Kyoungsook Park

Dkk3, downregulated in cervical cancer, functions as a negative regulator of β‐catenin

The Wnt/β‐catenin signaling pathway is activated during the malignant transformation of keratinocytes that originate from the human uterine cervix. Dkk1, 2 and 4 have been shown to modulate the Wnt‐induced stabilization of the β‐catenin signaling pathway. However, the function of Dkk3 in this pathway is unknown. Comparison of the Dkk3 gene expression profiles in cervical cancer and normal cervical tissue by cDNA microarray and subsequent real‐time PCR revealed that the Dkk3 gene is frequently downregulated in the cancer. Methylation studies showed that the promoter of Dkk3 was methylated in cervical cancer cell lines and 22 (31.4%) of 70 cervical cancer tissue specimens. This promoter methylation was associated with reduced expression of Dkk3 mRNA in the paired normal and tumor tissue samples. Further, the reintroduction of Dkk3 into HeLa cervical cancer cells resulted in reduced colony formation and retarded cell growth. The forced expression of Dkk3 markedly attenuated β‐catenin‐responsive luciferase activity in a dose‐dependent manner and decreased the β‐catenin levels. By utilizing a yeast two‐hybrid screen, βTrCP, a negative regulator of β‐catenin was identified as a novel Dkk3‐interacting partner. Coexpression with βTrCP synergistically enhanced the inhibitory function of Dkk3 on β‐catenin. The stable expression of Dkk3 blocks the nuclear translocation of β‐catenin, resulting in downregulation of its downstream targets (VEGF and cylcin D), whereas knockdown of Dkk3 abrogates this blocking. We conclude from our finding that Dkk3 is a negative regulator of β‐catenin and its downregulation contribute to an activation of the β‐catenin signaling pathway.

Activation of stress signaling molecules in bat brain during arousal from hibernation

Induction of glucose‐regulated proteins (GRPs) is a ubiquitous intracellular response to stresses such as hypoxia, glucose starvation and acidosis. The induction of GRPs offers some protection against these stresses inu2003vitro, but the specific role of GRPs inu2003vivo remains unclear. Hibernating bats present a good inu2003vivo model to address this question. The bats must overcome local high oxygen demand in tissue by severe metabolic stress during arousal thermogenesis. We used brain tissue of a temperate bat Rhinolopus ferrumequinum to investigate GRP induction by high metabolic oxygen demand and to identify associated signaling molecules. We found that during 30u2003min of arousal, oxygen consumption increased from nearly zero to 11.9/kg/h, which was about 8.7‐fold higher than its active resting metabolic rate. During this time, body temperature rose from 7°C to 35°C, and levels of TNF‐α and lactate in brain tissue increased 2–2.5‐fold, indicating a high risk of oxygen shortage. Concomitantly, levels of GRP75, GRP78 and GRP94 increased 1.5–1.7‐fold. At the same time, c‐Jun N‐terminal protein kinase (JNK) activity increased 6.4‐fold, and extracellular signal‐regulated protein kinase (ERK) activity decreased to a similar degree (6.1‐fold). p38 MAPK activity was very low and remained unchanged during arousal. In addition, survival signaling molecules protein kinaseu2003B (Akt) and protein kinaseu2003C (PKC) were activated 3‐ and 5‐fold, respectively, during arousal. Taken together, our results showed that bat brain undergoes high oxygen demand during arousal from hibernation. Up‐regulation of GRP proteins and activation of JNK, PKCγ and Akt may be critical for neuroprotection and the survival of bats during the repeated process.

Chemotherapeutic drug, adriamycin, restores the function of p53 protein in hepatitis B virus X (HBx) protein-expressing liver cells

Hepatitis B virus X (HBx) protein implicated in the development of liver cancer may inhibit the function of p53 tumor suppressor protein through cytoplasmic retention of p53 protein. Here, we attempt to investigate whether the functional inhibition of p53 protein by HBx protein is reversible. First, we provide the evidence for the association of endogenous p53 protein with HBx by co-immunoprecipitation in stable Chang cells that express HBx protein in an inducible manner (ChangX-34). By immunofluorescence microscopy, the major location of p53 protein of ChangX-34 cells was confirmed at the nuclear periphery as well as in the cytoplasm where HBx protein is mainly expressed. Surprisingly, anticancer drug, adriamycin induces the nuclear translocation of p53 protein sequestered in the cytoplasm. This change is accompanied by the restoration of p53 activity, which results in increased transcriptional activity at the p53-responsive DNA elements as well as increase of p21WAF1 mRNA expression. Further, we observed the induction of cell death and G1 arrest in these cells upon adriamycin treatment regardless of HBx expression. Together, we demonstrate that functional inhibition of p53 protein through its cytoplasmic retention by HBx protein is reversible. These results may be extended into other tumors of which p53 activity is modulated by viral oncoproteins.

