Homogentisic acid metabolism inhibits papillary thyroid carcinoma proliferation through ROS and p21-induced cell cycle arrest.

Thyroid cancer is one of the most common primary endocrine malignancies worldwide, and papillary thyroid carcinoma (PTC) is the predominant histological type observed therein. Although PTC has been studied extensively, our understanding of the altered metabolism and metabolic profile of PTC tumors is limited. We identified that the content of metabolite homogentisic acid (HGA) in PTC tissues was lower than that in adjacent non-cancerous tissues. We evaluated the potential of HGA as a novel molecular marker in the diagnosis of PTC tumors, as well as its ability to indicate the degree of malignancy. Studies have further shown that HGA contributes to reactive oxygen species (ROS) associated oxidative stress, leading to toxicity and inhibition of proliferation. In addition, HGA caused an increase in p21 expression levels in PTC cells and induced G1 arrest. Moreover, we found that the low HGA content in PTC tumors was due to the low expression levels of tyrosine aminotransferase (TAT) and p-hydroxyphenylpyruvate hydroxylase (HPD), which catalyze the conversion of tyrosine to HGA. The low expression levels of TAT and HPD are strongly associated with a higher probability of PTC tumor invasion and metastasis. Our study demonstrates that HGA could be used to diagnose PTC and provides mechanisms linking altered HGA levels to the biological behavior of PTC tumors.

Physiological aging and inflammation-induced cellular senescence may contribute to oligodendroglial dysfunction in MS.

Aging affects all cell types in the CNS and plays an important role in CNS diseases. However, the underlying molecular mechanisms driving these age-associated changes and their contribution to diseases are only poorly understood. The white matter in the aging brain as well as in diseases, such as Multiple sclerosis is characterized by subtle abnormalities in myelin sheaths and paranodes, suggesting that oligodendrocytes, the myelin-maintaining cells of the CNS, lose the capacity to preserve a proper myelin structure and potentially function in age and certain diseases. Here, we made use of directly converted oligodendrocytes (dchiOL) from young, adult and old human donors to study age-associated changes. dchiOL from all three age groups differentiated in an comparable manner into O4 + immature oligodendrocytes, but the proportion of MBP + mature dchiOL decreased with increasing donor age. This was associated with an increased ROS production and upregulation of cellular senescence markers such as CDKN1A, CDKN2A in old dchiOL. Comparison of the transcriptomic profiles of dchiOL from adult and old donors revealed 1324 differentially regulated genes with limited overlap with transcriptomic profiles of the donors' fibroblasts or published data sets from directly converted human neurons or primary rodent oligodendroglial lineage cells. Methylome analyses of dchiOL and human white matter tissue samples demonstrate that chronological and epigenetic age correlate in CNS white matter as well as in dchiOL and resulted in the identification of an age-specific epigenetic signature. Furthermore, we observed an accelerated epigenetic aging of the myelinated, normal appearing white matter of multiple sclerosis (MS) patients compared to healthy individuals. Impaired differentiation and upregulation of cellular senescence markers could be induced in young dchiOL in vitro using supernatants from pro-inflammatory microglia. In summary, our data suggest that physiological aging as well as inflammation-induced cellular senescence contribute to oligodendroglial pathology in inflammatory demyelinating diseases such as MS.

