A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation

The CD34⁺ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8⁺ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8⁺ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality.     

Differential Pharmacological Actions of Methadone and Buprenorphine in Human Embryonic Kidney 293 Cells Coexpressing Human μ-Opioid and Opioid Receptor-Like 1 Receptors

Methadone and buprenorphine are used in maintenance therapy for heroin addicts. In this study, we compared their effects on adenylate cyclase (AC) activity in human embryonic kidney (HEK) 293 cells stably overexpressing human μ-opioid receptor (MOR) and nociceptin/opioid receptor-like 1 receptor (ORL1) simultaneously. After acute exposure, methadone inhibited AC activity; however, buprenorphine induced compromised AC inhibition. When naloxone was introduced after 30 min incubation with methadone, the AC activity was enhanced. This was not observed in the case of buprenorphine. Enhancement of the AC activity was more significant when the incubation lasted for 4 h, and prolonged exposure to buprenorphine elevated the AC activity as well. The removal of methadone and buprenorphine by washing also obtained similar AC superactivation as that revealed by naloxone challenge. The study demonstrated that methadone and buprenorphine exert initially different yet eventually convergent adaptive changes of AC activity in cells coexpressing human MOR and ORL1 receptors.     

Traffic of Human α-Mannosidase in Plant Cells Suggests the Presence of a New Endoplasmic Reticulum-to-Vacuole Pathway without Involving the Golgi Complex

The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human α-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, α-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plant-secreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins.Acidic α-mannosidases (EC 3.2.1.24) are exoglycosidases responsible for the removal of α-linked Man residues in the catabolism of glycoproteins (Daniel et al., 1994). These enzymes are secretory proteins that perform their function within the lysosomes in mammalian cells and into the vacuoles of yeast (Saccharomyces cerevisiae) and plant cells. Moreover, acidic α-mannosidases have also been described in microorganisms (Santacruz-Tinoco et al., 2010). The secretory proteins normally move from the endoplasmic reticulum (ER) to the target compartment using either vesicles or direct connections between compartments (Vitale and Hinz, 2005). These types of proteins need an N-terminal signal peptide to be inserted into the ER, which is removed in the ER lumen by signal peptidases. Once in the ER, secretory proteins, in the absence of other types of sorting signals, are secreted out of the cell (Jurgens, 2004). With regard to acidic α-mannosidases, while the primary structure of these proteins is highly conserved among various kingdoms, the way in which they are targeted to their final compartment inside the cell differs in eukaryotic cells. In animal cells, these hydrolases are transported to lysosomes thanks to trans-Golgi mannose 6-phosphate receptors (MPRs) that recognize the phosphorylation of a specific residue of Man (Man-6-P) in the glucidic structure of the protein. Hence, the phosphorylated oligosaccharide side chains act as targeting signals for the lysosomal compartment (Thomas, 2001; Hansen et al., 2004). Two types of MPRs have been identified with molecular masses of 46 kD (cation-dependent MPR) and 300 kD (cation-independent MPR). MPRs are also present on the cell surface, and at least the cation-independent MPR is capable of endocytosing extracellular lysosomal hydrolases (Díaz and Pfeffer, 1998). In yeast, these enzymes reach the vacuolar localization by both cytoplasm-to-vacuole targeting and autophagy pathways (Hutchins and Klionsky, 2001). In plants, vacuolar α-mannosidase follows the classic secretory pathway involving the ER-Golgi system to reach their final destination (Faye et al., 1998).Recently, a functional human α-mannosidase (MAN2B1) has been expressed in stably transformed tobacco (Nicotiana tabacum) plants to develop an enzyme-replacement therapy for α-mannosidosis, which is a rare lysosomal storage disease caused by mutations in the MAN2B1 gene (De Marchis et al., 2011). In the human cells, MAN2B1 is synthesized as a high-M_r precursor that is posttranslationally modified by N-glycosylation, disulfide bridge formation, proteolysis, zinc binding, and homodimer formation (Tollersrud et al., 1997). Similarly, in transgenic plants, recombinant MAN2B1, provided with a plant signal peptide, is synthesized as a 110-kD precursor that undergoes specific posttranslational modifications including N-glycosylation and proteolytic maturation in the vacuole, producing four processed forms with apparent molecular masses of 70, 40, 32, and 18 kD. Unexpectedly, recombinant MAN2B1 in tobacco, instead of being secreted due to the absence in plants of MPRs (Gaudreault and Beevers, 1984), is targeted to the vacuole (De Marchis et al., 2011). Conversely, another human lysosomal enzyme, glucocerebrosidase, when produced in Arabidopsis (Arabidopsis thaliana) seeds, is mainly secreted in the apoplast, and only a minor fraction of the protein is detected in protein storage vacuoles (PSVs; He et al., 2012). Indeed, to facilitate glucocerebrosidase targeting to the vacuoles of carrot (Daucus carota) cells, Shaaltiel and colleagues (2007) added a seven-amino acid vacuole-targeting signal to the C terminus of the protein. Therefore, in this study, we tried to understand which route is used by the soluble lysosomal MAN2B1 in tobacco to reach the vacuoles.Mammalian lysosomes are considered equivalent to plant lytic vacuoles (LVs), but plants also contain PSVs for reserve accumulation, even if the distinction between different vacuoles is debated (Frigerio et al., 2008). In plants, regardless of the type of vacuole (LV or PSV), soluble vacuolar proteins reach the vacuole through the Golgi apparatus (Hwang, 2008). The transport of most secretory proteins from the ER to the Golgi complex is coat protein II mediated before reaching their final destinations. From the Golgi apparatus, vacuolar proteins reach the vacuole either through electron-opaque vesicles or via clathrin-coated vesicles (Vitale and Hinz, 2005). Plant vacuolar sorting signals and vacuolar sorting receptors that enable this traffic have recently been described (Hwang, 2008; De Marcos Lousa et al., 2012). There are certainly exceptions to this main vacuolar sorting mechanism, characterized by proteins that travel directly from the ER to the vacuole, bypassing the Golgi system, but these polypeptides are either membrane proteins or proteins that form insoluble aggregates. For example, the vacuolar storage proteins of pumpkin (Cucurbita maxima) reach PSVs via precursor-accumulating vesicles, bypassing the Golgi complex (Hara-Nishimura et al., 1998). In addition, the route that bypasses the Golgi system seems to be linked to the specific transport of proteins that form large aggregates (Herman and Schmidt, 2004; Herman, 2008). Cereal prolamins, when aggregated in the ER in large polymers, can also be transported directly from the ER to PSVs, apparently by autophagy (Levanony et al., 1992; Reyes et al., 2011). Moreover, many vacuolar enzymes are stored in ER-derived vesicles, which, under certain circumstances such as programmed cell death or seed germination, are directly fused with the vacuolar compartment (Hayashi et al., 2001; Rojo et al., 2003).We show that MAN2B1, when expressed in tobacco, reaches the vacuole of leaf cells while bypassing the Golgi and that the N-terminal domain of MAN2B1 has a cryptic vacuolar targeting signal. Indeed, the removal of 200 amino acids from the N terminus prevents MAN2B1 vacuolar delivery, and, when fused with a secreted protein, this N-terminal domain is able to redirect this protein to the vacuole by a transport mechanism without involving the Golgi apparatus. Therefore, this study describes an alternative route followed by plant soluble vacuolar proteins to reach the vacuole directly from the ER, without passing through the Golgi complex.     

