DNA damage signaling triggers degradation of histone methyltransferases through APC/C(Cdh1) in senescent cells.

Both the DNA damage response (DDR) and epigenetic mechanisms play key roles in the implementation of senescent phenotypes, but very little is known about how these two mechanisms are integrated to establish senescence-associated gene expression. Here we show that, in senescent cells, the DDR induces proteasomal degradation of G9a and GLP, major histone H3K9 mono- and dimethyltransferases, through Cdc14B- and p21(Waf1/Cip1)-dependent activation of APC/C(Cdh1) ubiquitin ligase, thereby causing a global decrease in H3K9 dimethylation, an epigenetic mark for euchromatic gene silencing. Interestingly, induction of IL-6 and IL-8, major players of the senescence-associated secretory phenotype (SASP), correlated with a decline of H3K9 dimethylation around the respective gene promoters and knockdown of Cdh1 abolished IL-6/IL-8 expression in senescent cells, suggesting that the APC/C(Cdh1)-G9a/GLP axis plays crucial roles in aspects of senescent phenotype. These findings establish a role for APC/C(Cdh1) and reveal how the DDR integrates with epigenetic processes to induce senescence-associated gene expression.

Expression of Ror2 Associated with Fibrosis of the Submandibular Gland.

The submandibular gland (SMG) is one of the major salivary glands that play important roles for variety of physiological functions, such as digestion of foods, prevention of infection, and lubrication of the mouth. Dysfunction of the SMG, often associated with a salivary inflammation, adversely influences a person's quality of life. However, the mechanism underlying inflammation-driven dysfunction of the SMG is largely unknown. Here, we used a mouse model in which the main excretory duct of the SMG is ligated unilaterally to induce inflammation of the gland and examined the expression of Wnt5a, Ror1 and Ror2 genes, encoding Wnt5a ligand and its cognate receptors, which have been implicated in tissue damage or inflammatory responses in variety of tissues. We show that expression levels of Ror1, Ror2, and Wnt5a are increased in the ligated SMG undergoing interstitial fibrosis, which is accompanied by robust expression of fibrosis-associated genes, such as TGF-ß1, TNF-α, IL-1ß, and MMP-2. Increased immunostaining signal of Ror2 was detected in the fibrotic tissues with abundant accumulation of fibroblasts and collagen fibers in the ligated SMG, suggesting that Ror2-mediated signaling might be activated in response to tissue damage and associated with progression of fibrosis in the SMG.Key words: submandibular gland, Ror2, Wnt5a, fibrosis, inflammation.

Critical role of Frizzled1 in age-related alterations of Wnt/ß-catenin signal in myogenic cells during differentiation.

Activation of Wnt/ß-catenin signal in muscle satellite cells (mSCs) of aged mice during myogenic differentiation has been appreciated as an important age-related feature of the skeletal muscles, resulting in impairment of their regenerative ability following muscle injury. However, it remains elusive about molecules involved in this age-related alteration of Wnt/ß-catenin signal in myogenic cells. To clarify this issue, we carried out expression analyses of Wnt receptor genes using real-time RT-PCR in mSCs isolated from the skeletal muscles of young and aged mice. Here, we show that expression of Frizzled1 (Fzd1) was detected at high levels in mSCs of aged mice. Higher expression levels of Fzd1 were also detected in mSC-derived myogenic cells from aged mice and associated with activation of Wnt/ß-catenin signal during their myogenic differentiation in vitro. We also provide evidence that suppressed expression of Fzd1 in myogenic cells from aged mice results in a significant increase in myogenic differentiation, and its forced expression in those from young mice results in its drastic inhibition. These findings indicate the critical role of Fzd1 in altered myogenic differentiation associated with aging.

Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence.

The p16(INK4a) cyclin-dependent kinase inhibitor has a key role in establishing stable G1 cell-cycle arrest through activating the retinoblastoma (Rb) tumour suppressor protein pRb in cellular senescence. Here, we show that the p16(INK4a) /Rb-pathway also cooperates with mitogenic signals to induce elevated intracellular levels of reactive oxygen species (ROS), thereby activating protein kinase Cdelta (PKCdelta) in human senescent cells. Importantly, once activated by ROS, PKCdelta promotes further generation of ROS, thus establishing a positive feedback loop to sustain ROS-PKCdelta signalling. Sustained activation of ROS-PKCdelta signalling irreversibly blocks cytokinesis, at least partly through reducing the level of WARTS (also known as LATS1), a mitotic exit network (MEN) kinase required for cytokinesis, in human senescent cells. This irreversible cytokinetic block is likely to act as a second barrier to cellular immortalization ensuring stable cell-cycle arrest in human senescent cells. These results uncover an unexpected role for the p16(INK4a)-Rb pathway and provide a new insight into how senescent cell-cycle arrest is enforced in human cells.

