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1.  Suppression of neuroinflammation in forebrain-specific Cdk5 conditional knockout mice by PPARγ agonist improves neuronal loss and early lethality 
Background
Cyclin-dependent kinase 5 (Cdk5) is essential for brain development and function, and its deregulated expression is implicated in some of neurodegenerative diseases. We reported earlier that the forebrain-specific Cdk5 conditional knockout (cKO) mice displayed an early lethality associated with neuroinflammation, increased expression of the neuronal tissue-type plasminogen activator (tPA), and neuronal migration defects.
Methods
In order to suppress neuroinflammation in the cKO mice, we first treated these mice with pioglitazone, a PPARγ agonist, and analyzed its effects on neuronal loss and longevity. In a second approach, to delineate the precise role of tPA in neuroinflammation in these mice, we generated Cdk5 cKO; tPA double knockout (dKO) mice.
Results
We found that pioglitazone treatment significantly reduced astrogliosis, microgliosis, neuronal loss and behavioral deficit in Cdk5 cKO mice. Interestingly, the dKO mice displayed a partial reversal in astrogliosis, but they still died at early age, suggesting that the increased expression of tPA in the cKO mice does not contribute significantly to the pathological process leading to neuroinflammation, neuronal loss and early lethality.
Conclusion
The suppression of neuroinflammation in Cdk5 cKO mice ameliorates gliosis and neuronal loss, thus suggesting the potential beneficial effects of the PPARγ agonist pioglitazone for the treatment for neurodegenerative diseases.
doi:10.1186/1742-2094-11-28
PMCID: PMC3931315  PMID: 24495352
Neuroinflammation; Cdk5; Pioglitazone; tPA; Cdk5 conditional knockout mice
2.  Regulation of an Autoimmune Model for Multiple Sclerosis in Th2-Biased GATA3 Transgenic Mice 
T helper (Th)2 cells have been proposed to play a neuroprotective role in multiple sclerosis (MS). This is mainly based on “loss-of-function” studies in an animal model for MS, experimental autoimmune encephalomyelitis (EAE), using blocking antibodies against Th2 related cytokines, and knockout mice lacking Th2-related molecules. We tested whether an increase of Th2 responses (“gain-of-function” approach) could alter EAE, the approach of novel GATA binding protein 3 (GATA3)-transgenic (tg) mice that overexpress GATA3, a transcription factor required for Th2 differentiation. In EAE induced with myelin oligodendrocyte glycoprotein (MOG)35–55 peptide, GATA3-tg mice had a significantly delayed onset of disease and a less severe maximum clinical score, compared with wild-type C57BL/6 mice. Histologically, GATA3-tg mice had decreased levels of meningitis and demyelination in the spinal cord, and anti-inflammatory cytokine profiles immunologically, however both groups developed similar levels of MOG-specific lymphoproliferative responses. During the early stage, we detected higher levels of interleukin (IL)-4 and IL-10, with MOG and mitogen stimulation of regional lymph node cells in GATA3-tg mice. During the late stage, only mitogen stimulation induced higher IL-4 and lower interferon-γ and IL-17 production in GATA3-tg mice. These results suggest that a preexisting bias toward a Th2 immune response may reduce the severity of inflammatory demyelinating diseases, including MS.
doi:10.3390/ijms15021700
PMCID: PMC3958817  PMID: 24463292
autoimmune demyelinating diseases; GATA3 transcription factor; autoimmunity; animal models; paraffin; histology; oligodendrocyte-myelin glycoprotein; Th1-Th2 assays; Luxol fast blue; Th17; incomplete Freund’s adjuvant
3.  Acute Optogenetic Silencing of Orexin/Hypocretin Neurons Induces Slow-Wave Sleep in Mice 
Orexin/hypocretin neurons have a crucial role in the regulation of sleep and wakefulness. To help determine how these neurons promote wakefulness, we generated transgenic mice in which orexin neurons expressed halorhodopsin (orexin/Halo mice), an orange light-activated neuronal silencer. Slice patch-clamp recordings of orexin neurons that expressed halorhodopsin demonstrated that orange light photic illumination immediately hyperpolarized membrane potential and inhibited orexin neuron discharge in proportion to illumination intensity. Acute silencing of orexin neurons in vivo during the day (the inactive period) induced synchronization of the electroencephalogram and a reduction in amplitude of the electromyogram that is characteristic of slow-wave sleep (SWS). In contrast, orexin neuron photoinhibition was ineffective during the night (active period). Acute photoinhibition of orexin neurons during the day in orexin/Halo mice also reduced discharge of neurons in an orexin terminal field, the dorsal raphe (DR) nucleus. However, serotonergic DR neurons exhibited normal discharge rates in mice lacking orexin neurons. Thus, although usually highly dependent on orexin neuronal activity, serotonergic DR neuronal activity can be regulated appropriately in the chronic absence of orexin input. Together, these results demonstrate that acute inhibition of orexin neurons results in time-of-day-dependent induction of SWS and in reduced firing rate of neurons in an efferent projection site thought to be involved in arousal state regulation. The results presented here advance our understanding of the role of orexin neurons in the regulation of sleep/wakefulness and may be relevant to the mechanisms that underlie symptom progression in narcolepsy.
