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1.  Notch1 counteracts WNT/β-catenin signaling through chromatin modification in colorectal cancer 
The Journal of Clinical Investigation  2012;122(9):3248-3259.
Crosstalk between the Notch and wingless-type MMTV integration site (WNT) signaling pathways has been investigated for many developmental processes. However, this negative correlation between Notch and WNT/β-catenin signaling activity has been studied primarily in normal developmental and physiological processes in which negative feedback loops for both signaling pathways are intact. We found that Notch1 signaling retained the capability of suppressing the expression of WNT target genes in colorectal cancers even when β-catenin destruction by the adenomatous polyposis coli (APC) complex was disabled. Activation of Notch1 converted high-grade adenoma into low-grade adenoma in an Apcmin mouse colon cancer model and suppressed the expression of WNT target genes in human colorectal cancer cells through epigenetic modification recruiting histone methyltransferase SET domain bifurcated 1 (SETDB1). Extensive microarray analysis of human colorectal cancers also showed a negative correlation between the Notch1 target gene, Notch-regulated ankyrin repeat protein 1 (NRARP), and WNT target genes. Notch is known to be a strong promoter of tumor initiation, but here we uncovered an unexpected suppressive role of Notch1 on WNT/β-catenin target genes involved in colorectal cancer.
doi:10.1172/JCI61216
PMCID: PMC3428081  PMID: 22863622
2.  Inactivation of Notch signaling in the renal collecting duct causes nephrogenic diabetes insipidus in mice 
The Journal of Clinical Investigation  2009;119(11):3290-3300.
The heterogeneous cellular composition of the mammalian renal collecting duct enables regulation of fluid, electrolytes, and acid-base homeostasis, but the molecular mechanism of its development has yet to be elucidated. The Notch signaling pathway is involved in cell fate determination and has been implicated in proximal-distal patterning in the mammalian kidney. To investigate the role of Notch signaling in renal collecting duct development, we generated mice in which Mind bomb-1 (Mib1), an E3 ubiquitin ligase required for the initiation of Notch signaling, was specifically inactivated in the ureteric bud of the developing kidney. Mice lacking Mib1 in the renal collecting duct displayed increased urinary production, decreased urinary osmolality, progressive hydronephrosis, sodium wasting, and a severe urinary concentrating defect manifested as nephrogenic diabetes insipidus. Histological analysis revealed a diminished number of principal cells and corresponding increase in the number of intercalated cells. Transgenic overexpression of Notch intracellular domain reversed the altered cellular composition of mutant renal collecting duct, with principal cells occupying the entire region. Our data demonstrate that Notch signaling is required for the development of the mammalian renal collecting duct and principal cell differentiation and indicate that pathway dysregulation may contribute to distal renal tubular disorders.
doi:10.1172/JCI38416
PMCID: PMC2769200  PMID: 19855135
3.  Mind bomb 1 in the lymphopoietic niches is essential for T and marginal zone B cell development 
The Journal of Experimental Medicine  2008;205(11):2525-2536.
Notch signaling regulates lineage decisions at multiple stages of lymphocyte development, and Notch activation requires the endocytosis of Notch ligands in the signal-sending cells. Four E3 ubiquitin ligases, Mind bomb (Mib) 1, Mib2, Neuralized (Neur) 1, and Neur2, regulate the Notch ligands to activate Notch signaling, but their roles in lymphocyte development have not been defined. We show that Mib1 regulates T and marginal zone B (MZB) cell development in the lymphopoietic niches. Inactivation of the Mib1 gene, but not the other E3 ligases, Mib2, Neur1, and Neur2, abrogated T and MZB cell development. Reciprocal bone marrow (BM) transplantation experiments revealed that Mib1 in the thymic and splenic niches is essential for T and MZB cell development. Interestingly, when BM cells from transgenic Notch reporter mice were transplanted into Mib1-null mice, the Notch signaling was abolished in the double-negative thymocytes. In addition, the endocytosis of Dll1 was impaired in the Mib1-null microenvironment. Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown. Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.
doi:10.1084/jem.20081344
PMCID: PMC2571928  PMID: 18824586
4.  Meteorin Regulates Mesendoderm Development by Enhancing Nodal Expression 
PLoS ONE  2014;9(2):e88811.
