Human pluripotent stem cells: Towards therapeutic development for the treatment of lifestyle diseases.

There are two types of human pluripotent stem cells: Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), both of which launched themselves on clinical trials after having taken measures to overcome problems: Blocking rejections by immunosuppressants regarding ESCs and minimizing the risk of tumorigenicity by depleting exogenous gene components regarding iPSCs. It is generally assumed that clinical applications of human pluripotent stem cells should be limited to those cases where there are no alternative measures for treatments because of the risk in transplanting those cells to living bodies. Regarding lifestyle diseases, we have already several therapeutic options, and thus, development of human pluripotent stem cell-based therapeutics tends to be avoided. Nevertheless, human pluripotent stem cells can contribute to the development of new therapeutics in this field. As we will show, there is a case where only a short-term presence of human pluripotent stem-derived cells can exert long-term therapeutic effects even after they are rejected. In those cases, immunologically rejections of ESC- or allogenic iPSC-derived cells may produce beneficial outcomes by nullifying the risk of tumorigenesis without deterioration of therapeutic effects. Another utility of human pluripotent stem cells is the provision of an innovative tool for drug discovery that are otherwise unavailable. For example, clinical specimens of human classical brown adipocytes (BAs), which has been attracting a great deal of attention as a new target of drug discovery for the treatment of metabolic disorders, are unobtainable from living individuals due to scarcity, fragility and ethical problems. However, BA can easily be produced from human pluripotent stem cells. In this review, we will contemplate potential contribution of human pluripotent stem cells to therapeutic development for lifestyle diseases.

Production of functional classical brown adipocytes from human pluripotent stem cells using specific hemopoietin cocktail without gene transfer.

Brown adipose tissue is attracting much attention due to its antiobestic effects; however, its development and involvement in metabolic improvement remain elusive. Here we established a method for a high-efficiency (>90%) differentiation of human pluripotent stem cells (hPSCs) into functional classical brown adipocytes (BAs) using specific hemopoietin cocktail (HC) without exogenous gene transfer. BAs were not generated without HC, and lack of a component of HC induced white adipocyte (WA) marker expressions. hPSC-derived BA (hPSCdBA) showed respiratory and thermogenic activation by ß-adrenergic receptor (AdrRß) stimuli and augmented lipid and glucose tolerance, whereas human multipotent stromal cell-derived WA (hMSCdWA) improved lipid but inhibited glucose metabolism. Cotransplantation of hPSCdBA normalized hMSCdWA-induced glucose intolerance. Surprisingly, hPSCdBAs expressed various hemopoietin genes, serving as stroma for myeloid progenitors. Moreover, AdrRß stimuli enhanced recovery from chemotherapy-induced myelosuppression. Our study enhances our understanding of BA, identifying roles in metabolic and hemogenic regulation.

Feeder-free and serum-free production of hepatocytes, cholangiocytes, and their proliferating progenitors from human pluripotent stem cells: application to liver-specific functional and cytotoxic assays.

We have established a serum- and feeder-free culture system for the efficient differentiation of multifunctional hepatocytes from human embryonic stem (ES) cells and three entirely different induced pluripotent stem (iPS) cells (including vector/transgene-free iPS cells generated using Sendai virus vector) without cell sorting and gene manipulation. The differentiation-inducing protocol consisted of a first stage; endoderm induction, second stage; hepatic initiation, and third stage; hepatic maturation. At the end of differentiation culture, hepatocytes induced from human pluripotent stem cells expressed hepatocyte-specific proteins, such as α-fetoprotein, albumin, α1 antitrypsin and cytochrome P450 (CYP3A4), at similar or higher levels compared with three control human hepatocyte or hepatic cell lines. These human iPS/ES cell-derived hepatocytes also showed mature hepatocyte functions: indocyanine green dye uptake (≈ 30%), storage of glycogen (>80%) and metabolic activity of CYP3A4. Furthermore, they produced a highly sensitive hepatotoxicity assay system for D-galactosamine as determined by the extracellular release of hepatocyte-specific enzymes. Hepatoprotective prostaglandin E1 attenuated this toxicity. Interestingly, bile duct-specific enzymes were also detected after drug treatment, suggesting the presence of bile-duct epithelial cells (cholangiocytes) in our culture system. Electron microscopic studies confirmed the existence of cholangiocytes, and an immunostaining study proved the presence of bipotential hepatoblasts with high potential for proliferation. Differentiated cells were transferrable onto new dishes, on which small-sized proliferating cells with hepatocyte markers emerged and expanded. Thus, our differentiation culture system provides mature functional hepatocytes, cholangiocytes, and their progenitors with proliferative potential from a wide variety of human pluripotent stem cells.

