department of developmental and moleculardepartment of developmental and molecular biology chanin...
TRANSCRIPT
DEPARTMENT OF DEVELOPMENTAL AND MOLECULAR BIOLOGY
The department consists of the laboratories of Drs. Ruth Angeletti, Nick Baker, Teresa Bowman, Dianne Cox, Ana Maria Cuervo, Antonio Di Cristofano, Andreas Jenny, Paraic Kenny, Umadas Maitra, Florence Marlow, Anne Müsch, Nicholas Sibinga, Richard Stanley, Amit Verma, Duncan Wilson, Fajun Yang, and Liang Zhu. Research interests in the Department cover four major areas: (1) Cell determination and regulation in blood, heart, kidney, muscle, nervous and reproductive systems development (Drs. Angeletti, Bowman, Jenny, Maitra, Marlow, Sibinga, Stanley and Verma); (2) Signal transduction pathways regulating cellular function or developmental interactions (Drs. Jenny, Müsch, Stanley, Verma, Yang and Zhu); (3) Cell cycle regulation in normal and neoplastic cells (Drs. Di Cristofano, Kenny, and Zhu); and (4) Protein synthesis, processing, targeting, intracellular vesicle trafficking and autophagy (Drs. Cuervo, Maitra, Müsch and Wilson). Faculties frequently collaborate on projects within and among these areas.
The strength of the Department derives from our outstanding group of PhD and MD/PhD graduate students, who have been encouraged to, and accepted the challenge of, working along side of our faculty in all aspects of department life. We have weekly departmental work-in-progress (Fridays at noon with lunch) and theme-focused journal clubs (in the morning with breakfast) that all graduate students participate along with post-doctoral fellows and our faculty. In these meetings, we enjoy not only superb presentations but also probing questioning-answering periods in an atmosphere of learning, appreciating, and being critical and caring at the same time. A dearly cherished asset of the department is our spirit of cooperation, which makes being a member of the department a fruitful and pleasant experience that you will take with you after you finish your Degrees.
The department’s outside speakers’ seminar program contains regularly scheduled
“student-invited speaker” seminars. Students poll and vote on their most favorite speakers every year and the students serve as hosts at the seminars and restaurant dinners.
The department retreats are another highlight of our department life. The retreats have
been held in a mountain lodge in the Shawangunk Mountains in upstate New York, Mystic Seaport in Connecticut and Montauk on the east tip of Long Island, among many others. In addition to formal presentations of research findings and strategies, we discuss ways of improving the function and atmosphere of the department as well as finding time to socialize!
Besides the scientific events, we are just as proud of our recreational activities, including
BBQ and picnic at Glen Island at the beginning of the school year, and the joyful but competitive presentations of skits by the students and the faculty at our Holidays parties, when students and faculty try to outwit each other in making the most fun of each other!
!
DR. NICHOLAS E. BAKER Department of Developmental and Molecular Biology Ullmann Building – Room 805 (718) 430-2854; [email protected]
Fundamental mechanisms of growth and development Growth and morphogenesis are regulated during the development of multicellular organisms. Growth stops with terminal cell cycle exit and must be up-regulated for regeneration, tissue regulation, or response to damage. Many diseases involve disorders in these events. To study these processes in intact animals, molecular and genetic manipulation of the fruitfly Drosophila melanogaster permits in vivo studies not yet possible in most organisms. We are also employing mathematical models to understand cell-cell interactions fully. Current projects include:
1) ‘Cell competition’ is a process that occurs between cells that differ in growth, for example because of different expression levels of ribosomal protein genes or of the proto-oncogene myc. It is thought that cell competition may exist to identify and eliminate aneuploid cells, or progenitor cells with reduced fitness, and that cell competition suppresses cancer and genome damage during aging. We have identified multiple genes that are required for cell competition and are studying the molecular mechanisms of this process and and its contributions to aging and cancer in flies and mice.
2) HLH proteins represent a well-known class of transcription factors that are important in development. Their ubiquitously-expressed heterodimer partners are implicated in a very wide variety of diseases. We are studying how they are controlled both to allow differentiation and to promote or suppress progenitor cell proliferation, processes that underly human diseases such as Pitt-Hopkins Syndrome, schizophrenia, Fuchs corneal dystrophy, Rett syndrome, and atherosclerosis.
These basic processes that balance growth against terminal differentiation are so fundamental to multicellular organisms that they contribute to understanding many normal and pathological processes. The processes we study are relevant to human diseases including many cancers, cellular senescence, diabetes, Diamond Blackfan Anemia, neuronal dendrite development, multiple neurodegenerative diseases, polycystic kidney disease, Sjogren’s Syndrome, and developmental changes associated with pregnancy and lactation.
For more details, and complete list of publications, please see our website at: http://fruitfly4.aecom.yu.edu/index.html
Selected Recent Publications
Baker, N.E. and Kale, A. (2015). Ribosomal protein mutations: apoptosis, cell competition and cancer. Mol Cell Oncol in press.
Wang, L.-H. and Baker, N.E. (2015) Salvador-Warts-Hippo pathway in a developmental checkpoint monitoring Helix-Loop-Helix proteins. Developmental Cell 32:.191-202.
Kale, A., Li, W., Lee, C.-H. and Baker, N.E (2015) Apoptotic mechanisms during competition of ribosomal protein mutant cells: roles of the initiator caspases Dronc and Dream/Strica. Cell Death Different 22:1300-1312.
Fullard, J.F. and Baker, N.E. (2015) Signaling by the engulfment receptor Draper: a screen in Drosophila melanogaster implicates cytoskeletal regulators, Jun N-terminal Kinase, and Yorkie. Genetics, 199:117-134.
Baker, N.E. and Jenny, A. (2014). Metabolism and the other fat: a protocadherin in mitochondria. Cell 158: 1240-1.
Ruggiero, R., Kale, A., Thomas, B. and Baker, N.E. (2012) Mitosis in neurons: Roughex and Anaphase Promoting Complex maintain cell cycle exit to prevent cytokinetic and axonal defects in Drosophila photoreceptor neurons. PLoS Genetics 8:e1003049.
Bhattacharya, A. and Baker, N.E. (2011). A network of broadly-expressed HLH genes regulates tissue-specific cell fates. Cell, 147, 881-892.
Baker, N.E. (2011). Cell competition. Curr Biol 21: R11-15.
Lubensky, D.K., Pennington, M.W., Shraiman, B., and Baker, N.E. (2011). A dynamical model of ommatidial crystal formation. Proc Natl Acad Sci, 108: 11145-11150.
!
DR. TERESA BOWMAN Department of Developmental and Molecular Biology Chanin Building – Room 501 (718) 430-4001; [email protected]
Bowman Laboratory Hematopoietic stem cells (HSCs) are one of the most widely utilized stem cell populations in the clinic today, in large part due to their robust ability to regenerate and replace the entire blood system after myeloablative injuries. Our research focuses on uncovering the molecular mechanisms underlying how HSCs respond to these injuries. Specifically, we are interested in identifying the key components for directing HSC fate decisions during regeneration. Our studies combine the advantages of zebrafish and mammalian models to explore the development and genetic regulation of HSC stress response. Zebrafish offer powerful genetic pliability, easily accessible in vivo imaging, numerous transplantation assays, and screening capabilities. Through these studies, we anticipate identifying factors that are critical in the HSC regenerative response, which can be used to inform therapeutic strategies to improve outcomes following myeloablative treatments as well as HSC derivation from pluripotent stem cells.
RNA regulation during HSC specification and regeneration: Proper mRNA processing is critical to HSC regeneration and development, but which components are involved is largely unknown. Recent identification of somatic mutations in spliceosomal components in patients with myelodysplastic syndrome (MDS) demonstrated how deregulation of splicing could lead to a disease state. We are combining zebrafish genetics and biochemical techniques to determine how mutations in spliceosomal factors result in aberrant hematopoiesis. Zebrafish is well-suited for this project as mutants in a subset of splicing factors display hematopoietic stem cell specification defects. Additionally, we are performing a synthetic lethal screen in these mutants to determine which epigenetic co-factors work with the splicing machinery during HSC formation and regeneration. Determining the cellular and contextual specificity of interactions between splicing and epigenetic factors would help reveal how mutations in such "general" machinery could elicit such specific phenotypic outcomes in vivo.
Drug discovery for regulators of HSC regeneration: Rapid hematopoietic recovery following myeloablative treatments, such as chemotherapy or radiotherapy, is critical to minimize complications from infection, bleeding, or anemia. Using a candidate approach, we previously showed that transient activation of the Wnt and BMP signaling pathways can accelerate hematopoietic regeneration. In order to find additional pathways involved in HSC regeneration, we plan to take advantage of the screening capabilities afforded in the zebrafish. We are developing new transgenic zebrafish models that allow hematopoietic regeneration to be followed in real time throughout development and into adulthood. These transgenics will be used to screen small molecule libraries for drugs that can block or accelerate hematopoietic recovery. The mechanism of action of the drugs will be tested using genetic, cell biological, and biochemical assays. One of the advantages to chemical screens is the ease at which chemicals can be tested in multiple assays. Thus, once confirmed in the zebrafish, compounds will be tested for functional conservation in murine and human models of hematopoietic regeneration. The identified compounds could have direct therapeutic potential in treating patients following myeloablative regimens.
Lab website: https://sites.google.com/site/thebowmanlaboratoryeinstein/
Selected Publications
Trompouki E*, Bowman TV*, Lawton LN, Fan Z, Wu D, DiBiase A, Martin CS, Cech JN, Sessa AK, Leblanc JL, Li P, Mosimann C, Durand E, Heffner GC, Daley GQ, Paulson RF, Young RA and Zon LI. BMP and Wnt pathway regulation of lineage-specific gene programs underlie Hematopoietic Regeneration. Cell, 2011; 147(3): 577-89. * These authors contributed equally.
Bowman TV, McCooey AJ, Merchant AA, Ramos CA, Fonseca P, Poindexter A, Bradfute SB, Oliveira D, Green R, Zheng Y, Jackson KA, Chambers SM, McKinney-Freeman S, Norwood KG, Darlington G, Gunaratne P, Steffen D, and Goodell MA. Differential mRNA Processing in Hematopoietic Stem Cells. Stem Cells, 2006; 24(3): 662-670.