doi:10.1523/JNEUROSCI.0784-11.2011
PMCID: PMC3864636  PMID: 21775598
4.  Myoepithelial Carcinoma of the Breast Treated with Surgery and Chemotherapy 
Myoepithelial carcinoma (malignant myoepithelioma) of the breast is a rare tumor, for which only a limited number of reports have been published. Most of the reports emphasized diagnosis and pathology but not biological behavior and treatment. We report a 61-year-old patient with breast myoepithelial carcinoma who developed locoregional and distant metastases and received many chemotherapy regimens. She presented with an elastic hard mass of the left breast. Breast conserving surgery was performed as part of both diagnosis and treatment. From the results of histological and immunohistochemical examinations, this case was considered to be a myoepithelial carcinoma. Fifteen months after the completion of adjuvant radiotherapy, distant metastasis of the left parasternal lymph node metastasis developed. She was treated by further excision and received a total of four regimens of chemotherapy including a combination of doxorubicin and cyclophosphamide. She received chemotherapy for 20 months after the diagnosis of metastasis.
doi:10.1155/2013/164761
PMCID: PMC3745899  PMID: 23984136
5.  In Vivo Function and Evolution of the Eutherian-Specific Pluripotency Marker UTF1 
PLoS ONE  2013;8(7):e68119.
Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. UTF1 is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the UTF1 gene during mouse development. We found that homozygous UTF1 mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of UTF1 expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of UTF1 expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the UTF1 gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the UTF1 genes.
doi:10.1371/journal.pone.0068119
PMCID: PMC3706607  PMID: 23874519
6.  Efficacy of Using Three-Tesla Magnetic Resonance Imaging Diagnosis of Capsule Invasion for Decision-Making About Neurovascular Bundle Preservation in Robotic-Assisted Radical Prostatectomy 
Korean Journal of Urology  2013;54(7):437-441.
Purpose
To evaluate the efficacy of using 3-tesla (T) magnetic resonance imaging (MRI) diagnosis of extracapsular extension (ECE) for decision-making about neurovascular bundle (NVB) preservation in robot-assisted radical prostatectomy (RARP) for prostate cancer (PC).
Materials and Methods
We prospectively collected data on PC patients (n=67) who underwent preoperative 3-T MRI before RARP. The choice between nerve sparing or resection was based on 3-T MRI findings of ECE. We compared the MRI findings with the pathological data on surgical margins. Our clinical staging in this study was defined only by MRI.
Results
When the data were divided by prostate lobe (right lobe or left lobe, n=134), 3-T MRI showed 28 positive cases of ECE in 134 prostate lobes, allowing NVB preservation in 42 cases (31.3%). Nerve-sparing surgery was achieved in 38.7% of cases in which clinical T2 staging by MRI was reported. The pathological data revealed that 10 of 134 prostate lobes had positive ECE. The overall sensitivity, specificity, positive predictive value, and negative predictive value for predicting stage T3 (positive ECE) by side were 60.0% (12 of 20 sides), 86.0% (98 of 114 sides), 42.9% (12 of 28 sides), and 92.5% (98 of 106 sides), respectively.
Conclusions
Three-T MRI prior to RARP enables the use of ECE diagnosis to guide decision-making about NVB preservation, with comparatively high specificity and negative predictive value. Further prospective studies are underway to reach more definitive conclusions.
doi:10.4111/kju.2013.54.7.437
PMCID: PMC3715706  PMID: 23878685
Diagnoses; Magnetic resonance imaging; Prostatectomy; Robotics
7.  Clinical Significance of Amyloid Precursor Protein in Patients with Testicular Germ Cell Tumor 
Advances in Urology  2013;2013:348438.
