Overexpression of satellite RNAs in heterochromatin induces chromosomal instability and reflects drug sensitivity in mouse cancer cells.

Overexpression of satellite RNAs in heterochromatin induces chromosomal instability (CIN) through the DNA damage response and cell cycle checkpoint activation. Although satellite RNAs may be therapeutic targets, the associated mechanisms underlying drug sensitivity are unknown. Here, we determined whether satellite RNAs reflect drug sensitivity to the topoisomerase I inhibitor camptothecin (CPT) via CIN induction. We constructed retroviral vectors expressing major satellite and control viruses, infected microsatellite stable mouse colon cancer cells (CT26) and MC38 cells harboring microsatellite instability, and assessed drug sensitivity after 48 h. Cells overexpressing satellite RNAs showed clear features of abnormal segregation, including micronuclei and anaphase bridging, and elevated levels of the DNA damage marker γH2AX relative to controls. Additionally, overexpression of satellite RNAs enhanced MC38 cell susceptibility to CPT [half-maximal inhibitory concentration: 0.814 µM (control) vs. 0.332 µM (MC38 cells with a major satellite), p = 0.003] but not that of CT26. These findings imply that MC38 cells, which are unlikely to harbor CIN, are more susceptible to CIN-induced CPT sensitivity than CT26 cells, which are characterized by CIN. Furthermore, CPT administration upregulated p53 levels but not those of p21, indicating that overexpression of major satellite transcripts likely induces CPT-responsive cell death rather than cellular senescence.

The role of MerTK in promoting cell migration is enhanced by the oncogenic Ras/IL-33 signaling axis.

Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras-induced cancer. Therefore, the downstream effectors of Ras signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF-HEV/IL-33, a member of the interleukin-1 family, and showed that intracellular IL-33 was required for oncogenic Ras-induced cellular transformation. In the present study, we demonstrated that the c-Mer proto-oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL-33 signaling. The expression of MerTK was enhanced in transformed NIH-3T3 cells by the expression of oncogenic Ras, H-Ras (G12V), in an IL-33-dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL-33 expression. The knockdown of IL-33 or MerTK effectively attenuated the migration of NIH-3T3 cells transformed by H-Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K-Ras mutation. Furthermore, the suppression of Ras-induced cell migration by the knockdown of IL-33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH-3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras-transformed cells.

K15 promoter-driven enforced expression of NKIRAS exhibits tumor suppressive activity against the development of DMBA/TPA-induced skin tumors.

NKIRAS1 and NKIRAS2 (also called as κB-Ras) were identified as members of the atypical RAS family that suppress the transcription factor NF-κB. However, their function in carcinogenesis is still controversial. To clarify how NKIRAS acts on cellular transformation, we generated transgenic mice in which NKIRAS2 was forcibly expressed using a cytokeratin 15 (K15) promoter, which is mainly activated in follicle bulge cells. The ectopic expression of NKIRAS2 was mainly detected in follicle bulges of transgenic mice with NKIRAS2 but not in wild type mice. K15 promoter-driven expression of NKIRAS2 failed to affect the development of epidermis, which was evaluated using the expression of K10, K14, K15 and filaggrin. However, K15 promoter-driven expression of NKIRAS2 effectively suppressed the development of skin tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA). This observation suggested that NKIRAS seemed to function as a tumor suppressor in follicle bulges. However, in the case of oncogenic HRAS-driven cellular transformation of murine fibroblasts, knockdown of NKIRAS2 expression drastically suppressed HRAS-mutant-provoked cellular transformation, suggesting that NKIRAS2 was required for the cellular transformation of murine fibroblasts. Furthermore, moderate enforced expression of NKIRAS2 augmented oncogenic HRAS-provoked cellular transformation, whereas an excess NKIRAS2 expression converted its functional role into a tumor suppressive phenotype, suggesting that NKIRAS seemed to exhibit a biphasic bell-shaped enhancing effect on HRAS-mutant-provoked oncogenic activity. Taken together, the functional role of NKIRAS in carcinogenesis is most likely determined by not only cellular context but also its expression level.

Oncogenic Ras mutant causes the hyperactivation of NF-κB via acceleration of its transcriptional activation.

