Mature Cell Surface Research Articles

A Pro-Inflammatory Gene Signature Characterizes a Better Risk Aged AML Patient Group in ECOG-ACRIN Cancer Research Group's Clinical Trial E3999

Background: Acute myeloid leukemia in patients over the age of 60 (aAML) is associated with poor prognosis. aAML patients are the majority diagnosed but are underrepresented in large molecular studies. We have identified two clinical subgroups of aAML patients with distinct survival outcomes (low-risk and high-risk) in ECOG-ACRIN Cancer Research Group's clinical trial E3999 (NCT00046930). We hypothesize that these two groups are characterized by distinct biological mechanisms that contribute to leukemogenesis. We also hypothesize that the low-risk group harbor molecular and phenotypic signatures characteristic of known better-risk AMLs. Methods: Diagnostic specimens were obtained from patients enrolled in NCT00046930. Disease enriched cells were isolated by negative selection of lymphocytes (magnetic beads). Flow cytometry was used to characterize the blast population. Bulk RNA-sequencing was performed on 220 blast enriched samples and 10 age matched normal controls (CD34+ bone marrow cells). Transcriptional differences were determined using DESeq2. Differentially expressed genes (DEGs) were identified as those with an absolute log2 fold change greater than 1 and q less than 0.05. Gene set enrichment analysis (GSEA) was performed against the MSigDB databases. Upstream regulators associated with gene expression changes were identified using Ingenuity Pathway Analysis (IPA). DESeq2 was used to identify genes that were differentially associated with overall survival times between the risk groups. Enrichment for transcription factor gene targets was calculated using EnrichR based on the CHEA Transcription Factor Target Dataset. Cytokines were quantified in a subset of patient serum samples using a Luminex assay. Results: We identified 2314 DEGs between the low-risk and high-risk aAMLs with high-risk as the baseline. The upregulated genes in the low-risk aAMLs were positively enriched for gene signatures derived from: 1. Favorable risk AML ( NPM1 mutated), 2. M4 and M5 AML patients, 3. Differentiated hematopoietic cells and 4. Inflammatory response pathways. On the contrary, upregulated genes in the high-risk aAMLs were enriched for gene signatures identified in hematopoietic and leukemic stem cells and known poor-risk AMLs (for example: EVI1 fusion). Accordingly, immunophenotypic data from flow cytometry results gated on the blast populations validated differences in the surface expression of hematopoietic cell markers between the two groups. Next, we sought to determine upstream regulators associated with the DEGs using IPA. We identified 107 potential upstream regulators that associated with the changes in gene expression observed between the low- and high-risk groups (87 activated and 20 inhibited). The results suggested that several pro-inflammatory cytokines (ex. tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ)) were predicted activators in the low-risk aAML group. Accordingly, we detected significantly higher levels of TNFα and IFNγ in the serum levels of a subset of low-risk aAML patients. Finally, we assessed for genes whose expression significantly associated with overall survival. Interestingly, in agreement with a previously proposed inflammation score (iScore), we identified higher iScore associated with the high-risk group. Of note, the upstream regulators identified do not overlap with those contributing to the calculation of iScore. Additionally, longer overall survival times were associated with higher levels of expression of genes associated with heme metabolism and erythroblast differentiation in the low-risk aAML patient group. Conclusions: Our results suggest that the low-risk aAML patients were characterized by a more mature hematopoietic cell gene expression signature and cell surface markers as well as a pro-inflammatory state. Our study supports the possibility that pro-inflammatory cytokines and/or signaling could mediate AML cell survival in the low-risk aAML patient group. The high-risk aAML patients were characterized by a more primitive hematopoietic and leukemic stem cell gene expression signature. Collectively, we propose that differences in the underlying molecular and/or immune-based mechanisms could contribute to leukemogenesis in the two risk groups. These findings suggest that distinct therapeutic approaches may need to be considered to improve clinical outcomes in the two aAML risk groups.

Biological Characteristics and Risk Factors for Prognosis of Myelodysplastic Syndrome

To investigate the biological characteristics and risk factors for prognosis of patients with myelodysplastic syndrome (MDS), to emphatically study the correlation of different chromosome karyotype and immune phenotype with the prognosis of MDS patients. All clinical data of 60 patients diagnosed as primary MDS over a period of 5 years were collected and compared. The bone marrow short-term culture and G banding technique were used to analyze the chromosome karyotypes of 60 patients, the flow cytometry was used to detect the immune phenotypes(CD10, CD13, CD15, CD7, CD34, CD117). SPSS 19.0 software was applied to deal with all data, the WHO2008 classification and IPSS-R score were used for grouping of MDS. There were 12 cases of low-risk, and 7 cases of high-risk in 60 patients with MDS; 35 out of 60 patients (58.33%) showed karyotypic abnormalities. The proportion of 5, 7, 8, 20, and Y chromosome abnormality were 22.89%, 20.0%, 17.14%, 11.43% and 5.71%, respectively, complex chromosome karyotype accounted for 14.29%. The percentage of normal karyotype transformed into leukemia was 8.0%, the percentage of other abnormal karyotype transformed into leukemia respectively was 80% for complex karyotype, 66.67% for 8 chromosome abnormality, 71.43% for 7 chromosome abnormality, 75.0% for 5 chromosome abnormality. The expression rate of immature myeloid cell surface antigen markers(CD13, CD33, CD117) in high-risk group of patients with MDS significantly higher than that in low-risk group(P<0.05). And the expression rate of mature myeloid cell surface antigen markers (CD15) in high-risk group was significantly lower than that in low-risk group(P<0.05). The expression rate of lymphoid cell surface antigen markers (CD7, CD10) in high-risk group was lower than that in low-risk group(P<0.05). Chromosome karyotypes and immune phenotypes have great influence on the prognosis of patients with MDS.

