Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1ß (IL-1ß) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.

The Mitochondrial Connection: The Nek Kinases' New Functional Axis in Mitochondrial Homeostasis.

Mitochondria provide energy for all cellular processes, including reactions associated with cell cycle progression, DNA damage repair, and cilia formation. Moreover, mitochondria participate in cell fate decisions between death and survival. Nek family members have already been implicated in DNA damage response, cilia formation, cell death, and cell cycle control. Here, we discuss the role of several Nek family members, namely Nek1, Nek4, Nek5, Nek6, and Nek10, which are not exclusively dedicated to cell cycle-related functions, in controlling mitochondrial functions. Specifically, we review the function of these Neks in mitochondrial respiration and dynamics, mtDNA maintenance, stress response, and cell death. Finally, we discuss the interplay of other cell cycle kinases in mitochondrial function and vice versa. Nek1, Nek5, and Nek6 are connected to the stress response, including ROS control, mtDNA repair, autophagy, and apoptosis. Nek4, in turn, seems to be related to mitochondrial dynamics, while Nek10 is involved with mitochondrial metabolism. Here, we propose that the participation of Neks in mitochondrial roles is a new functional axis for the Nek family.

Structural basis for the oligomerization-facilitated NLRP3 activation.

The NACHT-, leucine-rich-repeat-, and pyrin domain-containing protein 3 (NLRP3) is a critical intracellular inflammasome sensor and an important clinical target against inflammation-driven human diseases. Recent studies have elucidated its transition from a closed cage to an activated disk-like inflammasome, but the intermediate activation mechanism remains elusive. Here we report the cryo-electron microscopy structure of NLRP3, which forms an open octamer and undergoes a ~ 90° hinge rotation at the NACHT domain. Mutations on open octamer's interfaces reduce IL-1ß signaling, highlighting its essential role in NLRP3 activation/inflammasome assembly. The centrosomal NIMA-related kinase 7 (NEK7) disrupts large NLRP3 oligomers and forms NEK7/NLRP3 monomers/dimers which is a critical step preceding the assembly of the disk-like inflammasome. These data demonstrate an oligomeric cooperative activation of NLRP3 and provide insight into its inflammasome assembly mechanism.

Inhibition of the YAP-MMB interaction and targeting NEK2 as potential therapeutic strategies for YAP-driven cancers.

YAP activation in cancer is linked to poor outcomes, making it an attractive therapeutic target. Previous research focused on blocking the interaction of YAP with TEAD transcription factors. Here, we took a different approach by disrupting YAP's binding to the transcription factor B-MYB using MY-COMP, a fragment of B-MYB containing the YAP binding domain fused to a nuclear localization signal. MY-COMP induced cell cycle defects, nuclear abnormalities, and polyploidization. In an AKT and YAP-driven liver cancer model, MY-COMP significantly reduced liver tumorigenesis, highlighting the importance of the YAP-B-MYB interaction in tumor development. MY-COMP also perturbed the cell cycle progression of YAP-dependent uveal melanoma cells but not of YAP-independent cutaneous melanoma cell lines. It counteracted YAP-dependent expression of MMB-regulated cell cycle genes, explaining the observed effects. We also identified NIMA-related kinase (NEK2) as a downstream target of YAP and B-MYB, promoting YAP-driven transformation by facilitating centrosome clustering and inhibiting multipolar mitosis.

Attenuated cell cycle and DNA damage response transcriptome signatures and overrepresented cell adhesion processes imply accelerated progression in patients with lower-risk myelodysplastic neoplasms.