During gastrulation, distinct lineage specification into three germ layers, the mesoderm, endoderm and ectoderm, occurs through an elaborate harmony between signaling molecules along the embryonic proximo-distal and anterior-posterior axes, and Nodal signaling plays a key role in the early embryonic development governing embryonic axis formation, mesoderm and endoderm specification, and left-right asymmetry determination. However, the mechanism by which Nodal expression is regulated is largely unknown. Here, we show that Meteorin regulates Nodal expression and is required for mesendoderm development. It is highly expressed in the inner cell mass of blastocysts and further in the epiblast and extra-embryonic ectoderm during gastrulation. Genetic ablation of the Meteorin gene resulted in early embryonic lethality, presumably due to impaired lineage allocation and subsequent cell accumulation. Embryoid body culture using Meteorin-null embryonic stem (ES) cells showed reduced Nodal expression and concomitant impairment of mesendoderm specification. Meteorin-null embryos displayed reduced levels of Nodal transcripts before the gastrulation stage, and impaired expression of Goosecoid, a definitive endoderm marker, during gastrulation, while the proximo-distal and anterior-posterior axes and primitive streak formation were preserved. Our results show that Meteorin is a novel regulator of Nodal transcription and is required to maintain sufficient Nodal levels for endoderm formation, thereby providing new insights in the regulation of mesendoderm allocation.
doi:10.1371/journal.pone.0088811
PMCID: PMC3928293  PMID: 24558432
5.  Loss of BubR1 acetylation causes defects in spindle assembly checkpoint signaling and promotes tumor formation 
The Journal of Cell Biology  2013;202(2):295-309.
Failure of chromosome–spindle attachment and a weakened spindle assembly checkpoint lead to genetic instability and cancer in mice expressing acetylation-deficient BubR1.
BubR1 acetylation is essential in mitosis. Mice heterozygous for the acetylation-deficient BubR1 allele (K243R/+) spontaneously developed tumors with massive chromosome missegregations. K243R/+ mouse embryonic fibroblasts (MEFs) exhibited a weakened spindle assembly checkpoint (SAC) with shortened mitotic timing. The generation of the SAC signal was intact, as Mad2 localization to the unattached kinetochore (KT) was unaltered; however, because of the premature degradation of K243R-BubR1, the mitotic checkpoint complex disassociated prematurely in the nocodazole-treated condition, suggesting that maintenance of the SAC is compromised. BubR1 acetylation was also required to counteract excessive Aurora B activity at the KT for stable chromosome–spindle attachments. The association of acetylation-deficient BubR1 with PP2A-B56α phosphatase was reduced, and the phosphorylated Ndc80 at the KT was elevated in K243R/+ MEFs. In relation, there was a marked increase of micronuclei and p53 mutation was frequently detected in primary tumors of K243R/+ mice. Collectively, the combined effects of failure in chromosome–spindle attachment and weakened SAC cause genetic instability and cancer in K243R/+ mice.
doi:10.1083/jcb.201210099
PMCID: PMC3718975  PMID: 23878276
6.  Notch-dependent differentiation of adult airway basal stem cells 
Cell stem cell  2011;8(6):639-648.
Summary
The epithelium lining the airways of the adult human lung is composed of ciliated and secretory cells together with undifferentiated basal cells (BCs). The composition and organization of this epithelium is severely disrupted in many respiratory diseases. However, little is known about the mechanisms controlling airway homeostasis and repair after epithelial damage. Here, we exploit the mouse tracheobronchial epithelium, in which BCs function as resident stem cells, as a genetically tractable model of human small airways. Using a reporter allele we show that the low level of Notch signaling at steady state is greatly enhanced during repair and the generation of luminal progenitors. Loss-of-function experiments show that Notch signaling is required for the differentiation, but not self-renewal, of BCs. Moreover, sustained Notch activation in BCs promotes their luminal differentiation, primarily towards secretory lineages. We also provide evidence that this function of Notch signaling is conserved in BCs from human airways.
Notch signaling is active in steady state airways and increased during repairNotch is required for differentiation, but not self-renewal, of airway basal cellsNotch promotes luminal differentiation of mouse basal stem cellsOur data suggest this mechanism is conserved in human basal cells
doi:10.1016/j.stem.2011.04.003
PMCID: PMC3778678  PMID: 21624809
7.  Disruption of Sorting Nexin 5 Causes Respiratory Failure Associated with Undifferentiated Alveolar Epithelial Type I Cells in Mice 
PLoS ONE  2013;8(3):e58511.
