Nectin-4 regulates cellular senescence-associated enlargement of cell size.

Cellular senescence is defined as irreversible growth arrest induced by various stress, such as DNA damage and oxidative stress. Senescent cells exhibit various characteristic morphological changes including enlarged morphology. In our recent study, we identified Nectin-4 to be upregulated in cellular senescence by comparative transcriptomic analysis. However, there are few reports on the relationship between Nectin-4 and senescence. Therefore, we analyzed the function of Nectin-4 in senescence and its biological significance. When overexpressed with Nectin-4, the cells exhibited the enlarged cell morphology closely resembling senescent cells. In addition, the cell size enlargement during DNA damage-induced senescence was suppressed by knockdown of Nectin-4, while there were no significant changes in senescence induction. These results suggest that Nectin-4 is not involved in the regulation of senescence itself but contributes to the senescence-associated cell size increase. Furthermore, the Nectin-4-dependent cell size increase was found to be mediated by Src family kinase (SFK)/PI3 kinase (PI3K)/Rac1 pathway. To explore the functional consequences of cell size enlargement, we analyzed cell survival in Nectin-4-depleted senescent cells. Single-cell tracking experiments revealed that Nectin-4 knockdown induced apoptosis in senescent cells, and there is a strong positive correlation between cell size and survival rate. These results collectively indicate that Nectin-4 plays a causative role in the senescence-associated cell size enlargement via SFK/PI3K/Rac1, which can contribute to survival of senescent cells.

Integrin ß1 transduces the signal for LY6D-induced macropinocytosis and mediates senescence-inducing stress-evoked vacuole formation via FAK.

Cellular senescence is a highly stable cell-cycle arrest induced by DNA damage and various cellular stresses. Recently, we have revealed that lymphocyte antigen 6 complex, locus D (LY6D) is responsible for senescence-inducing stress-evoked vacuole formation through induction of Src family kinase (SFK)-mediated macropinocytosis. However, the signaling molecule(s) transducing the macropinocytosis signal from extracellular LY6D to the cytoplasmic SFK are unknown. In this study, we identified integrin ß1, a transmembrane signaling protein, as an interactor of LY6D by proteomic analysis and co-immunoprecipitation assays. Inhibition of integrin ß1 impaired LY6D-induced macropinocytosis, and integrin ß1 activated SFK through focal adhesion kinase to mediate macropinocytosis. These results indicate that integrin ß1 is a crucial mediator of the LY6D-induced vacuole formation in senescent cells.

Pathway Dependence of the Formation and Development of Prefibrillar Aggregates in Insulin B Chain.

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.

New Synthetic Lethality Re-Sensitizing Platinum-Refractory Cancer Cells to Cisplatin In Vitro: The Rationale to Co-Use PARP and ATM Inhibitors.

The pro-apoptotic tumor suppressor BIN1 inhibits the activities of the neoplastic transcription factor MYC, poly (ADP-ribose) polymerase-1 (PARP1), and ATM Ser/Thr kinase (ATM) by separate mechanisms. Although BIN1 deficits increase cancer-cell resistance to DNA-damaging chemotherapeutics, such as cisplatin, it is not fully understood when BIN1 deficiency occurs and how it provokes cisplatin resistance. Here, we report that the coordinated actions of MYC, PARP1, and ATM assist cancer cells in acquiring cisplatin resistance by BIN1 deficits. Forced BIN1 depletion compromised cisplatin sensitivity irrespective of Ser15-phosphorylated, pro-apoptotic TP53 tumor suppressor. The BIN1 deficit facilitated ATM to phosphorylate the DNA-damage-response (DDR) effectors, including MDC1. Consequently, another DDR protein, RNF8, bound to ATM-phosphorylated MDC1 and protected MDC1 from caspase-3-dependent proteolytic cleavage to hinder cisplatin sensitivity. Of note, long-term and repeated exposure to cisplatin naturally recapitulated the BIN1 loss and accompanying RNF8-dependent cisplatin resistance. Simultaneously, endogenous MYC was remarkably activated by PARP1, thereby repressing the BIN1 promoter, whereas PARP inhibition abolished the hyperactivated MYC-dependent BIN1 suppression and restored cisplatin sensitivity. Since the BIN1 gene rarely mutates in human cancers, our results suggest that simultaneous inhibition of PARP1 and ATM provokes a new BRCAness-independent synthetic lethal effect and ultimately re-establishes cisplatin sensitivity even in platinum-refractory cancer cells.