Venezia TA*, Merchant AM*, Ramos CA, Whitehouse NL, Young AS, Shaw CA, and Goodell MA. Molecular Signatures of Proliferation and Quiescence in Hematopoietic Stem Cells. PLoS Biology, 2004; 2(10): e301. * These authors contributed equally.
Selected Reviews
Bowman TV. Zebrafish Models to Study Stem Cells. The SAGE Encyclopedia of Stem Cell Research, Summer 2015.
Trompouki E, King KY, Will B, Lessard J, Flores-Figueroa E, Kokkaliaris K, and Bowman T. Bloody Signals: From birth to disease and death. Experimental Hematology, 2014; 42(12): 989-94.
Bowman TV. Getting to the ncor of hematopoietic stem cell emergence. Blood, 2014; 124(10): 1541-2.
Bowman TV and Zon LI. Swimming into the Future of Drug Discovery: In Vivo Chemical Screens in Zebrafish. ACS Chemical Biology, 2010; 5(2): 159-61.
5
MACROPHAGE PHAGOCYTOSIS AND MOTILITY
Dr. Dianne Cox, Ph.D. Laboratory: Gruss (MRRC) 306 Phone: 718-430-4005
E-mail: [email protected]
Macrophage Phagocytosis and Motility
Macrophages play an important role in host defense against invading micro-organisms and they are also key players in initiating and maintaining an immune response. However, macrophages can also play negative roles, such as in chronic inflammatory disease. Also, tumor-associated macrophages (TAMs), which are present in large numbers in many tumors, appear to play an important role in promoting the progression of solid tumors to an invasive, metastatic phenotype. Macrophages are therefore a prime target for therapies, but it is important to elucidate the mechanisms by which they are recruited to and activated in tissues.
Studying the molecular mechanisms of phagocytosis
Phagocytosis via receptors for the Fc portion of IgG (Fc!Rs) or for complement (CR3) requires actin assembly, pseudopod extension, and phagosome closure. Actin polymerization in response to particle binding requires the activation of members of the Rho GTPases, either Rac or Cdc42 for Fc!R-mediated phagocytosis or Rho in the case of CR3-mediated phagocytosis. Both Rac and Cdc42 regulate the cytoskeleton in part through the activation of the Wiskott Aldrich Syndrome/ WASP verprolin-homologous (WASP/WAVE) family of proteins. Several human pathogens, such as
Cryptococcus neoformans, and Legionella pneumophila and are internalized by macrophages, escape destruction and grow and divide inside the macrophage. The phagocytic processes by which these organisms are internalized are currently unknown. We are currently dissecting the downstream signaling pathways that mediate these processes during phagocytosis.
Studying the molecular mechanisms of chemotaxis
The directed movement of cells in response to chemoattractants involves several complex, interrelated processes, including directional or chemotactic sensing, polarity, and motility. These processes are mediated by complex, interacting signaling pathways that appear to have many similarities but yet have distinct characteristics depending on the chemoattractant and receptor. We are currently dissecting the signaling pathways required for macrophage chemotaxis towards;
1. CSF-1, a growth factor for macrophage survival and differentiation produced by many tumors and found in high concentrations in arthritic joints.
2. Chemokines that direct monocyte recruitment to different tissues
Understanding the specific signaling components required for migration towards each of these factors will allow us to test the importance of these signaling components in vivo. The ability of macrophages, altered in pathways identified in vitro, to migrate into the tissue space will be tested in animal models of peritoneal infection, atherosclerosis, and breast cancer.
The role of macrophages in the tumor microenvironment
It is now increasingly recognized that the tissue microenvironment plays a critical role in tumor progression, but most studies on
6
tumor metastasis involve the use of endpoint assays or in vitro studies on cell lines. It appears that macrophages and tumor cells participate in a paracrine interaction, with the tumor cells secreting CSF-1 and macrophages secreting EGF, but the precise roles of this paracrine interaction in tumor metastasis are unknown. We have developed a number of in vitro assays that reconstitute paracrine interaction between macrophage and carcinoma cells that mimic in vivo interactions of macrophages and tumor cells in the tumor microenvironment that have been observed by intravital imaging (Dovas et al., J. Microscopy 2012). We are currently using these assays to understand the roles of both soluble factors and direct interaction between macrophages and tumor cells through tunneling nanotubes to mediate long distance signaling to promote tumor metastasis.
Selected Publications (Students in bold) Abou-Kheir, W., Gevrey, J-C., Issac, B.M., Yamaguchi, H., and Cox, D. (2005) A WAVE2/Abi1 complex mediates CSF-1-induced F-actin rich membrane protrusions and migration in macrophages. J. Cell Science 118:5369-5379. Abou-Kheir, W.G., Isaac, B., Yamaguchi H., and Cox, D. (2008) Membrane targeting of WAVE2 is not sufficient for WAVE2 dependent actin polymerization: a role for IRSp53 in mediating the interaction between Rac and WAVE2. J. Cell Science 121:379-90. Luo, Y., Isaac, B.M., Casadevall, A., and Cox D. (2009) Macrophage phagocytosis suppresses F-actin enriched membrane protrusions stimulated by CX3CL1 and CSF-1. Infect. and Immun. 77:4487-4495. Isaac, B.M.*, Ishihara, D.*, Nusblat, L.M., Gevrey, J-C, Dovas A., Condeelis, J., and Cox, D. (2010) N-WASP has the Ability to Compensate for the Loss of WASP in Macrophage Podosome Formation and Function. Exp. Cell Res. 316:3406-16. *co-first authors Nusblat, L.M., Dovas, A., and Cox, D. (2011) The essential role of N-WASP in podosome-
mediated matrix degradation. Eur. J. Cell Biol. 90:205-212. Ishihara, D., Dovas A., Park, H., Isaac, B.M., and Cox, D. (2012) The chemotactic defect in Wiskott-Aldrich Syndrome macrophages is due to reduced persistence of directional protrusions. PLoS One 7(1):e30033 Ishihara, D.*, Dovas, A.*, Hernandez, L., Pozzuto, M., Wyckoff, J., Segall, J., Condeelis, J., Bresnick, B., and Cox, D. (2013) Wiskott-Aldrich syndrome protein regulates leukocyte-dependent breast cancer metastasis. Cell Reports 4:429-436 *co-first authors Rougerie, P., Miskolci, V. and Cox, D. (2013) Generation of membrane structures during phagocytosis and chemotaxis of macrophages: role and regulation of the actin cytoskeleton. Immunol. Reviews. 256:222-239. Park, H., Dovas, A., Hanna, S., Cougoule, C, Marridoneau-Parini, I., and Cox, D. (2014) Tyrosine phosphorylation of Wiskott-Aldrich Syndrome protein (WASP) by Hck regulates Macrophage Function. J. Biol. Chem. 289(11):7897-906. Hanna, S.*, Miskolci, V.*, Cox, D.# and Hodgson, L.# (2014) Development of a new single-chain genetically encoded Cdc42 FRET biosensor. PLoS One 9(5):e96469. *co-first authors, #co-corresponding authors Miskolci, V., Hodgson, L., Cox, D., and Vancurova, I. (2014) Western Analysis of Intracellular Interleukin-8 in Human Mononuclear Leukocytes. Methods in Molecular Biology: Cytokines. 1172:285-93. Miskolci, V., Spiering, D., Cox, D., and Hodgson, L. (2014) A mix-and-measure assay for determining the activation status of endogenous Cdc42 in cell lysates. Methods in Molecular Biology: Cytokines. 1172:173-84. Wu, B.*, Miskolci, V.*, Donnelly, S.K., Cox, D., Singer, R.H.% and Hodgson L.% (2015) Synonymous modification of repeated sequences in retroviral reporters. Genes Dev. 29 (8):876-86. *co-first authors
DR. ANA MARIA CUERVO Department of Developmental and Molecular Biology Chanin Building – Room 504 (718) 430-2689; [email protected]
Autophagy in Aging and Age-related diseases The main focus of our laboratory is on understanding how cytosolic proteins are transported into lysosomes for their degradation (autophagy) and how impaired autophagy contributes to aging and age-related diseases.
A common feature of senescent cells is the accumulation of abnormal or damaged proteins in their cytosol that, undoubtedly, impairs cellular function. Protein accumulation results, at least in part, from impaired protein degradation with age. Among the different systems that participate in the intracellular degradation of proteins, lysosomes are the most affected by age. We have previously identified in many tissues of aged animals a decrease with age in the activity of a selective pathway for the degradation of cytosolic proteins in lysosomes known as chaperone-mediated autophagy. The main goal of our research is to identify the defect(s) that lead to the decreased activity of chaperone-mediated autophagy with age and in age related pathologies, and to analyze if the correction of those defects and recovery of normal proteolytic activity in old cells leads to an improvement in cellular function.
Chaperone-mediated autophagy is responsible for the degradation of as much as 30% of cytosolic proteins, and it is mainly activated under conditions of stress, such as nutrient deprivation and oxidative stress. Substrate proteins are selectively recognized by cytosolic chaperones (hsc70 and cochaperones) that stimulate their binding to a glycoprotein receptor in the lysosomal membrane (LAMP-2A). The transport of the cytosolic proteins into lysosomes for their degradation requires also the presence of another chaperone in the lysosomal matrix (lys-hsc70).
Our current efforts are directed to:
1. Characterization of the different components involved in chaperone-mediated autophagy and identification of new players for this pathway.- We can isolate intact lysosomes from several tissues (liver, kidney and spleen) of rodents. For the identification of new CMA components we are using different immunochemical approaches and a global proteomic approach. We have also developed a photoswitchable reporter which allows us to identify changes in CMA in intact cells. We are currently using this reporter to perform RNAi screenings in order to discover novel unknown regulators of this pathway.
2 Understanding the consequences of the age-related defect in chaperone-mediated autophagy.- We have generate conditional and inducible transgenic mouse models incompetent for CMA in different tissues and have started to investigate possible tissue-dependence differences in the requirements for functional CMA. We are also analyzed the cellular response to CMA blockage and the different compensatory mechanisms elicited. We have found that blockage of CMA leads to important alterations in cellular quality control and cellular metabolism and deficiencies in the response to different stressors.