Early senescence is not an inevitable fate of human-induced pluripotent stem-derived cells.

Human-induced pluripotent stem cells (hiPSCs) are expected to become a powerful tool for regenerative medicine. Their efficacy in the use of clinical purposes is currently under intensive verification. It was reported that hiPSC-derived hemangioblasts had severely limited expansion capability due to an induction of early senescence: hiPSC-derived vascular endothelial cells (VECs) senesced after one passage and hiPSC-derived hematopoietic progenitor cells (HPCs) showed substantially decreased colony-forming activities. Here we show that early senescence is not an inevitable fate of hiPSC-derived cells. Applying our unique feeder-free culture methods for the differentiations of human embryonic stem cells (hESCs), we successfully generated VECs and HPCs from three lines of hiPSCs that were established by using a retrovirus vector system. All hiPS-derived VECs could be subcultured by 2:1∼3:1 dilutions up to 10∼20 passages, after which the cells underwent senescence. Among the three lines of hiPSCs, two lines generated HPCs that bore comparable granulocyte colony-forming units to those of hESCs. Moreover, one line effectively reproduced HPCs within the sac-like structures, the fields of in vitro hematopoiesis, as in the case of hESCs. Surprisingly, release of neutrophils into culture supernatant persisted even longer (∼60 days) than the case of hESCs (∼40 days). Thus, the problem of early senescence can be overcome by selecting appropriate lines of hiPSCs and applying proper differentiation methods to them.

High-efficiency production of subculturable vascular endothelial cells from feeder-free human embryonic stem cells without cell-sorting technique.

We previously reported a feeder-free culture method for pure production of subculturable vascular endothelial cells (VECs) from cynomolgus monkey embryonic stem cells (cmESCs) without as using cell-sorting technique. By this method, canonical vascular endothelial (VE)-cadherin/platelet-endothelial cell adhesion molecule 1 (PECAM1)-positive VECs (c-VECs) and atypical VE-cadherin/PECAM1-negative VECs (a-VECs) were generated without a contamination by pericytes, lymphatic endothelial cells, or immature ES cells. More recently, we established a unique culture technique to maintain human ESCs (hESCs) under a feeder-free and recombinant cytokine-free condition. Combining these two systems, we have successfully generated pure VECs from two lines of hESCs, khES-1 and khES-3, under a completely feeder-free condition. Our method is very simple: spheres generated from hESCs by floating culture using differentiation media supplemented with vascular endothelial growth factor, bone morphogenetic protein 4, stem cell factor, FMS-related tyrosine kinase-3 ligand, and interleukin 3 (IL3) and IL6 were cultured on gelatin-coated plates. Cell passage was performed by an ordinary enzymatic treatment. The hESC-derived differentiated cells demosntrated cord-forming activities and acetylated low-density lipoprotein-uptaking capacities. Moreover, they exclusively expressed von Willebrand factor and endothelial nitric oxide synthase. Flow cytometric analyses indicate that khES-3 generated both c-VECs and a-VECs as in the case of cmESCs. By contrast, khES-1 produced only a-VECs, which nonetheless demonstrated effective recruitment into neovascularity in vivo. Interestingly, a-VECs turned to express PECAM1 after transplantation into immunodeficient mice. The hESC-derived VECs were subculturable at least up to 10 passages without functional depression. Our method does not require a presorting processes to enrich progenitor fractions such as CD34-positive or kinase insert domain receptor (KDR)-positive cells, providing the most efficient and easiest technique for VEC production from hESCs.

