Genetic inactivation of ß-catenin is salubrious, whereas its activation is deleterious in desmoplakin cardiomyopathy.

AIMS: Mutations in the DSP gene encoding desmoplakin, a constituent of the desmosomes at the intercalated discs (IDs), cause a phenotype that spans arrhythmogenic cardiomyopathy (ACM) and dilated cardiomyopathy. It is typically characterized by biventricular enlargement and dysfunction, myocardial fibrosis, cell death, and arrhythmias. The canonical wingless-related integration (cWNT)/ß-catenin pathway is implicated in the pathogenesis of ACM. The ß-catenin is an indispensable co-transcriptional regulator of the cWNT pathway and a member of the IDs. We genetically inactivated or activated ß-catenin to determine its role in the pathogenesis of desmoplakin cardiomyopathy. METHODS AND RESULTS: The Dsp gene was conditionally deleted in the 2-week-old post-natal cardiac myocytes using tamoxifen-inducible MerCreMer mice (Myh6-McmTam:DspF/F). The cWNT/ß-catenin pathway was markedly dysregulated in the Myh6-McmTam:DspF/F cardiac myocytes, as indicated by a concomitant increase in the expression of cWNT/ß-catenin target genes, isoforms of its key co-effectors, and the inhibitors of the pathway. The ß-catenin was inactivated or activated upon inducible deletion of its transcriptional or degron domain, respectively, in the Myh6-McmTam:DspF/F cardiac myocytes. Genetic inactivation of ß-catenin in the Myh6-McmTam:DspF/F mice prolonged survival, improved cardiac function, reduced cardiac arrhythmias, and attenuated myocardial fibrosis, and cell death caused by apoptosis, necroptosis, and pyroptosis, i.e. PANoptosis. In contrast, activation of ß-catenin had the opposite effects. The deleterious and the salubrious effects were independent of changes in the expression levels of the cWNT target genes and were associated with changes in several molecular and biological pathways, including cell death programmes. CONCLUSION: The cWNT/ß-catenin was markedly dysregulated in the cardiac myocytes in a mouse model of desmoplakin cardiomyopathy. Inactivation of ß-catenin attenuated, whereas its activation aggravated the phenotype, through multiple molecular pathways, independent of the cWNT transcriptional activity. Thus, suppression but not activation of ß-catenin might be beneficial in desmoplakin cardiomyopathy.

PANoptosis is a prominent feature of desmoplakin cardiomyopathy.

Introduction: Arrhythmogenic cardiomyopathy (ACM) is hereditary cardiomyopathy caused by pathogenic variants (mutations) in genes encoding the intercalated disc (ID), particularly desmosome proteins. ACM caused by mutations in the DSP gene encoding desmoplakin (DSP) is characterized by the prominence of cell death, myocardial fibrosis, and inflammation, and is referred to as desmoplakin cardiomyopathy. Aim: The aim of this article was to gain insight into the pathogenesis of DSP cardiomyopathy. Methods and Results: The Dsp gene was exclusively deleted in cardiac myocytes using tamoxifen-inducible MerCreMer (Myh6-Mcm Tam) and floxed Dsp (Dsp F/F) mice (Myh6-Mcm Tam:Dsp F/F). Recombination was induced upon subcutaneous injection of tamoxifen (30 mg/kg/d) for 5 days starting post-natal day 14. Survival was analyzed by Kaplan-Meier plots, cardiac function by echocardiography, arrhythmias by rhythm monitoring, and gene expression by RNA-Seq, immunoblotting, and immunofluorescence techniques. Cell death was analyzed by the TUNEL assay and the expression levels of specific markers were by RT-PCR and immunoblotting. Myocardial fibrosis was assessed by picrosirius red staining of the myocardial sections, RT-PCR, and immunoblotting. The Myh6-Mcm Tam: Dsp F/F mice showed extensive molecular remodeling of the IDs and the differential expression of ~10,000 genes, which predicted activation of KDM5A, IRFs, and NFκB and suppression of PPARGC1A and RB1, among others in the DSP-deficient myocytes. Gene set enrichment analysis predicted activation of the TNFα/NFκB pathway, inflammation, cell death programs, and fibrosis. Analysis of cell death markers indicated PANoptosis, comprised of apoptosis (increased CASP3, CASP8, BAD and reduced BCL2), necroptosis (increased RIPK1, RIPK3, and MLKL), and pyroptosis (increased GSDMD and ASC or PYCARD) in the DSP-deficient myocytes. Transcript levels of the pro-inflammatory and pro-fibrotic genes were increased and myocardial fibrosis comprised ~25% of the myocardium in the DSP-deficient hearts. The Myh6-Mcm Tam:Dsp F/F mice showed severe cardiac systolic dysfunction and ventricular arrhythmias, and died prematurely with a median survival rate of ~2 months. Conclusion: The findings identify PANoptosis as a prominent phenotypic feature of DSP cardiomyopathy and set the stage for delineating the specific molecular mechanisms involved in its pathogenesis. The model also provides the opportunity to test the effects of pharmacological and genetic interventions on myocardial fibrosis and cell death.

