MicroRNAs in erythroid and megakaryocytic differentiation and megakaryocyte-erythroid progenitor lineage commitment.

MicroRNAs (miRNAs) are a class of small regulatory noncoding RNAs that modulate the expression of their target genes through either mRNA degradation or inhibition of protein translation. In recent years, miRNAs have been shown to be critical regulators of hematopoiesis and have important roles in the differentiation of specific lineages. Here, we summarize our current understanding of miRNAs involved in hematopoiesis with a focus on the role of miRNAs in regulating erythroid and megakaryocytic differentiation and megakaryocyte-erythroid progenitor lineage commitment.

In vitro erythropoiesis from bone marrow-derived progenitors provides a physiological assay for toxic and mutagenic compounds.

The goal of this study was to create an in vitro cell culture system that captures essential features of the in vivo erythroid micronucleus (MN) genotoxicity assay, thus enabling increased throughput and controlled studies of the hematopoietic DNA damage response. We show that adult bone marrow (BM) cultures respond to erythropoietin, the principal hormone that stimulates erythropoiesis, with physiological erythropoietic proliferation, differentiation, and enucleation. We then show that this in vitro erythropoietic system clearly signals exposure to genotoxicants through erythroid MN formation. Furthermore, we determined that DNA repair-deficient (MGMT(-/-)) BM displayed sensitivity to genotoxic exposure in vivo compared with WT BM and that this phenotypic response was reflected in erythropoietic cultures. These findings suggest that this in vitro erythroid MN assay is capable of screening for genotoxicity on BM in a physiologically reflective manner. Finally, responses to genotoxicants during erythroid differentiation varied with exposure time, demonstrating that this system can be used to study the effect of DNA damage at specific developmental stages.

Microenvironment-driven changes in the expression profile of hematopoietic cobblestone area-forming cells.

Studies with ex vivo cultures of bone marrow have indicated the importance of the adherent layer as a primary reservoir of the most primitive hematopoietic stem cells, from which derivative stem cells and more differentiated progenitors are continuously generated. We used the Affymetrix GeneChip to analyze the mRNA expressions between bone marrow-derived hematopoietic progenitor cells in the cobblestone areas (CA) and the free-floating cells released from the CA formations. Mouse bone marrow hematopoietic progenitor cell line FDCP-Mix and S17 stromal cells were used in this study. Of the 12,000 genes on the chip, only 29 showed more than fivefold higher in CAFC; and for cells in the supernatant, only 55 showed fivefold higher expressions than in the cobblestone area-forming cells (CAFC). The hematopoietic cells in CAFC expressed genes associated with homing, adhesion, and suppression of differentiation, while the free-floating hematopoietic cells showed mature lineage markers and differentiation-specific genes. This confirmed the more primitive nature of the hematopoietic cells in the adherent layer. Of interest in the findings were the discoveries of many secreted and surface protein expressions in CA hematopoietic cells. This may imply interactions among the hematopoietic cells, stromal cells, and the extracellular matrix in CA, which drive the growth, maturation, and differentiation of the hematopoietic cells.

Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts.

Erythropoietin (Epo) controls red cell production in the basal state and during stress. Epo binding to its receptor, EpoR, on erythroid progenitors leads to rapid activation of the transcription factor Stat5. Previously, fetal anemia and increased apoptosis of fetal liver erythroid progenitors were found in Stat5a(-/-)5b(-/-) mice. However, the role of Stat5 in adult erythropoiesis was not clear. The present study shows that some adult Stat5a(-/-)5b(-/-) mice have a near-normal hematocrit but are deficient in generating high erythropoietic rates in response to stress. Further, many adult Stat5a(-/-)5b(-/-) mice have persistent anemia despite a marked compensatory expansion in their erythropoietic tissue. Analysis of erythroblast maturation in Stat5a(-/-)5b(-/-) hematopoietic tissue shows a dramatic increase in early erythroblast numbers, but these fail to progress in differentiation. Decreased expression of bcl-x(L) and increased apoptosis in Stat5a(-/-)5b(-/-) early erythroblasts correlate with the degree of anemia. Hence, Stat5 controls a rate-determining step regulating early erythroblast survival.