Bmi-1 regulates mucin levels and mucin O-glycosylation in the submandibular gland of mice.

Mucins, the major components of salivary mucus, are large glycoproteins abundantly modified with O-glycans. Mucins present on the surface of oral tissues contribute greatly to the maintenance of oral hygiene by selectively adhering to the surfaces of microbes via mucin O-glycans. However, due to the complex physicochemical properties of mucins, there have been relatively few detailed analyses of the mechanisms controlling the expression of mucin genes and the glycosyltransferase genes involved in glycosylation. Analysis performed using supported molecular matrix electrophoresis, a methodology developed for mucin analysis, and knockout mice without the polycomb group protein Bmi-1 revealed that Bmi-1 regulates mucin levels in the submandibular gland by suppressing the expression of the mucin Smgc gene, and that Bmi-1 also regulates mucin O-glycosylation via suppression of the glycosyltransferase Gcnt3 gene in the submandibular gland.

Alteration of mucins in the submandibular gland during aging in mice.

OBJECTIVE: Mucins are large glycosylated glycoproteins that are produced in the salivary glands, and their changes may contribute to the development of xerostomia due to aging and the accompanying deterioration of oral hygiene. This study aimed to characterize the changes in the mucins produced in submandibular gland (SMG) during the aging process. METHODS: SMG mucins derived from mice of each age were separated using supported molecular matrix electrophoresis (SMME). Subsequently, the membranes were stained with Alcian blue (AB) or blotted with MAL-II lectin. The SMME membranes stained with AB were subjected to densitometric analysis and glycan analysis. The detailed structures of O-glycan were investigated by tandem mass spectrometry (MS/MS). RESULTS: The SMG of mice secreted three mucins with different glycan profiles: age-specific mucin, youth-specific mucin, and a mucin expressed throughout life, and the expression patterns of these mucins change during aging. Additionally, age-specific mucin began to be detected at about 12 months of age. A mucin expressed throughout life and age-specific mucin had the same mass of major glycans but different structures. Furthermore, the proportion of mucin glycan species expressed throughout life changed during the aging process, and aging tended to decrease the proportion of fucosylated glycans and increase the proportion of sialoglycans. CONCLUSION: There are three secretory mucins with different glycan profiles in the SMG of mice, and their expression patterns change according to the period of the aging process. The proportion of glycan species of mucin expressed throughout life also changes during the aging process.

Reduction of total E2F/DP activity induces senescence-like cell cycle arrest in cancer cells lacking functional pRB and p53.

E2F/DP complexes were originally identified as potent transcriptional activators required for cell proliferation. However, recent studies revised this notion by showing that inactivation of total E2F/DP activity by dominant-negative forms of E2F or DP does not prevent cellular proliferation, but rather abolishes tumor suppression pathways, such as cellular senescence. These observations suggest that blockage of total E2F/DP activity may increase the risk of cancer. Here, we provide evidence that depletion of DP by RNA interference, but not overexpression of dominant-negative form of E2F, efficiently reduces endogenous E2F/DP activity in human primary cells. Reduction of total E2F/DP activity results in a dramatic decrease in expression of many E2F target genes and causes a senescence-like cell cycle arrest. Importantly, similar results were observed in human cancer cells lacking functional p53 and pRB family proteins. These findings reveal that E2F/DP activity is indeed essential for cell proliferation and its reduction immediately provokes a senescence-like cell cycle arrest.

A rapid separation and characterization of mucins from mouse submandibular glands by supported molecular matrix electrophoresis.

Mucins are heavily glycosylated proteins with high molecular mass, and are involved in various diseases including infection, inflammation, and cancer. As easy separation method, such as gel electrophoresis, however, does not exist for mucins, due to their large molecular sizes and heterogeneities. In 2009, we published a supported molecular matrix electrophoresis (SMME) method that can be used to characterize mucins. For SMME analysis, mucins have been enriched by ultrafiltration of trypsin digests using a 100 KDa cutoff filter. However, this enrichment results in a loss of protein identification capability using proteomic approaches. In this study, we describe a simple enrichment of mucins without trypsinization for SMME analysis. The enrichment was developed using a porcine submandibular gland and then was applied to study and compare mouse submandibular glands between young and aged mice. From mouse submandibular glands, hyaluronic acid and some mucins were observed by SMME. One of the mucins was identified as MUC10 by proteomic analysis of the band on the SMME membrane and immunostaining using anti-MUC10 antibody. A major O-glycan of MUC10 was determined to be NeuAcα2-3Galß1-3GalNAc. Furthermore, our experiments revealed that the concentrations of these molecules were lower in aged mice than in young mice, and that an unknown mucin-like molecule was detected only from the aged mouse submandibular gland.

