Limited mitochondrial permeabilization causes DNA damage and genomic instability in the absence of cell death.

During apoptosis, the mitochondrial outer membrane is permeabilized, leading to the release of cytochrome c that activates downstream caspases. Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis. Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event. Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term "minority MOMP." Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death. Instead, this caspase activity leads to DNA damage that, in turn, promotes genomic instability, cellular transformation, and tumorigenesis. Our data demonstrate that, in contrast to its well-established tumor suppressor function, apoptosis also has oncogenic potential that is regulated by the extent of MOMP. These findings have important implications for oncogenesis following either physiological or therapeutic engagement of apoptosis.

IL-23 Promotes a Coordinated B Cell Germinal Center Program for Class-Switch Recombination to IgG2b in BXD2 Mice.

IL-23 promotes autoimmune disease, including Th17 CD4 T cell development and autoantibody production. In this study, we show that a deficiency of the p19 component of IL-23 in the autoimmune BXD2 (BXD2-p19-/- ) mouse leads to a shift of the follicular T helper cell program from follicular T helper (Tfh)-IL-17 to Tfh-IFN-γ. Although the germinal center (GC) size and the number of GC B cells remained the same, BXD2-p19-/- mice exhibited a lower class-switch recombination (CSR) in the GC B cells, leading to lower serum levels of IgG2b. Single-cell transcriptomics analysis of GC B cells revealed that whereas Ifngr1, Il21r, and Il4r genes exhibited a synchronized expression pattern with Cxcr5 and plasma cell program genes, Il17ra exhibited a synchronized expression pattern with Cxcr4 and GC program genes. Downregulation of Ighg2b in BXD2-p19-/- GC B cells was associated with decreased expression of CSR-related novel base excision repair genes that were otherwise predominantly expressed by Il17ra + GC B cells in BXD2 mice. Together, these results suggest that although IL-23 is dispensable for GC formation, it is essential to promote a population of Tfh-IL-17 cells. IL-23 acts indirectly on Il17ra + GC B cells to facilitate CSR-related base excision repair genes during the dark zone phase of GC B cell development.

Progressive Cellular Senescence Mediates Renal Dysfunction in Ischemic Nephropathy.

BACKGROUND: Peripheral vascular diseases may induce chronic ischemia and cellular injury distal to the arterial obstruction. Cellular senescence involves proliferation arrest in response to stress, which can damage neighboring cells. Renal artery stenosis (RAS) induces stenotic-kidney dysfunction and injury, but whether these arise from cellular senescenceand their temporal pattern remain unknown. METHODS: Chronic renal ischemia was induced in transgenic INK-ATTAC and wild type C57BL/6 mice by unilateral RAS, and kidney function (in vivo micro-MRI) and tissue damage were assessed. Mouse healthy and stenotic kidneys were analyzed using unbiased single-cell RNA-sequencing. To demonstrate translational relevance, cellular senescence was studied in human stenotic kidneys. RESULTS: Using intraperitoneal AP20187 injections starting 1, 2, or 4 weeks after RAS, selective clearance of cells highly expressing p16Ink4a attenuated cellular senescence and improved stenotic-kidney function; however, starting treatment immediately after RAS induction was unsuccessful. Broader clearance of senescent cells, using the oral senolytic combination dasatinib and quercetin, in C57BL/6 RAS mice was more effective in clearing cells positive for p21 (Cdkn1a) and alleviating renal dysfunction and damage. Unbiased, single-cell RNA sequencing in freshly dissociated cells from healthy and stenotic mouse kidneys identified stenotic-kidney epithelial cells undergoing both mesenchymal transition and senescence. As in mice, injured human stenotic kidneys exhibited cellular senescence, suggesting this process is conserved. CONCLUSIONS: Maladaptive tubular cell senescence, involving upregulated p16 (Cdkn2a), p19 (Cdkn2d), and p21 (Cdkn1a) expression, is associated with renal dysfunction and injury in chronic ischemia. These findings support development of senolytic strategies to delay chronic ischemic renal injury.

