Human cell senescence as a DNA damage response

It has been established that telomere-dependent replicative senescence of human fibroblasts is stress-dependent. First, it was shown that telomere shortening, which is a major contributor to telomere uncapping, is stress-dependent to a significant degree. Second, the signalling pathway connecting telomere uncapping and replicative senescence appears to be the same as the one that is activated by DNA damage: uncapped telomeres activate signalling cascades involving the protein kinases ATM, ATR and, possibly, DNA-PK. Furthermore, phosphorylation of histone H2A.X facilitates the formation of DNA damage foci around uncapped telomeres, and this in turn activates downstream kinases Chk1 and Chk2 and, eventually, p53. It appears that this signalling pathway has to be maintained in order to keep cells in a senescent state. Thus, cellular senescence can be regarded as a permanently maintained DNA damage response state. This suggests that antibodies against DNA damage foci components might be useful markers for senescent cells in vivo.     

Hes1 is a critical but context-dependent mediator of canonical Notch signaling in lymphocyte development and transformation

Although canonical Notch signaling regulates multiple hematopoietic lineage decisions including T?cell and marginal zone B cell fate specification, the downstream molecular mediators of Notch function are largely unknown. We showed here that conditional inactivation of Hes1, a well-characterized Notch target gene, in adult murine bone marrow (BM) cells severely impaired T?cell development without affecting other Notch-dependent hematopoietic lineages such as marginal zone B cells. Competitive mixed BM chimeras, intrathymic transfer experiments, and in?vitro culture of BM progenitors on Delta-like-expressing stromal cells further demonstrated that Hes1 is required for T?cell lineage commitment, but dispensable for Notch-dependent thymocyte maturation through and beyond the beta selection checkpoint. Furthermore, our data strongly suggest that Hes1 is essential for the development and maintenance of Notch-induced T?cell acute lymphoblastic leukemia. Collectively, our studies identify Hes1 as a critical but context-dependent mediator of canonical Notch signaling in the hematopoietic system.     

Cardiac myosin-binding protein-C is a critical mediator of diastolic function

Diastolic dysfunction prominently contributes to heart failure with preserved ejection fraction (HFpEF). Owing partly to inadequate understanding, HFpEF does not have any effective treatments. Cardiac myosin-binding protein-C (cMyBP-C), a component of the thick filament of heart muscle that can modulate cross-bridge attachment/detachment cycling process by its phosphorylation status, appears to be involved in the diastolic dysfunction associated with HFpEF. In patients, cMyBP-C mutations are associated with diastolic dysfunction even in the absence of hypertrophy. cMyBP-C deletion mouse models recapitulate diastolic dysfunction despite in vitro evidence of uninhibited cross-bridge cycling. Reduced phosphorylation of cMyBP-C is also associated with diastolic dysfunction in patients. Mouse models of reduced cMyBP-C phosphorylation exhibit diastolic dysfunction while cMyBP-C phosphorylation mimetic mouse models show enhanced diastolic function. Thus, cMyBP-C phosphorylation mediates diastolic function. Experimental results of both cMyBP-C deletion and reduced cMyBP-C phosphorylation causing diastolic dysfunction suggest that cMyBP-C phosphorylation level modulates cross-bridge detachment rate in relation to ongoing attachment rate to mediate relaxation. Consequently, alteration in cMyBP-C regulation of cross-bridge detachment is a key mechanism that causes diastolic dysfunction. Regardless of the exact molecular mechanism, ample clinical and experimental data show that cMyBP-C is a critical mediator of diastolic function. Furthermore, targeting cMyBP-C phosphorylation holds potential as a future treatment for diastolic dysfunction.     

Docking protein SNT1 is a critical mediator of fibroblast growth factor signaling during Xenopus embryonic development.

