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Research Overview
Research in my lab focuses on five important areas at the interface between molecular cell biology and medicine:
A. Red cell development and hematopoiesis.
Red blood cell development, especially on the role of the erythropoietin receptor and cell surface integrins in controlling terminal proliferation and differentiation of erythroid progenitor cells;
Hematopoietic stem cells, defining new cell surface proteins for their purification and new growth factors that support their expansion in culture;
C. MicroRNAs that regulate mammalian development.
MicroRNAs, defining their roles in lineage commitment of hematopoietic stem and progenitor cells, and regulating muscle and adipocyte differentiation;
D. Hormones controlling fatty acid and glucose metabolism.
Adiponectin, a hormone we cloned that is made exclusively by fat cells and that increases fatty acid and glucose metabolism by muscle, and several adiponectin ortholog proteins that share adiponectin’s biological activities;
E. New signaling pathways and new technologies.
Regulated cleavage and release of the extracellular domain ("ectodomain shedding") of transmembrane precursors of several secreted growth factors.
What ties all of these projects together is their focus on the basic cell and molecular biology of proteins important for human metabolism and disease. Another is the development and use of powerful tools of molecular biology including expression cloning strategies such as those we used to clone most of the receptors, transporters, and several signaling proteins we study, techniques for immunolocalizing proteins in cells and tissues, and generation and analysis of many types of gene- altered mice.
Erythropoietin receptor (EpoR) and red cell development:
Erythropoietin (Epo) controls production of red blood cells; it is produced by the kidney in response to low oxygen pressure in the blood. Epo binds to Epo receptors on the surface of committed erythroid late BFU-E and CFU-E progenitors, blocking the apoptosis (programmed cell death) that is their usual fate, and triggering them to undergo a two- day program of 4 - 5 terminal cell divisions and erythroid differentiation, forming red cells that are released into the blood. As evidenced by the properties of Epo- and EpoR- deficient mice we generated, 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 bclxL. Stat5-/- mice
exhibit fetal anemia and increased apoptosis of erythroid progenitors
caused by reduced bclxL 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. Additionally, we showed that activated Akt phosphorylates the erythroid important transcription factor GATA-1 both in vitro and in vivo and enhances GATA-1 activity in erythroid cells.
How Epo stimulation activates EpoR-associated JAK2 is a central question
in cytokine receptor signaling. Xiaohui Lu has taken two different approaches
to address this question. By screening libraries of EpoRs with random
mutations in the transmembrane domain Xiaohui 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. Furthermore, Xiaohui identified a single
amino acid substitution in the middle of the EpoR transmembrane domain
that constitutively activates JAK2 and EpoR signaling. To pinpoint the
exact structural changes in the EpoR transmembrane domain that render
the JAK2 activation, Xiaohui is embarking on structural studies on wild
type and mutant transmembrane peptides. To this end, he was able to express
the transmembrane peptides in a native membrane-embedded form in E. coli,
and purify isotope-labeled peptides to homogeneity. He is determining
the structure of peptides corresponding to these dimeric active alpha
helixes through NMR; this should shed light on the structure of the Epo-
activated receptor transmembrane domain.
Xiaohui is also investigating the hypothesized trans-phosphorylation of
JAK2 upon Epo stimulation, and the roles of the contributing structural
elements in EpoR that lead to JAK2 activation. In collaboration with Bruce
Tidor’s lab in the MIT Department of Bioengineering, he created
two sets of mutant EpoRs that are unable to bind to just one of the two
receptor-binding sites of Epo, Site-1 and Site-2. When expressed individually
in Ba/F3 cells, neither the Site-1 specific nor the Site-2 specific EpoR
can respond to Epo, since no productive dimers is formed. However, when
the mutant EpoRs were co-expressed in the same cells, Epo-response is
restored to the wild-type level. With these Site-1 specific and Site-2
specific- deficient EpoRs, Xiaohui determined that only one set of the
hydrophobic switch resides in the juxtamembrane domain of EpoR (L253,
I257, and W258) is required for the activation of JAK2, and it locates
on the Site-1 binding EpoR. This is the first indication that JAK2 activation
is initiated from one side of the asymmetric two-receptor one - Epo complex.
With this unique engineered heterodimeric system, Xiaohui is testing test
the hypothesis of trans-phosphorylation of JAK2 and dissecting how the
signal is transdued to STAT5 and other downstream molecules.
