• Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

      Raz, Limor; Zhang, Quan-Guang; Zhou, Cai-feng; Han, Dong; Gulati, Priya; Yang, Li-cai; Yang, Fang; Wang, Rui-min; Brann, Darrell W; Institute of Molecular Medicine and Genetics (2010-09-7)
      Background: Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.
    • Use of a microscope stage-mounted Nickel-63 microirradiator for real-time observation of the DNA double-strand break response.

      Cao, Zhen; Kuhne, Wendy W.; Steeb, Jennifer; Merkley, Mark A.; Zhou, Yunfeng; Janata, Jiri; Dynan, William S.; Institute of Molecular Medicine and Genetics (2010-08-12)
      Eukaryotic cells begin to assemble discrete, nucleoplasmic repair foci within seconds after the onset of exposure to ionizing radiation. Real-time imaging of this assembly has the potential to further our understanding of the effects of medical and environmental radiation exposure. Here, we describe a microirradiation system for targeted delivery of ionizing radiation to individual cells without the need for specialized facilities. The system consists of a 25-micron diameter electroplated Nickel-63 electrode, enveloped in a glass capillary and mounted in a micromanipulator. Because of the low energy of the beta radiation and the minute total amount of isotope present on the tip, the device can be safely handled with minimum precautions. We demonstrate the use of this system for tracking assembly of individual repair foci in real time in live U2OS human osteosarcoma cells. Results indicate that there is a subset of foci that appear and disappear rapidly, before a plateau level is reached approximately 30 min post-exposure. This subset of foci would not have been evident without real-time observation. The development of a microirradiation system that is compatible with a standard biomedical laboratory expands the potential for real-time investigation of the biological effects of ionizing radiation.
    • Prenatal alcohol exposure triggers ceramide-induced apoptosis in neural crest-derived tissues concurrent with defective cranial development

      Wang, G; Bieberich, Erhard; Institute of Molecular Medicine and Genetics (2010-05-27)
      Fetal alcohol syndrome (FAS) is caused by maternal alcohol consumption during pregnancy. The reason why specific embryonic tissues are sensitive toward ethanol is not understood. We found that in neural crest-derived cell (NCC) cultures from the first branchial arch of E10 mouse embryos, incubation with ethanol increases the number of apoptotic cells by fivefold. Apoptotic cells stain intensely for ceramide, suggesting that ceramide-induced apoptosis mediates ethanol damage to NCCs. Apoptosis is reduced by incubation with CDP-choline (citicoline), a precursor for the conversion of ceramide to sphingomyelin. Consistent with NCC cultures, ethanol intubation of pregnant mice results in ceramide elevation and increased apoptosis of NCCs in vivo. Ethanol also increases the protein level of prostate apoptosis response 4 (PAR-4), a sensitizer to ceramide-induced apoptosis. Prenatal ethanol exposure is concurrent with malformation of parietal bones in 20% of embryos at day E18. Meninges, a tissue complex derived from NCCs, is disrupted and generates reduced levels of TGF-b1, a growth factor critical for bone and brain development. Ethanol-induced apoptosis of NCCs leading to defects in the meninges may explain the simultaneous presence of cranial bone malformation and cognitive retardation in FAS. In addition, our data suggest that treatment with CDP-choline may alleviate the tissue damage caused by alcohol.
    • Extranuclear estrogen receptors mediate the neuroprotective effects of estrogen in the rat hippocampus.

