ABSTRACT
Healthy skin is a mosaic of wild-type and mutant clones1,2. Although injury can cooperate with mutated Ras family proteins to promote tumorigenesis3-12, the consequences in genetically mosaic skin are unknown. Here we show that after injury, wild-type cells suppress aberrant growth induced by oncogenic Ras. HrasG12V/+ and KrasG12D/+ cells outcompete wild-type cells in uninjured, mosaic tissue but their expansion is prevented after injury owing to an increase in the fraction of proliferating wild-type cells. Mechanistically, we show that, unlike HrasG12V/+ cells, wild-type cells respond to autocrine and paracrine secretion of EGFR ligands, and this differential activation of the EGFR pathway explains the competitive switch during injury repair. Inhibition of EGFR signalling via drug or genetic approaches diminishes the proportion of dividing wild-type cells after injury, leading to the expansion of HrasG12V/+ cells. Increased proliferation of wild-type cells via constitutive loss of the cell cycle inhibitor p21 counteracts the expansion of HrasG12V/+ cells even in the absence of injury. Thus, injury has a role in switching the competitive balance between oncogenic and wild-type cells in genetically mosaic skin.
Subject(s)
Cell Proliferation , Genes, ras , Mosaicism , Mutation , Skin , ras Proteins , Cell Cycle , Cell Proliferation/genetics , ErbB Receptors/metabolism , ras Proteins/genetics , ras Proteins/metabolism , Skin/cytology , Skin/injuries , Skin/metabolism , Skin/pathology , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolismABSTRACT
Type I IFNs, such as interferon alpha and interferon beta, are key regulators of the adaptive immune response during infectious diseases. Type I IFNs are induced upon infection, bind interferon α/ß receptors on T-cells and activate intracellular pathways. The activating and inhibitory consequences of type I IFN-signaling are determined by cell type and cellular environment. The neonatal immune system is associated with increased vulnerability to infectious diseases which could partly be explained by an immature CD4+ T-cell compartment. Here, we show low IFN-ß-mediated inhibition of CD4+ T-cell proliferation, phosphorylation of retinoblastoma protein and cytokine production in human newborns compared to adults. In addition, both naïve and total newborn CD4+ T-cells are unable to induce the cell-cycle inhibitor p21 upon exposure to IFN-ß in contrast to adults. The distinct IFN-ß-signaling in newborns provides novel insights into T cell functionality and regulation of T cell-dependent inflammation during early life immune responses.
Subject(s)
Adaptive Immunity/physiology , CD4-Positive T-Lymphocytes/immunology , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Interferon-beta/metabolism , Signal Transduction/immunology , Adaptive Immunity/drug effects , Adult , Age Factors , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Fetal Blood/cytology , Fetal Blood/immunology , Flow Cytometry , Humans , Immunomagnetic Separation , Infant, Newborn , Primary Cell Culture , Receptor, Interferon alpha-beta/antagonists & inhibitors , Receptor, Interferon alpha-beta/metabolism , Signal Transduction/drug effectsABSTRACT
The p53/p21 pathway is activated in response to cell stress. However, its role in acute lung injury has not been elucidated. Acute lung injury is associated with disruption of the alveolo-capillary barrier leading to acute respiratory distress syndrome (ARDS). Mechanical ventilation may be necessary to support gas exchange in patients with ARDS, however, high positive airway pressures can cause regional overdistension of alveolar units and aggravate lung injury. Here, we report that acute lung injury and alveolar overstretching activate the p53/p21 pathway to maintain homeostasis and avoid massive cell apoptosis. A systematic pooling of transcriptomic data from animal models of lung injury demonstrates the enrichment of specific p53- and p21-dependent gene signatures and a validated senescence profile. In a clinically relevant, murine model of acid aspiration and mechanical ventilation, we observed changes in the nuclear envelope and the underlying chromatin, DNA damage and activation of the Tp53/p21 pathway. Absence of Cdkn1a decreased the senescent response, but worsened lung injury due to increased cell apoptosis. Conversely, treatment with lopinavir and/or ritonavir led to Cdkn1a overexpression and ameliorated cell apoptosis and lung injury. The activation of these mechanisms was associated with early markers of senescence, including expression of senescence-related genes and increases in senescence-associated heterochromatin foci in alveolar cells. Autopsy samples from lungs of patients with ARDS revealed increased senescence-associated heterochromatin foci. Collectively, these results suggest that acute lung injury activates p53/p21 as an antiapoptotic mechanism to ameliorate damage, but with the side effect of induction of senescence.