Differences in Env and Gag protein expression patterns and epitope availability in feline immunodeficiency virus infected PBMC compared to infected and transfected feline model cell lines

Env and Gag are key components of the FIV virion that are targeted to the plasma membrane for virion assembly. They are both important stimulators and targets of anti-FIV immunity. To investigate and compare the expression pattern and antigenic changes of Gag and Env in various research models, infected PBMC (the natural FIV host cells) and GFox, and transfected CrFK were stained over time with various Env and Gag specific MAbs. In FIV infected GFox and PBMC, Env showed changes in epitope availability for antibody binding during processing and trafficking, which was not seen in transfected CrFK. Interestingly, epitopes exposed on intracellular Env and Env present on the plasma membrane of CrFK and GFox seem to be hidden on plasma membrane expressed Env of FIV infected PBMC. A kinetic follow up of Gag and Env expression showed a polarization of both Gag and Env expression to specific sites at the plasma membrane of PBMC, but not in other cell lines. In conclusion, mature trimeric cell surface expressed Env might be antigenically distinct from intracellular monomeric Env in PBMC and might possibly be unrecognizable by feline humoral immunity. In addition, Env expression is restricted to a small area on the plasma membrane and co-localizes with a large moiety of Gag, which may represent a preferred FIV budding site, or initiation of virological synapses with direct cell-to-cell virus transmission.

Analysis of Ikaros tumor suppressor function in BCR-ABL1+ pre-B ALL reveals conserved target genes and biological pathways

Abstract Inactivation of the transcriptional factor Ikaros (IKZF1) correlates with poor prognosis in progenitor B-cell acute lymphoblastic leukemia (pre-B ALL), and is a hallmark of the BCR-ABL1+ subgroup of pre-B ALL. Ikaros is a critical regulator of hematopoietic development and required for B-cell development, however the mechanisms by which Ikaros functions as a tumor suppressor in pre-B ALL remain poorly understood. We analyzed recently developed mouse models of BCR-ABL1+ pre-B ALL containing targeted deletions of Ikaros DNA-binding zinc finger domains together with a new model of inducible expression of WT Ikaros in IKZF1-mutant human BCR-ABL1+ pre-B ALL. We found that both the mouse and human Ikaros-mutated leukemic cells displayed a less mature cell surface phenotype and failed to downregulate the developmentally restricted cell surface receptors c-kit and CD34, respectively. In addition, Ctnnd1, a gene that is also expressed in earlier hematopoietic progenitor cells and normally downregulated as cells differentiate down the B-cell lineage, was found to be a conserved Ikaros target gene, with increased expression in Ikaros-mutated leukemic cells. RNA sequencing defined the Ikaros target genes in both mouse and human Ikaros-mutated pre-B ALL cells and revealed additional conserved target genes and biological functions. Loss of Ikaros tumor suppression was associated with deregulated adhesion pathways and stem-cell signatures. Furthermore, our results presented herein suggest that Ikaros mediates tumor suppressor function, at least in part, by enforcing proper developmental-stage specific expression of multiple genes involved in a network of cadherin-dependent, Rho-regulated Wnt/b-catenin pathways.

IL-1β and Inflammasome Activity Link Inflammation to Abnormal Fetal Airway Development.

Inflammation in the developing preterm lung leads to disrupted airway morphogenesis and chronic lung disease in human neonates. However, the molecular mechanisms linking inflammation and the pathways controlling airway morphogenesis remain unclear. In this article, we show that IL-1β released by activated fetal lung macrophages is the key inflammatory mediator that disrupts airway morphogenesis. In mouse lung explants, blocking IL-1β expression, posttranslational processing, and signaling protected the formation of new airways from the inhibitory effects ofEscherichia coliLPS. Consistent with a critical role for IL-1β, mice expressing a gain-of-functionNlrp3allele and subsequent overactive inflammasome activity displayed abnormal saccular-stage lung morphogenesis and died soon after birth. Although the early-stage fetal lung appeared capable of mounting an NF-κB-mediated immune response, airway formation became more sensitive to inflammation later in development. This period of susceptibility coincided with higher expression of multiple inflammasome components that could increase the ability to release bioactive IL-1β. Macrophages fromNlrp3gain-of-function mice also expressed higher levels of more mature cell surface markers, additionally linking inflammasome activation with macrophage maturation. These data identify developmental expression of the inflammasome and IL-1β release by fetal lung macrophages as key mechanisms and potential therapeutic targets for neonatal lung disease.

