Overview
Research in my lab focuses on five important areas at the interface between molecular cell biology and medicine:

1. Red blood cell development, especially on the role of three signal transduction pathways downstream of the erythropoietin receptor in controlling terminal proliferation and differentiation of erythroid progenitor cells;
2. Hematopoietic stem cells, defining new cell surface proteins for their purification and new growth factors that support their expansion in culture;
3. MicroRNAs, defining their roles in lineage commitment of hematopoietic stem and progenitor cells, and regulating muscle differentiation
4. Adiponectin, a hormone we cloned that is made exclusively by fat cells and that increases fatty acid and glucose metabolism by muscle, and four homologous proteins
5. Regulated cleavage and release of the extracellular domain ("ectodomain shedding") of transmembrane precursors of several secreted growth factors.

 

Research Summary
Erythropoietin receptor (EpoR) and red cell development: Epo and the EpoR are essential for proliferation and differentiation of committed erythroid progenitors, as is the cytosolic protein-tyrosine kinase JAK-2. JAK2 binds to the EpoR cytosolic domain in the endoplasmic reticulum and facilitates its folding to promote cell surface expression. EpoRs exist on the cell surface as inactive dimers; Epo binding changes their conformation, leading to JAK2 transphosphorylation and activation. JAK2 activates many signaling proteins including PI-3’ kinase, the transcription factor Stat5, and the Ras pathway. These pathways interact to prevent apoptosis of committed erythroid progenitors allowing them to undergo a predetermined program of terminal proliferation and erythroid differentiation. We showed that Stat5 directly activates transcription of the anti-apoptotic protein bcl_xL. Stat5^-/- mice exhibit fetal anemia and increased apoptosis of erythroid progenitors caused by reduced bcl_xL levels. Adult Stat5^-/- mice are anemic and deficient in generating high erythropoietic rates in response to stress. Thus Stat5 controls one rate-determining step regulating early erythroblast survival. Activation of the PI-3’ kinase pathway leads to activation of the Akt kinase and then phosphorylation and inhibition of FOXO3a, a member of the Forkhead transcription factor family. FOXO3a, in turn, activates transcription of Tumor Necrosis Factor Apoptosis-Inducing Ligand (TRAIL). We showed that inhibition of TRAIL production by Epo addition partially rescues cells from apoptosis, demonstrating the importance of this pathway in red cell formation.

By screening libraries of EpoRs with random mutations in the transmembrane domain Xiaohui Lu identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domains resides to cysteine. Xiaohui then performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at three positions activated EpoR signal transduction pathways and supported proliferation of hematopoietic cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Xiaohui is determining the structure of peptides corresponding to these dimeric active a- helixes; this should shed light on the structure of the Epo- activated receptor transmembrane domain.

Little is known concerning the degradation of Epo in the body – where this occurs or what may control it. Alec Gross is studying the mechanism of Epo degradation, both in erythroid cells expressing the EpoR and in mice expressing abnormal numbers of Epo receptors in various tissues. One goal is to explain why certain commercially-important mutant Epo’s with extra carbohydrate chains have a longer biological lifetime. Alec’s work using cell lines showed that Epo degradation requires expression of the EpoR. A fraction of the Epo bound to surface receptors is internalized by endocytosis and degraded in lysosomes. Most, however, either dissociates from the surface receptor into the medium or is internalized but resecreted. Long-lived mutant Epo binds slower and dissociates more rapidly from surface Epo receptors, but otherwise the kinetics of internalization, resecretion, and degradation are indistinguishable from normal Epo. As Alec’s kinetic modeling showed, these altered receptor-binding kinetics can explain its longer half life in vivo. To test this he will examine the fate of Epo and its long-lived variants in mice with altered numbers of Epo receptors in both hematopoietic and non-hematopoietic cells; in this way he should discern the role of surface EpoRs in normal Epo turnover.

Many of our current studies on EpoR signal transduction make use of a new culture system Jing Zhang developed where pure fetal liver erythroid progenitors (so-called CFU-Es) undergo normal terminal proliferation and differentiation; this can be followed on a cell-to-cell level by FACS. As example, Jing showed that expression of a dominant-negative H-ras in CFU-E progenitors, or addition of an inhibitor of the MAP kinase pathway, did not affect erythroid differentiation, indicating that activation of the Ras- MAPK pathway by Epo is not essential for erythroid development. To address the precise signaling pathway(s) regulated by K-ras Jing then studied K-ras signaling in K-ras ^-/- fetal liver erythroid progenitors. She found that K-ras ^-/- fetal liver cells showed a ~7-fold increase of apoptosis and significant delayed erythroid differentiation. Moreover, when K-ras^-/- erythroid progenitors were cultured in vitro, there is a significant delay in erythroid differentiation but little increase in apoptosis. She then examined the signaling pathways activated by Epo and stem cell factor (SCF) in K-ras -/- fetal liver cells. Epo- or SCF-dependent Akt activation was greatly reduced in these cells whereas other pathways including Stat5 and p44/p42 MAP kinase were activated normally. Taken together, her data identified K-ras as the major regulator for cytokine-dependent Akt activation in erythropoiesis in vivo.