Subject(s)
Acute Lung Injury/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Acids/administration & dosage , Acids/toxicity , Acute Lung Injury/etiology , Acute Lung Injury/pathology , Animals , Apoptosis , Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA Damage , Disease Models, Animal , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Respiration, Artificial/adverse effects , Respiratory Distress Syndrome/etiology , Respiratory Distress Syndrome/metabolism , Respiratory Distress Syndrome/pathology , Signal Transduction , Stress, Mechanical , Translational Research, Biomedical , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Oncolytic viruses are potent anticancer agents that replicate within and kill cancer cells rather than normal cells, and their selectivity is largely determined by oncogenic mutations. M1, a novel oncolytic virus strain, has been shown to target cancer cells, but the relationship between its cancer selectivity and oncogenic signaling pathways is poorly understood. Here, we report that RAS mutation promotes the replication and oncolytic effect of M1 in cancer, and we further provide evidence that the inhibition of the RAS/RAF/MEK signaling axis suppresses M1 infection and the subsequent cytopathic effects. Transcriptome analysis revealed that the inhibition of RAS signaling upregulates the type I interferon antiviral response, and further RNA interference screen identified CDKN1A as a key downstream factor that inhibits viral infection. Gain- and loss-of-function experiments confirmed that CDKN1A inhibited the replication and oncolytic effect of M1 virus. Subsequent TCGA data mining and tissue microarray (TMA) analysis revealed that CDKN1A is commonly deficient in human cancers, suggesting extensive clinical application prospects for M1. Our report indicates that virotherapy is feasible for treating undruggable RAS-driven cancers and provides reliable biomarkers for personalized cancer therapy.
Subject(s)
Neoplasms/metabolism , Neoplasms/virology , Oncolytic Viruses/physiology , Signal Transduction , ras Proteins/metabolism , Animals , Antiviral Agents/pharmacology , Biomarkers, Tumor/metabolism , Butadienes/pharmacology , Cell Line , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Female , Gene Expression Regulation, Viral/drug effects , Humans , Mice, Inbred BALB C , Mice, Nude , Mutation/genetics , Neoplasms/pathology , Nitriles/pharmacology , Oncolytic Viruses/drug effects , Oncolytic Viruses/genetics , RNA, Small Interfering/metabolism , Signal Transduction/drug effects , Up-Regulation/drug effects , Up-Regulation/genetics , Virus Replication/drug effects , Virus Replication/geneticsABSTRACT
p21 has emerged as an important protein involved in cardiovascular diseases, but its role remains controversial. Recently, p21 has been reported to mediate inflammatory responses. As inflammatory responses are a feature of sepsis, our study investigated whether p21 has a role in cardiac dysfunction induced by sepsis and analyzed the mechanisms involved. To establish a mouse sepsis model, p21 global knockout (p21KO) and C57BL/6J wild-type (WT) male mice were treated with 5 mg/kg LPS intraperitoneally for 6, 24, or 48 h. After LPS stimulation, the level of p21 had significantly increased in the WT mice and in cardiomyocytes. Cardiac dysfunction induced by LPS was markedly aggravated in p21KO mice relative to that of WT mice. Downregulation of p21 expression exacerbated the LPS-mediated inflammatory response, and it increased oxidative stress as well as mitochondrial damage in the heart and in cardiomyocytes. In contrast, overexpressing p21 attenuated the increase of TNFα and promoted the increase of SOD2. Moreover, p21 regulated the LPS-induced autophagy activation; that is, the increase in autophagy was impaired when p21 expression was decreased, whereas the increase was significant when p21 was overexpressed. The autophagy inducer rapamycin partially rescued the cardiac deterioration caused by p21 downregulation in the LPS-stimulated groups. In addition, p21 regulated the autophagy level by interacting with LC3B. These results revealed that p21 controls LPS-induced cardiac dysfunction by modulating inflammatory and oxidative stress, and it is partially dependent on regulating the autophagy level. This study is the first to show that p21 could interact with LC3B to promote autophagy for the improvement of cardiac function during sepsis.
