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Background & Aim Introduction Mesenchymal stromal cells (MSCs) are being investigated in clinical trials for treatment of a variety of veterinary conditions based on their anti-inflammatory actions and potential to induce functional tissue repair. However, outcomes of clinical trials have often fallen short of pre-clinical expectations. Heterogeneity of MSC preparations and lack of adequate characterization may be partial contributors to the variability in clinical outcomes. Purpose Identification of subpopulations within heterogenous MSC cultures may improve selection of cells with the desired functionality. Therefore, the functional heterogeneity of different subpopulations of MSCs that vary in size was evaluated, as size is hypothesized to relate to function. Methods, Results & Conclusion Methods A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size. Size sorted MSCs were then characterized and evaluated for the impact of culture conditions on functional heterogeneity. Results We found that larger subpopulations of MSCs with a mean diameter of ∼18 μm proliferate more slowly, senesce at earlier passages, and are inclined to form adipocytes compared to smaller MSC subpopulations (mean diameter of ∼15 μm). Most importantly, size-dependent functions are affected by culture conditions as well as time in culture. Cell surface staining for MSC-specific CD44 & CD90 antigens showed different sized subpopulations of MSCs had comparable expression levels suggesting that this criterion is not relevant to defining subpopulations of MSCs. However, transcriptome analysis showed that differently sized subpopulations of canine Ad-MSCs have distinct gene expression profiles, and culture medium and time in culture further affect gene expression. Conclusion This work provides a strategy for identifying functional subpopulations of canine MSCs based on size that could facilitate the development of quality cellular therapies through a better understanding of functional heterogeneity.

Catecholate type enterobactin, a prototype siderophore, comprises 2,3-dihydroxybenzoic acid (2,3-DHBA) cyclically linked to serine in E. coli . The existence of iron-chelating ligands in humans is a recent discovery, however, the basic chemical interactions between 2,5-dihydroxybenzoic acid and Fe(III) ion remain poorly understood. Achieving an accurate description of the fundamental Fe(III) binding properties of 2,5-DHBA is essential for understanding its role in iron transport mechanisms. Here, we show that 2,5-DHBA binds iron in a salicylate mode via a two-step kinetic mechanism by UV spectroscopy. Complexation between Fe(III) salt and 2,5-DHBA initially occurs at 1:1 ratio (of ligand to metal) and binding resulting in higher-order complexes continues at higher concentrations. Through potentiometric measurements we quantify the distribution of Fe(III)-2,5-DHBA complexes with 1:1, 1:2 and 1:3 stoichiometry. The formation of 1:3 complexes is further supported through high-resolution mass spectrometry. Further, using kinetic and equilibrium UV spectroscopy, we report Fe(III)-2,5-DHBA complex formation at a pH range of 2.5–9.0 at 298.15 K in water. Maximum complexation occurred at a pH range of 4.5–6.5 consistent with deprotonation of the carboxylic acid proton. Equilibrium measurements and stopped-flow kinetics show that complexation rate constants were independent of concentrations of 2,5-DHBA. Together the data supports a model in which the rate-determining step involves rearrangement of ligands on an initial complex formed by reversible binding between the carboxylate group of 2,5-DHBA and Fe(III).

Eukaryotes produce a siderophore-like molecule via a remarkably conserved biosynthetic pathway. 3-OH butyrate dehydrogenase (BDH2), a member of the short-chain dehydrogenase (SDR) family of reductases, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA). Depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of intracellular iron and mitochondrial iron deficiency in cultured mammalian cells, as well as in yeast cells and zebrafish embryos We disrupted murine bdh2 by homologous recombination to analyze the effect of bdh2 deletion on erythropoiesis and iron metabolism. bdh2 null mice developed microcytic anemia and tissue iron overload, especially in the spleen. Exogenous supplementation with 2,5-DHBA alleviates splenic iron overload in bdh2 null mice. Additionally, bdh2 null mice exhibit reduced serum iron. Although BDH2 has been proposed to oxidize ketone bodies, we found that BDH2 deficiency did not alter ketone body metabolism in vivo. In sum, our findings demonstrate a key role for BDH2 in erythropoiesis.