The inhibitory effect of human DEFA5 in growth of gastric cancer by targeting BMI1.

Defensins, a class of small cysteine-rich cationic polypeptides across cellular life, are identified as antimicrobial compounds that display direct antimicrobial and immune signaling activities that are involved in the host defense. In addition to their roles in the innate immune system, accumulating studies have reported that some members of defensins are expressed and involved in some cancer cells, such as colon cancer, colorectal cancer, lung cancer and renal cell carcinomas. However, the roles of α-Defensin 5 (DEFA5) in tumorigenesis and development remain unknown. In the present study, bioinformatics analysis and quantitative PCR results showed that the expression level of DEFA5 was dramatically downregulated in human gastric cancer. Overexpression of human DEFA5 in gastric cancer cell lines SGC7901 and BGC823 effectively diminished cell proliferation and reduced the colony forming ability. Moreover, DEFA5 overexpression induced cell cycle arrest by significantly increasing the number of G1-phase cells. Consistently, in vivo tumor formation experiments in nude mice showed the suppression of the tumor growth by DEFA5 overexpression, suggesting an inhibitory effect of DEFA5 in gastric cancer. Mechanistically, DEFA5 directly binds to BMI1, which subsequently decreased its binding at the CDKN2a locus and upregulated the expression of 2 cyclin-dependent kinase inhibitors, p16 and p19. Taken together, we concluded that DEFA5 showed an inhibitory effect in gastric cancer cell growth and may serve as a potential tumor suppressor in gastric cancer.

Phosphorylation-induced unfolding regulates p19INK4d during the human cell cycle.

Cell cycle progression is tightly regulated by cyclin-dependent kinases (CDKs). The ankyrin-repeat protein p19INK4d functions as a key regulator of G1/S transition; however, its molecular mode of action is unknown. Here, we combine cell and structural biology methods to unravel the mechanism by which p19INK4d controls cell cycle progression. We delineate how the stepwise phosphorylation of p19INK4d Ser66 and Ser76 by cell cycle-independent (p38) and -dependent protein kinases (CDK1), respectively, leads to local unfolding of the three N-terminal ankyrin repeats of p19INK4d This dissociates the CDK6-p19INK4d inhibitory complex and, thereby, activates CDK6. CDK6 triggers entry into S-phase, whereas p19INK4d is ubiquitinated and degraded. Our findings reveal how signaling-dependent p19INK4d unfolding contributes to the irreversibility of G1/S transition.

p19INK4d inhibits proliferation and enhances imatinib efficacy through BCR-ABL signaling pathway in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a kind of myeloproliferative disorder caused by a constitutively active BCR-ABL tyrosine kinase. Tyrosine kinase inhibitors (TKIs), imatinib and its derivatives, have achieved great progress in the treatment of CML. However, many CML patients do not respond to TKIs alone. p19INK4d, a cyclin-dependent kinase inhibitor, plays important roles in proliferation, DNA damage repair, apoptosis and cell differentiation, but its role in CML is unknown. Herein, we found that the expression of p19INK4d in CML patients was significantly lower than that in healthy controls. p19INK4d overexpression inhibits cell proliferation through cell cycle arrest, and cooperates with imatinib to inhibit CML more effectively in vitro and in vivo. Mechanistically, p19INK4d decreased the expression of BCR-ABL and its downstream molecules p-Mek1/2, moreover, the expression of Gli-1, c-myc, MUC1, Shh and TC48 also reduced significantly. Collectively, p19INK4d inhibits proliferation and enhances imatinib efficacy in the treatment of CML. These findings maybe have implications for developing potential targets to increase imatinib sensitivity for CML.

Development and regeneration of vestibular hair cells in mammals.