Disregulation of JAK2 signaling has been implicated in several hematological
malignancies. One example, an acquired point mutation in the JAK2, V617F,
was recently discovered in most patients with polycythemia vera and half
of those with other myeloproliferative disorders. It is unclear why this
oncogenic JAK2 mutation is reponsible for such related yet very distinct
diseases. In collaboration with Dr. Gary Gilliland’s lab in Harvard
Medical School, Xiaohui Liu and Wei Tong showed that both cell transformation
by JAK2V617F and constitutive activation of the JAK/STAT signaling pathway
requires the presence of cognate type I cytokine receptors. Using IL-3-dependent
Ba/F3 cells and 32D cells, Xiaohui and Wei showed that expression of JAK2V617F
confers factor-independent growth only in cells coexpressing homodimeric
cytokine receptors such as the erythropoietin receptor (EpoR), thrombopoietin
receptor, or granulocyte colony-stimulating factor receptor. Furthermore,
these cytokine receptors are also required for the constitutive phosphorylation
and activation of both JAK2 and STAT5. By coexpressing JAK2V617F together
with EpoR mutants, they demonstrated that EpoR provides an essential scaffold
for factor-independent activation of JAK2V617F and STAT5; in particular
phosphorylation of tyrosines in the cytosolic domain of the EpoR is essential
for activation of STAT5. We conclude that JAK2V617F transduces oncogenic
signals in conjunction with cytokine receptors, in a cytokine-independent
version of its normal signaling mechanisms. These findings provide a molecular
basis for the prevalence of JAK2V617F in diseases of myeloid cells that
express Type I cytokine receptors, and explain the overlapping clinical
observations in these diseases.
Wei Tong extended these results in a collaboration with Dr. Gary Gilliland’s
lab and that of Dr. Tony Green of the University of Cambridge. While the
JAK2 V617F mutation is found in many patients with myeloproliferative
disorders the molecular basis for most V617F-negative patients with a
myeloproliferative disorder was unclear. In patients diagnosed with V617F-negative
polycythemia vera or idiopathic erythrocytosis four different somatic
gain-of-function mutations affecting JAK2 exon 12 were identified. Patients
with a JAK2 exon 12 mutation presented with a histologically distinct
variant of polycythemia vera, characterized by an isolated erythrocytosis,
erythropoietin-independent erythroid colonies, and suppressed serum erythropoietin.
In contrast to the V617F mutation, exon 12 mutations were associated with
an absence of detectable mutation-homozygous erythroid progenitors. Exon
12 mutations resulted in cytokine-independent proliferation of BaF3 cells
expressing the EpoR cells, together with increased activation of downstream
signaling pathways. Thus JAK2 exon 12 mutations give rise to a distinct
variant of polycythemia vera. Our results emphasize the importance of
molecular genetics for the classification and diagnosis of the myeloproliferative
disorders, and suggest that the different clinical phenotypes associated
with JAK2 exon 12 and V617F mutations may reflect stronger signaling by
the former.
Most researchers agree that cognate cytokine receptors, such as EpoR,
are important for the oncogenic activities of JAK2V617F. However, others
reported contradictory results in Ba/F3 cells, suggesting JAK2V617F is
constitutively active by itself. To this end, Xiaohui demonstrated that
differences in JAK2V617F expression levels likely account for this critical
discrepancy, and overexpressed JAK2V617F functions through binding to
unidentified cytokine receptor dimers. Xiaohui further demonstrated that
receptor-mediated dimerization is critical for the activity of JAK2V617F,
and the EpoR intracellular hydrophobic motif (L253, I257 and W258) that
is essential for Epo-mediated JAK2 activation is not required for the
activation of JAK2V617F. These results support the notion that receptor-mediated
dimerization of JAK2V617F is required and sufficient for its activation.
Furthermore, the constitutively activated JAK2V617F may have subtle structural
differences from the Epo-activated wild-type JAK2, raising the possibility
of developing a JAK2V617F-specific inhibitor.
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 degradation of radioiodinated Epo 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. A long- lived commercial mutant Epo binds slower and dissociates
more rapidly from surface Epo receptors, but otherwise the kinetics of
internalization, resecretion, and degradation are indistinguishable from
that of normal Epo. As Alec’s kinetic modeling showed, these altered
receptor- binding kinetics can explain its longer half- life in vivo.
In an ongoing collaboration, Alec is using computational models generated
in Dr. Bruce Tidor’s laboratory to better understand how Epo binds
to EpoR in different cellular compartments and how this affects cellular
trafficking. Computational molecular modeling predicted that the EpoR
has a naturally occurring “Histidine Switch” in one of the
binding interfaces between Epo and the EpoR. That is to say, at the neutral
pH of the cell surface, this crucial histidine will be uncharged, but
at the more acidic pH in endosomes this histidine will become protonated
and a positive charge will be introduced into the binding interface. The
prediction is that the presence of a positive charge at this position
will decrease the affinity of Epo for EpoR, and thus this histidine will
play an important role in dissociating the Epo- EpoR complex and in the
cellular trafficking of Epo•EpoR complexes following their internalization.
