Binding Protein Epsilon Research Articles

CCAAT/Enhancer-Binding Protein ε27 Antagonism of GATA-1 Transcriptional Activity in the Eosinophil Is Mediated by a Unique N-Terminal Repression Domain, Is Independent of Sumoylation and Does Not Require DNA Binding

CCAAT/enhancer binding protein epsilon (C/EBPε) is required for eosinophil differentiation, lineage-specific gene transcription, and expression of C/EBPε32 and shorter 27kD and 14kD isoforms is developmentally regulated during this process. We previously defined the 27kD isoform (C/EBPε27) as an antagonist of GATA-1 transactivation of the eosinophil’s major basic protein-1 (MBP1) P2-promoter, showing C/EBPε27 and GATA-1 physically interact. In the current study, we used a Tat-C/EBPε27 fusion protein for cell/nuclear transduction of an eosinophil myelocyte cell line to demonstrate that C/EBPε27 is a potent repressor of MBP1 transcription. We performed structure-function analyses of C/EBPε27 mapping its repressor domains, comparing it to C/EBPε32 and C/EBPε14, using GATA-1 co-transactivation of the MBP1-P2 promoter. Results show C/EBPε27 repression of GATA-1 is mediated by its unique 68aa N-terminus combined with previously identified RDI domain. This repressor activity does not require, but is enhanced by, DNA binding via the basic region of C/EBPε27 but independent of sumoylation of the RDI core “VKEEP” sumoylation site. These findings identify the N-terminus of C/EBPε27 as the minimum repressor domain required for antagonism of GATA-1 in the eosinophil. C/EBPε27 repression of GATA-1 occurs via a combination of both C/EBPε27-GATA-1 protein–protein interaction and C/EBPε27 binding to a C/EBP site in the MBP1 promoter. The C/EBPε27 isoform may serve to titrate and/or turn off eosinophil granule protein genes like MBP1 during eosinophil differentiation, as these genes are ultimately silenced in the mature cell. Understanding the functionality of C/EBPε27 in eosinophil development may prove promising in developing therapeutics that reduce eosinophil proliferation in allergic diseases.

Brain Abscess as Severe Presentation of Specific Granule Deficiency

Severe invasive infections such as brain abscess in a child should prompt an immune evaluation. Specific granule deficiency (SGD) is a rare morphologic neutrophil granular defect characterized by reduced granules within neutrophils, absence of granule proteins, and bilobed nuclei. Patients are susceptible to invasive bacterial infections and Candida infections. Mutations in CCAT/enhancer binding protein epsilon (C/EBP-ε) are the most commonly described cause of SGD. The dihydrorhodamine assay is a quantitative and qualitative functional test that determines the oxidative burst and killing potential of neutrophils. Herein, we describe two brothers with specific granule deficiency. The index patient had a history of cellulitis twice in the first year of life and then presented at 13 months age with fever, leukocytosis, and right sided weakness. A large space occupying brain abscess was diagnosed. He underwent surgical drainage and cultures yielded Staphylococcus aureus. This infection prompted his diagnosis. His older brother had also been healthy but too had had several episodes of cellulitis. His brother too was diagnosed with SGD when family genetic screening was performed. Evaluation of the index patient included a peripheral smear that showed absent neutrophil granule presence. Forward and side scatter of whole blood via flow cytometry revealed a loss of granularity of neutrophils. A DHR was performed to rule out functional killing defects. After stimulation with PMA, neutrophils from the index patient displayed three distinct patterns, two with abnormal oxidase production, and two with reduced function. Both patients were ultimately diagnosed with SGD and remain on lifelong anti-bacterial prophylaxis. Diagnosis of SGD relies on establishing reduced or absent granularity within neutrophils. Lifelong anti-bacterial and anti-fungal prophylaxis is indicated. Hematopoietic cell transplantation has also been curative.

Ingenuity pathway analysis of human facet joint tissues: Insight into facet joint osteoarthritis.

Facet joint osteoarthritis (FJOA) is a common degenerative joint disorder with high prevalence in the elderly. FJOA causes lower back pain and lower extremity pain, and thus severely impacts the quality of life of affected patients. Emerging studies have focused on the histomorphological and histomorphometric changes in FJOA. However, the dynamic genetic changes in FJOA have remained to be clearly determined. In the present study, previously obtained RNA deep sequencing data were subjected to an ingenuity pathway analysis (IPA) and canonical signaling pathways of differentially expressed genes (DEGs) in FJOA were studied. The top 25 enriched canonical signaling pathways were identified and canonical signaling pathways with high absolute values of z-scores, specifically leukocyte extravasation signaling, Tec kinase signaling and osteoarthritis pathway, were investigated in detail. DEGs were further categorized by disease, biological function and toxicity (tox) function. The genetic networks between DEGs as well as hub genes in these functional networks were also investigated. It was demonstrated that C-X-C motif chemokine ligand 8, elastase, neutrophil expressed, growth factor independent 1 transcriptional repressor, Spi-1 proto-oncogene, CCAAT enhancer binding protein epsilon, GATA binding protein 1, TAL bHLH transcription factor 1, erythroid differentiation factor, minichromosome maintenance complex component 4, BTG anti-proliferation factor 2, BRCA1 DNA repair-associated, cyclin D1, chromatin assembly factor 1 subunit A, triggering receptor expressed on myeloid cells 1 and tumor protein p63 were hub genes in the top 5 IPA networks (with a score >30). The present study provides insight into the pathological processes of FJOA from a genetic perspective and may thus benefit the clinical treatment of FJOA.