Transfected SARS-CoV-2 spike DNA for mammalian cell expression inhibits p53 activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2 proteins in cancer cells and increases cancer cell viability after chemotherapy exposure.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 infection has led to worsened outcomes for patients with cancer. SARS-CoV-2 spike protein mediates host cell infection and cell-cell fusion that causes stabilization of tumor suppressor p53 protein. In-silico analysis previously suggested that SARS-CoV-2 spike interacts with p53 directly but this putative interaction has not been demonstrated in cells. We examined the interaction between SARS-CoV-2 spike, p53 and MDM2 (E3 ligase, which mediates p53 degradation) in cancer cells using an immunoprecipitation assay. We observed that SARS-CoV-2 spike protein interrupts p53-MDM2 protein interaction but did not detect SARS-CoV-2 spike bound with p53 protein in the cancer cells. We further observed that SARS-CoV-2 spike suppresses p53 transcriptional activity in cancer cells including after nutlin exposure of wild-type p53-, spike-expressing tumor cells and inhibits chemotherapy-induced p53 gene activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2. The suppressive effect of SARS-CoV-2 spike on p53-dependent gene activation provides a potential molecular mechanism by which SARS-CoV-2 infection may impact tumorigenesis, tumor progression and chemotherapy sensitivity. In fact, cisplatin-treated tumor cells expressing spike were found to have increased cell viability as compared to control cells. Further observations on γ-H2AX expression in spike-expressing cells treated with cisplatin may indicate altered DNA damage sensing in the DNA damage response pathway. The preliminary observations reported here warrant further studies to unravel the impact of SARS-CoV-2 and its various encoded proteins including spike on pathways of tumorigenesis and response to cancer therapeutics. More efforts should be directed at studying the effects of the SARS-CoV-2 spike and other viral proteins on host DNA damage sensing, response and repair mechanisms. A goal would be to understand the structural basis for maximal anti-viral immunity while minimizing suppression of host defenses including the p53 DNA damage response and tumor suppression pathway. Such directions are relevant and important including not only in the context of viral infection and mRNA vaccines in general but also for patients with cancer who may be receiving cytotoxic or other cancer treatments.

Exploring CDKN1A Upregulation Mechanisms: Insights into Cell Cycle Arrest Induced by NC2603 Curcumin Analog in MCF-7 Breast Cancer Cells.

Breast cancer stands out as one of the most prevalent malignancies worldwide, necessitating a nuanced understanding of its molecular underpinnings for effective treatment. Hormone receptors in breast cancer cells substantially influence treatment strategies, dictating therapeutic approaches in clinical settings, serving as a guide for drug development, and aiming to enhance treatment specificity and efficacy. Natural compounds, such as curcumin, offer a diverse array of chemical structures with promising therapeutic potential. Despite curcumin's benefits, challenges like poor solubility and rapid metabolism have spurred the exploration of analogs. Here, we evaluated the efficacy of the curcumin analog NC2603 to induce cell cycle arrest in MCF-7 breast cancer cells and explored its molecular mechanisms. Our findings reveal potent inhibition of cell viability (IC50 = 5.6 µM) and greater specificity than doxorubicin toward MCF-7 vs. non-cancer HaCaT cells. Transcriptome analysis identified 12,055 modulated genes, most notably upregulation of GADD45A and downregulation of ESR1, implicating CDKN1A-mediated regulation of proliferation and cell cycle genes. We hypothesize that the curcumin analog by inducing GADD45A expression and repressing ESR1, triggers the expression of CDKN1A, which in turn downregulates the expression of many important genes of proliferation and the cell cycle. These insights advance our understanding of curcumin analogs' therapeutic potential, highlighting not just their role in treatment, but also the molecular pathways involved in their activity toward breast cancer cells.

Absence of E2f1 Negates Pro-osteogenic Impacts of p21 Absence.

Loss of p21 leads to increased bone formation post-injury; however, the mechanism(s) by which this occurs remains undetermined. E2f1 is downstream of p21 and as a transcription factor can act directly on gene expression; yet it is unknown if E2f1 plays a role in the osteogenic effects observed when p21 is differentially regulated. In this study we aimed to investigate the interplay between p21 and E2f1 and determine if the pro-regenerative osteogenic effects observed with the loss of p21 are E2f1 dependent. To accomplish this, we employed knockout p21 and E2f1 mice and additionally generated a p21/E2f1 double knockout. These mice underwent burr-hole injuries to their proximal tibiae and healing was assessed over 7 days via microCT imaging. We found that p21 and E2f1 play distinct roles in bone regeneration where the loss of p21 increased trabecular bone formation and loss of E2f1 increased cortical bone formation, yet loss of E2f1 led to poorer bone repair overall. Furthermore, when E2f1 was absent, either individually or simultaneously with p21, there was a dramatic decrease of the number of osteoblasts, osteoclasts, and chondrocytes at the site of injury compared to p21-/- and C57BL/6 mice. Together, these results suggest that E2f1 regulates the cell populations required for bone repair and has a distinct role in bone formation/repair compared to p21-/-E2f1-/-. These results highlight the possibility of cell cycle and/or p21/E2f1 being potential druggable targets that could be leveraged in clinical therapies to improve bone healing in pathologies such as osteoporosis.