      Yang, Li-cai; Zhang, Quan-Guang; Zhou, Cai-feng; Yang, Fang; Zhang, Yi-dong; Wang, Rui-min; Brann, Darrell W; Institute of Molecular Medicine and Genetics (2010-05-18)
      BACKGROUND: 17beta-estradiol (E2) has been implicated to exert neuroprotective effects in the brain following cerebral ischemia. Classically, E2 is thought to exert its effects via genomic signaling mediated by interaction with nuclear estrogen receptors. However, the role and contribution of extranuclear estrogen receptors (ER) is unclear and was the subject of the current study. METHODOLOGY/PRINCIPAL FINDINGS: To accomplish this goal, we employed two E2 conjugates (E2 dendrimer, EDC, and E2-BSA) that can interact with extranuclear ER and exert rapid nongenomic signaling, but lack the ability to interact with nuclear ER due to their inability to enter the nucleus. EDC or E2-BSA (10 microM) was injected icv 60 min prior to global cerebral ischemia (GCI). FITC-tagged EDC or E2-BSA revealed high uptake in the hippocampal CA1 region after icv injection, with a membrane (extranuclear) localization pattern in cells. Both EDC and E2-BSA exerted robust neuroprotection in the CA1 against GCI, and the effect was blocked by the ER antagonist, ICI182,780. EDC and E2-BSA both rapidly enhanced activation of the prosurvival kinases, ERK and Akt, while attenuating activation of the proapoptotic kinase, JNK following GCI, effects that were blocked by ICI182,780. Administration of an MEK or PI3K inhibitor blocked the neuroprotective effects of EDC and E2-BSA. Further studies showed that EDC increased p-CREB and BDNF in the CA1 region in an ERK- and Akt-dependent manner, and that cognitive outcome after GCI was preserved by EDC in an ER-dependent manner. CONCLUSIONS/SIGNIFICANCE: In conclusion, the current study demonstrates that activation of extranuclear ER results in induction of ERK-Akt-CREB-BDNF signaling in the hippocampal CA1 region, which significantly reduces ischemic neuronal injury and preserves cognitive function following GCI. The study adds to a growing literature that suggests that extranuclear ER can have important actions in the brain.
    • Enhanced glutamatergic and decreased GABAergic synaptic appositions to GnRH neurons on proestrus in the rat: modulatory effect of aging.

      Khan, Mohammad; De Sevilla, Liesl; Mahesh, Virendra B; Brann, Darrell W; Institute of Molecular Medicine and Genetics (2010-04-26)
      BACKGROUND: Previous work by our lab and others has implicated glutamate as a major excitatory signal to gonadotropin hormone releasing hormone (GnRH) neurons, with gamma amino butyric acid (GABA) serving as a potential major inhibitory signal. However, it is unknown whether GABAergic and/or glutamatergic synaptic appositions to GnRH neurons changes on the day of the proestrous LH surge or is affected by aging. METHODOLOGY/PRINCIPAL FINDINGS: To examine this question, synaptic terminal appositions on GnRH neurons for VGAT (vesicular GABA transporter) and VGLUT2 (vesicular glutamate transporter-2), markers of GABAergic and glutamatergic synaptic terminals, respectively, was examined by immunohistochemistry and confocal microscopic analysis in young and middle-aged diestrous and proestrous rats. The results show that in young proestrous rats at the time of LH surge, we observed reciprocal changes in the VGAT and VGLUT2 positive terminals apposing GnRH neurons, where VGAT terminal appositions were decreased and VGLUT2 terminal appositions were significantly increased, as compared to young diestrus control animals. Interestingly, in middle-aged cycling animals this divergent modulation of VGAT and VGLUT2 terminal apposition was greatly impaired, as no significant differences were observed between VGAT and VGLUT2 terminals apposing GnRH neurons at proestrous. However, the density of VGAT and VGLUT2 terminals apposing GnRH neurons were both significantly increased in the middle-aged animals. CONCLUSIONS/SIGNIFICANCE: In conclusion, there is an increase in glutamatergic and decrease in GABAergic synaptic terminal appositions on GnRH neurons on proestrus in young animals, which may serve to facilitate activation of GnRH neurons. In contrast, middle-aged diestrous and proestrous animals show a significant increase in both VGAT and VGLUT synaptic terminal appositions on GnRH neurons as compared to young animals, and the cycle-related change in these appositions between diestrus and proestrus that is observed in young animals is lost.
    • LIM and SH3 protein-1 modulates CXCR2-mediated cell migration.

