大神 信孝

Noise stress generated in industry is one of the environmental factors that physically affects the functions of the inner ear. Exposure to noise can cause hearing loss, resulting in serious problems in occupational and daily life. At present, however, there are very limited ways to prevent hearing impairments. The inner ear consists of the organ of Corti, vestibule and semicircular canal. Functional or morphological damage of these tissues in the inner ear caused by genetic factors, aging or environmental factors can result in hearing or balance impairments. In this review, we first introduce a deafness-related molecule found by our clinical research. Our experimental research using genetically engineered mice further demonstrated that impaired activity of the target molecule caused congenital and age-related hearing loss with neurodegeneration of spiral ganglion neurons in the inner ears. We also describe impaired balance in mice caused by exposure to low-frequency noise under experimental conditions with indoor environmental monitoring. We believe that our approaches to pursue both experimental research and fieldwork research complementarily are crucial for the development of a method for prevention of impairments of the inner ear.

Due to the increased ultraviolet radiation, the incidence of melanoma is increasing worldwide more than that of any other cancer. In this study, the effects of irradiation of non-thermal atmospheric pressure plasmas (NEAPPs) on benign melanocytic tumors from our original hairless model mice (HL-RET-mice), in which benign melanocytic tumors and melanomas spontaneously develop in the skin stepwise, were examined. Expression levels of melanoma cell adhesion molecule (MCAM) and matrix metalloproteinase-2 (MMP-2) mRNA in melanomas were higher than those in benign melanocytic tumors in the mice. Repeated irradiation of non-thermal atmospheric pressure plasmas (NEAPPs) for the benign tumors decreased the expression levels of MCAM and MMP-2 mRNA in the tumors from the mice. Previous studies showed that MCAM sites are upstream of MMP-2, that MCAM regulates transcription of MMP-2 in melanoma cells and that MMP-2 is associated with the conversion of a benign tumor to a malignant tumor. Therefore, our results suggest that the NEAPP irradiation-mediated decrease in the expression level of MMP-2 in benign melanocytic tumors is associated with decreased expression levels of MCAM. Moreover, NEAPP irradiation might be a potential candidate for therapy to prevent melanoma development through suppression of malignant conversion in benign melanocytic tumors.

The incidence of cutaneous malignant melanoma is increasing at a greater rate than that of any other cancer in the world. However, an effective therapy for malignant melanoma has not been established. Recently, some studies have shown an antitumor effect of non-equilibrium atmospheric pressure plasmas (NEAPPs) in vitro. Here, we examined the in vivo effect of NEAPP on cell cycle regulators, key elements for malignant transformation, in spontaneously developed benign melanocytic tumors in a hairless animal model. NEAPP irradiation decreased expression levels of cell cycle promoters, Cyclin D1, E1 and E2, and increased expression level of a cell cycle repressor, p27KIP1. Cyclin D1, E1 and E2 and p27KIP expression levels were associated with malignant transformation of the benign tumor in the animal model. Our results suggest that NEAPP irradiation suppresses malignant transformation of a benign melanocytic tumor via control of the expression levels of cell cycle regulators.

Krishna et al. (Arch Toxicol 88(1): 47-64, 2014) recently published the results of a study in which adult C57BL/6 mice were subchronically exposed to 400,000 mu g/L manganese (Mn) using manganese chloride via drinking water for 8 weeks and examined the neurotoxic effects. After 5 weeks of Mn exposure, significant deposition of Mn in all of the brain regions examined by magnetic resonance imaging was detected. After 6 weeks of Mn exposure, neurobehavioral deficits in an open field test, a grip strength test, and a forced swim test were observed. Eight weeks of Mn exposure increased striatal 5-hydroxyindoleacetic acid (a serotonin metabolite) levels, but did not alter the levels of striatal dopamine, its metabolites and serotonin. Krishna et al. also reported significant increases in mRNA levels of GFAP (an astrocyte activation marker), HO-1 (an oxidative stress marker) and NOS2 (a nitrosative stress marker), and in protein expression level of GFAP in the substantia nigra pars reticulata after 8 weeks of Mn exposure. These results suggest that 400,000 mu g/L Mn exposure via drinking water in mice induces neurobehavioral deficits, serotonergic imbalance, and glial activation accompanied by an increase in brain Mn deposition. The report by Krishna et al. is interesting because the studies on the neurobehavioral effect of Mn exposure by drinking water in mice are very limited. However, Mn concentrations previously reported in well drinking water (Agusa et al. in Vietnam Environ Pollut 139(1): 95-106, 2006; Buschmann et al. in Environ Int 34(6): 756-764, 2008; Hafeman et al. in Environ Health Perspect 115(7): 1107-1112, 2007; Wasserman et al. in Bangladesh Environ Health Perspect 114(1): 124-129, 2006) were lower than 400,000 mu g/ L.