Vestibular sensation is essential for gaze stabilization, balance, and perception of gravity. The vestibular receptors in mammals, Type I and Type II hair cells, are located in five small organs in the inner ear. Damage to hair cells and their innervating neurons can cause crippling symptoms such as vertigo, visual field oscillation, and imbalance. In adult rodents, some Type II hair cells are regenerated and become re-innervated after damage, presenting opportunities for restoring vestibular function after hair cell damage. This article reviews features of vestibular sensory cells in mammals, including their basic properties, how they develop, and how they are replaced after damage. We discuss molecules that control vestibular hair cell regeneration and highlight areas in which our understanding of development and regeneration needs to be deepened.

Unexpected role for p19INK4d in posttranscriptional regulation of GATA1 and modulation of human terminal erythropoiesis.

Terminal erythroid differentiation is tightly coordinated with cell-cycle exit, which is regulated by cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors (CDKI), yet their roles in erythropoiesis remain to be fully defined. We show here that p19INK4d, a member of CDKI family, is abundantly expressed in erythroblasts and that p19INK4d knockdown delayed erythroid differentiation, inhibited cell growth, and led to increased apoptosis and generation of abnormally nucleated late-stage erythroblasts. Unexpectedly, p19INK4d knockdown did not affect cell cycle. Rather, it led to decreased expression of GATA1 protein. Importantly, the differentiation and nuclear defects were rescued by ectopic expression of GATA1. Because the GATA1 protein is protected by nuclear heat shock protein family (HSP) member HSP70, we examined the effects of p19INK4d knockdown on HSP70 and found that p19INK4d knockdown led to decreased expression of HSP70 and its nuclear localization. The reduced levels of HSP70 are the result of reduced extracellular signal-regulated kinase (ERK) activation. Further biochemical analysis revealed that p19INK4d directly binds to Raf kinase inhibitor PEBP1 and that p19INK4d knockdown increased the expression of PEBP1, which in turn led to reduced ERK activation. Thus we have identified an unexpected role for p19INK4d via a novel PEBP1-p-ERK-HSP70-GATA1 pathway. These findings are likely to have implications for improved understanding of disordered erythropoiesis.

Downregulated expression of microRNA-329 inhibits apoptosis of nigral dopaminergic neurons by regulating CDKN2D expression via the FoxO3a signaling pathway in rats with Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder due to the loss of dopaminergic neurons in the substantia nigra. This study focuses on the effect of microRNA-329 (miR-329) on nigral dopaminergic neurons in a rat model of PD via the FoxO3a signaling pathway by binding to CDKN2D. Brain tissues from the substantia nigra were taken from the rats in two groups. TUNEL staining was used to observe tyrosine hydroxylase (TH)-positive neurons. Nigral dopaminergic neurons were randomized into the normal, blank, negative control (NC), miR-329 mimics, miR-329 inhibitors, small interfering (siRNA)-CDKN2D, and miR-329 inhibitors + siRNA-CDKN2D groups. Expressions of miR-329, CDKN2D, FoxO3a, AKT, caspase-3 and Bcl-2 were determined using RT-qPCR and western blotting. Apoptosis rate of nigral dopaminergic neurons in 7 groups was determined by flow cytometry. Compared with the blank and NC groups, the miR-329 mimics group showed increased miR-329 and caspase-3 expressions as well as decreased expressions of CDKN2D, FoxO3a, AKT, and Bcl-2, the siRNA-CDKN2D group indicated enhanced expressions of caspase-3 and declined expressions of CDKN2D, FoxO3a, AKT, and Bcl-2, and the miR-329 inhibitors group revealed decreased miR-329 and caspase-3 expressions and increased expressions of CDKN2D, FoxO3a, AKT, and Bcl-2. The apoptosis rate of nigral dopaminergic neurons was significantly increased in the miR-329 mimics and siRNA-CDKN2D groups, but was decreased in the miR-329 inhibitors group. Our data suggested that downregulated miR-329 could inhibit apoptosis of nigral dopaminergic neurons in a rat model of PD by upregulating the expression of CDKN2D via the activation of the FoxO3a signaling pathway.