Riboflavin transporter SLC52A1, a target of p53, suppresses cellular senescence by activating mitochondrial complex II.

Cellular senescence is a state of permanent proliferative arrest induced by a variety of stresses, such as DNA damage. The transcriptional activity of p53 has been known to be essential for senescence induction. It remains unknown, however, whether among the downstream genes of p53, there is a gene that has antisenescence function. Our recent studies have indicated that the expression of SLC52A1 (also known as GPR172B/RFVT1), a riboflavin transporter, is up-regulated specifically in senescent cells depending on p53, but the relationship between senescence and SLC52A1 or riboflavin has not been described. Here, we examined the role of SLC52A1 in senescence. We found that knockdown of SLC52A1 promoted senescence phenotypes induced by DNA damage in tumor and normal cells. The senescence suppressive action of SLC52A1 was dependent on its riboflavin transport activity. Furthermore, elevation of intracellular riboflavin led to activation of mitochondrial membrane potential (MMP) mediated by the mitochondrial electron transport chain complex II. Finally, the SLC52A1-dependent activation of MMP inhibited the AMPK-p53 pathway, a central mediator of mitochondria dysfunction-related senescence. These results suggest that SLC52A1 contributes to suppress senescence through the uptake of riboflavin and acts downstream of p53 as a negative feedback mechanism to limit aberrant senescence induction.

Multistep Changes in Amyloid Structure Induced by Cross-Seeding on a Rugged Energy Landscape.

Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes, including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures, depending on species, each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new type of amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin seeds. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition analysis of the ultraviolet-visible absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation.

LY6D-induced macropinocytosis as a survival mechanism of senescent cells.

Although senescent cells display various morphological changes including vacuole formation, it is still unclear how these processes are regulated. We have recently identified the gene, lymphocyte antigen 6 complex, locus D (LY6D), to be upregulated specifically in senescent cells. LY6D is a glycosylphosphatidylinositol-anchored cell-surface protein whose function remains unknown. Here, we analyzed the functional relationship between LY6D and the senescence processes. We found that overexpression of LY6D induced vacuole formation and knockdown of LY6D suppressed the senescence-associated vacuole formation. The LY6D-induced vacuoles were derived from macropinocytosis, a distinct form of endocytosis. Furthermore, Src family kinases and Ras were found to be recruited to membrane lipid rafts in an LY6D-dependent manner, and inhibition of their activity impaired the LY6D-induced macropinocytosis. Finally, reduction of senescent-cell survival induced by glutamine deprivation was recovered by albumin supplementation to the culture media in an LY6D-dependent manner. Because macropinocytosis acts as an amino acid supply route, these results suggest that LY6D-mediated macropinocytosis contributes to senescent-cell survival through the incorporation of extracellular nutrients.

The complexity of intercellular localisation of alkaloids revealed by single-cell metabolomics.

Catharanthus roseus is a medicinal plant well known for producing bioactive compounds such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). Although the leaves of this plant are the main source of these antitumour drugs, much remains unknown on how TIAs are biosynthesised from a central precursor, strictosidine, to various TIAs in planta. Here, we have succeeded in showing, for the first time in leaf tissue of C. roseus, cell-specific TIAs localisation and accumulation with 10 µm spatial resolution Imaging mass spectrometry (Imaging MS) and live single-cell mass spectrometry (single-cell MS). These metabolomic studies revealed that most TIA precursors (iridoids) are localised in the epidermal cells, but major TIAs including serpentine and vindoline are localised instead in idioblast cells. Interestingly, the central TIA intermediate strictosidine also accumulates in both epidermal and idioblast cells of C. roseus. Moreover, we also found that vindoline accumulation increases in laticifer cells as the leaf expands. These discoveries highlight the complexity of intercellular localisation in plant specialised metabolism.

Autophosphorylation of MAP kinase disables the MAPK pathway in apoptotic Xenopus eggs.

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of various cellular processes, including cell survival and apoptosis. Here, we report that Xenopus p42 MAPK becomes phosphorylated in apoptotic eggs, however this modification does not activate the enzyme. Using phosphorylation residue-specific antibodies, we demonstrate that this modification occurs on the Tyr residue in the MAPK activation segment, pinpointing the autophosphorylation mechanism. Notably, MAPK phosphorylation in apoptotic Xenopus eggs coincides with prominent intracellular acidification accompanying apoptosis in these cells. Furthermore, autophosphorylation of recombinant Xenopus MAPK is stimulated and phosphorylation of a protein substrate is inhibited under low pH conditions. Thus, acidic intracellular conditions inactivate MAPK and effectively disable the MAPK-mediated survival pathway in the apoptotic eggs. Given that cell acidification is a rather common feature of apoptosis, we hypothesize that stimulation of MAPK autophosphorylation and shutdown of the MAPK pathway may represent universal traits of apoptotic cell death.