Sorting nexin 5 (Snx5) has been posited to regulate the degradation of epidermal growth factor receptor and the retrograde trafficking of cation-independent mannose 6-phosphate receptor/insulin-like growth factor II receptor. Snx5 has also been suggested to interact with Mind bomb-1, an E3 ubiquitin ligase that regulates the activation of Notch signaling. However, the in vivo functions of Snx5 are largely unknown. Here, we report that disruption of the Snx5 gene in mice (Snx5-/- mice) resulted in partial perinatal lethality; 40% of Snx5-/- mice died shortly after birth due to cyanosis, reduced air space in the lungs, and respiratory failure. Histological analysis revealed that Snx5-/- mice exhibited thickened alveolar walls associated with undifferentiated alveolar epithelial type I cells. In contrast, alveolar epithelial type II cells were intact, exhibiting normal surfactant synthesis and secretion. Although the expression levels of surfactant proteins and saturated phosphatidylcholine in the lungs of Snx5-/- mice were comparable to those of Snx5+/+ mice, the expression levels of T1α, Aqp5, and Rage, markers for distal alveolar epithelial type I cells, were significantly decreased in Snx5-/- mice. These results demonstrate that Snx5 is necessary for the differentiation of alveolar epithelial type I cells, which may underlie the adaptation to air breathing at birth.
doi:10.1371/journal.pone.0058511
PMCID: PMC3602295  PMID: 23526992
8.  Crif1 Deficiency Reduces Adipose OXPHOS Capacity and Triggers Inflammation and Insulin Resistance in Mice 
PLoS Genetics  2013;9(3):e1003356.
Impaired mitochondrial oxidative phosphorylation (OXPHOS) has been proposed as an etiological mechanism underlying insulin resistance. However, the initiating organ of OXPHOS dysfunction during the development of systemic insulin resistance has yet to be identified. To determine whether adipose OXPHOS deficiency plays an etiological role in systemic insulin resistance, the metabolic phenotype of mice with OXPHOS–deficient adipose tissue was examined. Crif1 is a protein required for the intramitochondrial production of mtDNA–encoded OXPHOS subunits; therefore, Crif1 haploinsufficient deficiency in mice results in a mild, but specific, failure of OXPHOS capacity in vivo. Although adipose-specific Crif1-haploinsufficient mice showed normal growth and development, they became insulin-resistant. Crif1-silenced adipocytes showed higher expression of chemokines, the expression of which is dependent upon stress kinases and antioxidant. Accordingly, examination of adipose tissue from Crif1-haploinsufficient mice revealed increased secretion of MCP1 and TNFα, as well as marked infiltration by macrophages. These findings indicate that the OXPHOS status of adipose tissue determines its metabolic and inflammatory responses, and may cause systemic inflammation and insulin resistance.
Author Summary
Type 2 diabetes is one of the most challenging health problems in the 21st century. Although insulin resistance is regarded as a fundamental defect that precedes the development of type 2 diabetes, the nature and cause of insulin resistance remain unknown. Adipose tissue is an important organ that determines whole-body energy metabolism, and its dysfunction is a critical element in the development of systemic insulin resistance. Adipose mitochondrial function is suppressed in the insulin-resistant state, and increased adipose mitochondrial biogenesis is associated with the reversal of insulin resistance by a PPARγ agonist. However, despite these important observations, little is known about how mitochondrial respiratory dysfunction in white adipose tissue (WAT) causes insulin resistance. To determine whether adipose deficiency of mitochondrial respiratory capacity plays an etiological role in systemic insulin resistance, the metabolic phenotype of mice with mitochondrial OXPHOS (oxidative phosphorylation)–deficient adipose tissue was examined. Crif1 is a protein required for the translation of mtDNA–encoded OXPHOS subunits. Interestingly, mice haploinsufficient for Crif1 in adipose tissue showed reduced OXPHOS capacity and developed marked insulin resistance.
doi:10.1371/journal.pgen.1003356
PMCID: PMC3597503  PMID: 23516375
9.  The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation 
The Journal of Clinical Investigation  2013;123(4):1475-1491.