Human embryonic stem cells with maintenance under a feeder-free and recombinant cytokine-free condition.

We previously reported that cynomolgus monkey embryonic stem (ES) cells could be maintained under a feeder-free condition without using recombinant cytokines if sizes and numbers of ES colonies were kept within an appropriate range. Here we show that this finding is also true with human ES cells (hESCs). The two lines of hESCs, khES-1 and khES-3, were appropriately maintained in the absence of feeder layers or exogenous cytokines such as fibroblast growth factors, Noggin, transforming growth factor beta, and Activin by closely controlling the size and number of hESC colonies. High-level expressions of immature markers including SSEA-4, Oct-4, and Nanog were detected in feeder-free and cytokine-free hESCs, and they formed teratomas when implanted into severe combined immunedeficiency (SCID) mice. No chromosomal abnormalities were observed over 20 passages, ruling out the possibility that special clones with growth advantages had been selected. Global protein expression profiles were quite similar among the hESCs maintained by our feeder- and cytokine-free method, by coculture with mouse embryonic fibroblasts (MEFs) and by a feeder-free method using conditioned media of MEFs. However, the activation level of Akt, an important player for the maintenance of ES cells, was highest and the activation level of extracellular signal-regulated kinase, a critical player for differentiation of ES cells, was lowest in the hESCs maintained by our cytokine-free method. Our results not only show a technical improvement for the maintenance of hESCs but also open a new avenue for the understanding of autocrine signaling networks of hESCs.

A feeder-free and efficient production of functional neutrophils from human embryonic stem cells.

A novel, feeder-free hematopoietic differentiation protocol was established for highly efficient production of neutrophils from human embryonic stem cells (hESCs). For the induction of differentiation, spheres were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin-coated plates. After approximately 2 weeks, a sac-like structure filled with abundant round cells emerged at the center of flattened spheres. After cutting off this sac-like structure, round cells actively proliferated, either floating in the supernatant or associated weakly with the adherent cells. Almost all of these round cells were CD45-positive hematopoietic cells with myeloid phagocytic markers (CD33 and CD11b), and approximately 30%-50% of the round cells were mature neutrophils, as judged from morphology, cytochemical characteristics (myeloperoxidase and neutrophil alkaline phosphatase), and neutrophil-specific cell surface markers (CD66b, CD16b, and GPI-80). In addition, hESC-derived neutrophils had chemotactic capacity in response to the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalanine and neutrophil-specific chemokine interleukin (IL)-8. Using "semipurified" neutrophils migrated to IL-8, both phagocytic and respiratory burst activities were demonstrated. Finally, it was shown that hESC-derived neutrophils had chemotactic activity in vivo in a murine air-pouch inflammatory model. The present results indicate successful induction of functional mature neutrophils from hESCs via highly efficient feeder-free differentiation culture system of human hematopoietic cells.

Highly efficient and feeder-free production of subculturable vascular endothelial cells from primate embryonic stem cells.

The vascular endothelial cell (VEC) differentiation from primate embryonic stem (ES) cells has critical problems: low differentiation efficiencies (<2%) and/or subculture incapability. We report a novel feeder-free culture method for high efficiency production of subculturable VECs from cynomolgus monkey ES cells. Spheres, which were generated from ES cells in the presence of cytokine cocktail, were cultured on gelatin-coated plates. Cobblestone-shaped cells spread out after a few days, which were followed by an emergence of a sac-like structure containing hematopoietic cells. All adherent cells including sac walls cells and surrounding cobblestone cells expressed vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Subculture of these cells resulted in a generation of homogeneous spindle-shaped population bearing cord-forming activities and a uniform acetylated low density lipoprotein-uptaking capacity with von Willbrand factor and endothelial nitric oxide synthetase expressions. They were freeze-thaw-tolerable and subculturable up to eight passages. Co-existence of pericytes or immature ES cells was ruled out. When introduced in a collagen sponge plug implanted intraperitoneally in mice, ES-derived cells recruited into neovascularity. Although percentages of surface VE-cadherin-positive population varied from 20% to 80% as assessed by flow cytometry, the surface VE-cadherin-negative population showed intracellular VE-cadherin expression and mature functions, as we call it as atypical VECs. When sorted, the surface VE-cadherin-positive population expanded as almost pure (>90%) VE-cadherin/PECAM-1-positive VECs by 160-fold after five passages. Thus, our system provides pure production of functional, subculturable and freeze-thaw-tolerable VECs, including atypical VECs, from primate ES cells.