Variations in take-off velocity of anuran amphibians: Relation to morphology, muscle contractile function and enzyme activity☆

The relationship between variability of take-off velocity and variation in skeletomuscular features was examined in three anuran species, Rana nigromaculata, R. rugosa and Bombina orientalis. Video analyses on “maximal” take-off trials of individuals indicated that average take-off velocity (m · s−1) of R. nigromaculata (2.35 ± 0.17 SD, n = 14) and R. rugosa (2.33 ± 0.11 SD, n = 8) was significantly greater than that of Bombina (1.74 ± 0.12, n = 8). Body mass (9.2 ± 3.3 SD for R. nigromaculata; 11.5 ± 5.6 SD for R. rugosa and 6.5 ± 0.8 SD for B. orientalis) did not affect take-off velocity within each species. Compared to Bombina, the two ranid species showed longer hindlimbs relative to body length, greater mass of thigh muscles relative to body mass and a narrower interilial width (at the sacral vertebra) relative to body length. The two ranid species also exhibited significantly shorter tetanic rise time (TRT) and greater rate of force production (dFdt) examined on the gastrocnemius muscle. Isometric tetanic force, ranging between 189 and 272 mN · mm−2, was essentially the same among the three species. Activity of lactate dehydrogenase, an enzyme involved in anaerobic metabolism, of the gastrocnemius muscle, was statistically indistinguishable among the three species. The activity of citrate synthase (CS), indicative of aerobic catalytic capacity, was significantly lower in R. nigromaculata than in either R. rubosa or B. orientalis, while the CS activity was not different between the latter two species. These results indicate that the faster jumpers have a more effective skeletomuscular system (relatively longer hindlimbs and more musculature, higher contraction rate) that can generate a faster out-velocity and a greater out-force against gravity. The enzyme assay study was not sufficient to support the observed relationships in these species.

Seasonal biochemical plasticity of a flight muscle in a bat, Murina leucogaster

Cellular and biochemical responses of the pectoral muscle to variation in seasonal activity were studied in the bat, Murina leucogaster ognevi. We collected bats in mid-hibernation (February), end-hibernation (April), and mid-summer (August) to track major activity periods in their annual cycle. Our findings indicated that myofiber cross-sectional area decreased to 68% between mid- and end-hibernation, but returned to the winter level in mid-summer. Total soluble protein and total RNA concentrations were not altered over these sampling periods. Oxidative potential gauged by citrate synthase activity increased 1.47-fold from mid- to end-hibernation and then remained at the similar level in mid-summer. Glycolytic potential gauged by lactate dehydrogenase activity changed little between mid- and end-hibernation but increased 1.42-fold in summer, compared with the winter level. Thus, the myofibers underwent disuse atrophy during hibernation, while enzymatic catalytic function recovered towards the level of mid-summer.

Vascular endothelial growth factor expression under ischemic stress in human meningiomas.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific antigen and angiogenic factor that plays a role in angiogenesis. We analyzed the expression of four VEGF mRNA isoforms in meningiomas. Among 35 meningiomas, 11 came from patients who underwent complete (n=4) or partial (n=7206=189 in all samples. However, the VEGF121 and 165 isoforms were significantly upregulated in samples from patients who underwent partial preoperative embolization. The diffusible VEGF121 isoform may be important for vascularity and edema formation in meningiomas.

Sustained torpidity following multi-dose administration of 3-iodothyronamine in mice

Despite significant medical benefits as in space exploration or emergency care, prolonged torpidity of non‐hibernator mammals remains unexplored to date. Here, we report that male Institute of Cancer Research mice could sustain two separate 2‐day torpor bouts and maintain body temperature of 28–33°C following repeated treatments of 3‐iodothyronamine (T1AM), a natural derivative of thyroid hormone. A 1‐day interbout arousal period, adopted to mimic the behavior of true hibernators, seemed critical for the subjects to restore physiological homeostasis. Molecular studies of neuron‐specific enolase, S100 calcium binding protein B and heat shock protein 72 suggested that the brain maintains functional and cytoprotective activities during sustained torpidity. Together, the results of this study propose a practical protocol using a torpor‐arousal cycle that can be applied to the extreme medical situations. J. Cell. Physiol. 226: 853–858, 2011.

