P21 Cip1 Expression Research Articles

Loss of myosin light chain kinase induces the cellular senescence associated secretory phenotype to promote breast epithelial cell migration

Overexpression or activation of oncogenes or loss of tumor-suppressor genes can induce cellular senescence as a defense mechanism against tumor development, thereby maintaining cellular homeostasis. However, cancer cells can circumvent this senescent state and continue to spread. Myosin light chain kinase (MLCK) is downregulated in many breast cancers. Here we report that downregulation of MLCK in normal breast epithelial cells induces a senescence-associated secretory phenotype and stimulates migration. The reduction of MLCK results in increased p21Cip1 expression, dependent on p53 and the AKT-mammalian target of rapamycin pathway. Subsequently, p21Cip1 promotes the secretion of soluble ICAM-1, IL-1α, IL-6 and IL-8, thereby enhancing collective cell migration in a non-cell-autonomous manner. These findings provide new mechanistic insights into the role of MLCK in cellular senescence and cancer progression.

Activation of ALDH2 alleviates hypoxic pulmonary hypertension in mice by upregulating the SIRT1/PGC-1α signaling pathway

To investigate whether activation of mitochondrial acetal dehydrogenase 2 (ALDH2) alleviates hypoxic pulmonary hypertension by regulating the SIRT1/PGC-1α signaling pathway. Thirty 8-week-old C57 BL/6 mice were randomized into control, hypoxia, and hypoxia +Alda-1 (an ALDH2 activator) group (n=10), and the mice in the latter two groups, along with 10 ALDH2 knockout (ALDH2-/-) mice, were exposed to hypoxia (10% O2, 90% N2) with or without daily intraperitoneal injection of Alda-1 for 4 weeks. The changes in right ventricular function and pressure (RVSP) of the mice were evaluated by echocardiography and right ventricular catheter test, and pulmonary artery pressure was estimated based on RVSP. Pulmonary vascular remodeling, right ventricular injury, myocardial α -SMA expression, distal pulmonary arteriole muscle normalization, right ventricular cross-sectional area, myocardial cell hypertrophy, and right cardiac hypertrophy index were assessed with HE staining, immunofluorescence staining and WGA staining, and the expressions of ALDH2, SIRT1, PGC-1α, P16INK4A and P21CIP1 were detected. In pulmonary artery smooth muscle cells with hypoxic exposure, the effect of Alda-1 and EX527 on cell senescence and protein expressions was evaluated using β-galactose staining and Western blotting. The wild-type mice with hypoxic exposure showed significantly increased RVSP, right ventricular free wall thickness and myocardial expressions of P16INK4A and P21CIP1, which were effectively lowered by treatment with Alda-1 but further increased in ALDH2-/- mice. In cultured pulmonary artery smooth muscle cells, hypoxic exposure significantly increased senescent cell percentage and cellular expressions of P16INK4A and P21CIP1, which were all lowered by treatment with Alda-1, but its effect was obviously attenuated by EX527 treatment. ALDH2 alleviates hypoxiainduced senescence of pulmonary artery smooth muscle cells by upregulating the SIRT1/PGC-1α signaling pathway to alleviate pulmonary hypertension in mice.

Lipid peroxidation products induce carbonyl stress, mitochondrial dysfunction, and cellular senescence in human and murine cells.

Lipid enals are electrophilic products of lipid peroxidation that induce genotoxic and proteotoxic stress by covalent modification of DNA and proteins, respectively. As lipid enals accumulate to substantial amounts in visceral adipose during obesity and aging, we hypothesized that biogenic lipid enals may represent an endogenously generated, and therefore physiologically relevant, senescence inducers. To that end, we identified that 4-hydroxynonenal (4-HNE), 4-hydroxyhexenal (4-HHE) or 4-oxo-2-nonenal (4-ONE) initiate the cellular senescence program of IMR90 fibroblasts and murine adipose stem cells. In such cells, lipid enals induced accumulation of γH2AX foci, increased p53 signaling, enhanced expression of p21Cip1, and upregulated the expression and secretion of numerous cytokines, chemokines, and regulatory factors independently from NF-κB activation. Concomitantly, lipid enal treatment resulted in covalent modification of mitochondrial proteins, reduced mitochondrial spare respiratory capacity, altered nucleotide pools, and increased the phosphorylation of AMP kinase. Lipid-induced senescent cells upregulated BCL2L1 (Bcl-xL) and BCL2L2 (Bcl-w). and were resistant to apoptosis while pharmacologic inhibition of BAX/BAK macropores attenuated lipid-induced senescence. Insitu, the 4-HNE scavenger L-carnosine ameliorated the development of the cellular senescence, while in visceral fat of obese C57BL/6J mice, L-carnosine reduced the abundance of 4-HNE-modified proteins and blunted the expression of senescence biomarkers CDKN1A (p21Cip1), PLAUR, BCL2L1, and BCL2L2. Taken together, the results suggest that lipid enals are endogenous regulators of cellular senescence and that biogenic lipid-induced senescence (BLIS) may represent a mechanistic link between oxidative stress and age-dependent pathologies.