Importantly, oncogenic mutations in ras genes frequently occur in patients with myeloid disorders and in these patients erythropoiesis is often affected. Last year Jing showed that overexpression of oncogenic H-ras in purified mouse primary fetal liver erythroid progenitors blocks terminal erythroid differentiation and supports Epo-independent proliferation. Jing showed that three major pathways are abnormally activated by oncogenic H-ras: Raf/ERK, PI3-kinase/Akt and RalGEF/RalA. However, only constitutive activation of the MEK/ERK pathway alone could recapitulate all of the effects of oncogenic H-ras expression in blocking erythroid differentiation and inducing Epo-independent proliferation. Moreover, all effects of oncogenic H-ras expression on primary erythroid cells were blocked by the addition of a specific inhibitor of MEK1/2, allowing normal terminal erythroid proliferation and differentiation. Jing’s data suggest that the interruption of constitutive MEK/ERK signaling is a potential therapeutic strategy to correct impaired erythroid differentiation in patients with myeloid disorders. But to avoid problems due to oncogenic Ras overexpression, Jing, assisted by Yangang Liu, is studying primary erythroid progenitors in which oncogenic Ras is expressed from the endogenous Ras promoter. Expression of oncogenic K-ras is induced using a rtTA/TetO-cre system for a short period of time, and oncogenic K-ras signaling will be assessed in highly purified primary erythroid progenitors. Initial focus will be on the signaling pathways constitutively activated by endogenous oncogenic K-ras and hyperactivated in response to cytokine stimulation. More importantly, the consequences of abnormal oncogenic K-ras signaling in erythroid cells will be evaluated at both the cellular and gene transcriptional levels.

The cytosolic adaptor protein Lnk has been implicated in cytokine receptor signaling. Recently Wei Tong, assisted by Sara Zarnegar, showed that Lnk-deficient mice have elevated numbers of erythroid progenitors, and that splenic CFU-e progenitors are hypersensitive to Epo. Lnk^-/- mice also exhibit superior recovery after erythropoietic stress. In addition, Lnk deficiency resulted in enhanced Epo-induced signaling pathways in splenic erythroid progenitors. Conversely, Lnk overexpression inhibits Epo-induced cell proliferation. In primary culture of fetal liver cells, Lnk overexpression inhibited Epo-dependent erythroblast differentiation and induced apoptosis; Lnk blocked all three major signaling pathways, Stat5, Akt, and MAPK, induced by Epo in primary erythroblasts. Wei showed that the Lnk SH2 domain is essential for its inhibitory function, whereas the conserved tyrosine near the C-terminus and the PH domain of Lnk are not critical. Thus Lnk, through its SH2 domain, negatively modulates EpoR signaling by attenuating JAK2 activation, and regulates Epo-mediated erythropoiesis. Determining the novel molecular mechanism by which Lnk inhibits signaling from the EpoR and other cytokine receptors is one of Wei’s current goals.

Another current project, conducted by Shilpa Hattangadi and Jing Zhang, involves determining all changes in gene expression that occur during terminal proliferation and differentiation of purified fetal liver erythroid cells. This involves assay of mRNAs by hybridization to DNA gene microarrays (“gene chips)”. Another, done by Shilpa in collaboration with members of Rick Young’s laboratory, involves immunoprecipitation of chromatin with antibodies specific for transcription factors, followed by hybridization of the recovered DNA to a genomic DNA microarray. This protocol will enable Shilpa to determine all of the genes that have critical erythroid-important transcription factors bound to their promoter/enhancer segments. Initial studies focus on transcriptional activation by Stat5 but other factors will soon be investigated. Shilpa’s long-term goal is to understand how the complex pattern of gene expression during erythroid development is controlled by transcription factors activated by signal transduction pathways downstream of the EpoR.

A third project focuses on the role of integrins in terminal proliferation and differentiation of purified fetal liver erythroid cells, since adhesion of these progenitors to fibronectin is essential for normal erythroid development. Shawdee Eshghi showed that both α₄β₁ and α₅β₁ integrins are present on erythroid progenitors, and that α₄β₁ and α₅β₁ integrins support binding of erythroid cells to different fibronectin domains. Shawdee also showed that loss of both α₄β₁ and α₅β₁ integrins during erythroid differentiation parallels the loss of adhesion of erythroid cells to fibronectin. She is now investigating the signal transduction pathways and transcriptional changes mediated by each of these integrins. She is also determining the pattern of integrin expression on purified hematopoietic stem cells.