A feeder-free hematopoietic differentiation system with generation of functional neutrophils from feeder- and cytokine-free primate embryonic stem cells.

We have established a novel feeder- and recombinant cytokine-free culture system for the maintenance of primate embryonic stem (ES) cells along with a feeder-free hematopoietic differentiation protocol for high efficiency CD45-positive cell production. In our system, cynomolgus monkey ES cells were properly maintained in an undifferentiated state with high immature marker expressions and teratoma-producing activities. Embryoid bodies (EBs) were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin-coated plates. After about 2 weeks, a sac-like structure filled with abundant round cells emerged at the center of flattened EB. Then total cells were collected and transferred onto new gelatin-coated plates, where cells were firmly attached and actively proliferated to confluence. After another few days culture, abundant floating cells were detected in the culture supernatant. These cells expressed high levels of CD45 (>90%), while adherent cells expressed low levels of CD45 (<10%). The former consisted of various differentiated stages of myeloid cells from immature myeloblasts to mature polymorphonuclear neutrophils and macrophages. Although the percentages of neutrophils varied between 10 to 20 depending on experiments, their mature phenotype was reproducibly confirmed by specific staining and functional assays. Our protocol provides the minimum essence for primate ES cell maintenance and hematopoietic differentiation that is beneficial from economical and clinical points of view.

Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells.

Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.

Identification of human neutrophils during experimentally induced inflammation in mice with transplanted CD34+ cells from human umbilical cord blood.

Nonobese diabetic/severe combined immunodeficiency/gamma chainnull (NOG) mice are excellent recipients for xenotrans-plantation and have been especially valuable for the evaluation of human hematopoietic stem cell (HSC) activities. Because human hematopoietic cells that developed in this mouse were mainly lymphoid cells and not myeloid cells, mature human myeloid cells such as neutrophils were hardly detectable in peripheral blood. We demonstrated that human neutrophils accumulated by means of a zymosan-induced air pouch inflammation technique could be identified with a fluorescence-activated cell sorter in NOG mice with transplanted CD34+ cells from human umbilical cord blood, which were putative hematopoietic progenitor cells including HSC. Our results indicate that human neutrophils with a chemotactic capacity can develop from human hematopoietic progenitor cells in vivo, suggesting that our system may be a useful tool for the evaluation of human HSC activities.

Peroxisome proliferator-activated receptor gamma ligands stimulate myeloid differentiation and lipogenensis in human leukemia NB4 cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in adipocyte and macrophage differentiation. Pioglitazone (Actos, AD4833), an antidiabetic drug, and 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2) have recently been identified as synthetic and natural ligands for PPARgamma, respectively. In this study, we examined the effects of PPARgamma ligands on differentiation and lipogenesis in promyelocytic leukemia NB4 cells, in which PPARgamma protein was expressed and ligand-stimulated PPARgamma-specific transcription of adipocyte fatty-acid binding protein was confirmed. Treatment with PPARgamma ligand (AD4833 or PGJ2) alone markedly suppressed proliferation but did not induce differentiation. The combined treatment of the cells with PPARgamma ligand and all-trans retinoic acid (ATRA) synergistically induced myelocytic differentiation, as determined by nitroblue tetrazolium reducing ability and cell morphology. During these processes of differentiation, we observed marked accumulation of lipid droplets in the cytoplasm. The cellular triacylglycerol levels increased 2.7-fold after treatment with the inducers. Simultaneously, BODIPY-fatty acid was incorporated into the cytosol and concentrated in lipid droplets. The biosynthesis of triacylglycerol-containing BODIPY-fatty acids was increased twofold in differentiated cells. These findings clearly demonstrate that treatment with PPARgamma ligands not only induced differentiation but also stimulated lipogenesis in NB4 cells, indicating a close association between differentiation and lipogenesis in PPARgamma-stimulated human myeloid cells.