Subject(s)
Autophagy , Cardiotonic Agents/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Heart/physiopathology , Animals , Cyclin-Dependent Kinase Inhibitor p21/deficiency , HEK293 Cells , Humans , Inflammation/pathology , Lipopolysaccharides , Male , Mice, Inbred C57BL , Mice, Knockout , Microtubule-Associated Proteins/metabolism , Mitochondria, Heart/pathology , Mitochondria, Heart/ultrastructure , Models, Biological , Oxidation-Reduction , Oxidative Stress , Protein Binding , Rats, Sprague-DawleyABSTRACT
Endometriosis is a common gynecological disorder that affects approximately 6-10% of the female population impairing the quality of life of patients. Several pathophysiologic pathways have been proposed as potential regulators of its severity; however, to date, the processes that trigger the onset and that influence the severity of the disease are not fully understood; hence, leading to disease recurrence in approximately 10-67% of cases. Cyclin-dependent kinase inhibitor 1 (p21/WAF1) is a protein that is a major target of p53 and is related to cell cycle arrest (it regulates transition from the G1 to the S phase) when DNA damage is detected. Its activity has been also linked to the angiogenic potential of tumors as it promotes the expression of various kinases that are responsible for endothelial development and function. Although several articles have underlined the importance of this protein in cancer cell development and tumor growth, there are no relevant data in the field of endometriosis. Indirect evidence suggests, however, that it may be involved in the pathogenesis of endometriosis as it inhibits the activity of various kinases which have been correlated with the course and severity of the disease. The present article investigates the background theory that implies the potential role of cyclin-dependent kinase inhibitor 1 (p21/WAF1) in the pathogenesis of endometriosis. Implications for future research are also provided given that indirect evidence seem to associate downregulation of p21 with decreased growth and invasiveness of human endometrial stromal cells.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/physiology , Endometriosis/etiology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis , Cell Cycle , Cyclin-Dependent Kinase Inhibitor p21/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Disease Models, Animal , Disease Progression , Drug Design , Endometriosis/metabolism , Female , Forecasting , Humans , Mice , Mice, Transgenic , Neoplasm Proteins/physiology , Neoplasms/blood supply , Neoplasms/drug therapy , Neovascularization, Pathologic/physiopathologyABSTRACT
p21(WAF1/CIP1/SDI1) is a critical sentinel of the cell cycle that plays an important role in determining cell fate with respect to proliferation, differentiation and apoptosis. Recent studies have demonstrated that inhibition/loss of p21 promotes osteo-chondro differentiation in progenitor/stem cells, and that p21 knockout (p21-/-) mice demonstrate enhanced bone regeneration compared to wild-type controls after a non-critical size defect. It was therefore hypothesized that the absence of p21 may also protect against bone loss through enhancing bone formation, tilting the balance away from bone resorption, in an ovariectomy-induced osteopenia mouse model, investigated via microCT imaging. While p21-/- mice demonstrated significantly less bone loss after ovariectomy compared to wild-type controls, no increase in the number osteoclasts or osteoblasts in the bone or bone marrow was observed, nor was there an increase in osteoclast activity. Therefore, while the absence of p21 protected mice against estrogen mediated bone loss, the mechanisms/pathways responsible remained elusive. This study demonstrates that p21 may play a significant role in bone remodeling, and a better understanding of how the p21 pathway regulates bone anabolism and catabolism could lead to novel therapies for osteoporosis in the future.