Epo prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases. The molecular mechanisms of this protection are incompletely understood. Using differentiated human neuroblastoma cells Moon Um confirmed the antiapoptotic activity of Epo and showed that Epo activates both the Stat5 and PI-3 kinase/ AKT signaling pathways. Studies using expression of chimeric mutant EpoRs able to activate neither or only one of these pathways showed that activation of both is required for EpoR activation to prevent neuronal death. In parallel Moon is studying apoptosis of primary adult neuronal cells genetically engineered to lack the Epo receptor. Once she elucidates how Epo prevents neuronal cell death in the brain, her findings could lead to a novel clinical application of Epo for limiting brain damage due to stroke or neurodegenerative diseases.

Joe Shuga, in collaboration with the laboratories of Profs Leona Samson and Linda Griffith, is extending our in vitro culture system for erythroid progenitors into an assay for genotoxicity. Assays that predict toxicity are an essential part of drug development and many drugs fail in phase I clinical trials; therefore, there is a demand for models that can better predict human responses. The mouse in vivo micronucleus (MN) assay is a robust toxicity test that assesses the genotoxic effect of drugs by detecting chromosome fragments that remain in the reticulocyte after enucleation; an in vitro correlate to this assay might allow extension to human cells and thus better predictive power in drug development. As first steps in developing a toxicity assay Joe is adapting our in vitro erythropoiesis culture system to induce optimized erythropoietic growth from Lin- populations from adult BM, and demonstrating that exposure to genotoxicants induces MN-formation in this culture system. In particular, Joe showed that addition of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to this culture system induces a global cytotoxic response and concomitant decreases in erythropoietic differentiation and increases in MN-formation. The increase in MN production in the presence of BCNU provides a clear signal of the clastogenic mechanism that likely induced the overall hematopoietic toxicity.

Hematopoietic stem cells: Hematopoietic stem cells (HSCs) are defined by their ability to self-renew and to differentiate into all blood cell types. These very rare cells form the basis of bone marrow transplantation for treatment of leukemia and other cancers, and are also a promising cell target for developing gene therapies for treating a broad variety of human diseases. However, development of these important clinical applications of HSCs are greatly hampered by the lack of understanding of the extracellular and intracellular signals that govern their fates and the difficulty in ex vivo expansion of these cells. We quantitate these cells by bone marrow transplantation, monitoring the long- term repopulation of the hematopoietic compartment of lethally irradiated mice. This assay thus requires several months to complete.

Several years ago Chang-Zheng Chen, a former fellow, identified Endoglin, an ancillary TGF-β receptor, as a surface marker for long-term repopulating mouse bone marrow HSCs. He showed that bone marrow cells purified by the Endoglin^Positive Sca-1^PositiveRhodamine^Low phenotype are a homogenous population of long- term repopulating HSCs. Shawdee Eshghi recently showed that these cells are morphologically homogenous and minute, only ~ 5 – 7 μm in diameter. Thus the Endoglin^Positive Sca-1^PositiveRhodamine^Low phenotype defines a simple and effective procedure for purifying a nearly homogenous stem cell population from mouse bone marrow.

No single known growth factor or combination of growth factors reproducibly supported HSC expansion in culture. Furthermore existing lines of “supportive stromal cells” did not support expansion of HSCs; at best they maintained the level of HSCs over time, presumably due to a steady state between generation of new HSCs by division and differentiation of “old” stem cells. Thus Chengcheng Zhang, assisted by Megan Kaba, turned to mouse fetal liver since the number of fetal HSCs normally increased markedly between embryonic Day 12 and Day 16. Chengcheng hypothesized that unknown growth proteins are produced by as-yet unidentified populations of fetal liver cells that stimulate the expansion of fetal liver HSCs. He then identified Embryonic Day 15 fetal liver CD3⁺ Ter119- cells as a completely novel cell population that supports a net expansion of HSC numbers in culture. Although CD3 is generally thought to be a specific T-cell marker, these fetal liver CD3⁺ Ter119^- cells do not express other characteristic T cell markers. By transcriptional profiling of these cells and several others that do not support HSC expansion, Chengcheng uncovered several novel growth factors that, together supported an unprecedented extent of ex vivo expansion of bone marrow HSCs. First he identified insulin-like growth factor 2 (IGF - 2), which is specifically produced by fetal liver CD3⁺ cells. Treatment of cocultures of HSCs and day 15 fetal liver CD3⁺ Ter119^- cells with anti- IGF-2 antisera showed that IGF-2 is a key molecule produced by these cells that stimulates HSC expansion. Furthermore, when combined with other growth factors IGF-2 is capable of markedly enhancing ex vivo expansion of long-term repopulating fetal liver and adult bone marrow HSCs. Systematic testing of combinations of growth factors led to the development of a serum-free culture medium containing low levels of SCF, TPO, IGF-2, and FGF-1. As measured by competitive repopulation analyses, there was a greater than 20-fold increase in numbers of long-term HSCs after a 10-day culture of total BM cells. Culture of a highly-enriched stem cell population, for 10 days resulted in an ~8 fold expansion of repopulating HSCs. Strikingly, the surface phenotype of ex vivo expanded HSCs was different from that of freshly isolated HSCs, but this plasticity of surface phenotype did not significantly alter their repopulation capability.