Deletion of the Lmna gene in fibroblasts causes senescence-associated dilated cardiomyopathy by activating the double-stranded DNA damage response and induction of senescence-associated secretory phenotype.

Introduction: Mutations in the LMNA gene, encoding Lamin A/C (LMNA), are established causes of dilated cardiomyopathy (DCM). The phenotype is typically characterized by progressive cardiac conduction defects, arrhythmias, heart failure, and premature death. DCM is primarily considered a disease of cardiac myocytes. However, LMNA is also expressed in other cardiac cell types, including fibroblasts. Aim: The purpose of the study was to determine the contribution of the fibroblasts to DCM caused by LMNA deficiency. Methods and Results: The Lmna gene was deleted by crossing the platelet-derived growth factor receptor α-Cre recombinase (Pdgfra-Cre) and floxed Lmna (Lmna F/F) mice. The LMNA protein was nearly absent in ~80% of the cardiac fibroblasts and ~25% of cardiac myocytes in the Pdgfra-Cre:Lmna F/F mice. The Pdgfra-Cre:Lmna F/F mice showed an early phenotype characterized by cardiac conduction defects, arrhythmias, cardiac dysfunction, myocardial fibrosis, apoptosis, and premature death within the first six weeks of life. The Pdgfra-Cre:Lmna wild type/F (Lmna W/F) mice also showed a similar but slowly evolving phenotype that was expressed within one year of age. RNA sequencing of LMNA-deficient and wild-type cardiac fibroblasts identified differential expression of ~410 genes, which predicted activation of the TP53 and TNFA/NFκB and suppression of the cell cycle pathways. In agreement with these findings, levels of phospho-H2AFX, ATM, phospho-TP53, and CDKN1A, markers of the DNA damage response (DDR) pathway, were increased in the Pdgfra-Cre:Lmna F/F mouse hearts. Moreover, expression of senescence-associated beta-galactosidase was induced and levels of the senescence-associated secretory phenotype (SASP) proteins TGFß1, CTGF (CCN2), and LGLAS3 were increased as well as the transcript levels of additional genes encoding SASP proteins in the Pdgfra-Cre:Lmna F/F mouse hearts. Finally, expression of pH2AFX, a bonafide marker of the double-stranded DNA breaks, was increased in cardiac fibroblasts isolated from the Pdgfra-Cre:Lmna F/F mouse hearts. Conclusion: Deletion of the Lmna gene in fibroblasts partially recapitulates the phenotype of the LMNA-associated DCM, likely through induction of double-stranded DNA breaks, activation of the DDR pathway, and induction of expression of the SASP proteins. The findings indicate that the phenotype in the LMNA-associated DCM is the aggregate consequence of the LMNA deficiency in multiple cardiac cells, including cardiac fibroblasts.

Alteration of miRNAs in Small Neuron-Derived Extracellular Vesicles of Alzheimer's Disease Patients and the Effect of Extracellular Vesicles on Microglial Immune Responses.