MiR-96-5p expression is sufficient to induce and maintain the senescent cell fate in the absence of stress

Senescence is a cell fate driven by different types of stress that results in exit from the cell cycle and expression of an inflammatory senescence-associated secretory phenotype (SASP). Here, we demonstrate that stable overexpression of miR-96-5p was sufficient to induce cellular senescence in the absence of genotoxic stress, inducing expression of certain markers of early senescence including SASP factors while repressing markers of deep senescence including LINE-1 and type 1 interferons. Stable miR-96-5p overexpression led to genome-wide changes in heterochromatin followed by epigenetic activation of p16Ink4a, p21Cip1, and SASP expression, induction of a marker of DNA damage, and induction of a transcriptional signature similar to other senescent lung and endothelial cell types. Expression of miR-96-5p significantly increased following senescence induction in culture cells and with aging in tissues from naturally aged and Ercc1-/Δ progeroid mice. Mechanistically, miR-96-5p directly suppressed expression of SIN3B and SIN3 corepressor complex constituents KDM5A and MORF4L2, and siRNA-mediated knockdown of these transcriptional regulators recapitulated the senescent phenotype. In addition, pharmacologic inhibition of the SIN3 complex suppressed senescence and SASP markers. These results clearly demonstrate that a single microRNA is sufficient to drive early senescence in the absence of genotoxic stress through targeting epigenetic and transcriptional regulators, identifying novel targets for the development of senotherapeutics.

Divergent Immediate and Delayed Effects of Juvenile Exposure to Doxorubicin on the Thymus in C57BL/6 Mice.

The understanding of alterations within the immune system following doxorubicin (DOX) chemotherapy, and subsequent restoration, in childhood cancer survivors remains limited. This investigation endeavors to elucidate the immediate and delayed changes in thymic immune cell populations and their phenotypes in response to clinically relevant low doses of DOX in a juvenile mouse model. Male mice underwent a regimen of repeated low-dose DOX intraperitoneal injections at 4 mg/kg/week for three consecutive weeks. One week after the last dose of DOX, a subset of mice was euthanized to assess the immediate effects of DOX administration. A second subset of mice was euthanized five weeks after the last DOX dose to evaluate the delayed effects. Thymic samples were collected for multiparameter flow cytometry analysis to evaluate alterations in immune cell composition and phenotype. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure gene expression of- cytokines and senescence markers. One week following DOX administration, DOX treatment resulted in significant decline in thymus weight, with notable alterations in immune cell subpopulations. Reduced frequencies of mature CD3+CD4+ and CD3+CD8+ T cells were observed, along with changes in proliferation and exhaustion markers. Gene expression analysis revealed upregulation of Foxn, Pax1, Ifnγ, and Il7 alongside decreased Il6 and Il17 expression. Furthermore, Cdkn1a (p21Cip1) expression was elevated, suggesting immunosenescence. Five weeks following DOX administration, delayed effects of DOX treatment manifested in rebound increase in thymus weight and altered frequencies of CD4+ and CD8+ T cell subsets, with distinct patterns of proliferation and exhaustion observed. Notably, central memory CD4+ T cells exhibited significant decrease in frequency, while naive and effector memory CD4+ T cells showed reduced proliferation (Ki67+) and PD1 expression. Similar trends were observed in CD8+ T cell subsets, indicating selective effects of DOX on T cell differentiation and function. Although expression of thymus-related genes was normalized, p21Cip1 gene expression remained elevated. DOX treatment elicits a multifaceted influence on immune cell subsets and thymic weight. Immediate effects included thymic atrophy and reductions in mature T cell populations, while delayed effects showed rebound thymic hyperplasia and selective changes in CD4+ and CD8+ T cell subsets. Notably, both central memory and effector memory T cells exhibited reduced proliferation and exhaustion, suggesting unique impacts of DOX on immune cell function. The enduring elevation in p21Cip1 gene expression 5 weeks after DOX treatment suggests an immunosenescent phenotype. These observations collectively illuminate the formidable task of preserving immune competence and overall well-being in childhood cancer survivors subjected to DOX therapy.