More recently Chengcheng identified a novel and unstudied protein specifically produced by day 15 fetal liver CD3⁺ Ter119^- cells that also stimulates ex vivo expansion of HSCs. Chengcheng showed that, when used in serum- free media in combination with other growth factors, this protein stimulates a greater than 20-fold expansion of HSCs following 10 days of culture of highly enriched stem cells. Its receptor(s) and the signal transduction pathway(s) it activates are unknown. A main focus of Chengcheng’s current research is deciphering the specific intracellular signal transduction pathway(s) and transcriptional activations induced by this protein in both cell lines and in HSCs.

Several years ago we identified 12 novel secreted and cell surface proteins expressed specifically by lines of stromal cells that support stem cell maintenance, including several novel cytokines. During the past year Alek Babic showed that one of these, pleiotrophin, also supports ex vivo expansion of long- term repopulating HSCs in culture. Currently Alek is investigating the receptors for pleiotrophin in HSCs, as well as the signal transduction pathways activated in these cells that stimulate HSC expansion.

Among the other proteins specifically expressed by Day 15 fetal liver CD3⁺ cells was the prion protein (PrP), a glycosylphosphatidylinositol (GPI)- anchored cell surface protein; despite many years of research the normal function of PrP was unknown. Chengcheng Zhang surmised that PrP would also be expressed on long-term repopulating hematopoietic stem cells and initiated a collaboration with Professor Susan Lindquist and her PhD student Andrew Steele. Not only did they quickly confirm this hypothesis, they went on to show that HSCs from PrP -/- bone marrow exhibit impaired activities in serial transplantation experiments. Most strikingly, ectopic expression of PrP in PrP -/- bone marrow cells rescued the defects in hematopoietic engraftment. Therefore, PrP is a novel marker for HSCs and supports their self-renewal during successive bone marrow transplantations. Together with Andrew, Chengcheng is trying to determine the molecular function of PrP. PrP might be the coreceptor for a hormone affecting HSC activity, possibly concentrating this as yet unidentified molecule on the cell surface and/or presenting it to the signaling receptor(s). Alternatively, PrP might interact with proteins in the BM extracellular matrix or on the surface of stromal cells, and possibly support retention of transplanted HSCs within the BM microenvironment.

MicroRNAs that modulate differentiation: MicroRNAs (miRNAs) are ~22-nt non-coding RNAs that can play important roles in development by targeting the messages of protein-coding genes for cleavage or repression of productive translation. Examples include lin-4 and let-7 miRNAs that control the timing of C. elegans larval development. As shown by the Bartel laboratory and others, humans have between 250 and 300 genes that encode miRNAs, an abundance corresponding to almost one percent of protein-coding genes. Based on the evolutionary conservation of many miRNAs among different animal lineages, it is reasonable to suspect that some mammalian miRNAs might also have important functions during development.

As a first step towards testing the idea that miRNAs might play roles in mammalian development, and more specifically hematopoiesis, Chang-Zheng Chen, in collaboration with Prof. David Bartel, cloned about 100 unique miRNAs from mouse bone marrow. Three, miR-181, miR-223, and miR-142s, were exclusively or preferentially expressed in hematopoietic tissues. miR-181 was very strongly expressed in thymus, the primary lymphoid organ, which mainly contains T-lymphocytes. Mature miR-181 expression in the bone marrow cells was detectable in undifferentiated Lin^- progenitor cells and up-regulated in differentiated B-lymphocytes, marked by the B220 surface antigen. In other differentiated lineages, miR-181 expression did not increase over that seen in Lin^- cells. Using retrovirus vectors he developed, Chang- Zheng ectopically expressed miR-181 in a population of bone marrow hematopoietic stem and progenitor cells. This led to an increased fraction of B-lineage cells both in tissue-culture differentiation assays and in transplanted adult mice; there was a corresponding decrease in CD-8⁺ T cells. Expression of miR-142s, in contrast, was most abundant in cells of the granulocyte and macrophage lineages. Overexpression of miR-142s in hematopoietic stem and progenitor cells led to an increase in the numbers of granulocytes and macrophages and a decrease in numbers of both mature CD-8⁺ and CD-4⁺ T cells. These results indicate that microRNAs are components of the molecular circuitry controlling mouse hematopoiesis and suggest that other microRNAs have similar regulatory roles during other facets of vertebrate development. Current projects aim to uncover the mRNAs downregulated by miR-181 and miR-142s, and much of this work is being done in Chang-Zheng’s new laboratory at the Stanford University School of Medicine.