Alec has experimentally tested these predictions and shown that there
is indeed a naturally occurring Histidine Switch in the EpoR. Binding
of Epo to EpoR is strongly pH-dependent, as shown by a greatly decreased
affinity of Epo for EpoR at pH 5.5 compared to pH 7.3. When Alec mutated
the histidine in question to amino acids with non-pH- sensitive side chains,
the affinity of Epo for EpoR was only slightly decreased at pH 5.5 compared
to pH 7.3. Thus, as predicted by molecular modeling, a single histidine
in the EpoR is primarily responsible for the pH-dependent binding of Epo.
Trafficking of Epo was altered in cells expressing the mutant EpoRs that
have decreased pH-dependent binding: intact Epo was retained intracellularly
for an abnormally extended time and Epo degradation was delayed. Our results
suggest that sorting steps in early recycling endosomes, where the pH
is only mildly acidic, are not affected by pH-dependent binding of Epo
to EpoR. However, pH-dependent binding of Epo plays an important role
in later sorting and degradation steps. Under this scenario, if Epo does
not dissociate from EpoR in the more acidic late endosomes, as with the
histidine-mutant EpoRs, there is a delay in trafficking to and/or the
actual degradation of Epo in lysosomes.
Due to the low surface expression and large intracellular pool of EpoR,
no one has been able to specifically label and follow the trafficking
of cell surface EpoR, where signaling is initiated, either before or after
Epo addition. Alec is currently applying a new technique to specifically
label EpoR at the cell surface with an affinity tag. This will allow him
to determine the relationship between EpoR signaling and receptor endocytosis
and trafficking, and to isolate and examine Epo•EpoR complexes and
associated proteins at different times after initiation of EpoR signaling.
Although Alec’s kinetic experiments show how Epo is degraded in
cells that express EpoR, it is possible that clearance of Epo from the
circulation and degradation in vivo may occur through more than one mechanism:
extracellular proteases and other enzymes could degrade or modify Epo,
or Epo could be taken up through unknown mechanisms and degraded in cells
that do not express the EpoR. To test if Epo degradation primarily occurs
by binding of Epo to its receptor, Alec will inject both wild- type Epo
with a mutant Epo that cannot bind to any surface EpoR and measure their
rates of degradation in vivo.
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. Two years ago 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 overexpression 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.
To avoid problems due to oncogenic Ras overexpression, Jing, assisted by Yangang Liu, studied primary erythroid progenitors in which oncogenic K-ras is expressed from its endogenous promoter. Expression of oncogenic K-ras is induced for a short period of time using a rtTA/TetO-cre system, and oncogenic K-ras signaling was assessed in highly purified primary erythroid progenitors. In contrast to results obtained when oncogenic Ras was overexpressed from retroviral vectors, endogenous levels of K-rasG12D fail to constitutively activate but rather hyperactivates cytokine-dependent signaling pathways, including Stat5, Akt, and p44/42 MAPK, in primary erythroid progenitors. This explains previous observations that hematopoietic progenitors expressing endogenous K-rasG12D display hypersensitivity to cytokine stimulation in various colony assays. In addition, unlike the overexpression situation, expression of endogenous K-rasG12D does not support primary erythroid progenitors to grow independent of cytokines. Rather, it leads to a partial block of terminal erythroid differentiation and mild hyperproliferation of erythroblasts.
Another current project, conducted by Shilpa Hattangadi, involves determining important 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)" isolated from progenitors in progressive stages of development and eventually, confirmation by RT-PCR and other methods. Initial results indicate that major changes in gene regulation occur midway during erythroblast differentiation. Upregulated genes include those involved in hemoglobin metabolism, heme or porphyrin ring metabolism, cell and nuclear membrane structure, iron ion homeostasis, negative regulators of cell proliferation, oxygen transport, and oxygen and reactive oxygen species metabolism, among others. Genes that were significantly downregulated included genes involved in glyoxylate metabolism, TNF-alpha production, NADP metabolism, NF-kappaB binding, actin binding, ubiquitin protein ligation, and non-erythroid specific functions such as immune response, antigen stimulation and response, and phagocytosis, among others. Further clustering analysis is ongoing, revealing interesting sets of genes for further study and future location analysis experiments.