Losmapimod ameliorates doxorubicin-induced cardiotoxicity through attenuating senescence and inflammatory pathways

Irreversible cardiotoxicity limits the clinical application of doxorubicin (DOX). DOX-induced cardiotoxicity has been associated with induction of senescence and activation of the p38 MAPK pathway. Losmapimod (LOSM), an orally active p38 MAPK inhibitor, is an anti-inflammatory agent with cardioprotective effects. Nevertheless, the effect of LOSM against DOX-induced cardiotoxicity has not been reported. In this study, we determined the effects of LOSM on DOX-induced chronic cardiotoxicity in C57BL/6 N mice. Five-week-old C57BL/6 N mice were fed diet containing LOSM (estimated daily intake 12 mg/kg/day) or a control diet for four days. Thereafter, mice were randomized to receive six weekly intraperitoneal injections of either DOX (4 mg/kg) or saline. Three days after the last injection, cardiac function was assessed by trans-thoracic echocardiography. Activation of p38, JNK, and ERK1/2 MAPKs were assessed by immunoblotting in the heart and liver. Gene expressions of senescence, inflammatory, oxidative stress, and mitochondrial function markers were quantified using real-time PCR and serum inflammatory markers were assessed by Luminex. Our results demonstrated that LOSM attenuated p38 MAPK activation, ameliorated DOX-induced cardiac dysfunction, and abrogated DOX-induced expression of the senescence marker p21Cip1. Additionally, LOSM demonstrated anti-inflammatory effects, with reduced cardiac Il-1α and Il-6 gene expression in DOX-treated mice. Systemic inflammation, assessed by serum cytokine levels, showed decreased IL-6 and CXCL1 in both DOX-treated mice and mice on LOSM diet. LOSM significantly increased mitofusin2 gene expression, which may enhance mitochondrial fusion. These findings underscore the potential therapeutic efficacy of p38 MAPK inhibition, exemplified by LOSM, in ameliorating DOX-induced cardiotoxicity, senescence, and inflammation.

Crocetin regulates cell cycle progression in N-nitroso-Nmethylurea(NMU)-induced breast cancer in rat: cyclin D1 suppression, p21Cip1 and p53 induction

Objectives: Crocetin, a saffron-derived carotenoid, inhibits tumor growth in some cancer types. However, its mechanism of action still needs to be clearly understood. Here, the Crocetin effect on the expression of some cell cycle regulators was investigated. Methods: The N-nitroso-N-methylurea (NMU)-induced rat mammary carcinomas were induced in a group of 35-day-old female Wistar Albino rats. Then, the expression of several cell cycle regulators was studied using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. After the tumor appearance, these rats were treated with Crocetin through weekly, intraperitoneal injections of 100 mg/kg body weight for four weeks. A control group has received the vehicle only. In the end, rats were sacrificed, and tumors were divided into two parts. A part was for pathologic investigation, and a part was retained at -70 °C to determine desired parameters. Results: Before Crocetin treatment, the tumor volumes were 13.27 ± 3.77 and 9.44 ± 4.39 cm3 , which was changed to 23.66 ± 8.82 and 4.71 ± 2.44 cm3 at the end of the experiment in the untreated and treated groups, respectively. The results showed that Crocetin markedly decreased the increased expression of cyclin D1 due to NMU administration. However, it further increased the expression of p53 and p21Cip1, with no significant effect on p27Kip1 expression. We previously showed Crocin-induced cell cycle arrest through a p53-dependent mechanism. Conclusion: Crocetin induced the cell cycle arrest in NMU-induced breast cancer in rats through a p53-independent mechanism. It is the second mechanism additional to apoptosis induction in cancer cells.

Cellular senescence mediates retinal ganglion cell survival regulation post-optic nerve crush injury.

