Mitsuyoshi Motizuki

Epithelial Splicing Regulatory Proteins 1 (ESRP1) and 2 (ESRP2) Suppress Cancer Cell Motility via Different Mechanisms

Background: The roles of ESRP1 and ESRP2 during carcinogenesis remain unknown. Results: ESRPs are up-regulated during carcinogenesis but down-regulated in invasive fronts. ESRP1 suppresses expression of the Rac1b isoform, whereas ESRP2 represses epithelial-mesenchymal transition-inducing transcription factors. Conclusion: ESRP1 and ESRP2 suppress cell motility through distinct transcriptional and/or post-transcriptional mechanisms. Significance: Our findings reveal a novel molecular network that regulates cancer cell motility. ESRP1 (epithelial splicing regulatory protein 1) and ESRP2 regulate alternative splicing events associated with epithelial phenotypes of cells, and both are down-regulated during the epithelial-mesenchymal transition. However, little is known about their expression and functions during carcinogenesis. In this study, we found that expression of both ESRP1 and ESRP2 is plastic: during oral squamous cell carcinogenesis, these proteins are up-regulated relative to their levels in normal epithelium but down-regulated in invasive fronts. Importantly, ESRP1 and ESRP2 are re-expressed in the lymph nodes, where carcinoma cells metastasize and colonize. In head and neck carcinoma cell lines, ESRP1 and ESRP2 suppress cancer cell motility through distinct mechanisms: knockdown of ESRP1 affects the dynamics of the actin cytoskeleton through induction of Rac1b, whereas knockdown of ESRP2 attenuates cell-cell adhesion through increased expression of epithelial-mesenchymal transition-associated transcription factors. Down-regulation of ESRP1 and ESRP2 is thus closely associated with a motile phenotype of cancer cells.

The effect of aging on protein synthesis in the yeast Saccharomyces cerevisiae

The protein synthetic rate in the yeast S. cerevisiae, measured by the incorporation of radioactive amino acids per unit amount of proteins, decreased linearly with age reaching 50% of the rate of 2nd generation cells (young cells) in 20th generation cells (old cells), whereas the RNA content of the old cells was increased three times. Using a cell-free system for poly(U)-directed poly-phenylalanine synthesis, the activity of run-off ribosomes from old cells was shown to be about 40% less than the activity of ribosomes from young cells and the polysome level in old cells was much decreased compared to that in young cells. However, as protein content was increased twice in 20 generations, the cell is considered to maintain a constant level of protein synthesis during the process of aging compensating the decrease in the activity of ribosomes. Thus, it is likely that the decrease in the synthesis of certain proteins whose requirement was raised by the increase in cell volume, which is twice the increase in protein content, causes prolongation of the unbudded phase in old cells.

Human trehalase is a stress responsive protein in Saccharomyces cerevisiae.

Three trehalases ATH1, NTH1, and NTH2 have been identified in Saccharomyces cerevisiae. ATH1, and NTH1 hydrolyze trehalose to glucose to provide energy and assist in recovery from stress. Human trehalase (TREH) is expressed in the intestine and kidney and probably hydrolyzes ingested trehalose in the intestine and acts as marker of renal tubular damage in kidney. Since trehalose is not present in circulation or kidney tubules, its renal effect suggests it has other yet unidentified actions. Here we examined the function of human trehalase in budding yeast. We constructed three yeast trehalase mutants (NTH1Delta, NTH2Delta, and ATH1Delta) and then transformed TREH into these mutants. NTH1Delta did not grow on media containing trehalose as the carbon source, and TREH did not rectify NTH1Delta dysfunction and also did not grow on trehalose medium, suggesting that TREH is not responsible for utilization of exogenous trehalose in yeast. In experiments involving exposure to heat, osmotic and oxidative stresses, NTH1Delta showed no recovery. Interestingly, ATH1Delta-TREH showed high sensitivity to all three stressors. ATH1Delta and NTH2Delta showed very low neutral trehalase activity and NTH1Delta did not show any neutral trehalase activity, and trehalose concentrations were higher. Increased neutral trehalase activity (equivalent to the wild type), reduction of trehalose content and brisk sensitivity to stressors were noted in TREH-ATH1Delta strain, but not in TREH-NTH1Delta or -NTH2Delta. Our results suggest that TREH acts as a stress-response protein in the kidney rather than involved in utilization of exogenous trehalose.