Loss of the tumor suppressor BIN1 enables ATM Ser/Thr kinase activation by the nuclear protein E2F1 and renders cancer cells resistant to cisplatin.

The tumor suppressor bridging integrator 1 (BIN1) is a corepressor of the transcription factor E2F1 and inhibits cell-cycle progression. BIN1 also curbs cellular poly(ADP-ribosyl)ation (PARylation) and increases sensitivity of cancer cells to DNA-damaging therapeutic agents such as cisplatin. However, how BIN1 deficiency, a hallmark of advanced cancer cells, increases cisplatin resistance remains elusive. Here, we report that BIN1 inactivates ataxia telangiectasia-mutated (ATM) serine/threonine kinase, particularly when BIN1 binds E2F1. BIN1 + 12A (a cancer-associated BIN1 splicing variant) also inhibited cellular PARylation, but only BIN1 increased cisplatin sensitivity. BIN1 prevented E2F1 from transcriptionally activating the human ATM promoter, whereas BIN1 + 12A did not physically interact with E2F1. Conversely, BIN1 loss significantly increased E2F1-dependent formation of MRE11A/RAD50/NBS1 DNA end-binding protein complex and efficiently promoted ATM autophosphorylation. Even in the absence of dsDNA breaks (DSBs), BIN1 loss promoted ATM-dependent phosphorylation of histone H2A family member X (forming γH2AX, a DSB biomarker) and mediator of DNA damage checkpoint 1 (MDC1, a γH2AX-binding adaptor protein for DSB repair). Of note, even in the presence of transcriptionally active (i.e. proapoptotic) TP53 tumor suppressor, BIN1 loss generally increased cisplatin resistance, which was conversely alleviated by ATM inactivation or E2F1 reduction. However, E2F2 or E2F3 depletion did not recapitulate the cisplatin sensitivity elicited by E2F1 elimination. Our study unveils an E2F1-specific signaling circuit that constitutively activates ATM and provokes cisplatin resistance in BIN1-deficient cancer cells and further reveals that γH2AX emergence may not always reflect DSBs if BIN1 is absent.

d-amino acid oxidase promotes cellular senescence via the production of reactive oxygen species.

d-amino acid oxidase (DAO) is a flavin adenine dinucleotide (FAD)-dependent oxidase metabolizing neutral and polar d-amino acids. Unlike l-amino acids, the amounts of d-amino acids in mammalian tissues are extremely low, and therefore, little has been investigated regarding the physiological role of DAO. We have recently identified DAO to be up-regulated in cellular senescence, a permanent cell cycle arrest induced by various stresses, such as persistent DNA damage and oxidative stress. Because DAO produces reactive oxygen species (ROS) as byproducts of substrate oxidation and the accumulation of ROS mediates the senescence induction, we explored the relationship between DAO and senescence. We found that inhibition of DAO impaired senescence induced by DNA damage, and ectopic expression of wild-type DAO, but not enzymatically inactive mutant, enhanced it in an ROS-dependent manner. Furthermore, addition of d-amino acids and riboflavin, a metabolic precursor of FAD, to the medium potentiated the senescence-promoting effect of DAO. These results indicate that DAO promotes senescence through the enzymatic ROS generation, and its activity is regulated by the availability of its substrate and coenzyme.

Identification of tyrosine autophosphorylation sites of Arabidopsis MEKK1 and their involvement in the regulation of kinase activity.

The MEKK1 kinase is a key regulator of stress signaling in Arabidopsis; however, little is known about the regulation of its kinase activity. Here, we found that recombinant MEKK1, expressed in both mammalian HEK293 cells and Escherichia coli, shows a mobility shift in SDS-PAGE, and immunoblotting detected phosphorylation of serine, threonine, and tyrosine residues. N-terminal deletions, site-directed mutagenesis, and protein phosphatase treatment revealed that the mobility shift results from autophosphorylation of the kinase domain. We identified the tyrosine autophosphorylation sites in the N-terminal region of MEKK1. Tyrosine to phenylalanine mutations decrease phosphorylation of the substrate MKK1, suggesting the important role of this residue in the regulation of MEKK1 kinase activity. The present study is the first to show that plant MAPKKKs are regulated by tyrosine phosphorylation.