Stem cell hunt in NHL.

Non-Hodgkin Lymphoma (NHL) is a heterogenous disease consisting of at least 40 different entities that are defined mainly by their clinical and histopathological presentation, their underlying genetic abnormalities but also their presumed cell of origin [1]. The latter can be of B- or T-cell type and derived from different stages of the differentiation process. B-cells are predisposed to DNA alterations including somatic hypermutation, class switch recombination and antigen receptor rearrangement. This innate genetic instability may be one of the reasons why B-cell NHL is the most frequently diagnosed lymphoma in the Western World; many B-cell NHLs arise from germinal center (GC) cells or cells that have just left the GC [2]. Hence, most NHL (including T-cell NHL) are considered to arise from mature, peripheral lymphoid cells. In contrast, both T- and B-lymphoblastic lymphoma/leukaemia cells arise from immature precursors that are found in the thymus and in the bone marrow, respectively. Based on these findings one may hypothesize that the differentiation status of the tumor cell defines their cell of origin in healthy tissue. However, it is also possible that mature lymphoid cells change differentiation status when they become neoplastic and/or that differentiation processes contribute to disease pathogenesis with the seeds of malignancy planted much earlier in their lifespan. We have shown in a specific entity of T-NHL that some of the tumor cells have a mature cell surface phenotype despite having the ‘ghostly’ genetic appearance of a primitive origin, supporting the idea of cell plasticity in the context of neoplasia [3]. Anaplastic Large Cell Lymphoma (ALCL) is a peripheral T-cell lymphoma; tumour cells are found in the periphery at nodal and extranodal sites, express proteins associated with a cytotoxic T-cell function and exhibit rearrangements of the T-cell receptor (TCR) at the molecular level (the TCR is rarely expressed on the surface of ALCL cells) [3]. However, contrary to popular belief, at least in paediatric ALCL, mediastinal involvement is not infrequent (50%) implicating the thymus, the organ of T-cell development in disease pathogenesis [4]. We show that a gene signature enriched in early thymic progenitors can be detected in a subset of ALCL tumour cells which functionally act as tumour propagating or cancer stem cells (CSC) [3]. The ALCL CSC were isolated using the Side Population (SP) technique which relies on the functional properties of stem cells, i.e. quiescence and expression of efflux pumps presumably evolutionarily conserved to protect the stem cell compartment. This technique has been applied to a number of cancers and was originally developed to enrich for haemopoietic stem cells in murine bone marrow [5]. We show that the ALCL SP cells not only give rise to the bulk tumour population whilst self-replicating to a discrete level but also produce as yet unidentified soluble factors that support the growth of the tumour as a whole [3]. Therefore we hypothesize that a thymic origin may apply to this disease and that these ‘primed’ T-cells egress the thymus and are able to survive and circulate in the periphery to eventually transform as a result of yet to be identified events giving rise to ALCL CSC. Whether distinct clones of ALCL CSC also exist remains to be determined and whilst to date we have been able to identify these cells by means of their functional attributes, in depth mechanistic analyses still need to be performed. It is clear now that at least a limited hierarchy exists and given the postulated thymic origin of these cells it is very likely that tumour propagating cells may reside in the thymus to seed disease relapse, an event that is common in children with ALCL [6]. Figure 1 ALCL cancer stem cells (CSC) have a potential thymic origin

Flow cytometric analysis of FSHR, BMRR1B, LHR and apoptosis in granulosa cells and ovulation rate in merino sheep

The aim of the present study was to determine the direct cause of the mutation-induced, increased ovulation rate in Booroola Merino (BB) sheep. Granulosa cells were removed from antral follicles before ovulation and post-ovulation from BB (n=5) and WT (n=12) Merino ewes. Direct immunofluorescence measurement of mature cell surface receptors using flow cytometry demonstrated a significant up-regulation of FSH receptor (FSHR), transforming growth factor beta type 1, bone morphogenetic protein receptor (BMPR1B), and LH receptor (LHR) in BB sheep. The increased density of FSHR and LHR provide novel evidence of a mechanism for increasing the number of follicles that are recruited during dominant follicle selection. The compounding increase in receptors with increasing follicle size maintained the multiple follicles and reduced the apoptosis, which contributed to a high ovulation rate in BB sheep. In addition, we report a mutation-independent mechanism of down-regulation to reduce receptor density of the leading dominant follicle in sheep. The suppression of receptor density coincides with the cessation of mitogenic growth and steroidogenic differentiation as part of the luteinization of the follicle. The BB mutation-induced attenuation of BMPR1B signaling led to an increased density of the FSHR and LHR and a concurrent reduction in apoptosis to increase the ovulation rate. The role of BMPs in receptor modulation is implicated in the development of multiple ovulations.