Oligodendrocyte Transcription Factor 1 (Olig1) Is a Smad Cofactor Involved in Cell Motility Induced by Transforming Growth Factor-β

Background: To date, the Smad cofactor involved in cell motility induced by transforming growth factor-β (TGF-β) has not been identified. Results: Knockdown of oligodendrocyte transcription factor-1 (Olig1), as well as inhibition of the Olig1-Smad interaction, resulted in attenuation of TGF-β-induced cell motility. Conclusion: Olig1 is involved in TGF-β-induced cell motility. Significance: This study enhances understanding of the regulation of TGF-β-induced cell motility. Transforming growth factor (TGF)-β plays crucial roles in embryonic development and adult tissue homeostasis by eliciting various cellular responses in target cells. TGF-β signaling is principally mediated through receptor-activated Smad proteins, which regulate expression of target genes in cooperation with other DNA-binding transcription factors (Smad cofactors). In this study, we found that the basic helix-loop-helix transcription factor Olig1 is a Smad cofactor involved in TGF-β-induced cell motility. Knockdown of Olig1 attenuated TGF-β-induced cell motility in chamber migration and wound healing assays. In contrast, Olig1 knockdown had no effect on bone morphogenetic protein-induced cell motility, TGF-β-induced cytostasis, or epithelial-mesenchymal transition. Furthermore, we observed that cooperation of Smad2/3 with Olig1 is regulated by a peptidyl-prolyl cis/trans-isomerase, Pin1. TGF-β-induced cell motility, induction of Olig1-regulated genes, and physical interaction between Smad2/3 and Olig1 were all inhibited after knockdown of Pin1, indicating a novel mode of regulation of Smad signaling. We also found that Olig1 interacts with the L3 loop of Smad3. Using a synthetic peptide corresponding to the L3 loop of Smad3, we succeeded in selectively inhibiting TGF-β-induced cell motility. These findings may lead to a new strategy for selective regulation of TGF-β-induced cellular responses.

The metabolism of ribosomal proteins microinjected into the oocytes of Xenopus laevis

When the total proteins from Xenopus laevis 60 S ribosomal subunits (TP60) were 3H-labeled in vitro and injected back into X. laevis oocytes, most 3H-TP60 are integrated into the cytoplasmic 60 S subunits via the nucleus during 16 h of incubation. In the oocytes whose rRNA synthesis is inhibited, 3H-TP60 are rapidly degraded with a half-life of 2-3 h. This degradation ceased as soon as rRNA synthesis was resumed, suggesting that ribosomal proteins unassociated with nascent rRNA are unstable in the oocytes. The degradation of 3H-TP60 in the absence of RNA synthesis was inhibited by iodoacetamide, a cysteine protease inhibitor, resulting in the accumulation of 3H-TP60 in the nucleus reaching about a threefold concentration in the cytoplasm. Considering the results with enucleated oocytes, we suggest that the X. laevis nucleus has a limited capacity to accumulate ribosomal proteins in an active manner but that those ribosomal proteins accumulated in excess over rRNA synthesis are degraded by a cysteine protease in the nucleus. By contrast, ribosomal proteins from Escherichia coli only equilibrate between the nucleus and the cytoplasm and are degraded by serine protease(s) in the cytoplasm without being integrated in the form of ribosomes in the nucleus.

Effect of 17β-estradiol on the generation time of old cells of the yeast Saccharomyces cerevisiae

The duration of unbudded period of the yeast Saccharomyces cerevisiae is known to be extended with age from unresolved causes; in this experiment the unbudded period of 20-generation-old cells was extended to be 1.6 times that of 6-generation-old cells. We found that the addition of 17β-estradiol into the culture medium reduced the age-related extension of the unbudded period reaching 1.35 times that of the young cells which was unaffected by the hormone. This effect of 17β-estradiol was not observed when the old cells were cultured in a glycerol-based medium instead of a glucose-based medium suggesting that the action of 17β-estradiol was mediated by facilitation of glycolysis. The administration of 17β-estradiol equally elevated the cAMP level of the old cells in either medium up to the level of the young cells but elevated the ATP level of only those in the glucose-based medium. Furthermore, the administration of cAMP shortens the unbudded period of the old cells cultured in the glucose-based medium. Therefore, it was suggested that 17β-estradiol causes the shortening of the unbudded period of the old cells by stimulating the energy metabolism through elevation of the cAMP level.

PSK2 coordinates glucose metabolism and utilization to maintain ultradian clock-coupled respiratory oscillation in Saccharomyces cerevisiae yeast.

