Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

Forming functional fat: a growing understanding of adipocyte differentiation.

Adipose tissue, which is primarily composed of adipocytes, is crucial for maintaining energy and metabolic homeostasis. Adipogenesis is thought to occur in two stages: commitment of mesenchymal stem cells to a preadipocyte fate and terminal differentiation. Cell shape and extracellular matrix remodelling have recently been found to regulate preadipocyte commitment and competency by modulating WNT and RHO-family GTPase signalling cascades. Adipogenic stimuli induce terminal differentiation in committed preadipocytes through the epigenomic activation of peroxisome proliferator-activated receptor-γ (PPARγ). The coordination of PPARγ with CCAAT/enhancer-binding protein (C/EBP) transcription factors maintains adipocyte gene expression. Improving our understanding of these mechanisms may allow us to identify therapeutic targets against metabolic diseases that are rapidly becoming epidemic globally.

The multi-functionality of UHRF1: epigenome maintenance and preservation of genome integrity.

During S phase, the cooperation between the macromolecular complexes regulating DNA synthesis, epigenetic information maintenance and DNA repair is advantageous for cells, as they can rapidly detect DNA damage and initiate the DNA damage response (DDR). UHRF1 is a fundamental epigenetic regulator; its ability to coordinate DNA methylation and histone code is unique across proteomes of different species. Recently, UHRF1's role in DNA damage repair has been explored and recognized to be as important as its role in maintaining the epigenome. UHRF1 is a sensor for interstrand crosslinks and a determinant for the switch towards homologous recombination in the repair of double-strand breaks; its loss results in enhanced sensitivity to DNA damage. These functions are finely regulated by specific post-translational modifications and are mediated by the SRA domain, which binds to damaged DNA, and the RING domain. Here, we review recent studies on the role of UHRF1 in DDR focusing on how it recognizes DNA damage and cooperates with other proteins in its repair. We then discuss how UHRF1's epigenetic abilities in reading and writing histone modifications, or its interactions with ncRNAs, could interlace with its role in DDR.

Long noncoding RNA CEBPA-DT promotes cisplatin chemo-resistance through CEBPA/BCL2 mediated apoptosis in oral squamous cellular cancer.

Intrinsic or developing resistance to chemotherapy drugs including cisplatin (CDDP) remains the major limitation of cancer therapeutic efficacy in cancers. Recently, increasing evidence suggested that long noncoding RNAs (lncRNAs) play a critical role in various biological processes of tumors, and have been implicated in resistance to various drugs. However, the role of lncRNAs in cisplatin resistance is poorly understood. Here, we found that the expression of lncRNA CEBPA-DT/CEBPA/BCL2 was upregulated in cisplatin resistance OSCC cells (Cal27-CisR and HSC4-CisR) compared with their parental cells (Cal27 and HSC4). CEBPA-DT overexpression could upregulated both cytoplasmic and nuclear CEBPA expression. Down-regulation of CEBPA-DT enhances cisplatin sensitivity, facilitates cell apoptosis in cisplatin-resistant OSCC cells. In addition, we identified that CEBPA-DT regulates cisplatin chemosensitivity through CEBPA/BCL2-mediated cell apoptosis. Knockdown of CEBPA and BCL2 could alleviate the increasement of cisplatin resistance induced by CEBPA-DT overexpression. Our findings indicate that downregulation of lncRNA CEBPA-DT may be a potential therapy to overcome cisplatin resistance in OSCC.

Identification of UHRF2 as a novel DNA interstrand crosslink sensor protein.