Patients with myelodysplastic neoplasms (MDS) are classified according to the risk of acute myeloid leukemia transformation. Some lower-risk MDS patients (LR-MDS) progress rapidly despite expected good prognosis. Using diagnostic samples, we aimed to uncover the mechanisms of this accelerated progression at the transcriptome level. RNAseq was performed on CD34+ ribodepleted RNA samples from 53 LR-MDS patients without accelerated progression (stMDS) and 8 who progressed within 20 months (prMDS); 845 genes were differentially expressed (ІlogFCІ > 1, FDR < 0.01) between these groups. stMDS CD34+ cells exhibited transcriptional signatures of actively cycling, megakaryocyte/erythrocyte lineage-primed progenitors, with upregulation of cell cycle checkpoints and stress pathways, which presumably form a tumor-suppressing barrier. Conversely, cell cycle, DNA damage response (DDR) and energy metabolism-related pathways were downregulated in prMDS samples, whereas cell adhesion processes were upregulated. Also, prMDS samples showed high levels of aberrant splicing and global lncRNA expression that may contribute to the attenuation of DDR pathways. We observed overexpression of multiple oncogenes and diminished differentiation in prMDS; the expression of ZEB1 and NEK3, genes not previously associated with MDS prognosis, might serve as potential biomarkers for LR-MDS progression. Our 19-gene DDR signature showed a significant predictive power for LR-MDS progression. In validation samples (stMDS = 3, prMDS = 4), the key markers and signatures retained their significance. Collectively, accelerated progression of LR-MDS appears to be associated with transcriptome patterns of a quiescent-like cell state, reduced lineage differentiation and suppressed DDR, inherent to CD34+ cells. The attenuation of DDR-related gene-expression signature may refine risk assessment in LR-MDS patients.

Targeting gut microbial nitrogen recycling and cellular uptake of ammonium to improve bortezomib resistance in multiple myeloma.

The gut microbiome has been found to play a crucial role in the treatment of multiple myeloma (MM), which is still considered incurable due to drug resistance. In previous studies, we demonstrated that intestinal nitrogen-recycling bacteria are enriched in patients with MM. However, their role in MM relapse remains unclear. This study highlights the specific enrichment of Citrobacter freundii (C. freundii) in patients with relapsed MM. Through fecal microbial transplantation experiments, we demonstrate that C. freundii plays a critical role in inducing drug resistance in MM by increasing levels of circulating ammonium. The ammonium enters MM cells through the transmembrane channel protein SLC12A2, promoting chromosomal instability and drug resistance by stabilizing the NEK2 protein. We show that furosemide sodium, a loop diuretic, downregulates SLC12A2, thereby inhibiting ammonium uptake by MM cells and improving progression-free survival and curative effect scores. These findings provide new therapeutic targets and strategies for the intervention of MM progression and drug resistance.

ZDHHC5-mediated NLRP3 palmitoylation promotes NLRP3-NEK7 interaction and inflammasome activation.

The nucleotide-binding domain (NBD), leucine-rich repeat (LRR), and pyrin domain (PYD)-containing protein 3 (NLRP3) inflammasome is a critical mediator of the innate immune response. How NLRP3 responds to stimuli and initiates the assembly of the NLRP3 inflammasome is not fully understood. Here, we found that a cellular metabolite, palmitate, facilitates NLRP3 activation by enhancing its S-palmitoylation, in synergy with lipopolysaccharide stimulation. NLRP3 is post-translationally palmitoylated by zinc-finger and aspartate-histidine-histidine-cysteine 5 (ZDHHC5) at the LRR domain, which promotes NLRP3 inflammasome assembly and activation. Silencing ZDHHC5 blocks NLRP3 oligomerization, NLRP3-NEK7 interaction, and formation of large intracellular ASC aggregates, leading to abrogation of caspase-1 activation, IL-1ß/18 release, and GSDMD cleavage, both in human cells and in mice. ABHD17A depalmitoylates NLRP3, and one human-heritable disease-associated mutation in NLRP3 was found to be associated with defective ABHD17A binding and hyper-palmitoylation. Furthermore, Zdhhc5-/- mice showed defective NLRP3 inflammasome activation in vivo. Taken together, our data reveal an endogenous mechanism of inflammasome assembly and activation and suggest NLRP3 palmitoylation as a potential target for the treatment of NLRP3 inflammasome-driven diseases.