Proline dehydrogenase promotes senescence through the generation of reactive oxygen species.

Cellular senescence is a complex stress response characterized by permanent loss of proliferative capacity and is implicated in age-related disorders. Although the transcriptional activity of p53 (encoded by TP53) is known to be vital for senescence induction, the downstream effector genes critical for senescence remain unsolved. Recently, we have identified the proline dehydrogenase gene (PRODH) to be upregulated specifically in senescent cells in a p53-dependent manner, and the functional relevance of this to senescence is yet to be defined. Here, we conducted functional analyses to explore the relationship between PRODH and the senescence program. We found that genetic and pharmacological inhibition of PRODH suppressed senescent phenotypes induced by DNA damage. Furthermore, ectopic expression of wild-type PRODH, but not enzymatically inactive forms, induced senescence associated with the increase in reactive oxygen species (ROS) and the accumulation of DNA damage. Treatment with N-acetyl-L-cysteine, a ROS scavenger, prevented senescence induced by PRODH overexpression. These results indicate that PRODH plays a causative role in DNA damage-induced senescence through the enzymatic generation of ROS.

Global decay of mRNA is a hallmark of apoptosis in aging Xenopus eggs.

Cytoplasmic mRNAs are specifically degraded in somatic cells as a part of early apoptotic response. However, no reports have been presented so far concerning mRNA fate in apoptotic gametes. In the present study, we analyzed the content of various cytoplasmic mRNAs in aging oocytes and eggs of the African clawed frog, Xenopus laevis. To circumvent large gene expression variation among the individual oocytes and eggs, single-cell monitoring of transcript levels has been implemented, using multiple cytoplasmic collections and reverse transcriptase quantitative PCR. It was found that numerous cytoplasmic mRNAs, coding for proteins classified in different functional types, are robustly degraded in apoptotic Xenopus eggs, but not in aging oocytes. mRNA degradation becomes evident in the eggs after meiotic exit at the time of cytochrome c release. A strong correlation between the length of PCR amplicon and specific transcript content was observed, suggesting endonucleolytic cleavage of mRNA. In addition, it was found that mRNA deadenylation also contributes to apoptotic mRNA degradation. Altogether, these findings indicate that the global decay of mRNA represents a hallmark of apoptosis in aging Xenopus eggs. To our knowledge, this is the first description of mRNA degradation in apoptotic gamete cells.

Reprogramming of somatic cells and nuclei by Xenopus oocyte and egg extracts.

Differentiated somatic cells and nuclei can be reprogrammed to a pluripotent undifferentiated state in the cytoplasm of oocytes and eggs. The ability of the gamete cells to induce reprogramming is not species-specific, so the extracts prepared from the oocytes and eggs of the African clawed frog Xenopus laevis can reprogram somatic mammalian cells. Thus, Xenopus egg extract-mediated reprogramming may constitute an alternative or complement other experimental reprogramming approaches, such as nuclear transfer, cell fusion, and transcription factor transduction. Here, we discuss the major reprogramming events induced by the extracts in somatic nuclei and cells, including remodeling of nuclear structure, replacement of somatic proteins with their embryonic counterparts, epigenetic modification of DNA and histones, transcriptional reprogramming, and initiation of DNA replication. We also address the advantages and limitations of the extract-based reprogramming approach.

High expression of Mcl-1L via the MEK-ERK-phospho-STAT3 (Ser727) pathway protects melanocytes and melanoma from UVB-induced apoptosis.

Ultraviolet (UV) B is a major factor in melanomagenesis. This fact is linked to the resistance of melanocytes to UVB-induced apoptosis. In this study, we characterized the involvement of Mcl-1L in the regulation of UVB-induced apoptosis in melanocytes and in melanoma cells. In melanocytes, apoptosis was not evident at 24 h after UVB irradiation. The Mcl-1L expression increased after UVB irradiation, and the high Mcl-1L expression continued for at least 24 h. This UVB-dependent increase in Mcl-1L was mediated by the MEK-ERK-pS-STAT3 (STAT3 phosphorylated at Ser727) pathway. The Ser727 phosphorylation facilitated nuclear localization of STAT3. In melanoma cells, the expression levels of Mcl-1L varied depending on the cell line. WM39 melanoma cells expressed high levels of Mcl-1L via the MEK-ERK-pS-STAT3 pathway and were resistant to UVB-induced apoptosis without up-regulation of Mcl-1L. In melanocytes and in WM39 cells, transfection with Mcl-1 siRNA promoted UVB-induced apoptosis. Immunohistochemical studies showed that melanoma cells in in situ lesions expressed high amounts of Mcl-1L. These results indicate that the high expression of Mcl-1L mediated by the MEK-ERK-pS-STAT3 pathway protects melanocytes and melanoma cells from UVB-induced apoptosis.