The Fanconi Anemia (FA) pathway is important for repairing interstrand crosslinks (ICLs) between the Watson-Crick strands of the DNA double helix. An initial and essential stage in the repair process is the detection of the ICL. Here, we report the identification of UHRF2, a paralogue of UHRF1, as an ICL sensor protein. UHRF2 is recruited to ICLs in the genome within seconds of their appearance. We show that UHRF2 cooperates with UHRF1, to ensure recruitment of FANCD2 to ICLs. A direct protein-protein interaction is formed between UHRF1 and UHRF2, and between either UHRF1 and UHRF2, and FANCD2. Importantly, we demonstrate that the essential monoubiquitination of FANCD2 is stimulated by UHRF1/UHRF2. The stimulation is mediating by a retention of FANCD2 on chromatin, allowing for its monoubiquitination by the FA core complex. Taken together, we uncover a mechanism of ICL sensing by UHRF2, leading to FANCD2 recruitment and retention at ICLs, in turn facilitating activation of FANCD2 by monoubiquitination.

Effect of Uhrf1 on intestinal development.

As a best-characterized epigenetic modification, DNA methylation plays an important role in mammalian development. Uhrf1 is a critical epigenetic regulator that can bind to hemimethylated DNA and recruit DNA methyltransferase 1 to maintain DNA methylation. So far, the role of Uhrf1-mediated DNA methylation in intestinal development is still unknown. In order to investigate the impact of Uhrf1 deletion in intestinal development, we have successfully constructed the epithelial-specific Uhrf1 knockout mouse model. After Uhrf1 ablation, we found the mutant mice exhibited abnormal epithlial structure with less and shorter villi and shrinked crypts compared with wild type mice via hematoxylin-eosin staining. Further analysis showed that Uhrf1 deletion in the intestinal epithelium significantly decreased the cell proliferation and induced cell apoptosis. In addition, Uhrf1 deletion inhibited the normal epithelial differentiation and the expression of intestinal stem cell marker genes. Preliminary mechanism study revealed that loss of Uhrf1 caused global DNA hypomethylation which induced DNA damage in crypt cells. Taken together, our data suggested that DNA methylation mediated by Uhrf1 is vital for the normal intestinal development. Our results enriched the in vivo role of Uhrf1 and laid the foundation for further epigenetic regulatory mechanism exploration.

CCAAT enhancer binding protein alpha (CEBPA) biallelic acute myeloid leukaemia: cooperating lesions, molecular mechanisms and clinical relevance.

Recent advances in sequencing technologies have allowed for the identification of recurrent mutations in acute myeloid leukaemia (AML). The transcription factor CCAAT enhancer binding protein alpha (CEBPA) is frequently mutated in AML, and biallelic CEBPA-mutant AML was recognised as a separate disease entity in the recent World Health Organization classification. However, CEBPA mutations are co-occurring with other aberrations in AML, and together these lesions form the clonal hierarchy that comprises the leukaemia in the patient. Here, we aim to review the current understanding of co-occurring mutations in CEBPA-mutated AML and their implications for disease biology and clinical outcome. We will put emphasis on patterns of cooperation, how these lesions cooperate with CEBPA mutations and the underlying potential molecular mechanisms. Finally, we will relate this to patient outcome and future options for personalised medicine.

Knockdown of DNA-binding protein A enhances the chemotherapy sensitivity of colorectal cancer via suppressing the Wnt/ß-catenin/Chk1 pathway.

DNA-binding protein A (dbpA) is reported to be upregulated in many cancers and associated with tumor progress. The present study aimed to investigate the role of dbpA in 5-fluorouracil (5-FU)-resistant and oxaliplatin (L-OHP)-resistant colorectal cancer (CRC) cells. We found that 5-FU and L-OPH treatment promoted the expression of dbpA. Enhanced dbpA promoted the drug resistance of SW620 cells to 5-FU and L-OHP. DbpA knockdown inhibited cell proliferation, induced cell apoptosis, and cell cycle arrested in SW620/5-FU and SW620/L-OHP cells. Besides, dbpA short hairpin RNA (shRNA) enhanced the cytotoxicity of 5-FU and L-OHP to SW620/5-FU and SW620/L-OHP cells. Meanwhile, dbpA shRNA inhibited the activation of the Wnt/ß-catenin pathway that induced by 5-FU stimulation in SW620/5-FU cells. Activation of the Wnt/ß-catenin pathway or overexpression of checkpoint kinase 1 (Chk1) abrogated the promoting effect of dbpA downregulation on 5-FU sensitivity of CRC cells. Importantly, downregulation of dbpA suppressed tumor growth and promoted CRC cells sensitivity to 5-FU in vivo. Our study indicated that the knockdown of dbpA enhanced the sensitivity of CRC cells to 5-FU via Wnt/ß-catenin/Chk1 pathway, and DbpA may be a potential therapeutic target to sensitize drug resistance CRC to 5-FU and L-OHP.