It is well established that nuclear factor κB (NF-κB) acts as one of the most important transcription factors for tumor initiation and progression, as it both protects cells from apoptotic/necrotic signals and accelerates angiogenesis and tumor metastasis, which is mediated via the expression of target genes. However, it has not yet been clarified how oncogenic signals accelerate the activation of NF-κB. In the current study, we utilized untransformed NIH-3T3 cells stably harboring a κB-driven luciferase gene to show that an oncogenic mutant of Ras GTPase augmented TNFα-induced NF-κB activation. Notably, enforced expression of cyclin-dependent kinase inhibitors, such as p27Kip1 and p21Cip1 , effectively canceled the accelerated activation of NF-κB, suggesting that oncogenic Ras-induced cell cycle progression is essential for the hyperactivation of NF-κB. Furthermore, we found that Ras (G12V) augmented the transcriptional activation of NF-κB, and this activation required the p38 MAP kinase. We observed that a downstream kinase of p38 MAP kinase, MSK1, was activated by Ras (G12V) and catalyzed the phosphorylation of p65/RelA at Ser-276, which is critical for its transcriptional activation. Significantly, phosphorylation of the p65/RelA subunit at Ser-276 was elevated in patient samples of colorectal cancer harboring oncogenic mutations of the K-Ras gene, and the expression levels of NF-κB target genes were drastically enhanced in several cancer tissues. These observations strongly suggest that oncogenic signal-induced acceleration of NF-κB activation is caused by activation of the p38 MAP kinase-MSK1 signaling axis and by cell cycle progression in cancer cells.

ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant.

The ST2 gene was originally identified as a primary responsive gene, and the expressions of its gene products are induced by stimulation with growth factors and by oncogenic stresses. In this study, we observed that oncogenic Ras mutant induced the expression of ST2 and ST2L proteins. Interestingly, the enforced expression of ST2 gene products in NIH-3T3 murine fibroblasts remarkably enhanced Ras (G12V)-induced cellular transformation. Furthermore, when the expression of ST2 gene products was silenced by RNA-interference technique, Ras (G12V)-induced cellular transformation was drastically suppressed. According to these observations, it was indicated that the oncogenic Ras-induced expression of ST2 gene products is required for the acceleration of cellular transformation, and this seems to be independent of the stimulation with IL-33, a ligand for ST2/ST2L. Interestingly, knockdown of ST2 gene products caused a reduction in Rb phosphorylation in transformed murine fibroblasts, suggesting the functional involvement of ST2 gene products in cell cycle progression during cellular transformation. Our current study strongly suggests the importance of ST2 gene products in cellular transformation, and the presence of novel mechanism how ST2 gene products affect the cellular transformation and cell proliferation.

ARIH2 Ubiquitinates NLRP3 and Negatively Regulates NLRP3 Inflammasome Activation in Macrophages.

The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a molecular platform that induces caspase-1 activation and subsequent IL-1ß maturation, and is implicated in inflammatory diseases; however, little is known about the negative regulation of NLRP3 inflammasome activation. In this article, we identified an E3 ligase, Ariadne homolog 2 (ARIH2), as a posttranslational negative regulator of NLRP3 inflammasome activity in macrophages. ARIH2 interacted with NLRP3 via its NACHT domain (aa 220-575) in the NLRP3 inflammasome complex. In particular, we found that while using mutants of ARIH2 and ubiquitin, the really interesting new gene 2 domain of ARIH2 was required for NLRP3 ubiquitination linked through K48 and K63. Deletion of endogenous ARIH2 using CRISPR/Cas9 genome editing inhibited NLRP3 ubiquitination and promoted NLRP3 inflammasome activation, resulting in apoptosis-associated speck-like protein containing a caspase recruitment domain oligomerization, pro-IL-1ß processing, and IL-1ß production. Conversely, ARIH2 overexpression promoted NLRP3 ubiquitination and inhibited NLRP3 inflammasome activation. Our findings reveal a novel mechanism of ubiquitination-dependent negative regulation of the NLRP3 inflammasome by ARIH2 and highlight ARIH2 as a potential therapeutic target for inflammatory diseases.

STAT3 and ERK pathways are involved in cell growth stimulation of the ST2/IL1RL1 promoter.