Bmi1 regulates murine intestinal stem cell proliferation and self-renewal downstream of Notch.

Genetic data indicate that abrogation of Notch-Rbpj or Wnt-ß-catenin pathways results in the loss of the intestinal stem cells (ISCs). However, whether the effect of Notch is direct or due to the aberrant differentiation of the transit-amplifying cells into post-mitotic goblet cells is unknown. To address this issue, we have generated composite tamoxifen-inducible intestine-specific genetic mouse models and analyzed the expression of intestinal differentiation markers. Importantly, we found that activation of ß-catenin partially rescues the differentiation phenotype of Rbpj deletion mutants, but not the loss of the ISC compartment. Moreover, we identified Bmi1, which is expressed in the ISC and progenitor compartments, as a gene that is co-regulated by Notch and ß-catenin. Loss of Bmi1 resulted in reduced proliferation in the ISC compartment accompanied by p16(INK4a) and p19(ARF) (splice variants of Cdkn2a) accumulation, and increased differentiation to the post-mitotic goblet cell lineage that partially mimics Notch loss-of-function defects. Finally, we provide evidence that Bmi1 contributes to ISC self-renewal.

Overexpression of WDFY2 inhibits prostate cancer cell growth and migration via inactivation of Akt pathway.

Prostate cancer is the most commonly diagnosed malignancy and is the second leading deadly reason among male cancer. WDFY2, which is found to be a cancer-specific fusion gene with CDKN2D in ovarian cancer, is a new gene with unknown function in carcinogenesis. In this study, we investigated the role of WDFY2 in prostate cancer development. We examined WDFY2 expression in human prostate tissue specimens and prostate cancer cell lines BPH-1, LNCaP, PC3, and DU-145. Overexpression of WDFY2 was performed to evaluate the role of WDFY2 in cell proliferation, migration, and colony formation of prostate cancer cells. We analyzed the clinical impact and prognosis of WDFY2 expression on the progress of prostate cancer through data from online datasets. Our results showed that WDFY2 had lower expression level in prostate tumors than in normal tissues. Overexpression of WDFY2 in prostate cancer cells DU145 and PC-3 led to the suppression of cancer cell migration and colony formation. Furthermore, we found that WDFY2 exerted its role by suppressing the activity of Akt pathway other than the epithelial-mesenchymal transition progression. In conclusion, we have uncovered WDFY2 as a tumor suppressor gene and a new potential biomarker for cancer progression. Our results showed that WDFY2 inhibited cancer cell colony formation and migration via suppressing Akt pathway, making it a potential new therapeutic target in prostate cancer.

CDKN2D-WDFY2 is a cancer-specific fusion gene recurrent in high-grade serous ovarian carcinoma.

Ovarian cancer is the fifth leading cause of cancer death in women. Almost 70% of ovarian cancer deaths are due to the high-grade serous subtype, which is typically detected only after it has metastasized. Characterization of high-grade serous cancer is further complicated by the significant heterogeneity and genome instability displayed by this cancer. Other than mutations in TP53, which is common to many cancers, highly recurrent recombinant events specific to this cancer have yet to be identified. Using high-throughput transcriptome sequencing of seven patient samples combined with experimental validation at DNA, RNA and protein levels, we identified a cancer-specific and inter-chromosomal fusion gene CDKN2D-WDFY2 that occurs at a frequency of 20% among sixty high-grade serous cancer samples but is absent in non-cancerous ovary and fallopian tube samples. This is the most frequent recombinant event identified so far in high-grade serous cancer implying a major cellular lineage in this highly heterogeneous cancer. In addition, the same fusion transcript was also detected in OV-90, an established high-grade serous type cell line. The genomic breakpoint was identified in intron 1 of CDKN2D and intron 2 of WDFY2 in patient tumor, providing direct evidence that this is a fusion gene. The parental gene, CDKN2D, is a cell-cycle modulator that is also involved in DNA repair, while WDFY2 is known to modulate AKT interactions with its substrates. Transfection of cloned fusion construct led to loss of wildtype CDKN2D and wildtype WDFY2 protein expression, and a gain of a short WDFY2 protein isoform that is presumably under the control of the CDKN2D promoter. The expression of short WDFY2 protein in transfected cells appears to alter the PI3K/AKT pathway that is known to play a role in oncogenesis. CDKN2D-WDFY2 fusion could be an important molecular signature for understanding and classifying sub-lineages among heterogeneous high-grade serous ovarian carcinomas.

