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3206 Blockade of the mineralocorticoid receptor (MR), the receptor for aldosterone (ALDO), improves cardiovascular morbidity and mortality. There is growing evidence for a critical role of ALDO in inflammation in addition to its well-described effects on sodium homeostasis. However, the role of ALDO on neutrophil activation is not entirely clear. We studied the role of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 3 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcript by quantitative RT-PCR using TaqMan detection probes in these cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence; an event not observed when cells were incubated with 10−8 M dexamethasone (DEXA). Consistent with these results, incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3); an event not observed when cells were incubated with either 10−8 or 10−7 M dexamethasone. The oxidative responses to ALDO were blunted by pre-incubation of cells with 1 uM canrenoic acid (CA), a well-described MR antagonist (P<0.03, n=3). We then studied the effect of ALDO on HL-60 transmigration and observed that 2 hr incubation at 37C with 10−8 M ALDO led to augmented migration (P<0.03, n=2) when compared to vehicle as estimated by CyQuant cell migration assays. We then isolated untouched circulating human neutrophils by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% neutrophils as these cells were positive for CD45, CD16 and CD66b. Live/dead cell automated analyses shows greater than 90% cell viability by acridine orange and propidium iodide fluorescence. These cells likewise express MR as determined by western blot analyses for MR as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8 M) showed a rise in cytosolic Ca2+ and an increase in the oxidative-respiratory burst (P<0.01, n=3); a response that was sensitive to 1 uM CA. We also observed that 4 hr 10−9M ALDO incubation led to augmented neutrophil transmigration (P<0.03, n=2). Thus our results suggest that activation of MR by ALDO leads to neutrophil activation that may contribute to the inflammatory responses associated with MR activation in vivo. Disclosures: No relevant conflicts of interest to declare.

3160 Recently, we reported that the neuropeptide, neuromedin U (NmU), functions as a novel extracellular cofactor with erythropoietin (EPO) to promote the expansion of early human erythroblasts. Because the expression of NmU is important during the early stages of erythropoiesis, we aimed to understand its temporal regulation during erythroid development. Although we have demonstrated that NmU is a target of the erythroid transcriptional regulator, c-Myb, our understanding of NmU regulation is incomplete. We hypothesized that microRNA (miRNA) molecules function to regulate NmU expression at the post-transcription level during erythropoiesis. Upon sequence analysis of the 3'-UTR of NmU using microCosm in the miRBase Targets database, 20 different miRNA molecules were predicted to interact with NmU's 3'-UTR. Among the 20 different miRNA molecules predicted to interact with NmU's 3'UTR, miR-101 was of interest, because in an independent study, its expression was elevated as measured by microarray analyses from primary human CD34+ cells cultured under erythroid inducing conditions. To determine the ability of miR-101 to directly interact with the 3'UTR of NmU, we used luciferase reporter assays. In a dose-dependent manner, miR-101 directly interacted with NmU's 3'-UTR. Also, 24-hours post-nucleofection of miR-101 into K562 cells, a hematopoietic cell line, the expression of NmU was decreased compared to control. Over-expression of miR-101 in primary human CD34+ cells decreased the growth of colony-forming unit-erythroid (CFU-E) ∼50% compared to control cells. In the presence of exogenously added NmU peptide, CFU-E growth from CD34+ cells over-expressing miR-101 was rescued to the level observed with control miRNA treated cells. To further determine the relationship between NmU, EPO, and miR-101, we cultured primary human CD34+ cells using a 2-phase liquid culture condition to induce erythroid development. During the first phase (days 0–6), the cells were cultured with IL-3, IL-6, and stem cell factor (SCF). The second phase of the erythroid inducing culture conditions began on day 6 when EPO was added to the culture. Erythroid differentiation was monitored using flow cytometry and fluorescent conjugated antibodies against CD34, transferrin receptor (CD71), and glycophorin A (GlyA). In parallel, primary cells were collected at regular intervals during culture to measure the expression of NmU mRNA and miR-101 by real time PCR (RT-PCR). Under our erythroid inducing culture conditions, NmU expression peaked between days 4 and 6 (before adding EPO) and between days 10 to 12. Also, between days 10 to 12 of culture in erythroid inducing conditions, we observed a dramatic increase in cell proliferation. Between days 13 to 15, cell proliferation reached a plateau, and the expression of miR-101 peaked. Erythroid progenitors purified from cord blood mononuclear cells by cell sorting revealed that NmU expression peaked in CD34-, CD71+, GlyA- (ERY2) cells, which is in good agreement with an independent microarray study, and miR-101 expression was not detected. By contrast, in CD34-, CD71lo, GlyA+ (ERY4) cells, miR-101 expression peaked while NmU expression decreased to the level observed in CD34-, CD71-, GlyA- cells. Combined, these data identify NmU as a novel miR-101 target and indicate that miR-101 regulates the temporal expression of NmU during the later stages of erythropoiesis. We hypothesize that the miR-101/NmU axis is a critical modulator of erythroid cell expansion that augments the effects of erythropoietin. Disclosures: Carroll: Glaxo Smith Kline, Inc.: Research Funding; Sanofi Aventis Corporation: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.