A related project, done by Shilpa in collaboration with members of Rick Young's laboratory, involves immunoprecipitation of chromatin with antibodies specific for various transcription factors, followed by hybridization of the recovered DNA to a promoter DNA microarray. This protocol will enable her 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, GATA1, FOG, and Foxo3 but other factors will soon be investigated. Shilpa's long- term goal is to understand how the complex pattern of gene expression during terminal erythroid differentiation is regulated 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. Fibronectin is an important part of the erythroid niche but its precise role in erythropoiesis is unknown. We showed some years ago that adhesion of these progenitors to fibronectin is essential for normal erythroid development. Shawdee Eshghi showed that both alpha4beta1 and alpha5beta1 integrins are present on erythroid progenitors and support binding of erythroid cells to different fibronectin domains. Shawdee also showed that loss of both alpha4beta1 and alpha5beta1 integrins during erythroid differentiation parallels the loss of adhesion of erythroid cells to fibronectin; this allows retention of immature erythroid cells in the bone marrow and then release of the enucleated reticulocytes into the circulation.
Using our in vitro culture system Shawdee showed that alpha4beta1 integrin is required for terminal proliferation of erythropoietic progenitors. More specifically, alpha4beta1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis. By culturing fetal liver erythroid progenitors she showed, in a collaboration with the lab of Prof. Richard Hynes, that fibronectin and Epo regulate erythroid proliferation in temporally distinct steps: an early Epo-dependent phase is followed by a fibronectin-dependent phase. In each phase, Epo and fibronectin promote expansion by preventing apoptosis, in part through enhancing expression of the antiapoptotic protein bcl-xL. By culturing erythroid progenitors on recombinant fibronectin fragments she established that only substrates that engage alpha4beta1 integrin support normal proliferation. Taken together, these data suggest a two-phase model for growth factor and extracellular matrix regulation of erythropoiesis, with an early Epo-dependent, integrin-independent phase followed by an Epo-independent, alpha4beta1 integrin-dependent phase. Shawdee is currently determining the signal transduction pathway that connects alpha4beta1 integrin to induction of bcl-xL expression, focusing on integrin- linked kinases, Src family tyrosine kinases, MAP kinases, Ras, Rac, and the Jak2/STAT5 and PI3K/Akt pathways.
Mammalian erythroid cells undergo enucleation in the late stages of differentiation, a process that does not occur in other vertebrates. This process has critical physiological and evolutional significance for the morphogenesis and hemoglobin enrichment of mature mammalian erythroid cells. Although enucleation has been known for decades the mechanisms that regulate the process remain obscure. Using the new in vitro culture system of fetal liver erythroid progenitors Jing developed, Peng Ji is investigating the mechanism of mammalian erythroid cell enucleation.
Since actin filaments have been show to be critical for enucleation, Peng determined the role of different Rho GTPases, the master regulators of actin nucleation, on enucleation. Peng showed that deregulation of Rac GTPase during the late stages of erythropoiesis completely blocks enucleation of cultured mouse fetal erythroblasts without affecting their normal proliferation and differentiation. The contractile actin ring formed on the plasma membrane of late-stage erythroblasts and the boundary between the cytoplasm and nucleus of enucleating cells was disrupted when Rac GTPase was inhibited in late stages of erythropoiesis. Peng further demonstrated that the Rac GTPase activity is mediated by a downstream target protein, a protein required for nucleation of unbranched actin filaments. These results reveal important roles for Rac GTPase and its effector proteins in enucleation of mammalian erythroblasts. Peng is also focusing on the role of histone deacetylases as well as different erythroid specific membrane and cytoskeletal proteins in mammalian erythroid cell enucleation.
Diamond Blackfan anemia (DBA) is a congenital anemia and broad developmental disease that develops at birth or soon after. The anemia is due to failure of production of erythrocytes and their precursors, with normal or near normal myeloid cells and platelets. DBA is inherited in about 10-20% of cases, mostly as an autosomal dominant. Recent genetic studies have led to the surprising identification of mutations in a ribosomal protein (RP) gene, RPS19, on chromosome 19q, in 20 - 25% of both familial and sporadic cases. Recently Colin Sieff and colleagues discovered a mutation in another ribosomal protein gene, RPS19, that co-segregates with affected family members in a large DBA pedigree. While experiments in yeast and recently in mammalian cells show that RPS19 depletion or mutation leads to a block in ribosomal RNA biosynthesis, this result does not explain why erythropoiesis is so severely affected in DBA. We hypothesize that during fetal development immature erythroid cells proliferate more rapidly than other lineages and therefore require very high ribosome synthetic rates to generate sufficient capacity for translation of erythroid specific transcripts that must take place before these unique cells enucleate; furthermore, we postulate that the relative deficiency in ribosomes and protein synthetic capacity that occurs in RPS19/RPS24 mutant DBA cells leads to loss of proliferation and cell death of rapidly dividing cells, but survival and normal differentiation of clones that are dividing more slowly, yielding fewer macrocytic erythrocytes.
