissue specimens were collected. Endothelial nitric oxide synthase (eNOS) expression in the ascending aortic intima epithelium was evaluated using immunostaining. In addition, eNOS expression and levels of cellular senescence markers were measured in RNA and proteins extracted from whole aortic tissue. KD-like vasculitis impaired vascular endothelial cells that produce eNOS, which maintains vascular homeostasis, and promoted macrophage infiltration into the tissue. Statins also restored vascular endothelial cell function by promoting eNOS expression. Statins may be used to prevent secondary cardiovascular events during the chronic phase of KD.

2-Deoxy-d-glucose induces deglycosylation of proinflammatory cytokine receptors and strongly reduces immunological responses in mouse models of inflammation.

Anti-proinflammatory cytokine therapies against interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1 are major advancements in treating inflammatory diseases, especially rheumatoid arthritis. Such therapies are mainly performed by injection of antibodies against cytokines or cytokine receptors. We initially found that the glycolytic inhibitor 2-deoxy-d-glucose (2-DG), a simple monosaccharide, attenuated cellular responses to IL-6 by inhibiting N-linked glycosylation of the IL-6 receptor gp130. Aglycoforms of gp130 did not bind to IL-6 or activate downstream intracellular signals that included Janus kinases. 2-DG completely inhibited dextran sodium sulfate-induced colitis, a mouse model for inflammatory bowel disease, and alleviated laminarin-induced arthritis in the SKG mouse, an experimental model for human rheumatoid arthritis. These diseases have been shown to be partially dependent on IL-6. We also found that 2-DG inhibited signals for other proinflammatory cytokines such as TNF-α, IL-1ß, and interferon -γ, and accordingly, prevented death by another inflammatory disease, lipopolysaccharide (LPS) shock. Furthermore, 2-DG prevented LPS shock, a model for a cytokine storm, and LPS-induced pulmonary inflammation, a model for acute respiratory distress syndrome of coronavirus disease 2019 (COVID-19). These results suggest that targeted therapies that inhibit cytokine receptor glycosylation are effective for treatment of various inflammatory diseases.

Virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis.

Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.

Cochlear supporting cells function as macrophage-like cells and protect audiosensory receptor hair cells from pathogens.

To protect the audiosensory organ from tissue damage from the immune system, the inner ear is separated from the circulating immune system by the blood-labyrinth barrier, which was previously considered an immune-privileged site. Recent studies have shown that macrophages are distributed in the cochlea, especially in the spiral ligament, spiral ganglion, and stria vascularis; however, the direct pathogen defence mechanism used by audiosensory receptor hair cells (HCs) has remained obscure. Here, we show that HCs are protected from pathogens by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs). In isolated murine cochlear sensory epithelium, we established Theiler's murine encephalomyelitis virus, which infected the SCs and GERCs, but very few HCs. The virus-infected SCs produced interferon (IFN)-α/ß, and the viruses efficiently infected the HCs in the IFN-α/ß receptor-null sensory epithelium. Interestingly, the virus-infected SCs and GERCs expressed macrophage marker proteins and were eliminated from the cell layer by cell detachment. Moreover, lipopolysaccharide induced phagocytosis of the SCs without cell detachment, and the SCs phagocytosed the bacteria. These results reveal that SCs function as macrophage-like cells, protect adjacent HCs from pathogens, and provide a novel anti-infection inner ear immune system.

DMPK is a New Candidate Mediator of Tumor Suppressor p53-Dependent Cell Death.

Tumor suppressor p53 plays an integral role in DNA-damage induced apoptosis, a biological process that protects against tumor progression. Cell shape dramatically changes when cells undergo apoptosis, which is associated with actomyosin contraction; however, it remains entirely elusive how p53 regulates actomyosin contraction in response to DNA-damaging agents. To identify a novel p53 regulating gene encoding the modulator of myosin, we conducted DNA microarray analysis. We found that, in response to DNA-damaging agent doxorubicin, expression of myotonic dystrophy protein kinase (DMPK), which is known to upregulate actomyosin contraction, was increased in a p53-dependent manner. The promoter region of DMPK gene contained potential p53-binding sequences and its promoter activity was increased by overexpression of the p53 family protein p73, but, unexpectedly, not of p53. Furthermore, we found that doxorubicin treatment induced p73 expression, which was significantly attenuated by downregulation of p53. These data suggest that p53 induces expression of DMPK through upregulating p73 expression. Overexpression of DMPK promotes contraction of the actomyosin cortex, which leads to formation of membrane blebs, loss of cell adhesion, and concomitant caspase activation. Taken together, our results suggest the existence of p53-p73-DMPK axis which mediates DNA-damage induced actomyosin contraction at the cortex and concomitant cell death.

Role of p53 in the Regulation of the Inflammatory Tumor Microenvironment and Tumor Suppression.

p53 has functional roles in tumor suppression as a guardian of the genome, surveillant of oncogenic cell transformation, and as recently demonstrated, a regulator of intracellular metabolism. Accumulating evidence has shown that the tumor microenvironment, accompanied by inflammation and tissue remodeling, is important for cancer proliferation, metastasis, and maintenance of cancer stem cells (CSCs) that self-renew and generate the diverse cells comprising the tumor. Furthermore, p53 has been demonstrated to inhibit inflammatory responses, and functional loss of p53 causes excessive inflammatory reactions. Moreover, the generation and maintenance of CSCs are supported by the inflammatory tumor microenvironment. Considering that the functions of p53 inhibit reprogramming of somatic cells to stem cells, p53 may have a major role in the inflammatory microenvironment as a tumor suppressor. Here, we review our current understanding of the mechanisms underlying the roles of p53 in regulation of the inflammatory microenvironment, tumor microenvironment, and tumor suppression.

Interleukin 6 enhances glycolysis through expression of the glycolytic enzymes hexokinase 2 and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3.

Enhanced glycolysis is important for oncogenesis and for the survival and proliferation of cancer cells in the tumor microenvironment. Recent studies have also shown that proinflammatory cytokine signaling, such as that mediated by nuclear factor kappaB and signal transducer and activator of transcription 3 (STAT3), is important for the generation of inflammation-associated tumors. However, the link between inflammation and enhanced glycolysis has not been identified. In the present study, we found that the proinflammatory cytokine interleukin (IL)-6 enhanced glycolysis in mouse embryonic fibroblasts and human cell lines. Moreover, STAT3 activated by IL-6 enhanced the expression of the glycolytic enzymes hexokinase 2 and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3). Ectopic expression of PFKFB3 enhanced glycolysis, suggesting that the IL-6-STAT3 pathway enhances glycolysis through the induction of these enzymes. Our findings may provide a novel mechanism for inflammation-associated oncogenesis.

5-Aza-2'-deoxycytidine restores proapoptotic function of p53 in cancer cells resistant to p53-induced apoptosis.

The expression of p53-target genes encoding the proapoptotic factor Noxa, but not PUMA, was not induced by p53 in HCT116 and SW480 cells, which show resistance to apoptosis in response to p53 overexpression. The lack of p53 inducibility of Noxa was restored by treatment with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-aza-CdR). Furthermore, p53 induced apoptosis in HCT116 and SW480 cells treated with 5-aza-CdR. Moreover, the inhibition of Noxa expression by RNAi in 5-aza-CdR-treated HCT116 cells resulted in the partial inhibition of p53-induced apoptosis. These results suggest that epigenetic cancer therapy is possible for some cancers in combination with forced p53 activation.