Autophagic pathways in mammalian cells
3. Consequences of impaired autophagy in age related-disorders.- In collaboration with the groups of David Sulzer (Columbia University) and Randolph Nixon (New York University), we have been analyzing changes in autophagy in specific neurodegenerative disorders, as example of age-related diseases. By combining metabolic assays, cellular fractionation procedures and our in vitro lysosomal transport assays in different animal and cellular models of these diseases, we have found a primary defect in CMA in some familial forms of Parkinson’s disease. We are currently analyzing other neurodegenerative disorders, Alzheimer’s and Huntington’s disease. We are interested in identifying the primary defect, the compensatory mechanisms elicited by the cell and the changes that lead to this general failure of the proteolytic systems in these and other age-related disorders.
References:
• Kaushik, S. Cuervo AM*. Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis. Nat. Cell. Biol. 17: 759-70, 2015
• Arias E., Koga H, Diaz A, Mocholi E, Patel B, Cuervo AM*. Lysosomal mTORC2/PHLPP1/Akt regulate chaperone-mediated autophagy. Mol. Cell 59, 270-84, 2015
• Park C, Shu Y, Cuervo AM*.Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage. Nat. Commun. 6:6823 doi: 10.1038/ncomms7823, 2015
• Rui Y-N, Xu Z, Patel B, Chen Z, Chen D, Tito A, David G, Sun Y, Stimming ER, Bellen H, Cuervo AM*, Zhang S*. Huntingtin functions as a scaffold for selective macroautophagy. Nat. Cell. Biol. 17: 262-75, 2015
• Schneider S, Villarroya J, Diaz A, Patel B, Urbanska AM, Thi MM, Villarroya F, Santambrogio L, Cuervo AM*. Loss of hepatic chaperone-mediated autophagy accelerates proteostasis failure in aging. Aging Cell, 14:249-64, 2015
• Schneider JL, Suh Y, Cuervo AM. Deficient chaperone-mediated autophagy in liver leads to metabolic disregulation. Cell Metab. 20:417-432, 2014
• Bejarano, E, Yuste, A, Patel B, Stout, RJ, Spary, D Cuervo AM. Connexins modulate autophagosome biogenesis. Nat. Cell. Biol. 16:401-14, 2014
• Pampliega O, Orhon I, Patel B, Sridhar S, Diaz-Carretero A, Beau I, Codogno P, Satir B, Satir P, Cuervo AM. Functional interaction between autophagy and ciliogenesis. Nature 502:194-200, 2013
• Orenstein SJ, Kuo SH, Tasset-Cuevas I, Arias E, Koga H, Fernandez-Carasa I, Cortes, E., Honig, L.S., Dauer, W., Consiglio A, Raya A, Sulzer, D, Cuervo AM. Interplay of LRRK2 with chaperone-mediated autophagy. Nat. Neurosci. 16:394-406, 2013
• Anguiano J, Gaerner T, Daas B, Gavathiotis E, Cuervo AM. Chemical modulation of Chaperone-mediated autophagy by novel retinoic acid derivatives. Nat. Chem. Biol. 9:374-82, 2013
Reviews:
• Schneider JL, Cuervo AM. Liver autophagy: much more than taking out trash. Nat Rev Gastroenterol Hepatol. 11:187-200, 2013
• Kauhsik S, Cuervo AM. Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends Cell Biol 2012, 22: 407-17, 2012
Failure of chaperone-mediated autophagy results in lipid accumulation (green)
DR. ANTONIO DI CRISTOFANO Department of Developmental and Molecular Biology Price Center – Room 302 (718) 678-1137; [email protected]
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
!!3C.#/(G)&0!-1-@')C.!#.+)+'-&/.!'(!48=:!)&5)B)'(#+!)&!'57#()1!/-&/.#!#0(7-08-,1!',;3-79<1!'0<!'<'&(-.9!798-8('0,9!'79!,/22/0!,/089;390,98!/6!,'0,97!,955!9G&/8379!(/!('7:9(9<!-0)-E-(/78=!!#0<99<1!*9!)'.9!6/30<!()'(!*)-59!"#$%!-0)-E-(/78!79<3,9!()9!:7/*()!7'(9!/6!BCH!,9558!).$8)%'(!'0<!-0!'55/:7'6(81!()-8!9669,(!-8!8)/7(?5-.9<!'0<!()989!,9558!-0.'7-'E5+!E9,/29!798-8('0(=!!I37!/EP9,(-.9!-8!(/!,)'7',(97-Q9! ).$ 8)8(! ()9! ,955! '3(/0/2/38! '0<! 0/0?'3(/0/2/38! 29,)'0-828! ()7/3:)! *)-,)! "#$%',(-.9!()+7/-<!(32/7!,9558!<9.95/&!'<'&(-.9!798-8('0,9!(/!"#$%!-0)-E-(/781!'0<!(/!(98(!()9!966-,',+!/6!,/2E-0'(-/0!()97'&-98!('7:9(-0:!O9+!29<-'(/78!/6!798-8('0,9=!!E./.&'!#.@#.+.&'-')C.!@?B<)/-')(&+;!A= R9':971! S=1! %59-0?DQ'0(/1! B=1! %-237'1! D=! '0<!@-! H7-8(/6'0/1! B=!<%#.! 5/88! -0! ()9!2/389! ()+7/-<! ,'3898!
:/-(97! '0<! 6/55-,35'7! '<90/2'8T! -08-:)(8! -0(/! ()+7/-<! 630,(-/0! '0<! H/*<90! <-89'89! &'()/:9098-8=!C-.0#'$F#/9!UVTWXW?WUU1!YZZV=!
Y= [79*971!H=1!R9':971!S=!'0<!@-!H7-8(/6'0/1!B=!C)+7/-<?8(-235'(-0:!)/72/09!-0-(-'(9<!&7/5-697'(-.9!8-:0'58!,/0.97:9!-0!.-./!/0!()9!2CIL!O-0'89!*-()/3(!',(-.'(-0:!B%C=!C-.0#'$F#/9!UVT\ZZU?\ZZU1!YZZV=!
$= R9':971!S=1![79*971!H=1!H'-1!%]1!J31!JJ=1!'0<!@-!H7-8(/6'0/1!B=!2CIL!-8!()9!O9+!9669,(/7!/6!"#$%?-0-(-'(9<!&7/5-697'(-.9!8-:0'58!-0!()9!()+7/-<!6/55-,35'7!9&-()95-32=!C-.0#'$F#/91!U\T^^^?^^W1!YZZ\9!
^= K-55971!%=1!R9':971!S=1
DR. ANDREAS JENNY Department of Developmental & Molecular Biology Chanin Building- Room 503 (718) 430-4183; [email protected]
Canonical and non-canonical Wnt signaling: patterning and cell polarization
Wnt/Wingless (Wg) growth factors commonly signal through either the canonical Wnt (Wg)-Frizzled (Fz)/!-catenin pathway or through non-canonical Wnt pathways such as the Wnt/Fx-planar cellular polarity (PCP) pathway, resulting the polarization of cells within the pane of the epithelium. These two pathways are highly conserved between humans, mice, fish, and flies. Canonical Wnt/!-catenin signaling is essential for many aspects of development. For example in vertebrates, it controls the specification of the dorsal-ventral (D-V) embryonic axis, cell proliferation in many tissues, and the maintenance of stem cells and during vascularization. In addition, aberrant canonical Wnt signaling in humans causes cancer. Our lab studies the function of Wnk kinases, for which we have identified a novel role in Wnt signaling in addition to their well-known role in the regulation of ion homeostasis in the kidney, where their lack causes hypertension (Gordon syndrome).
Non-canonical Wnt signaling established polarity within the plane o an epithelium, commonly referred to as epithelial planar cell polarity (PCP) and allows a cell to form structures that require not only positional, but also vectorial information. Examples of PCP in vertebrates can be very obvious, as in the ordered arrangement of scales on fish or hairs of mammalian skin. Less visible examples are the cilia of the respiratory tract and oviduct as well as the stereocilia of the sensory epithelium of the organ of Corti in the vertebrate inner ear. Aberrant PCP can lead to left/right asymmetry defects, open neural tubes, deafness and kidney disease. PCP signaling is, however, best studied in Drosophila melanogaster, mainly because of the versatility of the fly as model system. Our lab is particularly interested in how Rho kinase (Rock) is required for the migration aspect of PCP establishment which will help to understand tumor cell migration.
A genetic model for Endosomal Microautophagy
Autophagy delivers cytosolic components to lysosomes for degradation and is thus essential for
cellular homeostasis and to cope with different stressors. As such, autophagy counteracts various human diseases and its reduction leads to aging like phenotypes. Macroautophagy (MA) can selectively degrade organelles or aggregated proteins, but selective degradation of single proteins has only been described for Chaperone-mediated autophagy (CMA) and endosomal Microautophagy (eMI). These two autophagic pathways, described to date only in mammals, are specific for proteins containing KFERQ-related targeting motifs. In collaboration with the Cuervo lab, we have developed a fluorescent reporter to characterize an eMI or CMA-like process in Drosophila in vivo. Our data provide evidence for a novel, starvation inducible catabolic process resembling endosomal microautophagy in a non-mammalian species. We are thus for the first time able to perform genetic screens for regulatory components of eMI, this only recently identified form of autophagy about which barely anything is known.
It is our goal to use Drosophila as model system to address fundamental questions that are relevant for development and disease in general.
Lab homepage: http://jennylab.aecom.yu.edu/
Selected References: 1. Maung, S. M. T., Jenny, A. (2011). Planar Cell Polarity in Drosophila. Edited by Carroll, T. Organogenesis
7(3) 165-179. 2. Jenny, A. (2010) Planar Cell Polarity in the Drosophila Eye. Curr. Top. Dev. Bio. Edited by Reh, T. and
Cagan, R. Curr. Top. Dev. Bio. 93, 189-227. 3. Serysheva, E., Berhane, H., Grumolato, L., Demir, K., Balmer, S., Bodak, M. , Aaronson, S., Boutros, M.,
Mlodzik*, M., Jenny, A*. (2013). Wnk kinases are positive regulators of canonical Wnt/b-catenin signaling. EMBO Reports 14(8), 718-725.