After cell fate specification, differentiating cells must amplify the specific subcellular features required for their specialized function. How cells regulate such subcellular scaling is a fundamental unanswered question. Here, we show that the E3 ubiquitin ligase Mindbomb 1 (MIB1) is required for the apical secretory apparatus established by gastric zymogenic cells as they differentiate from their progenitors. When Mib1 was deleted, death-associated protein kinase–1 (DAPK1) was rerouted to the cell base, microtubule-associated protein 1B (MAP1B) was dephosphorylated, and the apical vesicles that normally support mature secretory granules were dispersed. Consequently, secretory granules did not mature. The transcription factor MIST1 bound the first intron of Mib1 and regulated its expression. We further showed that loss of MIB1 and dismantling of the apical secretory apparatus was the earliest quantifiable aberration in zymogenic cells undergoing transition to a precancerous metaplastic state in mouse and human stomach. Our results reveal a mechanistic pathway by which cells can scale up a specific, specialized subcellular compartment to alter function during differentiation and scale it down during disease.
doi:10.1172/JCI65703
PMCID: PMC3613919  PMID: 23478405
10.  Cardiomyocyte Specific Deletion of Crif1 Causes Mitochondrial Cardiomyopathy in Mice 
PLoS ONE  2013;8(1):e53577.
Mitochondria are key organelles dedicated to energy production. Crif1, which interacts with the large subunit of the mitochondrial ribosome, is indispensable for the mitochondrial translation and membrane insertion of respiratory subunits. To explore the physiological function of Crif1 in the heart, Crif1f/f mice were crossed with Myh6-cre/Esr1 transgenic mice, which harbor cardiomyocyte-specific Cre activity in a tamoxifen-dependent manner. The tamoxifen injections were given at six weeks postnatal, and the mutant mice survived only five months due to hypertrophic heart failure. In the mutant cardiac muscles, mitochondrial mass dramatically increased, while the inner structure was altered with lack of cristae. Mutant cardiac muscles showed decreased rates of oxygen consumption and ATP production, suggesting that Crif1 plays a critical role in the maintenance of both mitochondrial structure and respiration in cardiac muscles.
doi:10.1371/journal.pone.0053577
PMCID: PMC3537664  PMID: 23308255
11.  Mind bomb-1 is an essential modulator of long-term memory and synaptic plasticity via the Notch signaling pathway 
Molecular Brain  2012;5:40.
Background
Notch signaling is well recognized as a key regulator of the neuronal fate during embryonic development, but its function in the adult brain is still largely unknown. Mind bomb-1 (Mib1) is an essential positive regulator in the Notch pathway, acting non-autonomously in the signal-sending cells. Therefore, genetic ablation of Mib1 in mature neuron would give valuable insight to understand the cell-to-cell interaction between neurons via Notch signaling for their proper function.
Results
Here we show that the inactivation of Mib1 in mature neurons in forebrain results in impaired hippocampal dependent spatial memory and contextual fear memory. Consistently, hippocampal slices from Mib1-deficient mice show impaired late-phase, but not early-phase, long-term potentiation and long-term depression without change in basal synaptic transmission at SC-CA1 synapses.
Conclusions
These data suggest that Mib1-mediated Notch signaling is essential for long-lasting synaptic plasticity and memory formation in the rodent hippocampus.
doi:10.1186/1756-6606-5-40
PMCID: PMC3541076  PMID: 23111145
Mind bomb-1; Notch; Synaptic plasticity; Memory; Hippocampus
12.  Mapping a Dynamic Innate Immunity Protein Interaction Network Regulating Type I Interferon Production 
Immunity  2011;35(3):426-440.
SUMMARY
To systematically investigate innate immune signaling networks regulating production of type I interferon, we analyzed protein complexes formed after microbial recognition. Fifty-eight baits were associated with 260 interacting proteins forming a human innate immunity interactome for type I interferon (HI5) of 401 unique interactions; 21% of interactions were modulated by RNA, DNA, or LPS. Overexpression and depletion analyses identified 22 unique genes that regulated NF-κB and ISRE reporter activity, viral replication, or virus-induced interferon production. Detailed mechanistic analysis defined a role for mind bomb (MIB) E3 ligases in K63-linked ubiquitination of TBK1, a kinase that phosphorylates IRF transcription factors controlling interferon production. Mib genes selectively controlled responses to cytosolic RNA. MIB deficiency reduced antiviral activity, establishing the role of MIB proteins as positive regulators of antiviral responses. The HI5 provides a dynamic physical and regulatory network that serves as a resource for mechanistic analysis of innate immune signaling.