Bacteria can utilize a mammalian host siderophore to usurp host iron; however, the host can respond by down-regulating siderophore expression and up-regulating expression of an inhibitory siderophore-binding protein., Competition for iron influences host–pathogen interactions. Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron. In response, the host secretes siderophore-binding proteins, such as lipocalin 24p3, which limit siderophore-mediated iron import into bacteria. Mammals produce 2,5-dihydroxy benzoic acid, a compound that resembles a bacterial siderophore. Our data suggest that bacteria use both mammalian and bacterial siderophores. In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro. In addition, mice lacking the mammalian siderophore resist E. coli infection. Finally, we show that the host responds to infection by suppressing siderophore synthesis while up-regulating lipocalin 24p3 expression via TLR signaling. Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

Medium containing Fetal Bovine Serum (FBS) provides a supportive environment for isolation and expansion of mesenchymal stromal/stem cells (MSCs); however, the inherent variability of FBS may contribute to inconsistencies in cell growth and yield between batches of stem cell products. For this reason, we set out to develop a serum-free medium capable of supporting the in vitro expansion of MSCs. First a naïve serum-free medium was formulated by Sato's approach. Once it was established that the naïve serum-free medium supported the expansion of canine adipose-derived MSCs (Ad-MSCs), the serum-free medium was optimized by addition of growth factors. Combinations of growth factors were chosen and compared by their effect on cell proliferation and colony formation. Growth characteristics of canine adipose-derived MSCs cultured in the serum-free medium were comparable to those cultured in standard FBS containing medium. In addition, cell surface marker expression and differentiation potential of serum-free and FBS-based cultures were also comparable. However, a commercial serum-free medium developed for human MSC culture did not support growth of canine Ad-MSCs. In summary, canine Ad-MSCs isolated and cultured in serum-free medium retained the basic characteristics of MSCs cultured in FBS containing medium.

Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens for which siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions, but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. In this study, we show that neutrophils from mice genetically deficient for lipocalin 24p3 (24p3−/−) are defective in many neutrophil functions. Specifically, neutrophils in 24p3−/− mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3−/− neutrophils. Additionally, small regulatory RNAs (microRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3−/− neutrophils. Further, 24p3−/− neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3−/− mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans and Staphylococcus aureus) pathogens. Listeria does not secrete siderophores, and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3−/− mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function.

Mitochondria are the site of iron utilization, wherein imported iron is incorporated into heme or iron–sulfur clusters. Previously, we showed that a cytosolic siderophore, which resembles a bacterial siderophore, facilitates mitochondrial iron import in eukaryotes, including zebrafish. An evolutionarily conserved 3-hydroxy butyrate dehydrogenase, 3-hydroxy butyrate dehydrogenase 2 (Bdh2), catalyzes a rate-limiting step in the biogenesis of the eukaryotic siderophore. We found that inactivation of bdh2 in developing zebrafish embryo results in heme deficiency and delays erythroid maturation. The basis for this erythroid maturation defect is not known. Here we show that bdh2 inactivation results in mitochondrial dysfunction and triggers their degradation by mitophagy. Thus, mitochondria are prematurely lost in bdh2-inactivated erythrocytes. Interestingly, bdh2-inactivated erythroid cells also exhibit genomic alterations as indicated by transcriptome analysis. Reestablishment of bdh2 restores mitochondrial function, prevents premature mitochondrial degradation, promotes erythroid development, and reverses altered gene expression. Thus, mitochondrial communication with the nucleus is critical for erythroid development.

