ATP-induced apoptotic cell death in porcine ovarian theca cells through P2X7 receptor activation

Folliculogenesis modulation via distinct neurotransmitters is a well-documented phenomenon. Intraovarian purinergic signaling mechanisms have been identified previously in different species. However, the molecular elements involved and the physiological role of this purinergic signaling remain to be elucidated. Here, studies using RT-PCR amplification, immunoblotting, and immunofluorescence microscopy showed that murine and porcine ovaries express the P2X7 subtype receptor, a cationic receptor-channel operated by ATP. Using immunofluorescence it was demonstrated that P2X7 protein expression, in both mouse and pig, occurs specifically in the theca cells from antral follicles. Isolated porcine theca cells maintained in primary cultures and tested with 1 mM ATP or 250 microM Bz-ATP, a specific agonist of P2X7, responded with an increase in intracellular calcium concentration, as demonstrated in cells loaded with fluo-4 as calcium indicator. This strongly suggested that P2X7 receptors in theca cells are functional. Moreover, application for 24 hr of 1 mM ATP or 250 microM Bz-ATP induced apoptotic cell death as indicated by the DNA fragmentation pattern, positive TUNEL test, and annexin V binding. This ATP effect was antagonized by 300 microM PPADS and 200 microM oxidized ATP. Also, addition of 5 mM EGTA in the external medium to chelate free Ca++ decreased death cell to 24% of that produced by 200 microM Bz-ATP, suggesting that Ca++ influx participates in the phenomenon. The highly specific and functional expression of P2X7 receptors in theca cells suggest a role for ATP in modulating follicular physiology.     

Labeling of hematopoietic stem and progenitor cells in novel activatable EGFP reporter mice

Conditional activation and inactivation of genes using the Cre/loxP recombination system is a powerful tool for the analysis of gene function and for tracking cell fate. Here we report a novel silent EGFP reporter mouse line generated by enhancer trap technology using embryonic stem (ES) cells. Following transfection with the silent EGFP reporter construct, positive ES cell clones were treated with Cre recombinase. These "activated clones" were then further selected on the basis of ubiquitous EGFP expression during in vitro differentiation. The parental "silent" clones were then used for generating mice. Upon Cre-mediated activation in ovo tissues tested from these mice express EGFP. Long-term, strong and sustainable expression of EGFP is observed in most myeloid and lymphoid cells. As shown by in vivo transplantation assays, the majority of hematopoietic stem cells (HSCs) and spleen colony-forming units (CFU-S) reside within the EGFP positive fraction. Most in vitro colony-forming units (CFU-Cs) isolated from bone marrow also express EGFP. Thus, these reporter mice are useful for the analysis of Cre-mediated recombination in HSCs and hematopoietic progenitor cells. This, in combination with the high accessibility of the loxP sites, makes these mice a valuable tool for testing cell/tissue-specific Cre-expressing mice. .     

Osteoblast lineage cells expressing high levels of Runx2 enhance hematopoietic progenitor cell proliferation and function

Although osteoblasts (OB) play a key role in the hematopoietic stem cell (HSC) niche, little is known as to which specific OB lineage cells are critical for the enhancement of stem and progenitor cell function. Unlike hematopoietic cells, OB cell surface phenotypic definitions are not well developed. Therefore, to determine which OB lineage cells are most important for hematopoietic progenitor cell (HPC) function, we characterized OB differentiation by gene expression and OB function, and determined whether associations existed between OB and HPC properties. OB were harvested from murine calvariae, used immediately (fresh OB) or cultured for 1, 2, or 3 weeks prior to their co‐culture with Lin^?Sca1⁺c‐kit⁺ (LSK) cells for 1 week. OB gene expression, alkaline phosphatase activity, calcium deposition, hematopoietic cell number fold increase, CFU fold increase, and fold increase of Lin^?Sca1⁺ cells were determined. As expected, HPC properties were enhanced when LSK cells were cultured with OB compared to being cultured alone. Initial alkaline phosphatase and calcium deposition levels were significantly and inversely associated with an increase in the number of LSK progeny. Final calcium deposition levels and OB culture duration were inversely associated with all HPC parameters, while Runx2 levels were positively associated with all HPC properties. Since calcium deposition is associated with OB maturation and high levels of Runx2 are associated with less mature OB lineage cells, these results suggest that less mature OB better promote HPC proliferation and function than do more mature OB. J. Cell. Biochem. 111: 284–294, 2010. © 2010 Wiley‐Liss, Inc.     

