Harvey F. Lodish - US grants

We plan to characterize revertants of VSV ts mutants in the G gene, and determine the nature of the mutated amino acid. We will attempt to determine whether secretory and membrane glycoproteins follow the same intracellular route in maturation from the rough endoplasmic reticulum to the Golgi and thence to the surface. Also we will determine whether all VSV strains, in a number of cell types, inhibit synthesis of cell proteins by the same mechanism.

Most of our projects focus on the function of segments of the two polypeptides that constitute the human hepatic asialoglycoprotein receptor. They are very similar yet exhibit different properties in transfected cells. We will determine the cell-surface distribution of the receptor polypeptides with respect to coated pits, and determine whether ligand or antibody triggers receptor accumulation in coated pits and endocytosis. We showed that when one receptor polypeptide, H2, is expressed without the other, H1, in 3T3 cells, it is synthesized but rapidly degraded. We want to determine whether H2 is degraded in lysosomes, and whether it gets there from the ER without traversing the Golgi. We will make several deletion mutants of H1 and H2, and also chimeras of H1 and H2: by expressing these in 3T3 dells by themselves, or together with normal H1 or H2, we should define segments important in ligand binding, low pH-induced ligand dissociation, endocytosis, transport from ER to Golgi, and rapid degradation. Then we hope to use peptides corresponding to segments of the wild type and mutant proteins to search for cell proteins that bind specifically to that functional segment, and that might be a "transport receptor" or "sorting protein." Other studies focus on regulated transport or newly made hepatic secretory proteins from the ER to the Golgi, emphasizing retinol binding protein and alpha1 antitrypsin. Using 3T3 cells expressing wild type or mutant proteins we want to define the requirements for exit from the ER. We want to identify the putative ER-to-Golgi transport receptor by virtue of its specific binding to the holo, not the apo- form of RBP, and to the wild type and not mutant forms of these proteins. By making and expressing chimeras of RBP and alpha1-antitrypsin, and studying the requirements for their maturation from the ER, we hope to determine whether retention of unfolded proteins in the ER, or active export of folded proteins to the Golgi, is employed.

A major focus of our studies will be the regulation of transcription and of stability of mRNAs specific for prespore and prestalk cells, as well as mRNAs synthesized throughout growth and differentiation. These studies will include mapping and sequencing of genomic DNA clones; in vitro studies of regulated mRNA synthesis and destruction; in situ detection of mRNAs; and determination of the commitment of prespore and prestalk cells. We will attempt to elucidate the intracellular "second messenger(s)" that mediate the effect of the extracellular hormone, cAMP, on synthesis and stability of regulated mRNAs; and to detect specific cell surface proteins that are required for induction of gene expression in multicellular aggregates. Finally, we shall study the structure and regulation of transcription and mRNA processing of a class of repetitive DNA sequences that is expressed as mRNA only during differentiation of heat shock.

The Gordon Conference on the Red Cell will provide an informal forum for current advances and directions in this rapidly developing field. Sessions will be devoted to the structure and assembly of the erythrocyte cytoskeleton and membrane; the erythrocytic stage of the malaria parasite; erythropoiesis, especially the role of oncogenes and protein factors; the regulation of globin and non-globin gene expression; and manipulation of erythrocyte-specific gene expression and gene transfer. These topics are closely interrelated yet involve such diverse disciplines that investigators would benefit greatly from this meeting.

Mammalian tissues, including those affected by Cystic Fibrosis, contain several mRNAs that are homologous to the segment of Band 3 mRNA encoding the membrane-spanning (anion exchange) domain. We propose to clone and sequence a number of these mRNAs that we presume encode anion transport proteins. By expression of functional mRNAs in oocytes, we will determine the type(s) of channel activity of these encoded proteins: anion exchanger, symport or channel. In parallel, we will prepare antibodies against defined segments of the derived proteins, determine by immunocytochemistry their tissue and cellular localization, and test the ability of these antibodies to block anion channels, symports, and exchangers from various tissues. We will also obtain and characterize the genomic segments encoding these presumed channel proteins. Using cloned genomic DNAs or cDNAs we will search for abnormalities in mRNA levels in tissues from Cystic Fibrosis patients. These clones will also be provided to collaborators who will search for major gene alterations and for restriction fragment length polymorphisms in genomic DNA from Cystic Fibrosis family cohorts and from normal controls. In parallel, we will continue to explore aspects of the structure of the one anion transport protein - erythrocyte Band 3 - whose sequence is known. Using in vitro mutagenesis, peptide-specific antisera and immunocytochemistry, and proteolytic digestions, we will determine the membrane topology of the protein. In vitro mutagenesis of a few selected amino acids, such as the DIDS-binding lysine, together with expression of the cloned cDNAs in oocytes or, using retroviral vectors, in 3T3 cells should define the function of several amino acids in anion exchange.

