Impaired Plakophilin-2 in obesity breaks cell cycle dynamics to breed adipocyte senescence.
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ABSTRACT:
SUBMITTER: Lluch A
PROVIDER: S-EPMC10444784 | biostudies-literature | 2023 Aug
REPOSITORIES: biostudies-literature
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Impaired Plakophilin-2 in obesity breaks cell cycle dynamics to breed adipocyte senescence.
Lluch Aina A Latorre Jessica J Serena-Maione Angela A Espadas Isabel I Caballano-Infantes Estefanía E Moreno-Navarrete José M JM Oliveras-Cañellas Núria N Ricart Wifredo W Malagón María M MM Martin-Montalvo Alejandro A Birchmeier Walter W Szymanski Witold W Graumann Johannes J Gómez-Serrano María M Sommariva Elena E Fernández-Real José M JM Ortega Francisco J FJ
Nature communications 20230822 1
Plakophilin-2 (PKP2) is a key component of desmosomes, which, when defective, is known to promote the fibro-fatty infiltration of heart muscle. Less attention has been given to its role in adipose tissue. We report here that levels of PKP2 steadily increase during fat cell differentiation, and are compromised if adipocytes are exposed to a pro-inflammatory milieu. Accordingly, expression of PKP2 in subcutaneous adipose tissue diminishes in patients with obesity, and normalizes upon mild-to-inten ...[more]
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Project description:Plakophilin2 (PKP2) is a key component of desmosomes mostly recognized for its involvement in the fibro fatty infiltration of heart muscle under defective PKP2. While thoroughly explored in cardiomyocytes, less attention has been given to its role in fat cells. Here we report steadily increased PKP2 during adipogenesis, with expression levels reaching its apex in terminally differentiated adipocytes. Notably, the loss of this protein in adipocytes under a pro-inflammatory microenvironment demonstrates its commitment in maintaining the expression of genes required to nurture cell cycle. Then, we show that diminished expressions of this member of the armadillo repeat family in subcutaneous adipose tissue co-segregate with obesity, being normalized upon mild to intense weight loss. We also demonstrate that impaired PKP2 in human adipocytes breaks cell cycle dynamics to breed premature senescence, a key rheostat for stress induced adipose tissue dysfunction. Conversely, restoring PKP2 in inflamed adipocytes rewires E2F signalling towards re-activation of cell cycle and decreased senescence. Our findings bound the expression of PKP2 in fat cells to the physiopathology of obesity, and uncover a previously unknown defect in cell cycle and activated adipocyte senescence due to impaired PKP2.
2023-05-10 | MSV000091898 | MassIVE
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Project description:BackgroundLong non-coding RNAs (lncRNAs) can perform tasks of key relevance in fat cells, contributing, when defective, to the burden of obesity and its sequelae. Here, scrutiny of adipose tissue transcriptomes before and after bariatric surgery (GSE53378) granted identification of 496 lncRNAs linked to the obese phenotype. Only expression of linc-GALNTL6-4 displayed an average recovery over 2-fold and FDR-adjusted p-value <0.0001 after weight loss. The aim of the present study was to investigate the impact on adipocyte function and potential clinical value of impaired adipose linc-GALNTL6-4 in obese subjects.MethodsWe employed transcriptomic analysis of public dataset GSE199063, and cross validations in two large transversal cohorts to report evidence of a previously unknown association of adipose linc-GALNTL6-4 with obesity. We then performed functional analyses in human adipocyte cultures, genome-wide transcriptomics, and untargeted lipidomics in cell models of loss and gain of function to explore the molecular implications of its associations with obesity and weight loss.ResultsThe expression of linc-GALNTL6-4 in human adipose tissue is adipocyte-specific and co-segregates with obesity, being normalized upon weight loss. This co-segregation is demonstrated in two longitudinal weight loss studies and two cross-sectional samples. While compromised expression of linc-GALNTL6-4 in obese subjects is primarily due to the inflammatory component in the context of obesity, adipogenesis requires the transcriptional upregulation of linc-GALNTL6-4, the expression of which reaches an apex in terminally differentiated adipocytes. Functionally, we demonstrated that the knockdown of linc-GALNTL6-4 impairs adipogenesis, induces alterations in the lipidome, and leads to the downregulation of genes related to cell cycle, while propelling in adipocytes inflammation, impaired fatty acid metabolism, and altered gene expression patterns, including that of apolipoprotein C1 (APOC1). Conversely, the genetic gain of linc-GALNTL6-4 ameliorated differentiation and adipocyte phenotype, putatively by constraining APOC1, also contributing to the metabolism of triglycerides in adipose.ConclusionsCurrent data unveil the unforeseen connection of adipocyte-specific linc-GALNTL6-4 as a modulator of lipid homeostasis challenged by excessive body weight and meta-inflammation.