      Raman, Dayanidhi; Sai, Jiqing; Neel, Nicole F; Chew, Catherine S; Richmond, Ann; Institute of Molecular Medicine and Genetics (2010-04-26)
      BACKGROUND: The chemokine receptor CXCR2 plays a pivotal role in migration of neutrophils, macrophages and endothelial cells, modulating several biological responses such as angiogenesis, wound healing and acute inflammation. CXCR2 is also involved in pathogenesis of chronic inflammation, sepsis and atherosclerosis. The ability of CXCR2 to associate with a variety of proteins dynamically is responsible for its effects on directed cell migration or chemotaxis. The dynamic network of such CXCR2 binding proteins is termed as "CXCR2 chemosynapse". Proteomic analysis of proteins that co-immunoprecipitated with CXCR2 in neutrophil-like dHL-60 cells revealed a novel protein, LIM and SH3 protein 1 (LASP-1), binds CXCR2 under both basal and ligand activated conditions. LASP-1 is an actin binding cytoskeletal protein, involved in the cell migration. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that CXCR2 and LASP-1 co-immunoprecipitate and co-localize at the leading edge of migrating cells. The LIM domain of LASP-1 directly binds to the carboxy-terminal domain (CTD) of CXCR2. Moreover, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4. Using a site-directed and deletion mutagenesis approach, Iso323-Leu324 of the conserved LKIL motif on CXCR2-CTD was identified as the binding site for LASP-1. Interruption of the interaction between CXCR2-CTD and LIM domain of LASP-1 by dominant negative and knock down approaches inhibited CXCR2-mediated chemotaxis. Analysis for the mechanism for inhibition of CXCR2-mediated chemotaxis indicated that LASP-1/CXCR2 interaction is essential for cell motility and focal adhesion turnover involving activation of Src, paxillin, PAK1, p130CAS and ERK1/2. CONCLUSIONS/SIGNIFICANCE: We demonstrate here for the first time that LASP-1 is a key component of the "CXCR2 chemosynapse" and LASP-1 interaction with CXCR2 is critical for CXCR2-mediated chemotaxis. Furthermore, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4, suggesting that LASP-1 is a general mediator of CXC chemokine mediated chemotaxis. Thus, LASP-1 may serve as a new link coordinating the flow of information between chemokine receptors and nascent focal adhesions, especially at the leading edge. Thus the association between the chemokine receptors and LASP-1 suggests to the presence of a CXC chemokine receptor-LASP-1 pro-migratory module in cells governing the cell migration.
    • LGN regulates mitotic spindle orientation during epithelial morphogenesis

      Zheng, Zhen; Zhu, Huabin; Wan, Qingwen; Liu, Jing; Xiao, Zhuoni; Siderovski, David P.; Du, Quansheng; Institute of Molecular Medicine and Genetics; Department of Neurology (2010-04-19)
      Coordinated cell polarization and mitotic spindle orientation are thought to be important for epithelial morphogenesis. Whether spindle orientation is indeed linked to epithelial morphogenesis and how it is controlled at the molecular level is still unknown. Here, we show that the NuMA- and Ga-binding protein LGN is required for directing spindle orientation during cystogenesis of MDCK cells. LGN localizes to the lateral cell cortex, and is excluded from the apical cell cortex of dividing cells. Depleting LGN, preventing its cortical localization, or disrupting its interaction with endogenous NuMA or Ga proteins all lead to spindle misorientation and abnormal cystogenesis. Moreover, artificial mistargeting of endogenous LGN to the apical membrane results in a near 90° rotation of the spindle axis and profound cystogenesis defects that are dependent on cell division. The normal apical exclusion of LGN during mitosis appears to be mediated by atypical PKC. Thus, cell polarizationâ mediated spatial restriction of spindle orientation determinants is critical for epithelial morphogenesis.
    • Cleft palate is caused by CNS dysfunction in Gad1 and Viaat knockout mice.