Alzheimer's disease (AD) is one of the most severe neurodegenerative diseases observed in the elderly population. Although the hallmarks of AD have been identified, the methods for its definitive diagnosis and treatment are still lacking. Extracellular vesicles (EVs) have become a promising source for biomarkers since the identification of their content. EVs are released from multiple cell types and, when released from neurons, they pass from the brain to the blood with their cargo molecules. Hence, neuron-specific EV-resident microRNAs (miRNAs) are promising biomarkers for diagnosis of AD. This study aimed to identify altered miRNA content in small neuron-derived extracellular vesicles (sNDEVs) isolated from AD patients and healthy individuals. Furthermore, we examined the role of sNDEV-resident miRNAs in neuron-glia cellular interaction to understand their role in AD propagation. We identified 10 differentially expressed miRNAs in the sNDEVs of patients via next-generation sequencing and validated the most dysregulated miRNA, let-7e, with qRT-PCR. Let-7e was significantly increased in the sNDEVs of AD patients compared with those of healthy controls in a larger cohort. First, we evaluated the diagnostic utility of let-7e via ROC curve analysis, which revealed an AUC value of 0.9214. We found that IL-6 gene expression was increased in human microglia after treatment with sNDEVs of AD patients with a high amount of let-7e. Our study suggests that sNDEV-resident let-7e is a potential biomarker for AD diagnosis, and that AD patient-derived sNDEVs induce a neuroinflammatory response in microglia.

The WNT/ß-catenin pathway regulates expression of the genes involved in cell cycle progression and mitochondrial oxidative phosphorylation in the postmitotic cardiac myocytes.

INTRODUCTION: Aging is associated with cardiac myocyte loss, sarcopenia, and cardiac dysfunction. Adult cardiac myocytes are postmitotic cells with an insufficient proliferative capacity to compensate for myocyte loss. The canonical WNT (cWNT) pathway is involved in the regulation of cell cycle reentry in various cell types. The effects of the cWNT pathway on the expression of genes involved in cell cycle reentry in the postmitotic cardiac myocytes are unknown. AIM: The aim of the study was to identify genes whose expression is regulated by the ß-catenin, the indispensable component to the cWNT signaling, in the postmitotic myocytes. METHODS AND RESULTS: Cardiac myocyte-specific tamoxifen-inducible MerCreMer (Myh6-Mcm) mice were used to delete the floxed exon 3 or exons 8 to 13 of the Ctnnb1 gene to induce gain-of-function (GoF) or loss-of-function (LoF) the ß-catenin, respectively. Deletion of exon 3 leads to the expression of a stable ß-catenin. In contrast, deletion of exons 8-13 leads to the expression of transcriptionally inactive truncated ß-catenin, which is typically degraded. GoF or LoF of the ß-catenin was verified by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting, and immunofluorescence. Myocyte transcripts were analyzed by RNA-Sequencing (RNA-Seq) at 4 weeks of age. The GoF of ß-catenin was associated with differential expression of ~1700 genes, whereas its LoF altered expression of ~400 genes. The differentially expressed genes in the GoF myocytes were enriched in pathways regulating the cell cycle, including karyokinesis and cytokinesis, whereas the LoF was associated with increased expression of genes involved in mitochondrial oxidative phosphorylation. These findings were validated by RT-PCR in independent samples. Short-term GoF nor LoF of ß-catenin did not affect the number of cardiac myocytes, cardiac function, myocardial fibrosis, myocardial apoptosis, or adipogenesis at 4 weeks of age. CONCLUSION: Activation of the ß-catenin of the cWNT pathway in postmitotic myocytes leads to cell cycle reentry and expression of genes involved in cytokinesis without leading to an increase in the number of myocytes. In contrast, suppression of the ß-catenin modestly increases the expression of genes involved in oxidative phosphorylation. The findings provide insights into the role of ß-catenin of the cWNT pathway in the regulation of cell cycle reentry and oxidative phosphorylation in the postmitotic cardiac myocytes.

Effects of tamoxifen inducible MerCreMer on gene expression in cardiac myocytes in mice.