Progress of human CCAAT/enhancer binding protein epsilon on inflammation and infection

Inflammation and infection are main causes of death in critically ill patients. But traditional treatment are non-comprehensive and limited. Human CCAAT/enhancer binding protein epsilon (C/EBPε) is a key transcription factor regulating the terminal differentiation of neutrophils. It plays an important role in anti-inflammatory and anti-infective process by regulating inflammatory response cells. This article reviews the changes of C/EBPε in inflammation and infection, related regulatory mechanisms and targeted reversal measures, in order to provide references for exploring new directions and effective measures for inflammation and infection treatment.

C/EBPε ΔRS derived from a neutrophil-specific granule deficiency patient interacts with HDAC1 and its dysfunction is restored by trichostatin A

CCAAT/enhancer binding protein epsilon (C/EBPε), a myeloid-specific transcription factor, plays an important role in granulopoiesis. A loss-of-function mutation in this protein can result in an abnormal development of neutrophils and eosinophils, known as neutrophil-specific granule deficiency (SGD). The transcriptional activity of C/EBPε is regulated by interactions with other transcription factors and/or post-translational modification, including acetylation. Previously, we reported a novel SGD patient who had a homozygous mutation for two amino acids, arginine (R247) and serine (S248), which were deleted in the basic leucine zipper domain of C/EBPε (ΔRS) and exhibited loss of transcriptional activity with aberrant protein–protein interactions. In the present study, we found that a single amino acid deletion of either R247 (ΔR) or S248 (ΔS) was sufficient for the loss of C/EBPε transcriptional activity, while an amino acid substitution at S248 to alanine in C/EBPε (SA) had comparable transcriptional activity with the wild-type C/EBPε (WT). Although acetylation at lysine residues (K121 and K198) is indispensable for C/EBPε transcriptional activity, an acetylation mimic form of ΔRS (ΔRS-K121/198Q) did not exhibit the transcriptional activity. Interestingly, we discovered that ΔRS, ΔR, ΔS, and ΔRS-K121/198Q interacted with histone deacetylase 1 (HDAC1), whereas WT and SA did not. Furthermore, the proteoglycan 2/eosinophil major basic protein induction activity of ΔRS, ΔR, and ΔS could be restored by the HDAC inhibitor, trichostatin A (TSA), and protein–protein interactions between ΔRS and Gata1 could also be recovered by TSA treatment. Taken together, our results show that TSA has the potential to restore the transcriptional activity of ΔRS, indicating that the inhibition of HDAC1 could be a molecularly targeted treatment for SGD with ΔRS.

CEBPE (CCAAT/enhancer binding protein epsilon)

CEBPE (CCAAT/enhancer binding protein epsilon)

Abstract 1506: Characterization of fibronectin type III domain containing 3B (FNDC3B) as a novel fusion partner of retinoic acid receptor alpha in granulocytic differentiation