Subject(s)
Bone Diseases, Metabolic/prevention & control , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Animals , Bone Diseases, Metabolic/diagnostic imaging , Bone Diseases, Metabolic/metabolism , Bone Remodeling/genetics , Bone Remodeling/physiology , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Disease Models, Animal , Female , Humans , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteoblasts/metabolism , Osteoclasts/metabolism , Osteoporosis, Postmenopausal/prevention & control , Ovariectomy/adverse effects , X-Ray MicrotomographyABSTRACT
Mechanisms underlying the generation of induced pluripotent stem cells (iPSC) and keeping iPSC stability remain to be further defined. Accumulated evidences showed that iPSC reprogramming may be controlled by the cell-division-rate-dependent model. Here we reported effects of absence of mouse p27 or p18 on iPSC generation efficiency and genomic stability. Expression levels of cyclin-dependent kinases inhibitors (CDKIs), p21, p27, and p18 decreased during iPSC reprogramming. Like p21 loss, p27 or p18 deficiency significantly promoted efficiency of iPSC generation, whereas ectopic expression of p27, p18, or treatment with CDK2 or CDK4 inhibitors repressed the reprogramming rate, suggesting that CDKIs-regulated iPSC reprogramming is directly related with their functions as CDK inhibitors. However, unlike p21 deletion, absence of p27 or p18 did not increase DNA damage or chromosomal aberrations during iPSC reprogramming and at iPSC stage. Our data not only support that cell cycle regulation is critical for iPSC reprogramming, but also reveal the distinction of CDKIs in somatic cell reprogramming.
Subject(s)
Cellular Reprogramming/genetics , Cyclin-Dependent Kinase Inhibitor p18/deficiency , Cyclin-Dependent Kinase Inhibitor p27/deficiency , Genomic Instability/genetics , Induced Pluripotent Stem Cells/metabolism , Animals , Cell Division/genetics , Chromosome Aberrations , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p18/genetics , Cyclin-Dependent Kinase Inhibitor p18/metabolism , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Fibroblasts/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Transduction, GeneticABSTRACT
This study aimed to explore a role of p21Waf1 in γH2AX foci formation and DNA repair as assessed by a Host-Cell Reactivation Assay in wild-type (p21Waf+/+) and p21Waf1-deficient E1A+Ras-transformed cells. p21Waf1+/+ cells have low γH2AX background compared to p21Waf1-/- cells. The treatment with histone deacetylase inhibitor (HDI) sodium butyrate (NaBut) causes to accumulation of γH2AX in p21Waf+/+ cells with little effect in p21Waf-/- cells. Moreover, NaBut inhibits DNA repair in wt cells but not in p21Waf1-/- cells. This could be explained by the weakening of GADD45 and PCNA proteins binding in NaBut-treated p21Waf1-expressing cells but not in p21Waf1-/- cells. We suggest that in wt-ERas cells NaBut activates both p21Waf1 expression and a release of p21Waf1 from the complexes with E1A that leads to suppression of DNA repair and promotes γH2AX persistency. The absence of p21Waf1 is by itself considered by the cell as stressful factor with formation of γH2AX. But the lack of p21Waf1 interferes with an inhibitory effect of NaBut to inhibit DNA repair and thereby to stop concomitant accumulation of harmful mutations. We conclude that p21Waf1 is directly involved in control of genome integrity and DNA repair acting through modulation of the components of the DNA repair machinery.
Subject(s)
Butyric Acid/pharmacology , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA Repair , Histone Deacetylase Inhibitors/pharmacology , Animals , Cell Line, Transformed , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Mice , Mice, KnockoutABSTRACT
Many types of multinucleated cells (syncytia) generated by cell-cell fusion are post-mitotic, but it remains unclear how this state is maintained and why. Here, we utilized the fluorescent ubiquitination-based cell-cycle indicator (Fucci) reporter system to show that human placental trophoblast cells were all in the G0 phase before they fuse. Expression of the fusogenic protein (fusogen) Syncytin-2 was confined to G0 cells. Overexpression of Syncytin-2 in cycling cells overrode the cell-cycle restriction and enabled fusion of cells in the S/G2/M phases but resulted in the unstable syncytia retaining mitotic features. The Syncytin-2-induced syncytia were functionally compromised with respect to pathogen defense and hormone secretion. We found that, during trophoblast fusion, the cell-cycle inhibitor p21 interacted with the GCM1 transcription factor, and this complex bound to the promoter of Syncytin-2 and promoted its transcription. These findings demonstrate that G0-restricted Syncytin-2 expression is a prerequisite for development of functional post-mitotic syncytia.
Subject(s)
Giant Cells/metabolism , Placenta/metabolism , Pregnancy Proteins/metabolism , CRISPR-Cas Systems/genetics , Cadherins/metabolism , Cell Fusion , Cell Line , Colforsin/metabolism , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA-Binding Proteins , Female , Giant Cells/cytology , Humans , Microscopy, Fluorescence , Mitosis , Nuclear Proteins/metabolism , Pregnancy , Pregnancy Proteins/genetics , Promoter Regions, Genetic , Resting Phase, Cell Cycle , Time-Lapse Imaging , Transcription Factors/metabolism , Transcription, Genetic , Trophoblasts/cytology , Trophoblasts/metabolismABSTRACT
We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus.