To test this hypothesis Colin, assisted by Lilia Long infected primary mouse fetal liver erythroid progenitor cells with siRNAs to RPS19 and compared proliferation, differentiation, RNA biogenesis and cell cycle status in wild type and knockdown cells. They first measured the RNA content of wild type cells during the two days of erythroid differentiation. During the first 24 hours cell number increases 3-4 fold; remarkably, there is a 6-fold increase in RNA content during the same period, suggesting that the cells accumulate a relative excess of ribosomal RNA (80% of measured RNA) during early erythropoiesis. This was confirmed by quantitative real time PCR of rRNA. From 24-48 hours the cells decrease in size as they divide twice more and mature, and RNA content per cell decreases; however, cell numbers increase markedly so the net effect is that total RNA in the culture plateaus or decreases. Because the siRNAs are not expressed until 24-48 hours, we modified the culture system to allow expansion without differentiation of progenitor cells (in EPO, IGF-2 and dexamethasone). Under these conditions proliferation in RPS19 siRNA- expressing precursors is reduced significantly. We are currently using Hoechst 33342 in vitro and BrdU in vivo to examine cell cycle status at different stages of erythroid maturation. Taken together, these data suggest that RPS19 insufficient erythroid cells proliferate poorly because of inadequate accumulation of ribosome synthetic capacity, although it is not possible yet to exclude the possibility that apoptosis is also triggered by the lack of RPS19. The surviving cells differentiate normally but slowly, giving rise to macrocytes.
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 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. Using neuronal cells stably expressing an EpoR shRNA and lacking EpoR expression, she showed that high affinity binding of Epo to these neuronal cells is mediated by this EpoR and that it is essential for the antiapoptotic activity of Epo. From her studies on a dominant negative Epo mutant that binds only to one cell surface EpoR molecule, Moon concluded that the antiapoptotic activity of Epo in these neuronal cells is mediated through the “classical” Epo receptor signaling complex with the standard stoichiometry of one molecule of Epo binding to two EpoR subunits. Moon is currently studying the role of Epo and EpoR in neurodegenerative states, using several mouse models derived from disease and drug treatments. 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 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 has adapted our in vitro erythropoiesis culture system to induce optimized erythropoietic growth from the Lin- population of adult murine bone marrow. Using this system he demonstrated that exposure to genotoxicants induces micronucleus -formation in this culture system. In particular, Joe showed that addition of the alkylating agents BCNU, MNNG, and MMS to this culture system induces both a cytotoxic response and an increase in micronucleus frequency within the reticulocyte population. This increase in micronucleus production following exposure to these alkylating genotoxicants provides a clear signal of the genotoxic mechanism that likely induced the observed erythropoietic toxicity.
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-beta receptor, as a surface marker for long-term repopulating
mouse bone marrow HSCs. He showed that bone marrow cells purified by the
EndoglinPositive Sca-1Positive RhodamineLow
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
EndoglinPositive Sca-1Positive RhodamineLow
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 CD3M+ 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 showed that Angiopoietin-like 2 and 3, secreted proteins specifically produced by day 15 fetal liver CD3+ Ter119- cells, as novel hormones that also stimulate ex vivo expansion of HSCs. Chengcheng showed that, when used in serum- free media in combination with other growth factors, these proteins stimulate a 24-30-fold expansion of HSCs following 10 days of culture of highly enriched mouse stem cells. Ongoing work indicates that a similar “cocktail” of five growth factors in serum- free medium will support a ~30- fold expansion of human hematopoietic cord blood stem cells. The receptor(s) for these Angiopoietin-like proteins and the signal transduction pathway(s) they activate are unknown. A main focus of Chengcheng’s current research in his own laboratory at the University of Texas Southwestern Medical School is cloning these receptors, and also deciphering the specific intracellular signal transduction pathway(s) and transcriptional activations induced by these proteins both in cell lines and HSCs.
Hematopoietic stem cell environments or niches are very important in determination of HSC self-renewal and differentiation. Fibroblasts and osteoblasts have been described as important constituents and regulators of HSC niches. We are interested in characterizing additional cell types that contribute to regulation of HSC microenvironments and as noted we uncovered one such population from fetal liver. Megan Kaba and Alec Babic are studying different populations of T lymphocytes and their potential to interact with and regulate stem cell self renewal and expansion during fetal and adult hematopoiesis. Specifically, Megan and Alec are performing coculture experiments to further characterize which populations of fetal liver cells are able to enhance the ex vivo expansion of long-term repopulating adult bone marrow HSCs. Of interest are the embryonic day 15 fetal liver CD3+ Ter119- cells that express the T cell alphabeta or gammadelta receptors. Characterization of a specific type of supportive cell will aid in the identification of yet other new growth factors 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 and Megan are 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 (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 and Northern Blot analysis to identify several miRNAs specifically upregulated in isolated populations of thymic and splenic hematopoietic cells: 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 has confirmed these results by Northern blotting. Based on these results, five microRNAs (miR-195, miR-150, miR-106, miR-181a, and miR142) were chosen for further analysis. miR-150 and miR-181a were highly expressed in the thymus and spleen, the major secondary lymph organ, leading to our hypothesis that these two miRNAs are involved in lymphopoietic regulation.