      Oh, Won-Jong; Westmoreland, Joby J; Summers, Ryan; Condie, Brian G.; Institute of Molecular Medicine and Genetics (2010-03-24)
      BACKGROUND: Previous studies have shown that disruption of GABA signaling in mice via mutations in the Gad1, Gabrb3 or Viaat genes leads to the development of non-neural developmental defects such as cleft palate. Studies of the Gabrb3 and Gad1 mutant mice have suggested that GABA function could be required either in the central nervous system or in the palate itself for normal palatogenesis. METHODOLOGY/PRINCIPAL FINDINGS: To further examine the role of GABA signaling in palatogenesis we used three independent experimental approaches to test whether Gad1 or Viaat function is required in the fetal CNS for normal palate development. We used oral explant cultures to demonstrate that the Gad1 and Viaat mutant palates were able to undergo palatogenesis in culture, suggesting that there is no defect in the palate tissue itself in these mice. In a second series of experiments we found that the GABA(A) receptor agonist muscimol could rescue the cleft palate phenotype in Gad1 and Viaat mutant embryos. This suggested that normal multimeric GABA(A) receptors in the CNS were necessary for normal palatogenesis. In addition, we showed that CNS-specific inactivation of Gad1 was sufficient to disrupt palate development. CONCLUSIONS/SIGNIFICANCE: Our results are consistent with a role for Gad1 and Viaat in the central nervous system for normal development of the palate. We suggest that the alterations in GABA signaling lead to non-neural defects such as cleft palate as a secondary effect due to alterations in or elimination of fetal movements.
    • Cytotoxic effects of G(M1) ganglioside and amyloid beta-peptide on mouse embryonic neural stem cells.

      Yanagisawa, Makoto; Ariga, Toshio; Yu, Robert K.; Institute of Molecular Medicine and Genetics; Institute of Neuroscience (2010-03-22)
      AD (Alzheimer's disease) is a neurodegenerative disease and the most common form of dementia. One of the pathological hallmarks of AD is the aggregation of extracellular Abetas (amyloid beta-peptides) in senile plaques in the brain. The process could be initiated by seeding provided by an interaction between G(M1) ganglioside and Abetas. Several reports have documented the bifunctional roles of Abetas in NSCs (neural stem cells), but the precise effects of G(M1) and Abeta on NSCs have not yet been clarified. We evaluated the effect of G(M1) and Abeta-(1-40) on mouse NECs (neuroepithelial cells), which are known to be rich in NSCs. No change of cell number was detected in NECs cultured in the presence of either G(M1) or Abeta-(1-40). On the contrary, a decreased number of NECs were cultured in the presence of a combination of G(M1) and Abeta-(1-40). The exogenously added G(M1) and Abeta-(1-40) were confirmed to incorporate into NECs. The Ras-MAPK (mitogen-activated protein kinase) pathway, important for cell proliferation, was intact in NECs simultaneously treated with G(M1) and Abeta-(1-40), but caspase 3 was activated. NECs treated with G(M1) and Abeta-(1-40) were positive in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay, an indicator of cell death. It was found that G(M1) and Abeta-(1-40) interacted in the presence of cholesterol and sphingomyelin, components of cell surface microdomains. The cytotoxic effect was found also in NSCs prepared via neurospheres. These results indicate that Abeta-(1-40) and G(M1) co-operatively exert a cytotoxic effect on NSCs, likely via incorporation into NEC membranes, where they form a complex for the activation of cell death signalling.
    • Mitochondrial BNIP3 upregulation precedes endonuclease G translocation in hippocampal neuronal death following oxygen-glucose deprivation.