Subject(s)
Cell Nucleolus/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , Gene Expression Regulation , Organelles/metabolism , SUMO-1 Protein/metabolism , Cell Nucleolus/genetics , Cyclin E/genetics , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , DNA Damage , HCT116 Cells , Humans , Karyopherins/genetics , Karyopherins/metabolism , Organelle Biogenesis , Organelles/genetics , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Promyelocytic Leukemia Protein/genetics , Promyelocytic Leukemia Protein/metabolism , Protein Binding , Protein Multimerization , Protein Transport , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , SUMO-1 Protein/genetics , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Conjugating Enzymes/deficiency , Ubiquitin-Conjugating Enzymes/genetics , Exportin 1 ProteinABSTRACT
Loss of wild-type p53 function is widely accepted to be permissive for the development of multinucleated giant cells. However, whether therapy-induced multinucleation is associated with cancer cell death or survival remains controversial. Herein, we demonstrate that exposure of p53-deficient or p21WAF1 (p21)-deficient solid tumor-derived cell lines to ionizing radiation (between 2 and 8 Gy) results in the development of multinucleated giant cells that remain adherent to the culture dish for long times post-irradiation. Somewhat surprisingly, single-cell observations revealed that virtually all multinucleated giant cells that remain adherent for the duration of the experiments (up to three weeks post-irradiation) retain viability and metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and the majority (>60%) exhibit DNA synthesis. We further report that treatment of multinucleated giant cells with pharmacological activators of apoptosis (e.g., sodium salicylate) triggers their demise. Our observations reinforce the notion that radiation-induced multinucleation may reflect a survival mechanism for p53/p21-deficient cancer cells. With respect to evaluating radiosensitivity, our observations underscore the importance of single-cell experimental approaches (e.g., single-cell MTT) as the creation of viable multinucleated giant cells complicates the interpretation of the experimental data obtained by commonly-used multi-well plate colorimetric assays.
Subject(s)
Cell Survival/genetics , Cell Survival/radiation effects , DNA Replication/radiation effects , Genome, Human/radiation effects , Giant Cells/metabolism , Giant Cells/radiation effects , Radiation, Ionizing , Cell Cycle Checkpoints/genetics , Cell Cycle Checkpoints/radiation effects , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/genetics , Gene Knockdown Techniques , HCT116 Cells , Humans , Mutation , Radiation Tolerance/genetics , Tumor Stem Cell Assay , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/geneticsABSTRACT
Osteoarthritis (OA) is a multifactorial disease, and recent data suggested that cell cycle-related proteins play a role in OA pathology. Cyclin-dependent kinase (CDK) inhibitor 1 (p21) regulates activation of other CDKs, and recently, we reported that p21 deficiency induced susceptibility to OA induced by destabilization of the medial meniscus (DMM) surgery through STAT3-signaling activation. However, the mechanisms associated with why p21 deficiency led to susceptibility to OA by the STAT3 pathway remain unknown. Therefore, we focused on joint inflammation to determine the mechanisms associated with p21 function during in vitro and in vivo OA progression. p21-knockout (p21-/- ) mice were used to develop an in vivo OA model, and C57BL/6 (p21+/+ ) mice with the same background as the p21-/- mice were used as controls. Morphogenic changes were measured using micro-CT, IL-1ß serum levels were detected by ELISA, and histological or immunohistological analyses were performed. Our results indicated that p21-deficient DMM-model mice exhibited significant subchondral bone destruction and cartilage degradation compared with wild-type mice. Immunohistochemistry results revealed p21-/- mice susceptibility to OA changes accompanied by macrophage infiltration and enhanced MMP-3 and MMP-13 expression through IL-1ß-induced NF-κB signaling. p21-/- mice also showed subchondral bone destruction according to micro-CT analysis, and cathepsin K staining revealed increased numbers of osteoclasts. Furthermore, p21-/- mice displayed increased serum IL-1ß levels, and isolated chondrocytes from p21-/- mice indicated elevated MMP-3 and MMP-13 expression with phosphorylation of IκB kinase complex in response to IL-1ß stimulation, whereas treatment with a specific p-IκB kinase inhibitor attenuated MMP-3 and MMP-13 expression. Our results indicated that p21-deficient DMM mice were susceptible to alterations in OA phenotype, including enhanced osteoclast expression, macrophage infiltration, and MMP expression through IL-1ß-induced NF-κB signaling, suggesting that p21 regulation may constitute a possible therapeutic strategy for OA treatment. © 2017 American Society for Bone and Mineral Research.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/deficiency , Genetic Predisposition to Disease , Osteoarthritis/metabolism , Signal Transduction , Animals , Disease Models, Animal , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Matrix Metalloproteinase 13/biosynthesis , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 3/biosynthesis , Matrix Metalloproteinase 3/genetics , Mice , Mice, Knockout , Osteoarthritis/diagnostic imaging , Osteoarthritis/genetics , Osteoarthritis/pathology , X-Ray MicrotomographyABSTRACT