Introduction. The biological role of amyloid precursor protein (APP) is not well understood, especially in testicular germ cell tumors (TGCTs). Therefore, we aimed to investigate the immunoreactivity (IR) and expression of APP in TGCTs and evaluated its clinical relevance. Materials and Methods. We performed an analysis of immunohistochemistry and mRNA expression of APP in 64 testicular specimens and 21 snap-frozen samples obtained from 1985 to 2004. We then evaluated the association between APP expression and clinicopathological status in TGCTs. Results. Positive APP IR was observed in 9.8% (4/41) of seminomatous germ cell tumors (SGCTs) and 39.1% (9/23) of nonseminomatous germ cell tumors (NGCTs). NGCTs showed significantly more cases of positive IR (P = 0.00870) and a higher mRNA expression level compared with those of SGCTs (P = 0.0140). Positive APP IR was also significantly associated with α-fetoprotein (αFP) elevation (P = 0.00870) and venous invasion (P = 0.0414). Conclusion. We observed an elevated APP expression in TGCTs, especially in NGCTs. APP may be associated with a more aggressive cancer in TGCTs.
doi:10.1155/2013/348438
PMCID: PMC3639667  PMID: 23662100
8.  Bioluminescence Imaging of β Cells and Intrahepatic Insulin Gene Activity under Normal and Pathological Conditions 
PLoS ONE  2013;8(4):e60411.
In diabetes research, bioluminescence imaging (BLI) has been applied in studies of β-cell impairment, development, and islet transplantation. To develop a mouse model that enables noninvasive imaging of β cells, we generated a bacterial artificial chromosome (BAC) transgenic mouse in which a mouse 200-kbp genomic fragment comprising the insulin I gene drives luciferase expression (Ins1-luc BAC transgenic mouse). BLI of mice was performed using the IVIS Spectrum system after intraperitoneal injection of luciferin, and the bioluminescence signal from the pancreatic region analyzed. When compared with MIP-Luc-VU mice [FVB/N-Tg(Ins1-luc)VUPwrs/J] expressing luciferase under the control of the 9.2-kbp mouse insulin I promoter (MIP), the bioluminescence emission from Ins1-luc BAC transgenic mice was enhanced approximately 4-fold. Streptozotocin-treated Ins1-luc BAC transgenic mice developed severe diabetes concomitant with a sharp decline in the BLI signal intensity in the pancreas. Conversely, mice fed a high-fat diet for 8 weeks showed an increase in the signal, reflecting a decrease or increase in the β-cell mass. Although the bioluminescence intensity of the islets correlated well with the number of isolated islets in vitro, the intensity obtained from a living mouse in vivo did not necessarily reflect an absolute quantification of the β-cell mass under pathological conditions. On the other hand, adenovirus-mediated gene transduction of β-cell-related transcription factors in Ins1-luc BAC transgenic mice generated luminescence from the hepatic region for more than 1 week. These results demonstrate that BLI in Ins1-luc BAC transgenic mice provides a noninvasive method of imaging islet β cells and extrapancreatic activity of the insulin gene in the liver under normal and pathological conditions.
doi:10.1371/journal.pone.0060411
PMCID: PMC3617225  PMID: 23593212
9.  A Bacterial Artificial Chromosome Transgene with Polymorphic Cd72 Inhibits the Development of Glomerulonephritis and Vasculitis in MRL-Faslpr Lupus Mice 
Systemic lupus erythematosus is considered to be under the control of polygenic inheritance, developing according to the cumulative effects of susceptibility genes with polymorphic alleles; however, the mechanisms underlying the roles of polygenes based on functional and pathological genomics remain uncharacterized. In this study, we substantiate that a CD72 polymorphism in the membrane-distal extracellular domain impacts on both the development of glomerulonephritis and vasculitis in a lupus model strain of mice, MRL/MpJ-Faslpr, and the reactivity of BCR signal stimulation. We generated mice carrying a bacterial artificial chromosome transgene originating from C57BL/6 (B6) mice that contains the Cd72b locus (Cd72B6 transgenic [tg]) or the modified Cd72b locus with an MRL-derived Cd72c allele at the polymorphic region corresponding to the membrane-distal extracellular domain (Cd72B6/MRL tg). Cd72B6 tg mice, but not Cd72B6/MRL tg mice, showed a significant reduction in mortality following a marked improvement of disease associated with decreased serum levels of IgG3 and anti-dsDNA Abs. The number of splenic CD4−CD8− T cells in Cd72B6 tg mice was decreased significantly in association with a reduced response to B cell receptor signaling. These results indicate that the Cd72 polymorphism affects susceptibility to lupus phenotypes and that novel functional rescue by a bacterial artificial chromosome transgenesis is an efficient approach with wide applications for conducting a genomic analysis of polygene diseases.