Senescent characteristics of human corneal endothelial cells upon ultraviolet-A exposure.

PURPOSE: The objective of this study was to investigate the senescent phenotypes of human corneal endothelial cells (hCEnCs) upon treatment with ultraviolet (UV)-A. METHODS: We assessed cell morphology, senescence-associated ß-galactosidase (SA-ß-gal) activity, cell proliferation and expression of senescence markers (p16 and p21) in hCEnCs exposed to UV-A radiation, and senescent hCEnCs induced by ionizing radiation (IR) were used as positive controls. We performed RNA sequencing and proteomics analyses to compare gene and protein expression profiles between UV-A- and IR-induced senescent hCEnCs, and we also compared the results to non-senescent hCEnCs. RESULTS: Cells exposed to 5 J/cm2 of UV-A or to IR exhibited typical senescent phenotypes, including enlargement, increased SA-ß-gal activity, decreased cell proliferation and elevated expression of p16 and p21. RNA-Seq analysis revealed that 83.9% of the genes significantly upregulated and 82.6% of the genes significantly downregulated in UV-A-induced senescent hCEnCs overlapped with the genes regulated in IR-induced senescent hCEnCs. Proteomics also revealed that 93.8% of the proteins significantly upregulated in UV-A-induced senescent hCEnCs overlapped with those induced by IR. In proteomics analyses, senescent hCEnCs induced by UV-A exhibited elevated expression levels of several factors part of the senescence-associated secretory phenotype. CONCLUSIONS: In this study, where senescence was induced by UV-A, a more physiological stress for hCEnCs compared to IR, we determined that UV-A modulated the expression of many genes and proteins typically altered upon IR treatment, a more conventional method of senescence induction, even though UV-A also modulated specific pathways unrelated to IR.

C16, a PKR inhibitor, suppresses cell proliferation by regulating the cell cycle via p21 in colorectal cancer.

Double-stranded RNA-activated protein kinase R (PKR) is highly expressed in colorectal cancer (CRC). However, the role of PKR in CRC remains unclear. The aim of this study was to clarify whether C16 (a PKR inhibitor) exhibits antitumor effects and to identify its target pathway in CRC. We evaluated the effects of C16 on CRC cell lines using the MTS assay. Enrichment analysis was performed to identify the target pathway of C16. The cell cycle was analyzed using flow cytometry. Finally, we used immunohistochemistry to examine human CRC specimens. C16 suppressed the proliferation of CRC cells. Gene Ontology (GO) analysis revealed that the cell cycle-related GO category was substantially enriched in CRC cells treated with C16. C16 treatment resulted in G1 arrest and increased p21 protein and mRNA expression. Moreover, p21 expression was associated with CRC development as observed using immunohistochemical analysis of human CRC tissues. C16 upregulates p21 expression in CRC cells to regulate cell cycle and suppress tumor growth. Thus, PKR inhibitors may serve as a new treatment option for patients with CRC.

XPO1 blockade with KPT-330 promotes apoptosis in cutaneous T-cell lymphoma by activating the p53-p21 and p27 pathways.

Dysregulated nuclear-cytoplasmic trafficking has been shown to play a role in oncogenesis in several types of solid tumors and hematological malignancies. Exportin 1 (XPO1) is responsible for the nuclear export of several proteins and RNA species, mainly tumor suppressors. KPT-330, a small molecule inhibitor of XPO1, is approved for treating relapsed multiple myeloma and diffuse large B-cell lymphoma. Cutaneous T-cell lymphoma (CTCL) is an extranodal non-Hodgkin lymphoma with an adverse prognosis and limited treatment options in advanced stages. The effect of therapeutically targeting XPO1 with KPT-330 in CTCL has not been established. We report that XPO1 expression is upregulated in CTCL cells. KPT-330 reduces cell proliferation, induces G1 cell cycle arrest and apoptosis. RNA-sequencing was used to explore the underlying mechanisms. Genes associated with the cell cycle and the p53 pathway were significantly enriched with KPT-330 treatment. KPT-330 suppressed XPO1 expression, upregulated p53, p21WAF1/Cip1, and p27Kip1 and their nuclear localization, and downregulated anti-apoptotic protein (Survivin). The in vivo efficacy of KPT-330 was investigated using a bioluminescent xenograft mouse model of CTCL. KPT-330 blocked tumor growth and prolonged survival (p < 0.0002) compared to controls. These findings support investigating the use of KPT-330 and next-generation XPO1 inhibitors in CTCL.