To study the potential regulatory roles of these miRNAs in T cell function Beiyan and Stephanie Wang, a UROP student, have used retroviral infection of two clonal CD8+ T-cell lines to establish 8 stable clonal CD8+ T-cell lines that ectopically express either miR-106, miR-142, miR-150, or miR-195. She is currently establishing CD4 clonal cell lines that overexpress these selected miRNAs. These infected T-cell clones will be used for experiments to examine the effects of miRNAs on mature T-cell function.
Beiyan also studied the expression patterns of these selected microRNAs in detail during several various developmental stages of both B and T cells. miR-150, is mainly expressed in the lymph nodes and spleen, and is highly upregulated during the development of mature T and B cells. In particular, expression of miR-150 is sharply upregulated at the immature B cell stage. In contrast, expression of miR 181 is sharply downregulated during B cell development, and as noted we showed that overexpression of miR 181 in hematopoietic stem/ progenitor cells led to a significant increase in production of mature B cells. To explore the roles of miR-195 and miR-150 in lymphopoiesis she generated retroviral constructs that express each of these miRNAs. Ectopically expressed miR-150 (but not miR 195) elicited a significant inhibition of B cell development, and had little effect on other hematopoietic lineages. Further analysis showed that miR 150 overexpression did not affect B cell lineage commitment, as evidenced by unaltered pro-B cell numbers both 4 and 16 weeks after transplantation. Overexpression did block the pro-B to pre-B transition in the bone marrow. We hypothesize that premature expression of miR-150 in hematopoietic stem/progenitor cells inhibits production of proteins specifically required for early B cell development. Several potential mRNA targets were predicted by TargetScan screening. The mechanism of miR-150 regulation in B cell development is being further investigated by studying these potential targets, in particular B lineage specific transcription factors.
Acute lymphoblastic leukemia (ALL) is one of the most common and fatal
malignancies of children and young adults. Ai Kotani aims to identify
and characterize specific miRNAs that are critical in the development
and progression of MLL related leukemias that show poor prognosis. In
MLL related ALL, expression of many miRNAs is downregulated, raising the
possibility that downregulation of some miRNAs plays a critical role in
the pathogenesis of this leukemia. miR-128 is one of these. Ai Kotani
and Daon Ha, a UROP student, identified in the RS4; 11, a cell line, derived
from a MLL-AF4 ALL patient, a novel mutation in the miR-128b gene segment.
This mutation is transcribed in the primary miR- 128 transcript and blocks
the processing of the miR-128 precursor to mature miRNA. The RS4; 11 cell
line is resistant to glucocorticoid- induced apoptosis, and Ai showed
that overexpression of the wild- type miR-128 gene in these cells restored
glucocorticoid- induced apoptosis. She is currently determining whether
the same mutation is found in the miR-128 gene from several primary MLL-AF4
ALL tumors. These results indicate that the resultant downregulation of
the level of mature miR-128b is a major cause of the glucocorticoid resistance
associated with poor prognosis of these tumors. Presumably this is because
miR-128 normally downregulates certain mRNAs encoding proteins that in
some way inhibit or reduce glucocorticoid action, and she is trying to
elucidate the target mRNAs of this miRNA. Ai Kotani and Daon Ha are also
studying the functions of other miRNAs downregulated in MLL-AF4 ALL.
Prakash Rao has identified three microRNAs, miR-1, miR-133 and miR-206 that are upregulated during the differentiation of the C2C12 myoblast cell line into myotubes. Using gene-specific PCR, Prakash and Mina Farkhondeh, a UROP student, have identified the specific isoforms of these induced microRNAs. These studies led to the identification of myogenic transcription factors that appear to be responsible for their upregulation during C2C12 differentiation. In collaboration with a postdoctoral fellow, Dr. Roshan Kumar of the Young lab at Whitehead, Prakash and Mina determined that Myogenin and MyoD are bound to DNA specific DNA sequences upstream of the DNA encoding these microRNAs; this result neatly explains the upregulation observed during myogenic differentiation. They are extending these observations to other known microRNAs that are regulated during the myoblast to myotube transition.