doi:10.4049/jimmunol.1202196
PMCID: PMC3575566  PMID: 23365086
10.  Red Wine Consumption is inversely associated with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA Adduct Levels in Prostate 
In humans, genetic variation and dietary factors may alter the biologic effects of exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the major heterocyclic amines generated from cooking meats at high temperatures that has carcinogenic potential through the formation of DNA adducts. Previously, we reported grilled red meat consumption associated with PhIP-DNA adduct levels in human prostate. In the present study, we expanded our investigation to estimate the associations between beverage consumption and PhIP-DNA adduct levels in prostate for 391 prostate cancer cases. Of the 15 beverages analyzed, red wine consumption had the strongest association with PhIP-DNA adduct levels showing an inverse correlation in both tumor (p=0.006) and non-tumor (p=0.002) prostate cells. Red wine consumption differed significantly between African-American and white cases, but PhIP-DNA adduct levels in prostate did not vary by race. In African Americans compared with whites, however, associations between red wine consumption and PhIP-DNA adduct levels were not as strong as associations with specific (e.g., SULT1A1 and UGT1A10 genotypes) and non-specific (e.g., African ancestry) genetic variation. In a multivariable model, the covariate for red wine consumption explained a comparable percentage (13-16%) of the variation in PhIP-DNA adduct levels in prostate across the two racial groups, but the aforementioned genetic factors explained 33% of the PhIP-DNA adduct variation in African-American cases, while only 19% of the PhIPDNA adduct variation in whites. We conclude that red wine consumption may counteract biologic effects of PhIP exposure in human prostate, but genetic factors may play an even larger role, particularly in African Americans.
doi:10.1158/1940-6207.CAPR-11-0100
PMCID: PMC3188357  PMID: 21846795
compounds, heterocyclic; resveratrol; UDP-glucuronosyltransferase; sulfotransferases; African Americans; chemoprevention
11.  Hypomyelination Phenotype Caused by Impaired Differentiation of Oligodendrocytes in Emx1-cre Mediated Cdk5 Conditional Knockout Mice 
Neurochemical research  2011;36(7):1293-1303.
Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in neuronal migration and differentiation, and in axonal elongation. Although many studies have been conducted to analyze neuronal functions of Cdk5, its kinase activity has also been reported during oligodendrocyte differentiation, which suggests Cdk5 may play an important role in oligodendrocytes. Here, we describe a hypomyelination phenotype observed in Emx1-cre mediated Cdk5 conditional knockout (cKO) mice (Emx1-cKO), in which the Cdk5 gene was deleted in neurons, astrocytes and oligodendrocyte -lineage cells. In contrast, the Cdk5 gene in CaMKII cKO mice was deleted only in neurons. Because the development of mature oligodendrocytes from oligodendrocyte precursor cells is a complex process, we performed in situ hybridization using markers for the oligodendrocyte precursor cell and for the differentiated oligodendrocyte. Our results indicate that hypomyelination in Emx1-cKO is due to the impaired differentiation of oligodendrocytes, rather than to the proliferation or migration of their precursors. The present study confirmed the in vivo role of Cdk5 in oligodendrocyte differentiation.
doi:10.1007/s11064-010-0391-0
PMCID: PMC3431604  PMID: 21210220
Cyclin-dependent kinase 5; Oligodendrocyte differentiation; Hypomyelination; Conditional knockout mice
12.  Transcription factor GATA-3 is essential for lens development 
During vertebrate lens development, the anterior, ectoderm-derived lens vesicle cells differentiate into a monolayer of epithelial cells that retain proliferative potential. Subsequently, they exit the cell cycle and give rise to posterior lens fiber cells that form the lens body. In the present study, we demonstrate that the transcription factor GATA-3 is expressed in the posterior lens fiber cells during embryogenesis, and that GATA-3-deficiency impairs lens development. Interestingly, expression of E-cadherin, a premature lens vesicle marker, is abnormally prolonged in the posterior region of Gata3 homozygous mutant lenses. Furthermore, expression of γ-crystallin, a differentiation marker for fiber cells, is reduced. This suppressed differentiation is accompanied by an abnormal cellular proliferation, as well as with diminished levels of the cell-cycle inhibitors Cdkn1b/p27 and Cdkn1c/p57 and increased Ccnd2/cyclin D2 abundance. Thus, these observations suggest that GATA-3 is essential for lens cells differentiation and proper cell cycle control.
doi:10.1002/dvdy.22035
PMCID: PMC3408506  PMID: 19623612
GATA-3; crystallin; lens fiber; differentiation; cell cycle; apoptosis
13.  Two distinct origins for Leydig cell progenitors in the fetal testis 
Developmental biology  2011;352(1):14-26.