Abstract Acute promyelocytic leukemia (APL) is characterized by the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) fusion. Recently, we identified fibronectin type III domain containing 3B (FNDC3B) as a novel RARA fusion partner resulting from the t(3;17)(q26;q21) translocation in a variant APL patient. Unlike other RARA fusion partners, FNDC3B appears as the only partner involved in granulocytic differentiation but the molecular mechanisms underlying its actions remain to be characterized. FNDC3B mRNA expression in normal and malignant hematopoietic cells was determined by quantitative RT-PCR. Transcriptional control of FNDC3B was studied by reporter gene assays, site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays. FNDC3B-regulated pathways were identified by transcriptome analyses. Using BloodSpot, we observed that FNDC3B expression was progressively up-regulated as human hematopoietic stem cells (HSCs) differentiate along the granulocytic lineage, with the highest level in terminally differentiated polymorphonuclear cells. Concordantly, we demonstrated lower expression of FNDC3B in human cord blood CD34+ HSCs than HL-60 promyelocytes. The specific involvement of FNDC3B in granulocytic differentiation was further suggested by higher FNDC3B expression and promoter activity in human promyelocytic than monocytic cell lines. Mutational analyses indicated that two putative CCAAT-enhancer binding protein epsilon (CEBPE) and one Sp1 binding sites were critical for FNDC3B promoter activity in HL-60 cells. The specific association of CEBPE and Sp1 with the FNDC3B promoter could be confirmed by ChIP assays. Knockdown of CEBPE in HL60 cells by small interfering RNA (siRNA) reduced FNDC3B expression and promoter activity. Furthermore, we observed a strong positive correlation between CEBPE and FNDC3B expression in the Cancer Genome Atlas (TCGA)-acute myeloid leukemia (AML) dataset (n=173, r=0.66, P<0.0001). Collectively, these findings indicate CEBPE, which is critical in terminal granulopoiesis, as an important factor controlling FNDC3B transcription in granulocytic cells. Using RNA-seq, we found that FNDC3B knockdown in NB4 promyelocytes was associated with preferential downregulation of genes encoding granule and endoplasmic reticulum (ER) structural/functional proteins. Concordantly, DAVID functional annotation of genes that are positively correlated with FNDC3B in the TCGA-AML cohort revealed enrichment of Gene Ontology terms related to ER functions. Together, these findings suggest that FNDC3B mediates its functions primarily by targeting the ER/granule pathway, which is important for granulocytic maturation. In summary, our data indicate FNDC3B as a novel CEBPE target in the myeloid transcriptional hierarchy and may provide new clues in abnormal granulopoiesis. Citation Format: Zhe Wand, Chi Keung Cheng, Terry Wong, Kin Mang Lau, Thomas S.k. Wan, Kam Tong Leung, Margaret Heung-Ling Ng. Characterization of fibronectin type III domain containing 3B (FNDC3B) as a novel fusion partner of retinoic acid receptor alpha in granulocytic differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1506.

Role of the Leucine Zipper Domain of CCAAT/ Enhancer Binding Protein-Epsilon (C/EBPε) in Neutrophil-Specific Granule Deficiency.

Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by bilobed neutrophil nuclei and lack of neutrophil-specific granule proteins such as lactoferrin. A deficiency of a myeloid-specific transcription factor, CCAAT/enhancer binding protein-epsilon (C/EBPε), has been identified as a cause of SGD. C/EBPε binds to DNA though its basic leucine zipper (bZIP) domain, and regulates terminal differentiation of neutrophils and expression of specific granule genes. Homozygous frameshift mutations resulting in loss of the bZIP domain have been reported in two patients with SGD. A recent observation showed that a homozygous 2-aa deletion in the bZIP domain with normal DNA-binding and dimerization abilities causes SGD by impairing protein-protein interactions with other transcription factors, indicating that multiple molecular mechanisms can lead to SGD. Studies of patient-derived mutations and analysis of C/EBPε knockout mice have shown the importance of the bZIP domain for the essential functions of C/EBPε.

A Novel 2 Amino Acids Deletion in the Leucine Zipper Domain of C/EBPε Leads to Neutrophil-Specific Granule Deficiency (SGD)

Neutrophil-specific granule deficiency (SGD) is a rare congenital disorder. Neutrophils of SGD patients show abnormal bilobed nuclei and lack secondary and tertiary granule gene expression. Since neutrophils of SGD patients are deficient for bactericidal activity, the patients display immunodeficiency and suffer from frequent and severe infections. The transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) is one of the major regulators of granulopoiesis and is known to be the responsible gene for the disease. At least two homozygous germline mutations (5bp deletion and A-nucleotide insertion) of C/EBPε, which are functionally defective, have been reported.Here, we report a novel in-frame deletion of the C/EBPεgene in a 55 years-old female patient with a life-long history of recalcitrant skin infections with ulcer and scar formation. Peripheral blood smear showed characteristic changes, including reduction of cytoplasmic granules and increased bilobed nuclei of the neutrophils, monocytosis, absence of eosinophils and increased basophils. Flow cytometric examination revealed significant reduction of CD16 and abnormal expression of CD14 on the neutrophil surface.Sequencing of the C/EBPε gene revealed a homozygous deletion of 6 base-pairs (arginine and serine residues deletion; ΔRS) within the leucine zipper domain. Activation of luciferase driven by G-CSF receptor promoter was not induced by ΔRS; and endogenous expression of granule genes, including B9, NGAL and lactoferrin, was induced by wild-type C/EBPε but not by ΔRS in NIH3T3 cells, suggesting that transcriptional activity of ΔRS was diminished. GFP-tagged ΔRS, as well as wild-type C/EBPε, were localized to the nucleus in NIH3T3 cells, and DNA binding activity of ΔRS, which was determined by biotin-labeled DNA pulldown assay, was intact. Meanwhile, ΔRS together with Gata1 and PU.1 were not able to induce expression of eosinophil major basic protein (MBP) in NIH3T3 cells. Of note, wild-type C/EBPε associated with Gata1; however, ΔRS did not interact with Gata1, indicating that the ΔRS likely impairs protein-protein interaction of other transcription factors resulting in loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function and indicate multiple molecular mechanisms leading to SGD. DisclosuresNo relevant conflicts of interest to declare.