The Gordon Conference on Molecular Membrane Biology will provide an informa forum for current advances and directions in this rapidly developing field. Sessions will be devoted to the structure of membrane proteins; translocati n of proteins into and across membranes; sorting of proteins in the secretory pathway; interaction of membrane proteins with the cytoskeleton and mobilit of organelles; cell polarity; transmembrane signalling by cell surface receptors; structure and function of membrane lipids; receptors and endocytosis; and organelle structure and biosynthesis. These topics are closely interrelated yet involve such diverse disciplines that investigator would benefit greatly from this meeting. We will devote one session to abo t six 15 minute talks on exciting new developments, presented by young applicants to the meeting who submit abstracts to the chairman. This, together with two extensive poster sessions, should allow a maximum amount of dissemination of new data, and should allow workers to make important contacts with those in complementary disciplines. We request that this application be reviewed by the Molecular Cytology Stud Section, a group with special expertise in this area. We also would like this proposal considered for partial funding by several NIH institutes additional to NIGMS. The involvement of membrane proteins in disease and i novel therapeutic approaches will be a significant overall part of the meeting. For instance we will discuss the assembly of the influenza virus hemagluttinin and entry of icosahedral viruses into cells, subjects directl relevant to infectious diseases. A discussion of abnormalities in the insulin receptor in certain patients with type II diabetes will be part of the session on cell surface receptors, and new results on excitation- contraction coupling in striated muscle will be part of the organelle structure session. Receptors for growth facts and for matrix components wi l be discussed in some detail, and these have well-known involvement in aspec s of cell transformation and oncogenesis.

Our goal is to define by molecular cloning facilitated diffusion glucose transport proteins in the plasma membrane of several rat cells - adipocytes, striated muscle, hepatocytes, kidney, beta- cells of pancreatic islets, and intestine. Our molecular studies have shown that facilitated diffusion glucose transport proteins comprise a multi-gene family with 4-6 members; we have cloned, sequenced, and characterized two such cDNAs. We will attempt to clone all members of this multigene family by probing lambda gt11 expression cDNA libraries and genomic libraries by hybridization at several stringencies with existing clones, and by use of antibody probes. After cloning and sequencing, we will characterize the physiological importance of these putative transport proteins. Each cDNA will be functionally expressed in a suitable cell, initially in Escherichia coli and murine 3T3 fibroblasts. We will characterize the transport activity of the cloned proteins with respect to sugar specificity, KM ad pharmacological sensitivity. We want to determine the exact type of cell in the issue, and the type of membrane within the cell, that contains each of these transport proteins. We shall generate polyclonal antisera to chemically synthesized peptides that correspond to unique segments of the various glucose transporters. These antisera will be used to detect the presence of the corresponding protein by immunolight- and electron- microscopy and by Western blotting. Subcellular fractionation of adipocytes will also be employed to resolve plasma membrane from microsomes, as will biochemical assays to detect transporters on the cell surface. We want to determine the subcellular localization of all transporters in adipose and muscle cells, any translocation that might be induced by insulin, and also covalent modification of all transporters expressed in these tissues. We will also determine: the identity of the transporter(s) in the membrane of the islet cell that might allow the cells to respond to sense changes in extracellular glucose; and the transporter(s) in the hepatocyte membrane of the islet cell that might allow the cells to respond to sense changes in extracellular glucose;and the transporter(s)in the hepatocyte membrane that transports glucose into or oust of the cell. A long term aim is to understand defects in diabetes relating to sensing of glucose levels by beta cells, and to insulin-stimulated glucose uptake in adipocytes and muscle.