doi:10.1016/j.immuni.2011.06.014
PMCID: PMC3253658  PMID: 21903422
13.  Correction: Mind Bomb-1 in Dendritic Cells Is Specifically Required for Notch-mediated T Helper Type 2 Differentiation 
PLoS ONE  2012;7(7):10.1371/annotation/14bcb316-f12f-45ae-a1d3-d71a6f2014ce.
doi:10.1371/annotation/14bcb316-f12f-45ae-a1d3-d71a6f2014ce
PMCID: PMC3935727
14.  Survival and Differentiation of Mammary Epithelial Cells in Mammary Gland Development Require Nuclear Retention of Id2 Due to RANK Signaling▿  
Molecular and Cellular Biology  2011;31(23):4775-4788.
RANKL plays an essential role in mammary gland development during pregnancy. However, the molecular mechanism by which RANK signaling leads to mammary gland development is largely unknown. We report here that RANKL stimulation induces phosphorylation of Id2 at serine 5, which leads to nuclear retention of Id2. In lactating Id2Tg; RANKL−/− mice, Id2 was not phosphorylated and was localized in the cytoplasm. In addition, in lactating Id2S5ATg mice, Id2S5A (with serine 5 mutated to alanine) was exclusively localized in the cytoplasm of mammary epithelial cells (MECs), while endogenous Id2 was localized in the nucleus. Intriguingly, nuclear expression of Id2S5A rescued increased apoptosis and defective differentiation of MECs in RANKL−/− mice. Our results demonstrate that nuclear retention of Id2 due to RANK signaling plays a decisive role in the survival and differentiation of MECs during mammary gland development.
doi:10.1128/MCB.05646-11
PMCID: PMC3232922  PMID: 21947283
15.  Mind Bomb-1 in Dendritic Cells Is Specifically Required for Notch-mediated T Helper Type 2 Differentiation 
PLoS ONE  2012;7(4):e36359.
In dendritic cell (DC)-CD4+ T cell interaction, Notch signaling has been implicated in the CD4+ T cell activation, proliferation, and subset differentiation. However, there has been a lot of debate on the exact role of Notch signaling. Here, we observed that expression of Mind bomb-1 (Mib1), a critical regulator of Notch ligands for the activation of Notch signaling, increases gradually as precursor cells differentiate into DCs in mice. To clarify the role of Mib1 in DC-CD4+ T cell interactions, we generated Mib1-null bone marrow–derived DCs. These cells readily expressed Notch ligands but failed to initiate Notch activation in the adjacent cells. Nevertheless, Mib1-null DCs were able to prime the activation and proliferation of CD4+ T cells, suggesting that Notch activation in CD4+ T cells is not required for these processes. Intriguingly, stimulation of CD4+ T cells with Mib1-null DCs resulted in dramatically diminished Th2 cell populations, while preserving Th1 cell populations, both in vitro and in vivo. Our results demonstrate that Mib1 in DCs is critical for the activation of Notch signaling in CD4+ T cells, and Notch signaling reinforces Th2 differentiation, but is not required for the activation or proliferation of the CD4+ T cells.
doi:10.1371/journal.pone.0036359
PMCID: PMC3338679  PMID: 22558446
16.  Vaccinia-Related Kinase 1 Is Required for the Maintenance of Undifferentiated Spermatogonia in Mouse Male Germ Cells 
PLoS ONE  2010;5(12):e15254.
Vaccinia-related kinase 1 (VRK1) is a crucial protein kinase for mitotic regulation. VRK1 is known to play a role in germ cell development, and its deficiency results in sterility. Here we describe that VRK1 is essential for the maintenance of spermatogonial stem cells. To determine whether VRK1 plays a role in these cells, we assessed the population size of undifferentiated spermatogonia. Flow cytometry analyses showed that the number of undifferentiated spermatogonia was markedly reduced in VRK1-deficient testes. VRK1 was highly expressed in spermatogonial populations, and approximately 66% of undifferentiated spermatogonia that were sorted as an Ep-CAM+/c-kit−/alpha-6-integrin+ population showed a positive signal for VRK1. Undifferentiated stem cells expressing Plzf and Oct4 but not c-kit also expressed VRK1, suggesting that VRK1 is an intrinsic factor for the maintenance of spermatogonial stem cells. Microarray analyses of the global testicular transcriptome and quantitative RT-PCR of VRK1-deficient testes revealed significantly reduced expression levels of undifferentiated spermatogonial marker genes in early postnatal mice. Together, these results suggest that VRK1 is required for the proliferation and differentiation of undifferentiated spermatogonia, which are essential for spermatogenic cell maintenance.
doi:10.1371/journal.pone.0015254
PMCID: PMC3001494  PMID: 21179456
17.  Crucial Role for Mst1 and Mst2 Kinases in Early Embryonic Development of the Mouse▿  
Molecular and Cellular Biology  2009;29(23):6309-6320.