Differential Processing of Amyloid-�� Precursor Protein Directs Human Embryonic Stem Cell Proliferation and Differentiation into Neuronal Precursor Cells

The amyloid-β precursor protein (AβPP) is a ubiquitously expressed transmembrane protein whose cleavage product, the amyloid-β (Aβ) protein, is deposited in amyloid plaques in neurodegenerative conditions such as Alzheimer disease, Down syndrome, and head injury. We recently reported that this protein, normally associated with neurodegenerative conditions, is expressed by human embryonic stem cells (hESCs). We now report that the differential processing of AβPP via secretase enzymes regulates the proliferation and differentiation of hESCs. hESCs endogenously produce amyloid-β, which when added exogenously in soluble and fibrillar forms but not oligomeric forms markedly increased hESC proliferation. The inhibition of AβPP cleavage by β-secretase inhibitors significantly suppressed hESC proliferation and promoted nestin expression, an early marker of neural precursor cell (NPC) formation. The induction of NPC differentiation via the non-amyloidogenic pathway was confirmed by the addition of secreted AβPPα, which suppressed hESC proliferation and promoted the formation of NPCs. Together these data suggest that differential processing of AβPP is normally required for embryonic neurogenesis.The amyloid-β precursor protein (AβPP)⁵ is a ubiquitously expressed transmembrane protein whose cleavage product, the amyloid-β (Aβ) protein, is deposited in amyloid plaques in the aged brain, following head injury, and in the neurodegenerative conditions of Alzheimer disease (AD) and Down syndrome (DS). AβPP has structural similarity to growth factors (1) and modulates several important neurotrophic functions, including neuritogenesis, synaptogenesis, and synaptic plasticity (2). The function of AβPP during early embryogenesis and neurogenesis has not been well described.AβPP is processed by at least two pathways, the non-amyloidogenic and amyloidogenic pathways. Non-amyloidogenic processing of AβPP yields secreted AβPPα (sAβPPα), the secreted extracellular domain of AβPP that acts as a growth factor for many cell types and promotes neuritogenesis (3). Amyloidogenic processing of AβPP releases sAβPPβ, the AβPP intracellular domain, and Aβ proteins. The Aβ protein has both neurotoxic and neurotrophic properties (4) dependent on the differentiation state of the neuron; Aβ is neurotoxic to differentiating neurons via a mechanism involving differentiation-associated increases in the phosphorylation of the microtubule-associated protein tau (5) but neurotrophic to undifferentiated embryonic neurons. Evidence supporting a neurotrophic function for Aβ during development include its neurogenic activity toward rat neural stem cells (4–6). Consistent with these data, two studies have demonstrated increased hippocampal neurogenesis in young transgenic mice overexpressing human APP_Sw,Ind (7, 8).Recently we reported that human embryonic stem cells (hESCs) express AβPP and that both the stemness of the cells and the pregnancy-associated hormone human chorionic gonadotropin alter AβPP expression (9). These results suggest a functional role for AβPP during early human embryogenesis. To further investigate the function of AβPP and its cleavage products during early embryonic neurogenesis, we examined the expression and processing of this protein and its role in proliferation and differentiation of hESCs into neural precursor cells (NPCs). We found that amyloidogenic processing of AβPP promotes hESC proliferation whereas non-amyloidogenic processing induces hESC differentiation into NPCs. These data reveal an important function for AβPP during early human embryonic neurogenesis. Our data imply that any dysregulation in AβPP processing that leads to altered sAβPPα/Aβ production could result in aberrant neurogenesis as reported in the AD and DS brains.     