Microencapsulated human bone marrow cultures: a potential culture system for the clonal outgrowth of hematopoietic progenitor cells

Currently the most successful methods for culturing human hematopoietic cells employ some form of perfused bioreactor system. However, these systems do not permit the clonal outgrowth of single progenitor cells. Therefore, we have investigated the use of alginate-poly-L-lysine microencapsulation of human bone marrow, combined with rapid medium exchange, as a system that may overcome this limitation for the purpose of studying the kinetics of progenitor cell growth. We report that a 12 to 24-fold multilineage expansion of adult human bone marow cells was achieved in about 16 to 19 days with this system and that visually identifiable colonies within the capsules were responsible for the increase in cell number. The colonies that represented the majority of cell growth originated from cells that appeared to be present in a frequency of about 1 in 4000 in the encapsulated cell population. These colonies were predominantly granulocytic and contained greater than 40,000 cells each. Large erythroid colonies were also present in the capsules, and they often contained over 10,000 cells each. Time profiles of the erythroid progenitor cell density over time were obtained. Burst-forming units erythroid (BFU-E) peaked around day 5, and the number of morphologically identifiable erythroid cells (erythroblasts through reticulocytes) peaked on day 12. We also report the existence of a critical inoculum density and how growth was improved with the use of conditioned medium derived from a microcapsule culture initiated above the critical inoculum density. Taken together, these results suggest that microencapsulation of human hematopoietic cells allows for outgrowth of progenitor, and possible preprogenitor, cells and could serve as a novel culture system for monitoring the growth and differentiation kinetics of these cells.     

CAPE promotes the expansion of human umbilical cord blood-derived hematopoietic stem and progenitor cells in vitro

Due to the low number of collectable stem cells from single umbilical cord blood(UCB)unit,their initial uses were limited to pediatric therapies.Clinical applications of UCB hematopoietic stem and progenitor cells(HSPCs)would become feasible if there were a culture method that can effectively expand HSPCs while maintaining their self-renewal capacity.In recent years,numerous attempts have been made to expand human UCB HSPCs in vitro.In this study,we report that caffeic acid phenethyl ester(CAPE),a small molecule from honeybee extract,can promote in vitro expansion of HSPCs.Treatment with CAPE increased the percentage of HSPCs in cultured mononuclear cells.Importantly,culture of CD34+HSPCs with CAPE resulted in a significant increase in total colony-forming units and high proliferative potential colony-forming units.Burst-forming unit-erythroid was the mostly affected colony type,which increased more than 3.7-fold in 1μg mL 1CAPE treatment group when compared to the controls.CAPE appears to induce HSPC expansion by upregulating the expression of SCF and HIF1-α.Our data suggest that CAPE may become a potent medium supplement for in vitro HSPC expansion.     

Temporal molecular program of human hematopoietic stem and progenitor cells after birth

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Identification of primitive hematopoietic progenitor cells in the rhesus macaque

The close phylogenetic relationship of macaques to humans has resulted in their widespread use as a preclinical model for bone marrow transplantation and stem cell gene therapy. To facilitate further use of this model, we undertook analysis of hematopoietic cells using multiparametric flow cytometric analysis. Rhesus CD34+CD38- cells displayed a number of characteristics of primitive hematopoietic cells, including low forward and orthogonal scatter and the lack of expression of lineage-specific markers or human lymphocyte antigen-DR. Four-color flow cytometric analysis demonstrated that rhesus CD34+CD38- cells were heterogenous with respect to Thy-1 expression and were CD59dim. Quantitative limiting dilution long-term culture-initiating cell (LTC-IC) analysis demonstrated that CD34+CD38- cells were approximately 150-fold enriched for LTC-IC as compared with unfractionated bone marrow, and occurred at a frequency similar to that previously reported in humans. Thus, as in humans, the CD34+38- population of rhesus macaque bone marrow is enriched for primitive, multipotent hematopoietic progenitor cells.     

Inhibition of HIV‐1 infection by a unique short hairpin RNA to chemokine receptor 5 delivered into macrophages through hematopoietic progenitor cell transduction

Background

We recently expressed a potent and noncytotoxic short hairpin (sh)RNA directed against chemokine (c‐c motif) receptor 5 (CCR5) using lentiviral mediated transduction of CD34+ hematopoietic progenitor cells (HPCs) and demonstrated the stable reduction of CCR5 expression in T‐lymphocytes.

Methods

In the present study, we further assessed the activity of the shRNA through HPC transduction and differentiation into macrophages derived from fetal liver CD34+ (FL‐CD34+) HPCs. Transduced lentiviral vector encoding the human CCR5 shRNA was stably maintained in FL‐CD34+ cells and in the terminally differentiated macrophages using macrophage colony‐stimulating factor, granulocyte macrophage colony‐stimulating factor, interleukin‐3 and stem cell factor.

Results

Quantitative real‐time polymerase chain reaction for CCR5 mRNA indicated over 90% reduction of CCR5 mRNA levels in CCR5 shRNA‐transduced population. The cells with knockdown of CCR5 expression acquired resistance to R5 tropic HIV‐1 NFN‐SX strain. We also developed a novel approach utilizing a mCherry‐CCR5 chimeric reporter to assess the effectiveness of CCR5 target down‐regulation in macrophages directly. Both the shRNA and the reporter were maintained throughout HPC differentiation to macrophages without apparent cytotoxicity.