Beiyan Zhou has used microRNA microarrays developed in the Bartel laboratory to identify several miRNAs specifically upregulated in populations of thymic and splenic hematopoietic cells she isolated: B cells, immature CD-4^- CD-8^- and CD-4⁺ CD-8⁺ T cells, as well as in more mature thymic CD-4^- CD-8⁺ and CD-4⁺ CD-8^- T cells. She is confirming these results by Northern blotting. Overexpression of these miRNAs in cell lines and animals, as well as “knocking down” their expression in cultured cells, should shed light on the roles of these miRNAs in B- and T- cell development as well as in hematopoiesis more broadly.

Prakash Rao has identified three evolutionarily conserved muscle- specific several miRNAs that are upregulated during differentiation of cultured C2C12 myoblasts into differentiated myotubes. Prakash and Mina Farkhondeh, a UROP student, are collaborating with members of the Bartel laboratory to determine the direct targets of these miRNAs. They are also using several new technologies in an attempt to “knock down” expression of these miRNAs and thus learn more about their specific roles in muscle differentiation. Prakash is also working with Guangtao Ge, a former lab member, to predict the sequences upstream of these muscle- specific miRNA genes that regulate their transcription. They have identified several such sites and they are currently using multiple experimental approaches to determine whether these indeed regulate miRNA expression.

I-hung Shih, a postdoctoral fellow in the Bartel lab who has been working closely with us, has identified several micro RNAs that are upregulated during differentiation in culture of 3T3- L1 preadipocytes to adipocytes. I-hung is currently examining the effects on adipocyte differentiation of overexpressing these micro RNAs in preadipocytes, and knocking down their expression during adipogenesis. Using computational and experimental techniques developed in the Bartel lab she is trying to determine the mRNA target(s) of these miRNAs.

Adiponectin and its homologs: In 1995 we cloned adiponectin, originally called Acrp30, as novel adipocyte-specific secreted protein hormone. Adiponectin addition potently elevates fat and glucose catabolism by muscle, enhances glycogen accumulation in muscle, and inhibits gluconeogenesis in liver. Mutations in the adiponectin gene are linked to development of adult- onset diabetes and the levels of adiponectin in serum are reduced in obese and diabetic patients and mice. Circulating adiponectin levels negatively correlated with human plasma triglyceride and fasting insulin levels and several clinical studies showed those with low adiponectin levels are more likely to develop type II diabetes mellitus and cardiovascular disease. This data suggests that adiponectin is a potential genetic determinant of insulin sensitivity.

Adiponectin has four domains: a cleaved amino-terminal signal sequence, a region without homology to known proteins, a collagen-like region, and a globular segment at the carboxy-terminus. The three-dimensional structure of the globular domain is strikingly similar to that of TNF -a even though there is no homology at the primary sequence level. Like TNF -a the globular domain forms homotrimers, and intermolecular disulfide bonds generate hexameric and high molecular weight Adiponectin species.

In collaboration with the Ruderman laboratory at B.U. Medical School, Tsu-Shuen Tsao showed that treatment of rat striated muscle with trimeric adiponectin led to phosphorylation and activation of AMP-activated protein kinase (AMPK), an enzyme that when activated causes increases in muscle fatty acid oxidation, glucose uptake and oxidation, and insulin sensitivity. Adiponectin- mediated activation of AMPK caused phosphorylation and thus diminished activity of acetyl CoA carboxylase, a corresponding decrease in the concentration of malonyl CoA, and a corresponding increase in long- chain fatty acid oxidation. In addition, adiponectin caused an increase in glucose uptake. Both in vivo and in muscle culture adiponectin most likely exerts its actions on muscle fatty acid oxidation by inactivating ACC, via activation of AMPK and perhaps other signal transduction proteins.

AMPK is composed of three subunits – the a kinase subunit that undergoes regulated phosphorylation, the γ subunit that binds AMP, and the β subunit that is thought to act as a scaffold that binds to both the α and γ subunits. Cellular and physiological stresses that deplete ATP such as nutrient deprivation, hypoxia, ischemia, and exercise in muscle all lead to activation of AMPK. Kelly Wong has been redetermining the interactions of the three AMPK subunits and has shown that several key aspects of the current model are wrong. Most significantly, Kelly showed that the α-subunit binds directly to the γ-subunit, in striking contradiction to the “standard” model. He also showed that the “scaffolding” β-subunit does not bind directly to the γ-subunit; interactions of the β- and γ- subunits can be detected only if the α-subunit is also present. Thus his data suggests a model for AMPK structure in which the β-and the γ-subunit bind directly to the α-subunit, and in which the β-subunit does not bind directly to the AMP-sensing γ-subunit. Kelly is currently determining the precise subunit composition of intermediates in AMPK assembly and deciphering the molecular mechanism by which AMP binding to the γ-subunit allosterically activates the α-subunit kinase. He also aims to determine whether all three AMPK subunits are essential for kinase activation by adiponectin; in the process he hopes to identify other proteins that might connect the AMPK α-subunit to the elusive signaling adiponectin receptors.