Mammalian sterile 20-like kinases 1 and 2 (Mst1 and Mst2, respectively) are potent serine/threonine kinases that are involved in cell proliferation and cell death. To investigate the physiological functions of Mst1 and Mst2, we generated Mst1 and Mst2 mutant mice. Mst1−/− and Mst2−/− mice were viable and fertile and developed normally, suggesting possible functional overlaps between the two genes. A characterization of heterozygous and homozygous combinations of Mst1 and Mst2 mutant mice showed that mice containing a single copy of either gene underwent normal organ development; however, Mst1−/−; Mst2−/− mice lacking both Mst1 and Mst2 genes started dying in utero at approximately embryonic day 8.5. Mst1−/−; Mst2−/− mice exhibited severe growth retardation, failed placental development, impaired yolk sac/embryo vascular patterning and primitive hematopoiesis, increased apoptosis in placentas and embryos, and disorganized proliferating cells in the embryo proper. These findings indicate that both Mst1 and Mst2 kinases play essential roles in early mouse development, regulating placental development, vascular patterning, primitive hematopoiesis, and cell proliferation and survival.
doi:10.1128/MCB.00551-09
PMCID: PMC2786698  PMID: 19786569
18.  E2-25K/Hip-2 regulates caspase-12 in ER stress–mediated Aβ neurotoxicity 
The Journal of Cell Biology  2008;182(4):675-684.
Amyloid-β (Aβ) neurotoxicity is believed to contribute to the pathogenesis of Alzheimer's disease (AD). Previously we found that E2-25K/Hip-2, an E2 ubiquitin-conjugating enzyme, mediates Aβ neurotoxicity. Here, we report that E2-25K/Hip-2 modulates caspase-12 activity via the ubiquitin/proteasome system. Levels of endoplasmic reticulum (ER)–resident caspase-12 are strongly up-regulated in the brains of AD model mice, where the enzyme colocalizes with E2-25K/Hip-2. Aβ increases expression of E2-25K/Hip-2, which then stabilizes caspase-12 protein by inhibiting proteasome activity. This increase in E2-25K/Hip-2 also induces proteolytic activation of caspase-12 through its ability to induce calpainlike activity. Knockdown of E2-25K/Hip-2 expression suppresses neuronal cell death triggered by ER stress, and thus caspase-12 is required for the E2-25K/Hip-2–mediated cell death. Finally, we find that E2-25K/Hip-2–deficient cortical neurons are resistant to Aβ toxicity and to the induction of ER stress and caspase-12 expression by Aβ. E2-25K/Hip-2 is thus an essential upstream regulator of the expression and activation of caspase-12 in ER stress–mediated Aβ neurotoxicity.
doi:10.1083/jcb.200711066
PMCID: PMC2518707  PMID: 18710920
19.  Mind bomb-1 Is Essential for Intraembryonic Hematopoiesis in the Aortic Endothelium and the Subaortic Patches▿ †  
Molecular and Cellular Biology  2008;28(15):4794-4804.
Intraembryonic hematopoiesis occurs at two different sites, the floor of the aorta and subaortic patches (SAPs) of the para-aortic splanchnopleura (P-Sp)/aorta-gonad-mesonephros (AGM) region. Notch1 and RBP-jκ are critical for the specification of hematopoietic stem cells (HSCs) in Notch signal-receiving cells. However, the mechanism by which Notch signaling is triggered from the Notch signal-sending cells to support embryonic hematopoiesis remains to be determined. We previously reported that Mind bomb-1 (Mib1) regulates Notch ligands in the Notch signal-sending cells (B. K. Koo, M. J. Yoon, K. J. Yoon, S. K. Im, Y. Y. Kim, C. H. Kim, P. G. Suh, Y. N. Jan, and Y. Y. Kong, PLoS ONE 2:e1221, 2007). Here, we show that intraembryonic hematopoietic progenitors were absent in the P-Sp of Mib1−/− embryos, whereas they were partly preserved in the Tie2-cre; Mib1f/f P-Sps, suggesting that Mib1 plays a role in the endothelium and the SAPs. Interestingly, dll1 and dll4/Jag1 are expressed in the SAPs and the endothelium of the AGM, respectively, where mib1 is detected. Indeed, Notch signaling was activated in the nascent HSCs at both sites. In the P-Sp explant culture, the overexpression of Dll1 in OP9 stromal cells rescued the failed production of hematopoietic progenitors in the Mib1−/− P-Sp, while its activity was abolished by Mib1 knockdown. These results suggest that Mib1 is important for intraembryonic hematopoiesis not only in the aortic endothelium but also in the SAPs.