It is well known that heterotrimeric G protein is composed of a G alpha-subunit and a G beta Upsilon-dimer and promotes cancer characteristics. Our recent study showed reduced G protein.2 subunit (Gng2/GNG2) expression levels in malignant melanoma cells compared with those in benign melanocytic cells in both mice and humans. Our recent study also showed that reduced GNG2 alone augmented proliferation of malignant melanoma cells. To our knowledge, however, there is no evidence showing an effect of Gng2/GNG2 alone on metastasis of any cancers including malignant melanoma. In his study, we first prepared GNG2-overexpressed SK-Mel28 human malignant melanoma cells, in which GNG2 protein expression level was undetectably low. Migration and invasion activities of the GNG2overexpressed malignant melanoma cells were suppressed up to 1/10th, with decreased activity of focal adhesion kinase (FAK). We then found that the expression level of GNG2 in A375M, a highly metastatic cell line, was significantly lower than that in A375P, the parental cell line of A375M. We finally showed that knockdown of GNG2 alone in A375P cells enhanced migration and invasion with increased FAK activity. Taken together, our results suggest that overexpression of GNG2 alone inhibits metastasis in human malignant melanoma cells with decreased FAK activity. Thus, GNG2 might be a candidate of molecular targets of prevention and therapy for metastasis of malignant melanoma.

Impairments of hearing and balance are major problems in the field of occupational and environmental health. Such impairments have previously been reported to be caused by genetic and environmental factors. However, their mechanisms have not been fully clarified. On the other hand, the inner ear contains spiral ganglion neurons (SGNs) in the organ of Corti, which serve as the primary carriers of auditory information from sensory cells to the auditory cortex in the cerebrum. Inner ears also contain a vestibule in the vicinity of the organ of Corti-one of the organs responsible for balance. Thus, inner ears could be a good target to clarify the pathogeneses of sensorineural hearing losses and impaired balance. In our previous studies with c-Ret knock-in mice and Endothelin receptor B (Ednrb) knock-out mice, it was found that syndromic hearing losses involved postnatal neurodegeneration of SGNs caused by impairments of c-Ret and Ednrb, which play important roles in neuronal development and maintenance of the enteric nervous system. The organ of Corti and the vestibule in inner ears also suffer from degeneration caused by environmental stresses including noise and heavy metals, resulting in impairments of hearing and balance. In this review, we introduce impairments of hearing and balance caused by genetic and environmental factors and focus on impairments of SGNs and the vestibule in inner ears as the pathogeneses caused by these factors.

Health risk for well drinking water is a worldwide problem. Our recent studies showed increased toxicity by exposure to barium alone (≤700 µg/L) and coexposure to barium (137 µg/L) and arsenic (225 µg/L). The present edition of WHO health-based guidelines for drinking water revised in 2011 has maintained the values of arsenic (10 µg/L) and barium (700 µg/L), but not elements such as manganese, iron and zinc. Nevertheless, there have been very few studies on barium in drinking water and human samples. This study showed significant correlations between levels of arsenic and barium, but not its homologous elements (magnesium, calcium and strontium), in urine, toenail and hair samples obtained from residents of Jessore, Bangladesh. Significant correlation between levels of arsenic and barium in well drinking water and levels in human urine, toenail and hair samples were also observed. Based on these results, a high-performance and low-cost adsorbent composed of a hydrotalcite-like compound for barium and arsenic was developed. The adsorbent reduced levels of barium and arsenic from well water in Bangladesh and Vietnam to <7 µg/L within 1 min. Thus, we have showed levels of arsenic and barium in humans and propose a novel remediation system.

