MLKL associated with angiogenesis and osteolysis in periapical lesions
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PROVIDER: PRJNA1031590 | ENA |
REPOSITORIES: ENA
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Project description:OBJECTIVES: To detect the expression levels of MLKL and p-MLKL, and explore its potential roles in inflammatory cell infiltration, angiogenesis, and bone destruction, in human and mouse periapical lesions. METHODS: Forty-six human periapical tissues, including periapical granulomas (PGs, n =26), radicular cysts (RCs, n =20), and eight healthy control tissues were collected. Samples were fixed and analyzed by HE staining, RNA-Seq, western blot, and immunohistochemical staining. A periapical lesion mouse model was induced by pulp exposure in the first lower molars of 15 C57BL/6J mice. After lesion induction, the mice were sacrificed on days 0, 21, and 35. Mandibles were harvested for microcomputed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. Double immunofluorescence was utilized to assess the colocalization of phosphorylated MLKL (p-MLKL) with CD34, matrix metalloproteinase-9 (MMP-9), and Cathepsin K (CTSK) in human and mouse periapical lesions. RESULTS: RNA-Seq analysis showed that, in comparison with healthy gingiva tissues, MLKL was more significantly upregulated in periapical lesions (P-value < 0.05). Immunohistochemistry staining showed that, MLKL and p-MLKL were significantly overexpressed in the RC and PG groups compared with the control group (P-value < 0.05). However, the difference between the RC and PG groups was insignificant (P-value > 0.05). p-MLKL-positive cells were mainly lymphocytes, epithelial cells, and endothelial cells around the vascular wall. In mouse periapical lesions, the expression levels of p-MLKL were positively correlated with the bone defect area and tartrate-resistant acid phosphatase-positive (TRAP+) cell amounts (R2=0.4108, P-value < 0.05; R2=0.5668, P-value < 0.05, respectively). The double-labeling analysis showed that p-MLKL colocalized with CD34 and MMP-9 in human samples, and with CTSK adjacent to the bone in mouse periapical lesions. CONCLUSION: MLKL and p-MLKL were overexpressed in human periapical lesions. p-MLKL exhibited a close relationship with angiogenesis and alveolar bone resorption.
2024-10-24 | GSE246108 | GEO
Project description:The aim of this study was to identify macrophage-derived factors that promote osteoclast differentiation and periprosthetic bone destruction. To achieve this, we examined the effects of 12 macrophage-derived factors that were identified by RNA-seq analysis of stimulated macrophages on osteoclast differentiation. TYMP was found to trigger significant number of osteoclasts that exhibited resorbing activities on dentine slices. TYMP were detected in serum and synovial tissues of patients that had been diagnosed with aseptic loosening. RNA-seq for TYMP-induced-osteoclasts was then performed in an effort to understand action mode of TYMP. TYMP stimulation appeared to activate the tyrosine kinase FYN signaling associated with osteoclast formation. Oral administration of saracatinib, a FYN kinase inhibitor, significantly suppressed formation of bone osteolytic lesions in a polyethylene debris-induced osteolysis model. Our findings highlight a novel molecular target for therapeutic intervention in periprosthetic osteolysis.
2021-06-10 | GSE171542 | GEO
Project description:Caspase-8 is a protease with both pro-death and pro-survival functions: it is required for apoptosis induced by death receptors such as TNFR1 (tumour necrosis factor receptor 1), and it has a critical role in suppressing necroptosis mediated by the kinase RIPK3 (receptor interacting protein kinase 3) and the pseudokinase MLKL (mixed lineage kinase-like). Mice lacking caspase-8 display MLKL-dependent embryonic lethality, as do mice expressing catalytically inactive caspase-8 mutant C362A. However, Casp8C362A/C362A Mlkl-/- mice die in the perinatal period, whereas Casp8-/- Mlkl-/- mice are viable, indicating that inactive caspase-8 also has a pro-death scaffolding function. Here we show that caspase-8 C362A triggers ASC speck formation and caspase-1-dependent pyroptosis in MLKL-deficient intestinal epithelial cells (IECs) around embryonic day 18. Pyroptosis contributed to the perinatal lethal phenotype because a number of Casp8 C362A/C362A Mlkl-/- Casp1-/- mice survived beyond weaning. Transfection studies suggested inactive caspase-8 adopts a distinct conformation to wild-type caspase-8, enabling it to engage the caspase-1 adaptor ASC. Wild-type caspase-8 was found in the Triton X-100 soluble fraction, whereas wild-type caspase-8 inhibited with the pan-caspase inhibitor emricasan, or inactive caspase-8 mutant C362A, were detected in the insoluble fraction. Moreover, inhibited or inactive caspase-8 shifted ASC into the insoluble fraction. Perinatal lethality was recapitulated when expression of caspase-8 C362A was restricted to IECs, but intriguingly, only in the absence of MLKL. Hence, unanticipated plasticity in death pathways is revealed such that IECs can undergo caspase-1-dependent death when caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited.