During the differentiation of the mammalian embryonic testis, two compartments are defined: the testis cords and the interstitium. The testis cords give rise to the adult seminiferous tubules, whereas steroidogenic Leydig cells and other less well characterized cell types differentiate in the interstitium (the space between testis cords). Although the process of testis cord formation is essential for male development, it is not entirely understood. It has been viewed as a Sertoli-cell driven process, but growing evidence suggests that interstitial cells play an essential role during testis formation. However, little is known about the origin of the interstitium or the molecular and cellular diversity within this early stromal compartment. To better understand the process of mammalian gonad differentiation, we have undertaken an analysis of developing interstitial/stromal cells in the early mouse testis and ovary. We have discovered molecular heterogeneity in the interstitium and have characterized new markers of distinct cell types in the gonad: MAFB, C-MAF, and VCAM1. Our results show that at least two distinct progenitor lineages give rise to the interstitial/stromal compartment of the gonad: the coelomic epithelium and specialized cells along the gonad-mesonephros border. We demonstrate that both these populations give rise to interstitial precursors that can differentiate into fetal Leydig cells. Our analysis also reveals that perivascular cells migrate into the gonad from the mesonephric border along with endothelial cells and that these vessel-associated cells likely represent an interstitial precursor lineage. This study highlights the cellular diversity of the interstitial cell population and suggests that complex cell-cell interactions among cells in the interstitium are involved in testis morphogenesis.
doi:10.1016/j.ydbio.2011.01.011
PMCID: PMC3055913  PMID: 21255566
testis; interstitium; Maf; Leydig cell; vasculature; mesonephros
15.  Combinatorial motif analysis of regulatory gene expression in Mafb deficient macrophages 
BMC Systems Biology  2011;5(Suppl 2):S7.
Background
Deficiency of the transcription factor MafB, which is normally expressed in macrophages, can underlie cellular dysfunction associated with a range of autoimmune diseases and arteriosclerosis. MafB has important roles in cell differentiation and regulation of target gene expression; however, the mechanisms of this regulation and the identities of other transcription factors with which MafB interacts remain uncertain. Bioinformatics methods provide a valuable approach for elucidating the nature of these interactions with transcriptional regulatory elements from a large number of DNA sequences. In particular, identification of patterns of co-occurrence of regulatory cis-elements (motifs) offers a robust approach.
Results
Here, the directional relationships among several functional motifs were evaluated using the Log-linear Graphical Model (LGM) after extraction and search for evolutionarily conserved motifs. This analysis highlighted GATA-1 motifs and 5’AT-rich half Maf recognition elements (MAREs) in promoter regions of 18 genes that were down-regulated in Mafb deficient macrophages. GATA-1 motifs and MafB motifs could regulate expression of these genes in both a negative and positive manner, respectively. The validity of this conclusion was tested with data from a luciferase assay that used a C1qa promoter construct carrying both the GATA-1 motifs and MAREs. GATA-1 was found to inhibit the activity of the C1qa promoter with the GATA-1 motifs and MafB motifs.
Conclusions
These observations suggest that both the GATA-1 motifs and MafB motifs are important for lineage specific expression of C1qa. In addition, these findings show that analysis of combinations of evolutionarily conserved motifs can be successfully used to identify patterns of gene regulation.
doi:10.1186/1752-0509-5-S2-S7
PMCID: PMC3287487  PMID: 22784578
16.  Isolation and function of mouse tissue resident vascular precursors marked by myelin protein zero 
Newly identified tissue-resident vascular precursor cells are recruited into growing vessels and contribute to vasculogenesis in adult mice.
Vasculogenesis describes the process of de novo vessel formation from vascular precursor cells. Although formation of the first major vessels, such as the dorsal aorta and cardinal veins, occurs during embryonic vasculogenesis, the contribution of precursor cell populations to postnatal vessel development is not well understood. Here, we identified a novel population of postnatal vascular precursor cells in mice. These cells express the Schwann cell protein myelin protein zero (Po) and exhibit a CD45−CD31−VEcad−c-kit+CXCR4+ surface phenotype. Po+ vascular precursors (PVPs) are recruited into the growing vasculature, and comprise a minor population of arterial endothelial cells in adult mice. Recruitment of PVPs into growing vessels is mediated by CXCL12–CXCR4 signaling, and is enhanced during vascular expansion induced by Notch inhibition. Po-specific ablation of Flk1, a receptor for VEGF, results in branching defects and insufficient arterial patterning in the retina, as well as reduced neovascularization of tumors and ischemic tissues. Thus, in postnatal mice, although growing vessels are formed primarily by angiogenesis from preexisting vessels, a minor population of arterial endothelia may be derived from tissue-resident vascular precursor cells.
doi:10.1084/jem.20102187
PMCID: PMC3092348  PMID: 21536740
17.  The E3 Ubiquitin Ligase Activity of Trip12 Is Essential for Mouse Embryogenesis 
PLoS ONE  2011;6(10):e25871.