Entrectinib inhibits NLRP3 inflammasome and inflammatory diseases by directly targeting NEK7.

Excessive inflammation caused by abnormal activation of the NLRP3 inflammasome contributes to the pathogenesis of multiple human diseases, but clinical drugs targeting the NLRP3 inflammasome are still not available. In this study, we identify entrectinib (ENB), a US Food and Drug Administration (FDA)-approved anti-cancer agent, as a target inhibitor of the NLRP3 inflammasome to treat related diseases. ENB specifically blocks NLRP3 without affecting activation of other inflammasomes. Furthermore, we demonstrate that ENB directly binds to arginine 121 (R121) of NEK7 and blocks the interaction between NEK7 and NLRP3, thereby inhibiting inflammasome assembly and activation. In vivo studies show that ENB has a significant ameliorative effect on mouse models of NLRP3 inflammasome-related diseases, including lipopolysaccharide (LPS)-induced systemic inflammation, monosodium urate (MSU)-induced peritonitis, and high-fat diet (HFD)-induced type 2 diabetes (T2D). These data show that ENB is a targeted inhibitor of NEK7 with strong anti-NLRP3 inflammasome activity, making it a potential candidate drug for the treatment of inflammasome-related diseases.

The pharmacoepigenomic landscape of cancer cell lines reveals the epigenetic component of drug sensitivity.

Aberrant DNA methylation accompanies genetic alterations during oncogenesis and tumour homeostasis and contributes to the transcriptional deregulation of key signalling pathways in cancer. Despite increasing efforts in DNA methylation profiling of cancer patients, there is still a lack of epigenetic biomarkers to predict treatment efficacy. To address this, we analyse 721 cancer cell lines across 22 cancer types treated with 453 anti-cancer compounds. We systematically detect the predictive component of DNA methylation in the context of transcriptional and mutational patterns, i.e., in total 19 DNA methylation biomarkers across 17 drugs and five cancer types. DNA methylation constitutes drug sensitivity biomarkers by mediating the expression of proximal genes, thereby enhancing biological signals across multi-omics data modalities. Our method reproduces anticipated associations, and in addition, we find that the NEK9 promoter hypermethylation may confer sensitivity to the NEDD8-activating enzyme (NAE) inhibitor pevonedistat in melanoma through downregulation of NEK9. In summary, we envision that epigenomics will refine existing patient stratification, thus empowering the next generation of precision oncology.

Nek6 knockdown polarized macrophages into a pro-inflammatory phenotype via inhibiting STAT3 expression.

Recently macrophage polarization has emerged as playing an essential role in the oathogenesis of atherosclerosis, which is the most important underlying process in many types of cardiovascular diseases. Although Nek6 has been reported to be involved in various cellular processes, the effect of Nek6 on macrophage polarization remains unknown. Macrophages exposed to lipopolysaccharide (LPS) or IL-4 were used to establish an in vitro model for the study of regulation of classically (M1) or alternatively (M2) activated macrophage. Bone marrow-derived macrophages (BMDMs) transfected with short hairpin RNA-targeting Nek6 were then in functional studies. We observed that Nek6 expression was decreased in both peritoneal macrophages (PMs) and BMDMs stimulated by LPS. This effect was seen at both mRNA and protein level. The opposite results were obtained after administration of IL-4. Macrophage-specific Nek6 knockdown significantly exacerbated pro-inflammatory M1 polarized macrophage gene expression in response to LPS challenge, but the anti-inflammatory response gene expression that is related to M2 macrophages was attenuated by Nek6 silencing followed by treatment with IL-4. Mechanistic studies exhibited that Nek6 knockdown inhibited the phosphorylated STAT3 expression that mediated the effect on macrophage polarization regulated by AdshNek6. Moreover, decreased Nek6 expression was also observed in atherosclerotic plaques. Collectively, these evidences suggested that Nek6 acts as a crucial site in macrophage polarization, and that this operates in a STAT3-dependent manner.

