Zebrafish as a new model for rhododendrol-induced leukoderma.

Idiopathic leukoderma is a skin disorder characterized by patchy loss of skin pigmentation due to melanocyte dysfunction or deficiency. Rhododendrol (RD) was approved as a cosmetic ingredient in Japan in 2008. However, it was shown to induce leukoderma in approximately 20,000 customers. The prediction of cytotoxicity, especially to melanocytes in vivo, is required to avoid such adverse effects. Since the use of higher vertebrates is prohibited for medicinal and toxicological assays, we used zebrafish, whose melanocytes were regulated by mechanisms similar to mammals. Zebrafish larvae were treated with RD in breeding water for 3 days, which caused body lightening accompanied by a decrease in the number of melanophores. Interestingly, black particles were found at the bottom of culture dishes, suggesting that the melanophores peeled off from the body. In addition, RT-PCR analysis suggested that the mRNA levels of melanophore-specific genes were significantly low. An increase in the production of reactive oxygen species was found in larvae treated with RD. The treatments of the fish with other phenol compounds, which have been reported to cause leukoderma, also induced depigmentation and melanophore loss. These results suggest that zebrafish larvae could be used for the evaluation of leukoderma caused by chemicals, including RD.

Salt-inducible Kinases Are Critical Determinants of Female Fertility.

Follicle development is the most crucial step toward female fertility and is controlled mainly by follicle-stimulating hormone (FSH). In ovarian granulosa cells (GCs), FSH activates protein kinase A by increasing 3',5'-cyclic adenosine 5'-monophosphate (cAMP). Since cAMP signaling is impinged in part by salt-inducible kinases (SIKs), we examined the role of SIKs on the regulation of FSH actions. Here, we report that SIKs are essential for normal ovarian function and female fertility. All SIK isoforms are expressed in human and rodent GCs at different levels (SIK3>SIK2>SIK1). Pharmacological inhibition of SIK activity potentiated the stimulatory effect of FSH on markers of GC differentiation in mouse, rat, and human GCs and estradiol production in rat GCs. In humans, SIK inhibition strongly enhanced FSH actions in GCs of patients with normal or abnormal ovarian function. The knockdown of SIK2, but not SIK1 or SIK3, synergized with FSH on the induction of markers of GC differentiation. SIK inhibition boosted gonadotropin-induced GC differentiation in vivo, while the genomic knockout of SIK2 led to a significant increase in the number of ovulated oocytes. Conversely, SIK3 knockout females were infertile, FSH insensitive, and had abnormal folliculogenesis. These findings reveal novel roles for SIKs in the regulation of GC differentiation and female fertility, and contribute to our understanding of the mechanisms regulated by FSH. Furthermore, these data suggest that specific pharmacological modulation of SIK2 activity could be of benefit to treat ovulatory defects in humans and to increase the propagation of endangered species and farm mammals.

Potentiality of syringetin for preferential radiosensitization to cancer cells.

PURPOSE: To examine the enhancing effects of syringetin on the radiosensitivity of normal and cancer cells, and the related mechanism. MATERIALS AND METHODS: We used normal human lung and mouse fibroblasts as well as human lung and mouse cancer cells derived from the above normal fibroblasts. Cell radiosensitivity was measured using a colony formation assay. Apoptosis was analyzed with DAPI staining and Western blots. DNA lesions were analyzed with γH2AX immunofluorescent staining. RESULTS: The colony formation assay showed that syringetin enhanced radiosensitivity more effectively in cancer cells (H1299 and C3H/MCA clone 15) compared with normal cells (HFL-III and C3H/10T1/2). The radiosensitizing effect of syringetin was observed in mutated p53 and wild-type p53-transfected H1299 cells regardless of p53 status. Apoptosis was more frequently observed in X-ray-irradiated H1299 cells combined with syringetin compared with X-ray-only-treated cells. Enhanced apoptosis by syringetin was not observed in HFL-III cells. Western blot analysis showed that X-ray-induced Caspase-3 activation was enhanced by syringetin in H1299 cells. The number of X-ray-induced DNA double-strand breaks (DSB) measured by quantitative analysis of γH2AX foci was the same for H1299 cells treated with X-rays with or without syringetin. CONCLUSIONS: This study supports the hypothesis that syringetin enhances radiosensitivity more effectively in cancer cells than in normal cells through enhancement of the Caspase-3-mediated apoptosis pathway. Syringetin could be useful in the development of novel efficacious radiosensitizers.