Neutrophil-associated inflammation during Pseudomonas aeruginosa lung infection is a determinant of morbidity in cystic fibrosis (CF). Neutrophil apoptosis is a key factor in inflammation resolution and is controlled by cytosolic proliferating cell nuclear antigen (PCNA). p21/Waf1, a cyclin-dependent kinase inhibitor, is a partner of PCNA, and its mRNA is up-regulated in human neutrophils during LPS challenge. We show here that, after 7 days of persistent infection with P. aeruginosa, neutrophilic inflammation was more prominent in p21(-/-) compared with wild-type (WT) mice. Notably, no intrinsic defect in the phagocytosis of apoptotic cells by macrophages was found in p21(-/-) compared with WT mice. Inflammatory cell analysis in peritoneal lavages after zymosan-induced peritonitis showed a significantly increased number of neutrophils at 48 hours in p21(-/-) compared with WT mice. In vitro analysis was consistent with delayed neutrophil apoptosis in p21(-/-) compared with WT mice. Ectopic expression of p21/waf1 in neutrophil-differentiated PLB985 cells potentiated apoptosis and reversed the prosurvival effect of PCNA. In human neutrophils, p21 messenger RNA was induced by TNF-α, granulocyte colony-stimulating factor, and LPS. Neutrophils isolated from patients with CF showed enhanced survival, which was reduced after treatment with a carboxy-peptide derived from the sequence of p21/waf1. Notably, p21/waf1 was detected by immunohistochemistry in neutrophils within lungs from patients with CF. Our data reveal a novel role for p21/waf1 in the resolution of inflammation via its ability to control neutrophil apoptosis. This mechanism may be relevant in the neutrophil-dominated inflammation observed in CF and other chronic inflammatory lung conditions.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Neutrophils/metabolism , Pneumonia/metabolism , Pneumonia/microbiology , Pseudomonas Infections/metabolism , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/physiology , Adolescent , Animals , Apoptosis/drug effects , Cell Count , Cell Differentiation/drug effects , Cell Line , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cystic Fibrosis/complications , Cystic Fibrosis/microbiology , Cystic Fibrosis/pathology , Female , Granulocyte Colony-Stimulating Factor/pharmacology , Humans , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Models, Biological , Neutrophils/drug effects , Peritonitis/microbiology , Peritonitis/pathology , Phagocytosis/drug effects , Pneumonia/complications , Pneumonia/pathology , Proliferating Cell Nuclear Antigen/metabolism , Pseudomonas Infections/complications , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/pharmacology , ZymosanABSTRACT
Cell cycle is an integral part of cell proliferation, and consists mainly of four phases, G1, S, G2 and M. The p21 protein, a cyclin dependent kinase inhibitor, plays a key role in regulating cell cyclevia G1 phase control. Cells capable of epimorphic regeneration have G2/M accumulation as their distinctive feature, whilst the majority of somatic cells rest at G1 phase. To investigate the role played byp21 in antler regeneration, we studied the cell cycle distribution of antler stem cells (ASCs), via down-regulation of p21 in vitro using RNAi. The results showed that ASCs had high levels of p21 mRNA expression and rested at G1 phase, which was comparable to the control somatic cells. Down-regulation of p21 did not result in ASC cell cycle re-distribution toward G2/M accumulation, but DNA damage and apoptosis of the ASCs significantly increased and the process of cell aging was slowed. These findings suggest that the ASCs may have evolved to use an alternative, p21-independent cell cycle regulation mechanism. Also a unique p21-dependent inhibitory effect may control DNA damage as a protective mechanism to ensure the fast proliferating ASCs do not become dysplastic/cancerous. Understanding of the mechanism underlying the role played by p21 in the ASCs could give insight into a mammalian system where epimorphic regeneration is initiated whilst the genome stability is effectively maintained.