Mcph1, mutated in primary microcephaly, is also crucial for erythropoiesis.

Microcephaly is a common feature in inherited bone marrow failure syndromes, prompting investigations into shared pathways between neurogenesis and hematopoiesis. To understand this association, we studied the role of the microcephaly gene Mcph1 in hematological development. Our research revealed that Mcph1-knockout mice exhibited congenital macrocytic anemia due to impaired terminal erythroid differentiation during fetal development. Anemia's cause is a failure to complete cell division, evident from tetraploid erythroid progenitors with DNA content exceeding 4n. Gene expression profiling demonstrated activation of the p53 pathway in Mcph1-deficient erythroid precursors, leading to overexpression of Cdkn1a/p21, a major mediator of p53-dependent cell cycle arrest. Surprisingly, fetal brain analysis revealed hypertrophied binucleated neuroprogenitors overexpressing p21 in Mcph1-knockout mice, indicating a shared pathophysiological mechanism underlying both erythroid and neurological defects. However, inactivating p53 in Mcph1-/- mice failed to reverse anemia and microcephaly, suggesting that p53 activation in Mcph1-deficient cells resulted from their proliferation defect rather than causing it. These findings shed new light on Mcph1's function in fetal hematopoietic development, emphasizing the impact of disrupted cell division on neurogenesis and erythropoiesis - a common limiting pathway.

miRNA-142-3p aggravates hydrogen peroxide-induced human umbilical vein endothelial cell premature senescence by targeting SIRT1.

Vascular endothelial cell premature senescence plays an important part in stroke. Many microRNAs (miRNAs) are known to be involved in the pathological process of vascular endothelial cell premature senescence. The present study aimed to investigate the mechanism of hydrogen peroxide (H2O2)-induced premature senescence in human umbilical vein endothelial cells (HUVECs) and effect of miR-142-3p on hydrogen peroxide (H2O2)-induced premature senescence. HUVECs were exposed to H2O2 to establish a model premature senescence in endothelial cells. CCK-8 assay was performed to detect cell viability. Senescence-associated ß-galactosidase staining assay and senescence-related proteins p16 and p21 were used to detect changes in the degree of cell senescence. RT-qPCR and Western blot were conducted to measure mRNA and protein levels, respectively. The scratch wound-healing assay, transwell assay, and EdU assay were performed to evaluate the ability of migration and proliferation, respectively. miRNA-142-3p and silencing information regulator 2 related enzyme 1 (SIRT1) binding was verified using Targetscan software and a dual-luciferase assay. We found that miRNA-142-3p is abnormally up-regulated in HUVECs treated with H2O2. Functionally, miRNA-142-3p inhibition may mitigate the degree of HUVEC senescence and improve HUVEC migration and proliferation. Mechanistically, SIRT1 was validated to be targeted by miRNA-142-3p in HUVECs. Moreover, SIRT1 inhibition reversed the effects of miRNA-142-3p inhibition on senescent HUVECs exposed to H2O2. To our knowledge, this is the first study to show that miRNA-142-3p ameliorates H2O2-induced HUVECs premature senescence by targeting SIRT1 and may shed light on the role of the miR-142-3p/SIRT1 axis in stroke treatment.

