Two members of the C/EBP family of basic region‐leucine zipper proteins enriched in the liver, C/EBP (C/EBP alpha) and CRP2 (C/EBP beta), were previously shown to transactivate the albumin promoter in a cell type‐dependent manner. These proteins function efficiently in HepG2 hepatoma cells, but inefficiently in HeLa (epithelial) and L (fibroblastic) cells. Here we have investigated the mechanism for cell‐specific control of CRP2 activity. We show that CRP2 contains a negative regulatory region composed of two elements, RD1 and RD2. Deletions of RD2 relieve the inhibition of CRP2 activity in L cells, but do not affect CRP2 function in HepG2 cells. These deletions also increase the DNA binding activity of CRP2 approximately 3‐fold, suggesting that RD2‐mediated repression of DNA binding activity is responsible for CRP2 inhibition in L cells. The adjacent RD1 element functions independently of RD2 and modulates the CRP2 activation domain, which we show to be composed of three subdomains that are conserved within the C/EBP protein family. RD1 does not affect cell type specificity, but inhibits the transactivation potential of GAL4‐CRP2 hybrid proteins by 50‐fold. These findings suggest that CRP2 assumes a tightly folded conformation in which the DNA binding and activation domains are masked by interactions with the regulatory domain and that to function efficiently in HepG2 cells the protein must undergo an activation step. We propose that relief of inhibition conferred by the regulatory domains also accounts for CRP2 activation in response to extracellular signals.

Lipopolysaccharide (LPS) induces expression of tumor necrosis factor ␣ (TNF␣) and other pro-inflammatory cytokines in macrophages. Following its induction, TNF␣ gene transcription is rapidly attenuated, in part due to the accumulation of NF-B p50 homodimers that bind to three B sites in the TNF␣ promoter. Here we have investigated the inhibitory role of BCL-3, an IBlike protein that interacts exclusively with p50 and p52 homodimers. BCL-3 was induced by LPS with delayed kinetics and was associated with p50 in the nucleus. Forced expression of BCL-3 suppressed LPS-induced transcription from the TNF␣ promoter and inhibited two artificial promoters composed of TNF␣ B sites that preferentially bind p50 dimers. BCL-3-mediated repression was reversed by trichostatin A and was enhanced by overexpression of HDAC-1, indicating that transcriptional attenuation involves recruitment of histone deacetylase. Analysis of macrophages from p50 and BCL-3 knock-out mice revealed that both transcription factors negatively regulate TNF␣ expression and that BCL-3 inhibits IL-1␣ and IL-1␤. In contrast, induction of the anti-inflammatory cytokine IL-10 was reduced in BCL-3 null macrophages. BCL-3 was not required for the production of p50 homodimers but BCL-3 expression was severely diminished in p50-deficient cells. Together, these findings indicate that p50 and BCL-3 function as anti-inflammatory regulators in macrophages by attenuating transcription of pro-inflammatory cytokines and activating IL-10 expression.

Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-␣

Key Points• GM-CSF-dependent STAT5 hypersensitivity is detected in 90% of CMML samples and is enhanced by signaling mutations.• Treatment with a GM-CSF-neutralizing antibody and JAK2 inhibitors reveals therapeutic potential.Granulocyte-macrophage-colony-stimulating factor (GM-CSF) hypersensitivity is a hallmark of juvenile myelomonocytic leukemia (JMML) but has not been systematically shown in the related human disease chronic myelomonocytic leukemia (CMML). We find that primary CMML samples demonstrate GM-CSF-dependent hypersensitivity by hematopoietic colony formation assays and phospho-STAT5 (pSTAT5) flow cytometry compared with healthy donors. Among CMML patients, the pSTAT5 hypersensitive response positively correlated with high-risk disease, peripheral leukocytes, monocytes, and signalingassociated mutations. When compared with IL-3 and G-CSF, GM-CSF hypersensitivity was cytokine specific and thus a possible target for intervention in CMML. To explore this possibility, we treated primary CMML cells with KB003, a novel monoclonal anti-GM-CSF antibody, and JAK2 inhibitors. We found that an elevated proportion of immature GM-CSF receptor-a(R) subunit-expressing cells were present in the bone marrow myeloid compartment of CMML. In survival assays, we found that myeloid and monocytic progenitors were sensitive to GM-CSF signal inhibition. Our data indicate that a committed myeloid precursor expressing CD38 may represent the progenitor population with enhanced GM-CSF dependence in CMML, consistent with results in JMML. These preclinical data indicate that GM-CSF signaling inhibitors merit further investigation in CMML and that GM-CSFR expression on myeloid progenitors may be a biomarker for this therapy. (Blood. 2013;121(25):5068-5077)

Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute lung injury and mortality through the delivery of exotoxins by the type III secretion system (TTSS). PcrV is an important structural protein of the TTSS. An engineered human antibody Fab fragment that binds to the P. aeruginosa PcrV protein with high affinity has been identified and has potent in vitro neutralization activity against the TTSS. The instillation of a single dose of Fab into the lungs of mice provided protection against lethal pulmonary challenge of P. aeruginosa and led to a substantial reduction of viable bacterial counts in the lungs. These results demonstrate that blocking of the TTSS by a Fab lacking antibody Fc-mediated effector functions can be sufficient for the effective clearance of pulmonary P. aeruginosa infection.