The docking protein SNT1/FRS2 (fibroblast growth factor receptor substrate 2) is implicated in the transmission of extracellular signals from several growth factor receptors to the mitogen-activated protein (MAP) kinase signaling cascade, but its biological function during development is not well characterized. Here, we show that the Xenopus homolog of mammalian SNT1/FRS-2 (XSNT1) plays a critical role in the appropriate formation of mesoderm-derived tissue during embryogenesis. XSNT1 has an expression pattern that is quite similar to the fibroblast growth factor receptor-1 (FGFR1) during Xenopus development. Ectopic expression of XSNT1 markedly enhanced the embryonic defects induced by an activated FGF receptor, and increased the MAP kinase activity as well as the expression of a mesodermal marker in response to FGF receptor signaling. A loss-of-function study using antisense XSNT1 morpholino oligonucleotides (XSNT-AS) shows severe malformation of trunk and posterior structures. Moreover, XSNT-AS disrupts muscle and notochord formation, and inhibits FGFR-induced MAP kinase activation. In ectodermal explants, XSNT-AS blocks FGFR-mediated induction of mesoderm and the accompanying elongation movements. Our results indicate that XSNT1 is a critical mediator of FGF signaling and is required for early Xenopus development.     

Central amygdala ERK signaling pathway is critical to incubation of cocaine craving

Using a rat model of craving and relapse, we have previously found time-dependent increases in cue-induced cocaine seeking over the first months of withdrawal from cocaine, suggesting that drug craving incubates over time. Here, we explored the role of the amygdala extracellular signal-regulated kinase (ERK) signaling pathway in this incubation. Cocaine seeking induced by exposure to cocaine cues was substantially higher after 30 withdrawal days than after 1 withdrawal day. Exposure to these cues increased ERK phosphorylation in the central, but not the basolateral, amygdala after 30 d, but not 1 d, of withdrawal. After 30 d of withdrawal from cocaine, inhibition of central, but not basolateral, amygdala ERK phosphorylation decreased cocaine seeking. After 1 d of withdrawal, stimulation of central amygdala ERK phosphorylation increased cocaine seeking. Results suggest that the incubation of cocaine craving is mediated by time-dependent increases in the responsiveness of the central amygdala ERK pathway to cocaine cues.     

MEK/ERK signaling is a critical mediator for integrin-induced cell scattering in highly metastatic hepatocellular carcinoma cells

The human hepatocellular carcinoma (HCC)-derived cell line KYN-2 is thought to provide a good model for studying the molecular basis of invasion and metastasis of human HCC, because it often shows cell scattering in vitro and intrahepatic metastasis in vivo. We previously found that integrin-mediated extracellular signals inactivated E-cadherin in KYN-2, and caused loss of cell-cell contact with gain of cell motility, which is considered to be a critical step in the process of cancer cell invasion and metastasis. To further understand molecular mechanisms involved in biological aggressiveness of HCC, we investigated intracellular signaling involved in integrin-mediated scattering of KYN-2 cells. Cultured KYN-2 cells formed trabecular aggregates in suspension, but when adhering to integrin-stimulating substrata, they scattered according to phosphorylation of extracellular signal-regulated kinase (ERK). Upon treatment with ERK kinase (MEK) inhibitor PD98059, adhered KYN-2 cell scattering was inhibited, tight cell-to-cell contact was recovered, and both E-cadherin and actin filaments accumulated in the area of intercellular contact zone. In contrast, constitutively active MEK1-transfected KYN-2 cells showed reduced E-cadherin and actin filaments in the intercellular contact zone, showing a flattened phenotype with broad lamellipodia. Enforced signaling of MEK-ERK pathway in KYN-2 cells suppressed cadherin-mediated homotypic adhesion and increased the potential of cell motility. An antibody-based protein microarray analysis revealed that the cytoplasmic protein c-Cbl was significantly downregulated in MEK1-transfected KYN-2 cells, suggesting that c-Cbl might be a candidate downstream mediator of integrin/MEK/ERK-mediated cell scattering. In conclusion, cell scattering of the highly metastatic cell line KYN-2 is regulated through the integrin-MEK-ERK signaling cascade, suggesting that this molecular pathway may be critical in intrahepatic metastasis of human HCC.     