Low-Level Expression of p-S6 Is Associated with Nodal Metastasis in Patients with Head and Neck Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer. The incidence of metastasis for cSCC is estimated to be around 1.2-5%. Ribosomal protein S6 (p-S6) and the p21 protein (p21) are two proteins that play central roles in other cancers. These proteins may be equally important in cSCC, and together, these could constitute a good candidate for metastasis risk assessment of these patients. We investigate the relationship of p-S6 and p21 expression with the impact on the prognosis of head and neck cSCC (cSCCHN). p-S6 and p21 expression was analyzed by immunohistochemistry on paraffin-embedded tissue samples from 116 patients with cSCCHN and associations sought with clinical characteristics. Kaplan-Meier estimators and Cox proportional hazard regression models were also used. The expression of p-S6 was significantly inversely associated with tumor thickness, tumor size, desmoplastic growth, pathological stage, perineural invasion and tumor buds. p21 expression was significantly inversely correlated with >6 mm tumor thickness, desmoplastic growth, and perineural invasion. p-S6-negative expression significantly predicted an increased risk of nodal metastasis (HR = 2.63, 95% CI 1.51-4.54; p < 0.001). p21 expression was not found to be a significant risk factor for nodal metastasis. These findings demonstrate that p-S6-negative expression is an independent predictor of nodal metastasis. The immunohistochemical expression of p-S6 might aid in better risk stratification and management of patients with cSCCHN.

The ISG15-Protease USP18 Is a Pleiotropic Enhancer of HIV-1 Replication.

The innate immune response to viruses is formed in part by interferon (IFN)-induced restriction factors, including ISG15, p21, and SAMHD1. IFN production can be blocked by the ISG15-specific protease USP18. HIV-1 has evolved to circumvent host immune surveillance. This mechanism might involve USP18. In our recent studies, we demonstrate that HIV-1 infection induces USP18, which dramatically enhances HIV-1 replication by abrogating the antiviral function of p21. USP18 downregulates p21 by accumulating misfolded dominant negative p53, which inactivates wild-type p53 transactivation, leading to the upregulation of key enzymes involved in de novo dNTP biosynthesis pathways and inactivated SAMHD1. Despite the USP18-mediated increase in HIV-1 DNA in infected cells, it is intriguing to note that the cGAS-STING-mediated sensing of the viral DNA is abrogated. Indeed, the expression of USP18 or knockout of ISG15 inhibits the sensing of HIV-1. We demonstrate that STING is ISGylated at residues K224, K236, K289, K347, K338, and K370. The inhibition of STING K289-linked ISGylation suppresses its oligomerization and IFN induction. We propose that human USP18 is a novel factor that potentially contributes in multiple ways to HIV-1 replication.

The effects of resistance training on denervated myofibers, senescent cells, and associated protein markers in middle-aged adults.

Denervated myofibers and senescent cells are hallmarks of skeletal muscle aging. However, sparse research has examined how resistance training affects these outcomes. We investigated the effects of unilateral leg extensor resistance training (2 days/week for 8 weeks) on denervated myofibers, senescent cells, and associated protein markers in apparently healthy middle-aged participants (MA, 55 ± 8 years old, 17 females, 9 males). We obtained dual-leg vastus lateralis (VL) muscle cross-sectional area (mCSA), VL biopsies, and strength assessments before and after training. Fiber cross-sectional area (fCSA), satellite cells (Pax7+), denervated myofibers (NCAM+), senescent cells (p16+ or p21+), proteins associated with denervation and senescence, and senescence-associated secretory phenotype (SASP) proteins were analyzed from biopsy specimens. Leg extensor peak torque increased after training (p < .001), while VL mCSA trended upward (interaction p = .082). No significant changes were observed for Type I/II fCSAs, NCAM+ myofibers, or senescent (p16+ or p21+) cells, albeit satellite cells increased after training (p = .037). While >90% satellite cells were not p16+ or p21+, most p16+ and p21+ cells were Pax7+ (>90% on average). Training altered 13 out of 46 proteins related to muscle-nerve communication (all upregulated, p < .05) and 10 out of 19 proteins related to cellular senescence (9 upregulated, p < .05). Only 1 out of 17 SASP protein increased with training (IGFBP-3, p = .031). In conclusion, resistance training upregulates proteins associated with muscle-nerve communication in MA participants but does not alter NCAM+ myofibers. Moreover, while training increased senescence-related proteins, this coincided with an increase in satellite cells but not alterations in senescent cell content or SASP proteins. These latter findings suggest shorter term resistance training is an unlikely inducer of cellular senescence in apparently healthy middle-aged participants. However, similar study designs are needed in older and diseased populations before definitive conclusions can be drawn.

DEC1 is involved in circadian rhythm disruption-exacerbated pulmonary fibrosis.