Co-expression of C/EBPγ and ATF5 in mouse vomeronasal sensory neurons during early postnatal development.

The differentiation of sensory neurons involves gene expression changes induced by specific transcription factors. Vomeronasal sensory neurons (VSNs) in the mouse vomeronasal organ (VNO) consist of two major subpopulations of neurons expressing vomeronasal 1 receptor (V1r)/Gαi2 or vomeronasal 2 receptor (V2r)/Gαo, which differentiate from a common neural progenitor. We previously demonstrated that the differentiation and survival of VSNs were inhibited in ATF5 transcription factor-deficient mice (Nakano et al. Cell Tissue Res 363:621-633, 2016). These defects were more prominent in V2r/Gαo-type than in V1r/Gαi2-type VSNs; however, the molecular mechanisms responsible for the differentiation of V2r/Gαo-type VSNs by ATF5 remain unclear. To identify a cofactor involved in ATF5-regulated VSN differentiation, we investigated the expression and function of CCAAT/enhancer-binding protein gamma (C/EBPγ, Cebpg), which is a major C/EBP family member expressed in the mouse VNO and dimerizes with ATF5. The results obtained showed that C/EBPγ mRNAs and proteins were broadly expressed in the postmitotic VSNs of the neonatal VNO, and their expression decreased by the second postnatal week. The C/EBPγ protein was expressed in the nuclei of approximately 70% of VSNs in the neonatal VNO, and 20% of the total VSNs co-expressed C/EBPγ and ATF5 proteins. We examined the trans-acting effects of C/EBPγ and ATF5 on V2r transcription and found that the co-expression of C/EBPγ and ATF5, but not C/EBPγ or ATF5 alone, increased Vmn2r66 promoter reporter activity via the C/EBP:ATF response element (CARE) in Neuro2a cells. These results suggest the role of C/EBPγ on ATF5-regulated VSN differentiation in early postnatal development.

CARD10, a CEBPE target involved in granulocytic differentiation.

Maturation of granulocytes is dependent on controlled gene expression by myeloid lineage restricted transcription factors. CEBPE is one of the essential transcription factors required for granulocytic differentiation. Identification of downstream targets of CEBPE is vital to understand better its role in terminal granulopoiesis. In this study, we have identified Card10 as a novel target of CEBPE. We show that CEBPE binds to regulatory elements upstream of the murine Card10 locus, and expression of CARD10 is significantly reduced in Cebpe knock-out mice. Silencing Card10 in a human cell line and in murine primary cells impaired granulopoiesis, affecting expression of genes involved in myeloid cell development and function. Taken together, our data demonstrate for the first time that Card10 is expressed in granulocytes and is a direct target of CEBPE with functions extending to myeloid differentiation.

Signaling through the G-protein-coupled receptor Rickets is important for polarity, detachment, and migration of the border cells in Drosophila.