Characterization of the mature cell surface proteinase of Lactobacillus delbrueckii subsp. lactis CRL 581.

The cell envelope-associated proteinase (CEP) of Lactobacillus delbrueckii subsp. lactis CRL 581 (PrtL) has an essential role in bacterial growth, contributes to the flavor and texture development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. The genome of L. delbrueckii subsp. lactis CRL 581 possesses only one gene that encodes PrtL, which consists of 1924 amino acids and is a multidomain protein anchored to the cell via its W domain. PrtL was extracted from the cell under high ionic strength conditions using NaCl, suggesting an electrostatic interaction between the proteinase and the cell envelope. The released PrtL was purified and biochemically characterized; its activity was maximal at temperatures between 37 and 40°C and at pH between 7 and 8. Under optimal conditions, PrtL exhibited higher affinity for succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide than for succinyl-alanyl-glutamyl-prolyl-phenylalanine-p-nitroanilide, while methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide was not degraded. A similar α- and β-casein degradation pattern was observed with the purified and the cell envelope-bound proteinase. Finally, on the basis of its specificity towards caseins and the unique combination of amino acids at residues thought to be involved in substrate specificity, PrtL can be classified as a representative of a new group of CEP.

Exploration of a 3D Scaffold Culture System for Ex Vivo Blood Production

The human body produces 2.5 million red blood cells (RBC) every second in the bone marrow, where differentiation of immature erythroblasts to reticulocytes occurs largely within erythroblastic islands. Erythropoiesis can be routinely replicated in 2D liquid culture using hematopoietic stem cells (HSC) isolated from adult peripheral blood. However current 2D culture methodologies eventually exhaust the input of HSC. The use of 3D scaffolds to better mimic erythropoiesis in the bone marrow would increase the RBC yield and longevity of ex vivo cultures, whilst reducing use of exogenous cytokines and minimizing handling requirements.As a starting point we are utilizing a porous collagen coated synthetic polyurethane (PU) scaffold (0.5cm by 0.5cm) for static 3D cultures provided by the Mantalaris group at Imperial College London (Mortera-Blanco et al., 2011). The PU scaffolds were seeded with lineage depleted or CD34+ population isolated from adult peripheral blood and maintained in serum-free erythroid expansion media with SCF, IL-3 and Dexamethasone, alone, or with erythropoietin (EPO) or thrombopoietin (TPO). Advantages of using the lineage depleted population is that it provides a larger diversity of stem cells for establishment of the niche, potentially facilitating the use of rare patient blood samples which may only be available in small sample volumes with low numbers of CD34+ cells. Scaffolds are productive using both cellular inputs, with significant cellular egress for up to 5 weeks regardless of whether exogenous EPO or TPO were included. As anticipated, the highest increase in cell production from the scaffolds was observed using CD34+ in the presence of EPO, which also provided a significant reduction in cell death. Histology and immunofluorescence were used to explore the cell populations within the scaffold. No mature macrophages were detected but GPA+ cells within the scaffold was observed until the end of culture, suggesting that cellular expansion is occurring without establishment of the classical macrophage niche.We also characterized the cells that continually egress from the CD34+ scaffold cultures using flow cytometry. Typically the cellular output exhibited approximately 20-60% CD34 positivity dropping to <15% post day 24 of culture and 10-30% GPA positivity, the remainder of the population was largely CD61 positive, with a contingent of CD14 positive monocytes. Mature cell surface markers for erythroid and megakaryocytic lineages were detected in up to half of the population, when the harvested 3D egress cells were further cultured in erythroid or megakaryocyte 2D culture systems. This suggests a high proportion of the cells that egress from the scaffold are megakaryocyte erythroid progenitors (MEPs) that are consistently expanding within the scaffold environment for the entire culture period.Since CD34+ expansion here has been achieved in the absence of macrophages, we wanted to explore their effects in 2D with the intention of introducing macrophages or macrophage derived functionality into our next generation scaffolds. Importantly, macrophage inclusion significantly increased proliferation of expanding erythroblasts compared to erythroblasts alone. Expansion of CD34+ cells in co-culture gave a statistically significant average fold increase of 528 compared to 301 for the control at day 7 (p = 0.0126 and 0.0162 for days 5 and 7 respectively (n=5)). Flow cytometry at the endpoint of the experiment showed a larger CD34+ population and a reduced GPA+ population when cells are in co-culture, suggesting the CD34+ cells are maintained in a more primitive state for longer. Therefore, co-culture has the additional benefit of improving early erythroblast expansion, alongside the recently reported enhanced expansion of erythroblasts during terminal differentiation (Ramos et al., 2013).In summary, we have demonstrated that a basic static PU scaffold can be utilized to increase hematopoietic stem cell culture longevity and facilitate generation of megakaryocyte or erythroid progenitors with expansion potential. This occurs in the absence of any detectable macrophage niche generation. We have also shown that co-culture with macrophages enhances erythroblast expansion in 2D. Further work is needed to determine whether inclusion of macrophages or macrophage derived proteins in our scaffolds will effectively boost progenitor production. DisclosuresNo relevant conflicts of interest to declare.