BACKGROUND: The alveolar epithelial type II cell (AT2) and its senescence play a pivotal role in alveolar damage and pulmonary fibrosis. Cell circadian rhythm is strongly associated with cell senescence. Differentiated embryonic chondrocyte expressed gene 1 (DEC1) is a very important circadian clock gene. However, the role of DEC1 in AT2 senescence and pulmonary fibrosis was still unclear. RESULTS: In this study, a circadian disruption model of light intervention was used. It was found that circadian disruption exacerbated pulmonary fibrosis in mice. To understand the underlying mechanism, DEC1 levels were investigated. Results showed that DEC1 levels increased in lung tissues of IPF patients and in bleomycin-induced mouse fibrotic lungs. In vitro study revealed that bleomycin and TGF-ß1 increased the expressions of DEC1, collagen-I, and fibronectin in AT2 cells. Inhibition of DEC1 mitigated bleomycin-induced fibrotic changes in vitro and in vivo. After that, cell senescence was observed in bleomycin-treated AT2 cells and mouse models, but these were prevented by DEC1 inhibition. At last, p21 was confirmed having circadian rhythm followed DEC1 in normal conditions. But bleomycin disrupted the circadian rhythm and increased DEC1 which promoted p21 expression, increased p21 mediated AT2 senescence and pulmonary fibrosis. CONCLUSIONS: Taken together, circadian clock protein DEC1 mediated pulmonary fibrosis via p21 and cell senescence in alveolar epithelial type II cells.

Diphthamide deficiency promotes association of eEF2 with p53 to induce p21 expression and neural crest defects.

Diphthamide is a modified histidine residue unique for eukaryotic translation elongation factor 2 (eEF2), a key ribosomal protein. Loss of this evolutionarily conserved modification causes developmental defects through unknown mechanisms. In a patient with compound heterozygous mutations in Diphthamide Biosynthesis 1 (DPH1) and impaired eEF2 diphthamide modification, we observe multiple defects in neural crest (NC)-derived tissues. Knockin mice harboring the patient's mutations and Xenopus embryos with Dph1 depleted also display NC defects, which can be attributed to reduced proliferation in the neuroepithelium. DPH1 depletion facilitates dissociation of eEF2 from ribosomes and association with p53 to promote transcription of the cell cycle inhibitor p21, resulting in inhibited proliferation. Knockout of one p21 allele rescues the NC phenotypes in the knockin mice carrying the patient's mutations. These findings uncover an unexpected role for eEF2 as a transcriptional coactivator for p53 to induce p21 expression and NC defects, which is regulated by diphthamide modification.

Spatiotemporal transcriptomic changes of human ovarian aging and the regulatory role of FOXP1.

Limited understanding exists regarding how aging impacts the cellular and molecular aspects of the human ovary. This study combines single-cell RNA sequencing and spatial transcriptomics to systematically characterize human ovarian aging. Spatiotemporal molecular signatures of the eight types of ovarian cells during aging are observed. An analysis of age-associated changes in gene expression reveals that DNA damage response may be a key biological pathway in oocyte aging. Three granulosa cells subtypes and five theca and stromal cells subtypes, as well as their spatiotemporal transcriptomics changes during aging, are identified. FOXP1 emerges as a regulator of ovarian aging, declining with age and inhibiting CDKN1A transcription. Silencing FOXP1 results in premature ovarian insufficiency in mice. These findings offer a comprehensive understanding of spatiotemporal variability in human ovarian aging, aiding the prioritization of potential diagnostic biomarkers and therapeutic strategies.

The p21+ perinecrotic hepatocytes produce the chemokine CXCL14 after a severe acetaminophen overdose promoting hepatocyte injury and delaying regeneration.