      Zhao, Shen-Ting; Chen, Ming; Li, Shu-Ji; Zhang, Ming-Hai; Li, Bo-Xing; Das, Manas; Bean, Jonathan C; Kong, Ji-Ming; Zhu, Xin-Hong; Gao, Tian-Ming; et al. (2009-09-23)
      BACKGROUND: Caspase-independent apoptotic pathways are suggested as a mechanism for the delayed neuronal death following ischemic insult. However, the underlying signalling mechanisms are largely unknown. Recent studies imply the involvement of several mitochondrial proteins, including endonuclease G (EndoG) and Bcl-2/adenovirus E1B 19 kDa-interacting protein (BNIP3), in the pathway of non-neuronal cells. RESULTS: In this report, using western blot analysis and immunocytochemistry, we found that EndoG upregulates and translocates from mitochondria to nucleus in a time-dependent manner in cultured hippocampal neurons following oxygen-glucose deprivation (OGD). Moreover, the translocation of EndoG occurs hours before the observable nuclear pyknosis. Importantly, the mitochondrial upregulation of BNIP3 precedes the translocation of EndoG. Forced expression of BNIP3 increases the nuclear translocation of EndoG and neuronal death while knockdown of BNIP3 decreases the OGD-induced nuclear translocation of EndoG and neuronal death. CONCLUSION: These results suggest that BNIP3 and EndoG play important roles in hippocampal neuronal apoptosis following ischemia, and mitochondrial BNIP3 is a signal protein upstream of EndoG that can induce neuronal death.
    • Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes.

      Merkley, Mark A.; Hildebrandt, Ellen; Podolsky, Robert H.; Arnouk, Hilal; Ferris, Daron G.; Dynan, William S.; Stöppler, Hubert; Institute of Molecular Medicine and Genetics; Center for Biotechnology and Genomic Medicine; Department of Obstetrics and Gynecology; et al. (2009-09-16)
      BACKGROUND: Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. RESULTS: Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE). The median within-group coefficient of variation was 19-21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23%) of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2%) were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1); and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27). CONCLUSION: This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.
    • Mechanosensory Hair Cell Precursors in the Zebrafish Lateral Line

      Floyd, Tiffany L.; Institute of Molecular Medicine and Genetics (2009-07)
      The vertebrate inner ear mediates the senses of hearing and balance. Contained within both the auditory and vestibular compartments of the inner ear are specialized mechanosensory hair cells that function as receptors and transducers of environmental stimuli. In all vertebrates, these sensory hair cells are particularly susceptible to ototoxic insults resulting in cell death and, in mammals, the irreversible loss of hair cells underlies deafness and balance disorders. In contrast to mammals, several non-mammalian vertebrates (including zebrafish) possess the innate capacity to produce new hair cells throughout life as well as regenerate hair cells that have been lethally damaged. A long-term strategy of the hearing research field is to determine the molecular mechanisms of hair cell regeneration using regenerating model systems such as zebrafish, then to apply this information to mammalian models where sensory hair cell regeneration is limited or nonexistent. During embryogenesis, sensory hair cell fates are specified through a mechanism of Notch-Delta-mediated lateral inhibition. The gamma secretase complex is an upstream regulator of Notch signaling, responsible for proteolytic\ cleavage and activation of the Notch receptor. Recent evidence suggests that Notch signaling may also play a role during the process of hair cell regeneration in zebrafish (Ma et al., 2008). I used a chemical inhibitor of the gamma secretase complex to examine the role of Notch signaling in the regulation of hair cell number maintenance in larval zebrafish. Results presented in this thesis provide novel insight into the mechanisms regulating the maintenance of resident hair cell precursors within the sensory epithelium. Moreover, this new information is directly relevant to research efforts in mammalian models by providing the molecular framework for therapeutic strategies designed to replace or regenerate lethally damaged hair cells in the mammalian cochlea by reactivating resident precursors to differentiate into hair cells.
    • Characterization of Cervical and Head and Neck Squamous Cell Carcinomas by Proteomic Analysis