The ST2 gene was originally identified as a primary responsive gene induced by stimulation with growth factors and by oncogenic stress. The ST2 gene harbors two distinct promoters - a distal promoter and a proximal promoter. In this study, we identified a novel type of serum-responsive element in the ST2 proximal promoter using reporter gene analysis; this element includes a possible responsive element for STAT family proteins. Indeed, enforced expression of constitutively active STAT3 activated this promoter element and induced the expression of ST2 gene products. Furthermore, an oncogenic Ras (G12V) mutant also caused the expression of ST2 gene products by utilizing the proximal promoter. We also clarified that activation of the ST2 promoter by either growth stimulation or oncogenic Ras was suppressed by the inhibitors for STAT3 and ERK pathways. Our observations strongly suggest the importance of STAT family and ERK pathways for the induction of ST2 gene products by cell growth stimulation.

Clinical Practice Guideline (Update): Earwax (Cerumen Impaction)

Objective This update of the 2008 American Academy of Otolaryngology-Head and Neck Surgery Foundation cerumen impaction clinical practice guideline provides evidence-based recommendations on managing cerumen impaction. Cerumen impaction is defined as an accumulation of cerumen that causes symptoms, prevents assessment of the ear, or both. Changes from the prior guideline include a consumer added to the development group; new evidence (3 guidelines, 5 systematic reviews, and 6 randomized controlled trials); enhanced information on patient education and counseling; a new algorithm to clarify action statement relationships; expanded action statement profiles to explicitly state quality improvement opportunities, confidence in the evidence, intentional vagueness, and differences of opinion; an enhanced external review process to include public comment and journal peer review; and 3 new key action statements on managing cerumen impaction that focus on primary prevention, contraindicated intervention, and referral and coordination of care. Purpose The primary purpose of this guideline is to help clinicians identify patients with cerumen impaction who may benefit from intervention and to promote evidence-based management. Another purpose of the guideline is to highlight needs and management options in special populations or in patients who have modifying factors. The guideline is intended for all clinicians who are likely to diagnose and manage patients with cerumen impaction, and it applies to any setting in which cerumen impaction would be identified, monitored, or managed. The guideline does not apply to patients with cerumen impaction associated with the following conditions: dermatologic diseases of the ear canal; recurrent otitis externa; keratosis obturans; prior radiation therapy affecting the ear; previous tympanoplasty/myringoplasty, canal wall down mastoidectomy, or other surgery affecting the ear canal. Key Action Statements The panel made a strong recommendation that clinicians should treat, or refer to a clinician who can treat, cerumen impaction, defined as an accumulation of cerumen that is associated with symptoms, prevents needed assessment of the ear, or both. The panel made the following recommendations: (1) Clinicians should explain proper ear hygiene to prevent cerumen impaction when patients have an accumulation of cerumen. (2) Clinicians should diagnose cerumen impaction when an accumulation of cerumen, as seen on otoscopy, is associated with symptoms, prevents needed assessment of the ear, or both. (3) Clinicians should assess the patient with cerumen impaction by history and/or physical examination for factors that modify management, such as ≥1 of the following: anticoagulant therapy, immunocompromised state, diabetes mellitus, prior radiation therapy to the head and neck, ear canal stenosis, exostoses, and nonintact tympanic membrane. (4) Clinicians should not routinely treat cerumen in patients who are asymptomatic and whose ears can be adequately examined. (5) Clinicians should identify patients with obstructing cerumen in the ear canal who may not be able to express symptoms (young children and cognitively impaired children and adults), and they should promptly evaluate the need for intervention. (6) Clinicians should perform otoscopy to detect the presence of cerumen in patients with hearing aids during a health care encounter. (7) Clinicians should treat, or refer to a clinician who can treat, the patient with cerumen impaction with an appropriate intervention, which may include ≥1 of the following: cerumenolytic agents, irrigation, or manual removal requiring instrumentation. (8) Clinicians should recommend against ear candling for treating or preventing cerumen impaction. (9) Clinicians should assess patients at the conclusion of in-office treatment of cerumen impaction and document the resolution of impaction. If the impaction is not resolved, the clinician should use additional treatment. If full or partial symptoms persist despite resolution of impaction, the clinician should evaluate the patient for alternative diagnoses. (10) Finally, if initial management is unsuccessful, clinicians should refer patients with persistent cerumen impaction to clinicians who have specialized equipment and training to clean and evaluate ear canals and tympanic membranes. The panel offered the following as options: (1) Clinicians may use cerumenolytic agents (including water or saline solution) in the management of cerumen impaction. (2) Clinicians may use irrigation in the management of cerumen impaction. (3) Clinicians may use manual removal requiring instrumentation in the management of cerumen impaction. (4) Last, clinicians may educate/counsel patients with cerumen impaction or excessive cerumen regarding control measures.