doi:10.1128/MCB.00436-08
PMCID: PMC2493361  PMID: 18505817
20.  Molecule-level imaging of Pax6 mRNA distribution in mouse embryonic neocortex by molecular interaction force microscopy 
Nucleic Acids Research  2008;37(2):e10.
Detection of the cellular and tissue distributions of RNA species is critical in our understanding of the regulatory mechanisms underlying cellular and tissue differentiation. Here, we show that an atomic force microscope tip modified with 27-acid dendron, a cone shaped molecule with 27 monomeric units forming its base, can be successfully used to map the spatial distribution of mouse Pax6 mRNA on sectioned tissues of the mouse embryonic neocortex. Scanning of the sectioned tissue with a 30-mer DNA probe attached to the apex of the dendron resulted in detection of the target mRNA on the tissue section, permitting mapping of the mRNA distribution at nanometer resolution. The unprecedented sensitivity and resolution of this process should be applicable to identification of molecular level distribution of various RNAs in a cell.
doi:10.1093/nar/gkn965
PMCID: PMC2632921  PMID: 19043075
21.  An Obligatory Role of Mind Bomb-1 in Notch Signaling of Mammalian Development 
PLoS ONE  2007;2(11):e1221.
Background
The Notch signaling pathway is an evolutionarily conserved intercellular signaling module essential for cell fate specification that requires endocytosis of Notch ligands. Structurally distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), cooperatively regulate the endocytosis of Notch ligands in Drosophila. However, the respective roles of the mammalian E3 ubiquitin ligases, Neur1, Neur2, Mib1, and Mib2, in mammalian development are poorly understood.
Methodology/Principal Findings
Through extensive use of mammalian genetics, here we show that Neur1 and Neur2 double mutants and Mib2−/− mice were viable and grossly normal. In contrast, conditional inactivation of Mib1 in various tissues revealed the representative Notch phenotypes: defects of arterial specification as deltalike4 mutants, abnormal cerebellum and skin development as jagged1 conditional mutants, and syndactylism as jagged2 mutants.
Conclusions/Significance
Our data provide the first evidence that Mib1 is essential for Jagged as well as Deltalike ligand-mediated Notch signaling in mammalian development, while Neur1, Neur2, and Mib2 are dispensable.
doi:10.1371/journal.pone.0001221
PMCID: PMC2082076  PMID: 18043734
22.  Receptor Activator of NF-κB Ligand Regulates the Proliferation of Mammary Epithelial Cells via Id2 
Molecular and Cellular Biology  2006;26(3):1002-1013.
Receptor activator of NF-κB ligand (RANKL) is a key regulator for mammary gland development during pregnancy. RANKL-deficient mice display impaired development of lobulo-alveolar mammary structures. Similar mammary gland defects have been reported in mice lacking Id2. Here we report that RANKL induces the proliferation of mammary epithelial cells via Id2. RANKL triggers marked nuclear translocation of Id2 in mammary epithelial cells. In vivo studies further demonstrated the defective nuclear translocation of Id2, but the normal expression of cyclin D1, in the mammary epithelial cells of rankl−/− mice. In vitro studies with nuclear localization sequence-tagged Id2 revealed that the nuclear localization of Id2 itself is critical for the downregulation of p21 promoter activity. Moreover, RANKL stimulation failed to induce cell growth and to downregulate p21 expression in Id2−/− mammary epithelial cells. Our results indicate that the inhibitor of helix-loop-helix protein, Id2, is critical to control the proliferation of mammary epithelial cells in response to RANKL stimulation.
doi:10.1128/MCB.26.3.1002-1013.2006
PMCID: PMC1347028  PMID: 16428453
23.  Activating Signal Cointegrator 2 Required for Liver Lipid Metabolism Mediated by Liver X Receptors in Mice 
Molecular and Cellular Biology  2003;23(10):3583-3592.