      Merkley, Mark A.; Institute of Molecular Medicine and Genetics (2009-06)
      (First Paragraph) The oral cavity, oropharynx, larynx, esophagus, and ano-genital orifices are lined with stratified squamous nonkeratinized epithelium, which forms the barrier between the underlying tissue and the external environment. The proliferative nature of this epithelium, together with its potential exposure to environmental insults such as tobacco carcinogens, alcohol, or oncogenic viruses, makes it susceptible to carcinogenesis. Indeed, carcinomas of stratified squamous nonkeratinized epithelium are among the most common and deadly cancers worldwide. In particular head and neck squamous cell carcinoma and cervical squamous cell carcinoma together account for about 1 million new cases annually, worldwide. These cancers arise from similar tissues and share common risk factors, although they differ in that effective population-based screening exists only for cervical cancer.
    • The Current State of Proteomics in GI Oncology

      Lin, Ying; Dynan, William S.; Lee, Jeffrey R.; Zhu, Zhao-Hua; Schade, Robert R.; Institute of Molecular Medicine and Genetics; Department of Pathology (2009-03-23)
      Keywords: Clinical proteomics
    • The Clinical Phenotype of An Extended Pedigree with Late-onset Alzheimer's Disease

      Fennell, Eleanor M.; Institute of Molecular Medicine and Genetics (2009-03)
      Alzheimer's disease (AD) is a devastating neurodegenerative disease with a multifaceted etiology. This retrospective exploratory study used the family history method to develop a phenotype based on the personal history of cognitive decline, changes in behavioral characteristics, and the medical, social and environmental history of the five AD affected family members in one large extended family. Group family interviews provided a description of the phenotype and identified medical and environmental risk modifiers. Average age-of-onset for AD in 5 of 12 siblings, (all homozygous APOE4/4) was 69.2 years (range 66-72 years). Fisher's exact test identified neuropsychiatric behaviors as common phenotypic manifestations; delusions (p = 0.0455), hallucinations (p = 0.0101), irritability (p = 0.0455), personality change (p = 0.0013), pacing (p = 0.0013), aggressiveness (p = 0.0455), and poor judgment (p = 0.0013). Environmental variables that emerged as significant were a less than an eighth grade education (p — 0.0152), presence of stroke/TIA (p = 0.0455), presence of osteoarthritis (p = 0.0455), and vitamin B12 deficiency (p = 0.0013). Risk of stroke/TIA may be related to the increased risk from APOE4 while vitamin B12 deficiency may be associated with advanced age. There was no significant protective benefit from the management of hypertension (p = 0.5758), use of statins to control cholesterol (p = 0.9545), use of Vitamin C (p = 1.000), and/or Vitamin E (p = 0.9899) among family members. Potential modifiable health practices among the AD unaffected siblings demonstrated that 85% continued to engage in a sedentary lifestyle (p = 0.6818), 57% were overweight (p = 0.6894), 57% (p = 0.6894) consumed an unhealthy diet, and 56% smoked (p = 0.1591). Single nucleotide polymorphism (SNP) microarray Clinical Phenotype of Alzheimer's Disease analysis indicated that several SNPs in the gene, transient receptor potential cation channel, subfamily C, member 4 associated protein, (TRPC4AP), were significant. All five of the affected siblings and three unaffected siblings exhibited one haplotype; the unaffected siblings with the same haplotype as affected siblings were younger in age and did not have any cognitive problems at the time of the study (Poduslo, Huang, Huang, Smith, 2008).
    • Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide.