Novel genes associated with a placental phenotype in knockout mice also respond to cellular stressors in primary human trophoblasts.

INTRODUCTION: Placental insufficiency is a leading cause of intrauterine growth restriction, contributing to perinatal morbidity and mortality. The molecular regulation of placental development and what causes placental insufficiency is poorly understood. Recently, a panel of genes were found to cause significant placental dysmorphologies in mice with severely growth restricted off-spring. We aimed to assess whether these genes were also implicated in human intrauterine growth restriction. METHODS: We explored the expression of nine genes in primary cytotrophoblast cells in hypoxic (n = 6) and glucose starvation (n = 5) conditions in vitro. We also explored whether the genes were dysregulated in intrauterine growth restricted human placental samples (n = 11), with (n = 20) or without preeclampsia compared to gestationally matched controls (<34 weeks gestation) (n = 17). RESULTS: Hypoxic stress significantly upregulated the expressions of BRD2 (p = 0.0313), SMG9 (p = 0.0313) genes. In contrast, glucose starvation significantly suppressed Kif1bp (p = 0.0089) in primary cytotrophoblasts. The FRYL, NEK9, CHTOP, PSPH, ATP11A, HM13 genes did not change under hypoxia or glucose starvation conditions. The expression of these genes was not altered in placenta from patients with intrauterine growth restriction, compared to gestationally matched controls. DISCUSSION: We demonstrate that some of the genes that cause a placental phenotype in mice, respond to hypoxic and glucose mediated stress in human cytotrophoblast isolations. Despite this, they are unchanged in placenta from patients with intrauterine growth restriction. Therefore, dysregulation of these genes is less likely to contribute to preterm intrauterine growth restriction in humans.

Circular forms of dedicator of cytokinesis 1 promotes breast cancer progression by derepressing never in mitosis related kinase 2 via sponging miR-128-3p.

The conjecture of breast cancer is uncertain because of its explosive growth and the complicated molecular mechanisms. Circular RNAs (circRNAs) are regulatory RNA sequences present in the genome and their regulatory mechanism involves the sponging of microRNAs (miRNAs). In this study, we explored the regulation between circular forms of dedicator of cytokinesis 1 (circDOCK1) (hsa_circ_0007142) and miR-128-3p, and its implication on the pathogenesis of breast cancer modulated by never in mitosis (NIMA) related kinase 2 (NEK2). We revealed an increase in circDOCK1 and NEK2 expression, and a decrease in miR-128-3p expression in breast cancer tissues and cell lines. Bioinformatics analysis and experimental validation indicated a positive correlation between circDOCK1 and NEK2 expression but a negative correlation was recorded between miR-128-3p and circDOCK1 or NEK2, respectively. Furthermore, inhibition of circDOCK1 expression was followed by an increase in miR-128-3p and a decrease in NEK2 levels in vitro and in vivo. The luciferase assay concluded that miR-128-3p was a direct target of circDOCK1 while NEK2 was the direct target of miR-128-3p. Furthermore, circDOCK1 inhibition hindered breast cancer development by repressing NEK2 and thus promoting the increased expression of miR-128-3p both in vitro and in vivo. We therefore conclude that circDOCK1 promotes breast cancer progression by targeting miR-128-3p-mediated downregulation of NEK2 and that the circDOCK1/hsa-miR-128-3p/NEK2 axis may be a novel therapeutic target for breast cancer treatment.

Conserved NIMA kinases regulate multiple steps of endocytic trafficking.

Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint-DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C. elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease.

Never in mitosis gene A-related kinase-8 promotes proliferation, migration, invasion, and stemness of breast cancer cells via ß-catenin signalling activation.