Conclusions

The present study demonstrates a novel method to simply and directly assess the function of small interfering RNA and the effective inhibition of HIV‐1 infection by a potential potent shRNA to CCR5 delivered into macrophages derived from HPCs. Copyright © 2010 John Wiley & Sons, Ltd.     

Neural progenitor cell death is induced by extracellular ATP via ligation of P2X7 receptor

Neural progenitor cells (NPCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes, and have been used to treat several animal models of CNS disorders. In the present study, we show that the P2X7 purinergic receptor (P2X7R) is present on NPCs. In NPCs, P2X7R activation by the agonists extracellular ATP or benzoyl ATP triggers opening of a non-selective cationic channel. Prolonged activation of P2X7R with these nucleotides leads to caspase independent death of NPCs. P2X7R ligation induces NPC lysis/necrosis demonstrated by cell membrane disruption accompanied with loss of mitochondrial membrane potential. In most cells that express P2X7R, sustained stimulation with ATP leads to the formation of a non-selective pore allowing the entry of solutes up to 900 Da, which are reportedly involved in P2X7R-mediated cell lysis. Surprisingly, activation of P2X7R in NPCs causes cell death in the absence of pore formation. Our data support the notion that high levels of extracellular ATP in inflammatory CNS lesions may delay the successful graft of NPCs used to replace cells and repair CNS damage.     

A truncated P2X7 receptor variant (P2X7-j) endogenously expressed in cervical cancer cells antagonizes the full-length P2X7 receptor through hetero-oligomerization

A truncated naturally occurring variant of the human receptor P2X7 was identified in cancer cervical cells. The novel protein (P2X7-j), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X7 receptor. The P2X7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X7 receptor agonist benzoyl-ATP. The P2X7-j interacted with the full-length P2X7 in a manner suggesting heterooligomerization and blocked the P2X7-mediated actions. Interestingly, P2X7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but full-length P2X7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X7 immunoreactivity suggesting lack of P2X7 homo(tri)-oligomerization. These results identify a novel P2X7 variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.     

Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor

Background aimsMesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1α (SDF-1α) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro.MethodsWe monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor.ResultsCo-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34⁺ cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations—and conversely their hematopoiesis supportive functions—may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor.ConclusionsThese results support the notion that the CXCR4/SDF-1α axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.     

Point mutation in the mouse P2X7 receptor affects intercellular calcium waves in astrocytes

Purinergic P2 receptors and gap junctions are two groups of proteins involved in the transmission of ICWs (intercellular calcium waves) between astrocytes. The extent to which ICWs spread among these glial cells depends on the amount of ATP released, which can occur through membrane channels, as well as other pathways. Our previous studies have shown that the pore-forming P2X₇R (P2X₇ receptor) contributes to the amplification of ICW spread by providing sites of ATP release through Panx1 (Pannexin1) channels. To gain insight into the signal transduction events mediating this response we compared the properties of the P2X₇R–Panx1 complex in astrocytes from a mouse strain (C57Bl/6) containing a naturally occurring point mutation (P451L) in the C-terminus of the P2X₇R to that of non-mutated receptors (Balb/C mice). Electrophysiological, biochemical, pharmacological and fluorescence imaging techniques revealed that the P451L mutation located in the SH3 domain (a Src tyrosine kinase-binding site) of the C-terminus of the P2X₇R attenuates Panx1 currents, ATP release and the distance of ICW spread between astrocytes. Similar results were obtained when using the Src tyrosine inhibitor (PP2) and a membrane-permeant peptide spanning the P451L mutation of the P2X₇R of the C57Bl6 astrocytes. These results support the participation of a tyrosine kinase of the Src family in the initial steps mediating the opening of Panx1 channels following P2X₇R stimulation and in the transmission of calcium signals among astrocytes.     

Ontogenic shifts in cellular fate are linked to proteotype changes in lineage-biased hematopoietic progenitor cells

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The P2X7 receptor is a key modulator of aerobic glycolysis

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.     

P2X7 receptor involved in antitumor activity of atractylenolide I in human cervical cancer cells

Atractylenolide I (Atr-I) was found to sensitize a variety of human cancer cells in previous studies. Purinergic P2X7R plays important role in different cancers. However, whether Atr-I could generate antitumor activity in human cervical cancer cells and P2X7R get involved in this effect remain unclear. In this study, Hela (HPV 18 +) and SiHa (HPV 16 +) cells were treated with different doses of Atr-I. The results indicated that agonist and antagonist of P2X7 receptors, BzATP and JNJ-47965567 (JNJ), could suppress the proliferation of Hela and SiHa cells. Atr-I demonstrated a considerable antitumor effect in both human cervical cancer cells in vitro. Atr-I combined with P2X7R agonist, BzATP, restored Atr-I-induced growth inhibition in Hela cells but not in SiHa cells. However, the combinatorial treatment of P2X7R antagonist JNJ and Atr-I has an additive effect on cell growth inhibition in SiHa cells rather than in Hela cells. It implied that P2X7R would get involved in the anti-human cervical cancer cells effect of Atr-I.