Plastic induction of CD133AC133-positive cells in the microenvironment of glioblastoma spheroids.

Recent studies showed that the stemness of cancer stem cells is maintained under a hypoxic microenvironment. However, the relationship of the hypoxic microenvironment in a three-dimensional cell mass and the induction of cancer stem cell-like phenotype is not well known. We examined the relationship between CD133 expression and the hypoxic microenvironment using glioblastoma spheroids formed with the T98G cell line. CD133(AC133)- and HIF-1α-positive cells were observed in the marginal region of the central hypoxic area positive for HIF-1α 10 days after plating T98G cells. CD133(AC133)-positive cells were positive for nestin. Quantitative PCR analysis showed that the CD133 expression level is not different in spheroids during the tested period after spheroid formation, indicating that post-translational regulation of the CD133 protein mediates positivity to CD133(AC133). When spheroids were trypsinized and the dissociated cells were cultured under the adherent monolayer conditions, the CD133(AC133)-positive cells gradually disappeared. These results show that CD133(AC133)-positive cells, which may incline toward undifferentiated cells because of nestin positivity, are plastically induced under the different culture conditions, spheroid and monolayer. In this plasticity, HIF-1α is involved in the induction and maintenance of CD133(AC133)-positive cells. Spheroids as an in vitro tumor model are useful to study the dynamic changes in the tumor cell phenotype in the different cell microenvironments.

Involvement of SIK3 in glucose and lipid homeostasis in mice.

Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3(-/-) mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3(-/-) mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3(-/-) mice. Lipid metabolism disorders in Sik3(-/-) mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice.

SIK2 is a key regulator for neuronal survival after ischemia via TORC1-CREB.

The cAMP responsive element-binding protein (CREB) functions in a broad array of biological and pathophysiological processes. We found that salt-inducible kinase 2 (SIK2) was abundantly expressed in neurons and suppressed CREB-mediated gene expression after oxygen-glucose deprivation (OGD). OGD induced the degradation of SIK2 protein concomitantly with the dephosphorylation of the CREB-specific coactivator transducer of regulated CREB activity 1 (TORC1), resulting in the activation of CREB and its downstream gene targets. Ca(2+)/calmodulin-dependent protein kinase I/IV are capable of phosphorylating SIK2 at Thr484, resulting in SIK2 degradation in cortical neurons. Neuronal survival after OGD was significantly increased in neurons isolated from sik2(-/-) mice, and ischemic neuronal injury was significantly reduced in the brains of sik2(-)(/-) mice subjected to transient focal ischemia. These findings suggest that SIK2 plays critical roles in neuronal survival, is modulated by CaMK I/IV, and regulates CREB via TORC1.

Downregulation of SIK2 expression promotes the melanogenic program in mice.

cAMP response element-binding protein (CREB) promotes melanogenesis by inducing microphthalmia-associated transcription factor (Mitf ) gene expression. We report here that the CREB-specific coactivator TORC and its repressor, salt-inducible kinase 2 (SIK2), are fundamental determinants of the melanogenic program in mice. Exposure of B16 melanoma cells to ultraviolet (UV) light results in the immediate nuclear translocation of TORC1, which is inhibited by SIK2. Overexpression of dominant-negative TORC1 also inhibits UV-induced Mitf gene expression and melanogenesis. α-MSH signaling regulates hair pigmentation, and the decrease in α-MSH activity in hair follicle melanocytes switches the melanin synthesis from eumelanin (black) to pheomelanin (yellow). Mice with the lethal yellow allele of agouti (A(y)) have yellow hair because of impaired activation of the α-MSH receptor. To examine the involvement of SIK2 in the regulation of the melanogenesis switch in vivo, we prepared SIK2-knockout mice, and the Sik2(-/-) genotype was introduced into A(y)/a mice. The resultant Sik2(-/-); A(y)/a mice had brown hair, indicating that SIK2 represses eumelanogenesis in mice.