Subject(s)
Antlers/cytology , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/genetics , Down-Regulation/genetics , RNA Interference , Stem Cells/cytology , Animals , Apoptosis/genetics , Cell Proliferation , Cellular Senescence/genetics , DNA Damage/genetics , Deer , G1 Phase Cell Cycle Checkpoints/genetics , Gene Knockdown Techniques , MaleABSTRACT
The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO) mice and wild type (WT) mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. Cyclin D1 mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of MyoD, myogenin, and Pax7 indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the in vivo healing process in muscular injury.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/deficiency , Muscle, Skeletal/physiology , Regeneration/genetics , Animals , Antigens, Differentiation/metabolism , Cell Membrane/metabolism , Cyclin D1/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Ki-67 Antigen/metabolism , Mice , Mice, Knockout , Muscle Development/genetics , Muscle, Skeletal/anatomy & histology , Muscle, Skeletal/cytology , Muscle, Skeletal/injuries , Proliferating Cell Nuclear Antigen/metabolism , RNA, Messenger/genetics , Wound Healing/geneticsABSTRACT
Wild-type p53-induced phosphatase 1 (Wip1), a phosphatase previously considered as an oncogene, has been implicated in the regulation of thymus homeostasis and neutrophil maturation. However, the role of Wip1 in B-cell development is unknown. We show that Wip1-deficient mice exhibit a significant reduction of B-cell numbers in the bone marrow, peripheral blood, and spleen. A reciprocal transplantation approach revealed a cell-intrinsic defect in early B-cell precursors caused by Wip1 deficiency. Further experiments revealed that Wip1 deficiency led to a sustained activation of p53 in B cells, which led to increased level of apoptosis in the pre-B-cell compartment. Notably, the impairment of B-cell development in Wip1-deficient mice was completely rescued by genetic ablation of p53, but not p21. Therefore, loss of Wip1 phosphatase induces a p53-dependent, but p21-independent, mechanism that impairs B-cell development by enhancing apoptosis in early B-cell precursors. Moreover, Wip1 deficiency exacerbated a decline in B-cell development caused by aging as evidenced in mice with aging and mouse models with serial competitive bone marrow transplantation, respectively. Our present data indicate that Wip1 plays a critical role in maintaining antigen-independent B-cell development in the bone marrow and preventing an aging-related decline in B-cell development.
Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Phosphoprotein Phosphatases/metabolism , Tumor Suppressor Protein p53/metabolism , Aging/immunology , Aging/metabolism , Aging/pathology , Animals , Apoptosis , B-Lymphocytes/immunology , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cell Differentiation , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Female , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphoprotein Phosphatases/deficiency , Phosphoprotein Phosphatases/genetics , Precursor Cells, B-Lymphoid/cytology , Precursor Cells, B-Lymphoid/immunology , Precursor Cells, B-Lymphoid/metabolism , Protein Phosphatase 2C , Signal TransductionABSTRACT
Cell reprogramming technology has allowed the in vitro control of cell fate transition, thus allowing for the generation of highly desired cell types to recapitulate in vivo developmental processes and architectures. However, the precise molecular mechanisms underlying the reprogramming process remain to be defined. Here, we show that depleting p53 and p21, which are barriers to reprogramming, yields a high reprogramming efficiency. Deletion of these factors results in a distinct mitochondrial background with low expression of oxidative phosphorylation subunits and mitochondrial fusion proteins, including mitofusin 1 and 2 (Mfn1/2). Importantly, Mfn1/2 depletion reciprocally inhibits the p53-p21 pathway and promotes both the conversion of somatic cells to a pluripotent state and the maintenance of pluripotency. Mfn1/2 depletion facilitates the glycolytic metabolic transition through the activation of the Ras-Raf and hypoxia-inducible factor 1α (HIF1α) signaling at an early stage of reprogramming. HIF1α is required for increased glycolysis and reprogramming by Mfn1/2 depletion. Taken together, these results demonstrate that Mfn1/2 constitutes a new barrier to reprogramming, and that Mfn1/2 ablation facilitates the induction of pluripotency through the restructuring of mitochondrial dynamics and bioenergetics.