We have used an expression cloning strategy to isolate the cDNAs encoding the types III and II cell-surface receptors for transforming growth factor beta (TGF-beta). One major goal is to clone the multiple type I THP-1 cells, IEC-18 ras7 cells, and in HL-60 cells. Using the derived amino acid sequences of the types II and III receptors, we have generated anti-peptide sera that are specific for the encoded proteins. These reagents have allowed us to determine that the types of cell-surface TGF- beta binding-proteins expressed on a variety of human and animal tumors and cell lines that are or are not responsive to growth inhibition by TGF-beta is far more than previously appreciated. Thus a second major aim is to molecularly clone and characterize many of the other cell- surface TGF-beta receptors we and others have identified, and to study in detail their abilities to affect cell proliferation and gene expression. These include TGF-beta binding proteins attached to the plasma membrane through a glycosylphosphatidylinositol anchor, the major 70-74 kDa cell surface TGF-beta receptor in GH3 pituitary cells that also binds activin and inhibin, the major 38-kDa cell surface protein on several cells that binds TGF-beta2 at high affinity, and the about 50kDa proteins expressed on the surface of many cells that bind TGF-beta1, beta2, and beta3 with equal high affinity. As there are many cell- surface molecules that bind TGF-beta specifically, we need to understand how these several receptors interact with each other and with each other and with TGF-beta homo- and hetero-dimers, to understand why expression of certain receptor polypeptides enhances or inhibits binding of TGF-beta to other receptors, to determine the subcellular metabolism of the receptor polypeptides, and to determine whether receptor homo- or hetero- oligomerization is induced by TGF-beta and whether this is part of the signaling response. Finally, we and others have shown that a number of human tumor cell lines are resistant to growth-inhibition by TGF-beta and, as shown by affinity-labeling with [125I] TGF-beta, lack the type II and often other cell-surface receptors. We have shown, surprisingly, that several express mRNA encoding the type II TGF-beta receptor and that the encoded receptors contain the same point mutation in the predicted kinase domain, yet the cells express no surface receptor. We will clone and characterize these mutant receptor cDNAs, determine whether the mutations are functional and/or generate a dominant-negative phenotype, and cDNAs that we have and will clone. All of these studies are fundamental to our understanding of how TGF-beta exerts its multiple functions on a diverse array of cells, and in understanding how this novel class of cell-surface receptors transduces signals to the cell nucleus.

Our long-term goal is to understand how insulin causes an increase in glucose uptake by fat and muscle cells-specifically, to identify, characterize, and clone proteins that transduce the signal from the insulin-activated receptor protein-tyrosine kinase to the fusion of intracellular vesicles containing the GLUT-4 glucose transporter with the plasma membrane. We have cloned a new low-mw GTP binding protein, Rab3D, that is highly expressed in insulin-responsive tissues, and has many of the properties expected of a protein that regulates exocytosis of GLUT-4-rich vesicles. Indeed, we have generated a presumed dominant-negative Rab3D mutation, N135I, that cannot bind GTP; expression of this mutant but not the wild-type Rab3D in differentiated 3T3-L1 adipocytes inhibits the ability of insulin to increase glucose transport. Recently, we have cloned homologs of the synaptic vesicle proteins synaptophysin and synaptobrevin (VAMP) that, like Rab3D, are abundantly expressed in insulin responsive tissues and are induced during adipogenesis in vitro. We also showed that Rab3A, whose expression was thought confined to nerve and neuroendocrine cells, is also abundantly expressed in insulin responsive tissues and is induced during adipogenesis. We hypothesize that activation of the insulin receptor induces an exchange of GTP for GDP on Rab3A or 3D, much in the way that ras is activated by other receptor protein-tyrosine kinases, and that a Rab3GTP directly induces membrane fusion. Our specific goals are: (1) Elucidating the roles of Rab3D and Rab3A in exocytosis of GLUT4- and GLUT1- containing vesicles and in secretion of adipsin. This includes generation of other dominant-negative mutations in Rab3A and 3D and examining the effects on insulin-stimulation of glucose transport and translocation of GLUT1 and GLUT4 to the cell surface in transfected 3T3-L1 adipocytes; and examining the effects of GTPgammaS and synthetic peptides corresponding to the presumed effector domain of Rab3 on exocytosis of GLUT- 4 in two permeabilized adipocyte systems and also in permeabilized adipocytes expressing mutant Rab3 proteins; (2) Determining the subcellular localization of Rab3D, Rab3A, and the adipocyte-specific homologs of synaptophysin and synaptobrevin in basal and insulin-stimulated adipocytes; (3) Determining the roles of the adipocyte-specific homologs of synaptophysin and synaptobrevin in basal and insulin-stimulated glucose transport by generating clonal lies of 3T3-l1 adipocytes that express specific mutant forms of these proteins or that overexpress the wild-type protein, and examining them for insulin-stimulation of translocation of GLUT4 and GLUT1 to the cell surface and secretion of adipsin; and (4) Cloning Rab3D or RabA GDP-GTP exchange protein or GAP proteins that are induced during adipogenesis, using expression cloning, hybridization, or PCR and a suitable full-length subtractive, adipocyte-specific expression cDNA library. We will test whether these proteins are intermediates in the signal transduction pathway from the insulin receptor to exocytosis of glucose transporters.