Cell migration plays crucial roles during development. An excellent model to study coordinated cell movements is provided by the migration of border cell clusters within a developing Drosophila egg chamber. In a mutagenesis screen, we isolated two alleles of the gene rickets (rk) encoding a G-protein-coupled receptor. The rk alleles result in border cell migration defects in a significant fraction of egg chambers. In rk mutants, border cells are properly specified and express the marker Slbo. Yet, analysis of both fixed as well as live samples revealed that some single border cells lag behind the main border cell cluster during migration, or, in other cases, the entire border cell cluster can remain tethered to the anterior epithelium as it migrates. These defects are observed significantly more often in mosaic border cell clusters, than in full mutant clusters. Reduction of the Rk ligand, Bursicon, in the border cell cluster also resulted in migration defects, strongly suggesting that Rk signaling is utilized for communication within the border cell cluster itself. The mutant border cell clusters show defects in localization of the adhesion protein E-cadherin, and apical polarity proteins during migration. E-cadherin mislocalization occurs in mosaic clusters, but not in full mutant clusters, correlating well with the rk border cell migration phenotype. Our work has identified a receptor with a previously unknown role in border cell migration that appears to regulate detachment and polarity of the border cell cluster coordinating processes within the cells of the cluster themselves.

UHRF1 regulation of the Keap1-Nrf2 pathway in pancreatic cancer contributes to oncogenesis.

The cellular defence protein Nrf2 is a mediator of oncogenesis in pancreatic ductal adenocarcinoma (PDAC) and other cancers. However, the control of Nrf2 expression and activity in cancer is not fully understood. We previously reported the absence of Keap1, a pivotal regulator of Nrf2, in ∼70% of PDAC cases. Here we describe a novel mechanism whereby the epigenetic regulator UHRF1 suppresses Keap1 protein levels. UHRF1 expression was observed in 20% (5 of 25) of benign pancreatic ducts compared to 86% (114 of 132) of pancreatic tumours, and an inverse relationship between UHRF1 and Keap1 levels in PDAC tumours (n = 124) was apparent (p = 0.002). We also provide evidence that UHRF1-mediated regulation of the Nrf2 pathway contributes to the aggressive behaviour of PDAC. Depletion of UHRF1 from PDAC cells decreased growth and enhanced apoptosis and cell cycle arrest. UHRF1 depletion also led to reduced levels of Nrf2-regulated downstream proteins and was accompanied by heightened oxidative stress, in the form of lower glutathione levels and increased reactive oxygen species. Concomitant depletion of Keap1 and UHRF1 restored Nrf2 levels and reversed cell cycle arrest and the increase in reactive oxygen species. Mechanistically, depletion of UHRF1 reduced global and tumour suppressor promoter methylation in pancreatic cancer cell lines, and KEAP1 gene promoter methylation was reduced in one of three cell lines examined. Thus, methylation of the KEAP1 gene promoter may contribute to the suppression of Keap1 protein levels by UHRF1, although our data suggest that additional mechanisms need to be explored. Finally, we demonstrate that K-Ras drives UHRF1 expression, establishing a novel link between this oncogene and Nrf2-mediated cellular protection. Since UHRF1 over-expression occurs in other cancers, its ability to regulate the Keap1-Nrf2 pathway may be critically important to the malignant behaviour of these cancers.

The Glutamine-Alanine Repeat Domain of TCERG1 is Required for the Inhibition of the Growth Arrest Activity of C/EBPα.

TCERG1 was characterized previously as a repressor of the transcription factor C/EBPα through a mechanism that involved relocalization of TCERG1 from nuclear speckles to pericentromeric regions. The inhibitory activity as well as the relocalization activity has been demonstrated to lie in the amino terminal half of the protein, which contains several discrete motifs including an imperfect glutamine-alanine (QA) repeat. In the present study, we showed that deletion of this domain completely abrogated the ability of TCERG1 to inhibit the growth arrest activity of C/EBPα. Moreover, the QA repeat deletion mutant of TCERG1 lost the ability to be relocalized from nuclear speckles to pericentromeric regions, and caused an increase in the average size of individual speckles. We also showed that deletion of the QA repeat abrogated the complex formation between TCERG1 and C/EBPα. Examination of mutants with varying numbers of QA repeats indicated that a minimal number of repeats are required for inhibitory activity as well as relocalization ability. These data contribute to our overall understanding of how TCERG1 can have gene-specific effects in addition to its more general roles in coordinating transcription elongation and splicing.

ELANE mutant-specific activation of different UPR pathways in congenital neutropenia.