Tsu-Shuen Tsao and Christopher Hug assessed if adiponectin is a signaling molecule by searching for promoter or enhancer elements that respond to hormone addition. Addition of hexameric and larger isoforms of adiponectin to C2C12 myocytes or myotubes leads to activation of NF-κB transcription factor in a manner dependent upon phosphorylation and degradation of the IκB-α subunit; trimeric adiponectin has no effect. In contrast, only trimeric adiponectin but not hexameric and larger isoforms, activates AMPK in muscle. Their data indicates that oligomerization of adiponectin is important for at least some of its biological activities, and that changes in the relative abundance of each oligomeric isoform in plasma may regulate adiponectin activity. These results also suggest that hexameric and trimeric adiponectin might bind to different receptors and/or activate different intracellular signal transduction pathways.

Christopher Hug, assisted by Jin Wang, used an expression cloning strategy to identify T-cadherin as a receptor for hexameric and high molecular weight forms of adiponectin. T-cadherin is highly and specifically expressed in the vasculature, where it is predominantly found in endothelial and smooth muscle cells in the blood vessel intima. T-cadherin is attached to the membrane via a GPI anchor at the C-terminus. Chris’ preliminary studies indicate that it is the major adiponectin binding protein in the body, as deletion of T-cadherin results in a many-fold increase in the level of high molecular weight adiponectin in the circulation. T- cadherin is upregulated following vascular injury and he hypothesizes that, by binding to adiponectin, it may play a role in atherosclerosis progression. Adiponectin also binds to extracellular collagens exposed during vessel wall injury; adiponectin also contains a KGD sequence that we hypothesize to bind to integrins, a point that will be examined by testing whether or not thrombosis is affected in adiponectin -/- mice. Currently Chris is determining the role of T-cadherin in adiponectin activation of the AMPK and NF-κB signal transduction pathways. Chris and Jin are also cloning other cell surface adiponectin receptors including those that directly activate AMPK.

Guang Wong, with the assistance of Sarah Krawczyk, used multiple genomic approaches to identify a family of seven highly conserved human and mouse proteins homologous in sequence and presumed structure to adiponectin. These are designated as C1q/TNF-arelated proteins (CTRP)-1 to 7 Expression of CTRP1, 2, 3, and 7 mRNAs, like that of adiponectin, is far higher in adipose tissue that in any other tissue tested. Like that of adiponectin, expression of CTRP1, 2, 3, and 7 mRNAs in 3T3- L1 adipocytes is upregulated by treatment with a thiazolidinedione agonist of PPAR-g. CTRP2 is the closest paralog of adiponectin; Guang’s data show that CTRP2 is structurally homologous to adiponectin in that both form higher order structures including trimers and hexamers. Moreover, CTRP1, 2, 3, and 7 are functionally homologous to adiponectin in their ability to activate the key metabolic sensor AMP-activated protein kinase (AMPK) in muscle and lung cells. Similar to adiponectin, treatment of C2C12 myotubes with CTRP2 (the others have not yet been tested) resulted in increased accumulation of glycogen and enhanced oxidation of long chain fatty acids, the latter due to phosphorylation of Acetyl CoA carboxylase (ACC) by AMPK. Taken together, these results suggest significant metabolic functions for CTRP1, 2, 3, and 7, but the natural target cells of these hormones and the functions they control are not known. An understanding of the natural metabolic functions of these hormones will likely emerge from analysis of the CTRP- overexpressing transgenic mice and CTRP gene knock- out mice Guang is now generating. Guang is also using expression cloning strategies to identify the receptors for these novel proteins. This discovery of a family of adiponectin paralogs has implications for understanding the control of energy homeostasis and could provide new targets for pharmacologic intervention in metabolic diseases such as diabetes and obesity.

Regulated cleavage and release of the extracellular domain of transmembrane precursors of several secreted growth factors. Protease cleavage and release of the extracellular domain (ECD, "ectodomain shedding") of a multitude of transmembrane proteins has been linked to the activation of many signaling pathways including the MAPK pathway. Cleavage of the ECD is mostly carried out by metalloproteases (MMPs) of the ADAM family (“a disintegrin and metalloprotease”). ECD cleavage is often followed by and is a prerequisite for intramembranous cleavage of the intracellular domain (ICD) of the same protein by γ-secretase; some of the cleaved ICDs translocate to the nucleus, where they may regulate gene transcription.