Our work focuses on three interrelated aspects of the mechanisms by which transforming growth factor beta (TGF-beta) triggers cessation of the cell cycle and induction of synthesis of extracellular matrix proteins, and the ways that these processes can be annulled in human malignancies. First, we recently identified novel types of mutations in both the types I and II TGF-beta receptors in human hematopoietic tumors, and we now want to find out how these mutations affect receptor structure and function. Specifically, we will determine the functional defects of the dominant negative D404G mutant type II TGF-beta receptor (Tbeta-RII) we recently identified in a cutaneous T cell lymphoma. Also, we showed recently that resistance to TGF-beta 1 in chronic lymphocytic leukemia (CLL) was associated with the presence of Tbeta-RI mRNA but loss of functional surface Tbeta-RI; expression of surface Tbeta-RII was normal. We will determine the mutation(s) in the Tbeta-RI or possibly in the Tbeta-RII genes in several isolates of TGF-beta- resistant CLL, and will determine the effects of these mutations on Tbeta-RI function. In the course of our studies on the biosynthesis, and oligomerization of Tbeta-RII and Tbeta-RI, we showed that Zn++ions bind to the cytosolic domain of Tbeta-RI and that Zn++ is essential for Tbeta-RI function. To explore this further, we will determine the number of Zn++ bound to the cytosolic domain of Tbeta-RI and the amino acids that ligate them, and will determine the role of bound Zn++ in the function of Tbeta-RI. Our major focus is using several robust expression cloning strategies we have developed to isolate novel genes encoding proteins that either activate or inhibit specific TGF-beta signal transduction pathways. In particular, we will clone proteins that affect only a subset of the processes normally induced by TGF-beta, such as expression of genes encoding extracellular matrix proteins or genes encoding cell cycle regulatory proteins. We may also clone by expression protein fragments that act in a dominant-negative fashion to block the function of the corresponding wild-type protein. Specifically, we will isolate and sequence cDNAs encoding proteins that (i) abolish the ability of TGF-beta to induce growth inhibition and/or apoptosis, (ii) that stimulate expression, in the absence of TGF-beta, of specific promoters, JunB, PAI, p15, and/or p21 - that are normally induced by TGF-beta in cells we are using, and (iii) that abolish the ability of TGF-beta to induce these promoters. Some of these strategies utilize green florescent protein and cell surface reporter genes that are activated by the indicated promoter. Also, we will clone by complementation the genes defective in three mutant lines of HT1080 cells in which TGF-beta is unable to induce the PAI promoter. Most importantly, we will isolate and sequence full-length clones of these novel proteins, and determine their function in TGF-beta signaling.