Identification of functional nucleotide and haplotype variants in the promoter of the CEBPE gene

The current study examined the promoter activity of an association signal in a 5'-upstream region of the gene encoding CCAAT/enhancer binding protein epsilon (CEBPE) identified from a recent genome-wide association study (GWAS) for complex acute lymphoblastic leukemia (ALL). This follow-up study first compared the activity of reporter constructs with three haplotypes estimated with the rs2239633 and its proximity nucleotide variants in strong linkage. The most frequent haplotype was CTTTTGT (H1), and the second most frequent haplotype consisted of entirely opposite alleles to H1 (TCGCACC, H2). Their luciferase activity revealed the strongest expression with H2 and the weakest with H1. Subsequent analysis revealed that different luciferase activity was found by the single-nucleotide substitution at rs2239632 and rs2239633 (P<0.05). Especially, the difference in luciferase activity between two alleles of rs2239632 corresponded to the difference between H1 and H2. We concluded that rs2239632 could regulate the expression of the CEBPE gene. We suggest a hypothesis that its risk allele (G) might increase the gene product and lead to leukemogenesis. As a result, a person with the allele or the corresponding haplotype might have increased susceptibility to ALL. Further research is warranted to investigate this hypothesis and the underlying mechanisms.

CCAAT/Enhancer Binding Protein Epsilon Mediates Nicotinamide-Enhanced Clearance of Staphylococcus Aureus Infection

AbstractAbstract 925Staphylococcus aureus in community and healthcare settings commonly causes serious and potentially life-threatening infections. Widespread use of antibiotics is responsible for the emergence and rapid spread of resistant pathogens, including methicillin-resistant S. aureus (MRSA), and highlights a pressing need for development of novel antimicrobial therapies. The myeloid-specific transcription factor, CCAAT/enhancer binding protein epsilon (C/EBPε) serves as a critical regulator of the terminal differentiation and functional maturation of neutrophils and macrophages, both crucial components of the innate immune system.Comparable to humans with neutrophil specific granule deficiency (SGD) carrying a causative mutation in this transcription factor, we showed that C/EBPε-deficient (C/EBPε—/—) mice were severely affected by in vivo infection with S. aureus. Paradoxically, depletion of the defective neutrophils attenuated disease pathology and even improved the outcome of infection. During subcutaneous infection with S. aureus, C/EBPε—/— mice treated with mouse anti-polymorphonuclear neutrophil antibody showed significantly smaller skin lesions, fewer CFU within the lesion, and reduced systemic spread of bacteria. In addition, whole blood from C/EBPε—/— mice was less effective at killing S. aureus compared to their cell-free plasma. Therefore, ineffective clearance of S. aureus by C/EBPε—/— neutrophils, even compared to extracellular killing mechanisms, likely permitted S. aureus to thrive within neutrophils, which further aggravated the infection.Because C/EBPε plays a critical role in the host immune response against S. aureus infection, we further hypothesized that increased activity of C/EBPε could enhance immune killing of bacteria. Using a zink-inducible expression vector, we induced overexpression of C/EBPε in U937-macrophages, and thereby enhanced bacterial clearance including MRSA by up to 1.5 log10 CFU/mL. Interestingly, we found that the epigenetic modulator, nicotinamide (NAM; vitamin B3), increased activity of C/EBPε as well as downstream antimicrobial targets. Upon exposing bone-marrow derived macrophages or mononuclear cells from wildtype mice to NAM (1 or 10 mM), increased levels of lysine acetylation on core histone H3 were detected at the promoter region of CEBPE. This was associated with elevated mRNA and protein levels of C/EBPε, and increased expression of downstream antimicrobials such as cathelicidin(-related) antimicrobial peptide (CAMP) and Lactoferrin. In an in vitro, as well as in vivo infection model, moderately concentrated NAM enhanced killing of S. aureus by up to 3 log10, but had no effect when administered to C/EBPε-deficient mice. This again points to C/EBPε as an important target to boost killing of bacteria by the innate immune system. Strikingly, and consistent with our murine data, NAM treatment reduced the ability of S. aureus to survive in whole human blood obtained from 12 healthy humans by 2–3 log10. In line with our findings on S. aureus, we were able to demonstrate similar immune boosting effects of NAM in human blood infected with other important human pathogens such as K. pneumoniae and P. aeruginosa.In an age when the number of antibiotics in the pipeline is limited and development of resistance occurs rapidly, use of complementary strategies to antibiotic treatment provides a promising method of limiting development of antibiotic resistance. Here, we demonstrated that C/EBPε is a regulatory factor that critically impacts the host's ability to fight bacterial infections. Compounds exerting modulatory effects on this myeloid-specific transcription factor may emerge as important antimicrobial therapeutics against frequent pathogens such as S. aureus. Disclosures:No relevant conflicts of interest to declare.