Never in mitosis gene A (NIMA)-related kinase-8 (NEK8) is involved in cell cycle progression, cytoskeleton development, and DNA damage repair. However, its role in breast cancer has not yet been explored. To investigate this, NEK8 was knocked down in MDA-MB-231, BT549, and HCC38 breast cancer cell lines. We observed a decrease in cell proliferation and colony formation owing to regulation of the G1/S and G2/M transitions. Furthermore, the expression of several cell cycle regulatory proteins was altered, including that of cyclin D1, cyclin B1, CDK4, CDK2, and surviving. NEK8 knockdown impaired cell migration and invasion as well as reduced the expression of epithelial-mesenchymal transition markers. Regarding stem-cell characteristics, NEK8 knockdown decreased the tumour sphere formation, aldehyde dehydrogenase activity, and stem-cell marker expression, including that of CD44, Sox2, Oct4a, and Nanog. Further analysis revealed that NEK8 interacts with ß-catenin. Also, NEK8 knockdown promoted ß-catenin degradation. NEK8-silenced MDA-MB-231 cells inhibited xenograft tumour growth, metastasis, and tumour initiation in vivo. Using the Oncomine and TNMplot public databases, we found a significant correlation between NEK8 overexpression and poor clinical outcomes in breast cancer patients. Thus, NEK8 may be a crucial regulator of breast cancer progression and a potential therapeutic target.

LncRNA SNHG3 Promotes Sevoflurane-Induced Neuronal Injury by Activating NLRP3 via NEK7.

BACKGROUND: Early exposure to sevoflurane may cause brain tissue degeneration; however, the mechanism involved in this process has not been explored. In this study, we investigated the role of long non-coding RNA small nucleolar RNA host gene 3 (lncRNA SNHG3) in sevoflurane-induced neuronal injury. METHODS: The injury models of HT22 and primary cultures of neurons were constructed using sevoflurane treatment. The WST-8 reduction was detected by CCK-8 assay, the level of inflammatory factors was detected by enzyme-linked immunosorbent assay (ELISA), and cell pyroptosis was detected by flow cytometry. The expression of genes and proteins was detected by qRT-PCR and Western blot, respectively. The level of ß-tubulin III in primary cultures of hippocampal neurons was analyzed by immunofluorescence. The relationship among SNHG3, PTBP1 and NEK7 was confirmed by RIP assay. RESULTS: The expression of SNHG3 and NEK7 were enhanced in sevoflurane-treated HT22 cells. Sevoflurane inhibited the WST-8 reduction in a concentration-dependent manner, promoted the pyroptosis, and increased pyroptosis-related protein expression. SNHG3 knockdown significantly inhibited sevoflurane-induced pyroptosis and inflammatory injury in HT22 cells and primary cultures of neurons. Furthermore, SNHG3 regulated NEK7 expression by binding to PTBP1. NEK7 knockdown reversed the decrease in WST-8 reduction, inhibited pyroptosis, and decreased the release of inflammatory factors and pyroptosis-related protein expression by inactivation of NLRP3 signaling in sevoflurane-induced HT22 cells. Moreover, NEK7 overexpression attenuated the effect of SNHG3 knockdown on neuronal pyroptosis and inflammation injury. CONCLUSION: Downregulation of SNHG3 attenuates sevoflurane-induced neuronal inflammation and pyroptosis by mediating the NEK7/NLRP3 axis, suggesting that SNHG3 could be a potential target gene for neuronal injury.

Revisiting the inhibitory potential of protein kinase inhibitors against NEK7 protein via comprehensive computational investigations.