Effects of 2‐Chloro‐2′‐Deoxyadenosine on the Cell Cycle in the Human Leukemia EHEB Cell Line

To explain why 2‐chloro‐2′‐deoxyadenosine (CdA) is unable to block DNA synthesis and cell cycle progression, and paradoxically enhances progression from G1 into S phase in the CdA‐resistant leukemia EHEB cell line, we studied its metabolism and effects on proteins regulating the transition from G1 to S phase. A low deoxycytidine kinase activity and CdATP accumulation, and a lack of p21 induction despite p53 phosphorylation and accumulation may account for the inability of CdA to block the cell cycle. An alternative pathway involving pRb phosphorylation seems implicated in the CdA‐induced increase in G1 to S phase progression.     

Characterization of Stimulus-Secretion Coupling in the Human Pancreatic EndoC-βH1 Beta Cell Line

Aims/Hypothesis

Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.

Methods

Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca²⁺ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.

Results

Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.

Conclusions/Interpretation

Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca²⁺ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.     

A Dual Reporter Mouse Model of the Human β-Globin Locus: Applications and Limitations

The human β-globin locus contains the β-like globin genes (i.e. fetal γ-globin and adult β-globin), which heterotetramerize with α-globin subunits to form fetal or adult hemoglobin. Thalassemia is one of the commonest inherited disorders in the world, which results in quantitative defects of the globins, based on a number of genome variations found in the globin gene clusters. Hereditary persistence of fetal hemoglobin (HPFH) also caused by similar types of genomic alterations can compensate for the loss of adult hemoglobin. Understanding the regulation of the human γ-globin gene expression is a challenge for the treatment of thalassemia. A mouse model that facilitates high-throughput assays would simplify such studies. We have generated a transgenic dual reporter mouse model by tagging the γ- and β-globin genes with GFP and DsRed fluorescent proteins respectively in the endogenous human β-globin locus. Erythroid cell lines derived from this mouse model were tested for their capacity to reactivate the γ-globin gene. Here, we discuss the applications and limitations of this fluorescent reporter model to study the genetic basis of red blood cell disorders and the potential use of such model systems in high-throughput screens for hemoglobinopathies therapeutics.     

Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated α-synuclein (αS^D) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T αS and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of αS^D (disease-associated αS) in these mice, an ELISA was designed to specifically quantify αS^D in sick mice. Analysis of the central nervous system in this mouse model showed the presence of αS^D mainly in the caudal brain regions and the spinal cord. There were no differences in αS^D distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of αS^D immunoreactivity using an antibody against Ser129 phosphorylated αS by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of αS. The propagation of αS^D, suggesting the involvement of a “prion-like” mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.     

Strychnos nux-vomica Root Extract Induces Apoptosis in the Human Multiple Myeloma Cell Line—U266B1

Multiple myeloma (MM) is an incurable B cell neoplasm causing significant morbidity and mortality. Despite recent advances in the MM-treatment, the disease still remains incurable; necessitating a search for new therapeutic agents. Therefore, Strychnos nux-vomica L. root extract (SN) was screened using the human MM-cell line, U266B1. SN-extract demonstrated anti-proliferative effect in a time- and dose-dependent manner. Morphological studies, cell cycle analysis by FACS indicate apoptosis. Furthermore, assessment of signaling molecules like IL-6 (interleukin-6) and CD-138 (Sydencan-1) confirmed apoptosis. The anti-proliferative activity of SN-extract is associated with apoptosis, which is likely due to the presence of alkaloids, strychnine and brucine, which have been identified by MALDI-TOF-MS and RP-HPLC analysis