Protein ubiquitination is a post-translational protein modification that regulates many biological conditions [1], [2], [3], [4]. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1 [5], [6]. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12mt/mt) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12mt/mt embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16 [7], [8], [9], [10]. In contrast, Trip12mt/mt ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12mt/mt ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.
doi:10.1371/journal.pone.0025871
PMCID: PMC3196520  PMID: 22028794
18.  Insulin Transactivator MafA Regulates Intrathymic Expression of Insulin and Affects Susceptibility to Type 1 Diabetes 
Diabetes  2010;59(10):2579-2587.
OBJECTIVE
Tissue-specific self-antigens are ectopically expressed within the thymus and play an important role in the induction of central tolerance. Insulin is expressed in both pancreatic islets and the thymus and is considered to be the primary antigen for type 1 diabetes. Here, we report the role of the insulin transactivator MafA in the expression of insulin in the thymus and susceptibility to type 1 diabetes.
RESEARCH DESIGN AND METHODS
The expression profiles of transcriptional factors (Pdx1, NeuroD, Mafa, and Aire) in pancreatic islets and the thymus were examined in nonobese diabetic (NOD) and control mice. Thymic Ins2 expression and serum autoantibodies were examined in Mafa knockout mice. Luciferase reporter assay was performed for newly identified polymorphisms of mouse Mafa and human MAFA. A case-control study was applied for human MAFA polymorphisms.
RESULTS
Mafa, Ins2, and Aire expression was detected in the thymus. Mafa expression was lower in NOD thymus than in the control and was correlated with Ins2 expression. Targeted disruption of MafA reduced thymic Ins2 expression and induced autoantibodies against pancreatic islets. Functional polymorphisms of MafA were newly identified in NOD mice and humans, and polymorphisms of human MAFA were associated with susceptibility to type 1 diabetes but not to autoimmune thyroid disease.
CONCLUSIONS
These data indicate that functional polymorphisms of MafA are associated with reduced expression of insulin in the thymus and susceptibility to type 1 diabetes in the NOD mouse as well as human type 1 diabetes.
doi:10.2337/db10-0476
PMCID: PMC3279543  PMID: 20682694
19.  Phosphorylation of p27Kip1 at Thr187 by Cyclin-dependent Kinase 5 Modulates Neural Stem Cell Differentiation 
Molecular Biology of the Cell  2010;21(20):3601-3614.
Cdk5 plays a role in nervous system development; its role in the initial stages of neural differentiation is poorly understood. We isolated neural stem cells from E13 Cdk5 WT and KO mouse and observed them as they switched from proliferating stage to neural differentiation. We show that Cdk5 phosphorylation of p27kip1 at Thr187 is crucial to neural differentiation.
Cyclin-dependent kinase 5 (Cdk5) plays a key role in the development of the mammalian nervous system; it phosphorylates a number of targeted proteins involved in neuronal migration during development to synaptic activity in the mature nervous system. Its role in the initial stages of neuronal commitment and differentiation of neural stem cells (NSCs), however, is poorly understood. In this study, we show that Cdk5 phosphorylation of p27Kip1 at Thr187 is crucial to neural differentiation because 1) neurogenesis is specifically suppressed by transfection of p27Kip1 siRNA into Cdk5+/+ NSCs; 2) reduced neuronal differentiation in Cdk5−/− compared with Cdk5+/+ NSCs; 3) Cdk5+/+ NSCs, whose differentiation is inhibited by a nonphosphorylatable mutant, p27/Thr187A, are rescued by cotransfection of a phosphorylation-mimicking mutant, p27/Thr187D; and 4) transfection of mutant p27Kip1 (p27/187A) into Cdk5+/+ NSCs inhibits differentiation. These data suggest that Cdk5 regulates the neural differentiation of NSCs by phosphorylation of p27Kip1 at theThr187 site. Additional experiments exploring the role of Ser10 phosphorylation by Cdk5 suggest that together with Thr187 phosphorylation, Ser10 phosphorylation by Cdk5 promotes neurite outgrowth as neurons differentiate. Cdk5 phosphorylation of p27Kip1, a modular molecule, may regulate the progress of neuronal differentiation from cell cycle arrest through differentiation, neurite outgrowth, and migration.
doi:10.1091/mbc.E10-01-0054
PMCID: PMC2954124  PMID: 20810788
20.  Impairment of Host Defense against Disseminated Candidiasis in Mice Overexpressing GATA-3▿  
Infection and Immunity  2010;78(5):2302-2311.