Subject(s)
Cellular Reprogramming , Mitochondria/metabolism , Animals , Cell Line , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/genetics , GTP Phosphohydrolases/antagonists & inhibitors , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mice , Mice, Knockout , Mitochondrial Dynamics , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , raf Kinases/metabolismABSTRACT
We previously reported that the functional deletion of p21, a cyclin-dependent kinase inhibitor, in mice attenuated renal cell senescence in streptozotocin (STZ)-induced type 1 diabetic mice. In the present study, we investigated the effect of iron chelation on renal cell senescence and inflammation in the type 1 diabetic kidney. STZ-treated mice showed increase in iron accumulation, tubular cell senescence and macrophage infiltration at week 28 in the kidney. Administering deferasirox, which removes only dietary iron, significantly attenuated iron accumulation in proximal tubules and the number of infiltrating F4/80-positive cells without effecting blood glucose, hematocrit or hemoglobin levels. In contrast however, deferasirox did not influence renal cell senescence. The lack of p21 decreased the renal tubular iron accumulation and did not change tubular cell senescence. Interestingly, the STZ-treated animals showed an increase in p16, another cyclin-dependent kinase inhibitor. The results suggest that type 1 diabetes increases renal tubular iron accumulation and macrophage infiltration through a p21-dependent mechanism, and that the chelation of dietary iron attenuates these responses.
Subject(s)
Benzoates/pharmacology , Diabetes Mellitus, Type 1/complications , Diabetic Nephropathies/drug therapy , Iron Chelating Agents/pharmacology , Iron, Dietary , Iron/metabolism , Macrophages/drug effects , Triazoles/pharmacology , Animals , Benzoates/therapeutic use , Cellular Senescence/drug effects , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/genetics , Deferasirox , Diabetic Nephropathies/immunology , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Gene Knockout Techniques , Iron Chelating Agents/therapeutic use , Kidney/drug effects , Kidney/immunology , Kidney/metabolism , Kidney/pathology , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Triazoles/therapeutic useABSTRACT
Cyclin-dependent kinase inhibitors p21(Cip1) (CDKN1A) and p27(Kip1) (CDKN1B) are expressed in Leydig cells. Previously, we reported that Cdkn1b knockout in the mouse led to increased Leydig cell proliferative capacity and lower steroidogenesis. However, the relative importance of CDKN1A and CDKN1B in these regulations was unclear. In the present study, we examined the relative importance of CDKN1A and CDKN1B in regulation of Leydig cell proliferation and steroidogenesis by whole-body knockout of CDKN1A (Cdkn1a(-/-)) and CDKN1A/CDKN1B double knockout (DBKO). The cell number, 5-bromo-2-deoxyuridine incorporation rate, steroidogenesis, and steroidogenic enzyme mRNA levels and activities of Leydig cells were compared among wild-type (WT), Cdkn1a(-/-), and DBKO mice. Relative to WT mice, Leydig cell number per testis was doubled in the DBKO and unchanged in the Cdkn1a(-/-) mice. Testicular testosterone levels and mRNA levels for luteinizing hormone receptor (Lhcgr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), 17alpha-hydroxylase/17,20-lyase (Cyp17a1), and 17beta-hydroxysteroid dehydrogenase 3 (Hsd17b3) and their respective proteins were significantly lower in the DBKO mice. However, testicular testosterone level was unchanged in the Cdkn1a(-/-) mice, although Lhcgr mRNA levels were significantly lower relative to those in the WT control. We conclude that Cdkn1a(-/-) did not increase Leydig cell numbers (although a defect of Leydig cell function was noted), whereas DBKO caused a significant increase of Leydig cell numbers but a decrease of steroidogenesis.