C/EBPε directs granulocytic-vs-monocytic lineage determination and confers chemotactic function via Hlx

Mutations in the CCAAT enhancer binding protein epsilon (C/EBPepsilon) gene have been identified in the cells of patients with neutrophil specific granule deficiency, a rare congenital disorder marked by recurrent bacterial infections. Their neutrophils, in addition to lacking specific granules required for normal respiratory burst activity, also lack normal phagocytosis and chemotaxis. Although the specific granule deficiency phenotype has been replicated in C/EBPepsilon(-/-) (knockout [KO]) mice, the mechanisms by which C/EBPepsilon mutations act to decrease neutrophil function are not entirely clear. In order to determine the role of C/EBPepsilon in neutrophil differentiation and migration, we generated immortalized progenitor cell lines from C/EBPepsilon KO and wild-type mice and performed expression and flow cytometric analysis and functional studies. Expression of lineage-specific cell surface antigens on our in vitro differentiated cell lines revealed persistent expression of monocytic markers on KO granulocytes. We verified this in primary murine peripheral blood and bone marrow cells. In addition, KO bone marrow had an increase in immature myeloid precursors at the common myeloid progenitor and granulocyte/monocyte progenitor levels, suggesting a critical role for C/EBPepsilon not only in granulocyte maturation beyond the promyelocyte stage, but also in the monocyte/granulocyte lineage decision. We found that restoration of Hlx (H2.0-like homeo box 1) expression, which was decreased in C/EBPepsilon KO cells, rescued chemotaxis, but not the other defects of C/EBPepsilon KO neutrophils. We show two new regulatory functions of C/EBPepsilon in myelopoiesis: in the absence of C/EBPepsilon, there is not only incomplete differentiation of granulocytes, but myelopoiesis is disrupted with the appearance of an intermediate cell type with monocyte and granulocyte features, and the neutrophils have abnormal chemotaxis. Restoration of expression of Hlx provides partial recovery of function; it has no effect on neutrophil maturation, but can completely ameliorate the chemotaxis defect in C/EBPepsilon KO cells.

Modulation of DNA binding properties of CCAAT/enhancer binding protein epsilon by heterodimer formation and interactions with NFkappaB pathway

AbstractC/EBP epsilon is a transcription factor involved in myeloid cell differentiation. Along with C/EBP-α, -β, -γ, -δ, and -ζ, C/EBP-ϵ belongs to the family of CCAAT/enhancer binding proteins that are implicated in control of growth and differentiation of several cell lineages in inflammation and stress response. We have previously shown that C/EBP-ϵ preferentially binds DNA as a heterodimer with other C/EBP family members such as C/EBP-δ, CHOP (C/EBP-ζ), and the b-zip family protein ATF4. In this study, we define the consensus binding sites for C/EBP-ϵ dimers and C/EBP-ϵ–ATF4 heterodimers. We show that the activated NFkappaB pathway promotes interaction of the C/EBP-ϵ subunit with its cognate DNA binding site via interaction with RelA. RelA-C/EBP interaction is enhanced by phosphorylation of threonine at amino acid 75 and results in increased DNA binding compared with the wild-type nonphosphorylated C/EBP both in vitro and in vivo. We suggest that interaction of the activated NFkappaB pathway and C/EBP-ϵ may be important in selective activation of a subset of C/EBP-ϵ–responsive genes.

Expression of Bactericidal/Permeability-Increasing Protein Requires C/EBPϵ

Bactericidal/permeability-increasing protein (BPI) is a 55-kd cationic protein found mainly in neutrophil primary granules. BPI shows cytotoxicity against Gram-negative bacteria. In this study, we studied the role of a myeloid-specific transcription factor, CCAAT/enhancer binding protein epsilon (C/EBP epsilon), in the regulation of BPI gene expression. A patient with neutrophil-specific granule deficiency with a homozygous inactivating mutation in the CEBP epsilon gene showed severely impaired expression of both BPI messenger RNA (mRNA) and BPI protein. Both U937 and NB4 cells treated with 10-7 M all-trans retinoic acid (ATRA) for 6 days displayed increased levels of BPI protein and accompanying up-regulated C/EBP epsilon expression. Chromatin-immunoprecipitation analysis and electrophoretic mobility shift assays revealed binding of the C/EBP epsilon protein to the C/EBP-binding site in the BPI gene promoter. U937 cells stably transfected with a zinc-inducible C/EBP epsilon expression vector showed a 30-fold increase in BPI mRNA levels compared with cells transfected with control empty vector after culturing for 48 hours with 100 microM ZnSO4. BPI mRNA expression was severely reduced in the bone marrow of C/EBP epsilon-deficient mice compared with wild-type mice. Expression of BPI in human cord blood cells was increased by incubation with 10-7 MATRA for 48 hours. These results demonstrate the requirement for C/EBP epsilon in mediating BPI gene expression in myeloid cells in vitro and in vivo.