Activating signal cointegrator 2 (ASC-2), a cancer-amplified transcriptional coactivator of nuclear receptors and many other transcription factors, contains two LXXLL-type nuclear receptor interaction domains. Interestingly, the second LXXLL motif is highly specific to the liver X receptors (LXRs). In cotransfection, DN2, an ASC-2 fragment encompassing this motif, exerts a potent dominant-negative effect on transactivation by LXRs, which is rescued by ectopic coexpression of the full-length ASC-2 but not by other LXXLL-type coactivators, such as SRC-1 and TRAP220. In contrast, DN2/m, in which the LXXLL motif is mutated to LXXAA to abolish the interactions with LXRs, is without any effect. Accordingly, expression of DN2, but not DN2/m, in transgenic mice results in phenotypes that are highly homologous to those previously observed with LXRα−/− mice, including a rapid accumulation of large amounts of cholesterol and down-regulation of the known lipid-metabolizing target genes of LXRα in the liver upon being fed a high-cholesterol diet. These results identify ASC-2 as a physiologically important transcriptional coactivator of LXRs and demonstrate its pivotal role in the liver lipid metabolism.
doi:10.1128/MCB.23.10.3583-3592.2003
PMCID: PMC164762  PMID: 12724417
24.  Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection  
Periodontitis, a prime cause of tooth loss in humans, is implicated in the increased risk of systemic diseases such as heart failure, stroke, and bacterial pneumonia. The mechanisms by which periodontitis and antibacterial immunity lead to alveolar bone and tooth loss are poorly understood. To study the human immune response to specific periodontal infections, we transplanted human peripheral blood lymphocytes (HuPBLs) from periodontitis patients into NOD/SCID mice. Oral challenge of HuPBL-NOD/SCID mice with Actinobacillus actinomycetemcomitans, a well-known Gram-negative anaerobic microorganism that causes human periodontitis, activates human CD4+ T cells in the periodontium and triggers local alveolar bone destruction. Human CD4+ T cells, but not CD8+ T cells or B cells, are identified as essential mediators of alveolar bone destruction. Stimulation of CD4+ T cells by A. actinomycetemcomitans induces production of osteoprotegerin ligand (OPG-L), a key modulator of osteoclastogenesis and osteoclast activation. In vivo inhibition of OPG-L function with the decoy receptor OPG diminishes alveolar bone destruction and reduces the number of periodontal osteoclasts after microbial challenge. These data imply that the molecular explanation for alveolar bone destruction observed in periodontal infections is mediated by microorganism-triggered induction of OPG-L expression on CD4+ T cells and the consequent activation of osteoclasts. Inhibition of OPG-L may thus have therapeutic value to prevent alveolar bone and/or tooth loss in human periodontitis.
This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org. J. Clin. Invest. 106:R59–R67 (2000).
PMCID: PMC3102542  PMID: 10995794
25.  Vav Regulates Peptide-specific Apoptosis in Thymocytes  
The Journal of Experimental Medicine  1998;188(11):2099-2111.
The protooncogene Vav functions as a GDP/GTP exchange factor (GEF) for Rho-like small GTPases involved in cytoskeletal reorganization and cytokine production in T cells. Gene-targeted mice lacking Vav have a severe defect in positive and negative selection of T cell antigen receptor transgenic thymocytes in vivo, and vav−/− thymocytes are completely resistant to peptide-specific and anti-CD3/anti-CD28–mediated apoptosis. Vav acts upstream of mitochondrial pore opening and caspase activation. Biochemically, Vav regulates peptide-specific Ca2+ mobilization and actin polymerization. Peptide-specific cell death was blocked both by cytochalasin D inhibition of actin polymerization and by inhibition of protein kinase C (PKC). Activation of PKC with phorbol ester restored peptide-specific apoptosis in vav−/− thymocytes. Vav was found to bind constitutively to PKC-θ in thymocytes. Our results indicate that peptide-triggered thymocyte apoptosis is mediated via Vav activation, changes in the actin cytoskeleton, and subsequent activation of a PKC isoform.
PMCID: PMC2212394  PMID: 9841924
Vav; negative selection; actin cytoskeleton; signaling transduction; protein kinase C

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