Expression of aromatase CYP19 and its relationship with parameters in NSCLC.

Human aromatase (CYP19) responsible for the conversion of androgens to estrogens is expressed not only in gonads and adrenals but also in many other tissues, including normal lungs and lung cancers. To investigate the involvement of CYP19 in lung cancer development, purified CYP19 protein and antibody are required. In this study, we have developed an efficient expression method of human aromatase in E. coli (>1000 nmol/L culture). The protein purified from E. coli was used to raise an antibody against the human CYP19 in rabbits. The resulting antibody showed a high titer judged by ELISA, which allowed us to determine the expression of CYP19 in non-small cell lung cancer (NSCLC). Of 78 NSCLC specimens from Japanese patients, 50 (64%) NSCLC aberrantly expressed CYP19. This CYP19 expression in NSCLC was independent of any clinical and pathological parameters as well as the expression of other P450s, except tumor stage. The results suggest that the aromatase inhibitors might be useful for the management of non-small cell lung cancer in postmenopausal women.

The spatial and temporal expression of delta-like protein 1 in the rat pituitary gland during development.

An analysis of secreted proteins by the signal sequence trap method using a cDNA library of the rat pituitary anlage at embryonic days (E) 13.5 revealed the abundant expression of delta-like protein 1 (Dlk1) in the pituitary gland. Dlk1, an epidermal growth factor-like repeat protein in preadipocytes, functions in maintaining the preadipose state. Expression of Dlk1 mRNA in the pituitary at E13.5 and in the adult pituitary was confirmed by in situ hybridization. The expression pattern of Dlk1 during pituitary development was also studied by immunohistochemistry. Dlk1 protein first appeared in Rathke's pouch and the infundibulum at E11.5; as development proceeded, expression became restricted to the pars distalis and pars tuberalis (PT). Dlk1 was expressed in most ACTH cells during the embryonic stages, but its expression was limited to only a few ACTH cells in the adult pituitary. It was also expressed in a small population of TSH, GTH, and PRL cells throughout development, whereas it was present in the cytoplasm of most GH cells at all developmental stages. Similarly, Dlk1 was localized in the cytoplasm of PT cells during development. These findings provide new insights into the mechanism of Dlk1 regarding its regulation of pituitary hormone-secreting cells during development.

ER stress is the initial response to polyglutamine toxicity in PC12 cells.

Persistent endoplasmic reticulum (ER) stress and impairment of the ubiquitin-proteasome system (UPS) cause neuronal cell death. However, the relationship between these two phenomena remains controversial. In our current study, we have utilized an expanded polyglutamine fusion protein (polyQ81) expression system in PC12 cells to further examine the involvement of ER stress and UPS impairment in cell death. The expression of polyQ81-induced ER stress and cell death. PolyQ81 also induced the activation of c-Jun N-terminal kinase (JNK) and caspase-3 and an increase in polyubiquitin immunoreactivity, suggesting UPS impairment. ER stress was induced prior to the accumulation of polyubiquitinated proteins. Low doses of lactacystin had almost similar effects on cell viability and on the activation of JNK and caspase-3 between normal cells and polyQ81-expressing cells. These results suggest that ER stress mediates polyglutamine toxicity prior to UPS impairment during the initial stages of these toxic effects.

Dephosphorylation of TORC initiates expression of the StAR gene.