Traumatic optic neuropathy refers to optic nerve (ON) injury by trauma, including explosion and traffic accident. Retinal ganglion cell (RGC) death is the critical pathological cause of irreversible visual impairment and blindness in ON injury. We previously investigated the patterns of 11 modes of cell death in mouse retina post-ON injury. Here we aimed to identify additional signalling pathways regulating RGC survival in rodents post-ON injury. RNA sequencing analysis identified the upregulation of inflammation and cellular senescence-related genes in retina post-ON injury, which were confirmed by immunoblotting and immunofluorescence analyses. Increased expression of senescence-associated β-galactosidase (SA-βgal) in RGCs and activation of microglia were also found. Transforming growth factor-β receptor type II inhibitor (LY2109761) treatment suppressed p15Ink4b and p21Cip1 protein and SA-βgal expression and promoted RGC survival post-ON injury with decreasing the expression of cell death markers in retina. Consistently, senolytics (dasatinib and quercetin) treatments can promote RGC survival and alleviate the reduction of ganglion cell complex thickness and pattern electroretinography activity post-ON injury with reducing SA-βgal, p15Ink4b, p21Cip1, microglial activation and cell death marker expression. In summary, this study revealed the activation of cellular senescence in rodent retina post-ON injury and contribute to RGC survival regulation. Targeting cellular senescence can promote RGC survival after ON injury, suggesting a potential treatment strategy for traumatic optic neuropathy.

Hypoxia-induced immortalization of primary cells depends on Tfcp2L1 expression

Cellular senescence is a stress response mechanism that induces proliferative arrest. Hypoxia can bypass senescence and extend the lifespan of primary cells, mainly by decreasing oxidative damage. However, how hypoxia promotes these effects prior to malignant transformation is unknown. Here we observed that the lifespan of mouse embryonic fibroblasts (MEFs) is increased when they are cultured in hypoxia by reducing the expression of p16INK4a, p15INK4b and p21Cip1. We found that proliferating MEFs in hypoxia overexpress Tfcp2l1, which is a main regulator of pluripotency and self-renewal in embryonic stem cells, as well as stemness genes including Oct3/4, Sox2 and Nanog. Tfcp2l1 expression is lost during culture in normoxia, and its expression in hypoxia is regulated by Hif1α. Consistently, its overexpression in hypoxic levels increases the lifespan of MEFs and promotes the overexpression of stemness genes. ATAC-seq and Chip-seq experiments showed that Tfcp2l1 regulates genes that control proliferation and stemness such as Sox2, Sox9, Jarid2 and Ezh2. Additionally, Tfcp2l1 can replicate the hypoxic effect of increasing cellular reprogramming. Altogether, our data suggest that the activation of Tfcp2l1 by hypoxia contributes to immortalization prior to malignant transformation, facilitating tumorigenesis and dedifferentiation by regulating Sox2, Sox9, and Jarid2.

Repression of YEATS2 induces cellular senescence in hepatocellular carcinoma and inhibits tumor growth

ABSTRACT Hepatocellular carcinoma (HCC) stands as the third leading cause of cancer-related fatalities globally. In this study, we observed a significant increase in the expression level of the YEATS2 gene in HCC patients, and it is negatively correlated with the patients’ survival rate. While we have previously identified the association between YEATS2 and the survival of pancreatic cancer cells, the regulatory mechanisms and significance in HCC are still to be fully elucidated. Our study shows that knockdown (KD) of YEATS2 expression leads to DNA damage, which in turn results in an upregulation of γ-H2A.X expression and activation of the canonical senescence-related pathway p53/p21Cip1. Moreover, our transcriptomic analysis reveals that YEATS2 KD cells can enhance the expression of p21Cip1 via the c-Myc/miR-93-5p pathway, consequently fostering the senescence of HCC cells. The initiation of cellular senescence through dual-channel activation suggests that YEATS2 plays a pivotal regulatory role in the process of cell proliferation. Ultimately, our in vivo research utilizing a nude mouse tumor model revealed a notable decrease in both tumor volume and weight after the suppression of YEATS2 expression. This phenomenon is likely attributable to the attenuation of proliferative cell activity, coupled with a concurrent augmentation in the population of natural killer (NK) cells. In summary, our research results have supplemented the understanding of the regulatory mechanisms of HCC cell proliferation and indicated that targeting YEATS2 may potentially inhibit liver tumor growth.

Small airway fibroblasts from patients with chronic obstructive pulmonary disease exhibit cellular senescence.