      Krishnamurthy, Kannan; Wang, Guanghu; Rokhfeld, Dmitriy; Bieberich, Erhard; Institute of Molecular Medicine and Genetics; Student Research and Training (STAR) Program, School of Graduate Studies (2009-02-23)
      INTRODUCTION: At physiologic concentration in serum, the bile acid sodium deoxycholate (DC) induces survival and migration of breast cancer cells. Here we provide evidence of a novel mechanism by which DC reduces apoptosis that is induced by the sphingolipid ceramide in breast cancer cells. METHODS: Murine mammacarcinoma 4T1 cells were used in vitro to determine apoptosis and alteration of sphingolipid metabolism by DC, and in vivo to quantify the effect of DC on metastasis. RESULTS: We found that DC increased the number of intestinal metastases generated from 4T1 cell tumors grafted into the fat pad. The metastatic nodes contained slowly dividing cancer cells in immediate vicinity of newly formed blood vessels. These cells were positive for CD44, a marker that has been suggested to be expressed on breast cancer stem cells. In culture, a subpopulation (3 +/- 1%) of slowly dividing, CD44+ cells gave rise to rapidly dividing, CD44- cells. DC promoted survival of CD44+ cells, which was concurrent with reduced levels of activated caspase 3 and ceramide, a sphingolipid inducing apoptosis in 4T1 cells. Z-guggulsterone, an antagonist of the farnesoid-X-receptor, obliterated this anti-apoptotic effect, indicating that DC increased cell survival via farnesoid-X-receptor. DC also increased the gene expression of the vascular endothelial growth factor receptor 2 (Flk-1), suggesting that DC enhanced the initial growth of secondary tumors adjacent to blood vessels. The Flk-1 antagonist SU5416 obliterated the reduction of ceramide and apoptosis by DC, indicating that enhanced cell survival is due to Flk-1-induced reduction in ceramide. CONCLUSIONS: Our findings show, for the first time, that DC is a natural tumor promoter by elevating Flk-1 and decreasing ceramide-mediated apoptosis of breast cancer progenitor cells. Reducing the level or effect of serum DC and elevating ceramide in breast cancer progenitor cells by treatment with Z-guggulsterone and/or vascular endothelial growth factor receptor 2/Flk-1 antagonists may thus be a promising strategy to reduce breast cancer metastasis.
    • alpha-Actinin interacts with rapsyn in agrin-stimulated AChR clustering.

      Dobbins, G Clement; Luo, Shiwen; Yang, Zhihua; Xiong, Wen C; Mei, Lin; Institute of Molecular Medicine and Genetics; Department of Neuroscience and Regenerative Medicine (2009-01-13)
      : AChR is concentrated at the postjunctional membrane at the neuromuscular junction. However, the underlying mechanism is unclear. We show that alpha-actinin, a protein known to cross-link F-actin, interacts with rapsyn, a scaffold protein essential for neuromuscular junction formation. alpha-Actinin, rapsyn, and surface AChR form a ternary complex. Moreover, the rapsyn-alpha-actinin interaction is increased by agrin, a factor known to stimulate AChR clustering. Downregulation of alpha-actinin expression inhibits agrin-mediated AChR clustering. Furthermore, the rapsyn-alpha-actinin interaction can be disrupted by inhibiting Abl and by cholinergic stimulation. Together these results indicate a role for alpha-actinin in AChR clustering.
    • Ceramide-mediated Regulation of Cell Polarity in Primitive Ectoderm Cells: A novel role for sphingolipids in morphogenesis