The NEK7 protein is required for spindle formation, cell division, and the activation of the NLRP3 inflammasome receptor. The aberrant expression of NEK7 has been implicated to the growth of metastasis and severe inflammatory conditions like rheumatoid arthritis, liver cirrhosis, and gout. An emergent target for the development of anti-cancer drugs is NEK7. In this context, the PubChem database was used to retrieve the 675 compound library and FDA-approved protein kinase inhibitors, which were then thoroughly examined via in-silico experiments. Computational studies investigated the binding orientation, electronic, and thermodynamic characteristics of drug candidates related to target protein. Drugs were investigated using density functional theory and molecular docking to find binding interactions with NEK7. Molecular dynamic simulations assessed interactions and stability of protein-ligand complex. DFT analyses showed that selected compounds maintained a significant amount of chemical reactivity in both liquid and gaseous states. Alectinib, Crizotinib, and compound 146476703 all displayed promising molecular interactions, according to molecular docking studies, with docking scores of - 32.76, - 30.54, and - 34.34 kJ/mol, respectively. Additionally, MD simulations determined the stability and dynamic characteristics of the complex over a 200 ns production run. The current study's findings indicate that the drugs Alectinib, Crizotinib, and compound 146476703 can successfully inhibit the overexpression of the NEK7 protein. To discover more potent drugs against NEK7, it is recommended to synthesize the derivatives of Alectinib and Crizotinib and carry out additional in-vitro and in-vivo studies at the molecular level.

ALK-JNK signaling promotes NLRP3 inflammasome activation and pyroptosis via NEK7 during Streptococcus pneumoniae infection.

Streptococcus pneumoniae (S. pneumoniae), a clinically important pathogen worldwide, causes serious invasive diseases, such as pneumonia, otitis media, and meningitis. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome, an important component of the innate immune system, plays a key role in defense against pathogen infection; however the specific activation mechanism induced by S. pneumoniae infection is not fully understood. Here, primary mouse macrophages were selected as the in vitro cell model, and the effect of kinases on S. pneumoniae infection-induced NLRP3 inflammasome activation was investigated in vivo and in vitro using the western blot/RT-PCR/Co-IP/immunofluorescence staining/ELISA with or without kinase inhibitor or siRNA pretreatment. In this study, we found that the formation of the NEK7-NLRP3 complex significantly increased during S. pneumoniae infection and that anaplastic lymphoma kinase (ALK) and Jun N-terminal kinase (JNK) were phosphorylated rapidly. ALK and JNK inhibitors significantly reduced the ability of bacterial killing, the gene expression of NLRP3 inflammasome, the formation of apoptosis-associated speck-like protein containing caspase-recruitment domain (ASC) specks and the NEK7-NLRP3 complex, which in turn decreased the activation level of NLRP3 inflammasome-associated molecules and the maturation of interleukin-1ß (IL-1ß). In addition, ALK regulated the phosphorylation of JNK. Interestingly, the ALK/JNK/NEK7-NLRP3 signaling pathway is also involved in regulating pyroptosis and IL-1ß secretion triggered by S. pneumoniae infection. In conclusion, our data suggest, for the first time, that the ALK/JNK/NEK7-NLRP3 signaling pathway may play an important role in NLRP3 inflammasome activation and pyroptosis and consequently regulate the host immune response upon S. pneumoniae infection.

Phosphate NIMA-Related Kinase 2-Dependent Epigenetic Pathways in Dorsal Root Ganglion Neurons Mediates Paclitaxel-Induced Neuropathic Pain.