We have cloned and characterized five mammalian fatty acid transport proteins (FATPs). FATP1 is expressed at high levels in heart, adipocytes, muscle, and brain. FATP4 is expressed predominantly in the brush border of the absorptive small intensive epithelial cells and is essential for uptake of LCFAs. Human FATP6 is expressed exclusively in heart muscle and its level is elevated in hypertrophic tissue. Our overall goal is to understand the molecular mechanism of fatty acid transport and its regulation. We focus on the FATPs as well as other proteins implicated in fatty acid transport: the "scavenger" receptors CD36 and SRI-BI; LACS (long chain fatty acyl CoA synthetase); and cytosolic FABPs (fatty acid binding proteins), and their roles in fatty acid transport in heart, adipocytes, and intestinal epithelial cells. Our specific aims are to 1) Determine whether these proteins functional synergize to enhance the transports of LCFAs by cultured cells and whether, depending on the transport mechanism involved, fatty acids are esterified to CoA upon entry. 2) Determine whether stable physical interactions occur between various FATPs, FABPs, SR-BI/CD36, and LACS. 3) Determine by antisense experiments which protein is rate-limiting for adipocyte LCFA uptake, both in the absence and presence of insulin. 4) Determine how insulin stimulates and TNF-alpha inhibits LCFA uptake by adipocytes 5) Clone and study the putative purine homologue of the cardiac-specific human FATP6. 6) Depending on the results obtained, use expression model systems to test the roles of FATP1, FATP4, and other proteins in LCFA uptake and determine the role of these proteins in whole-mouse lipid metabolism. Thus we will 1) Determine the expression pattern of these candidate fatty acid transport proteins within the developing and adult murine heart. 2) Using both FATP1 knockout and transgenic FATP1 over-expressing mice, determine the role of FATP1 in LCFA, uptake and lipid metabolism by adipose tissue and heart and striated muscle. 3) Using gene knock-out mice, determine the role of FAT4, SR- BI, and CD36 in uptake of LCFAs from the small intestine by the absorptive epithelial cells.

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are an abundant and highly conserved class of endogenous ~22-base RNAs that play crucial roles in cell function and development by base pairing to sites within target mRNAs, triggering either translational repression or mRNA degradation, or both. Our long-term goal is to discover the regulatory roles of several conserved miRNAs in critical and well-defined stages in formation of erythrocytes - uncovering not only their specific mRNA targets at both the BFU-E and CFU-E developmental stages but also the underlying network of miRNAs and their mRNA targets essential for these developmental transitions. Our specific aims are 1) to investigate the functions of miR-144, 451, 221, 222, and 223 in terminal proliferation and differentiation of CFU-E progenitors. We will determine the cellular effects on erythroid differentiation of ectopically overexpressing or knocking down expression these miRNAs in cultured CFU-E cells. Next we will determine the developmentally important mRNA targets downregulated by each of these miRNAs during specific stages of CFU-E proliferation and differentiation using a combination of experimental and computational approaches, and we will determine the roles of selected key miRNA target interactions during erythroid differentiation in culture. In Aim 2 we will use similar techniques to determine the functions of miR- 221, miR-222, miR-223 and other developmentally regulated miRNAs in the proliferation of BFU-E progenitors and the formation of CFU-Es. These studies will provide important information on the gene regulatory circuitry that controls hematopoiesis, and provide insights into pathological states caused by aberrant expression of certain miRNAs.

DESCRIPTION (provided by applicant): Our principal hypotheses are that many as yet unidentified morphogens/growth factors control fetal and adult hematopoietic stem cell (HSC) survival and proliferation, and that genetic modifications of stromal cell lines can enhance their ability to support HSC expansion in culture without undergoing differentiation to lineage-restricted progenitors. We identified several potentially important secreted proteins specifically expressed in AFT024, a mouse fetal liver stromal cell line that maintains HSC stem cell activity: Pleiotrophin; Deltalike; the fibrillin-like protein T16; Cytokine Receptor-like Factor, and three members of the Proliferin gene family, Proliferin- 1; Mrp4, and proliferin- related protein (PRP). Using stable expression of siRNAs to block their production in AFT024 and AFT024 - FIt3L cells we will determine the function of these and other signaling proteins, such as M-CSF, in HSC expansion and differentiation. Using Fc fusion proteins we will determine whether HSCs have receptors for these novel growth/differentiation factors and whether their receptors might provide additional HSC- specific surface markers. Our preliminary data indicates positive effects of added Flt3L and IGF-2 on HSC maintenance and expansion in vitro. Thus, in parallel we will determine whether forced overexpression in AFT024 and AFT024 - Flt3L cells of several proteins including thrombopoietin; Stromal cell - derived factor 1; stem cell factor; IL-6; 3 Wnts; and IGF-2 enhances their ability to support expansion of HSCs in culture. As appropriate knock- out mice selectively missing one or more of these factors will be made and analyzed for HSCs and hematopoiesis. By comparing the ability of other stromal cell lines, including OP-9, MS-5, and S 17, to support maintenance and expansion of fetal liver and adult bone marrow HSCs, together with our existing transcriptional profiling data, we should identify other secreted/ surface proteins that potentially affect HSC expansion or differentiation positively or negatively, and in later years will test their role in HSC biology. Our long- term aim is to engineer a cloned line that supports robust, continuous expansion of fetal liver or bone marrow HSCs in culture. In continued collaboration with Prof. George Daley, we will test the ability of our novel growth factors/morphogens and genetically altered stromal cell lines to support the generation of transplantable HSCs from cultured human and mouse ES cell lines.