Background: Previous studies have revealed that heterotrimeric G protein is composed of a G alpha-subunit and a G beta-gamma-dimer and is correlated with c-Src and AKT activities. Our recent study showed reduced G protein gamma 2 subunit (Gng2/GNG2) expression levels in malignant melanoma cells compared with those in benign melanocytic cells in both mice and humans. At present, however, there is no evidence showing an effect of Gng2/GNG2 alone on cancer biology. Objective: The purpose of this study was to examine the biological significance of GNG2 in human malignant melanoma cells. Methods: Levels of proliferation and activities of signal transduction molecules were examined in both GNG2-overexpressed and -depleted human malignant melanoma cells. Results: Proliferation of GNG2-overexpressed SK-Mel28 human malignant melanoma cells was suppressed with decreased c-SRC and AKT activities and increased p21(Cip/WAF1) expression level in vitro. In contrast, proliferation of GNG2-depleted A375P human malignant melanoma cells was enhanced with increased c-SRC and AKT activities and decreased p21(Cip/WAF1) expression level in vitro. In the in vivo experiment, the mean tumor size of GNG2-overexpressed SK-Mel28 cells was less than 1/45th of that of control SK-Mel28 cells in nude mice at 95 days after inoculation. Conclusion: We demonstrated for the first time that increased protein expression level of GNG2 alone inhibits proliferation of malignant melanoma cells in vitro and in vivo, suggesting that GNG2 could be a novel molecular target for malignant melanoma therapy. (C) 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Purpose: We continuously ingest barium as a general element by drinking water and foods in our daily life. Exposure to high-dose barium (&gt;100 mg/kg/day) has been shown to cause physiological impairments. Direct administration of barium to inner ears by vascular perfusion has been shown to cause physiological impairments in inner ears. However, the toxic influence of oral exposure to low-dose barium on hearing levels has not been clarified in vivo. We analyzed the toxic influence of oral exposure to low-dose barium on hearing levels and inner ears in mice. Experimental design: We orally administered barium at low doses of 0.14 and 1.4 mg/kg/day to wild-type ICR mice by drinking water. The doses are equivalent to and 10-fold higher than the limit level (0.7 mg/l) of WHO health-based guidelines for drinking water, respectively. After 2-week exposure, hearing levels were measured by auditory brain stem responses and inner ears were morphologically analyzed. After 2-month exposure, tissue distribution of barium was measured by inductively coupled plasma mass spectrometry. Results: Low-dose barium in drinking water caused severe hearing loss in mice. Inner ears including inner and outer hair cells, stria vascularis and spiral ganglion neurons showed severe degeneration. The Barium-administered group showed significantly higher levels of barium in inner ears than those in the control group, while barium levels in bone did not show a significant difference between the two groups. Barium levels in other tissues including the cerebrum, cerebellum, heart, liver and kidney were undetectably low in both groups. Conclusions: Our results demonstrate for the first time that low-dose barium administered by drinking water specifically distributes to inner ears resulting in severe ototoxicity with degeneration of inner ears in mice. (C) 2012 Elsevier Inc. All rights reserved.

We are routinely exposed to low frequency noise (LFN; below 0.5 kHz) at moderate levels of 60-70 dB sound pressure level (SPL) generated from various sources in occupational and daily environments. LFN has been reported to affect balance in humans. However, there is limited information about the influence of chronic exposure to LFN at moderate levels for balance. In this study, we investigated whether chronic exposure to LFN at a moderate level of 70 dB SPL affects the vestibule, which is one of the organs responsible for balance in mice. Wild-type ICR mice were exposed for 1 month to LFN (0.1 kHz) and high frequency noise (HFN; 16 kHz) at 70 dB SPL at a distance of approximately 10-20 cm. Behavior analyses including rotarod, beam-crossing and footprint analyses showed impairments of balance in LFN-exposed mice but not in non-exposed mice or HFN-exposed mice. Immunohistochemical analysis showed a decreased number of vestibular hair cells and increased levels of oxidative stress in LFN-exposed mice compared to those in non-exposed mice. Our results suggest that chronic exposure to LFN at moderate levels causes impaired balance involving morphological impairments of the vestibule with enhanced levels of oxidative stress. Thus, the results of this study indicate the importance of considering the risk of chronic exposure to LFN at a moderate level for imbalance.

c-Ret has been shown to be crucial for neural development and survival. We have recently shown that complete impairment of tyrosine 1062 (Y1062)-phosphorylation in c-Ret causes congenital hearing loss with neurodegeneration of spiral ganglion neurons (SGNs) in homozygous c-Ret knockin mice (c-Ret-KIY1062F/Y1062F-mice). However, there is no information to link c-Ret and age-related hearing loss. Here we show that partial impairment of Y1062-phosphorylation in c-Ret accelerates age-related hearing loss in heterozygous c-Ret Y1062F knockin mice (c-Ret-KIY1062F/+-mice). In contrast, complete impairment of serine 697 (S697)-phosphorylation in c-Ret did not affect hearing levels in 10-month-old homozygous c-Ret S697A knockin mice (c-Ret-KIS697A/S697A-mice). The hearing loss involved late-onset neurodegeneration of spiral ganglion neurons in c-Ret-(KIY1062F/+)-mice. Morphological abnormalities in inner-and outer-hair cells and the stria vascularis in c-Ret-KIY1062F/+-mice were undetectable. The acceleration of age-related hearing loss in c-Ret-KIY1062F/(+)-mice was rescued by introducing constitutively activated RET. Thus, our results suggest that c-Ret is a novel age-related hearing loss-related molecule in mice. Our results suggest that these hearing losses partially share a common pathogenesis that is monogenetically caused by a single point mutation (Y1062F) in c-Ret. (C) 2012 Elsevier Inc. All rights reserved.