Ultradian clock-coupled respiratory oscillation (UCRO) in an aerobic continuous culture of Saccharomyces cerevisiae S288C is principally regulated by control of certain redox reactions of energy metabolism. It is also modulated by the metabolism of storage carbohydrates during adaptation to environmental change. However, the mechanism of cell sensing and response to environmental nutrients in UCRO is unknown. The purpose of the present study was to determine the role of PSK2 kinase in UCRO in yeast. S. cerevisiae in culture showed oscillation in PSK2 mRNA levels with a definite phase relationship to the respiratory oscillation. Furthermore, inactivation of Psk2 by gene disruption severely affected UCRO and its decline to undetectable levels within 2days. In addition, the extracellular and intracellular glucose concentrations of PSK2 deletion mutants in culture were higher and lower, respectively, than those of the wild type. PSK2 mutant cells showed no alteration in redox state. Furthermore, the levels of storage carbohydrates such as glycogen and trehalose fluctuated in PSK2 mutants with attenuated amplitudes comparable to those in the wild type. The results indicated that PSK2 kinase is important for the uptake of glucose and regulation of storage-carbohydrate synthesis and hence the maintenance of an unperturbed continuously oscillating state.

Importance of Polarisome Proteins in Reorganization of Actin Cytoskeleton at Low pH in Saccharomyces cerevisiae

The actin cytoskeleton of the yeast Saccharomyces cerevisiae can be altered rapidly in response to external cues. We reported previously that S. cerevisiae responds to low-pH stress by transiently depolarizing its actin cytoskeleton, and that this step requires a mitogen-activated protein kinase, high osmolarity glycerol 1 (Hog1p). This study further investigated the components involved in this actin reorganization at pH 3.0. Gene deletions on the Sln1p branch of the HOG pathway completely blocked actin depolarization, suggesting that Hog1p activation depends mainly on the osmosensor Sln1p. The protein-synthesis inhibitor cycloheximide did not influence the time course of actin depolarization, suggesting that the depolarization is a direct effect of the HOG pathway. Deletion of the scaffolding protein, Spa2p, or the Spa2p-interacting protein Pea2p, markedly inhibited the depolarization, and further deletion of the formin protein, Bni1p, notably delayed actin repolarization. Our results suggest the involvement of polarisome proteins, such as Spa2p, Pea2p and Bni1p, but not Bud6p, in Hog1p-dependent reorganization of the yeast actin cytoskeleton at low pH.

The DLP1 mutant of the yeast Saccharomyces cerevisiae with an increased copy number of the 2μ plasmid shows a shortened lifespan

We isolated and characterized a recessive mutant, named dlp1, which shows the Dlp phenotype (delayed loss of proliferation activity) during the autophagic death of cdc28. The dip1 mutant was found to consist of two subtypes of cells based on colony morphology. One subtype with the Dlp phenotype, named dlp1-1, became large, red, and nibbled during the incubation, suggesting that the cells on the surface of the colonies were dying. The other without the Dlp phenotype, named dlp1-s, retained small, white colonies even after a prolonged incubation and was found to be a petite mutant. The change from dlp1-1 to dlp1-s (petite) occurred much more frequently (about 15%) than that from the wild-type to petite mutant (less than 1%). The lifespan of both subtypes of cells was severely shortened. The copy number of the endogenous 2micron plasmid of dlp1-1 was 68-fold that of the original cdc28, and decreased by half after the conversion to dlp1-s (petite). A 4.0-kbp fragment of the 2micron plasmid containing REP2 decreased the copy number of the endogenous 2micron plasmid to 8-fold that of the original cdc28 cells and partially rescued the shortened lifespan, in addition to resulting in the complete complementation of the Dlp and nibbled-colony phenotypes. These results suggest that DLP1 is a chromosomal gene that regulates the copy number of the 2micron plasmid, and that the shortening of the lifespan and other effects of the dlp1 mutation are likely caused by the increased copy number of the endogenous 2micron plasmid.

Maid is a negative regulator of transforming growth factor-β-induced cell migration.

Maternal Id-like molecule (Maid) is a dominant negative helix-loop-helix protein that has been implicated in regulating gene expression as well as cell-cycle progression. Overexpressed Maid was previously shown to inhibit certain cellular responses induced by transforming growth factor-β (TGF-β), such as TGF-β-induced cytostasis and cell motility, but not epithelial-mesenchymal transition (EMT). The role of endogenous Maid in regulating TGF-β signalling, however, has not been elucidated. We have found evidence that endogenous Maid negatively regulates TGF-β-induced cell motility. Maid knockdown enhanced TGF-β-induced cell motility as measured by chamber migration and wound healing assays but did not affect cell motility induced by bone morphogenetic protein (BMP)-4. Endogenous Maid does not appear to be involved in regulating TGF-β-induced cytostasis, resistance to apoptosis or EMT. Notably, Maid expression was induced in the delayed phase (later than 24 h) after TGF-β stimulation whereas the expression of two other negative feedback regulators, Smad7 and SnoN, was induced as early as 1 h after stimulation. These findings indicate that Maid is a unique negative feedback regulator of TGF-β signalling in its mode of action as well as the timing of its induction.