Human Resistin Is a Systemic Immune-Derived Proinflammatory Cytokine Targeting both Leukocytes and Adipocytes

The characteristics of human resistin (RETN) are unclear and controversial despite intensive adipose-focused research. Its transcriptional and functional similarity with the murine myeloid-specific and CCAAT/enhancer binding protein epsilon (Cebpe)-dependent gene, resistin-like gamma (Retnlg), is unexplored. We examined the human CEBPE-regulatory pathway by unbiased reference and custom gene expression assays. Real-time RT-PCR analysis demonstrated lack of both the transcriptional factor CEBPE and RETN expression in adipose and muscle cells. In contrast, primary myelocytic samples revealed a concerted CEBPE-RETN transcription that was significantly elevated in inflammatory synoviocytes relative to intact peripheral blood mononuclear cells (PBMC). Mouse Cebpe and Retnlg were predictably expressed in macrophages, whereas Retn was abundant in adipocytes. Quite the opposite, a low and inconsistent RETN transcription was seen in some human white adipose tissue (WAT) biopsies without any relationship to body mass index, insulin sensitivity, or fat depot. However, in these cases, RETN was co-detected with CEBPE and the leukocyte-specific marker, EMR1, indicating the presence of inflammatory cells and their possible resistin-mediated effect on adipocytes. Indeed, addition of human resistin to WAT in culture induced, like in PBMC, the inflammatory cytokines IL6, IL8 and TNF. Importantly, the expression of the adipose-specific markers CEBPA, FABP4 and SLC2A4 was unchanged, while the expected inhibitory effect was seen with TNF. Both cytokines increased the mRNA level of CCL2 and MMP3, which may further promote inflammation in WAT. Thus, the myeloid-restricted nature of CEBPE precludes the expression of RETN in human adipocytes which, however, are targeted by this innate immune-derived proinflammatory cytokine.

Identification of Common Molecular Pathways in Granulopoiesis Associated with C/EBP Epsilon and Gfi-1-Deficient Neutrophil Specific Granule Deficiency (SGD).

Neutrophil specific granule deficiency (SGD) is a rare congenital disorder marked by recurrent bacterial infections of the skin and respiratory system. Neutrophils from SGD patients lack secondary and tertiary granules and their content proteins and exhibit defects in chemotaxis and bactericidal activity. A mouse model deficient for the transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) manifests a similar phenotype to SGD patients, and functional mutations in the C/EBPε gene have been identified in two patients with SGD. However, other patients with a similar disease phenotype do not have functional C/EBPε mutations, suggesting that other genetic abnormalities in myelopoiesis can lead to SGD. Studies in our laboratory on one such patient lacking a functional C/EBPε mutation demonstrated elevated protein levels of C/EBPε and significantly decreased levels of the transcription factor Gfi-1 (Growth factor independent-1) in peripheral blood neutrophils from this SGD patient. However, no mutation has been found thus far in the coding region of the Gfi-1 gene from the SGD patient. SGD can therefore be classified as C/EBPε negative or C/EBPε positive. We hypothesize that during granulopoiesis defects in a common molecular pathway occur in both C/EBPε- positive and -negative SGD resulting in the SGD phenotype. In order to gain insight into the molecular features of the two SGD phenotypes in the face of severe limitations on primary material from SGD patients, we have generated and characterized two cell line models. Using bone marrow from both C/EBPε−/− and Gfi-1+/− mice and their corresponding wildtype (WT) littermates, we generated EML (erythroid, myeloid, lymphoid) cell lines by transducing bone marrow with a retroviral vector expressing the dominant negative RARα and selecting for immortalized cells. Upon induction of the knock out and WT EML cell lines with all-trans retinoic acid (ATRA) to the neutrophil stage, we confirmed that both C/EBPε−/− and Gfi-1+/− cells share morphologic and functional abnormalities corresponding to their respective knockout mice, and hence to SGD. To further characterize the molecular defects in SGD, we have initiated microarray and proteomics analyses using RNA and protein prepared from both knockout cell lines. Using this approach, we may delineate defective pathways in both knock out cell types and correlate these observations to the specific SGD type. Our study should shed light on the relationship between Gfi-1 and C/EBPε during neutrophil development and should address the issue as to whether common or divergent molecular pathways are responsible for C/EBPε-negative and -positive SGD.

Differential Re-Programming of Myeloid Stem Cell Development by Activator Versus Repressor Isoforms of Human CCAAT/Enhancer Binding Protein Epsilon (C/EBPε).