3876 The proto-oncogene c-myb encodes the transcription factor c-Myb, which is predominantly expressed in immature hematopoietic cells where it plays an obligate role in definitive hematopoiesis. Given the critical functions of c-Myb in lineage commitment, proliferation, and differentiation, c-Myb regulatory factors are of great interest but remain incompletely defined. In recent years, c-Myb has been shown to regulate the expression of microRNA (miRNA) molecules in hematopoietic cells. MiRNA molecules are noncoding RNA molecules that are 21–23 nucleotides in length and function to hybridize to the 3′UTR region of its target mRNA to stimulate/repress translation or induce mRNA degradation. For example, in hematopoietic cells, miR-15a and c-Myb form an autoregulatory negative feedback loop in that over-expression of miR-15a in hematopoietic cells was determined to block erythroid and myeloid colony formation. In megakaryocytes, the hormone thrombopoietin induced miR-150 expression which subsequently functioned to degrade c-myb mRNA through direct interaction with c-myb's 3′-UTR. Our studies have focused on determining the physiologic function of the neuroendocrine Neuromedin U (NmU) during the early stages of erythropoiesis, because we recently determined that silencing NmU in primary human CD34+ cells impairs burst-forming units-erythroid and colony-forming unit-erythroid formation. In subsequent studies, we determined that c-Myb directly interacts with the NmU promoter at Myb Response Elements (MREs) distal to its transcription start site. Also, the expression profiles of NmU and c-myb are similar in CD34+ cells cultured under erythroid inducing conditions for 10 days, and silencing c-myb expression in hematopoietic cells inhibits NmU expression. To gain insight into the regulatory mechanism involved in NmU expression during the early stages of erythropoiesis, we hypothesized that miRNA molecules regulated by c-Myb would inhibit NmU expression through a negative feedback loop. To address this hypothesis, we first scanned the 3′-UTR of NmU and identified 24 different miRNA molecules predicted to interact with NmU's 3′-UTR. Second, we used luciferase reporter assays to determine which of the miRNA molecules interacted with NmU's 3′-UTR. Of the three miRNA molecules we tested, miR-101 directly interacted with NmU's 3′-UTR in a dose-dependent manner. Third, we determined the expression profile of miR-101 in primary CD34+ cells cultured under erythroid inducing conditions. The gene expression of miR-101 was inversely correlated with NmU and c-myb. Finally, because miR-101 contained 6 MREs, we determined the ability of c-Myb to directly interact with the promoter of miR-101 using chromatin immunoprecipitation (ChIP) assays. Using primers that flank the MREs proximal to miR-101's transcription start site, we observed a greater than 2-fold increase in the amplification of DNA recovered from ChIP assays completed with c-Myb antibody compared to ChIP assays completed with irrelevant antibody. Studies are underway to confirm by luciferase-reporter assays that c-Myb directly binds to and transactivates the miR-101 promoter. Collectively, these data identify a regulatory loop comprised of c-Myb, NmU, and miR-101 that could be of potential importance during human erythropoiesis. Disclosures: No relevant conflicts of interest to declare.