Background: An appropriate animal model for malignant melanoma could be a strong tool to develop biomarkers through analysis of melanomagenesis. Objective: Development of a novel animal model that spontaneously develops malignant melanoma with a high percentage. Methods: We crossed oncogenic RET (RFP-RET)-carrying transgenic mice of line 304/B6 (RET-mice) with hairless mice (hr/hr) and newly established hairless RFP-RET-transgenic mice of line 304-hr/hr (HL-RET-mice). Results: The HL-RET-mice developed hyperpigmented skin and benign melanocytic tumors without exception. More importantly, 63.8% (46/72) of the benign tumors were transformed to malignant melanoma in the HL-RET-mice. Mean time until the development of benign melanocytic tumors (2.4 months; n = 102) in the HL-RET-mice was about half of that in the original RET-mice (4.6 months; n = 20). Mean life span in the HL-RET-mice (9.7 months; n = 38) was also significantly (p &lt; 0.01) shorter than that in the original RET-mice (10.8 months; n = 20). Since early development of tumors could contribute to shortening of the research period, HL-RET-mice could be a useful model for analysis of melanomagenesis. We then found that the expression level of Mps one binder kinase activator-like-2B (Mobkl2b) in benign tumors was higher than that in malignant melanoma in HL-RET-mice. Expression level of MOBKL2B in malignant melanoma cell lines was also lower than that in non-malignant melanocytic cells in mice and humans, suggesting that MOBKL2B could be a novel marker for malignant melanoma. Conclusion: We established a novel hairless RET-transgenic mouse line spontaneously developing cutaneous malignant melanomas from benign melanocytic tumors. This mouse model may be useful to find new candidates of melanoma-related molecule. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Cutaneous malignant melanoma is one of the most serious skin cancers and is highly invasive and markedly resistant to conventional therapy. Melanomagenesis is initially triggered by environmental agents including ultraviolet (UV), which induces genetic/epigenetic alterations in the chromosomes of melanocytes. In human melanomas, the RAS/RAF/MEK/ERK (MAPK) and the PI3K/PTEN/AKT (AKT) signaling pathways are two major signaling pathways and are constitutively activated through genetic alterations. Mutations of RAF, RAS, and PTEN contribute to antiapoptosis, abnormal proliferation, angiogenesis, and invasion for melanoma development and progression. To find better approaches to therapies for patients, understanding these MAPK and AKT signaling mechanisms of melanoma development and progression is important. Here, we review MAPK and AKT signaling networks associated with melanoma development and progression.

About 120 million people worldwide suffer from congenital (early-onset) hearing loss. Thirty percent of them have syndromic hearing loss and the remaining 70% have non-syndromic hearing loss. In addition, a large number of elderly people worldwide suffer from age-related (late-onset) hearing loss. c-Ret and c-RET have been shown to be essential for the development and maintenance of neurons including the enteric nervous system (ENS) in mice and humans. Impairments of endothelin receptor B (EDNRB) and SOX10 have been shown to cause a significantly increased risk of dominant sensorineural deafness in Hirschsprung disease (HSCR) patients. We have recently shown that impairments of tyrosine 1062 (Y1062) phosphorylation in c-Ret causes syndromic congenital deafness in mice and humans and non-syndromic age-related hearing loss with neurodegeneration of spiral ganglion neurons (SGNs) in mice. This review focuses on the pathogenesis of hearing loss caused by impairments of c-Ret.

Impairments of endothelin receptor B (Ednrb/EDNRB) cause the development of Waardenburg-Shah syndrome with congenital hearing loss, hypopigmentation, and megacolon disease in mice and humans. Hearing loss in WaardenburgShah syndrome has been thought to be caused by an Ednrbmediated congenital defect of melanocytes in the stria vascularis (SV) of inner ears. Here we show that Ednrb expressed in spiral ganglion neurons (SGNs) in inner ears is required for postnatal development of hearing in mice. Ednrb protein was expressed in SGNs from WT mice on postnatal day 19 (P19), whereas it was undetectable in SGNs from WT mice on P3. Correspondingly, Ednrb homozygously deleted mice (Ednrb(-/-) mice) with congenital hearing loss showed degeneration of SGNs on P19 but not on P3. The congenital hearing loss involving neurodegeneration of SGNs as well as megacolon disease in Ednrb(-/-) mice were markedly improved by introducing an Ednrb transgene under control of the dopamine beta-hydroxylase promoter (Ednrb(-/-); DBH-Ednrb mice) on P19. Neither defects of melanocytes nor hypopigmentation in the SV and skin in Ednrb(-/-) mice was rescued in the Ednrb(-/-); DBH-Ednrb mice. Thus, the results of this study indicate a novel role of Ednrb expressed in SGNs distinct from that in melanocytes in the SV contributing partially to postnatal hearing development.