Efficient and repetitive production of hematopoietic and endothelial cells from feeder-free monolayer culture system of primate embryonic stem cells.

We have established an innovative culture system for the efficient differentiation of hematopoietic and endothelial cells from primate embryonic stem (ES) cells without feeder cells, embryoid bodies, or cell-sorting processes. After several days' culture in murine stromal OP9-conditioned medium supplemented with a cytokine cocktail on collagen-coated dishes, ES cells differentiated into a very unique population of cells with a finger-like appearance. These finger-like cells were positive for mesodermal and/or hemangioblastic markers of kinase insert domain receptor (KDR) and T-cell acute lymphocytic leukemia 1 (TAL1), and produced large amounts of protein tyrosine phosphatase, receptor type, C-positive hematopoietic cells. These hematopoietic cells showed the morphology of immature hematopoietic cells, formed blast cell colonies with high efficiency, and were positive for CD34 antigen, KDR, TAL1, and GATA binding protein 1, suggesting that these blast cells are equivalent to the multipotent hematopoietic progenitor cells. Moreover, they produced functional macrophages in murine stromal MS-5-conditioned medium and primitive erythroblasts in the presence of erythropoietin. The finger-like cells, putative mesodermal progenitors and/or hemangioblasts, actively proliferated and repetitively produced hematopoietic cells as long as they were maintained on the original dish. By contrast, the majority of the finger-like cells differentiated into endothelial cells with specific markers and specific functions after transfer to fresh dishes, indicating that conditions established in the original dish supported the proliferation and hematopoietic differentiation of the finger-like cells. Our method provides a highly controllable culture protocol for repetitive production of hematopoietic and endothelial cells from feeder-free monolayer cultivation of primate ES cells.

Proteomic analysis on insulin signaling in human hematopoietic cells: identification of CLIC1 and SRp20 as novel downstream effectors of insulin.

Insulin/IGF-I-dependent signals play important roles for the regulation of proliferation, differentiation, metabolism, and autophagy in various cells, including hematopoietic cells. Although the early protein kinase activation cascade has been intensively studied, the whole picture of intracellular signaling events has not yet been clarified. To identify novel downstream effectors of insulin-dependent signals in relatively early phases, we performed high-resolution two-dimensional electrophoresis (2-DE)-based proteomic analysis using human hematopoietic cells 1 h after insulin stimulation. We identified SRp20, a splicing factor, and CLIC1, an intracellular chloride ion channel, as novel downstream effectors besides previously reported effectors of Rho-guanine nucleotide dissociation inhibitor 2 and glutathione S-transferase-pi. Reduction in SRp20 was confirmed by one-dimensional Western blotting. Moreover, MG-132, a proteasome inhibitor, prevented this reduction. By contrast, upregulation of CLIC1 was not observed in one-dimensional Western blotting, unlike the 2-DE results. As hydrophilic proteins were predominantly recovered in 2-DE, the discrepancy between the 1-DE and 2-DE results may indicate a certain qualitative change of the protein. Indeed, the nuclear localization pattern of CLIC1 was remarkably changed by insulin stimulation. Thus insulin induces the proteasome-dependent degradation of SRp20 as well as the subnuclear relocalization of CLIC1.

A prostacyclin analog prevents radiocontrast nephropathy via phosphorylation of cyclic AMP response element binding protein.