4. Fagan, J.K., Dollar, G., Lu, Q., Barnett, A., Pechuan Jorge, J., Schlosser, A., Pfleger, C., Adler, P., Jenny, A..* (2014) Combover/ CG10732, a novel PCP effector for Drosophila wing hair formation. PLoS One, 9(9):e107311. doi: 10.1371. PMID: 25207969.
5. Forbes, M.M., Rothhämel, S., Jenny, A., Marlow FL. (2015). Maternal dazap2 regulates germ granules via counteracting Dynein in zebrafish primordial germ cells. Cell Reports,12 (1), 49-57.
6. Gombos, R., Migh, E., Antal, O., Mukherjee, A., Jenny, A., Mihály, J. (2015) The Formin DAAM Functions as Molecular Effector of the Planar Cell Polarity Pathway during Axonal Development in Drosophila. J. Neuroscience, 35 (28),10154-67.
DR. FLORENCE MARLOW Department of Developmental and Molecular Biology Chanin Building – Room 514 (718) 430-4208; [email protected]
Polarity in the Zebrafish Ovary
Every animal starts out as a single fertilized cell, yet we do not fully understand the events that are essential for producing that cell because they take place within the ovary of the mother. Failure to form an egg that is capable of embryonic development can result in profound birth defects or miscarriage. In addition, cancers of the ovary can arise from uncontrolled proliferation of the germ cells, those cells that can become eggs, or the somatic cells, the cells that do not develop as eggs, of the ovary. In normal ovaries, these two types of cells communicate with one another to regulate the growth and survival of both cell populations. In most animals, the germ line stem cells undergo an asymmetric division to generate daughter cells that will remain stem cells and others, cystoblasts that divide and eventually form eggs. The divisions of the cystoblasts are unique because the cells do not completely separate from one another, but instead remain attached to each other. Studies in mammals show that the connections between cystoblasts prevent too many cells from becoming oocytes, and in humans uncontrolled and complete separation of cystoblasts correlates with germ cell neoplasias. However, since these events occur before or at the time of fertilization we understand little about how the genes that are involved. Therefore, understanding how the growth and survival of these cells is regulated has important consequences to both fertility and cancer formation. To study relationships between interacting cells within adjacent tissues, such as germline and somatic follicle cells, we need to analyze an animal system in which we can manipulate genes and study early development. The zebrafish system has advantages that allow us to use embryological, biochemical, and genetic techniques to access maternally controlled processes during vertebrate animal development. Our studies exploit the powerful genetics and cell biological access in the zebrafish system to unravel the mechanisms that regulate oocyte polarization and follicle cell fate in a vertebrate. Many features of primary oocyte development are evolutionarily conserved, including humans; thus this architecture is likely fundamental for germline development and fertility. Our genetic and biochemical studies of oocyte polarity have led us to genes involved in mRNA localization and polarized transport, including motor and RNA binding proteins. Like oocytes, neurons are highly polarized cells, which rely on trafficking and post-transcriptional regulation of mRNAs to ensure that gene products are only expressed in discrete locations. Inappropriate accumulation of proteins and organelles due to failed trafficking and post-transcriptional regulation is associated with neuronal loss underlying devastating neurodegenerative diseases. The rapid development, optical clarity, and ease of generating transgenic and mutant zebrafish strains make it an ideal system for live cell tracking and visualization of fluorescently labeled organelles, motor proteins, mRNA cargos, and the cytoskeleton in the living animal. To better understand how trafficking is regulated in distinct polarized cell types, we are also using the zebrafish model system to examine the cellular and molecular basis of transport in neurons. Knowledge of how the individual transport mechanisms operating in neurons contribute to their function has potential to uncover novel pathological mechanisms underlying neurodegenerative diseases. Selected Publications: Zebrafish vasa is required for germ cell differentiation and maintenance. Hartung O, Forbes MM, Marlow FL. Molecular Reproduction and Development. In press.
Oocyte polariry requires a Bucky ball dependent feedback amplification loop. Heim A, Rothhämel S, Hartung O, Ferriera, E Jenny A, Marlow FL. Development. 2014 Feb;141(4):842-54. doi: 10.1242/dev.090449.PMID: 24496621. notch3 is essential for oligodendrocyte development and vascular integrity in zebrafish. Zaucker A, Mercurio S, Sternheim N, Talbot WS, Marlow FL. Disease Models and Mechanisms. 2013 Sep;6(5):1246-59. doi: 10.1242/dmm.012005. Epub: 2013 May 29.
Analysis of zebrafish kinesin-1 kif5 heavy chain genes during zebrafish development. Campbell, PD, Marlow FL. Gene Expression Patterns. Epub 2013 May 15. 2013 Oct;13(7):271-9. doi: 10.1016/j.gep.2013.05.002.
The adhesion GPCR Gpr125 modulates Dishevelled distribution and planar cell polarity signaling.Li X, Roszko I, Sepich DS, Ni M, Hamm H, Marlow FL*, Solnica-Krezel L.* Development. 2013 Jul;140(14):3028-39. doi: 10.1242/dev.094839. PMID: 23821037 *cocorresponding authors.
DR. ANNE MÜSCH Department of Developmental and Molecular Biology Chanin Building – Room 517 (718) 430-3508; [email protected]
Signaling Mechanisms Involved in the Establishment of Epithelial Cell Polarity
Loss of epithelial cell polarity is associated with the overwhelming majority of human cancers. This has prompted intense research on mechanisms for epithelial polarity. Over the last decades, we have gained insight into how epithelial cell establish polarized surface domains and carry out vectorial protein transport, how cell-cell adhesion is mediated and how simple epithelia maintain a monolayer organization. This groundwork has set the stage for a new challenge: to identify and characterize the signaling mechanisms that integrate these individual aspects of epithelial polarity and architecture during morphogenesis and in pathology. My lab is interested in characterizing such novel polarity signaling cascades for epithelial polarity by means of cell biological approaches in established model cell lines for non-stratified epithelia of the kidney and liver.
Most of our current effort is focused on Par1, a serine/threonine kinase that was identified as polarity determinant in the one-cell embryo of C. elegans. We have determined that dysfunction of Par1 inhibits various aspects of epithelial polarization, notably the establishment of an apical surface domain and protein trafficking to the apical surface, the development of a columnar cell shape and the organization of an epithelial-specific microtubule array. Moreover, we found that Par1 is targeted by Helicobacter pylori, a bacterium that destroys the epithelial lining of the stomach, resulting in gastritis and gastric ulcers and is implicated in gastric cancer. We are now working to identify the Par1 substrates that regulate individual aspects of epithelial morphology and polarity. We anticipate that this will give us mechanistic insight into how these different aspects of polarization are linked. It is known, for instance, that protein trafficking to the apical domain is microtubule-dependent. Thus, we expect that the Par1 substrate(s) involved in epithelial microtubule-organization also regulate apical protein targeting and the formation of the apical cell surface.
As few Par1 substrates are known so far, we have developed an unbiased proteome-wide screen for novel substrates of the kinase in epithelial cells. Putative Par1 targets are tested for their Par1-dependent phosphorylation in vitro and in vivo. Subsequently, we are characterizing their cell biological roles in cultured epithelial cells.
Our repertoire of approaches include the analysis of microtubule-dynamics and of apical surface formation in intact cells by time lapse imaging of GFP and RFP-tagged proteins, immunofluorescence and confocal laser microscopy to study cell morphology and sub-cellular protein distribution, biochemical assays that measure the kinetics of different protein trafficking steps and biochemical assays that measure protein-protein interactions and enzyme activities in cell lysates. Selected Publications: Treyer A., Müsch, A. “Hepatiocyte Polarity” (2013), Comprehensive Physiology, 3: 243-287. Ispolatov, I., Müsch, A. " A model for the self-organization of vesicular flux and protein distributions in the Golgi apparatus" (2013), PLoS Comput Biol. 2013 Jul;9(7):e1003125. doi: 10.1371/journal.pcbi.1003125. Cohen D., Fernandez D., Lazaro-Dieguez, F. and Anne Müsch,. “The serine/threonine kinase Par1b regulates epithelial lumen polarity via IRSp53-mediated cell-ECM signaling”, (2011) J Cell Biol. 192, 525-540 Zaher Zeaiter, David Cohen, Anne Müsch, Fabio Bagnoli, Antonello Covacci, and Markus Stein “Analysis of detergent resistant membranes of Helicobacter pylori infected gastric adenocarcinoma cells reveals a
role for MARK2/Par1b in CagA-mediated disruption of cellular polarity”, (2008) J Cellular Microbiology, 10, 781-94. Cohen D., Tian Y and Müsch, A (2007): “Par1b promotes hepatic-type lumen polarity in MDCK cells via myosin II and E-cadherin dependent signaling”, Mol. Biol. Cell 18, 2203-15.
Elbert, M., Cohen D.and Müsch, A, (2006): “ Par1b promotes cell-cell adhesion and inhibits Dishevelled-mediated transformation of MDCK cells”, Mol.Biol.Cell, 17, 3345-55. Cohen, D., Rodriguez-Boulan, E. and Müsch, A. (2004). "Par-1 promotes a hepatic mode of apical protein trafficking in MDCK cells". PNAS 101, 13792-97. Cohen, D., Brennwald, P.J., Rodriguez-Boulan, E. and Müsch, A. (2004).“Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton”. Journal Cell Biology, 164, 717-727.
DR. NICHOLAS E. S. SIBINGA Department of Developmental & Molecular Biology Forchheimer Building- Room G35 S 718 430-2881; [email protected]
Key Words: Cardiovascular disease, inflammation, growth regulation, mouse models
CONTROL OF VASCULAR CELL GROWTH, DIFFERENTIATION, AND INJURY RESPONSE
Vascular disease, the greatest single cause of morbidity and mortality in developed societies, results from interactions between circulating inflammatory cells, the endothelium that lines the vasculature, and underlying vascular smooth muscle cells (VSMCs) that comprise most of the arterial wall. We seek to identify new factors and pathways that regulate disease-associated activation of these cell types.