The Cre-LoxP technology, including the tamoxifen (TAM) inducible MerCreMer (MCM), is increasingly used to delineate gene function, understand the disease mechanisms, and test therapeutic interventions. We set to determine the effects of TAM-MCM on cardiac myocyte transcriptome. Expression of the MCM was induced specifically in cardiac myocytes upon injection of TAM to myosin heavy chain 6-MCM (Myh6-Mcm) mice for 5 consecutive days. Cardiac function, myocardial histology, and gene expression (RNA-sequencing) were analyzed 2 weeks after TAM injection. A total of 346 protein coding genes (168 up- and 178 down-regulated) were differentially expressed. Transcript levels of 85 genes, analyzed by a reverse transcription-polymerase chain reaction in independent samples, correlated with changes in the RNA-sequencing data. The differentially expressed genes were modestly enriched for genes involved in the interferon response and the tumor protein 53 (TP53) pathways. The changes in gene expression were relatively small and mostly transient and had no discernible effects on cardiac function, myocardial fibrosis, and apoptosis or induction of double-stranded DNA breaks. Thus, TAM-inducible activation of MCM alters cardiac myocytes gene expression, provoking modest and transient interferon and DNA damage responses without exerting other discernible phenotypic effects. Thus, the effects of TAM-MCM on gene expression should be considered in discerning the bona fide changes that result from the targeting of the gene of interest.

MicroRNAs in Genetic Etiology of Human Diseases.

Since their first discovery more than 20 years ago, miRNAs have been subject to deliberate research and analysis for revealing their physiological or pathological involvement. Regulatory roles of miRNAs in signal transduction, gene expression, and cellular processes in development, differentiation, proliferation, apoptosis, and homeostasis also imply their critical role in disease pathogenesis. Their roles in cancer, neurodegenerative diseases, and other systemic diseases have been studied broadly. In these regulatory pathways, their mutations and target sequence variations play critical roles to determine their functional repertoire. In this chapter, we summarize studies that investigated the role of mutations, polymorphisms, and other variations of miRNAs in respect to pathological processes.

Inhibitory effects of sulforaphane on NLRP3 inflammasome activation.

SFN, a dietary phytochemical, is a significant member of isothiocyanates present in cruciferous vegetables at high levels in broccoli. It is a well-known activator of the Nrf2/ARE antioxidant pathway. Long since, the therapeutic effects of SFN have been widely studied in several different diseases. Other than the antioxidant effect, SFN also exhibits an anti-inflammatory effect through suppression of various mechanisms, including inflammasome activation. Considerably, SFN has been demonstrated to inhibit multiple inflammasomes, including NLRP3 inflammasome. NLRP3 inflammasome induces secretion of pro-inflammatory cytokines and promotes inflammatory cell death. The release of pro-inflammatory cytokines enhances the inflammatory response, in turn leading to tissue damage. These self-propelling inflammatory responses would need modulation with exogenous therapeutic agents to suppress them. SFN is a promising candidate molecule for the mitigation of NLRP3 inflammasome activation, which has been related to the pathogenesis of numerous disorders. In this review, we have provided fundamental knowledge about Sulforaphane, elaborated its characteristics, and evidentially focused on its mechanisms of action with regard to its anti-inflammatory, anti-oxidative, and neuroprotective features. Thereafter, we have summarized both in vitro and in vivo studies regarding SFN effect on NLRP3 inflammasome activation.

Lamin A/C Is Dispensable to Mechanical Repression of Adipogenesis.

Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.

The Role of Melatonin on NLRP3 Inflammasome Activation in Diseases.

NLRP3 inflammasome is a part of the innate immune system and responsible for the rapid identification and eradication of pathogenic microbes, metabolic stress products, reactive oxygen species, and other exogenous agents. NLRP3 inflammasome is overactivated in several neurodegenerative, cardiac, pulmonary, and metabolic diseases. Therefore, suppression of inflammasome activation is of utmost clinical importance. Melatonin is a ubiquitous hormone mainly produced in the pineal gland with circadian rhythm regulatory, antioxidant, and immunomodulatory functions. Melatonin is a natural product and safer than most chemicals to use for medicinal purposes. Many in vitro and in vivo studies have proved that melatonin alleviates NLRP3 inflammasome activity via various intracellular signaling pathways. In this review, the effect of melatonin on the NLRP3 inflammasome in the context of diseases will be discussed.