A number of studies have demonstrated induction of the unfolded protein response (UPR) in patients with severe congenital neutropenia (CN) harbouring mutations of ELANE, encoding neutrophil elastase. Why UPR is not activated in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations is unclear. We evaluated the effects of ELANE mutants on UPR induction in myeloid cells from CN and CyN patients, and analysed whether additional CN-specific defects contribute to the differences in UPR induction between CN and CyN patients harbouring identical ELANE mutations. We investigated CN-specific p.C71R and p.V174_C181del (NP_001963.1) and CN/CyN-shared p.S126L (NP_001963.1) ELANE mutants. We found that transduction of haematopoietic cells with p.C71R, but not with p.V174_C181del or p.S126L ELANE mutants induced expression of ATF6, and the ATF6 target genes PPP1R15A, DDIT3 and HSPA5. Recently, we found that levels of secretory leucocyte protease inhibitor (SLPI), a natural ELANE inhibitor, are diminished in myeloid cells from CN patients, but not CyN patients. Combined knockdown of SLPI by shRNA and transduction of ELANE p.S126L in myeloid cells led to elevated levels of ATF6, PPP1R15A and HSPA5 RNA, suggesting that normal levels of SLPI in CyN patients might protect them from the UPR induced by mutant ELANE. In summary, different ELANE mutants have different effects on UPR activation, and SLPI regulates the extent of ELANE-triggered UPR.

Co-operative leukemogenesis in acute myeloid leukemia and acute promyelocytic leukemia reveals C/EBPα as a common target of TRIB1 and PML/RARA.

The PML/RARA fusion protein occurs as a result of the t(15;17) translocation in the acute promyelocytic leukemia subtype of human acute myeloid leukemia. Gain of chromosome 8 is the most common chromosomal gain in human acute myeloid leukemia, including acute promyelocytic leukemia. We previously demonstrated that gain of chromosome 8-containing MYC is of central importance in trisomy 8, but the role of the nearby TRIB1 gene has not been experimentally addressed in this context. We have now tested the hypothesis that both MYC and TRIB1 have functional roles underlying leukemogenesis of trisomy 8 by using retroviral vectors to express MYC and TRIB1 in wild-type bone marrow and in marrow that expressed a PML/RARA transgene. Interestingly, although MYC and TRIB1 readily co-operated in leukemogenesis for wild-type bone marrow, TRIB1 provided no selective advantage to cells expressing PML/RARA. We hypothesized that this lack of co-operation between PML/RARA and TRIB1 reflected a common pathway for their effect: both proteins targeting the myeloid transcription factor C/EBPα. In support of this idea, TRIB1 expression abrogated the all-trans retinoic acid response of acute promyelocytic leukemia cells in vitro and in vivo Our data delineate the common and redundant inhibitory effects of TRIB1 and PML/RARA on C/EBPα providing a potential explanation for the lack of selection of TRIB1 in human acute promyelocytic leukemia, and highlighting the key role of C/EBPs in acute promyelocytic leukemia pathogenesis and therapeutic response. In addition, the co-operativity we observed between MYC and TRIB1 in the absence of PML/RARA show that, outside of acute promyelocytic leukemia, gain of both genes may drive selection for trisomy 8.

Low-level expression of human ACAT2 gene in monocytic cells is regulated by the C/EBP transcription factors.

Acyl-coenzyme A:cholesterol acyltransferases (ACATs) are the exclusive intracellular enzymes that catalyze the formation of cholesteryl/steryl esters (CE/SE). In our previous work, we found that the high-level expression of human ACAT2 gene with the CpG hypomethylation of its whole promoter was synergistically regulated by two transcription factors Cdx2 and HNF1α in the intestine and fetal liver. Here, we first observed that the specific CpG-hypomethylated promoter was correlated with the low expression of human ACAT2 gene in monocytic cell line THP-1. Then, two CCAAT/enhancer binding protein (C/EBP) elements within the activation domain in the specific CpG-hypomethylation promoter region were identified, and the expression of ACAT2 in THP-1 cells was evidently decreased when the C/EBP transcription factors were knock-downed using RNAi technology. Furthermore, ChIP assay confirmed that C/EBPs directly bind to their elements for low-level expression of human ACAT2 gene in THP-1 cells. Significantly, the increased expressions of ACAT2 and C/EBPs were also found in macrophages differentiated from both ATRA-treated THP-1 cells and cultured human blood monocytes. These results demonstrate that the low-level expression of human ACAT2 gene with specific CpG-hypomethylated promoter is regulated by the C/EBP transcription factors in monocytic cells, and imply that the lowly expressed ACAT2 catalyzes the synthesis of certain CE/SE that are assembled into lipoproteins for the secretion.