The rat CYP2D5 P-450 gene is activated in the liver during postnatal development. We previously showed that liver-specific transcription of the CYP2D5 gene is dictated by a proximal promoter element, termed 2D5, that is composed of a binding site for Sp1 or a related factor, and an adjacent cryptic C/EBP (CCAAT/ enhancer-binding protein) site. Despite the fact that both C/EBP␣ and C/EBP␤ are expressed abundantly in liver, only C/EBP␤ is capable of stimulating the 2D5 promoter in HepG2 hepatocarcinoma cells. In addition, activation of the 2D5 promoter by C/EBP␤ is completely dependent on the presence of the Sp1 site. Domain switch experiments reveal that C/EBP␤ proteins containing either the leucine zipper or the activation domain of C/EBP␣ are unable to stimulate the 2D5 promoter yet are fully capable of transactivating an artificial promoter bearing a high-affinity C/EBP site. Thus, the leucine zipper and the activation domain of C/EBP␤ are absolutely required to support transactivation of the 2D5 promoter. Using Drosophila cells that lack endogenous Sp1 activity, we show that the serine/threonine-and glutamine-rich activation domains A and B of Sp1 are required for efficient cooperatively with C/EBP␤. Furthermore, analysis of c/ebp␤-deficient mice shows that mutant animals are defective in expression of a murine CYP2D5 homolog in hepatic cells, confirming the selective ability of C/EBP␤ to activate this liver-specific P-450 gene in vivo. Our findings illustrate that two members of a transcription factor family can achieve distinct target gene specificities through differential interactions with a cooperating Sp1 protein.Transcription of most eukaryotic genes is regulated by several activator proteins that bind to cis-regulatory sites flanking the gene. Single regulatory elements are rarely sufficient to promote high levels of transcription or to direct proper cellspecific or inducible expression. Usually two or more cis elements are required, and frequently these sites function synergistically (superadditively) to activate gene transcription (14). Synergism can occur at the level of transcriptional activation, where two activators may have independent targets in the transcription initiation complex or enhance different rate-limiting steps in the initiation pathway (2,15,23). Alternatively, cooperativity may be achieved through facilitated DNA-binding interactions in which binding of one protein increases the affinity of a second activator for its binding site (9,35,39). DNA-binding cooperativity involves either protein-protein interactions between two factors or, less likely, a conformational change in the DNA induced by one protein that promotes binding of the second. Although synergism between activators is an important aspect of gene activation in eukaryotic organisms, relatively little is known of the molecular basis for such cooperativity.We previously reported evidence for cooperative interactions between C/EBP␤ (also named NF-IL6, IL-6DBP, LAP, AGP/EBP, CRP2, and NF-M [reviewed in reference 16]) and Sp1...

C/EBP␣, ␤, and ␦ are all expressed by bone marrowderived macrophages. Ectopic expression of any of these transcription factors is sufficient to confer lipopolysaccharide (LPS)-inducible expression of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) to a B lymphoblast cell line, which normally lacks C/EBP factors and does not display LPS induction of proinflammatory cytokines. Thus, the activities of C/EBP␣, ␤, and ␦ are redundant in regard to expression of IL-6 and MCP-1. Surprisingly, the bZIP region of C/EBP␤, which lacks any previously described activation domains, can also confer LPS-inducible expression of IL-6 and MCP-1 in stable transfectants. Transient transfections reveal that the bZIP regions of C/EBP␤, C/EBP␦, and, to a lesser extent, C/EBP␣ can activate the IL-6 promoter and augment its induction by LPS. Furthermore, the transdominant inhibitor, LIP, can activate expression from the IL-6 promoter. The ability of the C/EBP␤ bZIP region to activate the IL-6 promoter in transient transfections is completely dependent upon an intact NF-B-binding site, supporting a model where the bZIP protein primarily functions to augment the activity of NF-B. Replacement of the leucine zipper of C/EBP␤ with that of GCN4 yields a chimeric protein that can dimerize and specifically bind to a C/EBP consensus sequence, but shows a markedly reduced ability to activate IL-6 and MCP-1 expression. These results implicate the leucine zipper domain in some function other than dimerization with known C/EBP family members, and suggest that C/EBP redundancy in regulating cytokine expression may result from their highly related bZIP regions.

Eph receptor tyrosine kinases are critical for cell-cell communication during normal and oncogenic tissue patterning and tumor growth. Somatic mutation profiles of several cancer genomes suggest EphA3 as a tumor suppressor, but its oncogenic expression pattern and role in tumorigenesis remain largely undefined. Here, we report unexpected EphA3 overexpression within the microenvironment of a range of human cancers and mouse tumor xenografts where its activation inhibits tumor growth. EphA3 is found on mouse bone marrow-derived cells with mesenchymal and myeloid phenotypes, and activation of EphA3stromal cells with an EphA3 agonist leads to cell contraction, cell-cell segregation, and apoptosis. Treatment of mice with an agonistic a-EphA3 antibody inhibits tumor growth by severely disrupting the integrity and function of newly formed tumor stroma and microvasculature. Our data define EphA3 as a novel target for selective ablation of the tumor microenvironment and demonstrate the potential of EphA3 agonists for anticancer therapy. Cancer Res; 74(16); 4470-81. Ó2014 AACR.