Human C/EBPε is expressed as four isoforms (32, 30, 27, 14kD) through alternative promoter usage, RNA splicing, and translational start sites. We are studying the C/EBPε isoforms in vivo in cord blood (CB) CD34+ progenitors to define their roles in granulocyte differentiation and hematopoietic lineage specification. The C/EBPε32/30 isoforms function as transcriptional activators. We reported that C/EBPε27 interacts with and is a repressor of GATA-1 transactivation of eosinophil-specific genes (Du J et al. JBC 2002; 277:43481). C/EBPε14, which contains DNA binding and bZIP domains but lacks a transactivation domain, may function as a dominant negative repressor of C/EBPε32,30 or other C/EBPs through heterodimerization or competition for C/EBP binding sites. We assessed mRNA expression of the C/EBPε isoforms during eosinophil differentiation of CB CD34+ progenitors to determine their temporal patterns of expression. CD34+ progenitors (>95% pure) induced to differentiate into eosinophils by SCF, IL-3, and IL-5 showed differentiation to >90% eosinophils by 17–19 days. Semi-quantitative RT-PCR using C/EBPε isoform-specific primers showed that CD34+ progenitors initially express only the repressor C/EBPε14 isoform, with induction of all isoforms by day 3 to different peak levels of expression by days 9–11 during the promyelocyte to myelocyte transition. These results define temporal changes in the expression ratios of the C/EBPε activator vs. repressor isoforms during eosinophil development that may differentially regulate gene transcription in this process. We subcloned the C/EBPε isoform cDNAs into an MSCV-based bicistronic retroviral vector (pGCDNsam IRES-EGFP) and ectopic expression was induced in CB CD34+ progenitors by retroviral transduction for 72hrs. The CD34+/GFP+ cells were sorted by FACS and plated in Collagen Cult™ media containing SCF, IL-3 and a lineage-specific cytokine (i.e. EPO, G-CSF, or IL-5), or in suspension culture containing SCF, IL-3 and IL-5 to drive eosinophil differentiation. Total and differential colony and cell counts were performed after 15–17 days based on colony morphology, histochemical and enzyme staining of the cells. The activator C/EBPε32 isoform significantly altered myeloid development, favoring eosinophil over neutrophil or erythroid development. Even in cultures containing EPO, cells transduced with C/EBPε32 failed to develop into erythroid colonies (BFU-E). C/EBPε27, a potent repressor of GATA-1 in vitro, inhibited erythroid colony growth by ~50%, and doubled the numbers of granulocyte-macrophage colonies compared to C/EBPε14 or empty vector control. C/EBPε32 strongly induced eosinophil colony formation at the expense of neutrophil other myeloid lineages regardless of the cytokines used, inducing ~90% eosinophil colonies. C/EBPε27 reduced eosinophil colonies by >50%. Likewise, >90% of cells transduced with C/EBPε32 and grown in suspension culture were eosinophils, whereas C/EBPε27 and C/EBPε14 inhibited eosinophil differentiation by ~50% and >98%, respectively. Thus, the C/EBPε isoforms: (1) are differentially expressed during eosinophil development, (2) have the capacity to reprogram stem cells to different myeloid lineages consistent with their predicted activator vs. repressor activities and interactions with hematopoietic transcription factors such as GATA-1 or other C/EBPs, and (3) may be useful in their ability to reprogram myeloid terminal differentiation for the development of novel approaches to treat myeloid or other leukemias.

Haptoglobin is synthesized during granulocyte differentiation, stored in specific granules, and released by neutrophils in response to activation

Haptoglobin (Hp) is a plasma protein synthesized primarily by hepatocytes. It exerts a broad range of anti-inflammatory activities and acts indirectly as a bacteriostatic agent and an antioxidant by virtue of its ability to bind free hemoglobin (Hb) and to facilitate its immediate clearance by macrophages. We identified Hp as a novel specific granule protein of neutrophils by means of immunoelectron microscopy, subcellular fractionation, and exocytosis studies. Consistent with these findings, blood cells from a patient with specific granule deficiency (SGD) lacked neutrophil-derived Hp. Neutrophils contained a large amount of highly glycosylated Hp (beta-chain 45-65 kDa) synthesized in neutrophil precursors and stored in specific granules and a small amount of Hp (beta-chain 39 kDa) endocytosed from plasma and stored in secretory vesicles. Subsequent binding studies revealed that Hp from specific granules binds to Hb. Finally, the CCAAT enhancer binding protein-epsilon (C/EBPepsilon) induced Hp transcription in a myeloid cell line, suggesting that Hp expression in myeloid cells, as in hepatocytes, is at least partially regulated by members of the C/EBP transcription factor family. Collectively, these findings demonstrate that Hp is stored in specific granules and is released by neutrophils in response to activation. Hence, neutrophil-derived Hp might reduce tissue damage and bacterial growth at sites of infection or injury by propagating anti-inflammatory activities and Hb clearance.