Fifty percent of all acute liver failure (ALF) cases in the United States are due to acetaminophen (APAP) overdose. Assessment of canonical features of liver injury, such as plasma alanine aminotransferase activities are poor predictors of acute liver failure (ALF), suggesting the involvement of additional mechanisms independent of hepatocyte death. Previous work demonstrated a severe overdose of APAP results in impaired regeneration, the induction of senescence by p21, and increased mortality. We hypothesized that a discrete population of p21+ hepatocytes acquired a secretory phenotype that directly impedes liver recovery after a severe APAP overdose. Leveraging in-house human APAP explant liver and publicly available single-nuclei RNAseq data, we identified a subpopulation of p21+ hepatocytes enriched in a unique secretome of factors, such as CXCL14. Spatial transcriptomics in the mouse model of APAP overdose confirmed the presence of a p21+ hepatocyte population that directly surrounded the necrotic areas. In both male and female mice, we found a dose-dependent induction of p21 and persistent circulating levels of the p21-specific constituent, CXCL14, in the plasma after a severe APAP overdose. In parallel experiments, we targeted either the putative senescent hepatocytes with the senolytic drugs, dasatinib and quercetin, or CXCL14 with a neutralizing antibody. We found that targeting CXCL14 greatly enhanced liver recovery after APAP-induced liver injury, while targeting senescent hepatocytes had no effect. These data support the conclusion that the sustained induction of p21 in hepatocytes with persistent CXCL14 secretion are critical mechanistic events leading to ALF in mice and human patients.

CDK-independent role of D-type cyclins in regulating DNA mismatch repair.

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.

SIRT6-mediated vascular smooth muscle cells senescence participates in the pathogenesis of abdominal aortic aneurysm.

BACKGROUND AND AIMS: In this study, we carried out a clinical sample study, and in vivo and in vitro studies to evaluate the effect of SIRT6 and SIRT6-mediated vascular smooth muscle senescence on the development of abdominal aortic aneurysm (AAA). METHOD AND RESULTS: AAA specimen showed an increased P16, P21 level and a decreased SIRT6 level compared with control aorta. Time curve study of Ang II infusion AAA model showed similar P16, P21 and SIRT6 changes at the early phase of AAA induction. The in vivo overexpression of SIRT6 significantly prevented AAA formation in Ang II infusion model. The expression of P16 and P21 was significantly reduced after SIRT6 overexpression. SIRT6 overexpression also attenuated chronic inflammation and neo-angiogenesis in Ang II infusion model. The overexpression of SIRT6 could attenuate premature senescence, inflammatory response and neo-angiogenesis in human aortic smooth muscle cells (HASMC) under Ang II stimulation. CONCLUSIONS: SIRT6 overexpression could limit AAA formation via attenuation of vascular smooth muscle senescence, chronic inflammation and neovascularity.

Multifaceted roles of Meg3 in cellular senescence and atherosclerosis.

BACKGROUND AND AIMS: Long noncoding RNAs are involved in the pathogenesis of atherosclerosis. As long noncoding RNAs maternally expressed gene 3 (Meg3) prevents cellular senescence of hepatic vascular endothelium and obesity-induced insulin resistance, we decided to examine its role in cellular senescence and atherosclerosis. METHODS AND RESULTS: By analyzing our data and human and mouse data from the Gene Expression Omnibus database, we found that Meg3 expression was reduced in humans and mice with cardiovascular disease, indicating its potential role in atherosclerosis. In Ldlr-/- mice fed a Western diet for 12 weeks, Meg3 silencing by chemically modified antisense oligonucleotides attenuated the formation of atherosclerotic lesions by 34.9% and 20.1% in male and female mice, respectively, revealed by en-face Oil Red O staining, which did not correlate with changes in plasma lipid profiles. Real-time quantitative PCR analysis of cellular senescence markers p21 and p16 revealed that Meg3 deficiency aggravates hepatic cellular senescence but not cellular senescence at aortic roots. Human Meg3 transgenic mice were generated to examine the role of Meg3 gain-of-function in the development of atherosclerosis induced by PCSK9 overexpression. Meg3 overexpression promotes atherosclerotic lesion formation by 29.2% in Meg3 knock-in mice independent of its effects on lipid profiles. Meg3 overexpression inhibits hepatic cellular senescence, while it promotes aortic cellular senescence likely by impairing mitochondrial function and delaying cell cycle progression. CONCLUSIONS: Our data demonstrate that Meg3 promotes the formation of atherosclerotic lesions independent of its effects on plasma lipid profiles. In addition, Meg3 regulates cellular senescence in a tissue-specific manner during atherosclerosis. Thus, we demonstrated that Meg3 has multifaceted roles in cellular senescence and atherosclerosis.