Iron is vital for almost all organisms because of its ability to donate and accept electrons with relative ease. It serves as a cofactor for many proteins and enzymes necessary for oxygen and energy metabolism, as well as for several other essential processes. Mammalian cells utilize multiple mechanisms to acquire iron. Disruption of iron homeostasis is associated with various human diseases: iron deficiency resulting from defects in the acquisition or distribution of the metal causes anemia, whereas iron surfeit resulting from excessive iron absorption or defective utilization causes abnormal tissue iron deposition, leading to oxidative damage. Mammals utilize distinct mechanisms to regulate iron homeostasis at the systemic and cellular levels. These involve the hormone hepcidin and iron regulatory proteins, which collectively ensure iron balance. This review outlines recent advances in iron regulatory pathways as well as in mechanisms underlying intracellular iron trafficking, an important but less studied area of mammalian iron homeostasis.

Intracellular iron homeostasis is critical for survival and proliferation. Lipocalin 24p3 is an iron trafficking protein that binds iron through association with a bacterial siderophore, such as enterobactin, or a postulated mammalian siderophore. Here we show that the iron-binding moiety of the 24p3-associated mammalian siderophore is 2,5-dihydroxybenzoic acid (2,5-DHBA), which is similar to 2,3-DHBA, the iron-binding component of enterobactin. We find that the murine enzyme responsible for 2,5-DHBA synthesis is the homologue of bacterial EntA, which catalyzes 2,3-DHBA production during enterobactin biosynthesis. RNA interference-mediated knockdown of the murine homologue of EntA results in siderophore depletion. Mammalian cells lacking the siderophore accumulate abnormally high amounts of cytoplasmic iron, resulting in elevated levels of reactive oxygen species, whereas the mitochondria are iron deficient. Siderophore-depleted mammalian cells and zebrafish embryos fail to synthesize heme, an iron-dependent mitochondrial process. Our results reveal features of intracellular iron homeostasis that are conserved from bacteria through humans.

SummaryThe lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis due to interleukin-3 (IL-3) deprivation and iron transport. Here we report cloning of the 24p3 cell-surface receptor (24p3R). Ectopic 24p3R expression confers on cells the ability to undergo either iron uptake or apoptosis, dependent upon the iron content of the ligand: Iron-loaded 24p3 increases intracellular iron concentration without promoting apoptosis; iron-lacking 24p3 decreases intracellular iron levels, which induces expression of the proapoptotic protein Bim, resulting in apoptosis. Intracellular iron delivery blocks Bim induction and suppresses apoptosis due to 24p3 addition or IL-3 deprivation. We find, unexpectedly, that the BCR-ABL oncoprotein activates expression of 24p3 and represses 24p3R expression, rendering BCR-ABL+ cells refractory to secreted 24p3. By inhibiting BCR-ABL, imatinib induces 24p3R expression and, consequently, apoptosis. Our results reveal an unanticipated role for intracellular iron regulation in an apoptotic pathway relevant to BCR-ABL-induced myeloproliferative disease and its treatment.

The mammalian ATF/CREB family of transcription factors comprises a large group of basic-region leucine zipper (bZIP) proteins whose members mediate diverse transcriptional regulatory functions. Here we report that expression of a specific mouse ATF gene,ATFx, is down-regulated in a variety of cells undergoing apoptosis following growth factor deprivation. When stably expressed in an interleukin 3 (IL-3)-dependent cell line, ATFx suppresses apoptosis resulting from cytokine deprivation. Conversely, a dominant-negative ATFx mutant induces apoptosis of cells cultured in the presence of growth factors. We also show that 24p3, a secreted lipocalin that induces apoptosis when added to hematopoietic cells, represses ATFx expression. However, constitutive expression ofATFx renders cells resistant to 24p3-mediated apoptosis. Collectively, our results indicate that ATFx is an anti-apoptotic factor, a novel role for an ATF protein.