The underlying pathogenesis is complex: factors that impinge on these cell types include reactivated developmental pathways, innate and acquired immune responses, and changes in cell function that result from physical stresses, perturbed blood flow, and biochemical stimuli. Our general approach is to characterize these responses at the molecular level, in cell culture, and in mouse models that reflect specific types of vascular injury, including atherosclerosis, restenosis after angioplasty, saphenous vein graft disease, and transplant-associated arteriosclerosis.
One project focuses on the atypical cadherin adhesion molecule Fat1, which is strongly induced in injured arteries. Fat1 is a member of the ancient Fat cadherin subfamily – in Drosophila, these proteins are important regulators of growth and planar cell polarity. Our work in mammalian VSMCs shows that Fat1 interacts with and limits the transcriptional activity of beta-catenin, the major downstream mediator of Wnt signaling, a core developmental pathway that regulates many aspects of metazoan embryogenesis. Fat1 also interacts with Atrophin proteins to control VSMC directional migration. Interestingly, beta-catenin-mediated signaling in smooth muscle cells is required for vessel wall assembly during vascular development. While beta-catenin is essential in development, Fat1 is an important regulator of VSMC growth and differentiation in injured vessels in the adult. A second project involves the allograft inflammatory factor-1 (Aif-1), a 17kD EF-hand protein expressed primarily in activated macrophages. We have found that Aif-1 promotes macrophage migration, phagocytosis, survival, and selected cytokine production. We are currently evaluating how Aif-1 contributes to integrated immune responses and metabolism, and how loss of Aif-1 function in the mouse affects the pathogenesis of multiple inflammatory diseases, including atherosclerosis, in which this factor is strongly induced.
In collaborative work with the Stanley lab, we have characterized a role for colony stimulating factor-1 (CSF-1), the main regulator of macrophage survival, proliferation, and differentiation, in control of transplant-associated arteriosclerosis, the major barrier to longterm success of organ transplants. Surprisingly, this effect appears to involve VSMC-associated CSF-1 in an autocrine/juxtacrine mechanism that is largely independent of macrophages.
Ongoing work in these areas involves defining the molecular bases for these effects. By identifying such novel mechanisms, we hope to find new targets for therapeutic intervention to regulate VSMC activities and improve vascular disease prevention and treatment. Selected References:
1. Chinnasamy P, Lutz S, Casimiro I, Brosnan C, Sibinga NES. Loss of Allograft inflammatory factor-1 ameliorates experimental autoimmune encephalomyelitis by limiting encephalitogenic CD4 T cell expansion. Molecular Medicine, 2015 Jan 6;21(1):233-41.
2. Bruder-Nascimento T, Chinnasamy P, Riascos-Bernal DF, Cau SB, Callera GE, Touyz RM, Tostes RC, Sibinga NE. Angiotensin II induces Fat1 expression/activation and vascular smooth muscle cell migration via Nox1-dependent reactive oxygen species generation. J Mol Cell Cardiol. 2014 Jan;66:18-26.
3. Hiroyasu S, Chinnasamy P, Hou R, Hotchkiss K, Casimiro I, Dai XM, Stanley ER, Sibinga NES. Donor and Recipient Cell Surface Colony Stimulating Factor-1 Promote Neointimal Formation in Transplant-Associated Arteriosclerosis. Arterioscler Thromb Vasc Biol. 2013 Jan;33(1):87-95.
4. Hou R, Sibinga NES. Atrophin proteins interact with the Fat1 cadherin and regulate migration and orientation in vascular smooth muscle cells. J Biol Chem, 2009; 284(11):6955-65.
5. Hou R, Liu L, Anees S, Hiroyasu S, Sibinga NES. The Fat1 cadherin integrates vascular smooth muscle cell growth and migration signals. J Cell Biol, 2006; 173(3):417-29.
DR. E. RICHARD STANLEY Department of Developmental and Molecular Biology Chanin Building – Room 507 (718) 430-2344; [email protected]
Growth Factors and Signaling in Development: CSF-1 Biology: Colony stimulating factor-1 (CSF-1) is a growth factor which regulates the production of macrophages, osteoclasts, Paneth cells, microglia, neuronal cells and the function of certain non-myeloid cell types in the female reproductive tract. It is expressed as a secreted glycoprotein, secreted proteoglycan or a membrane-spanning, cell-surface glycoprotein. Its effects are mediated via a receptor tyrosine kinase, the c-fms protooncoprotein. CSF-1-deficient mice are osteopetrotic due to a lack of osteoclasts, have poor fertility and several other defects related to CSF-1 regulation of macrophages that have critical scavenger and trophic roles in the development, maintenance or function of tissues in which they reside. CSF-1R-deficient mice have a more severe phenotype than CSF-1-deficient mice, due to the existence of a second CSF-1R ligand, interleukin-34 (IL-34), which we have recently shown also acts through another receptor, protein tyrosine phosphatase-zeta. IL-34 is highly expressed in brain where, with CSF-1, it regulates the development and maintenance of microglia and the differentiation of neural progenitor cells. CSF-1R regulation of macrophages is also important in innate immunity, gut development, inflammatory diseases, atherosclerosis, obesity, cognitive disorders and within tumors, for tumor progression and metastasis. Autocrine regulation by CSF-1 has been demonstrated in leukemia and solid tumors. Mouse genetic and other approaches are being taken to investigate the developmental and physiological roles of CSF-1 and IL-34, as well as their roles in disease development. CSF-1 signal transduction: Since phosphorylation of specific CSF-1R intracellular domain tyrosine residues initiate particular signaling pathways, detailed structure-function studies of the CSF-1R are being carried out in macrophages and macrophage progenitor cells. In the analysis of very early post-receptor events, proteins that are rapidly phosphorylated in tyrosine in response to CSF-1 or associated with tyrosine-phosphorylated proteins, approximately 800 in all, have been identified by mass spectrometry. A combination of genetic, proteomic, biochemical and analytical imaging approaches are being used to elucidate the roles and interactions of these signaling proteins in the immediate post-receptor events in CSF-1 signal transduction. Previous studies have identified and elucidated the actions of the protein tyrosine phosphatase SHP-1, that negatively affects cell survival in the absence of CSF-1, the cbl proto-oncogene product, that negatively regulates CSF-1 proliferation signaling by enhancing CSF-1R endocytosis and the protein tyrosine phosphatase, PTP-phi, that mediates CSF-1-induced morphological changes, adhesion and motility, via its action on a specific substrate, paxillin. Current work is focused on Dok-1, that regulates macrophage motility and the macrophage F-actin associated and tyrosine phosphorylated protein (MAYP/PSTPIP2). PSTPIP2 is an F-BAR protein that coordinates processes involving the cell membrane and actin cytoskeleton. It is a negative feedback regulator of CSF-1R-mediated osteoclast and macrophage production and loss-of-function mutations in PSTPIP2 lead to an autoinflammatory disease involving these cell types. Signaling by the Shark tyrosine kinase: Embryonic dorsal closure in Drosophila is a series of morphogenetic movements involving the bilateral dorsal movement of the epidermis (cell stretching) and dorsal suturing of the leading edge cells to enclose the viscera. The Syk family tyrosine kinase, Shark, is expressed in the epidermis and plays a crucial role in this Jun kinase-dependent process where, with the adapter, Ddok, it acts upstream of
JNK in leading edge cells. Shark is also expressed by glial cells, where it functions downstream of the Draper receptor and Src42A to regulate the recognition and engulfment of dying neuronal cells. Mutations in the genes for Shark-interacting proteins, coupled with cell biological approaches, are being used to define Shark function and the Shark signaling pathways. Selected Publications: Cecchini, M.G., Dominguez, M.G., Mocci, S., Wetterwald, A., Felix, R., Fleisch, H., Chisholm, O., Pollard, J.W., Hofstetter, W. and Stanley, E.R. (1994) Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during post natal development of the mouse. Development 120:1357-1372. Dai, X-M., Ryan, G.R., Hapel, A.J., Dominguez, M.G., Kapp, S., Sylvestre, V. and Stanley, E.R. (2001) Targeted disruption of the mouse CSF-1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased splenic progenitor cell frequencies and reproductive defects. Blood 99:111-120. Dai X-M, Zong XH, Sylvestre V and Stanley ER. (2004) Incomplete restoration of colony stimulating factor-1 (CSF-1) function in CSF-1-deficient Csf1op/Csf1op mice by transgenic expression of cell surface CSF-1. Blood 103: 1114-1123. Nandi, S., Akhter, M.P., Seifert, M.F., Dai, X-M. and Stanley, E.R. (2006). Developmental and functional significance of the CSF-1 proteoglycan chondroitin sulfate chain. Blood 107(2):786-795. Huynh, D., Dai, X-M., Nandi, S., Lightowler, S., Trivett, M., Chan, C-K., Bertoncello, I., Ramsay, R.G. and Stanley, E.R. (2009). CSF-1 dependence of Paneth cell development in the mouse small intestine. Gastroenterology 137(1):136-144. Yeung, Y-G. and Stanley, E.R. (2003) Proteomic approaches to analysis of the early events in CSF-1 signal transduction. Molecular and Cellular Proteomics. 2: 1143-1155 Wei, S., Nandi, S., Chitu, V., Yeung, Y-G., Yu, W., Huang, M., Williams, L.T., Lin, H. and Stanley, E.R. (2010). Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells. J Leukoc. Biol. 88(3):495-505. Ginhoux, F., Greter, M., Leboeuf, M., Nandi, S., See, P., Gokhan, S., Mehler, M.F., Ng, L.G., Stanley, E.R., Samokhvalov, I.M. and Merad, M. Fate mapping studies reveal that adult microglia derive from primitive macrophages. Science 330(6005):841-845. Nandi, S., Gokhan,S., Dai, X-M., Wei, S., Enikolopov, G., Lin,H., Mehler, M.F. and Stanley E.R. (2012). The CSF-1 receptor ligands IL-34 and CSF-1 exhibit distinct developmental brain expression patterns and regulate neural progenitor cell maintenance and maturation. Developmental Biology 367:100-13. PMID: 22542597. Nandi, S., Cioce, M., Yeung, Y.G., Nieves, E., Tesfa, L., Lin, H., Hsu, A.W., Halenbeck R., Cheng, H.Y., Gokhan, S., Mehler, M.F., Stanley, E.R.(2013).Receptor-type protein tyrosine phosphatase zeta is a functional receptor for interleukin-34. J. Biol. Chem., 288:21972-86. PMID: 23744080 Wang, Y., Yeung, Y.G., Langdon, W.Y. and Stanley, E.R. (1996) c-Cbl Is Transiently Tyrosine phosphorylated, Ubiquitinated, and Membrane-targeted following CSF-1 Stimulation of Macrophages. J. Biol. Chem. 271:17-20. Lee, P.S.W., Wang, Y., Dominguez, M.G., Yeung, Y-G., Murphy, M.A. Bowtell, D.D.L. and Stanley, E.R. (1999) The Cbl protooncoprotein stimulates CSF-1 receptor multiubiquitination and endocytosis, and attenuates macrophage proliferation. EMBO J. 18:3616-3628.