Inhibition of C2C12 myotube atrophy by a novel HSP70 inducer, celastrol, via activation of Akt1 and ERK1/2 pathways

Celastrol (CEL) is known as a potent inducer of heat shock protein (HSP) in non-muscle cells and exhibits cytoprotective function and inhibitory effects on proteasome and glucocorticoid receptor activities. To investigate an anti-atrophic effect of CEL on skeletal muscle cells, C2C12 myotubes were treated with 150 μM dexamethasone (DEX) for 24h and 1.5 μM CEL was added for the last 6h during the 24h DEX treatment. Compared to the control, the myotube diameter was reduced by a factor of 0.30 by DEX, but CEL treatment almost abrogated the DEX-induced atrophy. CEL treatment also increased expression of HSP72 and phosphorylation of heat shock transcription factor 1 (p-HSF1) 11-fold and 3.4-fold, respectively, as well as accumulation of p-HSF1 in the nucleus. Furthermore, CEL treatment elevated activities of Akt1, p70/S6K and ERK1/2 2.0- to 4.4-fold whereas DEX had no effect on these signaling activities. Inhibition of Akt1 and ERK1/2 pathways by specific inhibitors confirmed CEL-induced anti-atrophic effect. Moreover, DEX-mediated downregulation of FoxO3 phosphorylation and upregulation of MuRF1 expression and proteasome activity were abrogated by CEL treatment. These results demonstrate a novel anti-atrophic function of CEL in muscle cells via both activation of protein anabolic signals and suppression of catabolic signaling activities.

A Developmentally Controlled Competitive STAT5–PU.1 DNA Binding Mechanism Regulates Activity of the Ig κE3′ Enhancer

Stage-specific rearrangement of Ig H and L chain genes poses an enigma because both processes use the same recombinatorial machinery, but the H chain locus is accessible at the pro-B cell stage, whereas the L chain loci become accessible at the pre-B cell stage. Transcription factor STAT5 is a positive-acting factor for rearrangement of distal VH genes, but attenuation of IL-7 signaling and loss of activated STAT5 at the pre-B cell stage corresponds with Igκ locus accessibility and rearrangement, suggesting that STAT5 plays an inhibitory role at this locus. Indeed, loss of IL-7 signaling correlates with increased activity at the Igκ intron enhancer. However, the κE3′ enhancer must also be regulated as this enhancer plays a role in Igκ rearrangement. We show in this study that STAT5 can repress κE3′ enhancer activity. We find that STAT5 binds to a site that overlaps the κE3′ PU.1 binding site. We observed reciprocal binding by STAT5 and PU.1 to the κE3′ enhancer in primary bone marrow cells, STAT5 and PU.1 retrovirally transduced pro-B cell lines, or embryonic stem cells induced to differentiate into B lineage cells. Binding by STAT5 corresponded with low occupancy of other enhancer binding proteins, whereas PU.1 binding corresponded with recruitment of IRF4 and E2A to the κE3′ enhancer. We also find that IRF4 expression can override the repressive activity of STAT5. We propose a novel PU.1/STAT5 displacement model during B cell development, and this, coupled with increased IRF4 and E2A activity, regulates κE3′ enhancer function.

3-Iodothyronamine-mediated metabolic suppression increases the phosphorylation of AMPK and induces fuel choice toward lipid mobilization.

Despite broad medical application, induction of artificial hypometabolism in vitro and its biochemical consequence have been rarely addressed. This study aimed to elucidate whether 3-iodothyronamine (T1AM) induces hypometabolism in an in vitro model with activation of AMP-activated protein kinase (AMPK) and whether it leads to a switch in primary fuel from carbohydrates to lipids as observed in in vivo models. Mouse C2C12 myotube and T1AM, a natural derivative of thyroid hormone, were used in this study. The oxygen consumption rate (OCR) decreased in a dose-dependent manner in response to 0-100u2009μM T1AM for up to 10u2009h. Upon 6-h of exposure to 75u2009μM T1AM, the OCR was reduced to 60 vs. ~u200995% for the control. The intracellular [AMP]/[ATP] was 1.35-fold higher in T1AM-treated cells. RT-PCR and immunoblotting analyses revealed that treated cells had upregulated p-AMPK/AMPK (1.8-fold), carnitine palmitoyl transferase 1 mRNA, and pyruvate dehydrogenase kinase, and downregulated acetyl CoA carboxylase (0.4-fold) and pyruvate dehydrogenase phosphatase. The treated cells had darker periodic acid-Schiff staining with 1.2-fold greater glycogen content than controls. Taken together, the hypometabolic response of myotubes to T1AM was dramatic and accompanied by increases in both the relative abundance of AMP and AMPK activation, and fuel choice favoring lipids over carbohydrates. These results are consistent with the general trends observed for rodent models and true hibernators.