Small airway disease (SAD) is a key early-stage pathology of chronic obstructive pulmonary disease (COPD). COPD is associated with cellular senescence whereby cells undergo growth arrest and express the senescence-associated secretory phenotype (SASP) leading to chronic inflammation and tissue remodeling. Parenchymal-derived fibroblasts have been shown to display senescent properties in COPD, however small airway fibroblasts (SAFs) have not been investigated. Therefore, this study investigated the role of these cells in COPD and their potential contribution to SAD. To investigate the senescent and fibrotic phenotype of SAF in COPD, SAFs were isolated from nonsmoker, smoker, and COPD lung resection tissue (n = 9-17 donors). Senescence and fibrotic marker expressions were determined using iCELLigence (proliferation), qPCR, Seahorse assay, and ELISAs. COPD SAFs were further enriched for senescent cells using FACSAria Fusion based on cell size and autofluorescence (10% largest/autofluorescent vs. 10% smallest/nonautofluorescent). The phenotype of the senescence-enriched population was investigated using RNA sequencing and pathway analysis. Markers of senescence were observed in COPD SAFs, including senescence-associated β-galactosidase, SASP release, and reduced proliferation. Because the pathways driving this phenotype were unclear, we used cell sorting to enrich senescent COPD SAFs. This population displayed increased p21CIP1 and p16INK4a expression and mitochondrial dysfunction. RNA sequencing suggested these senescent cells express genes involved in oxidative stress response, fibrosis, and mitochondrial dysfunction pathways. These data suggest COPD SAFs are senescent and may be associated with fibrotic properties and mitochondrial dysfunction. Further understanding of cellular senescence in SAFs may lead to potential therapies to limit SAD progression.NEW & NOTEWORTHY Fibroblasts and senescence are thought to play key roles in the pathogenesis of small airway disease and COPD; however, the characteristics of small airway-derived fibroblasts are not well explored. In this study we isolate and enrich the senescent small airway-derived fibroblast (SAF) population from COPD lungs and explore the pathways driving this phenotype using bulk RNA-seq.

NUAK1 coordinates growth factor-dependent activation of mTORC2 and Akt signaling

BackgroundmTORC2 is a critical regulator of cytoskeleton organization, cell proliferation, and cancer cell survival. Activated mTORC2 induces maximal activation of Akt by phosphorylation of Ser-473, but regulation of Akt activity and signaling crosstalk upon growth factor stimulation are still unclear.ResultsWe identified that NUAK1 regulates growth factor-dependent activation of Akt by two mechanisms. NUAK1 interacts with mTORC2 components and regulates mTORC2-dependent activation of Akt by controlling lysosome positioning and mTOR association with this organelle. A second mechanism involves NUAK1 directly phosphorylating Akt at Ser-473. The effect of NUAK1 correlated with a growth factor-dependent activation of specific Akt substrates. NUAK1 induced the Akt-dependent phosphorylation of FOXO1/3a (Thr-24/Thr-32) but not of TSC2 (Thr-1462). According to a subcellular compartmentalization that could explain NUAK1’s differential effect on the Akt substrates, we found that NUAK1 is associated with early endosomes but not with plasma membrane, late endosomes, or lysosomes. NUAK1 was required for the Akt/FOXO1/3a axis, regulating p21CIP1, p27KIP1, and FoxM1 expression and cancer cell survival upon EGFR stimulation. Pharmacological inhibition of NUAK1 potentiated the cell death effect induced by Akt or mTOR pharmacological blockage. Analysis of human tissue data revealed that NUAK1 expression positively correlates with EGFR expression and Akt Ser-473 phosphorylation in several human cancers.ConclusionsOur results showed that NUAK1 kinase controls mTOR subcellular localization and induces Akt phosphorylation, demonstrating that NUAK1 regulates the growth factor-dependent activation of Akt signaling. Therefore, targeting NUAK1, or co-targeting it with Akt or mTOR inhibitors, may be effective in cancers with hyperactivated Akt signaling.

Exploring the anti-aging effects of fisetin in telomerase-deficient progeria mouse model.

Aging is a natural and complex process characterized by the gradual deterioration of tissue and physiological functions in the organism over time. Cell senescence, a hallmark of aging, refers to the permanent and irreversible cell cycle arrest of proliferating cells triggered by endogenous stimuli or environmental stresses. Eliminating senescent cells has been shown to extend the healthy lifespan. In this study, we established a progeria mouse model with telomerase deficiency and confirmed the presence of shortened telomere length and increased expression of aging markers p16INK4a and p21CIP1 in the organ tissues of G3 Tert-/- mice. We identified fisetin as a potent senolytic drug capable of reversing premature aging signs in telomerase-deficient mice. Fisetin treatment effectively suppressed the upregulation of aging markers p16INK4a and p21CIP1 and reduced collagen fiber deposition. Furthermore, we observed a significant elevation in the mRNA level of Stc1 in G3Tert-/- mice, which was reduced after fisetin treatment. Stc1 has been implicated in anti-apoptotic processes through the upregulation of the Akt signaling pathway. Our findings reveal that fisetin exerts its anti-aging effect by inhibiting the Akt signaling pathway through the suppression of Stc1 expression, leading to the apoptosis of senescent cells.