Previous studies have shown that activities of tyrosine kinases and secretion of the active form of matrix metalloproteinase-2 (MMP-2) are correlated with promotion of tumor growth, while apoptotic cell death in cancer cells is correlated with anti-cancer effects. Although arsenic has been reported to have both cancer-promoting and anti-cancer effects, the mechanisms of the arsenic-mediated bidirectional effects remain unknown. We examined the effects of arsenic on both proto-oncogene c-RET-transfected NIH3T3 cells with benign characters and oncogenic RET-MEN2A-transfected NIH3T3 cells with malignant characters. Arsenic promoted not only c-RET tyrosine kinase activity but also genetically activated RET-MEN2A kinase activity with promotion of dimer formation of RET proteins. Arsenic also increased secretion of the active form of MMP-2 in both RET-MEN2A-transfectants and c-RET-transfectants. On the other hand, arsenic promoted poly-(ADP-ribose) polymerase (PARP) degradation and cell death in both malignant and non-malignant cells. Interestingly, L-cysteine inhibited the arsenic-mediated tumor-promoting effects (activation of kinases and MMP-2 secretion) but not arsenic-mediated anti-cancer effects (PARP degradation and cell death). Our results suggest redox-linked regulation of arsenic-mediated activities of kinases and MMP-2 secretion but not arsenic-mediated cell death. Our results also suggest that L-cysteine is an ideal supplement that inhibits arsenic-mediated tumor-promoting effects without affecting arsenic-mediated anti-cancer effects. (c) 2011 Elsevier Ltd. All rights reserved.

There have been few reports showing a correlation between hearing levels and life style in young people. In this study, we succeeded in sensitively evaluating hearing levels in 51 young male adults of 21 - 23 years in age by 12 k Hz extra-high-frequency auditory thresholds, which cannot be measured by usual audiometry devices for clinical use. Noise exposure, alcohol consumption and sleeping time did not affect hearing levels in young adults. Auditory thresholds of 12 kHz frequency in smokers were significantly (p &lt; 0.05) higher than those in non-smokers, while there were no differences in 1 kHz, 4 kHz and 8 kHz frequencies of hearing levels between smokers and non-smokers. Since the Brinkman Index (BI; cigarettes/day multiplied by number of years) of smokers in this study was from 12 to 60, our results suggest that even light smoking of less than 20 cigarettes/day for 3 years can result in the development of hearing loss of 12 kHz frequency in young adults. Binary logistic regression analysis again showed a correlation between hearing loss (&gt;= 40 dB of auditory thresholds in 12 kHz frequency) and light smoking (12 &lt;= BI &lt;= 60). Thus, this study showed that auditory threshold at 12 kHz frequency could be a sensitive marker for hearing in young adults. More importantly, we for the first time provided epidemiological evidence that light smoking might affect hearing level at 12 kHz frequency and revealed a new risk of light smoking.

Various environmental and genetic factors affect the development and progression of skin cancers including melanoma. Melanoma development is initially triggered by environmental factors including ultraviolet (UV) light, and then genetic/epigenetic alterations occur in skin melanocytes. These first triggers alter the conditions of numerous genes and proteins, and they induce and/or reduce gene expression and activate and/or repress protein stability and activity, resulting in melanoma progression. Microphthalmia-associated transcription factor (MITF) is a master regulator gene of melanocyte development and differentiation and is also associated with melanoma development and progression. To find better approaches to molecular-based therapies for patients, understanding MITF function in skin melanoma development and progression is important. Here, we review the molecular networks associated with MITF in skin melanoma development and progression.

A significantly increased risk for dominant sensorineural deafness in patients who have Hirschsprung disease (HSCR) caused by endothelin receptor type B and SOX10 has been reported. Despite the fact that c-RET is the most frequent causal gene of HSCR, it has not been determined whether impairments of c-Ret and c-RET cause congenital deafness in mice and humans. Here, we show that impaired phosphorylation of c-Ret at tyrosine 1062 causes HSCR-linked syndromic congenital deafness in c-Ret knockin (KI) mice. The deafness involves neurodegeneration of spiral ganglion neurons (SGNs) with not only impaired phosphorylation of Akt and NF-kappa B but decreased expression of calbindin D28k in inner ears. The congenital deafness involving neurodegeneration of SGNs in c-Ret KI mice was rescued by introducing constitutively activated RET. Taken together with our results for three patients with congenital deafness with c-RET-mediated severe HSCR, our results indicate that c-Ret and c-RET are a deafness-related molecule in mice and humans.