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. Bovine herpesvirus 1 (BHV-1) induces PCD in peripheral blood mononuclear cells at the G 0 /G 1 phase of the cell cycle (E. Hanon, S. Hoornaert, F. Dequiedt, A. Vanderplasschen, J. Lyaku, L. Willems, and P.-P. Pastoret, Virology 232:351–358, 1997). However, penetration of virus particles is not required for PCD (E. Hanon, G. Meyer, A. Vanderplasschen, C. Dessy-Doize, E. Thiry, and P. P. Pastoret, J. Virol. 72:7638–7641, 1998). The mechanism by which BHV-1 induces PCD in peripheral blood mononuclear cells is not understood, nor is it clear whether nonlymphoid cells undergo PCD following infection. This study demonstrates that infection of bovine kidney (MDBK) cells with BHV-1 leads to PCD, as judged by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, DNA laddering, and chromatin condensation. p53 appears to be important in this process, because p53 levels and promoter activity increased after infection. Expression of proteins that are stimulated by p53 (p21 Waf1 and Bax) is also activated after infection. Cleavage of Bcl-x L , a protein that inhibits PCD, occurred after infection, suggesting that caspases (interleukin-1β-converting enzyme-like proteases) were activated. Other caspase substrates [poly(ADP-ribose) polymerase and actin] are also cleaved during the late stages of infection. Inhibition of caspase activity delayed cytotoxic activity and virus release but increased the overall virus yield. Taken together, these results indicate that nonlymphoid cells undergo PCD near the end of productive infection and further suggest that caspases enhance virus release.

Human JC virus (JCV) is a neurotropic human polyomavirus that was found in the plaques and oligodendroglial cells of the brains of patients with the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Transgenic mice expressing JCV large tumor (T)-antigen from integrated DNA showed dysmyelination in the central nervous system. However, the role of T-antigen from episomal DNA in the demyelination in PML remains unclear. In this report, we examined the effect of episomally expressed JCV T-antigen on the expression of myelin basic protein (MBP) in U-87 MG human glioblastoma cells to study the mechanism of demyelination. Expression assays of the MBP promoter in U-87 MG detected a 2.5-fold reduction in cells expressing intact T-antigen. Next, U-87 MG expressing T-antigen were examined by RNase protection assays for mRNA accumulation from the endogenous MBP promoter. Also, the expression of the MBP promoter plasmid was determined using in vitro transcription assays with extracts from T-antigen expressing cells. Both assays found a similar down-regulation of the MBP promoter by T-antigen, confirming that negative regulation occurred at the transcriptional level for the endogenous and exogenous MBP promoters. Furthermore, in situ immunofluorescence assays and quantitative Western blot analysis provided convincing evidence of a similar reduction in the level of MBP produced from the functional endogenous gene in U-87 MG glioblastoma cells expressing T-antigen. Thus, we provide evidence for the role of T-antigen in a transcriptional control mechanism for the demyelination that is caused by JCV in PML patients.

The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis, iron trafficking, development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However, a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3, we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid, myeloid, and erythroid cells, which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead, apoptotic defects were unique to many mature hematopoietic cell types, including neutrophils, cytokine-dependent mast cells, thymocytes, and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells, thus explaining their resistance to the ensuing cell death. The results of these studies, in conjunction with those of previous studies, reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly, these functions are limited to relatively mature blood cell compartments.