A sialo-oligosaccharide-rich mucin-like molecule specifically detected in the submandibular glands of aged mice.

OBJECTIVES: Mucins are heavily glycosylated large glycoproteins produced in the salivary glands that contribute to salivary viscosity. This study aimed to characterize age-related changes in mucin production in mouse submandibular salivary gland (SMG). METHODS: The paraffin sections of the SMGs of the young and aged mice were stained with HE or Alcian blue (AB). SMGs mucins derived from the young and aged mice were separated using supported molecular matrix electrophoresis (SMME). After SMME, the membranes were stained with AB and subsequent glycan analysis or subjected to immunoblotting. The expression of 18 mucin genes and 4 sialyltransferase genes in the young and aged SMGs were determined by qPCR. The neuraminidase activity in the SMG homogenates was determined using Neuraminidase Assay Kit. RESULTS: The mouse SMG is more strongly stained by AB with increasing age. On SMME, a characteristic band not found in the young SMG is detected in aged SMG. Based on migration position and the MALDI MS, the band that appeared specifically with aging was determined to be acidic mucin. Additionally, most glycans of this acidic mucin were sialo-oligosaccharides. Furthermore, there was an increase in the expression of sialyltransferase genes ST6GalNAc I and ST6GalNAc II, but not a decrease in neuraminidase activity, in the SMG of aged mice. CONCLUSION: A sialomucin or sialylated mucin-like molecule not found in the SMGs of young WT mice is expressed in aged WT mice. The increase in the sialo-oligosaccharide content in this aging-associated molecule may be attributed to the increased expression of the sialyltransferase genes.

Generation of aberrant transcripts of and free DNA ends in zebrafish no tail gene.

The zebrafish no tail gene (ntl) is indispensable for the formation of the notochord and the tail structure. In a wild-type zebrafish population, we occasionally observed adult zebrafish with a narrow or no tailfin. This led us to examine the hypothesis that the activity of ntl was somehow genetically unstable. Here we present two findings regarding the gene. First, approximately 3% of ntl transcripts were aberrant; most of them carried deletions at various positions. Second, free, DNA double-stranded ends (DSEs) were formed at an AT dinucleotide repeat in ntl. DSEs were also generated in another zebrafish gene, noggin2 (nog2). DSEs in ntl and nog2 had common characteristics, which suggested that the AT repeats in these genes elicited DSEs by blocking progression of the replication.

Dysregulation of the Bmi-1/p16(Ink4a) pathway provokes an aging-associated decline of submandibular gland function.

Bmi-1 prevents stem cell aging, at least partly, by blocking expression of the cyclin-dependent kinase inhibitor p16(Ink4a) . Therefore, dysregulation of the Bmi-1/p16(Ink4a) pathway is considered key to the loss of tissue homeostasis and development of associated degenerative diseases during aging. However, because Bmi-1 knockout (KO) mice die within 20 weeks after birth, it is difficult to determine exactly where and when dysregulation of the Bmi-1/p16(Ink4a) pathway occurs during aging in vivo. Using real-time in vivo imaging of p16(Ink4a) expression in Bmi-1-KO mice, we uncovered a novel function of the Bmi-1/p16(Ink4a) pathway in controlling homeostasis of the submandibular glands (SMGs), which secrete saliva into the oral cavity. This pathway is dysregulated during aging in vivo, leading to induction of p16(Ink4a) expression and subsequent declined SMG function. These findings will advance our understanding of the molecular mechanisms underlying the aging-related decline of SMG function and associated salivary gland hypofunction, which is particularly problematic among the elderly.

Ablation of the p16(INK4a) tumour suppressor reverses ageing phenotypes of klotho mice.

The p16(INK4a) tumour suppressor has an established role in the implementation of cellular senescence in stem/progenitor cells, which is thought to contribute to organismal ageing. However, since p16(INK4a) knockout mice die prematurely from cancer, whether p16(INK4a) reduces longevity remains unclear. Here we show that, in mutant mice homozygous for a hypomorphic allele of the α-klotho ageing-suppressor gene (kl(kl/kl)), accelerated ageing phenotypes are rescued by p16(INK4a) ablation. Surprisingly, this is due to the restoration of α-klotho expression in kl(kl/kl) mice and does not occur when p16(INK4a) is ablated in α-klotho knockout mice (kl(-/-)), suggesting that p16(INK4a) is an upstream regulator of α-klotho expression. Indeed, p16(INK4a) represses α-klotho promoter activity by blocking the functions of E2Fs. These results, together with the observation that the expression levels of p16(INK4a) are inversely correlated with those of α-klotho throughout ageing, indicate that p16(INK4a) plays a previously unrecognized role in downregulating α-klotho expression during ageing.