The orphan nuclear receptor Ear-2 (Nr2f6) is a novel negative regulator of T cell development

We describe a novel role for the orphan nuclear receptor Ear-2 in regulating T cell development. Retrovirus-mediated overexpression of Ear-2 (EAR-2++) in a bone marrow (BM) transplantation assay resulted in limited T cell development and a greater than tenfold decrease in thymus size and cellularity relative to controls. Ear-2-transduced murine BM hematopoietic stem cells (HSCs) in OP9-DL1 cultures showed a proliferation deficit during days 1-5 after induction of differentiation, which corresponded to increased expression of the cell cycle regulators p21 (cdkn1a) and p27 (cdkn1b), as well as increased expression of Hes1, Notch3, Egr1, and Scl (Tal1) and decreased expression of Gli1, Gfi-1, HoxA9, PU.1, Nrarp, and Tcf1. In addition, there was a block in differentiation at the DN4 to double-positive (DP) transition accompanied by an increase in apoptosis, similar to the deficit seen in the RORγt null mouse. Gene expression profiling revealed that, like the RORγt-deficient mouse, EAR-2++ DP cells had decreased expression of BclXL and increased expression of the proapoptosis gene Bad. In addition, EAR-2++ DP cells had decreased expression of Bcl11b, PU.1, and HoxA9, and increased expression of Id2. Based on these findings, we conclude that EAR-2++ cells were able to migrate to, but not fully repopulate, the thymus because of a cell-intrinsic defect in the proliferation of DN1 cells followed by a block in differentiation from the DN4 to DP stage of T cell development. We conclude that Ear-2 is a novel negative regulator of T-cell development and that downregulation of Ear-2 is indispensable for the proliferation of DN1 cells and the survival of DN4-DP cells.

Megakaryopoiesis in the mammalian embryo is associated with rapid lineage maturation and synthesis of platelets with altered activation

We have previously determined that megakaryocyte (MK) progenitors first emerge in the yolk sac of E7.5 mouse embryos and are subsequently found in the E10.5 liver, concomitant with initiation of platelet release into the bloodstream (Tober, Blood 2007). While megakaryopoiesis is established prior to hematopoietic stem cell emergence, little is known regarding the differentiation and function of the MK lineage during embryogenesis. We have now compared E12.5 fetal liver versus the adult marrow and find that primary embryonic MKs have lower mean ploidy than adult MKs. Unlike adult MKs, low-ploidy embryonic MKs reveal evidence of maturation, including mature cell surface marker expression and the presence of a punctate pattern of a-granule components and demarcation membranes in the cytoplasm. These differences between embryonic and adult MKs appear to be cell intrinsic, since MKs derived from the in vitro culture of E9.5 yolk sac versus adult marrow have lower ploidy and late-stage cell surface marker expression. While we had previously shown that embryonic platelets are larger, more reticulated, with small a-granules than adult platelets, our recent histochemical studies reveal similar distribution patterns of the a-granule components VEGF and endostatin. To begin to determine if there are functional differences between embryonic and adult platelets, we first compared expression of transcripts associated with signaling cascades regulating platelet activation. While the transcripts for many components are similar, embryonic platelets express much lower levels of ADP-receptors (P2Y12, P2Y1) and P-selectin. Consistent with these results, E12.5 platelet activation failed to increase P-selectin on the platelet surface. Taken together, our findings indicate that embryonic MKs are characterized by low ploidy and rapid maturation leading to the generation of platelets with marked differences in size, structure and function compared to adult platelets. Hypothesizing that differentiating ES cells recapitulate early embryonic events, we find that MKs generated from ES cells have the low ploidy and rapid maturation characteristics of primary embryonic MKs. A better understanding of embryonic megakaryopoiesis will lay a foundation for the development of clinically useful platelet therapies from ES/iPS cell sources.

β-Adrenergic Agonists Mediate Enhancement of β1-Adrenergic Receptor N-terminal Cleavage and Stabilization In Vivo and In Vitro

The β(1)-adrenergic receptor (β(1)AR) is the predominant βAR in the heart and is the main target for β-adrenergic antagonists, widely used in the treatment of cardiovascular diseases. Previously, we have shown that the human (h) β(1)AR is cleaved in its N terminus by a metalloproteinase, both constitutively and in a receptor activation-dependent manner. In this study, we investigated the specific events involved in β(1)AR regulation, focusing on the effects of long-term treatment with β-adrenergic ligands on receptor processing in stably transfected human embryonic kidney 293(i) cells. The key findings were verified using the transiently transfected hβ(1)AR and the endogenously expressed receptor in neonatal rat cardiomyocytes. By using flow cytometry and Western blotting, we demonstrated that isoproterenol, S-propranolol, CGP-12177 [4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one], pindolol, and timolol, which displayed agonistic properties toward the β(1)AR in either the adenylyl cyclase or the mitogen-activated protein kinase signaling pathways, induced cleavage of the mature cell-surface receptor. In contrast, metoprolol, bisoprolol, and CGP-20712 [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol], which showed no agonistic activity, had only a marginal or no effect. Importantly, the agonists also stabilized intracellular receptor precursors, possibly via their pharmacological chaperone action, and they stabilized the receptor in vitro. The opposing effects on the two receptor forms thus led to an increase in the amount of cleaved receptor fragments at the plasma membrane. The results underscore the pluridimensionality of β-adrenergic ligands and extend this property from receptor activation and signaling to the regulation of β(1)AR levels. This phenomenon may contribute to the exceptional resistance of β(1)ARs to downregulation and tendency toward upregulation following long-term ligand treatments.