DESCRIPTION (provided by applicant): Understanding and manipulating hematopoietic differentiation requires knowing the regulatory circuitry that orchestrates the programs of gene expression during this process. One class of gene regulatory molecules are the microRNAs (miRNAs) - tiny endogenous RNAs, about 22 nucleotides in length, that are thought to use the elements of the RNA-interference pathway to post transcriptionally down-regulate the expression of protein-coding genes. Starting with the hypothesis that miRNAs are playing important regulatory roles during hematopoietic differentiation, the Lodish and Bartel labs have collaborated to clone about 100 different miRNAs from mouse bone marrow. These include five miRNAs referred to as "hematopoietic miRNAs", because they are highly or preferentially expressed in hematopoietic cell lineages. Three of the five also derive from loci associated with chromosomal breakpoints or aberrations previously linked to leukemias. Preliminary studies show that ectopic expression of one of these miRNAs in bone marrow progenitors modulates hematopoietic differentiation both in cell culture and in transplanted mice. The experiments of this proposal focus on the hematopoietic miRNAs with the broad, long-term objective of understanding the gene regulatory events needed for hematopoietic stem cell and progenitor maintenance and differentiation. The specific aims are: 1) To examine the consequences of altered miRNA expression during hematopoiesis. 2) To identify the regulatory targets of hematopoietic miRNAs and examine the consequences of disrupting miRNA regulation of these targets. 3) To identify additional hematopoietic miRNAs. These experiments include the ectopic expression of hematopoietic miRNAs in hematopoietic stem cells and lineage-committed progenitors, knock-outs of miRNA genes, in vitro validation of predicted miRNA regulatory targets, in vivo substitution of target genes with versions unresponsive to miRNA regulation, cloning of additional miRNAs from hematopoietic tissues, and further expression analyses. They seek to place miRNAs within specific gene regulatory pathways needed for hematopoietic stem cell maintenance and lymphoid and myeloid differentiation. They will also address more fundamental issues regarding miRNA regulation, such as the combinatorial control of expression by miRNAs and the features of functional miRNA complementary sites within vertebrate mRNAs. Thus, these experiments will provide important insights for understanding the action of miRNAs in mammals and how their dysfunction might contribute to both hematological and other human diseases.

Our overall goal to understand the role of the adipokine adiponectin in vascular function, atherosclerosis, and coronary heart disease. Adiponectin is produced exclusively by adipocytes and is found at high levels in the intima surrounding several types of blood vessels. Adiponectin plays a key role in regulating hepatic and muscle fat and glucose metabolism and also the metabolism and proliferation of vascular smooth muscle and possibly endothelial cells. We identified several adiponectin orthologs, expressed mainly by adipocytes that share biological activities and signaling properties with adiponectin;these, like adiponectin, may regulate metabolism of vascular cells. The identities of the adiponectin signaling receptors and signal transduction pathways are not known;our and other labs have unequivocally shown that previously reported, putative signaling receptors for adiponectin do not function in that capacity. We identified T-cadherin, a GPI- anchored surface protein, as an adiponectin binding protein that is highly and specifically expressed by cells in the blood vessel intima. Deletion of T-cadherin in mice results in a decrease in adiponectin in the vasculature and a major increase in the circulation, indicating it is a major adiponectin receptor. However, additional cell surface receptors are necessary to mediate adiponectin signaling. We will clone these signaling adiponectin receptors, analyze their structures and functions in vitro and in vivo, and determine the signal transduction pathways activated in cultured vascular endothelial and smooth muscle cells by the three isoforms of adiponectin, focusing initially on the AMP- activated protein kinase, and NF-kB, MAP kinase, and NO pathways. Cells from T-cadherin -/- mice will allow us to continue to explore the role of this receptor in adiponectin signaling and localization in the vasculature. Importantly, with Projects I and II we will determine the effects of the various isoforms and orthologs of adiponectin and of T-cadherin on blood vessel endothelial and smooth muscle cells and on atherosclerosis and CHD in apoE -/- and SR-BI/apoE double knockout (dKO) mice. Thus, over the coming five years we hope to elucidate the roles of adiponectin and its principal vascular binding protein, T-cadherin, in maintaining the normal state of vascular endothelial and smooth muscle cells, and understand whether and how deletion of either of these proteins leads to atherosclerosis, thrombosis and CHD.