We reported previously that radiocontrast medium induces caspase-dependent apoptosis and that cAMP analogs inhibit cell injury in cultured renal tubular cells. In the present study, cellular mechanisms underlying the protective effects of cAMP were determined. Ioversol, a radiocontrast medium, caused cell injury accompanied by decreases in Bcl-2, increases in Bax, and caspase activation in LLC-PK1 cells. Both cell injury and cellular events induced by ioversol were inhibited by dibutyryl cAMP and the prostacyclin analog beraprost. Dibutyryl cAMP increased phosphorylation of Akt and CREB, both of which were reversed by H89, wortmannin and the Akt inhibitor SH-6. The protective effect of dibutyryl cAMP was also reversed by these kinase inhibitors. In dominant-negative CREB-transfected cells, dibutyryl cAMP no longer prevented cell injury or inhibited changes in mRNA expression of Bcl-2 and Bax. In mice with unilateral renal occlusion, ioversol increased urinary excretion of N-acetyl-beta-d-glucosaminidase with concomitant decreases in Bcl-2 mRNA, increases in Bax mRNA, activation of caspase-3, and induction of apoptosis in tubular and interstitial cells. Beraprost completely reversed these in vivo effects of ioversol. These findings suggest that elevation of endogenous cAMP effectively prevents radiocontrast nephropathy through activation of A kinase/PI 3-kinase/Akt followed by CREB phosphorylation and enhanced expression of Bcl-2.

Synergistic effects of dehydroepiandrosterone and retinoic acid on granulocytic differentiation of human promyelocytic NB4 cells.

We report a novel effect of dehydroepiandrosterone (DHEA) on human granulocyte differentiation: DHEA enhances the all-trans-retinoic acid (ATRA)-induced differentiation of promyelocytic NB4 cells. DHEA (100 microM) significantly augmented the respiratory burst activity of NB4 cells treated with 1 nM ATRA, whereas DHEA alone did not induce respiratory burst activity. The protein and message expressions of p67phox, the gene for the dose-limiting component of phagocyte NADPH oxidase, were significantly enhanced by the coexistence of DHEA and ATRA. The protein expression of p47phox, another component of phagocyte NADPH oxidase, was also up-regulated by DHEA and ATRA. Moreover, the ATRA-induced increment of CCAAT/enhancer-binding protein beta (C/EBPbeta) and the reciprocal reduction in C/EBPUalpha expression were also potentiated by DHEA. In contrast, the expression of PU.1, a transcription factor reportedly involved in the basal expression of p67phox in monocytic cells, was only slightly up-regulated by DHEA and ATRA. Interestingly, DHEA sulfate (DHEAS), the sulfate ester of DHEA that exists in peripheral blood at a concentration approximately 3 orders of magnitude larger than that of DHEA, did not stimulate the ATRA-induced differentiation of NB4 cells. Thus, DHEA, but not DHEAS, plays important roles in synergy with ATRA during granulocyte differentiation of human promyelocytic NB4 cells.

Brap2 functions as a cytoplasmic retention protein for p21 during monocyte differentiation.

The cell cycle inhibitor p21 plays an important role in monocytic cell differentiation, during which it translocates from the nucleus to cytoplasm. This process involves the negative regulation of the p21 nuclear localization signal (NLS). Here, we sought to determine the relationship between the cytoplasmic translocation of p21 and another molecule, Brap2, a cytoplasmic protein which binds the NLS of BRCA1 and was recently reported to inactivate KSR in the Ras-activating signal pathway under the name of IMP. We report that p21 and Brap2 directly interact, both in vitro and in vivo, in a manner requiring the NLS of p21 and the C-terminal portion of Brap2. When it is cotransfected with Brap2, p21 is expressed in the cytoplasm. Monocytic differentiation of the promyelomonocytic cell lines U937 and HL60 is associated with the upregulation of Brap2 expression concomitantly with the upregulation and cytoplasmic relocalization of p21. Our results underscore the role played by Brap2 in the process of cytoplasmic translocation of p21 during monocyte differentiation.

Insulin-dependent signaling regulates azurophil granule-selective macroautophagy in human myeloblastic cells.