Over‐Expression of Sphingosine Kinase‐1 Enhances a Progenitor Phenotype in Human Endothelial Cells

The use of endothelial progenitor cells in vascular therapies has been limited due to their low numbers present in the bone marrow and peripheral blood. The aim of this study was to investigate the effect of sphingosine kinase on the de-differentiation of mature human endothelial cells toward a progenitor phenotype. The lipid enzyme sphingosine kinase-1 was lentivirally over-expressed in human umbilical vein endothelial cells and cells were analyzed for progenitor phenotype and function. Sphingosine kinase-1 mRNA expression was induced approximately 150-fold with a resultant 20-fold increase in sphingosine kinase-1 enzymatic activity. The mRNA expression of the progenitor cell markers CD34, CD133, and CD117 and transcription factor NANOG increased, while the endothelial cell markers analyzed were largely unchanged. The protein level of mature endothelial cell surface markers CD31, CD144, and von Willebrand factor significantly decreased compared to controls. In addition, functional assays provided further evidence for a de-differentiated phenotype with increased viability, reduced uptake of acetylated low-density lipoprotein and decreased tube formation in Matrigel in the cells over-expressing sphingosine kinase-1. These findings suggest that over-expression of sphingosine kinase-1 in human endothelial cells promotes, in part, their de-differentiation to a progenitor cell phenotype, and is thus a potential tool for the generation of a large population of vascular progenitor cells for therapeutic use.

TWEAK Induces Apoptosis through a Death-signaling Complex Comprising Receptor-interacting Protein 1 (RIP1), Fas-associated Death Domain (FADD), and Caspase-8

The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TNFSF12, CD255) (TWEAK) can stimulate apoptosis in certain cancer cells. Previous studies suggest that TWEAK activates cell death indirectly, by inducing TNFα-mediated autocrine signals. However, the underlying death-signaling mechanism has not been directly defined. Consistent with earlier work, TWEAK assembled a proximal signaling complex containing its cognate receptor FN14, the adaptor TRAF2, and cellular inhibitor of apoptosis protein 1 (cIAP1). Neither the death domain adaptor Fas-associated death domain nor the apoptosis-initiating protease caspase-8 associated with this primary complex. Rather, TWEAK induced TNFα secretion and TNF receptor 1-dependent assembly of a death-signaling complex containing receptor-interacting protein 1 (RIP1), FADD, and caspase-8. Knockdown of RIP1 by siRNA prevented TWEAK-induced association of FADD with caspase-8 but not formation of the FN14-TRAF2-cIAP1 complex and inhibited apoptosis activation. Depletion of the RIP1 E3 ubiquitin ligase cIAP1 enhanced assembly of the RIP1-FADD-caspase-8 complex and augmented cell death. Conversely, knockdown of the RIP1 deubiquitinase CYLD inhibited these functions. Depletion of FADD, caspase-8, BID, or BAX and BAK but not RIP3 attenuated TWEAK-induced cell death. Pharmacologic inhibition of the NF-κB pathway or siRNA knockdown of RelA attenuated TWEAK induction of TNFα and association of RIP1 with FADD and caspase-8. These results suggest that TWEAK triggers apoptosis by promoting assembly of a RIP1-FADD-caspse-8 complex via autocrine TNFα-TNFR1 signaling. The proapoptotic activity of TWEAK is modulated by cIAP1 and CYLD and engages both the extrinsic and intrinsic signaling pathways.

Reticulocyte membrane remodeling: contribution of the exosome pathway

In addition to the loss of all the internal compartments, reticulocyte maturation is characterized by an extensive membrane remodeling. Exosomal secretion contributes to this process by eliminating specific proteins. Regulation in the exosomal sorting of the water channel aquaporin-1 represents a newly described mechanism by which reticulocytes could adapt to environmental modifications. The extracellular osmotic conditions found in the peripheral circulation vs. the bone marrow could dictate a lower level of expression of aquaporin-1 on the mature red cell surface. In addition, a new mechanism for protein sorting to exosomes, involving an endogenous lectin, has been pinpointed in rat reticulocytes. Galectin-5 is secreted by a so-called alternative pathway, and could be involved in the sorting of galactoside-bearing glycoconjugates, since it was found associated with the surface of released exosomes. Secretion of exosomes during reticulocyte maturation is an integral part of the red cell differentiation program and illustrates specific mechanisms in terms of biogenesis, protein sorting and fate, which are far from completely understood and open new paths for research in the red cell physiology field.