Clinical Practice Guideline (Update): Earwax (Cerumen Impaction) Executive Summary

The American Academy of Otolaryngology-Head and Neck Surgery Foundation (AAO-HNSF) has published a supplement to this issue of Otolaryngology-Head and Neck Surgery featuring the updated Clinical Practice Guideline: Earwax (Cerumen Impaction). To assist in implementing the guideline recommendations, this article summarizes the rationale, purpose, and key action statements. The 11 recommendations emphasize proper ear hygiene, diagnosis of cerumen impaction, factors that modify management, evaluating the need for intervention, and proper treatment. An updated guideline is needed due to new evidence (3 guidelines, 5 systematic reviews, and 6 randomized controlled trials) and the need to add statements on managing cerumen impaction that focus on primary prevention, contraindicated intervention, and referral and coordination of care.

Intracellular NF-HEV/IL-33 harbors essential roles in Ras-induced cellular transformation by contributing to cyclin D1 protein synthesis.

A member of the interleukin-1 family, interleukin-33 (NF-HEV/IL-33), is a ligand for the receptor, ST2L and stimulates the production of Th2 cytokines. Although IL-33 localizes to the nucleus and may be involved in the regulation of transcription independent of ST2L, its functions in the nucleus currently remain unclear. We herein demonstrated that the expression of IL-33 was markedly enhanced in NIH-3T3 cells transformed by an oncogenic H-Ras mutant (H-Ras (G12V)), and the induced IL-33 was mainly located in the nuclei of these cells. The enforced expression of IL-33 accelerated H-Ras (G12V)-induced transformation in NIH-3T3 cells, and this transforming activity was markedly reduced by the knockdown of IL-33 with shRNA. We subsequently analyzed several signaling molecules regulated by Ras in order to elucidate the mechanism by which IL-33 contributes to Ras (G12V)-induced transformation. We found that the knockdown of IL-33 effectively attenuated the Ras (G12V)-induced expression of cyclin D1. However, the knockdown of IL-33 failed to affect cyclin D1 mRNA expression levels, and epoxomicin, a proteasome inhibitor, did not cancel the IL-33 knockdown-induced down-regulation of its protein levels. We showed that Ras (G12V)-induced cyclin D1 protein synthesis was markedly suppressed by the knockdown of IL-33. Taken together, the results of the present study strongly suggest a novel role for IL-33 in cellular transformation.

Anti-inflammatory activity of flavonoids in Nepalese propolis is attributed to inhibition of the IL-33 signaling pathway.

Propolis has been used in folk medicine to improve health and prevent inflammatory diseases; however, the components that exhibit its anti-inflammatory activity remain unknown. We herein investigated the effects of flavonoids isolated from Nepalese propolis on the IL-33 signaling pathway to clarify the anti-inflammatory mechanism involved. Of the 8 types of flavonoids isolated from Nepalese propolis, 4 types of compounds, such as 3',4'-dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, cearoin, and chrysin, markedly inhibited the IL-33-induced mRNA expression of inflammatory genes including IL-6, TNFα and IL-13 in bone marrow-derived mast cells (BMMC). These four flavonoids also inhibited the IL-33-induced activation of nuclear factor κB (NF-κB), which was consistent with their inhibitory effects on cytokine expression. The effects of these flavonoids are attributed to inhibition of IL-33-induced activation of IKK, which leads to the degradation of IκBα and nuclear localization of NF-κB. On the other hand, other flavonoids isolated from Nepalese propolis, such as isoliquiritigenin, plathymenin, 7-hydroxyflavanone, and (+)-medicarpin, had no effect on the IL-33 signaling pathway or cytokine expression. Therefore, these results indicate that 3',4'-dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, cearoin, and chrysin are the substances responsible for the anti-inflammatory activity of Nepalese propolis.

NLRP3 regulates neutrophil functions and contributes to hepatic ischemia-reperfusion injury independently of inflammasomes.