Erythropoiesis in the absence of janus-kinase 2: BCR-ABL induces red cell formation in JAK2(-/-) hematopoietic progenitors.

The receptor-associated protein tyrosine kinase janus-kinase 2 (JAK2) is essential for normal red cell development and for erythropoietin receptor (EpoR) signaling. JAK2(-/-) embryos are severely deficient in erythropoiesis and die at an early stage of development from fetal anemia. The binding of erythropoietin (Epo) to the EpoR triggers the activation of JAK2, the phosphorylation of the EpoR, and the initiation of the EpoR signaling cascade. In addition to Epo binding to its receptor, signaling pathways downstream of the EpoR can also be stimulated by the BCR-ABL oncoprotein. This study explored whether JAK2 is required for BCR-ABL-mediated stimulation of erythropoiesis. Here, it is shown that JAK2 is constitutively tyrosine phosphorylated in cultured and primary erythroid cells expressing BCR-ABL. However, BCR-ABL effectively supports normal erythroid proliferation, differentiation, and maturation in JAK2-deficient fetal liver cells. Using mutants of BCR-ABL, this study shows that certain signaling pathways activated by BCR-ABL segments distinct from its tyrosine kinase domain are essential for rescue of erythropoiesis in JAK2(-/-) progenitors. The consequences of these multiple signaling pathways for normal erythroid development are discussed.

A novel mechanism for regulating transforming growth factor beta (TGF-beta) signaling. Functional modulation of type III TGF-beta receptor expression through interaction with the PDZ domain protein, GIPC.

Transforming growth factor beta (TGF-beta) mediates its biological effects through three high-affinity cell surface receptors, the TGF-beta type I, type II, and type III receptors, and the Smad family of transcription factors. Although the functions of the type II and type I receptors are well established, the precise role of the type III receptor in TGF-beta signaling remains to be established. While expression cloning signaling molecules downstream of TGF-beta, we cloned GIPC (GAIP-interacting protein, C terminus), a PDZ domain-containing protein. GIPC binds a Class I PDZ binding motif in the cytoplasmic domain of the type III receptor resulting in regulation of expression of the type III receptor at the cell surface. Increased expression of the type III receptor mediated by GIPC enhanced cellular responsiveness to TGF-beta both in terms of inhibition of proliferation and in plasminogen-activating inhibitor (PAI)-based promoter gene induction assays. In all cases, deletion of the Class I PDZ binding motif of the type III receptor prevented the type III receptor from binding to GIPC and abrogated the effects of GIPC on type III receptor expressing cells. These results establish, for the first time, a protein that interacts with the cytoplasmic domain of the type III receptor, determine that expression of the type III receptor is regulated at the protein level and that increased expression of the type III receptor is sufficient to enhance TGF-beta signaling. These results further support an essential, non-redundant role for the type III receptor in TGF-beta signaling.

TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has a principal role in growth control through both its cytostatic effect on many different epithelial cell types and its ability to induce programmed cell death in a variety of other cell types. Here we have used a screen for proteins that interact physically with the cytoplasmic domain of the type II TGF-beta receptor to isolate the gene encoding Daxx - a protein associated with the Fas receptor that mediates activation of Jun amino-terminal kinase (JNK) and programmed cell death induced by Fas. The carboxy-terminal portion of Daxx functions as a dominant-negative inhibitor of TGF-beta-induced apoptosis in B-cell lymphomas, and antisense oligonucleotides to Daxx inhibit TGF-beta-induced apoptosis in mouse hepatocytes. Furthermore, Daxx is involved in mediating JNK activation by TGF-beta. Our findings associate Daxx directly with the TGF-beta apoptotic-signalling pathway, and make a biochemical connection between the receptors for TGF-beta and the apoptotic machinery.

Nucleocytoplasmic shuttling of Smad1 conferred by its nuclear localization and nuclear export signals.