Calcium signaling and meiotic exit at fertilization in Xenopus egg.

Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation.

Monitoring gene expression in a single Xenopus oocyte using multiple cytoplasmic collections and quantitative RT-PCR.

Oocytes and eggs of the African clawed frog, Xenopus laevis, are commonly used in gene expression studies. However, monitoring transcript levels in the individual living oocytes remains challenging. To address this challenge, we used a technique based on multiple repeated collections of nanoliter volumes of cytoplasmic material from a single oocyte. Transcript quantification was performed by quantitative RT-PCR. The technique allowed monitoring of heterologous gene expression in a single oocyte without affecting its viability. We also used this approach to profile the expression of endogenous genes in living Xenopus oocytes. Although frog oocytes are traditionally viewed as a homogenous cell population, a significant degree of gene expression variation was observed among the individual oocytes. A lognormal distribution of transcript levels was revealed in the oocyte population. Finally, using this technique, we observed a dramatic decrease in the content of various cytoplasmic mRNAs in aging unfertilized eggs but not in oocytes, suggesting a link between mRNA degradation and egg apoptosis.

Unlaid Xenopus eggs degrade by apoptosis in the genital tract.

BACKGROUND: In several species with external fertilization, including frogs, laid unfertilized eggs were found to die by apoptosis outside of the animal body. However, there is no apparent reason for the externally laid eggs to degrade by this process, considering that apoptosis developed as a mechanism to reduce the damaging effect of individual cell death to the whole organism. RESULTS: Here, we demonstrate that a number of eggs are retained in the genital tract of the African clawed frog Xenopus laevis after gonadotropin-induced ovulation. The majority of these eggs exit meiotic arrest within 24 hours of hormone administration. Subsequently, post-meiotic eggs die in the frog genital tract by a well-defined apoptotic process. The hallmarks of egg degradation include prominent morphological changes, cytochrome c release, caspase activation, increase in ADP/ATP ratio, progressive intracellular acidification, egg swelling and all-out proteolysis of egg proteins. The sustained presence of post-apoptotic eggs in the genital tract of ageing frogs evidenced age-associated worsening of apoptotic clearance. CONCLUSIONS: The direct observation of egg degradation in the Xenopus genital tract provides a clue to the physiological relevance of frog egg apoptosis. It works to eliminate the mature unlaid eggs retained in the animal body after ovulation. Our findings establish egg apoptosis as a major physiological process accompanying ovulation in frogs.

Membrane microdomain-associated uroplakin IIIa contributes to Src-dependent mechanisms of anti-apoptotic proliferation in human bladder carcinoma cells.

Our previous study demonstrated that tyrosine phosphorylation of p145(met)/ß-subunit of hepatocyte growth factor receptor by epidermal growth factor receptor and Src contributes to the anti-apoptotic growth of human bladder carcinoma cell 5637 under serum-starved conditions. Here, we show that some other cell lines of human bladder carcinoma, but not other types of human cancer cells, also exhibit Src-dependent, anti-apoptotic proliferation under serum-starved conditions, and that low-density, detergent-insoluble membrane microdomains (MD) serve as a structural platform for signaling events involving p145(met), EGFR, and Src. As an MD-associated molecule that may contribute to bladder carcinoma-specific cellular function, we identified uroplakin IIIa (UPIIIa), an urothelium-specific protein. Results obtained so far revealed: 1) UPIIIa undergoes partial proteolysis in serum-starved cells; 2) a specific antibody to the extracellular domain of UPIIIa inhibits the proteolysis of UPIIIa and the activation of Src, and promotes apoptosis in serum-starved cells; and 3) knockdown of UPIIIa by short interfering RNA also promotes apoptosis in serum-starved cells. GM6001, a potent inhibitor of matrix metalloproteinase (MMP), inhibits the proteolysis of UPIIIa and promotes apoptosis in serum-starved cells. Furthermore, serum starvation promotes expression and secretion of the heparin-binding EGF-like growth factor in a manner that depends on the functions of MMP, Src, and UPIIIa. These results highlight a hitherto unknown signaling network involving a subset of MD-associated molecules in the anti-apoptotic mechanisms of human bladder carcinoma cells.