Ikaros limits basophil development by suppressing C/EBP-α expression.

The Ikaros gene (Ikzf1) encodes a family of zinc-finger transcription factors implicated in hematopoietic cell differentiation. Here we show that Ikaros suppresses the development of basophils, which are proinflammatory cells of the myeloid lineage. In the absence of extrinsic basophil-inducing signals, Ikaros(-/-) (Ik(-/-)) mice exhibit increases in basophil numbers in blood and bone marrow and in their direct precursors in bone marrow and the spleen, as well as decreased numbers of intestinal mast cells. In vitro culture of Ik(-/-) bone marrow under mast cell differentiation conditions also results in predominance of basophils. Basophil expansion is associated with an increase in basophil progenitors, increased expression of Cebpa and decreased expression of mast cell-specifying genes Hes1 and microphthalmia-associated transcription factor (Mitf). Ikaros directly associates with regulatory sites within Cebpa and Hes1 and regulates the acquisition of permissive H3K4 tri-methylation marks at the Cebpa locus and reduces H3K4 tri-methylation at the Hes1 promoter. Ikaros blockade in cultured cells or transfer of Ik(-/-) bone marrow into irradiated Ik(+/+) recipients also results in increased basophils confirming a cell-intrinsic effect of Ikaros on basophil development. We conclude that Ikaros is a suppressor of basophil differentiation under steady-state conditions and that it acts by regulating permissive chromatin modifications of Cebpa.

Lenalidomide-mediated enhanced translation of C/EBPα-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia.

Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.

An AKI biomarker lipocalin 2 in the blood derives from the kidney in renal injury but from neutrophils in normal and infected conditions.

BACKGROUND: Lipocalin 2 (LCN2 or neutrophil gelatinase-associated lipocalin) is a secretory protein discovered from neutrophils, which accumulates in the blood and urine during acute kidney injury (AKI) and in the blood by bacterial infection. Little is known about the tissue source and molecular forms of this protein under normal and pathophysiologic conditions. METHODS: By sandwich ELISA, serum and urinary LCN2 levels were measured in 36 patients with hematologic malignancies who transiently became neutropenic by stem cell transplantation (SCT). To evaluate contribution of neutrophil-derived LCN2 in the physiologic blood LCN2 concentrations, we examined CCAAT/enhancer-binding protein ε (C/EBPε) knockout mice, which lack mature neutrophils. RESULTS: In patients without AKI and bacterial infection, at 1 week after SCT, the median blood neutrophil counts became zero and serum LCN2 levels were decreased by 76 ± 6 % (p < 0.01), but urinary LCN2 levels were not altered. During neutropenic conditions, bacterial infection caused only a modest rise of serum LCN2 but AKI produced a marked rise of serum and urinary LCN2 levels. Serum LCN2 concentrations in C/EBPε knockout mice were reduced by 66 ± 11 % compared to wild-type mice (p < 0.05). Blood LCN2 existed predominantly in high molecular weight forms (>100 kDa), while urinary LCN2 was mainly in low molecular weight forms. CONCLUSION: Our findings suggest that neutrophils are the major source of circulating LCN2 in normal and infected conditions, whereas blood and urinary LCN2 mainly derive from the kidney during AKI, and that the molecular forms and regulation of blood and urinary LCN2 are clearly distinct.