Small Molecule Inhibition of MAGE-A3 Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma

Background: The Type I Melanoma Antigen Gene-A3 (MAGE-A3) is commonly expressed in multiple myeloma (MM), a cancer of plasma cells. MAGE-A3 binds to Kap1 to form ubiquitin (Ub) ligase complexes and is associated with inhibition of apoptosis, resistance to chemotherapy, and cell cycle dysregulation. The highly conserved MAGE Homology Domain (MHD) undergoes a conformation change from “closed” to “open” in order to complex with Kap1, activate Ub ligase activity, and bind substrates. RNA interference (RNAi) knockdown of MAGE-A3 in MM cells results in upregulation of the endogenous CDK4/6 inhibitor P21 Cip1 and pro-apoptotic BIM, resulting in cell cycle arrest and intrinsic apoptosis. Knockdown of MAGE-A3 also increased cell death in response to melphalan and panobinostat, but not to bortezomib or cis-platin. These findings lead to the hypothesis that small molecule inhibition (SMI) of MAGE-A3 Ub ligase activity will have therapeutic application in this disease. Methods: We used the Schrodinger suite to perform in silico screening of a custom designed library of compounds using the crystal structure of the MAGE-A3 MHD to identify potential binding molecules that stabilize the closed MHD conformation. Candidates were then screened on human myeloma cell lines (HMCL) by CellTiter-Glo assay to identify compounds that reduced cell viability. Isothermal titration calorimetry was used to identify candidate SMI that bound to the MHD. HMCL were co-incubated with SMI; cell cycling was assessed by BrdU assay and apoptosis by Annexin V staining. Protein expression of p21 Cip1, BIM, and MAGE-A3 was assessed by Western blot. Combinatorial indexes in co-incubation experiments with ergotamine and chemotherapy agents were calculated with the Chou-Talalay method using CompuSyn. Results: Structure-based and biochemical screening nominated ergotamine as a candidate MAGE-A3 SMI. Ergotamine bound to the MAGE-A3 MHD and induced cell cycle arrest and apoptosis in MAGE-A3-expressing human myeloma and lymphoma cell lines and increased expression of pro-apoptotic BIM and p21 Cip1, similar to results with RNAi knockdown. These effects were not observed in negative control MAGE-A3(-) lymphoma cells. Co-incubation of ergotamine with chemotherapy agents melphalan or panobinostat resulted in synergistic killing of MAGE-A3+ HMCL. Ergotamine did not synergize with bortezomib or cis-platin under these conditions. Conclusions: These results support the combined structural and biochemical methods as an efficient means of screening for candidate inhibitors for MAGE-A3. Ergotamine recapitulated the results of RNAi knockdown of MAGE-A3 in cells that express the target, supporting a tool compound for further pharmacologic development. MAGE-A3 SMI have the potential to be a novel class of MM therapy that may be effective alone or in combination with chemotherapy or immune oncology agents. MAGE inhibitors may have broader application in other target-expressing cancers, including lung, melanoma, breast, and ovarian cancers, which are among the most common causes of cancer death.

The involvement of let-7 in hCG-induced progesterone synthesis via regulating p27Kip1 and p21Cip1 expression

Progesterone is essential in females to maintain a regular menstrual cycle and pregnancy. The luteinizing hormone (LH) surge induces the luteinization of granulosa cells and thecal cells to form the corpus luteum, which is responsible for progesterone synthesis. However, the specific mechanism of how hCG, the analog of LH, regulates progesterone synthesis has yet to be fully discovered. In this study, we found that progesterone level was increased in adult wild-type pregnant mice 2 and 7 days post-coitum, along with a decrease in let-7 expression compared with the estrus stage. Besides, the let-7 expression was negatively correlated with progesterone level in post-delivery day 23 wild-type female mice after being injected with PMSG and hCG. Then, using let-7 transgenic mice and a human granulosa cell line, we found that overexpression of let-7 antagonized progesterone level via targeting p27Kip1 and p21Cip1 and steroidogenic acute regulatory protein (StAR) expression, which is a rate-limiting enzyme in progesterone synthesis. Furthermore, hCG suppressed let-7 expression by stimulating the MAPK pathway. This study elucidated the role of microRNA let-7 in regulating hCG-induced progesterone production and provided new insights into its role in clinical application.

MicroRNA-19a-3p Decreases with Age in Mice and Humans and Inhibits Osteoblast Senescence.