Apoptosis of mature T lymphocytes preserves immune system homeostasis by counteracting transient increases in T-cell number. This process is regulated, at least in part, by the cytokine interleukin 2 (IL-2): T cells deprived of IL-2 undergo apoptosis. The mechanism of apoptosis induction by IL-2 deprivation remains to be determined but is known to require RNA synthesis, implying the existence of transcriptionally activated genes whose products induce cell death. To identify such genes, we have performed expression profiling in IL-2-dependent T cells following cytokine deprivation. Our results reveal an intricate transcriptional program entailing the induction of known proapoptotic factors and the simultaneous repression of known antiapoptotic factors. Surprisingly, one gene whose transcription substantially increased was RC3 (also called neurogranin), which encodes a calmodulin binding protein thought to be a neural-specific factor involved in learning and memory. We show that ectopic expression of RC3 in IL-2-dependent T cells increases the intracellular Ca(2+) concentration and induces apoptosis even in the presence of cytokine. Buffering the Ca(2+) increase with the cytoplasmic Ca(2+) chelator BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N1,N-tetraacetic acid] blocks RC3-induced apoptosis, indicating that the rise in intracellular Ca(2+) is required for apoptotic death. RC3 mutants unable to bind calmodulin fail to increase intracellular Ca(2+) levels and to induce apoptosis. Based upon these results, we propose that IL-2 deprivation raises the level of RC3 and other apoptotic factors, which induce apoptosis by increasing the intracellular Ca(2+) concentration.

Many hematopoietic cells undergo apoptosis when deprived of specific cytokines, and this process requires de novo RNA/protein synthesis. Using DNA microarrays to analyze interleukin-3 (IL-3)–dependent murine FL5.12 pro–B cells, we found that the gene undergoing maximal transcriptional induction after cytokine withdrawal is24p3, which encodes a secreted lipocalin. Conditioned medium from IL-3–deprived FL5.12 cells contained 24p3 and induced apoptosis in naı̈ve FL5.12 cells even when IL-3 was present. 24p3 also induced apoptosis in a wide variety of leukocytes but not other cell types. Apoptotic sensitivity correlated with the presence of a putative 24p3 cell surface receptor. We conclude that IL-3 deprivation activates24p3transcription, leading to synthesis and secretion of 24p3, which induces apoptosis through an autocrine pathway.

Transcription of the human polyomavirus JC virus (JCV) genome is regulated by cellular proteins and the large tumour (T) antigen. Earlier studies led to the identification of nuclear factor-1 (NF-1)-binding sites in the JCV enhancer by DNase I protection assays of extracts from retinoic acid (RA)-differentiated P19 embryonal carcinoma (EC) cells. In this study, a cDNA clone that encodes a protein capable of binding to the JCV NF-1 sites was isolated from an RA-differentiated EC cell cDNA library. Sequence analysis revealed that the cDNA isolated was identical to the previously described Bcl-2-interacting protein BAG-1 (Bcl-2-associated athano gene-1). Results from RNA studies indicated that BAG-1 is expressed in several cell types. Co-transfection of a recombinant BAG-1 expression plasmid with JCV promoters indicated that BAG-1 stimulates transcription of the JCVE promoter and to a lesser extent the JCVL promoter. Mutations in the NF-1 sites in the JCVE promoter eliminated the activation by BAG-1. Thus, BAG-1 is a novel transcription factor that may play a role in JCV expression.

Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in sensory neurons of infected individuals. Infected cell protein 0 (ICP0) is important for productive infection and reactivation from latency. Thus, activation of ICP0 expression in neurons is likely to be important for reactivation from latency. In a mouse neuroblastoma cell line, ICP0 promoter activity is high compared with other strong viral promoters. In contrast, promoter activity is low in non-neuronal cells. DNase I footprinting assays indicated that three distinct motifs in the ICP0 promoter are bound by nuclear factors. One of these motifs contains a binding site for a novel helix-loop-helix olfactory neuron-specific transcription factor (Olf-1). Gel shift assays and supershift assays using an Olf-1-specific antibody demonstrated that mouse neuroblastoma cells express Olf-1, which is bound to the Olf-1-like site in the ICP0 promoter. Deletion of the putative Olf-1 motif reduced ICP0 promoter activity more than 5-fold in mouse neuroblastoma cells and prevented trans-activation by an Olf-1 expression vector. We hypothesize that the Olf-1-binding site activates ICP0 promoter activity in neurons during reactivation from latency.