2019-09-20 | GSE132133 | GEO
Project description:Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase 8-independent form of programmed cell death called necroptosis. Once Receptor Interacting serine/threonine Protein Kinase 3 (RIPK3) is activated by upstream cell death signals, it phosphorylates MLKL and triggers the oligomerization and membrane translocation required for MLKL induced membrane disruption. Besides phosphorylation, MLKL also undergoes ubiquitylation during the early stages of necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here we show that necroptosis-specific, multi-mono-ubiquitylation of MLKL occurs on biological membranes, and requires its activation and oligomerisation. Inactive MLKL mutants recruited to membranes during necroptosis are ubiquitylated but this results in their proteasome and lysosome dependent turnover. We identified several ubiquitylated lysines however mutation of these did not affect MLKL ubiquitylation in response to a necroptotic stimulus.
2021-04-28 | PXD015537 | Pride
Project description:Purpose: Necroptosis as been implicated in various deseases. The goal of this study is to invastigate the impact of RIPK3 and MLKL in the lipid metabolism of adipocytes. Methods: 3T3-L1 preadipocytes invalidated or not for RIPK3 or MLKL were exposed differenciated into mature adipocytes and the mRNA profiles of wild type (WT), RIPK3-/- knockout (RIPK3-KO) or MLKL-/- knockout 3T3-L1 cells control (J0) or differenciated into mature adipocytes (J7) were generated by deep sequencing, in 3 copies, using Illumina NOVAseq 6000 plateform. Differential expression analysis between two conditions/groups (five biological replicates per condition) was performed using DESeq2 R package. Genes with an adjusted P value < 0.05 found by DESeq2 were assigned as differentially expressed. qRT–PCR validation was performed using SYBR Green assays Results: The DEGs were clustered using a hierarchical clustering algorithm, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis unveiled a clear reduction in the expression of genes involved in the early or late stages of adipogenesis in MLKL-KO cells Conclusions: Taken together, these data suggest that Mlkl but not Ripk3 deficiency impaired adipogenesis of 3T3-L1 cells by reducing the expression of pro-adipogenic factors and genes involved in fatty acid metabolism.
2022-10-10 | GSE201450 | GEO
Project description:Neuroinflammatory processes are a prominent contributor to the pathology of Parkinson’s disease (PD), characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and deposits of α-synuclein aggregates. MLKL-mediated cell necroptosis might occur in the onset of PD and lead to neuronal dopaminergic degeneration. However, the link between α-synuclein, neuroinflammatory processes, and neurodegeneration in PD remains unclear. Here, our in vitro study indicated that inhibition of MLKL exerted a protective effect against 6-OHDA- and TNF-α-induced neuronal cell death. Furthermore, we created a mouse model (Tg-Mlkl-/-) with typical progressive Parkinson traits by crossbreeding SNCA A53T transgenic mice with MLKL knockout mice. Tg-Mlkl-/ mice displayed dramatically improved motor symptoms and reduced hyperphosphorylated α-synuclein expression. More data suggested that MLKL deficiency protected dopaminergic neurons, blocked neuronal cell death, and attenuated neuroinflammation by inhibiting the activation of the microglia and astrocytes. Single-cell RNA-seq analysis revealed reduced microglial cells and damped neuron death in the SN of the Tg-Mlkl-/- mice. Subcluster analysis identified a unique cell type-specific transcriptome profiling in the MLKL deficiency mice. Thus, MLKL represents a critical therapeutic target for reducing neuroinflammation and preventing dopaminergic neuron degeneration.