The p16INK4a-RB pathway: molecular link between cellular senescence and tumor suppression.

The p16INK4a tumor suppressor protein functions as an inhibitor of CDK4 and CDK6, the D-type cyclin-dependent kinases that initiate the phosphorylation of the retinoblastoma tumor suppressor protein, RB. Thus, p16INK4a has the capacity to arrest cells in the G1-phase of the cell cycle and its probable physiological role is in the implementation of irreversible growth arrest termed cellular senescence. Cellular senescence is a state of permanent growth arrest that can be induced by a variety of stresses such as DNA-damage and aberrant mitogenic signaling in human primary cells. In contrast to normal cells, the function of the p16INK4a gene or its downstream mediators is frequently deregulated in many types of human cancers, illustrating the importance of cellular senescence in tumor suppression. Here we discuss the molecular mechanisms that direct cellular senescence and reveal its potential for tumor suppression.

Real-time in vivo imaging of p16gene expression: a new approach to study senescence stress signaling in living animals.

Oncogenic proliferative signals are coupled to a variety of growth inhibitory processes. In cultured primary human fibroblasts, for example, ectopic expression of oncogenic Ras or its downstream mediator initiates cellular senescence, the state of irreversible cell cycle arrest, through up-regulation of cyclin-dependent kinase (CDK) inhibitors, such as p16(INK4a). To date, much of our current knowledge of how human p16(INK4a )gene expression is induced by oncogenic stimuli derives from studies undertaken in cultured primary cells. However, since human p16(INK4a )gene expression is also induced by tissue culture-imposed stress, it remains unclear whether the induction of human p16(INK4a )gene expression in tissue-cultured cells truly reflects an anti-cancer process or is an artifact of tissue culture-imposed stress. To eliminate any potential problems arising from tissue culture imposed stress, we have recently developed a bioluminescence imaging (BLI) system for non-invasive and real-time analysis of human p16(INK4a )gene expression in the context of a living animal. Here, we discuss the molecular mechanisms that direct p16(INK4a )gene expression in vivo and its potential for tumor suppression.

Intrinsic cooperation between p16INK4a and p21Waf1/Cip1 in the onset of cellular senescence and tumor suppression in vivo.

Although the p16(INK4a) and p21Waf1/Cip1 cyclin-dependent kinase (CDK) inhibitors are known to play key roles in cellular senescence in vitro, their roles in senescence remain rather poorly understood in vivo. This situation is partly due to the possibility of compensatory effect(s) between p16INK4a and p21Waf1/Cip1 or to the upregulation of functionally related CDK inhibitors. To directly address the cooperative roles of p16INK4a and p21Waf1/Cip1 in senescence in vivo, we generated a mouse line simply lacking both p16INK4a and p21Waf1/Cip1 genes [double-knockout (DKO)]. Mouse embryonic fibroblasts (MEF) derived from DKO mice displayed no evidence of cellular senescence when cultured serially in vitro. Moreover, DKO MEFs readily escaped Ras-induced senescence and overrode contact inhibition in culture. This was not the case in MEFs lacking either p16INK4a or p21Waf1/Cip1, indicating that p16(INK4a) and p21Waf1/Cip1 play cooperative roles in cellular senescence and contact inhibition in vitro. Notably, we found the DKO mice to be extremely susceptible to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin carcinogenesis that involves oncogenic mutation of the H-ras gene. Mechanistic investigations suggested that the high incidence of cancer in DKO mice likely reflected a cooperative effect of increased benign skin tumor formation caused by p21Waf1/Cip1 loss, with increased malignant conversion of benign skin tumors caused by p16(INK4a) loss. Our findings establish an intrinsic cooperation between p16INK4a and p21Waf1/Cip1 in the onset of cellular senescence and tumor suppression in vivo.

Real-time in vivo imaging of p16Ink4a reveals cross talk with p53.