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Project description:DNA double strand breaks (DSBs) elicit prompt activation of DNA damage response (DDR), which arrests cell-cycle either in G1/S or G2/M in order to avoid entering S and M phase with damaged DNAs. Since mammalian tissues contain both proliferating and quiescent cells, there might be fundamental difference in DDR between proliferating and quiescent cells (or G0-arrested). To investigate these differences, we studied recruitment of DSB repair factors and resolution of DNA lesions induced at site-specific DSBs in asynchronously proliferating, G0-, or G1-arrested cells. Strikingly, DSBs occurring in G0 quiescent cells are not repaired and maintain a sustained activation of the p53-pathway. Conversely, re-entry into cell cycle of damaged G0-arrested cells, occurs with a delayed clearance of DNA repair factors initially recruited to DSBs, indicating an inefficient repair when compared to DSBs induced in asynchronously proliferating or G1-synchronized cells. Moreover, we found that initial recognition of DSBs and assembly of DSB factors is largely similar in asynchronously proliferating, G0-, or G1-synchronized cells. Our study thereby demonstrates that repair and resolution of DSBs is strongly dependent on the cell-cycle state.
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Project description:Background and objectivesPlakophilin-2 (PKP2) is an intracellular desmosomal anchoring protein that has been implicated in a genome-wide association study, in which genetic variants of PKP2 are associated with Porphyromonas gingivalis (P.gingivalis) -dominant periodontal dysbiosis. In this study, we compared the ex vivo PKP2 expression in periodontitis gingival biopsies to periodontitis-free subjects and assessed the in vitro role of PKP2 in gingival epithelial barrier function and the mechanism by which P.gingivalis modulates PKP2 expression.Material and methodsUsing reverse transcription quantitative real-time PCR (RT-qPCR), we determined PKP2 mRNA expression levels in gingival biopsies collected from 11 periodontally healthy, 10 experimental gingivitis, and 10 chronic periodontitis subjects. PKP2 protein expression in gingival biopsies was detected by immunohistochemistry. We then challenged primary gingival epithelial cells with bacteria including P.gingivalis, Campylobacter rectus, and various Toll-like receptor agonists. Western blot and immunofluorescence staining were used to detect protein expression. Inhibitors blocking proteases pathways were tested for P.gingivalis-mediated PKP2 protein degradations. We also knocked down endogenous epithelial PKP2 using lentiviral short-hairpin RNA (shRNA) and evaluated cell proliferation, spreading, and barrier function.ResultsPeriodontitis gingival biopsies had approximately twofold less PKP2 mRNA than did healthy controls (p < .05). PKP2 protein was predominantly expressed in gingival epithelium. In primary gingival epithelial cells, P.gingivalis challenge increased PKP2 mRNA levels, while protein expression decreased, which suggests that P.gingivalis has a protein degradation mechanism. Cysteine proteases inhibitors greatly attenuated P.gingivalis-mediated PKP2 protein degradation. Epithelial cells with deficient PKP2 exhibited inhibited cell proliferation and spreading and failed to form monolayers. Finally, P.gingivalis impaired gingival epithelial barrier function.ConclusionsPKP2 appears to be critical in maintaining gingival epithelial barrier function and is susceptible to degradation by cysteine proteases produced by P.gingivalis. Our findings have identified a mechanism by which P.gingivalis impairs epithelial barrier function by promoting PKP2 degradation.