Smad1 mediates signaling by bone morphogenetic proteins (BMPs). In the resting state, Smad1 is found in both the nucleus and cytosol. BMP addition triggers Smad1 serine phosphorylation, binding of Smad4, and its accumulation in the nucleus. Mutations in the Smad1 N-terminal basic nuclear localization signal (NLS)-like motif, conserved among all Smad proteins, eliminated its ligand-induced nuclear translocation without affecting its other functions, including DNA binding and complex formation with Smad4. Addition of leptomycin B, an inhibitor of nuclear export, induced rapid nuclear accumulation of Smad1, whereas overexpression of CRM1, the receptor for nuclear export, resulted in Smad1 re-localization to the cytoplasm and inhibition of BMP-induced nuclear accumulation. Thus, in addition to the NLS, Smad1 also contains a functional nuclear export signal (NES). We identified a leucine-rich NES motif in the C terminus of Smad1; its disruption led to constitutive Smad1 nuclear distribution. Reporter gene activation assays demonstrated that both the NLS and NES are required for optimal transcriptional activation by Smad1. Despite its constitutive nuclear accumulation, a Smad1 NES mutant did not display higher basal reporter gene activity. We conclude that Smad1 is under constant nucleocytoplasmic shuttling conferred by its NLS and NES; nuclear accumulation after ligand-induced phosphorylation represents a change in the balance of the activities of these opposing signals and is essential for transcriptional activation.

Fatty acid transport proteins: a current view of a growing family.

Long-chain fatty acids (LCFAs) are a major caloric component of our diet and are key metabolites for energy generation and storage. Physiological uptake of LCFAs across cell membranes is a saturable and competable process occurring at low concentrations, indicative of protein-mediated transport. Fatty acid transport proteins are a family of transmembrane proteins that enhance LCFA uptake and are produced in all fatty acid-utilizing tissues. Here, we review our current understanding of the function, expression patterns and regulation and subcellular localization of this interesting family of proteins.

Insulin-responsive compartments containing GLUT4 in 3T3-L1 and CHO cells: regulation by amino acid concentrations.

In fat and muscle, insulin stimulates glucose uptake by rapidly mobilizing the GLUT4 glucose transporter from a specialized intracellular compartment to the plasma membrane. We describe a method to quantify the relative proportion of GLUT4 at the plasma membrane, using flow cytometry to measure a ratio of fluorescence intensities corresponding to the cell surface and total amounts of a tagged GLUT4 reporter in individual living cells. Using this assay, we demonstrate that both 3T3-L1 and CHO cells contain intracellular compartments from which GLUT4 is rapidly mobilized by insulin and that the initial magnitude and kinetics of redistribution to the plasma membrane are similar in these two cell types when they are cultured identically. Targeting of GLUT4 to a highly insulin-responsive compartment in CHO cells is modulated by culture conditions. In particular, we find that amino acids regulate distribution of GLUT4 to this kinetically defined compartment through a rapamycin-sensitive pathway. Amino acids also modulate the magnitude of insulin-stimulated translocation in 3T3-L1 adipocytes. Our results indicate a novel link between glucose and amino acid metabolism.

Functional cloning of the proto-oncogene brain factor-1 (BF-1) as a Smad-binding antagonist of transforming growth factor-beta signaling.

Using the plasminogen activator inhibitor (PAI) promoter to drive the expression of a reporter gene (mouse CD2), we devised a system to clone negative regulators of the transforming growth factor-beta (TGF-beta) signaling pathway. We infected a TGF-beta-responsive cell line (MvLu1) with a retroviral cDNA library, selecting by fluorescence-activated cell sorter single cells displaying low PAI promoter activity in response to TGF-beta. Using this strategy we cloned the proto-oncogene brain factor-1 (BF-1). BF-1 represses the PAI promoter in part by associating with both unphosphorylated Smad3 (in the cytoplasm) and phosphorylated Smad3 (in the nucleus), thus preventing its binding to DNA. BF-1 also associates with Smad1, -2, and -4; the Smad MH2 domain binds to BF-1, and the C-terminal segment of BF-1 is uniquely and solely required for binding to Smads. Further, BF-1 represses another TGF-beta-induced promoter (p15), it up-regulates a TGF-beta-repressed promoter (Cyclin A), and it reverses the growth arrest caused by TGF-beta. Our results suggest that BF-1 is a general inhibitor of TGF-beta signaling and as such may play a key role during brain development.

Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain.

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Although it is well established that the Epo-bound EpoR is a dimer, it is not clear whether, in the absence of ligand, the intact EpoR is a monomer or oligomer. Using antibody-mediated immunofluorescence copatching (oligomerizing) of epitope-tagged receptors at the surface of live cells, we show herein that a major fraction of the full-length murine EpoR exists as preformed dimers/oligomers in BOSC cells, which are human embryo kidney 293T-derived cells. This observed oligomerization is specific because, under the same conditions, epitope-tagged EpoR did not oligomerize with several other tagged receptors (thrombopoietin receptor, transforming growth factor beta receptor type II, or prolactin receptor). Strikingly, the EpoR transmembrane (TM) domain but not the extracellular or intracellular domains enabled the prolactin receptor to copatch with EpoR. Preformed EpoR oligomers are not constitutively active and Epo binding was required to induce signaling. In contrast to tyrosine kinase receptors (e.g., insulin receptor), which cannot signal when their TM domain is replaced by the strongly dimerizing TM domain of glycophorin A, the EpoR could tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo.

Ski/Sno and TGF-beta signaling.

Transforming growth factor-beta is a potent inhibitor of epithelial cell proliferation. Proteins involved in TGF-beta signaling are bona fide tumor suppressors and many tumor cells acquire the ability to escape TGF-beta growth inhibition through the loss of key signaling transducers in the pathway or through the activation of oncogenes. Recent studies indicate that there is a specific connection between the TGF-beta signaling pathway and the Ski/SnoN family of oncoproteins. We summarize evidence that Ski and SnoN directly associate with Smad proteins and block the ability of the Smads to activate expression of many if not all TGF-beta-responsive genes. This appears to cause abrogation of TGF-beta growth inhibition in epithelial cells.

Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling.

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-beta signaling.

The erythropoietin receptor cytosolic juxtamembrane domain contains an essential, precisely oriented, hydrophobic motif.

We report that the erythropoietin receptor cytosolic juxtamembrane region is conformationally rigid and contains a hydrophobic motif, composed of residues L253, I257, and W258, that is crucial for Janus kinase 2 (JAK2) activation and receptor signaling. Alanine insertion mutagenesis shows that the orientation of this motif and not its distance from the membrane bilayer is critical. Intragenic complementation studies suggest that L253 is contained within an alpha helix functionally continuous to the transmembrane alpha helix. The alpha-helical orientation of L53 is required not for JAK2 activation but for activated JAK2 to induce phosphorylation of the erythropoietin receptor. This motif is highly conserved among cytokine receptors and couples ligand-induced conformational changes in the receptor to intracellular activation of JAK2.

Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice.

Adipocyte complement-related protein (30 kDa) (Acrp30), a secreted protein of unknown function, is exclusively expressed in differentiated adipocytes; its mRNA is decreased in obese humans and mice. Here we describe novel pharmacological properties of the protease-generated globular head domain of Acrp30 (gAcrp30). Acute treatment of mice with gAcrp30 significantly decreased the elevated levels of plasma free fatty acids caused either by administration of a high fat test meal or by i.v. injection of Intralipid. This effect of gAcrp30 was caused, at least in part, by an acute increase in fatty acid oxidation by muscle. As a result, daily administration of a very low dose of gAcrp30 to mice consuming a high-fat/sucrose diet caused profound and sustainable weight reduction without affecting food intake. Thus, gAcrp30 is a novel pharmacological compound that controls energy homeostasis and exerts its effect primarily at the peripheral level.

The N-terminal domain of Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin receptor.

We show that Janus kinase 2 (JAK2), and more specifically just its intact N-terminal domain, binds to the erythropoietin receptor (EpoR) in the endoplasmic reticulum and promotes its cell surface expression. This interaction is specific as JAK1 has no effect. Residues 32 to 58 of the JAK2 JH7 domain are required for EpoR surface expression. Alanine scanning mutagenesis of the EpoR membrane proximal region reveals two modes of EpoR-JAK2 interaction. A continuous block of EpoR residues is required for functional, ligand-independent binding to JAK2 and cell surface receptor expression, whereas four specific residues are essential in switching on prebound JAK2 after ligand binding. Thus, in addition to its kinase activity required for cytokine receptor signaling, JAK is also an essential subunit required for surface expression of cytokine receptors.