Yeung, Y.G., Soldera, S. and Stanley, E.R. (1998) A novel macrophage actin-associated protein (MAYP) is tyrosine phosphorylated following CSF-1 stimulation. J. Biol. Chem. 273:30638 30642. Chitu, V., Pixley, F.J., Yeung, Y.G., Macaluso, F., Condeelis, J and Stanley, E.R. (2005). The PCH family member MAYP/PSTPIP2 directly regulates F-actin bundling and enhances filopodia formation and motility in macrophages. Mol. Biol. Cell. 16(6):2947-2959. Grosse, J., Chitu, V., Marquardt, A., Hanke, P., Schmittwolf, C., Zeitlmann, L., Schropp, P., Barth, B., Yu, P., Paffenholz, R., Stumm, G., Nehls M., and Stanley E. R. (2006). Mutation of mouse MAYP/PSTPIP2 causes a macrophage autoinflammatory disease. Blood 107(8):3350-3358. Chitu, V. and Stanley E. R. (2006). Colony stimulating factor-1 in immunity and inflammation. Current Opinion in Immunology 18(1):39-48. Chitu, V. and Stanley, E.R. (2007). Pombe Cdc15 homology (PCH) proteins: coordinators of membrane-cytoskeletal interactions. Trends in Cell Biol. 17(3):145-156. Chitu V., Nacu, V., Charles, J.F., Henne, W.M., McMahon, H.T., Nandi, S., Ketchum, H., Harris, R., Nakamura, M.C., Stanley, E.R. (2012) PSTPIP2 deficiency in mice causes osteopenia and increased differentiation of multipotent myeloid precursors into osteoclasts. Blood, in press. Pixley, F.J., Lee, P.S.W., Condeelis, J. and Stanley, E.R. (2001) Protein tyrosine phosphatase phi regulates paxillin tyrosine phosphorylation and mediates Colony Stimulating Factor 1 induced morphological changes in macrophages. Mol. Cell. Biol. 21:1795-1809. Wyckoff, J., Wang, W., Lin, E.L., Wang, Y., Pixley, F.J., Stanley, E.R., Graf, T., Pollard, J.W., Segall, J. and Condeelis, J. (2004). A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res. 64(19):7022-7029. Patsialou, A., Wyckoff, J., Wang, Y., Goswami, S., Stanley, E.R. and Condeelis J.S. (2009). Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony stimulating factor-1 receptor. Cancer Research 69(24):9498-9506. Pixley, F.J. and Stanley, E.R. (2004) CSF-1 Regulation of the Wandering Macrophage: Complexity in action. Trends in Cell Biol. 14:628-638. Xiong, Y., Song, D., Cai, Y., Yu, W., Yeung, Y.G., and Stanley, E.R. (2010). A CSF-1 receptor phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation. J. Biol. Chem. 286(2):952-960. Sampaio, N., Yu, W., Cox, D., Wyckoff, J., Condeelis, J., Stanley, E.R. and Pixley, F.J. (2011). Phosphorylation of Y721 of the CSF-1R mediates PI3K association to regulate macrophage motility and enhancement of tumor cell invasion. J. Cell Sci. 124(Pt 12):2021-2031. Yu, W., Chen, J., X. Ying, Pixley, F.J., Yeung, Y.G., and Stanley, E.R. (2012). Macrophage proliferation is regulated through CSF-1 receptor tyrosines 544, 559 and 807. J. Biol.Chem. 287:13694-704. PMID: 22375015. Fernandez, R., Takahashi, F., Liu, Z., Steward, R., Stein, D. and Stanley, E.R. (2000) The Drosophila Shark tyrosine kinase is required for embryonic dorsal closure. Genes and Development 14:604-614.
Biswas, R., Stein, D. and Stanley, E.R (2006). Drosophila Dok is required for embryonic dorsal closure. Development 133(2):217-227. Zeigenfuss, J.S.*, Biswas, R.*, Avery, M.A., Sheehan, A.E., Hong, K., Yeung Y-G., Stanley, E.R.** and Freeman, M.R.** (2008). Draper-dependent glial phagocytic activity is mediated by Src and Syk family kinase signaling. Nature 453(7197):935-939.
DR. AMIT K. VERMA Department of Developmental and Molecular Biology Chanin Building – Room 302B (718) 430-8761; [email protected]
Key Words: cytokines, hematopoiesis, epigenetics, mds, leukemia, esophageal cancer, pancreatic cancer
1. Targeting signal transduction in hematologic malignancies: Cytokines play important roles in the regulation of normal hematopoiesis and a balance between the actions of hematopoietic growth factors and myelosuppressive factors is required for optimal production of different hematopoietic cell lineages. We study the role of MAP kinases in the regulation of hematopoiesis and have shown that the p38 MAPK signaling pathway is the dominant cytokine regulated inhibitory pathway in human hematopoiesis and is overactivated in MDS. The myelodysplastic syndromes (MDS) are collections of heterogeneous hematologic diseases characterized by refractory cytopenias due to ineffective hematopoiesis. These preleukemic disorders are common causes of anemia in the elderly and are rapidly increasing in incidence. We have also demonstrated that the TGF-b pathways is overactivated in MDS. We are trying to study the molecular mechanisms that lead to the activation of these pathways in MDS and are using small molecule inhibitors in mouse models to target these pathways.
2. Epigenomic analysis of tumors: We are using high throughput assays to analyze DNA methylation across the genome and have optimized these assays to work with low amounts of DNA. We have successfully used these assays in conducting an integrative analysis of epigenetic and genetic alterations in MDS and esophageal cancer. We are now exploring alterations in DNA methylation in pancreatic cancer, myeloproliferative neoplasms and renal cell cancer.
3. Clinical studies in Myelodysplastic syndromes: We have a “center of excellence” clinic for patients with Myelodysplatic syndromes. We offer a variety of clinical trials for these patients (www.mdstreatment.com).
Selected References: 1. Zhou L, Opalinska J, Sohal D, Yu Y, Mo Y, Bhagat T, Abdel-Wahab O, Fazzari M, Figueroa M, Alencar C,
Zhang J, Kanbhampati S, Parmar S, Nischal S, Heuck C, Suzuki M, Friedman E, Pellagatti A, Boultwood J, Steidl U, Sauthararajah Y, Yajnik V, Gore SD, Platanias LC, Levine R, Melnick A, Greally JM, Verma A. Aberrant epigenetic and genetic marks are seen in myelodysplastic leucocytes and reveal DOCK4 as a candidate pathogenic gene on chr7q. J Biol Chem. 2011 Apr 30. PMID: 21532034
2. Alvarez H, Opalinska J, Zhou L, Sohal D, Fazzari M, Yu Y, Montagna C, Montgomery E, Canto M, Dunbar K, Wang J, Roa J, Mo Y, Bhagat T, Ramesh K, Cannizzaro L, Mollenhauer J, Thompson R, Suzuki M, Meltzer S, Melnick A, Greally JM, Maitra A, Verma A Widespread hypomethylation occurs early and synergizes with gene amplification during esophageal carcinogenesis. PLOS Genetics 2011 Mar;7(3) PMID: 21483804
4. L Zhou, C McMahon, T Bhagat, C Alencar, Y Yu, M Fazzari, D Sohal, C Heuck, K Gundabolu, C Ng, Y Mo, W Shen, A Wickrema, G Kong, E Friedman, L Sokol, G Mantzaris, A Pellagatti , J Boultwood, LC. Platanias. U Steidl, L Yan, JM Yingling, MM Lahn, A List, M Bitzer and A Verma Reduced SMAD7 leads to overactivation of TGF-beta signaling in MDS that can be reversed by a specific inhibitor of TGF-beta receptor I kinase. Cancer Research 2011 Feb 1;71(3):955-63.PMID: 21189329
5. Sohal D, Yeatts A, Ye K, Pellagatti A, Zhou L, Pahanish P, Mo Y, Bhagat T, Mariadason J, Boultwood J, Melnick A, Greally JM, Verma A. Meta-analysis of microarray studies reveals a novel hematopoietic progenitor cell signature and demonstrates feasibility of inter-platform data integration. PLOS One 2008 Aug 13;3(8):e2965 PMID: 18698424
6. Zhou L, Nguyen AN, Sohal D, Ma JY, Pahanish P, Gundabolu K, Hayman J, Chubak A, Mo Y, Bhagat T, Das B, Haghnazari E, Navas T, Parmar S, Kambhampati S, Pellagati A, Braunchweig I, Zhang YE, Wickrema A, Boultwood J, Platanias LC, Higgins LS, List A, Bitzer M, Verma A. Inhibition of Transforming Growth Factor beta receptor I kinase can stimulate hematopoiesis in MDS Blood 2008 Oct 15;112(8):3434-43 PMID: 18474728
7. Navas T, Mohindru M, Estes M, Ma JY, Sokol L, Pahanish P, Parmar S, Haghnazari E, Zhou L, Collins RC, Kerr I, Nguyen A, Xu Y, Platanias LC, List AA, Higgins LS, Verma A. Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors Blood 2006, 15;108(13):4170-7 PMID: 16940419
!