Candida species are the most common source of nosocomial invasive fungal infections. Previous studies have indicated that T-helper immune response is the critical host factor for susceptibility to Candida infection. The transcription factor GATA-3 is known as the master regulator for T-helper type 2 (Th2) differentiation. We therefore investigated the role of GATA-3 in the host defense against systemic Candida infection using GATA-3-overexpressing transgenic mice. The survival of GATA-3-overexpressing mice after Candida infection was significantly lower than that of wild-type mice. Candida outgrowth was significantly increased in the kidneys of GATA-3-overexpressing mice, compared with wild-type mice. The levels of various Th2 cytokines, including interleukin-4 (IL-4), IL-5, and IL-13, were significantly higher while the level of Th1 cytokine gamma interferon was significantly lower in the splenocytes of GATA-3-overexpressing mice after Candida infection. Recruitment of macrophages into the peritoneal cavity in response to Candida infection and their phagocytic activity were significantly lower in GATA-3-overexpressing mice than in wild-type mice. Exogenous administration of gamma interferon to GATA-3-overexpressing mice significantly reduced Candida outgrowth in the kidney and thus increased the survival rate. Administration of gamma interferon also increased the recruitment of macrophages into the peritoneal cavity in response to Candida infection. These results indicate that overexpression of GATA-3 modulates macrophage antifungal activity and thus enhances the susceptibility to systemic Candida infection, possibly by reducing the production of gamma interferon in response to Candida infection.
doi:10.1128/IAI.01398-09
PMCID: PMC2863530  PMID: 20231412
21.  Maf promotes osteoblast differentiation in mice by mediating the age-related switch in mesenchymal cell differentiation 
The Journal of Clinical Investigation  2010;120(10):3455-3465.
Aging leads to the disruption of the homeostatic balance of multiple biological systems. In bone marrow multipotent mesenchymal cells undergo differentiation into various anchorage-dependent cell types, including osteoblasts and adipocytes. With age as well as with treatment of antidiabetic drugs such as thiazolidinediones, mesenchymal cells favor differentiation into adipocytes, resulting in an increased number of adipocytes and a decreased number of osteoblasts, causing osteoporosis. The mechanism behind this differentiation switch is unknown. Here we show an age-related decrease in the expression of Maf in mouse mesenchymal cells, which regulated mesenchymal cell bifurcation into osteoblasts and adipocytes by cooperating with the osteogenic transcription factor Runx2 and inhibiting the expression of the adipogenic transcription factor Pparg. The crucial role of Maf in both osteogenesis and adipogenesis was underscored by in vivo observations of delayed bone formation in perinatal Maf–/– mice and an accelerated formation of fatty marrow associated with bone loss in aged Maf+/– mice. This study identifies a transcriptional mechanism for an age-related switch in cell fate determination and may provide a molecular basis for novel therapeutic strategies against age-related bone diseases.
doi:10.1172/JCI42528
PMCID: PMC2947225  PMID: 20877012
22.  Loss of the BMP antagonist USAG-1 ameliorates disease in a mouse model of the progressive hereditary kidney disease Alport syndrome 
The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney. Mutations involving any of the collagen IV genes (COL4A3, COL4A4, and COL4A5) affect GBM assembly and cause Alport syndrome, a progressive hereditary kidney disease with no definitive therapy. Previously, we have demonstrated that the bone morphogenetic protein (BMP) antagonist uterine sensitization–associated gene-1 (USAG-1) negatively regulates the renoprotective action of BMP-7 in a mouse model of tubular injury during acute renal failure. Here, we investigated the role of USAG-1 in renal function in Col4a3–/– mice, which model Alport syndrome. Ablation of Usag1 in Col4a3–/– mice led to substantial attenuation of disease progression, normalization of GBM ultrastructure, preservation of renal function, and extension of life span. Immunohistochemical analysis revealed that USAG-1 and BMP-7 colocalized in the macula densa in the distal tubules, lying in direct contact with glomerular mesangial cells. Furthermore, in cultured mesangial cells, BMP-7 attenuated and USAG-1 enhanced the expression of MMP-12, a protease that may contribute to GBM degradation. These data suggest that the pathogenetic role of USAG-1 in Col4a3–/– mice might involve crosstalk between kidney tubules and the glomerulus and that inhibition of USAG-1 may be a promising therapeutic approach for the treatment of Alport syndrome.
doi:10.1172/JCI39569
PMCID: PMC2827946  PMID: 20197625
23.  Cyclooxygenase 2 and Prostaglandin E2 are not Involved in N-Nitrosodiethylamine-Initiated Early Rat Hepatocarcinogenesis 
Journal of Toxicologic Pathology  2009;22(4):263-271.