Disruption of TGF-beta growth inhibition by oncogenic ras is linked to p27Kip1 mislocalization.

Expression of oncogenic Ras in epithelial tumor cells is linked to the loss of transforming growth factor-beta (TGF-beta) anti-proliferative activity, and was proposed to involve inhibition of Smad2/3 nuclear translocation. Here we studied several epithelial cell lines expressing oncogenic N-RasK61 and show that TGF-beta-induced nuclear translocation of and transcriptional activation by Smad2/3 were unaffected. In contrast, oncogenic Ras mediated nuclearto-cytoplasmic mislocalization of p27KiP1 (p27) and of the cyclin-dependent kinase (CDK) CDK6, but not CDK2. Concomitantly, oncogenic Ras abrogated the ability of TGF-beta to release p27 from CDK6, to enhance its binding to CDK2 and to inhibit CDK2 activity. Inactivation of Ras by a specific antagonist restored the growth inhibitory response to TGF-beta with concurrent normalization of p27 and CDK6 localization. Therefore, the disruption of TGF-beta-mediated growth inhibition by oncogenic Ras appears to be due to lack of inhibition of CDK2, caused by the sequestration of p27 and CDK2 in different subcellular compartments and by the loss of TGF-beta-induced partner switching of p27 from CDK6 to CDK2.

Tankyrase is a golgi-associated mitogen-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles.

The poly(ADP-ribose) polymerase tankyrase was originally described as a telomeric protein whose catalytic activity was proposed to regulate telomere function. Subsequent studies revealed that most tankyrase is actually extranuclear, but a discordant pattern of cytoplasmic targeting was reported. Here we used fractionation and immunofluorescence to show in 3T3-L1 fibroblasts that tankyrase is a peripheral membrane protein associated with the Golgi. We further colocalized tankyrase with GLUT4 storage vesicles in the juxtanuclear region of adipocytes. Consistent with this colocalization, we found that tankyrase binds specifically to a resident protein of GLUT4 vesicles, IRAP (insulin-responsive amino peptidase). The binding of tankyrase to IRAP involves the ankyrin repeats of tankyrase and a defined sequence ((96)RQSPDG(101)) in the IRAP cytosolic domain (IRAP(1-109)). Tankyrase is a novel signaling target of mitogen-activated protein kinase (MAPK); it is stoichiometrically phosphorylated upon insulin stimulation. Phosphorylation enhances the poly(ADP-ribose) polymerase activity of tankyrase but apparently does not mediate the acute effect of insulin on GLUT4 targeting. Taken together, tankyrase is a novel target of MAPK signaling in the Golgi, where it is tethered to GLUT4 vesicles by binding to IRAP. We speculate that tankyrase may be involved in the long term effect of the MAPK cascade on the metabolism of GLUT4 vesicles.

Synergism between transcription factors TFE3 and Smad3 in transforming growth factor-beta-induced transcription of the Smad7 gene.

Activation of transforming growth factor-beta (TGF-beta) receptors triggers phosphorylation of Smad2 and Smad3. After binding to Smad4, the complex enters the nucleus and interacts with other transcription factors to activate gene transcription. Unlike other Smads, Smad7 inhibits phosphorylation of Smad2 and Smad3, and its transcription is induced by TGF-beta, suggesting a negative feedback loop. Here, we show that TFE3 and Smad3 synergistically mediate TGF-beta-induced transcription from the Smad7 promoter by binding to an E-box and two adjacent Smad binding elements (SBEs), respectively. A precise 3-base pair spacer between one SBE and the E-box is essential. Previously, we showed that a similar arrangement between a SBE and an E-box of an element is essential for TGF-beta-dependent transcription of the plasminogen activator inhibitor-1 gene (PAI-1) and that TGF-beta-induced phosphorylation of Smad3 triggers its association with TFE3. Thus, TFE3-Smad3 response elements may represent a common target for TGF-beta-induced gene expression.