We examined the biochemical effects of arsenic on the activities of RET proto-oncogene (c-RET protein tyrosine kinases) and RET oncogene (RET-MEN2A and RET-PTC1 protein tyrosine kinases) products. Arsenic activated c-RET kinase with promotion of disulfide bond-mediated dimerization of c-RET protein. Arsenic further activated RET-MEN2A kinase, which was already 3- to 10-fold augmented by genetic mutation compared with c-RET kinase activity, with promotion of disulfide bond-mediated dimerization of RET-MEN2A protein (superactivation). Arsenic also increased extracellular domain-deleted RET-PTC1 kinase activity with promotion of disulfide bond-mediated dimerization of RET-PTC1 protein. Arsenic increased RET-PTC1 kinase activity with cysteine 365 (C365) replaced by alanine with promotion of dimer formation but not with cysteine 376 (C376) replaced by alanine. Our results suggest that arsenic-mediated regulation of RET kinase activity is dependent on conformational change of RET protein through modulation of a special cysteine sited at the intracellular domain in RET protein (relevant cysteine of C376 in RET-PTC1 protein). Moreover, arsenic enhanced the activity of immunoprecipitated RET protein with increase in thiol-dependent dimer formation. As arsenic (14.2 mu M) was detected in the cells cultured with arsenic (100 direct association between arsenic and RET in the cells might modulate dimer formation. Thus, we demonstrated a novel redox-linked mechanism of activation of arsenic-mediated RET proto-oncogene and oncogene products. J. Cell. Biochem. 110: 399-407, 2010. (C) 2010 Wiley-Liss, Inc.

While the genetic control of renal branching morphogenesis has been extensively described, the cellular basis of this process remains obscure. GDNF/RET signaling is required for ureter and kidney development, and cells lacking Ret are excluded from the tips of the branching ureteric bud in chimeric kidneys. Here, we find that this exclusion results from earlier Ret-dependent cell rearrangements in the caudal Wolffian duct, which generate a specialized epithelial domain that later emerges as the tip of the primary ureteric bud. By juxtaposing cells with elevated or reduced RET activity, we find that Wolffian duct cells compete, based on RET signaling levels, to contribute to this domain. At the same time, the caudal Wolffian duct transiently converts from a simple to a pseudostratified epithelium, a process that does not require Ret. Thus, both Ret-dependent cell movements and Ret-independent changes in the Wolffian duct epithelium contribute to ureteric bud formation.

Recently, we crossed an original haired RET-transgenic mouse of line 242 with a hairless mouse and established a hairless RET-(HL/RET)-transgenic mouse line (242-hr/hr) with hyperpigmented skin but no tumors. In this study, we examined the effect of hyperpigmented skin in HL/RET-transgenic mice on UV irradiation-mediated cutaneous cancer development. UV irradiation to this mouse line never induced melanoma despite the presence of melanoma-inducible transgenic RET oncogenes. On the contrary, the hyperpigmented skin efficiently protected UV-mediated squamous carcinoma development in the skin. Probably underlying this result, hyperpigmentation protected the skin from damage and blocked the accompanying signal transduction for tyrosine phosphorylation of multiple cellular proteins and activation/phosphorylation of extracellular signal-regulated, c-Jun N-terminal, and p38 kinases. Thus, we demonstrated hyperpigmentation-mediated in vivo protection against UV irradiation-induced skin cancer.

The effect(s) of endogenously synthesized cholesterol (endo-CHOL) on the endosomal system in mammalian cells has not been examined. Here we treated Chinese hamster ovary cell lines with lovastatin (a hydroxymethylglutaryl-CoA reductase inhibitor) and mevalonate (a precursor for isoprenoids) to block endo-CHOL synthesis and then examined its effects on the fate of cholesterol liberated from low density lipoprotein (LDL-CHOL). The results showed that blocking endo-CHOL synthesis for 2 h or longer does not impair the hydrolysis of cholesteryl esters but partially impairs the transport of LDL-CHOL to the plasma membrane. Blocking endo-CHOL synthesis for 2 h or longer also alters the localization patterns of the late endosomes/lysosomes and retards their motility, as monitored by time-lapse microscopy. LDL-CHOL overcomes the effect of blocking endo-CHOL synthesis on endosomal localization patterns and on endosomal motility. Overexpressing Rab9, a key late endosomal small GTPase, relieves the endosomal cholesterol accumulation in Niemann-Pick type C1 cells but does not revert the reduced endosomal motility caused by blocking endo-CHOL synthesis. Our results suggested that endo-CHOL contributes to the cholesterol content of late endosomes and controls its motility, in a manner independent of NPC1. These results also supported the concept that endosomal motility plays an important role in controlling cholesterol trafficking activities.