Ethyl Pyruvate Attenuates Microglial NLRP3 Inflammasome Activation via Inhibition of HMGB1/NF-κB/miR-223 Signaling.

Ethyl pyruvate is a molecule with anti-inflammatory and pro-metabolic effects. Ethyl pyruvate has been shown to ameliorate the clinical and pathological findings of neurodegenerative diseases such as Alzheimer's and Parkinson's Diseases in rodents. Its anti-inflammatory and neuroprotective effects are widely investigated in animal and cellular models. Our study aimed to investigate the mechanism of the impact of Ethyl pyruvate on NLRP3 inflammasome activation in the N9 microglial cell line. Our results indicated that ethyl pyruvate significantly suppressed LPS and ATP-induced NLRP3 inflammasome activation, decreased active caspase-1 level, secretion of IL-1ß and IL-18 cytokines, and reduced the level of pyroptotic cell death resulting from inflammasome activation. Furthermore, ethyl pyruvate reduced the formation of total and mitochondrial ROS and suppressed inflammasome-induced HMGB1 upregulation and nuclear NF-κB translocation and reversed the inflammasome activation-induced miRNA expression profile for miR-223 in N9 cells. Our study suggests that ethyl pyruvate effectively suppresses the NLRP3 inflammasome activation in microglial cells regulation by miR-223 and NF-κB/HMGB1 axis.

Proteome profiling of neuron-derived exosomes in Alzheimer's disease reveals hemoglobin as a potential biomarker.

Alzheimer's disease is a chronic and progressive neurodegenerative disorder, which is the most common cause of dementia worldwide. Although amyloid plaques and neurofibrillary tangles are identified as the hallmarks of the disease, the only valid diagnostic method yet is post-mortem imaging of these molecules in brain sections. Exosome is a type of extracellular vesicles secreted into extracellular space and plays fundamental roles in healthy and pathological conditions, including cell-to-cell communication. In this study, we aimed to investigate the proteomic contents of neuron-derived exosomes (NDEs) from AD patients and healthy controls (HCs) to identify a possible marker for AD diagnosis. We identified alpha-globin, beta-globin, and delta-globin increase in neuron-derived exosomes of AD patients compared to HCs with LC-MS/MS proteomics analysis. Then, we confirmed the high hemoglobin (Hb) level in NDEs of AD patients with ELISA. We found the area under the curve of hemoglobin level as 0.6913 with ROC analysis. Cargo proteins of NDEs may be useful diagnostic biomarker for AD.

Sulforaphane inhibits NLRP3 inflammasome activation in microglia through Nrf2-mediated miRNA alteration.

The NLRP3 inflammasome is a multiprotein complex that activates caspase-1 and triggers the release of the proinflammatory cytokines IL-1ß and IL-18 in response to diverse signals. Although inflammasome activation plays critical roles against various pathogens in host defense, overactivation of inflammasome contributes to the pathogenesis of inflammatory diseases, including acute CNS injuries and chronic neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In the current study, we demonstrated that Sulforaphane (SFN), a dietary natural product, inhibits NLRP3 inflammasome mediated IL-1ß and IL-18 secretion and pyroptosis in murine microglial cells. SFN decreased the secretion of IL-1ß and IL-18, and their mRNA levels in LPS primed microglia triggered by ATP. SFN suppressed the overexpression of cleaved caspase-1 and NLRP3 protein expressions as measured by caspase activity assay and western blot, respectively. SFN also prevented caspase-1 dependent pyroptotic cell death in microglia. Our data indicate that SFN suppresses NLRP3 inflammasome via the inhibition of NF-κB nuclear translocation and Nrf2 mediated miRNAs expression modulation in murine microglia.

Inhibitory effects of phytochemicals on NLRP3 inflammasome activation: A review.