The latency-related transcript (LRT) of bovine herpesvirus 1 (BHV-1) is the only abundant viral RNA detected during latency. A previous study (A. Hossain, L. M. Schang, and C. Jones, J. Virol. 69:5345–5352, 1995) concluded that splicing of polyadenylated [poly(A) + ] and splicing of nonpolyadenylated [poly(A) − ] LRT are different. In this study, splice junction sites of LRT were identified. In trigeminal ganglia of acutely infected calves (1, 7, or 15 days postinfection [p.i.]) or in latently infected calves (60 days p.i.), alternative splicing of poly(A) + LRT occurred. Productive viral gene expression in trigeminal ganglia is readily detected from 2 to 7 days p.i. but not at 15 days p.i. (L. M. Schang and C. Jones, J. Virol. 71:6786–6795, 1997), suggesting that certain aspects of a lytic infection occur in neurons and that these factors influence LRT splicing. Splicing of poly(A) − LRT was also detected in transfected COS-7 cells or infected MDBK cells. DNA sequence analysis of spliced LRT cDNAs, poly(A) + or poly(A) − , revealed nonconsensus splice signals at exon/intron and intron/exon boundaries. The GC-AG splicing signal utilized by the herpes simplex virus type 1 latency-associated transcript in latently infected mice is also used by LRT in latently infected calves. Taken together, these results led us to hypothesize that (i) poly(A) + LRT is spliced in trigeminal ganglia by neuron-specific factors, (ii) viral or virus-induced factors participate in splicing, and (iii) alternative splicing of LRT may result in protein isoforms which have novel biological properties.

The nuclear factor 1 (NF-1) motifs, NF-1 II/III, in the two 98-bp repeats of the transcription-regulatory region of JC virus (JCV), have a critical role in brain-specific transcription from the JCV early promoter-enhancer. In this study, the role of these motifs in transactivation of the JCV late promoter-enhancer (JCV L ) was examined in differentiating glial P19 embryonal carcinoma cells. The expression of papovaviral large tumor antigen (T-Ag) in the glial cells was shown by double immunofluorescence assays. By using site-directed mutagenesis and in vivo assays, the two wild-type NF-1 II/III sites, but not the third site, were found to be essential for the transactivation of JCV L by JCV T-Ag. In vitro transcription assays confirmed this specific transactivation and the transactivation was abolished by T-Ag antibody. In electrophoretic mobility shift assays, expression of JCV T-Ag increased the binding of a factor(s) to the 98-bp repeat. T-Ag antibody abolished the increase of binding. Binding assays with oligonucleotides of NF-1 II/III motifs showed that the increased binding specifically required the wild-type NF-1 II/III sequences and confirmed the requirement of T-Ag. To determine the region of T-Ag necessary for transactivation of JCV L , the coding sequences were mutated. The amino-terminal region of JCV Ag in amino acids 1-437 was essentially required for efficient transactivation. These results indicated that transactivation of JCV L and increased binding require a factor(s) found specifically in glial cells, the JCV NF-I II/III sites, and the T-Ag amino-terminal region.

Abstract 833 Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores are chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins of secondary granules. Neutrophils from SGD patients lack normal functions and are prone to bacterial infections, but the importance of 24p3 for neutrophil dysfunction in SGD is not known. Here we show that neutrophils of 24p3−/− mice are defective for many neutrophil functions. Specifically, neutrophils in 24p3−/− mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3−/− neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3−/− neutrophils. Further, 24p3−/− neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3−/− mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3−/− mice to these pathogens is not due to sequestration of siderophores to limit the iron availability but as a consequence of impaired neutrophil functions. Disclosures: No relevant conflicts of interest to declare.