[unreadable] DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) reside in specialized niches and receive signals from surrounding stromal cells, but their identities and the effects they have on HSCs are largely unknown. Our work focuses on defining secreted growth factors that are produced by stromal cells and that trigger HSC expansion ex vivo. We identified a novel cell population from Embryonic Day 15 (E15) fetal livers with the surface phenotype of early T cells and that supports ex vivo expansion of HSCs. Major aims of this grant are to purify and characterize several E15.5 FL T cell subpopulations for their ability to expand HSCs in culture, to determine whether similar cells are found in the adult mouse bone marrow and spleen, to determine whether these are part of the HSC niche, and to examine differences and similarities between gene expression in adult and fetal populations of T cells capable of supporting HSCs in order to develop a cellular and molecular model of HSC expansion. Our DNA microarray experiments showed that, among other proteins, IGF - 2 and Angiopoietin- like proteins 2 (Angptl2) and 3 (AngptIS) are specifically expressed in these E15 FL early T cells. We then developed a serum- free HSC culture system containing low levels of SCF, TPO, FGF-1, IGF- 2 and Angptl2 or AngptIS; as measured by competitive repopulation analyses there was a >24 increase in numbers of long-term repopulating HSCs (LT-HSC) after 10 days of culture. Angptl2 and AngptIS are largely unstudied and have not previously been suggested to act on hematopoiesis or stem cells. Here we will test whether IGF-2, TPO, Angptl2, and other growth factors are each required for maximum HSC expansion ex vivo and thus whether each of these likely activates different signaling pathways in HSCs. In parallel we will adopt a high-resolution cell tracking procedure to determine whether Angptl2, IGF-2, Tpo, and later other factors directly stimulates HSC self- renewal or prevents HSC apoptosis or differentiation. The receptors for Angptl2 or AngptIS and signal transduction pathway(s) are unknown; we will use two expression cloning protocols developed and extensively utilized in my laboratory to isolate the Angptl2 and AngptIS cell surface receptor(s), and then to characterize them extensively. Our studies can lead to major improvements in the clinical use of HSCs in bone marrow transplantation for treatment of leukemia and other cancers; many applications, especially transplantation using cord blood, have been hindered by the low numbers of HSCs and by the unavailability of compatible donors. HSCs are also a promising cell target for developing gene therapies for treating a broad variety of inherited immunodeficiency syndromes and inborn errors of metabolism. The ability to expand HSCs ex vivo would greatly enhance these clinical applications, in the case of gene therapy allowing selection of those HSCs that have integrated a transgene in a specific chromosomal location. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We showed that deletion of T-cadherin results in a ~10 - fold increase in circulating adiponectin, indicating it is the major adiponectin receptor in the body, and obese mice lacking T-cadherin are diabetic due to an abnormal enhancement of hepatic gluconeogenesis. We will determine whether T- cadherin is important for adiponectin binding and signaling in liver and muscle using cultured tissues from T-cadherin -/- mice, and also C2C12 myoblasts and myotubes in which T-cadherin has been overexpressed or knocked down. In parallel we will investigate the ability of different adiponectin isoforms to activate AMPK in these tissues. How adiponectin receptors are coupled to activation of AMPK is unknown but adiponectin signaling is accompanied by an increase in 5' AMP. Our preliminary data strongly suggest that acyl CoA synthetases transduce adiponectin signals to AMPK by producing 5'AMP. We will test the hypothesis that Fatty Acid Transporters (FATPs) and/or acyl-CoA synthetases (Acsls) are signal transduction proteins essential for AMPK activation by adiponectin. Initially we will focus on FATP1 and Acsl1 in muscle; we will stably overexpress Acsl and FATPs proteins, individually and together, in C2C12 myoblasts and myotubes, and also use shRNA to knock down their expression. We will monitor effects of adiponectin activation of AMPK, ACC phosphorylation, and malonyl CoA levels. We will also determine whether adiponectin signals translocation of acyl-CoA synthetases or FATPs to the plasma membrane. As receptors and signaling proteins other than T-cadherin and AdipoR1 and R2 are certainly involved in adiponectin signaling, we will use new biotin-containing tri-functional cross-linking reagents and mass spectrometry- based proteomic approaches to determine whether T-cadherin, Acsl or FATP isoforms, AdipoR1 or R2, or unknown proteins are part of the signaling adiponectin receptor complex in C2C12 myoblasts and myotubes, muscle and liver. Recently we cloned a family of ten adiponectin paralogs conserved in human and mouse that share biological activities and signaling properties with adiponectin. We focused on CTRP9 since it, like adiponectin, is made predominantly by adipocytes, activates AMPK in cultured muscle, and forms heterooligomers with adiponectin. We hypothesize that CTRP9, like adiponectin, normally regulates metabolism of muscle and liver. Mainly by studying adiponectin -/- mice, CTRP9-/- mice, and double mutant CTRP9-/- adiponectin -/- mice we will determine the role of CTRP9 in AMPK activation in muscle and liver and in the regulation of whole- body glucose and fatty acid metabolism. Receptors for CTRP9 are unknown and thus we will use several approaches to identify and clone the CTRP9 receptor(s).