BACKGROUND: The NLRP3 inflammasome formation and following cytokine secretion is a crucial step in innate immune responses. Internal and external factors may trigger inflammasome activation and result in inflammatory cytokine secretion. Inflammasome formation and activity play critical roles in several disease pathologies such as cardiovascular, metabolic, renal, digestive, and CNS diseases. Underlying pathways are not yet clear, but phytochemicals as alternative therapies have been extensively used for suppression of inflammatory responses. PURPOSE: In this review, we aimed to summarize in vivo and in vitro effects on NLRP3 inflammasome activation of selected phytochemicals. METHOD: Three phytochemicals; Sulforaphane, Curcumin, and Resveratrol were selected, and studies were reviewed to clarify their intracellular signaling mechanism in NLRP3 inflammasome activity. PubMed and Scopus databases are used for the search. For sulforaphane, 8 articles, for curcumin, 25 articles, and for resveratrol, 41 articles were included in the review. CONCLUSION: In vitro and in vivo studies pointed out that the selected phytochemicals have inhibitory properties on NLRP3 inflammasome activity. However, neither the mechanism is clear, nor the study designs and doses are standardized.

Microglial NLRP3 inflammasome activation in multiple sclerosis.

Multiple sclerosis (MS) is a chronic, autoimmune and neuroinflammatory disease of the central nervous system (CNS) mediated by autoreactive T cells directed against myelin antigens. Although the crucial role of adaptive immunity is well established in MS, the contribution of innate immunity has only recently been appreciated. Microglia are the main innate immune cells of the CNS. Similar to other myeloid cells, microglia recognize both exogenous and host-derived endogenous danger signals through pattern recognition receptors (PRRs) localized on their cell surface such as Toll Like receptor 4, or in the cytosol such as NLRP3. The second one is the sensor protein of the multi-molecular NLRP3 inflammasome complex in activated microglia that promotes the maturation and secretion of proinflammatory cytokines, interleukin-1ß and interleukin-18. Overactivation of microglia and aberrant activation of the NLRP3 inflammasome have been implicated in the pathogenesis of MS. Indeed, experimental data, together with post-mortem and clinical studies have revealed an increased expression of NLRP3 inflammasome complex elements in microglia and other immune cells. In this review, we focus on microglial NLRP3 inflammasome activation in MS. First, we overview the basic knowledge about MS, microglia and the NLRP3 inflammasome. Then, we summarize studies about microglial NLRP3 inflammasome activation in MS and its animal models. We also highlight experimental therapeutic approaches that target different steps of NLRP inflammasome activation. Finally, we discuss future research avenues and new methods in this rapidly evolving area.

Melatonin Attenuates LPS-Induced Acute Depressive-Like Behaviors and Microglial NLRP3 Inflammasome Activation Through the SIRT1/Nrf2 Pathway.

Inflammation is a crucial component of various stress-induced responses that contributes to the pathogenesis of major depressive disorder (MDD). Depressive-like behavior (DLB) is characterized by decreased mobility and depressive behavior that occurs in systemic infection induced by Lipopolysaccharide (LPS) in experimental animals and is considered as a model of exacerbation of MDD. We assessed the effects of melatonin on behavioral changes and inflammatory cytokine expression in hippocampus of mice in LPS-induced DLB, as well as its effects on NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation, oxidative stress and pyroptotic cell death in murine microglia in vitro. Intraperitoneal 5 mg/kg dose of LPS was used to mimic depressive-like behaviors and melatonin was given at a dose of 500 mg/kg for 4 times with 6 h intervals, starting at 2 h before LPS administration. Behavioral assessment was carried out at 24 h post-LPS injection by tail suspension and forced swimming tests. Additionally, hippocampal cytokine and NLRP3 protein levels were estimated. Melatonin increased mobility time of LPS-induced DLB mice and suppressed NLRP3 expression and interleukin-1ß (IL-1ß) cleavage in the hippocampus. Immunofluorescence staining of hippocampal tissue showed that NLRP3 is mainly expressed in ionized calcium-binding adapter molecule 1 (Iba1) -positive microglia. Our results show that melatonin prevents LPS and Adenosine triphosphate (ATP) induced NLRP3 inflammasome activation in murine microglia in vitro, evidenced by inhibition of NLRP3 expression, Apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, caspase-1 cleavage and interleukin-1ß (IL-1ß) maturation and secretion. Additionally, melatonin inhibits pyroptosis, production of mitochondrial and cytosolic reactive oxygen species (ROS) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. The beneficial effects of melatonin on NLRP3 inflammasome activation were associated with nuclear factor erythroid 2-related factor 2 (Nrf2) and Silent information regulator 2 homolog 1 (SIRT1) activation, which were reversed by Nrf2 siRNA and SIRT1 inhibitor treatment.