Inflammation plays a key role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury. However, the mechanism by which hepatic I/R induces inflammatory responses remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by I/R is mediated through a multiple-protein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in hepatic I/R injury and found that hepatic I/R stimuli upregulated the inflammasome-component molecule, nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), but not apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). NLRP3(-/-) mice, but not ASC(-/-) and caspase-1(-/-) mice, had significantly less liver injury after hepatic I/R. NLRP3(-/-) mice showed reduced inflammatory responses, reactive oxygen species production, and apoptosis in I/R liver. Notably, infiltration of neutrophils, but not macrophages, was markedly inhibited in the I/R liver of NLRP3(-/-) mice. Bone marrow transplantation experiments showed that NLRP3 not only in bone marrow-derived cells, but also in non-bone marrow-derived cells contributed to liver injury after I/R. In vitro experiments revealed that keratinocyte-derived chemokine-induced activation of heterotrimeric G proteins was markedly diminished. Furthermore, NLRP3(-/-) neutrophils decreased keratinocyte-derived chemokine-induced concentrations of intracellular calcium elevation, Rac activation, and actin assembly formation, thereby resulting in impaired migration activity. Taken together, NLRP3 regulates chemokine-mediated functions and recruitment of neutrophils, and thereby contributes to hepatic I/R injury independently of inflammasomes. These findings identify a novel role of NLRP3 in the pathophysiology of hepatic I/R injury.

Presence of a novel exon 2E encoding a putative transmembrane protein in human IL-33 gene.

Interleukin-33 (IL-33) is a dual-function molecule that regulates gene expression in nuclei and, as a cytokine, conveys proinflammatory signals from outside of cells via its specific receptor ST2L. There are still a lot of questions about localization and processing of IL-33 gene products. In the course of re-evaluating human IL-33 gene, we found distinct promoter usage depending on the cell type, similar to the case in the ST2 gene. Furthermore, we found a novel exon 2E in the conventional intron 2 whose open reading frame corresponded to a transmembrane protein of 131 amino acids. Dependence of exon 2E expression on differentiation of HUVEC cells is of great interest in relation to human IL-33 function.

YM-175 induces apoptosis of human native monocyte-lineage cells via inhibition of prenylation.

Nitrogen-containing bisphosphonates (NCBPs) have been widely used as standard supportive therapy to reduce skeletal-related events (SREs) in myeloma patients through suppression of osteoclast activity. In various prospective randomized trials that were performed following preliminary reports concerning efficacy, NCBPs have shown a significant beneficial effect on myeloma bone disease through both suppression of bone resorption and direct antimyeloma activity. Thus, NCBPs have an influence on many types of human cells. In this study, we examined the effect of an NCBP (YM-175) on an apoptosis of a monocytic cell line and of human native monocytes/macrophages and dendritic cells (DCs). We confirmed that monocytes, monocyte-derived macrophages, DCs, and a monoblastic cell line (THP-1) showed dose-dependent and time-dependent apoptosis related to the activation of caspases after exposure to YM-175 at concentrations below that at which the apoptosis of myeloma cell lines was induced. Such apoptosis of monocytic cells was suppressed by the addition of farnesol or geranylgeraniol. These findings suggest that the inhibition of monocyte-lineage cells or DCs by NCBPs might interfere with phagocytic activity or pathogen-presenting activity.

Soluble ST2 protein inhibits LPS stimulation on monocyte-derived dendritic cells.

ST2 protein is a soluble splicing variant of ST2L protein, which is the receptor for interleukin-33 (IL-33). Previously, we reported that soluble ST2 suppressed the signal transduction of lipopolysaccharide (LPS) and cytokine production in monocytic cells. To investigate whether or not this inhibitory effect occurs in dendritic cells, which are the key players in innate and adaptive immunity, human monocyte-derived dendritic cells were pre-treated with soluble ST2 protein before LPS stimulation. Although soluble ST2 did not attenuate the LPS-induced maturation of dendritic cells, pre-treatment with soluble ST2 suppressed cytokine production and inhibited LPS signaling. Moreover, the proliferation of naive T cells was inhibited significantly by soluble ST2 pre-treatment. IL-33 had little effect on the cytokine production of immature monocyte-derived dendritic cells. Furthermore, soluble ST2 protein was internalized into dendritic cells, suggesting that soluble ST2 protein acts by a noncanonical mechanism other than the sequestration of IL-33.