DR. MARGARITA VIGODNER Department of Developmental and Molecular Biology Chanin Building – Room 501 (718) 430-4001; [email protected]
Understanding the role of sumoylation during spermatogenesis Post-translational modification by Small Ubiquitin-like Modifiers or SUMO proteins has been identified as an important regulatory event that is implicated in several cellular processes, seemingly based on the cell type. Recent findings from our laboratory suggest diverse and potentially multiple roles of SUMO in testicular function and spermatogenesis however, SUMO targets remained uncharacterized in the testis due to the complex multicellular nature of testicular tissue, the inability to maintain and manipulate spermatogenesis in vitro, and the technical challenges involved in identifying low-abundance endogenous SUMO targets. My laboratory performed cell-specific identification of sumoylated proteins using concentrated cell lysates prepared with de-sumoylation inhibitors from germ cells and sperm. Hundred and twenty proteins were uniquely identified in specific germ cell fractions. The identified proteins are involved in the regulation of transcription, stress response, microRNA biogenesis, regulation of major enzymatic pathways, nuclear-cytoplasmic transport, cell cycle control, acrosome biogenesis, and other processes. Several proteins with important roles during spermatogenesis were chosen for further characterization by co-immunoprecipitation, co-localization and in-vitro sumoylation studies. Many of these proteins have a unique role in spermatogenesis, particularly during meiotic progression. Interestingly, several kinases have been identified as sumoylated targets. This research opens a novel avenue for numerous further studies of SUMO at the level of individual targets. Studies are currently underway to understand how sumoylation affects activity of several targets, in particular the activity of kinases and phosphatases important for spermatogenesis. Characterization of new regulators of WNT signaling in lung diseases Wnt signaling is required for airway epithelial differentiation and healing. A decrease in Wnt target genes and an increase in Wnt inhibitors was reported in bronchial epithelial cells of smokers and COPD (Chronic obstructive pulmonary disease) patients, and in experimental mouse models of emphysema. Notably, preventive and therapeutic WNT/!-catenin activation led to a significant reduction of experimental emphysema. Therefore, targeting the Wnt pathway is a promising approach to the development of therapies for lung diseases; however, the question of where and how to target Wnt pathway remains open. CDK14 was identified by our group as a gene significantly down-regulated by tobacco exposure in vivo in testes ands lungs. It has recently been discovered that Wnt signaling is activated by Cdk14/ cyclin Y complex through phosphorylation of LRP6 co-receptor, a key regulator of the WNT pathway. We have recently shown that CDK14 is expressed in bronchial and alveolar epithelium and that treatment of lung cell lines including primary normal human bronchial cells (NHBE) with CS decreased the level of CDK14; COPD-derived human bronchial cell (DHBE) showed reduced CDK14 levels versus NHBE. Furthermore, preliminary observations suggest a lower number of CDK14 positive cells in emphysema versus normal lungs. The research in our laboratory currently focuses on 1) performing a screening and quantitative analysis of COPD samples as well as smokers versus non-smokers for the levels of CDK14/ Cyc Y and Wnt pathway activity; 2).Testing whether down-regulation of CDK14 and/or CycY gene expression inhibits Wnt signaling in NHBE and small airway epithelial cells (SAEC), and whether restoration of the normal level of CDK14/CycY in COPD-derived cells would activate Wnt signaling.3)
comparative analysis of quantitative changes in phosphoproteome of NHBE before and after the down-regulation of CDK14 and/or CycY in order to identify CDK14 /CycY downstream targets and molecular pathways. The results from this research will lead to a better understanding of the molecular pathology of COPD in order to use the WNT pathway as a diagnostic and therapeutic target for lung diseases.
Lab homepage: http://einstein.yu.edu/labs/margarita-vigodner/research-projects/
Selected Publications
• Vigodner, M., Shrivastava V, Gutstein LE, *Schneider J, Nieves E, Goldstein M, Feliciano M, Callaway M., Localization and identification of sumoylated proteins in human sperm: excessive sumoylation is a marker of defective spermatozoa. Hum Reprod, 2013. 28(1): p. 210-23. PMID:23077236.
• Xiao Y, Pollack D, Nieves E, Winchell A, Callaway M, Vigodner M. Can your protein be sumoylated? A quick summary and important tips to study SUMO-modified proteins. Anal Biochem. 2014 Nov 29. pii: S0003-2697(14)00527-2. PMID:25454506.
• Pollack D, XiaoY, Shrivasatava V, Levy A, Andrusier M, Jeanine D'Armiento J, Holz MK, and Vigodner M.; CDK14 expression is down-regulated by cigarette smoke in vivo and in vitro, Toxicology Letters, 2015; 234 :120-30. PMID: 25680692
!
DR. DUNCAN W. WILSON Department of Developmental and Molecular Biology Chanin Building – Room 513 (718) 430-2305; [email protected]
Viruses, cancer and the nervous system
Humans are infected by at least eight different species of herpes viruses. These pathogens cause several forms of cancer, severe birth defects or miscarriage, and are a leading cause of organ transplant rejection. Our interests are focused on the Kaposi’s sarcoma herpes virus (KSHV) and Herpes Simplex Virus (HSV). KSHV: a cancer-causing virus of humans Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of AIDS-associated and endemic Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease. In all these cancers the KSHV-encoded protein K15p plays a pivotal role. In KSHV-infected B cells, the principal reservoir of the virus in infected individuals, K15p drives cell proliferation and B cell-transformation to primary effusion lymphomas. K15p then keeps these lymphoma cells alive by “pretending” to be an activated B-cell receptor (BCR), ensuring continued cell-proliferation and suppressing apoptosis. In Kaposi’s sarcoma (the archetypal malignancy of AIDS patients) K15p adopts a different disguise; this time impersonating an activated vascular endothelial growth factor receptor (VEGF-R) in KSHV-infected endothelial cells. This drives angiogenesis and results in the extensive vascularization that is characteristic of KS tumors. We are studying the unusual and dramatic mechanism by which K15p is processed to its active receptor-mimicking form (Fig. 1).
Fig. 1. K15p: the “Great Pretender” of Kaposi’s sarcoma virus. K15p is initially synthesized as a ~45kDa protein with twelve membrane-spanning domains and a C-terminal cytoplasmic tail that recruits cellular signaling molecules (orange ovals). Lipid bilayer is in pale green. An unknown processing mechanism, the focus of our interests, cleaves the carboxy-terminal region of the molecule (in blue) from the rest of the molecule (in green) to generate an active mimic of the B-cell and VEGF-Receptors (see text).
HSV: a pathogen of the nervous system Herpes Simplex Virus (HSV) is a leading cause of blindness, fetal mortality and severe neurodevelopmental and other birth defects. These diseases are a direct result of the ability of the virus to invade, manipulate and traffic within the human nervous system. Our laboratory is dissecting the molecular machinery that HSV uses to achieve assembly and transport within neurons (Fig. 2) (1, 2).
Fig. 2. HSV makes a break for it. Left hand image: HSV initially assembles clusters of capsids in the neuronal cell nucleus. These then fill with viral DNA, visible as a dense core (arrow at right). They then traffic to, and bud into, the nuclear membranes (left hand arrow) to enter the cytoplasm and neuronal axons. Right hand image: Similar to left hand but at higher magnification, showing a DNA-containing HSV capsid punching through the nuclear membranes. Once in the cell cytoplasm HSV capsids dock with, and become enveloped by, cytoplasmic organelles to assemble the mature infectious virus. These then traffic along neuronal microtubules to travel within the nervous system. The events of virus assembly, and the detailed molecular structure of assembly and trafficking intermediates are very poorly understood. Most recently, in collaboration with the analytical imaging facility (AIF) here at the Albert Einstein College of Medicine we have pioneered the application of Correlative Light and Electron Microscopy to the study of HSV assembly in the neuronal cell body (Fig. 3) (3).
Fig. 3. What to do when more light doesn’t help you see better. Correlative light and electron microscopy makes it possible to observe HSV assembly simultaneously by fluorescence microscopy and electron microscopy. Rows A, B and C represent three different examples of HSV capsids assembling onto cellular organelles in the cytoplasm of infected neurons. In each row a light-microscopic fluorescence image is shown in the left hand panel, an electron-microscopic image of the same field is in the right hand panel and an alignment of the two is in the middle panel. Green light is being emitted by HSV capsids engineered to contain molecules of Green Fluorescent Protein (GFP). Red light is coming from the membranes of cellular organelles that have been stained by a fluorescent lipophilic dye. Yellow shows where Green and Red light overlap. Until the application of this technology our understanding of HSV assembly was limited to the level of resolution shown in the leftmost panels. Scale bars: 500nM.
Selected References
1. Kharkwal H., Smith C.G., Wilson D.W. (2014). Blocking ESCRT-mediated envelopment inhibits
microtubule-dependent trafficking of alphaherpesviruses in vitro. Journal of Virology 88:14467-14478. 2. Shanda S.K., Wilson D.W. (2008). UL36p is required for efficient transport of membrane-associated
herpes simplex virus type 1 along microtubules. Journal of Virology 82:7388-7394. 3. Kharkwal H., Shanda S.K., Wilson D.W. (2015). HSV capsid/organelle association in the absence of
the tegument protein UL36p. Journal of Virology. Manuscript Submitted.
DR. FAJUN YANG Department of Developmental & Molecular Biology Price Center - Room 377 (718) 678-1142; [email protected]
Key Words: gene expression, lipid metabolism, metabolic syndrome, obesity Dysregulation of lipid homeostasis is common in major human diseases, including type 2 diabetes, obesity, fatty liver diseases, atherosclerosis and some types of cancer. Our laboratory is interested in the transcriptional control of lipid metabolism. In addition to DNA-binding transcription factors, gene expression in eukaryotic cells often requires a series of transcriptional cofactors. We use biochemical and genetic approaches to study the physiological and pathophysiological functions and regulation of the network of transcription factors and their cofactors in the liver (hepatocytes) and adipose tissues (white, brown and beige adipocytes). A focus of our work is the multi-subunit Mediator complex, which is a highly conserved transcriptional cofactor. The mammalian Mediator is one of the cofactors for the sterol regulatory element -binding proteins (SREBP), the master regulators of both fatty acid/triglyceride and cholesterol biosynthesis. SREBP proteins are synthesized as inactive precursors that are tethered to the endoplasmic reticulum membrane. When lipid biosynthesis is demanded, SREBP precursors are proteolytically processed to mature forms of transcription factors that migrate into the nucleus, interact with cofactors including the Mediator complex, and activate transcription of target genes, which encode the rate-limiting enzymes in synthesizing fatty acids/triglycerides and cholesterol. Through the protein-protein interactions of different subunits with SREBP as well as other transcription factors, the Mediator complex critically regulates lipid metabolism. Moreover, dynamic remodeling of the Mediator complex conveys the metabolic signals to transcriptional outputs, providing another layer of regulation of gene expression. With the ultimate goal of identifying novel targets for preventing or treating metabolic syndrome (including type 2 diabetes, obesity, fatty liver disease, hyperlipidemia and hypertension), our research will advance our understanding of how lipid homeostasis is regulated at the molecular level.