Expression of the p16(Ink4a) tumor suppressor gene, a sensor of oncogenic stress, is up-regulated by a variety of potentially oncogenic stimuli in cultured primary cells. However, because p16(Ink4a) expression is also induced by tissue culture stress, physiological mechanisms regulating p16(Ink4a) expression remain unclear. To eliminate any potential problems arising from tissue culture-imposed stress, we used bioluminescence imaging for noninvasive and real-time analysis of p16(Ink4a) expression under various physiological conditions in living mice. In this study, we show that oncogenic insults such as ras activation provoke epigenetic derepression of p16(Ink4a) expression through reduction of DNMT1 (DNA methyl transferase 1) levels as a DNA damage response in vivo. This pathway is accelerated in the absence of p53, indicating that p53 normally holds the p16(Ink4a) response in check. These results unveil a backup tumor suppressor role for p16(Ink4a) in the event of p53 inactivation, expanding our understanding of how p16(Ink4a) expression is regulated in vivo.

Visualizing the dynamics of p21(Waf1/Cip1) cyclin-dependent kinase inhibitor expression in living animals.

Although the role of p21(Waf1/Cip1) gene expression is well documented in various cell culture studies, its in vivo roles are poorly understood. To gain further insight into the role of p21(Waf1/Cip1) gene expression in vivo, we attempted to visualize the dynamics of p21(Waf1/Cip1) gene expression in living animals. In this study, we established a transgenic mice line (p21-p-luc) expressing the firefly luciferase under the control of the p21(Waf1/Cip1) gene promoter. In conjunction with a noninvasive bioluminescent imaging technique, p21-p-luc mice enabled us to monitor the endogenous p21(Waf1/Cip1) gene expression in vivo. By monitoring and quantifying the p21(Waf1/Cip1) gene expression repeatedly in the same mouse throughout its entire lifespan, we were able to unveil the dynamics of p21(Waf1/Cip1) gene expression in the aging process. We also applied this system to chemically induced skin carcinogenesis and found that the levels of p21(Waf1/Cip1) gene expression rise dramatically in benign skin papillomas, suggesting that p21(Waf1/Cip1) plays a preventative role(s) in skin tumor formation. Surprisingly, moreover, we found that the level of p21(Waf1/Cip1) expression strikingly increased in the hair bulb and oscillated with a 3-week period correlating with hair follicle cycle progression. Notably, this was accompanied by the expression of p63 but not p53. This approach, together with the analysis of p21(Waf1/Cip1) knockout mice, has uncovered a novel role for the p21(Waf1/Cip1) gene in hair development. These data illustrate the unique utility of bioluminescence imaging in advancing our understanding of the timing and, hence, likely roles of specific gene expression in higher eukaryotes.

Identification of a gene required for de novo DNA methylation of the zebrafish no tail gene.

The zebrafish no tail gene (ntl) is indispensable for tail and notochord development. We have shown previously that ntl is de novo methylated during early embryogenesis. To find the gene that de novo methylates ntl and understand the meaning of this methylation, we cloned seven genes that encode the conserved catalytic domain of methyltransferases. We found that injection of antisense morpholino oligonucleotides against one of them, termed dnmt7, into eggs significantly reduced the level of ntl methylation, although no apparent phenotype was induced by the injection. Inhibition of Dnmt7 activity did not change the level of genome-wide methylation nor did it affect de novo methylation of injected plasmid DNA, indicating that Dnmt7 specifically methylates ntl in the genome.

PCR-based cloning of an intronless zebrafish no tail gene.

The zebrafish no tail gene (ntl) is indispensable for the formation of the notochord and tail structure. Here, we show the presence of an intronless ntl gene in zebrafish, which we designated cryptail (ctl). ctl could not be found in any zebrafish genomic resources examined and was only just cloned by a PCR-based approach that relied on its lack of introns and homology to ntl. The amplifiable region of ctl was confined to the transcribed region of ntl. ctl thus appeared to have been generated by reverse transcription of ntl mRNA, like a processed pseudogene. ctl was very polymorphic even in individual fish, but had no missense mutations. This may suggest that ctl has a physiological function in zebrafish.

De novo DNA methylation at the CpG island of the zebrafish no tail gene.

The zebrafish no tail gene (ntl) is indispensable for the formation of the notochord and the tail structure. Here we showed that de novo DNA methylation occurred at the CpG island of ntl. The methylation started at the segmentation stage and continued after the larval stage. However, it occurred predominantly between 14 and 48 h postfertilization, which overlaps the period in which ntl expression disappears in the notochord and the tailbud. This inverse correlation, together with the methylation-associated formation of an inaccessible chromatin structure at the ntl CpG island region, suggested the involvement of the de novo methylation in ntl repression. Since no changes in methylation patterns were observed at the CpG islands of four other zebrafish genes, there must be a mechanism in zebrafish for specific methylation of the ntl CpG island.