DNA damage, vascular senescence and atherosclerosis

Atherosclerosis, an intrinsically age-related disease, is attributed to an excessive inflammatory and fibroproliferative process that selectively affects arteries. However, premature atherosclerosis is a feature of several human diseases that are known to be defective in DNA repair pathways and characterised by predisposition to early onset of age-related diseases. Accordingly, there is a growing amount of data demonstrating that oxidative-stress-induced DNA damage and dysfunctional telomeres play an important role in the pathogenesis of atherosclerosis. This review examines the evidence that an activated DNA response pathway induced by both oxidative DNA damage and telomere dysfunction is the crucial mediator for vascular replicative or premature senescence via activation of the p21(cip1) (via p53) and p16(ink4) pathways. Prevention of DNA-damage-induced cellular vascular senescence may be a novel target for the clinical treatment of atherosclerosis.     

Tim-3 signaling pathway as a novel negative mediator in lipopolysaccharide-induced endotoxic shock

Sepsis is a complex clinical condition caused by a dysregulated immune response to an infection. However, the mechanism by which our immune system controls this amplified inflammation is largely unknown. In this study, we investigated whether Tim-3 pathway could serve as a negative mediator in lipopolysaccharide (LPS)-induced endotoxic shock. Our results showed that Tim-3 was expressed on CD4⁺ T cells, CD8⁺ T cells, and NK cells, and was significantly increased in the peritoneal cavity of septic mice. Tim-3 acted as a marker of immune exhaustion and Tim-3-positive T cells and NK cells had a lower interferon (IFN)-γ production. Furthermore, blockade of Tim-3 pathway significantly accelerated mortality in septic mice, while activation of this pathway prolonged survival time. In vitro administration of Tim-3 blocking antibody restored the release of IFN-γ from splenocytes and decreased splenocyte apoptosis, and increased levels of IFN-γ and tumor necrosis factor (TNF)-α were also detected in septic mice at 24 h post in vivo administration of the antibody. In contrast, activation of Tim-3 pathway prevented cell proliferation. Thus, Tim-3 signaling pathway acts as a novel negative mediator in LPS-induced endotoxic shock and could be a potential therapeutic target for the treatment of sepsis.     

Tollip is a critical mediator of cerebral ischaemia–reperfusion injury

Toll‐like receptor (TLR) signalling plays an important role in regulating cerebral ischaemia–reperfusion (I/R) injury. Toll‐interacting protein (Tollip) is an endogenous negative modulator of TLR signalling that is involved in several inflammatory diseases. Our previous study showed that Tollip inhibits overload‐induced cardiac remodelling. However, the role of Tollip in neurological disease remains unknown. In the present study, we proposed that Tollip might contribute to the progression of stroke and confirmed this hypothesis. We found that Tollip expression was significantly increased in I/R‐challenged brain tissue of humans, mice and rats in vivo and in primary neurons subjected to oxygen and glucose deprivation in vitro, indicating the involvement of Tollip in I/R injury. Next, using genetic approaches, we revealed that Tollip deficiency protects mice against I/R injury by attenuating neuronal apoptosis and inflammation, as demonstrated by the decreased expression of pro‐apoptotic and pro‐inflammatory genes and the increased expression of anti‐apoptotic genes. By contrast, neuron‐specific Tollip over‐expression exerted the opposite effect. Mechanistically, the detrimental effects of Tollip on neuronal apoptosis and inflammation following I/R injury were largely mediated by the suppression of Akt signalling. Additionally, to further support our findings, a Tollip knockout rat strain was generated via CRISPR‐Cas9‐mediated gene inactivation. The Tollip‐deficient rats were also protected from I/R injury, based on dramatic decreases in neuronal apoptosis and ischaemic inflammation through Akt activation. Taken together, our findings demonstrate that Tollip acts as a novel modulator of I/R injury by promoting neuronal apoptosis and ischaemic inflammation, which are largely mediated by suppression of Akt signalling. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