BACKGROUND: The microtubule-stabilizing drug paclitaxel (PTX) is an important chemotherapeutic agent for cancer treatment and causes peripheral neuropathy as a common side effect that substantially impacts the functional status and quality of life of patients. The mechanistic role for NIMA-related kinase 2 (NEK2) in the progression of PTX-induced neuropathic pain has not been established. METHODS: Adult male Sprague-Dawley rats intraperitoneally received PTX to induce neuropathic pain. The protein expression levels in the dorsal root ganglion (DRG) of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by von Frey tests and hot plate tests. RESULTS: PTX increased phosphorylation of the important microtubule dynamics regulator NEK2 in DRG neurons and induced profound neuropathic allodynia. PTX-activated phosphorylated NEK2 (pNEK2) increased jumonji domain-containing 3 (JMJD3) protein, a histone demethylase protein, to specifically catalyze the demethylation of the repressive histone mark H3 lysine 27 trimethylation (H3K27me3) at the Trpv1 gene, thereby enhancing transient receptor potential vanilloid subtype-1 (TRPV1) expression in DRG neurons. Moreover, the pNEK2-dependent PTX response program is regulated by enhancing p90 ribosomal S6 kinase 2 (RSK2) phosphorylation. Conversely, intrathecal injections of kaempferol (a selective RSK2 activation antagonist), NCL 00017509 (a selective NEK2 inhibitor), NEK2-targeted siRNA, GSK-J4 (a selective JMJD3 inhibitor), or capsazepine (an antagonist of TRPV1 receptor) into PTX-treated rats reversed neuropathic allodynia and restored silencing of the Trpv1 gene, suggesting the hierarchy and interaction among phosphorylated RSK2 (pRSK2), pNEK2, JMJD3, H3K27me3, and TRPV1 in the DRG neurons in PTX-induced neuropathic pain. CONCLUSIONS: pRSK2/JMJD3/H3K27me3/TRPV1 signaling in the DRG neurons plays as a key regulator for PTX therapeutic approaches.

The phosphorylation of PHF5A by TrkA-ERK1/2-ABL1 cascade regulates centrosome separation.

During interphase, the newly duplicated pairs of centrosomes are held together by a centrosome linker, and the centrosome separation needs the disruption of this linker to induce the duplicated centrosomes separating into two distinct microtubule organization centers. The mechanism of regulating centrosome separation is however poorly understood. Here, we demonstrated that the phosphorylation of PHF5A at Y36 by the TrkA-ERK1/2-ABL1 cascade plays a critical role in regulating centrosome separation. PHF5A, a well-characterized spliceosome component, is enriched in the centrosome. The pY36-PHF5A promotes the interaction between CEP250 and Nek2A in a spliceosomal-independent manner, which leads to premature centrosome separation. Furthermore, the unmatured centrosome remodels the microtubule and subsequently regulates cell proliferation and migration. Importantly, we found that the phosphorylation cascade of TrkA-ERK1/2-ABL1-PHF5A is hyper-regulated in medulloblastoma. The inhibition of this cascade can induce senescence and restrict the proliferation of medulloblastoma. Our findings on this phosphorylation cascade in regulating centrosome separation could provide a series of potential targets for restricting the progress of medulloblastoma.

NEK6 Regulates Redox Balance and DNA Damage Response in DU-145 Prostate Cancer Cells.

NEK6 is a central kinase in developing castration-resistant prostate cancer (CRPC). However, the pathways regulated by NEK6 in CRPC are still unclear. Cancer cells have high reactive oxygen species (ROS) levels and easily adapt to this circumstance and avoid cell death by increasing antioxidant defenses. We knocked out the NEK6 gene and evaluated the redox state and DNA damage response in DU-145 cells. The knockout of NEK6 decreases the clonogenic capacity, proliferation, cell viability, and mitochondrial activity. Targeting the NEK6 gene increases the level of intracellular ROS; decreases the expression of antioxidant defenses (SOD1, SOD2, and PRDX3); increases JNK phosphorylation, a stress-responsive kinase; and increases DNA damage markers (p-ATM and γH2AX). The exogenous overexpression of NEK6 also increases the expression of these same antioxidant defenses and decreases γH2AX. The depletion of NEK6 also induces cell death by apoptosis and reduces the antiapoptotic Bcl-2 protein. NEK6-lacking cells have more sensitivity to cisplatin. Additionally, NEK6 regulates the nuclear localization of NF-κB2, suggesting NEK6 may regulate NF-κB2 activity. Therefore, NEK6 alters the redox balance, regulates the expression of antioxidant proteins and DNA damage, and its absence induces the death of DU-145 cells. NEK6 inhibition may be a new strategy for CRPC therapy.