Abstract 1560 The lipocalin mouse 24p3 has been implicated in diverse physiological processes including apoptosis, iron trafficking, development and innate immunity. Studies from our lab as well as others demonstrated the pro-apoptotic activity of 24p3 in a variety of cultured models. However, a general role for the lipocalin 24p3 in the homeostasis of the hematopoietic system has not been tested in vivo. To study the role of 24p3 in apoptosis, we derived 24p3 null mice on 129 SVE and C57BL/6 genetic backgrounds, which enabled us to test, for the first time, the in vivo role of 24p3 without any confounders. We find that homozygous 24p3-/- mice have apoptotic defects in several hematopoietic lineages, including neutrophils, cytokine-dependent mast cells, thymocytes and erythroid cells. The progressive accumulation of lymphoid, myeloid and erythroid cells in 24p3-/- mice demonstrate the importance of 24p3 in the regulation of homeostasis of hematopoietic cells in adults. Thymocytes isolated from 24p3 null mice displayed resistance to apoptosis-induced by Dexamethasone. In addition, Dexamethasone-induced apoptosis is also reduced in 24p3-/- mice. Collectively, these results indicate that 24p3 is an important target gene involved in GC-mediated apoptosis of lymphocytes and other hematopoietic cell types. Finally, our results also indicate that the genetic background of mice can modulate the effects of 24p3 deficiency. In summary, the results presented here, in conjunction with those of previous studies, reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Disclosures: No relevant conflicts of interest to declare.

Programmed cell death or Apoptosis, is a critical aspect of normal physiology as well as the genesis and treatment of cancer. Certain apoptotic pathways are transcriptionally regulated; in these cases, apoptosis is induced by the transcriptional activation of genes encoding proapoptotic proteins. We originally identified lipocalin 24p3 as the gene undergoing maximum transcriptional stimulation following induction of apoptosis by cytokine-deprivation of interleukin 3 (IL-3) dependent cells. 24p3 is a member of the lipocalin family of carrier proteins – a group of small-secreted molecules that bind and transport low-molecular weight ligands. By delivering this cargo via cell-surface receptors they are known to influence many responses. 24p3 is a secreted lipocalin, which we have found induces apoptosis when added to a variety of lymphoid cells. These and other results revealed a model in which IL-3 deprivation activates 24p3 transcription, leading to synthesis and secretion of 24p3, which induces apoptosis through an autocrine/paracrine pathway. We have isolated the 24p3 cell surface receptor (24p3R) and found that 24p3 induces apoptosis through a novel pathway culminating in a decrease in intracellular iron levels (a biological iron chelator). Interestingly, iron chelators inhibit cellular proliferation and induce apoptosis, and are under active investigation as chemotherapeutic agents. The basis by which decreased intracellular iron induces apoptosis is not well understood. We performed expression-profiling experiments to identify differentially regulated genes in 24p3 and as a control in Deferoxamine (DFO), a synthetic iron chelator, treated cells. Our preliminary results suggest that 24p3 activates the expression of a novel gene, ING-2 (inhibitor of growth-2). ING-2 prevents cell growth by inducing cell cycle arrest at the G2/M phase. In contrast, the synthetic iron chelator, DFO activates the expression of NDRG1 (n-Myc downstream-regulated gene 1), which induces cell cycle arrest at G0/G1 phase. These results suggest that 24p3 induces cell death by activating regulators of the cell cycle. Finally, we have also found that the oncogene BCR-ABL counteracts the 24p3 proapoptotic pathway by misregulating expression of 24p3 and 24p3R. To study the contribution of 24p3 apoptotic pathway in the progression of CML, we have performed CML modeling experiments in mice. BCR-ABL transformed 24p3 deficient bone marrow cells failed to induce myeloproliferative disease in recipients upon transplantation. However, wild-type bone marrow cells when transduced with BCR-ABL oncogene readily induced CML-like disease in transplanted mice. Therefore, the secretion of 24p3 by BCR-ABL transformed cells facilitates the progression of CML. We have also demonstrated that 24p3 plays an important role in Gleevec-induced cell death in BCR-ABL transformed cells. These studies have therapeutic implications for Gleevec resistant CML.