The observation that miR-1, miR-133 and miR-206 are induced during the differentiation of a myoblastic line have led them to consider their potential mRNA targets in muscle differentiation. In particular, they have focused on the ability of miR’s to target components of conserved regulatory cascades that inhibit myogenesis, and on the regions within the 3` UTRs of these mRNAs that possesses miR-1 binding sites. They showed that miR-1 can inhibit reporter genes bearing regions of some of these potential target mRNAs. Overall, these studies point to the importance of miR-1 in inhibiting expression of important myogenic mRNAs and they have initiated studies in rhabdomyosarcoma—a muscle derived tumor—in which the deregulation of miR-1 could play an important role in oncogenicity.
Prakash, along with Anna Poukchanski, another UROP student, is also cloning a seed-based library of microRNAs for use in functional assays. These function-based assays will serve to further refine the roles of microRNAs within the context of a well-defined biological pathway. Greg Hyde, who is a visiting scientist on a sabbatical from Massasoit Community College, is building reporter constructs to assess the possible impact of these induced miRNAs on the expression of genes associated with muscle cell differentiation. Prakash is also studying the impact of the global loss of all microRNAs by specifically deleting in muscle tissue a gene required for microRNA biogenesis.
Following the work of I-hung Shih, a former postdoctoral fellow in the
Bartel lab who worked closely with us, Huangming Xie is examining the
role of miRNAs in adipogenesis using several adipocyte cell culture differentiation
systems. Using microRNA microarrays he has identified several microRNAs
that are regulated during adipocyte differentiation and has confirmed
the results by real time RT-PCR. Currently, Huangming is characterizing
the role in adipogenesis of these miRNAs by both gain-of-function and
loss-of-function approaches. He is also investigating the mRNA targets
of these candidate miRNAs and examining the regulation of miRNA expression
using bioinformatics tools followed by experimental validation. Understanding
the role of miRNAs in adipose biology will allow us to utilize them in
the future as diagnostic markers and/or therapeutic targets for metabolic
diseases such as diabetes.
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-alpha even
though there is no homology at the primary sequence level. Like TNF-alpha
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 by muscle. 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. Tsu-Shuen has moved to the University of Arizona where he is an assistant professor.
AMPK is composed of three subunits - the alpha kinase subunit that undergoes
regulated phosphorylation, the gamma subunit that binds AMP, and the beta
subunit that is thought to act as a scaffold that binds to both the alpha
and gamma 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 re-determining the
interactions of the three AMPK subunits. Most significantly, Kelly showed
that the alpha -subunit binds directly to the gamma-subunit, in striking
contradiction to the “standard” model. He also showed that
the “scaffolding” beta-subunit does not bind directly to the
gamma-subunit; interactions of the beta- and gamma- subunits can be detected
only if the alpha-subunit is also present. Thus his data suggests a model
for AMPK structure in which the beta-and the gamma-subunit bind directly
to the alpha-subunit, and in which the beta-subunit does not bind directly
to the AMP- sensing gamma-subunit. Kelly is also working on determining
the physiological role of the beta-subunit and its importance in glycogen
binding. To this end he has undertaken an in vivo loss of function approach
to create and analyze mice deficient in the beta-subunit, and also mice
that harbor a specific germ line point mutation in the beta-gene that
deletes its glycogen binding ability. Kelly also aims to determine how
adiponectin and other cytokines such as IL-6 are able to stimulate AMPK
activation and in the process identify other proteins that connect AMPK
to the elusive adiponectin signaling receptors.
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. At its C-terminus T-cadherin
is attached to the membrane via a GPI anchor. Chris’ preliminary
studies indicate that T-cadherin 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.
Immunohistochemical localization of adiponectin demonstrated that mice
lacking T-cadherin had no detectable binding of adiponectin to the vascular
endothelium, in contrast to wild-type animals that had substantial binding
of adiponectin to the endothelium. T- cadherin is upregulated following
vascular injury and Chris hypothesizes that, by binding to adiponectin,
it may play a role in atherosclerosis progression as well as blood vessel
formation and endothelial cell function.
Furthermore, T-cadherin null mice demonstrate hepatic insulin resistance,
a phenotype virtually identical to that of adiponectin knockout animals.
In conjunction with Dr. Gerry Shulman’s lab at Yale Medical School,
Chris is characterizing the metabolic and physiologic abnormalities of
mice lacking T-cadherin, which may mimic those of the metabolic syndrome.
Using the hyperinsulinemic euglycemic clamp technique on mice fed a high-fat
diet for three weeks, they demonstrated that after an overnight fast there
was no difference in insulin stimulated whole body glucose uptake, glycolysis,
and glycogen synthesis. However, hepatic glucose production rates in T-cadherin
deficient animals during the clamp were significantly higher than those
of the control animals. Thus, T-cadherin deficient mice demonstrate hepatic,
but not peripheral, insulin resistance. These results confirm that T-cadherin
is a bonafide receptor for high-molecular weight forms of adiponectin,
and that loss of T-cadherin causes a metabolic phenotype similar to that
reported for loss of adiponectin.