Membrane-spanning pro-hormone ligands of the epidermal growth factor receptor (HER) family are well studied examples of proteins that undergo ectodomain shedding and are physiologically important in many cellular contexts in organisms from Drosophila to mammals. But how the ectodomain cleavage machinery is regulated is largely unknown, as only a few specific stimuli which induce ectodomain shedding have been identified. Activation of the cardiac β-adrenergic receptor leads to HB-EGF-cleavage-mediated development of cardiac hypertrophy. Andreas Herrlich showed that another HER-ligand, neuregulin1β (NRG1β), is cleaved by an MMP in response to hypertonic stress and subsequently activates EGF-family receptors in an autocrine fashion. This signaling step leads to MAPK activation followed by enhanced expression of genes encoding water channels (aquaporins). Regulation of ectodomain cleavage could occur at least two levels - at the level of the MMP or via covalent modifications of the target protein, such as phosphorylation or ubiquitination on the cytosolic face.

Andreas, and Cameron Sadegh, a UROP student, are cloning novel genes that regulate ectodomain shedding using a high-throughput expression cloning strategy. They can detect cleavage of all chosen HER-ligands by either hypertonic stress, phorbol ester addition, or stimulation with lysophosphatidic acid in a FACS-based assay using mouse lung epithelial (MLE) cell clones stably expressing one of the chosen pro-hormone ligands. The ligands are tagged at the extracellular domain with one of several epitope tags; at its cytosol-facing terminus the proteins have been fused with EGFP. The extracellular epitope of the transmembrane pro-hormone ligand is detected with a fluorochrome-coupled (red) anti-epitope antibody, while the intracellular domain EGFP-fusion is detected by green fluorescence. Stimulation of cleavage results in a decrease of the red to green fluorescence ratio, while inhibition of basal or induced cleavage is reflected by an increase in this ratio. Reporter cell clones have been infected with a retroviral library generated from a cleavage competent cell line and cells that exhibit altered cleavage of the doubly tagged HER ligands. are being sorted and cloned. Genomic PCR followed by cloning will allow detection of the particular cDNA library insert in the isolated cell clones that encodes a protein that either activates or inhibits regulated ectodomain shedding. This protocol should enable Andreasand Cameron to identify and clone novel proteins that regulate shedding of the ectodomain of members of the EGF family of hormones. Hideshiro Saito-Benz will be using similar technologies to identify novel proteins that regulate cleavage of APP, a transmembrane protein whose cleavage products are thought to precipitate certain cases of Alzheimer’s disease.

 

Selected Publications
Tong, W., J. Zhang, and H. F. Lodish, Lnk inhibits erythropoiesis and Epo-dependent JAK2 activation and downstream signaling pathways Blood in the press (2005).

Marszalek, J., and H. F. Lodish. Docosahexaenoic Acid, Fatty Acid Interacting Proteins, and Neuronal Function: Breastmilk and Fish are Good For You. Ann. Rev. Cell. Dev. Biol in the press (2005)

Ruan, H. and H. F. Lodish. Dyslipidemia and Atherosclerosis: mechanism, transcriptional regulation, consequences, and treatment. Lipid Disorder Updates in the press (2005)

Marszalek, J., C. Kitidis, C. DiRusso and H. F. Lodish Long-chain Acyl CoA Synthetase 6 preferentially promotes DHA metabolism J. Biol. Chem. 280: 10817 - 10826 (2005).

Chen, C-Z., and H. F. Lodish. microRNAs as regulators of mammalian hematopoiesis. Semin. Immunol. 17: 155–165 (2005)

Hug, C. and H. F. Lodish. The role of the adipocyte hormone Adiponectin in cardiovascular disease. Current Opinions in Pharmacology 5: 129 - 134 (2005)

Zhang, C-C. and H. F. Lodish. Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood 105: 4314 - 4320 (2005).

Zhang, C-C and H. F. Lodish. Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells. Blood 103: 2513 - 2521 (2004)

Lodish, H. F., A. Berk, P. Matsudaira, C. Kaiser, M. Krieger, M. Scott, L. Zipursky, and J. E. Darnell. Molecular Cell Biology, 5th ed. Scientific American Press, N.Y. (2004).

Chen, C-Z., L. Li, H. F. Lodish, and D. P. Bartel. MicroRNAs Modulate Hematopoietic Lineage Differentiation. Science 303, 83-86 (2004)

Luo, B., A. Heard, and H. F. Lodish. siRNA production by enzymatic engineering of DNA (SPEED) Proc. Natl. Acad. Sci. USA 101: 5494 - 5499 (2004)

Ruan, H. and H. F. Lodish. Role of Adipose-Tissue-Derived Hormones and Inflammatory Cytokines in Obesity-Linked Type 2 Diabetes Curr. Opin Lipidology 15:297-302 (2004).

Marszalek, J. R., C. Kitidis, A. Dararutana and H. F. Lodish Acyl CoA Synthetase 2 (ACS2) Over-expression Enhances Fatty Acid Internalization and Neurite Outgrowth J. Biol. Chem. 279: 23882 - 23891 (2004).