p19-INK4d inhibits neuroblastoma cell growth, induces differentiation and is hypermethylated and downregulated in MYCN-amplified neuroblastomas.

Uncontrolled cell cycle entry, resulting from deregulated CDK-RB1-E2F pathway activity, is a crucial determinant of neuroblastoma cell malignancy. Here we identify neuroblastoma-suppressive functions of the p19-INK4d CDK inhibitor and uncover mechanisms of its repression in high-risk neuroblastomas. Reduced p19-INK4d expression was associated with poor event-free and overall survival and neuroblastoma risk factors including amplified MYCN in a set of 478 primary neuroblastomas. High MYCN expression repressed p19-INK4d mRNA and protein levels in different neuroblastoma cell models with conditional MYCN expression. MassARRAY and 450K methylation analyses of 105 primary neuroblastomas uncovered a differentially methylated region within p19-INK4d. Hypermethylation of this region was associated with reduced p19-INK4d expression. In accordance, p19-INK4d expression was activated upon treatment with the demethylating agent, 2'-deoxy-5-azacytidine, in neuroblastoma cell lines. Ectopic p19-INK4d expression decreased viability, clonogenicity and the capacity for anchorage-independent growth of neuroblastoma cells, and shifted the cell cycle towards the G1/0 phase. p19-INK4d also induced neurite-like processes and markers of neuronal differentiation. Moreover, neuroblastoma cell differentiation, induced by all-trans retinoic acid or NGF-NTRK1-signaling, activated p19-INK4d expression. Our findings pinpoint p19-INK4d as a neuroblastoma suppressor and provide evidence for MYCN-mediated repression and for epigenetic silencing of p19-INK4d by DNA hypermethylation in high-risk neuroblastomas.

p19INK4d is involved in the cellular senescence mechanism contributing to heterochromatin formation.

BACKGROUND: During evolution, organisms with renewable tissues have developed mechanisms to prevent tumorigenesis, including cellular senescence and apoptosis. Cellular senescence is characterized by a permanent cell cycle arrest triggered by both endogenous stress and exogenous stress. The p19INK4d, a member of the family of cyclin-dependent kinase inhibitors (INK4), plays an important role on cell cycle regulation and in the cellular DNA damage response. We hypothesize that p19INK4d is a potential factor involved in the onset and/or maintenance of the senescent state. METHODS: Senescence was confirmed by measuring the cell cycle arrest and the senescence-associated ß-galactosidase activity. Changes in p19INK4d expression and localization during senescence were determined by Western blot and immunofluorescence assays. Chromatin condensation was measured by microccocal nuclease digestion and histone salt extraction. RESULTS: The data presented here show for the first time that p19INK4d expression is up-regulated by different types of senescence. Changes in senescence-associated hallmarks were driven by modulation of p19 expression indicating a direct link between p19INK4d induction and the establishment of cellular senescence. Following a senescence stimulus, p19INK4d translocates to the nucleus and tightly associates with chromatin. Moreover, reduced levels of p19INK4d impair senescence-related global genomic heterochromatinization. Analysis of p19INK4d mRNA and protein levels in tissues from differently aged mice revealed an up-regulation of p19INK4d that correlates with age. CONCLUSION: We propose that p19INK4d participates in the cellular mechanisms that trigger senescence by contributing to chromatin compaction. GENERAL SIGNIFICANCE: This study provides novel insights into the dynamics process of cellular senescence, a central tumor suppressive mechanism.