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Project description:Plakophilin 2 (PKP2), a desmosome component, modulates the activity and localization of the small GTPase RhoA at sites of cell-cell contact. PKP2 regulates cortical actin rearrangement during junction formation, and its loss is accompanied by an increase in actin stress fibers. We hypothesized that PKP2 may regulate focal adhesion dynamics and cell migration. Here we show that PKP2-deficient cells bind efficiently to the extracellular matrix, but upon spreading display total cell areas ≈ 30% smaller than control cells. Focal adhesions in PKP2-deficient cells are ≈ 2 × larger and more stable than in control cells, and vinculin displays an increased time for fluorescence recovery after photobleaching. Furthermore, β4 and β1 integrin protein and mRNA expression is elevated in PKP2-silenced cells. Normal focal adhesion phenotypes can be restored in PKP2-null cells by dampening the RhoA pathway or silencing β1 integrin. However, integrin expression levels are not restored by RhoA signaling inhibition. These data uncover a potential role for PKP2 upstream of β1 integrin and RhoA in integrating cell-cell and cell-substrate contact signaling in basal keratinocytes necessary for the morphogenesis, homeostasis, and reepithelialization of the stratified epidermis.
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Project description:Aims: Peroxisome is a critical organelle for fatty acid oxidation (FAO) and metabolism of reactive oxygen species (ROS). Increased oxidative stress in adipose tissue contributes to the development of insulin resistance and metabolic syndrome in obesity. This study aimed to investigate the role of peroxisomal fitness in maintaining adipocyte function, which has been under-rated in the obesity research area. Results: Reduced peroxisomal gene expressions in white adipose tissue (WAT) of obese mice suggested a close correlation between peroxisomes and obesity. Peroxisomal biogenesis factor 5 siRNA increased cellular ROS and inflammatory mediators in 3T3-L1 adipocytes. On the contrary, hydrogen peroxide or tumor necrosis factor-α treatment significantly decreased biogenesis- and function-related peroxisomal proteins, suggesting a positive feedback loop of ROS/inflammation and peroxisomal dysfunction. Correspondingly, catalase (a major peroxisomal antioxidant)-knockout mice fed with high-fat diet (HFD) exhibited suppressed peroxisomal proteins along with increased oxidative stress and accelerated obesity. In response to fenofibrate (a peroxisomal proliferator) treatment, WAT of HFD-fed wild-type mice showed not only increases in peroxisomal biogenesis and FAO but also attenuated features of adipocyte dysfunction and obesity. However, these results were not observed in peroxisome proliferator-activated receptor-alpha null obese mice. Innovation: Impaired peroxisomal fitness enhanced oxidative stress and inflammation in adipocytes, which exacerbates obesity. Conclusion: Adipose tissue peroxisomal homeostasis plays an important role in attenuating the features of obesity, and it can be a potential therapeutic target of obesity.
| S-EPMC6859694 | biostudies-literature
Project description:Loss-of-function mutations in PKP2, which encodes plakophilin-2, cause arrhythmogenic cardiomyopathy (AC). Restoration of deficient molecules can serve as upstream therapy, thereby requiring a human model that recapitulates disease pathology and provides distinct readouts in phenotypic analysis for proof of concept for gene replacement therapy. Here, we generated isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with precisely adjusted expression of plakophilin-2 from a patient with AC carrying a heterozygous frameshift PKP2 mutation. After monolayer differentiation, plakophilin-2 deficiency led to reduced contractility, disrupted intercalated disc structures, and impaired desmosome assembly in iPSC-CMs. Allele-specific fluorescent labeling of endogenous DSG2 encoding desmoglein-2 in the generated isogenic lines enabled real-time desmosome-imaging under an adjusted dose of plakophilin-2. Adeno-associated virus-mediated gene replacement of PKP2 recovered contractility and restored desmosome assembly, which was sequentially captured by desmosome-imaging in plakophilin-2-deficient iPSC-CMs. Our isogenic set of iPSC-CMs recapitulates AC pathology and provides a rapid and convenient cellular platform for therapeutic development.
| S-EPMC8828557 | biostudies-literature