Kim, J., R. E. Gimeno, T. Higashimori, H-J. Kim, H. Cho, ,S. Punreddy, R. Mozell, G. Tan, A. Stricker-Krongrad, D. J. Hirsch, J. J. Fillmore, Z-X. Liu, J. Dong, G. Cline, A. Stahl, H. F. Lodish, and G. I. Shulman. Inactivation of Fatty Acid Transport Protein 1 Prevents Fat-Induced Insulin Resistance In Skeletal Muscle J. Clinical Investigation 113: 756 - 763 (2004).

Zhang, J., and H. F. Lodish Constitutive activation of the MEK/ERK pathway mediates all effects of oncogenic H-ras expression in primary erythroid progenitors. Blood 104: 1679 – 1687 (2004)

Hug, C., J. Wang, N. Ahmad, J. Bogan, T.-S. Tsao, and H. F. Lodish. T-cadherin is a receptor for hexameric and high molecular weight forms of Acrp30/adiponectin. Proc. Natl. Acad. Sci. USA 101: 10308 - 10313 (2004)

Tong, W., and H. F. Lodish. Lnk inhibits Tpo/mpl signaling and Tpo-mediate megakaryocytopoiesis. J. Exp. Med. 200: 569 - 580 (2004)

Wong, G., J. Wang, C. Hug, T.-S. Tsao, and H. F. Lodish. A family of Acrp30/adiponectin structural and functional paralogs Proc. Natl. Acad. Sci. USA 101: 10302 - 10307 (2004)

Ruan, H. and H. F. Lodish. Insulin Resistance in Adipose Tissue: Direct and Indirect Effects of Tumor Necrosis Factor-a. Cytokine and Growth Factor Reviews 14: 447-455 (2003).

Gimeno, R. E., A. Ortegon. S. Patel, S. Punreddy, P. Ge, Y. Sun, H. F. Lodish, and A. Stahl Identification of a Heart-specific Fatty Acid Transport Protein J. Biol. Chem. 278: 16039 - 16044 (2003)

Zhang, J., M. Socolovsky, A. W. Gross, and H. F. Lodish. Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry-based novel culture system. Blood 102: 3938 - 3946 (2003)

Tsao, T-s., E. Tomas, H. E. Murrey, C. Hug, D. H. Lee, N. B. Ruderman, J. E. Heuser, and H. F. Lodish. Role of Disulfide Bonds in Acrp30/Adiponectin Structure and Signaling Specificity: Different Oligomers Activate Different Signal Transduction Pathways. J. Biol. Chem. 278: 50810 - 50817 (2003).

Ghaffari, S., Z. Jagani, C. Kitidis, H. F. Lodish and R. Khosravi-Far. Cytokines and BCR-ABL Mediate Suppression of TRAIL-Induced Apoptosis through Inhibition of FOXO3a Transcription Factor. Proc. Natl. Acad. Sci. USA 100: 6523 - 6528 (2003).

Gimeno, R. E., D. J. Hirsch, S. Punreddy, Y. Sun, A. M. Ortegon, H. Wu, T. Daniels, A. Stricker-Krongrad, H. F. Lodish, and A. Stahl. Targeted Deletion of Fatty Acid Transport Protein-4 Results in Early Embryonic Lethality. J. Biol. Chem. 278: 49512 – 49516 (2003)

Ruan, H., M. Zarnowski, S. Cushman, and H. F. Lodish. Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte-genes. J. Biol. Chem. 278: 47585 - 47593 (2003)

Chen, C-Z., L. Li, M. Li, and H. F Lodish The Endoglin^Positive Sca-1^PositiveRhodamine^Lowphenotype defines a near homogeneous population of long–term repopulating hematopoietic stem cells. Immunity 19: 525 - 533 (2003).

Ruan, H., H. J. Pownall, and H. F. Lodish. Troglitazone antagonizes TNF-a-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-kB J. Biol. Chem. 278: 28181 - 28192 (2003)

Bogan, J., N. Hendon, A. McKee, T-s Tsao, and H.F. Lodish Functional cloning of TUG as a regulator of GLUT4 glucose transporter trafficking. Nature 425: 727 – 733 (2003)

Ketteler, R., C. S. Moghraby, J. G. Hsiao, O. Sandra, H. F. Lodish, and U. Klingmüller The cytokine-inducible SH2 domain containing protein CIS negatively regulates signaling by promoting apoptosis in erythroid progenitor cells. J. Biol. Chem. 278: 2654 - 2660 (2003)

Ghaffari S., L. J. S. Huang, J. Zhang and H. F. Lodish. Erythropoietin Receptor Signaling Processes, in Erythropoietins and Erythropoiesis: Molecular, Cellular, Preclinical, and Clinical Biology, Graham Molineux, MaryAnn Foote, and Steven Elliott, editors, Birkhauser Publishing, (2003)

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photo credit: Justin Allardyce Knight