The present study was undertaken to investigate the effect of dietary supplementation with nimesulide or eugenol on N-nitrosodiethylamine (DEN)-initiated early hepatocarcinogenesis in F344 male rats. Both compounds did not alter the expression of cytochrome P450 (CYP) 2E1, the enzyme that plays a major role in the activation of DEN to genotoxic products; however, nimesulide induced the expression of CYP1A1. Western blot analysis revealed that COX-1 and COX-2 protein expressions were not modulated by DEN compared with normal controls. Furthermore, post-initiation feeding with nimesulide or eugenol did not modulate COX-2 protein expression in normal or DEN-treated rats, whereas eugenol significantly increased the liver prostaglandin E2 (PGE2) levels of DEN-injected animals compared with the DEN controls. Ultimately, nimesulide or eugenol did not modify DEN-induced hepatocarcinogenesis as evidenced by insignificant changes in the number and size of preneoplastic placental glutathione S-transferase (GST-P) positive liver foci compared with the DEN controls. These results suggest that COX-2, as well as prostaglandin E2, may play no role in the post-initiation development of DEN-induced rat hepatocarcinogenesis at an early stage.
doi:10.1293/tox.22.263
PMCID: PMC3234601  PMID: 22272001
rat; liver; nimesulide; eugenol; cyclooxygenases
24.  Aire controls the differentiation program of thymic epithelial cells in the medulla for the establishment of self-tolerance 
The Journal of Experimental Medicine  2008;205(12):2827-2838.
The roles of autoimmune regulator (Aire) in the expression of the diverse arrays of tissue-restricted antigen (TRA) genes from thymic epithelial cells in the medulla (medullary thymic epithelial cells [mTECs]) and in organization of the thymic microenvironment are enigmatic. We approached this issue by creating a mouse strain in which the coding sequence of green fluorescent protein (GFP) was inserted into the Aire locus in a manner allowing concomitant disruption of functional Aire protein expression. We found that Aire+ (i.e., GFP+) mTECs were the major cell types responsible for the expression of Aire-dependent TRA genes such as insulin 2 and salivary protein 1, whereas Aire-independent TRA genes such as C-reactive protein and glutamate decarboxylase 67 were expressed from both Aire+ and Aire− mTECs. Remarkably, absence of Aire from mTECs caused morphological changes together with altered distribution of mTECs committed to Aire expression. Furthermore, we found that the numbers of mTECs that express involucrin, a marker for terminal epidermal differentiation, were reduced in Aire-deficient mouse thymus, which was associated with nearly an absence of Hassall's corpuscle-like structures in the medulla. Our results suggest that Aire controls the differentiation program of mTECs, thereby organizing the global mTEC integrity that enables TRA expression from terminally differentiated mTECs in the thymic microenvironment.
doi:10.1084/jem.20080046
PMCID: PMC2585853  PMID: 19015306
25.  Frequently increased epidermal growth factor receptor (EGFR) copy numbers and decreased BRCA1 mRNA expression in Japanese triple-negative breast cancers 
BMC Cancer  2008;8:309.
Background
Triple-negative breast cancer (estrogen receptor-, progesterone receptor-, and HER2-negative) (TNBC) is a high risk breast cancer that lacks specific therapy targeting these proteins.
Methods
We studied 969 consecutive Japanese patients diagnosed with invasive breast cancer from January 1981 to December 2003, and selected TNBCs based on the immunohistochemical data. Analyses of epidermal growth factor receptor (EGFR) gene mutations and amplification, and BRCA1 mRNA expression were performed on these samples using TaqMan PCR assays. The prognostic significance of TNBCs was also explored. Median follow-up was 8.3 years.
Results
A total of 110 (11.3%) patients had TNBCs in our series. Genotyping of the EGFR gene was performed to detect 14 known EGFR mutations, but none was identified. However, EGFR gene copy number was increased in 21% of TNBCs, while only 2% of ER- and PgR-positive, HER2-negative tumors showed slightly increased EGFR gene copy numbers. Thirty-one percent of TNBCs stained positive for EGFR protein by immunohistochemistry. BRCA1 mRNA expression was also decreased in TNBCs compared with controls. Triple negativity was significantly associated with grade 3 tumors, TP53 protein accumulation, and high Ki67 expression. TNBC patients had shorter disease-free survival than non-TNBC in node-negative breast cancers.
Conclusion
TNBCs have an aggressive clinical course, and EGFR and BRCA1 might be candidate therapeutic targets in this disease.
doi:10.1186/1471-2407-8-309
PMCID: PMC2612006  PMID: 18950515

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