2023-12-14 | GSE197679 | GEO
Project description:Caspase-8 is a protease with both pro-death and pro-survival functions: it is required for apoptosis induced by death receptors such as TNFR1 (tumor necrosis factor receptor 1) 1, and it has a critical role in suppressing necroptosis mediated by the kinase RIPK3 (receptor interacting protein kinase 3) and the pseudokinase MLKL (mixed lineage kinase-like) 2-4. Mice lacking caspase-8 display MLKL-dependent embryonic lethality 4, as do mice expressing catalytically inactive caspase-8 mutant C362A. However, Casp8C362A/C362A Mlkl-/- mice die in the perinatal period, whereas Casp8-/- Mlkl-/- mice are viable 4, indicating that inactive caspase-8 also has a pro-death scaffolding function. Here we show that inactive caspase-8 activates pyroptosis in MLKL-deficient intestinal epithelial cells around embryonic day 18, triggering the formation of ASC specks. Accordingly, intestinal atrophy and perinatal lethality in Casp8C362A/C362A Mlkl-/- mice was prevented by loss of caspase-1. In transfection studies, inactive caspase-8 mutants C362A or C362S were found in both the triton X-100 insoluble and soluble fractions, whereas wild-type caspase-8 existed only in the soluble fraction. Moreover, inactive caspase-8 shifted co-transfected ASC into the insoluble fraction, whereas wild-type caspase-8 did not. Thus, a defense mechanism is revealed that would allow intestinal epithelial cell death in the face of pathogens expressing virulence factors to inhibit caspase-8-dependent apoptosis and necroptosis.
2019-09-20 | GSE125625 | GEO
Project description:Osteolysis is a serious postoperative complication of total joint arthroplasty that leads to aseptic loosening and surgical revision. Osteolysis is a chronic destructive process occurs when host macrophages recognize the implant particles and release inflammatory mediators that increase bone-resorbing osteoclastic activity and attenuate bone-formation osteoblastic activity. Although much progress has been made on understanding molecular responses of macrophage to implant particles, pathways/signals initiating osteolysis remain poorly characterized. Transcriptomics and gene-expression profiling of these macrophages may unravel key mechanism in pathogenesis of osteolysis and aid in identifying molecular candidates for therapeutic intervention. To this end, we analyzed the transcriptional profiling of macrophages exposed to UHMWPE particles of the most common components used in bearing materials of orthopedic implants. Regulated genes in stimulated macrophages were involved in cytokine, chemokine, growth factor and receptor activities. Gene enrichment analysis suggested that stimulated macrophages elicited common gene expression signatures for inflammation and rheumatoid arthritis. Among the regulated genes, TNFSF15 and CCL20 were further characterized as molecular targets involved pathogenesis of osteolysis. Treatment of monocyte cultures with TNFSF15 and CCL20 resulted in an increase in osteoclastogenesis and bone-resorbing osteoclastic activity, suggesting their potential contribution to loosening between implant and bone tissue.
2019-05-06 | GSE104589 | GEO
Project description:Wear debris-induced osteolysis, especially titanium (Ti) particles-induced osteolysis, is the most common cause of arthroplasty failure with no effective therapy. Previous studies have suggested that inflammation and impaired osteogenesis are associated with Ti particles -induced osteolysis. Selenium (Se) is an essential trace element in the human body, which forms selenomethionine (Se-Met) in nature, and selenoproteins has strong anti-inflammatory and antioxidant stress effects. In this study, the effects of Se-Met on Ti particles-induced osteolysis were observed and the potential mechanism was explored. We found that exogenous Se-Met relieved osteolysis induced by Ti particles in two animal models and MC3T3-E1 cells. We found that the addition of Se-Met effectively inhibited Ti particle-induced inflammation by regulating ROS-dependent NLRP3 inflammasome activation. These therapeutic effects were abrogated in MC3T3-E1 cells that had received a β-catenin antagonist, suggesting that Se-Met alleviates inflammatory osteolysis via the β-catenin signaling pathway. Collectively, these findings indicated that Se-Met may serve as a potential therapeutic agent for treating Ti particle-induced osteolysis.
2023-08-09 | GSE236911 | GEO
Project description:Chronic apical periodontitis (CAP) is a unique dynamic interaction between microbial invasions and host defense mechanisms, resulting in bone absorption, infiltration of immune cells and sporadic periapical granuloma. In this study, we constituted a single-cell atlas for 26,737 high-quality cells from hyperplastic periapical tissue using single-cell RNA sequencing. Identifying cell types and signatures at the single-cell level might generate novel insights into the clinical pathogenesis of CAP. A histological analysis to verify the gene signatures of nonimmune cells was combined with immunohistochemistry staining. We then discovered the diversity and heterogeneity of nonimmune cells in regional CAP lesions. The temporal profiling of genomic alterations from common CAP to typical periapical granuloma provided predictions for key transcription factors and biological processes. Our study also inferred that the marked shift of inflammatory cytokines, chemokines, proteases and growth factors enables the initiation of polymorphic cell differentiation, lymphangiogenesis and angiogenesis during CAP.
2021-08-31 | GSE181688 | GEO