Antibodies to the Desmoglein 1 Precursor Proprotein but Not to the Mature Cell Surface Protein Cloned from Individuals without Pemphigus

In pemphigus foliaceus (PF), autoantibodies against desmoglein 1 (Dsg1) cause blisters. Using Ab phage display, we have cloned mAbs from a PF patient. These mAbs, like those from a previous patient, were directed against mature Dsg1 (matDsg1) on the cell surface of keratinocytes and precursor Dsg1 (preDsg1) in the cytoplasm. To determine whether individuals without pemphigus have B cell tolerance to Dsg1, we cloned mAbs from two patients with thrombotic thrombocytopenic purpura and a healthy person. We found mAbs against preDsg1, but not matDsg1. All but 1 of the 23 anti-preDsg1 mAbs from PF patients and those without PF used the VH3-09 (or closely related VH3-20) H chain gene, whereas no PF anti-matDsg1 used these genes. V(H) cDNA encoding anti-preDsg1 had significantly fewer somatic mutations than did anti-matDsg1 cDNA, consistent with chronic Ag-driven hypermutation of the latter compared with the former. These data indicate that individuals without PF do not have B cell tolerance to preDsg1 and that loss of tolerance to matDsg1 is not due to epitope shifting of anti-preDsg1 B cells (because of different V(H) gene usage). However, presentation of peptides from Dsg1 by preDsg1-specific B cells may be one step in developing autoimmunity in PF.

Major Histocompatibility Complex Class II-Peptide Complexes Internalize Using a Clathrin- and Dynamin-independent Endocytosis Pathway

Major histocompatibility complex (MHC) class II molecules (MHC-II) function by binding antigenic peptides and displaying these peptides on the surface of antigen presenting cells (APCs) for recognition by peptide-MHC-II (pMHC-II)-specific CD4 T cells. It is known that cell surface MHC-II can internalize, exchange antigenic peptides in endosomes, and rapidly recycle back to the plasma membrane; however, the molecular machinery and trafficking pathways utilized by internalizing/recycling MHC-II have not been identified. We now demonstrate that unlike newly synthesized invariant chain-associated MHC-II, mature cell surface pMHC-II complexes internalize following clathrin-, AP-2-, and dynamin-independent endocytosis pathways. Immunofluorescence microscopy of MHC-II expressing HeLa-CIITA cells, human B cells, and human DCs revealed that pMHC enters Arf6(+)Rab35(+)EHD1(+) tubular endosomes following endocytosis. These data contrast the internalization pathways followed by newly synthesized and peptide-loaded MHC-II molecules and demonstrates that cell surface pMHC-II internalize and rapidly recycle from early endocytic compartments in tubular endosomes.

Identification and Characterization of 6-Benzylthioinosine as a Novel Myeloid Differentiation-Inducing Compound.

Acute myeloid leukemia (AML) is the most common form of leukemia in adults and despite advances in treatment the 5 year survival is less than 20–50% in adults and significantly lower in the elderly. The remarkable success in treating one relatively uncommon subset of AML, APL, with all trans-retinoic acid (ATRA) illustrates the great promise for agents with greater efficacy and less toxicity. Utilizing ATRA, the presumed cure of 75–85% of patients is possible. ATRA's remarkable success stems from the fact that AML is a disease characterized by the arrest of differentiation of immature myeloid cells. ATRA overcomes this block in differentiation by forcing leukemic cells to mature. Unfortunately, ATRA is not clinically useful for patients with AML that do not have the APL subtype. Through a compound library screen, we have recently identified several novel leukemia differentiation-inducing compounds structurally unrelated to previously described differentiation-inducers including a particularly promising compound, 6-benzylthioinosine (6BT). 6BT is a nucleoside analogue that induces monocytic differentiation of multiple AML cell lines as well as primary leukemic patient samples as measured by immunophenotyping, morphology, and NBT reduction. 6BT can induce terminal differentiation of leukemic cells as evidenced by complete prevention of colony formation in soft agar assays after pretreatment with low doses of 6BT (5μM) for 72 hours. In addition 6BT has potent in vivo activity. 6BT completely prevents subcutaneous tumor formation in myeloid leukemia mouse xenograft models in nude mice using HL-60 or MV4-11 leukemic cell lines (n=5) and significantly inhibits myeloid leukemia tumor growth in an HL-60 established tumor xenograft model (n=5). In the established tumor model at the end of the 4 week study period, tumors were significantly smaller after 6BT treatment as compared to vehicle treated mice (0.16g +/− 0.05g vs 0.73g +/− 0.28g). Evidence of 6BT-mediated in vivo differentiation exists as 92% of the tumor cells expressed the mature myeloid cell surface marker CD11b as compared to only 16% in the vehicle control after 4 weeks. Early studies suggest 6BT partially depletes purine nucleotide stores leading to growth inhibition and subsequent myeloid differentiation. Treatment of HL-60 cells with 6BT (5μM) leads to rapid depletion of approximately 75% of ATP stores within 24 hours. The depletion of ATP is not due to the differentiation process itself as after 6BT treatment HL-60 cells contain 24.6% +/− 2.8 of the ATP levels of vehicle treated cells compared to 91.2% +/− 7.2 in ATRA (1μM) treated cells. Preliminary studies also indicate that 6BT exhibits relatively low toxicity with an LD50 at 5 days greater than 100μM in human umbilical vein endothelial cells, mouse embryonic fibroblasts, and primary human lymphocytes. The low toxicity is likely related to its unique chemical structure that prevents its uptake into most mammalian cells as it acts as an inhibitor of one of the main nucleoside transporters, ent1. It is known that leukemic cells can express nucleoside transporters that are not widely expressed by other cell types. As differentiation therapy has already been demonstrated in the case of ATRA to significantly improve the prognosis of patients with AML, further characterization of 6BT could lead to a novel AML therapy that is more efficacious, less toxic, and better tolerated especially for elderly patients.