细胞衰老：阻碍癌基因致癌的机制

细胞衰老是生物体中普遍存在的一种永久性生长抑制现象,能够防止老化的或非正常细胞的进一步生长,对抗细胞的无限增殖能力而对机体起到保护作用。近年来,体内外研究表明,癌基因诱导的细胞衰老能够抑制肿瘤的发生,但一旦衰老机制缺陷,癌基因就可能刺激细胞无限增殖而导致恶性肿瘤,引起细胞衰老的机制目前还不是很清楚,但已发现ARF-P53和P16INK4 a-RB这两条途径的活化与细胞衰老的启动和调节过程密切相关。对细胞衰老的信号传导通路及其调节因子等方面的深入研究可能为肿瘤等重大疾病的预防和治疗提供帮助。     

Replicative senescence: a critical review

Human cells in culture have a limited proliferative capacity. After a period of vigorous proliferation, the rate of cell division declines and a number of changes occur in the cells including increases in size, in secondary lysosomes and residual bodies, nuclear changes and a number of changes in gene expression which provide biomarkers for senescence. Although human cells in culture have been used for over 40 years as models for understanding the cellular basis of aging, the relationship of replicative senescence to aging of the organism is still not clear. In this review, we discuss replicative senescence in the light of current information on signal transduction and mitogenesis, cell stress, apoptosis, telomere changes and finally we discuss replicative senescence as a model of aging in vivo.     

Tulp3 is a critical repressor of mouse hedgehog signaling

Precise regulation of the morphogen sonic hedgehog (Shh) and modulation of the Shh signaling pathway is required for proper specification of cell fate within the developing limbs and neural tube, and resultant tissue morphogenesis. Tulp3 (tubby‐like protein 3) is a protein of unknown function which has been implicated in nervous system development through gene knockout studies. We demonstrate here that mice lacking the Tulp3 gene develop abnormalities of both the neural tube and limbs consistent with improper regulation of Shh signaling. Tulp3^?/? embryos show expansion of Shh target gene expression and display a ventralization of neural progenitor cells in the caudal neural tube. We further show that Tulp3^?/?/Shh^?/? compound mutant embryos resemble Tulp3 mutants, and express Shh target genes in the neural tube and limbs which are not expressed in Shh^?/? embryos. This work uncovers a novel role for Tulp3 as a negative regulatory factor in the Hh pathway. Developmental Dynamics 238:1140–1149, 2009. © 2009 Wiley‐Liss, Inc.     

Increased expression of oncogene-induced senescence markers during cervical squamous cell cancer development

Purpose: To investigate the expression of p15^INK4b, p16^INK4a and p21^Waf1/Cip1 in specimens from cases of normal cervical epithelium (NCE), cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma (SCC), and to evaluate whether there is evidence implicating oncogene-induced senescence (OIS) in cervical squamous cell cancer development. Methods: The immunohistochemical expression of p15^INK4b, p16^INK4a and p21^Waf1/Cip1 were investigated in formalin-fixed paraffin-embedded specimens from 19 NCE, 51 CIN and 21 SCC cases, respectively. Comparisons among different groups for each marker were performed with Chi-square test. Results: The expression of p15^INK4b, p16^INK4a and p21^Waf1/Cip1 were significantly higher in both CIN and SCC compared to NCE. Furthermore, the expression of p15^INK4b and p21^Waf1/Cip1 was significantly higher in CIN П compared to CIN І, and these expressions were statistically higher in CIN Ш compared to CIN П, respectively. The p16^INK4a expression was significantly higher in CIN Ш compared to CIN І. Conclusions: The results suggested that the senescence programs mediated by p15^INK4b, p16^INK4a and p21^Waf1/Cip1 were activated during the stage of CIN and SCC, and demonstrated that senescence may play important role in preventing from NCE to SCC.     

SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway

The ATR (ATM [ataxia telangiectasia-mutated]- and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR–Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11–RAD50–NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway.     

Activation of p53/ATM-dependent DNA damage signaling pathway by shiga toxin in mammalian cells

In this report, we studied the role of DNA damage signaling pathway in shiga toxin (STX)-induced mammalian cell death. Shiga toxin 1 exhibited cytotoxic activity in different mammalian cells such as HeLa cells, mouse embryo fibroblasts, and Caco-2 cells (a human intestinal primary fibroblast cell line). STX-1 was found to induce the release of cytochrome c from the mitochondria, nuclear condensation, and fragmentation of chromosomal DNA. STX-1 activated DNA damage signaling as determined by induction of H2AX phosphorylation and cleavage of PARP. Inhibition of caspase-3 reduced STX-1-induced phosphorylation of H2AX and nuclear condensation. It was also found that STX-1-induced p53 expression, and activated ATM in mammalian cells. STX-1-induced nuclear condensation significantly reduced in p53-, and ATM-knockout cells suggesting an involvement of p53 and ATM in transducing signals produced by STX in inducing apoptosis in mammalian cells. This is the first demonstration of involvement of ATM/p53 in STX-inducing mammalian cell death.     

Hyphal differentiation induced via a DNA damage checkpoint-dependent pathway engaged in crosstalk with nutrient stress signaling in Schizosaccharomyces japonicus

DNA damage response includes DNA repair, nucleotide metabolism and even a control of cell fates including differentiation, cell death pathway or some combination of these. The responses to DNA damage differ from species to species. Here we aim to delineate the checkpoint pathway in the dimorphic fission yeast Schizosaccharomyces japonicus, where DNA damage can trigger a differentiation pathway that is a switch from a bidirectional yeast growth mode to an apical hyphal growth mode, and the switching is regulated via a checkpoint kinase, Chk1. This Chk1-dependent switch to hyphal growth is activated with even low doses of agents that damage DNA; therefore, we reasoned that this switch may depend on other genes orthologous to the components of the classical Sz. pombe Chk1-dependent DNA checkpoint pathway. As an initial test of this hypothesis, we assessed the effects of mutations in Sz. japonicus orthologs of Sz. pombe checkpoint genes on this switch from bidirectional to hyphal growth. The same set of DNA checkpoint genes was confirmed in Sz. japonicus. We tested the effect of each DNA checkpoint mutants on hyphal differentiation by DNA damage. We found that the Sz. japonicus hyphal differentiation pathway was dependent on Sz. japonicus orthologs of Sz. pombe checkpoint genes—_SP rad3, _SP rad26, _SP rad9, _SP rad1, _SP rad24, _SP rad25, _SP crb2, and _SP chk1—that function in the DNA damage checkpoint pathway, but was not dependent on orthologs of two Sz. pombe genes—_SP cds1 or _SP mrc1—that function in the DNA replication checkpoint pathway. These findings indicated that although the role of each component of the DNA damage checkpoint and DNA replication checkpoint is mostly same between the two fission yeasts, the DNA damage checkpoint was the only pathway that governed DNA damage-dependent hyphal growth. We also examined whether DNA damage checkpoint signaling engaged in functional crosstalk with other hyphal differentiation pathways because hyphal differentiation can also be triggered by nutritional stress. Here, we discovered genetic interactions that indicated that the cAMP pathway engaged in crosstalk with Chk1-dependent signaling.     

Toll-like receptor 2 signaling is a mediator of apoptosis in herpes simplex virus-infected microglia

Background  

Information regarding the response of brain cells to infection with herpes simplex virus (HSV)-1 is needed for a complete understanding of viral neuropathogenesis. We have recently demonstrated that microglial cells respond to HSV infection by producing a number of proinflammatory cytokines and chemokines through a mechanism involving Toll-like receptor 2 (TLR2). Following this cytokine burst, microglial cells rapidly undergo cell death by apoptosis. We hypothesized that TLR2 signaling might mediate the cell death process as well.