Currently Chris is determining the role of T-cadherin in adiponectin activation
of the AMPK and NF-kB signal transduction pathways, and is studying the
downstream pathways activated by adiponectin binding to T-cadherin. As
T-cadherin lacks a transmembrane domain, and is thus not likely to be
a signaling receptor, Chris and Jin are also cloning other cell surface
adiponectin receptors including those that directly activate AMPK. Finally,
as adiponectin shares significant structural but not sequence similarities
with TNF-alpha, Chris is working with Michael Xiang, an MIT undergraduate,
to test all the known TNF-alpha superfamily receptors for possible binding
to recombinant adiponectin.
Adiponectin has important roles in enhancement of insulin sensitivity and these beneficial effects are closely associated with the activation of AMP-activated protein kinase (AMPK) in muscle and liver. How adiponectin activates AMPK is not known. Interestingly, AMPK activation by adiponectin is accompanied by an increase in concentration of 5’AMP, which implies the presence of signal transduction proteins in a pathway connecting the adiponectin signal with AMPK. Qingqing Liu is determining, first, what metabolic or signaling pathway(s) downstream of adiponectin receptors leads to this rise in 5’AMP. Second, she is determining whether this rise in 5’AMP is necessary for activation of AMPK. Activation of long-chain fatty acids to the CoA derivative is one important metabolic process that directly generates 5’AMP, and at least some Acyl CoA synthase isoforms are on the plasma membrane. She therefore proposes that one or more of Acyl CoA synthase enzymes are directly coupled to adiponectin receptors, possibly to T- cadherin. Her preliminary data show that free fatty acids, essential substrates for the production of 5’AMP by acyl-CoA synthetases, are required for AMPK activation by adiponectin in C2C12 myocytes. Using a cell-permeant acyl-CoA synthetase inhibitor, triacsin C, she confirmed a role for acyl-CoA synthetases in adiponectin activation of AMPK. She is currently determining which acyl-CoA synthetases are involved in adiponectin activation of AMPK.
Although the effects of adiponectin on glucose and lipid metabolism in
liver and skeletal muscle were reported to be mediated by two receptor
isoforms, AdipoR1 and AdipoR2, work in several laboratories including
our own failed to confirm these as receptors. As T-cadherin is attached
to the plasma membrane by a glycosylphosphatidylinositol anchor and lacks
any transmembrane or cytoplasmic domain, it is also unlikely to be a signaling
receptor. Qingqing intends to identify adiponectin receptors using a biotin-label-transfer
method. These studies are essential to understand the mechanisms underlying
adiponectin activity and potentially exploit these to develop new strategies
to treat metabolic diseases.
Guang William Wong, with the assistance of Claire Kitidis, used multiple
genomic approaches to identify a family of ten highly conserved human
and mouse adiponectin paralogs. These are designated as C1q/TNF-alpha
related proteins (CTRP)-1 to 10. Expression of CTRP1, 2, 3, 7, and 9 mRNAs,
like that of adiponectin, are 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-gamma. CTRP2 and CTRP9 are the closest
paralogs of adiponectin; Guang’s data show that CTRP1, 2, 3, 5,
6, 7, 9, and 10 are structurally homologous to adiponectin in that all
form higher order structures including trimers, hexamers, and HMW oligomers.
In addition to forming homo-oligomers, some of these CTRPs can also form
hetero-oligomers. 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 3T3-L1 adipocytes. 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.
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 gamma-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 that induce ectodomain shedding have been identified. Activation of the cardiac beta-adrenergic receptor leads to HB-EGF-cleavage-mediated development of cardiac hypertrophy. Andreas Herrlich showed that another HER-ligand, neuregulin1beta (NRG1beta), 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, with the assistance of Jonathan Fu, an MIT undergraduate, is
cloning novel genes that regulate ectodomain shedding using a high-throughput
expression cloning strategy. They can detect cleavage of all chosen HER-ligands
either by hypertonic stress, phorbol ester addition, or stimulation with
lysophosphatidic acid in a FACS-based assay using mouse lung epithelial
(MLE) or human breast cancer (MCF7) 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 their cytosol-facing C- termini
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 of the 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 cDNA 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 Andreas and Jonathan to identify and clone novel
proteins that regulate shedding of the ectodomain of members of the EGF
family of hormones. Recently Andreas has adapted his screen to a 96-well
format and is testing the effect of lentiviral shRNA constructs on ectodomain
cleavage of his reporter genes. This approach allows direct identification
of the candidate genes and complements his cDNA overexpression approach.