Mammalian cells acquire cholesterol from low-density lipoprotein (LDL) and from endogenous biosynthesis. The roles of the Niemann-Pick type C1 protein in mediating the endosomal transport of LDL-derived cholesterol and endogenously synthesized cholesterol are discussed. Excess cellular cholesterol is converted to cholesteryl esters by the enzyme acyl-coenzyrne A:cholesterol acyltransferase (ACAT) I or is removed from a cell by cellular cholesterol efflux at the plasma membrane. A close relationship between the ACXF substrate pool and the cholesterol efflux pool is proposed. Sterol-sensing domains (SSDs) are present in several membrane proteins, including NPC1, HMG-CoA reductase, and the SREBP cleavage-activating protein. The functions of SSDs are described. ACAT1 is an endoplasmic reticulum cholesterol sensor and contains a signature motif characteristic of the membraric-bound acyltransferase family. The nonvesicular cholesterol translocation processes involve the START domain proteins and the oxysterol binding protein-related proteins (ORPs). The properties of these proteins are summarized.

Niemann-Pick type C (NPC) 1 protein plays important roles in moving cholesterol and other lipids out of late endosomes by means of vesicular trafficking, but it is not known whether NPC1 directly interacts with cholesterol. We performed photoaffinity labeling of intact cells expressing fluorescent protein (FP)-tagged NPC1 by using [H-3]7,7-azocholestanol ([H-3]AC). After immunoprecipitation, H-3-labled NPC1-GFP appeared as a single band. Including excess unlabeled sterol to the labeling reaction significantly diminished the labeling. Altering the NPC1 sterol-sensing domain (SSD) with loss-of-function mutations (P692S and Y635C) severely reduced the extent of labeling. To further demonstrate the specificity of labeling, we show that NPC2, a late endosomal/lysosomal protein that binds to cholesterol with high affinity, is labeled, whereas mutant NPC2 proteins inactive in binding cholesterol are not. Vamp7, an abundant late endosomal membrane protein without an SSD but with one transmembrane domain, cannot be labeled. Binding between [H-3]AC and NPC1 does not require NPC2. Treating cells with either U-18666A, a compound that creates an NPC-like phenotype, or with bafilomycin All, a compound that raises late endosomal pH, has no effect on labeling of NPC1-YFP, suggesting that both drugs affect processes other than NPC1 binding to cholesterol. We also developed a procedure to label the NPC1-YFP by [H-3]AC in vitro and showed that cholesterol is more effective in protection against labeling than its analogs epicholesterol or 5-alpha-cholestan. Overall, the results demonstrate that there is direct binding between NPC1 and azocholestanol; the binding does not require NPC2 but requires a functional SSD within NPC1.

We previously studied the early trafficking of low density lipoprotein (LDL)-derived cholesterol in mutant Chinese hamster ovary cells defective in Niemann-Pick type C1 (NPC1) using cyclodextrin ( CD) to monitor the arrival of cholesterol from the cell interior to the plasma membrane ( PM) (Cruz, J. C., Sugii, S., Yu, C., and Chang, T.-Y. ( 2000) J. Biol. Chem. 275, 4013 - 4021). We found that newly hydrolyzed cholesterol derived from LDL first appears in certain CD-accessible pool(s), which we assumed to be the PM, before accumulating in the late endosome/lysosome, where NPC1 resides. To determine the identity of the early CD-accessible pool( s), in this study, we performed additional experiments, including the use of revised CD incubation protocols. We found that prolonged incubation with CD (&gt; 30 min) caused cholesterol in internal membrane compartment( s) to redistribute to the PM, where it became accessible to CD. In contrast, a short incubation with CD ( 5 - 10 min) did not cause such an effect. We also show that one of the early compartments contains acid lipase (AL), the enzyme required for liberating cholesterol from cholesteryl ester in LDL. Biochemical and microscopic evidence indicates that most of the AL is present in endocytic compartment( s) distinct from the late endosome/lysosome. Our results suggest that cholesterol is liberated from LDL cholesteryl ester in the hydrolytic compartment containing AL and then moves to the NPC1-containing late endosome/lysosome before reaching the PM or the endoplasmic reticulum.

BCtheta is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin 0 (theta-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCtheta can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCtheta signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCtheta. Under this condition, BCtheta mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCtheta staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to beta-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCtheta staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCtheta is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.