DESCRIPTION (provided by applicant): We have identified and partially characterized several BAT- specific miRNAs and Long Intergenic Noncoding (Linc) RNAs. This grant is focused on understanding their roles in BAT development and function with the long- term goals of determining their mechanisms of action and, their roles in regulatory mechanisms governing BAT development. We have shown that a BAT-enriched microRNA cluster, miR-193b-365, is an essential regulator for brown adipocyte differentiation and lineage determination: knockdown of both miR-193-365 loci in BAT progenitors blocked BAT differentiation and ectopic expression of miR-193b in C2C12 myoblasts induced differentiation into brown adipocytes that undergo adaptive thermogenesis11. At least one other miRNA, miR203 is also highly and specifically enriched in brown fat and our preliminary data indicates that it is essential for development of mature brown adipocytes. We need to determine the mechanisms by which these miRNAs function. Thus we will determine their target mRNAs and the global changes in mRNA levels and translation induced by miR-193- 365 or miR-203 overexpression and knockdown in murine cells; we do this by determining the changes in mRNA composition and by analysis of changes in ribosome occupancy. This will allow us to construct a network of miRNA- mRNA interactions that ultimately modify expression of key human and murine BAT, muscle, and WAT proteins, and also to determine the mechanism by which these miRNAs inhibit synthesis of specific proteins. . We will determine the function of key target mRNAs as targets by reporter gene assays and then by appropriate knockdown or overexpression experiments, with the goal of identifying additional genes that regulate BAT development. To determine the roles of miR-193-365 and miR- 203 during brown fat development in vivo we will generate adipocyte- specific and whole body deletions and then characterize the effects of miR deletion on BAT development, metabolic homeostasis, and thermogenesis. In collaboration with the Broad Institute, we performed RNA-seq to profile the transcriptome of primary brown and white adipocytes, pre-adipocytes and cultured white and brown adipocytes. We identified 481 LincRNAs that are specifically regulated during adipogenesis; RNAi-mediated loss of function screens identified 10 functional LincRNAs required for adipogenesis. Seven other LncRNAs are specifically expressed in BAT cells. Here we will test our hypothesis that these and other BAT- specific LincRNAs are essential for BAT development and we will determine their molecular mechanisms of action. We will focus on nuclear- localized lincRNAs, and determine the effects of their overexpression and knockdown on BAT development and on BAT, WAT, and muscle- specific gene expression. For selected LncRNAs, we will develop RNA pull- down and yeast three- hybrid assays to identify bound proteins as a first step in determining their mechanism of action. In the long- term, we will use Chip experiments to determine how these nuclear LincRNA- protein complexes function to regulate gene expression.