CDK5-mediated phosphorylation of p19INK4d avoids DNA damage-induced neurodegeneration in mouse hippocampus and prevents loss of cognitive functions.

DNA damage, which perturbs genomic stability, has been linked to cognitive decline in the aging human brain, and mutations in DNA repair genes have neurological implications. Several studies have suggested that DNA damage is also increased in brain disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. However, the precise mechanisms connecting DNA damage with neurodegeneration remain poorly understood. CDK5, a critical enzyme in the development of the central nervous system, phosphorylates a number of synaptic proteins and regulates dendritic spine morphogenesis, synaptic plasticity and learning. In addition to these physiological roles, CDK5 has been involved in the neuronal death initiated by DNA damage. We hypothesized that p19INK4d, a member of the cell cycle inhibitor family INK4, is involved in a neuroprotective mechanism activated in response to DNA damage. We found that in response to genotoxic injury or increased levels of intracellular calcium, p19INK4d is transcriptionally induced and phosphorylated by CDK5 which provides it with greater stability in postmitotic neurons. p19INK4d expression improves DNA repair, decreases apoptosis and increases neuronal survival under conditions of genotoxic stress. Our in vivo experiments showed that decreased levels of p19INK4d rendered hippocampal neurons more sensitive to genotoxic insult resulting in the loss of cognitive abilities that rely on the integrity of this brain structure. We propose a feedback mechanism by which the neurotoxic effects of CDK5-p25 activated by genotoxic stress or abnormal intracellular calcium levels are counteracted by the induction and stabilization of p19INK4d protein reducing the adverse consequences on brain functions.

Nuclear translocation of p19INK4d in response to oxidative DNA damage promotes chromatin relaxation.

DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site.

Lung tumourigenesis in a conditional Cul4A transgenic mouse model.

Cullin4A (Cul4A) is a scaffold protein that assembles cullin-RING ubiquitin ligase (E3) complexes and regulates many cellular events, including cell survival, development, growth and cell cycle control. Our previous study suggested that Cul4A is oncogenic in vitro, but its oncogenic role in vivo has not been studied. Here, we used a Cul4A transgenic mouse model to study the potential oncogenic role of Cul4A in lung tumour development. After Cul4A over-expression was induced in the lungs for 32 weeks, atypical epithelial cells were observed. After 40 weeks, lung tumours were visible and were characterized as grade I or II adenocarcinomas. Immunohistochemistry (IHC) revealed decreased levels of Cul4A-associated proteins p21(CIP1) and tumour suppressor p19(ARF) in the lung tumours, suggesting that Cul4A regulated their expression in these tumours. Increased levels of p27(KIP1) and p16(INK4a) were also detected in these tumours. Moreover, the protein level of DNA replication licensing factor CDT1 was decreased. Genomic instability in the lung tumours was further analysed by the results from pericentrin protein expression and array comparative genomic hybridization analysis. Furthermore, knocking down Cul4A expression in lung cancer H2170 cells increased their sensitivity to the chemotherapy drug cisplatin in vitro, suggesting that Cul4A over-expression is associated with cisplatin resistance in the cancer cells. Our findings indicate that Cul4A is oncogenic in vivo, and this Cul4A mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A.

Increased Th17 cells in the tumor microenvironment is mediated by IL-23 via tumor-secreted prostaglandin E2.