Cyclic AMP responsive element (CRE) binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. However, transducer of regulated CREB activity (TORC), a CREB specific co-activator, upregulates CREB activity in a phospho-Ser133-independent manner. Interestingly, TORC is also regulated by phosphorylation; the phospho-form is inactive, and the dephospho-form active. When PKA phosphorylates CREB, it inhibits TORC kinases simultaneously and accelerates dephosphorylation of TORC. We show in this report that staurosporine, a kinase inhibitor, induces the expression of the StAR gene in Y1 adrenocortical cells, possibly a result of an increase in the population of dephospho-TORC. The expression of the StAR gene is known to be regulated by SF-1 and CREB, and the co-activators CBP/p300 may mediate the actions of both factors. Our experiments using KG501, a disruptor of the interaction between phospho-CREB and CBP/p300, also support the importance of TORC in the regulation of StAR gene expression.

Regional morphogenesis in the hypothalamus: a BMP-Tbx2 pathway coordinates fate and proliferation through Shh downregulation.

A central challenge in embryonic development is to understand how growth and pattern are coordinated to direct emerging new territories during morphogenesis. Here, we report on a signaling cascade that links cell proliferation and fate, promoting formation of a distinct progenitor domain within the developing chick hypothalamus. We show that the downregulation of Shh in floor plate-like cells in the forebrain governs their progression to a distinctive, proliferating hypothalamic progenitor domain. Shh downregulation occurs via a local BMP-Tbx2 pathway, Tbx2 acting to repress Shh expression. We show in vivo and in vitro that forced maintenance of Shh in hypothalamic progenitors prevents their normal morphogenesis, leading to maintenance of the Shh receptor, ptc, and preventing progression to an Emx2(+)-proliferative progenitor state. Our data identify a molecular pathway for the downregulation of Shh via a BMP-Tbx2 pathway and provide a mechanism for expansion of a discrete progenitor domain within the developing forebrain.

Tetracycline protects myocardium against ischemic injury.

Stress pretreatments protect myocardium from ischemic injury. We hypothesized that tetracycline, an antibiotic, may induce a stress response via the inhibition of mitochondrial translation as it induces the cold stress response by translational inhibition in E. coli. If so, tetracycline may protect myocardium from ischemic injury as stress pretreatments do. Thus, we investigated the effects of tetracycline on myocardial ischemia and its association with stress response. In a dog model of acute ischemia, 4mg/kg tetracycline injected 30 min prior to the occlusion improved the functional recovery from stunning of myocardium caused by ischemia. The same dosage of tetracycline dramatically reduced the size of infarct area in murine hearts analyzed by tetrazolium staining. In HeLa cells, tetracycline induced molecules that were increased by cold stress, which suggests that tetracycline may induce a cold stress-like response in mammalian cells. These molecules were also induced by ischemic stress in murine hearts, suggesting that the stress response caused by translational inhibition in mitochondria may be associated with the cardioprotection by tetracycline. Our results suggest that a subclinical dosage of tetracycline may protect heart from ischemic injury. Therefore, tetracycline may be of great use in suppressing the development of infarction caused by myocardial ischemia. This study is also important for providing new insights into the non-antimicrobial effects of tetracycline and its derivatives.

Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors.

The adult brain is extremely vulnerable to various insults. The recent discovery of neural progenitors in adult mammals, however, raises the possibility of repairing damaged tissue by recruiting their latent regenerative potential. Here we show that activation of endogenous progenitors leads to massive regeneration of hippocampal pyramidal neurons after ischemic brain injury. Endogenous progenitors proliferate in response to ischemia and subsequently migrate into the hippocampus to regenerate new neurons. Intraventricular infusion of growth factors markedly augments these responses, thereby increasing the number of newborn neurons. Our studies suggest that regenerated neurons are integrated into the existing brain circuitry and contribute to ameliorating neurological deficits. These results expand the possibility of novel neuronal cell regeneration therapies for stroke and other neurological diseases.