Aging is a major risk factor for most chronic diseases, including osteoporosis, and is characterized by an accumulation of senescent cells in various tissues. MicroRNAs (miRNAs) are critical regulators of bone aging and cellular senescence. Here, we report that miR-19a-3p decreases with age in bone samples from mice as well as in posterior iliac crest bone biopsies of younger versus older healthy women. miR-19a-3p also decreased in mouse bone marrow stromal cells following induction of senescence using etoposide, H2O2, or serial passaging. To explore the transcriptomic effects of miR-19a-3p, we performed RNA sequencing of mouse calvarial osteoblasts transfected with control or miR-19a-3p mimics and found that miR-19a-3p overexpression significantly altered the expression of various senescence, senescence-associated secretory phenotype-related, and proliferation genes. Specifically, miR-19a-3p overexpression in nonsenescent osteoblasts significantly suppressed p16 Ink4a and p21 Cip1 gene expression and increased their proliferative capacity. Finally, we established a novel senotherapeutic role for this miRNA by treating miR-19a-3p expressing cells with H2O2 to induce senescence. Interestingly, these cells exhibited lower p16 Ink4a and p21 Cip1 expression, increased proliferation-related gene expression, and reduced SA-β-Gal+ cells. Our results thus establish that miR-19a-3p is a senescence-associated miRNA that decreases with age in mouse and human bones and is a potential senotherapeutic target for age-related bone loss. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

FIBRONECTIN TYPE III DOMAIN CONTAINING 5 MITIGATES CHONDROCYTE SENESCENCE, SUBCHONDRAL OSTEOCLAST FORMATION, AND OSTEOARTHRITIS DEVELOPMENT THROUGH INHIBITING INTERLEUKIN-6

Senescent chondrocyte and subchondral osteoclast overburden aggravate inflammatory cytokine and pro-catabolic proteinase overproduction, accelerating extracellular matrix degradation and pain during osteoarthritis (OA). Fibronectin type III domain containing 5 (FNDC5) is found to promote tissue homeostasis and alleviate inflammation. This study aimed to characterize what role Fndc5 may play in chondrocyte aging and OA development.Serum and macroscopically healthy and osteoarthritic cartilage were biopsied from patients with knee OA who received total knee replacement. Murine chondrocytes were transfected with Fndc5 RNAi or cDNA. Mice overexpressing Fndc5 (Fndc5Tg) were operated to have destabilized medial meniscus mediated (DMM) joint injury as an experimental OA model. Cellular senescence was characterized using RT-PCR analysis of p16INK4A, p21CIP1, and p53 expression together with ß-galactosidase activity staining. Articular cartilage damage and synovitis were graded using OARSI scores. Osteophyte formation and mechanical allodynia were quantified using microCT imaging and von Frey filament, respectively. Osteoclast formation was examined using tartrate-resistant acid phosphatase staining.Senescent chondrocyte and subchondral osteoclast overburden together with decreased serum FNDC5 levels were present in human osteoarthritic cartilage. Fndc5 knockdown upregulated senescence program together with increased IL-6, MMP9 and Adamts5 expression, whereas Alcian blue-stained glycosaminoglycan production were inhibited. Forced Fndc5 expression repressed senescence, apoptosis and IL-6 expression, reversing proliferation and extracellular matrix production in inflamed chondrocytes. Fndc5Tg mice showed few OA signs, including articular cartilage erosion, synovitis, osteophyte formation, subchondral plate sclerosis and mechanical allodynia together with decreased IL-6 production and few senescent chondrocytes and subchondral osteoclast formation during DMM-induced joint injury. Mechanistically, Fndc5 reversed histone H3K27me3-mediated IL-6 transcription repression to reduce reactive oxygen species production.Fndc5 loss correlated with OA development. It was indispensable in chondrocyte growth and anabolism. This study sheds light onto the anti-ageing and anti-inflammatory actions of Fndc5 to chondrocytes; and highlights the chondroprotective function of Fndc5 to compromise OA.