Endoplasmic Reticulum-Associated Degradation of Growth Hormone Receptor in Janus Kinase 2-Deficient Cells

A key factor governing cellular sensitivity to GH is cell surface GH receptor (GHR) abundance, which is affected transcriptionally and posttranscriptionally. Mature cell surface GHR abundance is regulated by constitutive and inducible metalloproteolysis and constitutive endosomal/lysosomal degradation. We previously found that Janus kinase 2 (JAK2)-deficient GHR-expressing cells have a greater precursor/mature GHR ratio, exhibit diminished inducible metalloproteolysis, and have a cytoplasmic domain-containing GHR fragment called the basal remnant (by virtue of comigration on SDS-PAGE with the inducible, metalloprotease-generated remnant). Herein we examined the mechanism of generation of basal remnant in JAK2-deficient cells, asking whether it originates from precursor vs. mature receptor and which protease(s) catalyzes its appearance. Prolonged metalloprotease inhibitor treatment or small interfering RNA knockdown of TNF-alpha converting enzyme (TACE) and a disintegrin and metalloprotease-10 (ADAM10) (both implicated in inducible GHR proteolysis) did not reduce basal remnant, indicating its generation is not metalloprotease dependent. However, a mutant GHR resistant to metalloprotease cleavage did not yield basal remnant when expressed in JAK2-deficient cells, suggesting common structural determinants for generation of the inducible remnant and the non-metalloprotease-generated basal remnant seen in JAK2-deficient cells. Treatment of JAK2-deficient cells with a proteasome inhibitor, but not two separate lysosome inhibitors, dramatically decreased basal remnant, accompanied by decreased precursor GHR and increased mature GHR abundance. Disruption of endoplasmic reticulum-to-Golgi transport with brefeldin A (BFA) also reduced basal remnant, and washout of BFA allowed regeneration of basal remnant along with GHR precursor. Notably, BFA washout in the presence of cycloheximide blocked both basal remnant and precursor GHR reappearance, but BFA washout in the presence of lactacystin blocked only basal remnant reappearance, suggesting that basal remnant is generated proteasome dependently from precursor GHR. Collectively, our data suggest that JAK2, by association with GHR in the secretory pathway, blunts proteasome activity-dependent discrete GHR cleavage and endoplasmic reticulum-dependent degradation of the precursor receptor. In so doing, JAK2 enables efficient processing of precursor receptor to mature GHR.

Impaired trafficking and maturation of hERG K+ channel protein by Hypoxia. Role of Hsp90 and Hsp70 chaperones

Human ether-a-go-go (hERG) K+ channels regulate resting membrane potential in a variety of cells. We have reported that hypoxia decreases hERG protein expression and attenuates hERG current densities. The effects of hypoxia were reversed by ROS scavengers. In the present study we examined whether the effects of hypoxia on hERG protein are due to defective maturation and/or trafficking of the protein. To test this possibility, we studied the impact of hypoxia on hERG processing in HEK 293 cells stably expressing hERG protein. Pulse chase studies showed that hypoxia blocks maturation of the core glycosylated form in the endoplasmic reticulum (ER) to the fully glycosylated form on the cell surface. Hypoxia also inhibited hERG protein synthesis by 50%. However, the turnover rate of the ER form was not affected by hypoxia. To test if the effects of hypoxia on maturation are due to chaperone-hERG interactions, we determined the association of Hsp90/Hsp70 with hERG protein under hypoxia. Coimmunoprecipitation and radio labelling experiments revealed that under normoxic conditions both Hsp90 and Hsp70 interacted exclusively with newly synthesized core-glycosylated hERG protein but not with fully glycosylated mature cell surface protein. The chaperone interactions were completely inhibited under hypoxia. These results demonstrate that hypoxia prevents maturation of the hERG protein by inhibiting chaperone interactions. Supported by NIH-HLBI-25830