We show that insulin-dependent signals regulate azurophil granule-selective macroautophagy in human myeloid cells. Depletion of insulin from an insulin-transferrin-supplemented serum-free medium caused growth retardation of myeloblastic HL-60 cells, in which sequestration of electronic-dense cytoplasmic materials by autophagosomes was observed. Positive immunoreactivity with anti-CD68, anti-cathepsin D, and anti-myeloperoxidase antibodies indicated that the sequestrated materials were azurophil granules, the granulocyte/macrophage lineage-specific lysosome-like particles. By contrast, other organelles, including the mitochondria, endoplasmic reticulum, and Golgi apparatus remained intact, indicating that the macroautophagy selectively targeted azurophil granules. The addition of insulin induced rapid activations of p70S6K and Akt, and the cells were rescued from macroautophagy. Rapamycin, an inhibitor of mammalian target of rapamycin, did not block the insulin-mediated rescue from macroautophagy, although it nullified the activation of p70S6K and cell growth. Low doses of LY294002, a phosphatidyl-inositol-3-kinase inhibitor, which abolished cell growth and p70S6K activity but did not influence Akt activity, did not block the insulin-mediated rescue either. By contrast, low doses of Akt-specific inhibitors, which inhibited neither cell growth nor p70S6K activity, completely blocked the insulin-mediated rescue from macroautophagy. Thus, insulin-dependent signals are responsible for the control of azurophil granule-selective macroautophagy via Akt-dependent pathways, while p70S6K-dependent pathways promote cell growth.

Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce cell cycle progression through the synthesis of c-Myc protein by internal ribosome entry site-mediated translation via phosphatidylinositol 3-kinase pathway in human factor-dependent leukemic cells.

To investigate the roles of c-myc during hematopoietic proliferation induced by growth factors, we used factor-dependent human leukemic cell lines (MO7e and F36P) in which proliferation, cell cycle progression, and c-Myc expression were strictly regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3). In these cell lines, both c-myc mRNA and c-Myc protein stability were not affected by GM-CSF and IL-3, suggesting a regulation of c-Myc protein at the translational level. However, rapamycin, an inhibitor of cap-dependent translation, did not block c-myc induction by GM-CSF and IL-3. Thus, we studied the cap-independent translation, the internal ribosome entry site (IRES), during c-Myc protein synthesis using dicistronic reporter gene plasmids and found that GM-CSF and IL-3 activated c-myc IRES to initiate translation. c-myc IRES activation, c-Myc protein expression, and cell cycle progression were all blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002. In another factor-dependent cell line, UT7, we observed the cell cycle progression and up-regulation of c-Myc protein, c-myc mRNA, and c-myc IRES simultaneously, which were all inhibited by LY294002. Results indicate that hematopoietic growth factors induce cell cycle progression via IRES-mediated translation of c-myc though the PI3K pathway in human factor-dependent leukemic cells.

Distinct involvement of cAMP-response element-dependent transcriptions in functional and morphological maturation during retinoid-mediated human myeloid differentiation.

We evaluated the involvement of cyclic adenosine monophosphate-response element (CRE)-dependent transcriptions in all-trans retinoic acid (ATRA)-induced myeloid differentiation using human monoblastic U937 cells. ATRA treatment caused an increment in the CRE-dependent transcription activity and induced a wide variety of differentiation phenotypes including functional and morphological maturation. Indeed, ATRA treatment induced the expression of CCAAT/enhancer-binding protein beta (C/EBPbeta), a CRE-dependent transcription factor important in monocytic differentiation, and the inhibition of CRE-enhancer activity by the expression of a dominant-negative CRE-binding protein (dn-CREB) abolished the induction of C/EBPbeta. Functional maturation, such as the enhancement of cell adhesion and respiratory burst activity, was dramatically suppressed by the expression of dn-CREB. In addition, the differentiation-dependent induction of an adhesion molecule (CD11b), the phagocyte oxidase required for respiratory burst, and the transcription factor PU.1 responsible for phagocyte oxidase induction were all abolished by dn-CREB. Surprisingly, morphological maturation, including nuclear convolution and cytoplasmic vacuolar formation, was augmented by dn-CREB. Under the same conditions, the differentiation-associated cell-growth arrest was not affected by the expression of dn-CREB. Our results clearly indicate that CRE-driven transcription plays at least three distinct roles during myeloid differentiation: It stimulates functional maturation but suppresses morphological maturation and has no effects on cell-growth arrest.