Sun-mediated mechanical LINC between nucleus and cytoskeleton regulates ßcatenin nuclear access.

ßcatenin acts as a primary intracellular signal transducer for mechanical and Wnt signaling pathways to control cell function and fate. Regulation of ßcatenin in the cytoplasm has been well studied but ßcatenin nuclear trafficking and function remains unclear. In a previous study we showed that, in mesenchymal stem cells (MSC), mechanical blockade of adipogenesis relied on inhibition of ßcatenin destruction complex element GSK3ß (glycogen synthase kinase 3ß) to increase nuclear ßcatenin as well as the function of Linker of Cytoskeleton and Nucleoskeleton (LINC) complexes, suggesting that these two mechanisms may be linked. Here we show that shortly after inactivation of GSK3ß due to either low intensity vibration (LIV), substrate strain or pharmacologic inhibition, ßcatenin associates with the nucleoskeleton, defined as the insoluble nuclear fraction that provides structure to the integrated nuclear envelope, nuclear lamina and chromatin. Co-depleting LINC elements Sun-1 and Sun-2 interfered with both nucleoskeletal association and nuclear entry of ßcatenin, resulting in decreased nuclear ßcatenin levels. Our findings reveal that the insoluble structural nucleoskeleton actively participates in ßcatenin dynamics. As the cytoskeleton transmits applied mechanical force to the nuclear surface to influence the nucleoskeleton and its LINC mediated interaction, our results suggest a pathway by which LINC mediated connectivity may play a role in signaling pathways that depend on nuclear access of ßcatenin.

Daily application of low magnitude mechanical stimulus inhibits the growth of MDA-MB-231 breast cancer cells in vitro.

INTRODUCTION: Mechanical loads can regulate cell proliferation and differentiation at various stages of development and homeostasis. However, the extension of this regulatory effect of mechanical loads on cancer cells is largely unknown. Increased physical compliance is one of the key features of cancer cells, which may hamper the transmission of mechanical loads to these cells within tumor microenvironment. Here we tested whether brief daily application of an external low magnitude mechanical stimulus (LMMS), would impede the growth of MDA-MB-231 aggressive type breast cancer cells in vitro for 3 wks of growth. METHODS: The signal was applied in oscillatory form at 90 Hz and 0.15 g, a regimen that would induce mechanical loads on MDA-MB-231 cells via inertial properties of cells rather than matrix deformations. Experimental cells were exposed to LMMS 15 min/day, 5 days/week in ambient conditions while control cells were sham loaded. Cell proliferation, viability, cycle, apoptosis, morphology and migration were tested via Trypan Blue dye exclusion, MTT, PI, Annexin V, Calcein-AM and phalloidin stains and scratch wound assays. RESULTS: Compared to sham controls, daily application of LMMS reduced the number and viability of cancerous MDA-MB-231 cells significantly after first week in the culture, while non-cancerous MCF10A cells were found to be unaffected. Flow cytomety analyses suggested that the observed decrease for the cancer cells in the LMMS group was due to a cell cycle arrest rather than apoptosis. LMMS further reduced cancer cell circularity and increased cytoskeletal actin in MDA-MB-231 cells. CONCLUSION: Combined, results suggest that direct application of mechanical loads negatively regulate the proliferation of aggressive type cancer cells. If confirmed, this non-invasive approach may be integrated to the efforts for the prevention and/or treatment of cancer.