We report the chemical synthesis of a new photo-activatable cholesterol analog 7,7-azocholestanol (AC) and its linoleate ester (ACL). We also examined the biochemical properties of the sterol and its ester by employing several different mutant Chinese hamster ovary (CHO) cell lines with defined abnormalities in cholesterol metabolism as tools. AC mimics cholesterol in supporting the growth of a mutant cell line (M19) that requires cholesterol for growth. In normal cells, tritiated ACL present in low-density lipoprotein (LDL) was hydrolyzed and reesterified in a manner similar to tritiated cholesteryl linoleate (CL) in LDL. Also, in the mutant cell line (AC29) lacking the enzyme acyl-coenzyme A:cholesterol acyltransferase or in the mutant cell line (CT60) defective in the Niemann-Pick type C1 protein, the hydrolysis of ACL in LDL was normal, but the reesterification of the liberated AC was defective. Therefore, the metabolism of ACL in LDL is very similar to that of CL in LDL. Tritium-labeled AC delivered to intact CHO cells as a cyclodextrin complex was shown to photoaffinity label several discrete polypeptides, including caveolin-1.jlr These results demonstrate AC as an effective reagent for studying cholesterol-protein interactions involved in intracellular cholesterol trafficking.

Cellular interactions of advanced glycation end products (AGE) are mediated by AGE receptors, We demonstrated previously that class A scavenger receptor types I and II (SR-A) and CD36, a member of class B scavenger receptor family, serve as the AGE receptors, In this study, we investigated whether scavenger receptor class B type I (SR-BI), another receptor belonging to class B scavenger receptor family, was also an AGE receptor. We used Chinese hamster ovary (CHO) cells overexpressed hamster SR-BI (CHO-SR-BI cells). I-125-AGE-bovine serum albumin (AGE-BSA) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-SR-BI cells. I-125-AGE-BSA exhibited saturable binding to CHO-SR-BI cells (K-d = 8.3 mug/ml). Endocytic uptake of I-125-AGE-BSA by CHO-SR-BI cells was completely inhibited by oxidized low density lipoprotein (LDL) and acetylated LDL, whereas LDL exerted only a weak inhibitory effect (&lt;20%). Cross-competition experiments showed that AGE-BSA had no effect on HDL binding to these cells and vice verse. Interestingly, however, SR-BI-mediated selective uptake of HDL-CE was completely inhibited by AGE-BSA in a dose-dependent manner (IC50 &lt;10 mug/ml). Furthermore, AGE-BSA partially inhibited (by &lt;30%) the selective uptake of HDL-CE in human hepatocarcinoma HepG2 cells (IC50 &lt;30 mug/ml). In addition, [H-3]cholesterol efflux from CHO-SR-BI cells to HDL was significantly inhibited by AGE-BSA in a dose-dependent manner (IC50 &lt;30 &lt;mu&gt;g/ml), Our results indicate that AGE proteins, as ligands for SR-BI, effectively inhibit both SR-BI-mediated selective uptake of HDL-CE and cholesterol efflux from peripheral cells to HDL, suggesting that AGE proteins might modulate SR-BI-mediated cholesterol metabolism in vivo.

Sulfonylureas are generally used in the therapeutic treatment of non- insulin-dependent diabetes mellitus. Little is known, however, whether they also affect the lipid metabolism. Using glibenclamide (GB), a typical sulfonylurea, we have investigated its effects on the lipid metabolism in macrophages, J774 and phorbol ester-treated THP-1 cells. In the whole-cell assay system for cholesteryl ester (CE) accumulation that is inducedby addition of chemically modified low-density lipoprotein (LDL), such as Ac-LDL and ox-LDL, GB effectively inhibited the CE accumulation of J774 cells in dose-dependent manners. The inhibition was resulted from increase in free cholesterol but not from change in total cholesterol amount. The results suggest that GB acts on acyl-CoA: cholestrol acytransferase (ACAT) and inhibits its activity. To confirm the possibility, we then tested GB by another assay system using ACAT that was solubilized from the cells and reconstituted into the liposome composed of phosphatidyl choline- cholesterol. GB inhibition was not so much effective as those by CI-976 and NTE-122, known ACAT inhibitors, but the inhibition was complete in the presence of 100 μM GB. Using cell homogenates of PMA-stimulated THP-1 cells, GB also inhibited the ACAT activity to the level of undifferentiated THP-1 cells. These results indicate that GB acts as ACAT inhibitor but the chemical structure is quite different from the conventional ACAT inhibitors, suggesting it can be a seed to generate potential ACAT inhibitors which do not exhibit toxicity in adrenal gland.