A novel anti-HBV agent, E-CFCP, restores Hepatitis B virus (HBV)-induced senescence-associated cellular marker perturbation in human hepatocytes

Cellular senescence is a cellular state with a broad spectrum of age-related physiological conditions that can be affected by various infectious diseases and treatments. Therapy of hepatitis B virus (HBV) infection with nucleos(t)ide analogs [NA(s)] is well established and benefits many HBV-infected patients, but requires long-term, perhaps lifelong, medication. In addition to the effects of HBV infection, the effects of NA administration on hepatocellular senescence are still unclear. This study investigated how HBV infection and NA treatment influence cellular senescence in human hepatocytes and humanized-liver chimeric mice chronically infected with live HBV. HBV infection upregulates or downregulates multiple cellular markers including senescence-associated β-galactosidase (SA-β-Gal) activity and cell cycle regulatory proteins (e.g., p21CIP1) expression level in hepatocellular nuclei and humanized-mice liver. A novel highly potent anti-HBV NA, E-CFCP, per se did not have significant disturbance on markers evaluated. Besides, E-CFCP treatment restored HBV-infected cells to their physiological phenotypes that are comparable to the HBV-uninfected cells. The results reported here demonstrate that, regardless of the mechanism(s), chronic HBV infection perturbates multiple senescence-associated markers in human hepatocytes and humanized-mice liver, but E-CFCP can restore this phenomenon.

Implications of Senescent Cell Burden and NRF2 Pathway in Uremic Calcification: A Translational Study.

Increased senescent cell burden and dysregulation of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway have been associated with numerous age-related pathologies; however, their role in promoting vascular calcification (VC) in chronic kidney disease (CKD) has yet to be determined. We investigated whether senescence and NRF2 pathways may serve as drivers of uremia-induced VC using three complementary approaches: a novel model of induced VC in 5/6-nephrectomized rats supplemented with high phosphate and vitamin D; epigastric arteries from CKD patients with established medial calcification; and vascular smooth muscle cells (VSMCs) incubated with uremic serum. Expression of p16Ink4a and p21Cip1, as well as γ-H2A-positive cells, confirmed increased senescent cell burden at the site of calcium deposits in aortic sections in rats, and was similarly observed in calcified epigastric arteries from CKD patients through increased p16Ink4a expression. However, uremic serum-induced VSMC calcification was not accompanied by senescence. Expression of NRF2 and downstream genes, Nqo1 and Sod1, was associated with calcification in uremic rats, while no difference was observed between calcified and non-calcified EAs. Conversely, in vitro uremic serum-driven VC was associated with depleted NRF2 expression. Together, our data strengthen the importance of senescence and NRF2 pathways as potential therapeutic options to combat VC in CKD.

Effects of Ginsenoside Rg1 on the Biological Behavior of Human Amnion-Derived Mesenchymal Stem/Stromal Cells (hAD-MSCs).

Ginsenoside Rg1 (Rg1) is purified from ginseng with various pharmacological effects, which might facilitate the biological behavior of human amnion-derived mesenchymal stem/stromal cells (hAD-MSCs). This study is aimed at investigating the effects of Rg1 on the biological behavior, such as viability, proliferation, apoptosis, senescence, migration, and paracrine, of hAD-MSCs. hAD-MSCs were isolated from human amnions. The effects of Rg1 on the viability, proliferation, apoptosis, senescence, migration, and paracrine of hAD-MSCs were detected by CCK-8, EdU, flow cytometry, SA-β-Gal staining, wound healing, and ELISA assays, respectively. The protein expression levels were detected by western blot. Cell cycle distribution was evaluated using flow cytometry. We found that Rg1 promoted hAD-MSC cycle progression from G0/G1 to S and G2/M phases and significantly increased hAD-MSC proliferation rate. Rg1 activated PI3K/AKT signaling pathway and significantly upregulated the expressions of cyclin D, cyclin E, CDK4, and CDK2 in hAD-MSCs. Inhibition of PI3K/AKT signaling significantly downregulated the expressions of cyclin D, cyclin E, CDK4, and CDK2, prevented cell cycle progression, and reduced hAD-MSC proliferation induced by Rg1. hAD-MSC senescence rate was significantly increased by D-galactose, while the elevated hAD-MSC senescence rate induced by D-galactose was significantly decreased by Rg1 treatment. D-galactose significantly induced the expressions of senescence markers, p16INK4a, p14ARF, p21CIP1, and p53 in hAD-MSCs, while Rg1 significantly reduced the expressions of those markers induced by D-galactose in hAD-MSCs. Rg1 significantly promoted the secretion of IGF-I in hAD-MSCs. Rg1 reduced the hAD-MSC apoptosis rate. However, the difference was not significant. Rg1 had no influence on hAD-MSC migration. Altogether, our results demonstrate that Rg1 can promote the viability, proliferation, and paracrine and relieve the senescence of hAD-MSCs. PI3K/AKT signaling pathway is involved in the promotive effect of Rg1 on hAD-MSC proliferation. The protective effect of Rg1 on hAD-MSC senescence may be achieved via the downregulation of p16INK4A and p53/p21CIP1 pathway.