Tumor cell-derived molecules such as cytokines and lipid mediators play a critical role in inducing chronic inflammation in the tumor microenvironment. We found that Th17 cells were increased in the peripheral blood, spleen, and tumor tissues of mammary gland tumor-bearing mice. The Th17 cell survival factor, IL-23, was also overexpressed in tumor tissues isolated from mice and human breast cancer patients. Soluble molecules secreted from breast tumor cells, but not normal breast epithelial cells, induced IL-23 protein secretion in dendritic cells via induction of p19 mRNA expression. Our data further indicate that tumor-secreted PGE2 through EP2 and EP4 receptors enhanced IL-23 p19 gene transcription through binding to the cAMP-response element in the p19 promoter. Blocking PGE2 synthesis by NS398, a COX2 inhibitor, abrogated the enhancement of p19 expression both in vitro and in vivo. Furthermore, blocking protein kinase A (PKA) by H89 completely abrogated the inductive effects of tumor-conditioned medium and PGE2 on p19 transcription, whereas the cAMP active analog, Forskolin, mimics the PGE2 effect. Taken together, our results indicate that tumor-secreted PGE2 induces IL-23, but not IL-12, production in the tumor microenvironment, leading to Th17 cell expansion. This inductive effect of PGE2 on IL-23 p19 transcription is mediated through cAMP/PKA signaling transduction pathway.

Varied manifestations of persistent hyperplastic primary vitreous with graded somatic mosaic deletion of a single gene.

PURPOSE: Persistent hyperplastic primary vitreous (PHPV) represents a developmental eye disease known to have diverse manifestations ranging from a trivial remnant of hyaloid vessels to a dense fibrovascular mass causing lens opacity and retinal detachment. PHPV can be modeled in mice lacking individual genes, but certain features of such models differ from the clinical realm. For example, mice lacking the Arf gene have uniformly severe disease with consistent autosomal recessive disease penetrance. We tested whether the graded somatic loss of Arf in a subset of cells in chimeric mice mimics the range of disease in a non-heritable manner. METHODS: Wild type ↔ Arf(-/-) mouse chimeras were generated by morulae fusion, and when the mice were 10 weeks old, fundoscopic, slit-lamp, and histological evaluations were performed. The relative fraction of cells of the Arf(-/-) lineage was assessed with visual, molecular genetic, and histological analysis. Objective quantification of various aspects of the phenotype was correlated with the genotype. RESULTS: Sixteen chimeras were generated and shown to have low, medium, and high contributions of Arf(-/-) cells to tail DNA, the cornea, and the retinal pigment epithelium (RPE), with excellent correlation between chimerism in the tail DNA and the RPE. Phenotypic differences (coat color and severity of eye disease) were evident, objectively quantified, and found to correlate with the contribution of Arf(-/-) cells to the RPE and tail-derived DNA, but not the cornea. CONCLUSIONS: Generating animals composed of different numbers of Arf(-/-) cells mimicked the range of disease severity observed in patients with PHPV. This establishes the potential for full manifestations of PHPV to be caused by somatic mutations of a single gene during development.

Cellular senescence occurred widespread to multiple selective sites in the fetal tissues and organs of mice.

Cellular senescence protects multicellular organisms from tissue overgrowth including cancer, and contributes to tissue ageing. With stable cell cycle arrests, cellular senescence has been mostly studied in the adult tissues of mammals. In the present study, we report widespread cellular senescence within certain time windows of late-phase normal development of mouse embryos. Using in situ senescence-associated ß-galactosidase (SA-ß-gal) staining, we showed SA-ß-gal activity in selected cell populations of the brain, stomach, interdigital webs, tail, ear, limbs and nasal mouth area on gestation day 14.5 of the mouse embryos. On day 18.5 of gestation, selected cells in the intestines and bone developmental areas showed SA-ß-gal activity. The chondrocytes in ossification zones were significantly marked by the activities of SA-ß-gal, p21, p15 and Hp1Y, suggesting activation of the cell cycle checkpoint by the p53 and Rb pathways, and development of senescence-associated heterochromatic foci. Throughout gestation days 14.5-18.5, the trophoblast cells in the labyrinth layer of the placentas also showed strong activities of SA-ß-gal, p53 and p21. Increased expressions of p19, p16 and Rb of the p16/Rb pathway, and reduced expressions of Ki67 were also observed in the placentas. Taken together, the present findings suggest that cellular senescence represents an essential mechanism at multiple sites including the fetal bone forming zones and placenta during mammalian embryonic development, playing potential roles in the full embryonic development of tissue growth and organ formation.