Modulation of DNA Binding Properties of CCAAT/Enhancer Binding Protein Epsilon by Heterodimer Formation and Interactions with NFkappaB Pathway.

Specific regulation of gene expression is achieved through interaction of multiple transcriptional activators and repressors with their cognate DNA sites. This activity is subject to several levels of control, including post-translational modification by phosphorylation, acetylation, selective degradation and interaction with co-activators and co-repressors.Recent studies directed at discovering C/EBP- responsive genes resulted in identification of a diverse and degenerate collection of putative C/EBP binding sites. Identification of binding sites for each individual member of the C/EBP family is complicated by common co-expression of several of these proteins in the same cell and their ability to form hetero-dimeric complexes with each other and structurally-related ATF/CREB proteins. Activity of C/EBP proteins is regulated by phosphorylation mediated through several major kinase pathways, that affect DNA-binding, subcellular localization in the nucleus, and interaction with cell cycle proteins.Our studies of C/EBP-e phosphorylation identified several sites, including one for p38 MAPK within the transactivation domain of C/EBP-e. As a result of phosphorylation at Threonine-75 (T75), C/EBP-e DNA binding and specific transcriptional activity was significantly enhanced.In this study, we identified consensus binding sequences for C/EBP-e and for heterodimers of C/EBP-e with its most common dimerization partner ATF4. Furthermore, we find that C/EBP-e- DNA interaction is highly upregulated by interaction with activated NFkappaB pathway protein p65-RelA. This interaction depends on the T75 phosphorylation status of C/EBP-e and can be enhanced by introduction of phosphomimetic Thr to Asp mutation. By using selective removal of p65-RelA and siRNA mediated p65 knock-down, we demonstrate that activated NFkappaB pathway is required for high level expression of genes with C/EBP-e responsive promoter. This novel interaction may play an important role in regulation of a C/EBP-e - dependent genes in myeloid cells, for example at sites of infection or other inflammations.

Knock-Down of CCAAT Displacement Protein (CDP) Restores Specific Granule Protein (SGP) Gene Expression in NB4 Acute Promyelocytic Leukemia Cells.

CCAAT displacement protein (CDP) is a highly conserved, ubiquitously expressed homeodomain protein with extensive homology to the Drosophila cut protein. CDP contains three highly conserved DNA-binding repeats called cut repeats, as well as a conserved homeodomain sequence. CDP is a transcriptional repressor of several developmentally regulated genes involved in neutrophil maturation, including the phagocyte-specific cytochrome heavy chain gene, gp91-phox, and CCAAT enhancer binding protein epsilon (C/EBPε ). It also represses expression of neutrophil secondary granule proteins (SGPs), including LF, which are downstream targets of C/EBPε . We have previously shown that CDP binds to and represses both the C/EBPε and lactoferrin (LF) gene promoters thereby preventing expression of secondary granule proteins (SGPs) both directly and indirectly. Additionally, we have demonstrated that overexpression of CDP represses expression of SGPs in 32Dcl3 cells, an IL-3 dependent murine myeloid cell line that undergoes differentiation in response to IL-3 withdrawal and G-CSF stimulation. Conversely, knocking down CDP expression by RNA interference in 32D cells upregulates levels of LF mRNA in uninduced cells. NB4 cells are an acute promyelocytic cell line that contains the t(15;17) PML-RARα translocation and can be induced toward neutrophil maturation with all-trans-retinoic Acid (ATRA). We have previously demonstrated that NB4 cells uniformly fail to express LF and all the SGP genes upon ATRA induction. In further studies, we showed that this failure of expression occurs despite appropriate binding of C/EBP factors known to upregulate SGP expression. We further demonstrated persistent binding of CDP to the LF promoter in NB4 cells upon ATRA induction, suggesting that failure of LF expression was due to persistent binding of CDP rather than a failure of C/EBP transactivation. In order to test this hypothesis, we knocked down CDP expression in NB4 cells using short hairpin RNA (shRNA) constructs and examined the effect of reduced CDP levels on SGP expression. ShRNA CDP constructs were introduced into NB4 cells by nucleofection using an Amaxa® nucleofector. Half the transfected cells were induced with ATRA, while the other half remained uninduced. After 24-hours of induction, total RNA was extracted from the cells and RTqPCR performed to assess the levels of SGP mRNA. We demonstrate that decreased CDP expression in ATRA- induced NB4 cells is sufficient to restore LF, collagenase, and gelatinase expression in the leukemic cell line. SGP expression remains absent in uninduced CDP-shRNA NB4 cells, as well as in both uninduced and induced cells transfected with an empty vector. Our observations reaffirm the critical role played by CDP in regulating the expression of LF and other SGP proteins during neutrophil development. How the PML-RARα gene product functions to alter CDP activity in NB4 cells at the molecular level, thereby restricting SGP expression, is currently being addressed.