Selected Publications:
Abdulla A, Zhang Y, Hsu FN, Xiaoli AM, Zhao X, Yang ES, Ji JY, Yang F. Regulation of lipogenic gene expression by lysine-specific histone demethylase-1 (LSD1). J Biol Chem. 2014, 289(43): 29937-47. Zhao X, Feng D, Wang Q, Abdulla A, Xie XJ, Zhou J, Sun Y, Yang ES, Liu LP, Vaitheesvaran B, Bridges L, Kurland IJ, Strich R, Ni JQ, Wang C, Ericsson J, Pessin JE, Ji JY, Yang F. Regulation of lipogenesis by cyclin-dependent kinase 8-mediated control of SREBP-1. J Clin Invest. 2012, 122(7): 2417-27. Mulligan P, Yang F, Di Stefano L, Ji JY, Ouyang J, Nishikawa JL, Toiber D, Kulkarni M, Wang Q, Najafi-Shoushtari SH, Mostoslavsky R, Gygi SP, Gill G, Dyson NJ, Näär AM. A SIRT1-LSD1 Corepressor Complex Regulates Notch Target Gene Expression and Development. Mol Cell. 2011, 42(5): 689-699. Walker AK, Yang F, Jiang K, Ji JY, Watts JL, Purushotham A, Boss O, Hirsch ML, Ribich S, Smith JJ, Israelian K, Westphal CH, Rodgers JT, Shioda T, Elson SL, Mulligan P, Najafi-Shoushtari H, Black JC, Thakur JK, Kadyk LC, Whetstine JR, Mostoslavsky R, Puigserver P, Li X, Dyson NJ, Hart AC, Näär AM. Conserved role of SIRT1 orthologs in fasting-dependent inhibition of the lipid/cholesterol regulator SREBP. Genes & Development, 2010, 24(13): 1403-1417. Morris EJ!, Ji JY!, Yang F, Di Stefano L, Herr A, Moon N, Kwon E, Haigis KM, Näär AM, Dyson NJ. E2F1 represses b-catenin transcription and is antagonized by both pRB and CDK8. Nature, 2008, 455(7212): 552-556. (!Equal contribution) Thakur JK!, Arthanari H!, Yang F!, Pan SJ, Fan X, Breger J, Frueh DP, Gulshan K, Li DK, Mylonalis E, Struhl K, Moye-Rowley WS, Cormack BP, Wagner G, Näär AM. A nuclear receptor-like pathway regulating multidrug resistance in fungi. Nature (Article), 2008, 452(7187): 604-609. (!Equal contribution) Yang F!, Vought BW!, Satterlee JS, Walker AK, Sun ZY, Watts JL, DeBeaumont R, Saito RM, Hyberts SG, Yang S, Macol C, Lyer L, Tjian R, van den Heuvel S, Hart AC, Wagner G, Näär AM. An ARC/Mediator subunit required for SREBP control of cholesterol and lipid homeostasis. Nature, 2006, 442(7103): 700-7004. (!Equal contribution)
DR. LIANG ZHU Department of Developmental and Molecular Biology Ullmann Building – Room 521 (718) 430-3320; [email protected]
Key Words: tumor suppressor, cell cycle, cell death, cancer, inhibiting cancer, metabolism and obesity, mouse models
CONTROL OF TUMORIGENESIS, OBESITY, AND REGENERATION BY TUMOR SUPPRESSORS AND ONCOPROTEINS
A common feature shared by cancer, obesity, and tissue regeneration is deregulation of cellular homeostasis, including cell proliferation, cell growth, cell metabolism, and cell death. Tumor suppressors such as pRb and p53, oncoproteins such as Ras and Myc, and organ size regulators such as YAP, are the major regulators of these aspects of cellular homeostasis. We are studying how these regulators regulate these aspects.
In the cancer field, we are identifying treatment strategies for tumors that have permanently lost the tumor suppressor pRb or both pRb and p53 using mouse tumor models. pRb and p53 are the two major tumor suppressors. Their functions are activated by oncogenic events and they implement most and best cells’ antitumor mechanisms to safeguard against tumorigenesis. When tumorigenesis succeeds, pRb, p53, or both is frequently inactivated. In reverse, reactivation of pRb and p53 are rationale for most antitumor therapeutics. However, when pRb and p53 are genetically inactivated, cells irreparably lose the antitumor mechanisms afforded by them, which may explain why advanced cancers are difficult to treat. We generated mouse tumor models in which the genes for pRb or both pRb and p53 are knocked out to identify mechanisms that can still inhibit these tumors. We showed that combining deletion of Skp2, which is a target of repression by pRb, completely blocked tumorigenesis in the absence of pRb or both pRb and p53 (refs 2, 5, 7, 8). Ongoing studies aim to target functions of Skp2 in regulating p27 degradation, regulating cancer cell metabolism, and regulating epithelial-to-mesenchymal transition to inhibit pRb and p53 doubly deficient tumors (ref 9).
In the obesity field, we are studying the function of pRb in hypothalamus POMC neurons. These neurons negatively regulate feeding, and it has been suggested that high fat feeding could damage these neurons, which results in more desire to eat. This feed-forward circle may explain why obesity is difficult to treat. We determined that high fat feeding induced or activated a kinase in POMC neurons to phosphorylate and, therefore, functionally inactivate pRb. Interestingly, in this context, inactivation of pRb did not induce tumorigenesis. On the contrary, it induced degenerative changes in POMC neurons. When we deleted pRb in POMC neurons, the mice increased their food intake and become obese, demonstrating that pRb functions to maintain POMC neuron homeostasis to suppress obesity. To our surprise, in AGRP neurons, which reside together with POMC neurons in hypothalamus and functionally antagonize POMC neurons to reduce the desire to feed, pRb function is not important. Deletion of pRb in AGRP neurons did not harm AGRP neuron homeostasis. Based on these findings (ref 6, in collaboration of with Dr. Chua of the Einstein Diabetes Center), we are identifying the kinases that phosphorylate pRb in POMC neurons after high fat feeding, and establishing techniques to prevent pRb phosphorylation following high fat feeding. Through these studies, we aim to block the feed-forward circle to treat obesity.
The liver has remarkable ability to regenerate when more than 70% of it is resected. This regenerative capability has important implication to tumorigenesis and regenerative medicine. Since the supply of donor liver is far smaller than the demand, hepatocyte transplantation is the best approach to treating liver failure and liver defects. We showed that by deleting the cyclin-dependent kinase inhibitor p27, hepatocytes were stimulated to proliferate more in the host liver to save mice from liver failure (ref 1). Unfortunately, deleting p27 also increased liver cancer burden during liver carcinogenesis by chronic HBV or chemical DEN (ref 3, 4). In collaboration with Dr. Shafritz of the Einstein Liver Center, we are now studying the liver size regulator YAP to determine its ability to increase hepatocyte proliferation following transplantation and its associated liver cancer risk.
Selected References: 1. Karnezis, A.N., Dorokhov, M., Grompe, M. and Zhu, L. (2001) Loss of p27Kip1 enhances the
transplantation efficiency of hepatocytes transferred into diseased livers. J Clin Invest. 108:383-390.
2. Ji, P., Jiang, H., Rekhtman, K., Bloom, J., Ichetovkin, M., Pagano, M., and Zhu, L. (2004) An Rb-Skp2-p27 pathway mediates acute cell cycle inhibition by Rb and is retained in a partial penetrance Rb mutant. Mol Cell. 16:47-58.
3. Sun, D., Ren, H., Oertel, M., Sellers, R.S., and Zhu, L. (2007) Loss of p27Kip1 enhances tumor progression in chronic hepatocyte injury-induced liver tumorigenesis with widely ranging effects on Cdk2 or Cdc2 activation. Carcinogenesis. 28:1859-1866.
4. Sun, D., Ren, H., Oertel, M., Sellers, R.S., Shafritz, D.A., and Zhu, L. (2008) Inactivation of p27Kip1 promotes chemical mouse liver tumorigenesis in the resistant strain C57BL/6J. Mol Carcinog. 47:47-55.
5. Wang, H., Bauzon, F., Ji, P., Xu, X., Sun, D., Locker, J., Sellers, R.S., Nakayama, K., Nakayama, K.I., Cobrinik, D., and Zhu, L. (2010) Skp2 is required for survival of aberrantly proliferating Rb1-deficient
cells and for tumorigenesis in Rb1+/-
mice. Nat Genet. 42:83-887.
6. Lu, Z., Marcelin, G., Bauzon, F., Wang, H., Fu, H., Dun, S.L., Zhao, H., Li, X., Jo, Y.H., Wardlaw, S., Dun, N., Chua, S. Jr., and Zhu, L. (2013) pRb is an obesity suppressor in hypothalamus and high-fat diet inhibits pRb in this location. EMBO J. 32:844-857.
7. Zhao, H., Bauzon, F., Fu, H., Lu, Z., Cui, J., Nakayama, K., Nakayama, K.I., Locker, J., and Zhu, L.
(2013) Skp2 deletion unmasks a p27 safeguard that blocks tumorigenesis in the absence of pRb and p53 tumor suppressors. Cancer Cell. 24:645-659.
8. Lu, Z., Bauzon, F., Fu, H., Cui, J., Zhao, H., and Zhu, L. (2014) Skp2 suppresses apoptosis in Rb1
deficient tumors by limiting E2F1 activity. Nat Commun. 5:3463.
9. Zhu, L., Lu, Z., and Zhao, H. (2014) Antitumor mechanisms when pRb and p53 are genetically inactivated. Oncogene. in press