      Krishnamurthy, Kannan; Institute of Molecular Medicine and Genetics (2009-01)
      Ceramide is considered a key sphingolipid, regulating a variety of critical cellular processes. To facilitate the study of ceramide localization and its interaction with cellular proteins, we have developed a novel antibody against ceramide, raised in rabbit (rabbit IgG). The novel antibody specifically recognizes ceramide in lipid overlay assays and detects ceramide containing different fatty acid chain lengths (i.e. C2-, C16-, C18-, C20- and C24 ceramide). The new antibody was compared with the commercially available anti-ceramide mouse IgM antibody in immunocytochemistry experiments to study the localization of ceramide. Although both antibodies stain similar regions on the cell membrane, the rabbit IgG reveals the distribution of ceramide in intracellular compartments that are not well identified with the commercially available antibody. Pharmacological depletion or increase of ceramide levels results in a corresponding change in staining intensity, confirming the specificity of the antibody. These results indicate that the rabbit IgG is a suitable antibody to determine both the localization of ceramide, and its interaction with proteins by immunocytochemistry. To investigate the role of ceramide in early embryonic development, we used embryoid bodies (EBs) differentiated from mouse embryonic stem cells as a model. The primitive ectoderm cell layer of EBs represents the primitive ectoderm of the early embryo. In mammals, the primitive ectoderm is an epithelium of polarized cells that undergoes gastrulation and differentiates into all embryonic tissues. We find that in primitive ectoderm cells, ceramide was elevated and asymmetrically distributed to the apico-lateral cell membrane, where it was co-distributed with Cdc42 and F-actin. Pharmacological or siRNAmediated inhibition of ceramide biosynthesis impaired primitive ectoderm formation and concomitantly increased apoptosis in EBs. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog, indicating that the observed defect was due to loss of ceramide. Ceramide depletion also prevented membrane translocation of atypical PKC (aPKC), interaction of aPKC with Cdc42, and phosphorylation of GSK-3|3. Recombinant aPKC, when bound to ceramide-containing lipid vesicles, formed a complex with the polarity protein Par6 and Cdc42. Taken together, our data suggest a novel mechanism by which a ceramide-induced, apico-lateral polarity complex with aPKC regulates primitive ectoderm cell polarity and morphogenesis.
    • Welcome to molecular brain.

      Mei, Lin; Cho, Kei; Lee, C Justin; Li, Xiao-Jiang; Zhuo, Min; Kaang, Bong-Kiun; Institute of Molecular Medicine and Genetics (2008-09-22)
      ABSTRACT: We are delighted to announce the arrival of a brand new journal dedicated to the ever-expanding field of neuroscience. Molecular Brain is a peer-reviewed, open-access online journal that aims at publishing high quality articles as rapidly as possible. The journal will cover a broad spectrum of neuroscience ranging from molecular/cellular to behavioral/cognitive neuroscience and from basic to clinical research. Molecular Brain will publish not only research articles, but also methodology articles, editorials, reviews, and short reports. It will be a premier platform for neuroscientists to exchange their ideas with researchers from around the world to help improve our understanding of the molecular mechanisms of the brain and mind.
    • Energy Balance, Myostatin, and GILZ: Factors Regulating Adipocyte Differentiation in Belly and Bone.

      Shi, Xing-Ming; Hamrick, Mark; Isales, Carlos M; Institute of Molecular Medicine and Genetics; Department of Pathology; Department of Cellular Biology and Anatomy; Department of Orthopaedic Surgery (2008-02-29)
      Peroxisome proliferator-activated receptor gamma (PPAR-gamma) belongs to the nuclear hormone receptor subfamily of transcription factors. PPARs are expressed in key target tissues such as liver, fat, and muscle and thus they play a major role in the regulation of energy balance. Because of PPAR-gamma's role in energy balance, signals originating from the gut (e.g., GIP), fat (e.g., leptin), muscle (e.g., myostatin), or bone (e.g., GILZ) can in turn modulate PPAR expression and/or function. Of the two PPAR-gamma isoforms, PPAR-gamma2 is the key regulator of adipogenesis and also plays a role in bone development. Activation of this receptor favors adipocyte differentiation of mesenchymal stem cells, while inhibition of PPAR-gamma2 expression shifts the commitment towards the osteoblastogenic pathway. Clinically, activation of this receptor by antidiabetic agents of the thiazolidinedione class results in lower bone mass and increased fracture rates. We propose that inhibition of PPAR-gamma2 expression in mesenchymal stem cells by use of some of the hormones/factors mentioned above may be a useful therapeutic strategy to favor bone formation.