Toxoplasma gondii tachyzoites inhibit proinflammatory cytokine induction in infected macrophages by preventing nuclear translocation of the transcription factor NF-kappa B.

Control of microbial infection requires regulated induction of NF-kappaB-dependent proinflammatory cytokines such as IL-12 and TNF-alpha. Activation of this important transcription factor is driven by phosphorylation-dependent degradation of the inhibitory IkappaB molecule, an event which enables NF-kappaB translocation from the cytoplasm to the nucleus. In this study, we show that intracellular infection of macrophages with the protozoan parasite Toxoplasma gondii induces rapid IkappaB phosphorylation and degradation. Nevertheless, NF-kappaB failed to translocate to the nucleus, enabling the parasite to invade cells without triggering proinflammatory cytokine induction. Infected cells subsequently subjected to LPS triggering were severely crippled in IL-12 and TNF-alpha production, a result of tachyzoite-induced blockade of NF-kappaB nuclear translocation. Our results are the first to demonstrate the ability of an intracellular protozoan to actively interfere with the NF-kappaB activation pathway in macrophages, an activity that may enable parasite survival within the host.

CCAAT/enhancer-binding protein beta mediates interferon-gamma-induced p48 (ISGF3-gamma ) gene transcription in human monocytic cells.

Previous studies have identified a novel interferon-stimulated response element-like element, termed gamma-interferon-activating transcription element, within the interferon-stimulating gene factor-3gamma (p48) promoter region that is bound by novel transcription factors in response to stimulation with interferons (IFNs) (Weihua, X., Kolla, V., and Kalvakolanu, D. V. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 103-108). In the present study, we have identified CCAAT/enhancer-binding protein beta (C/EBP-beta) as one of the gamma-interferon-activating transcription element cognate transcription factors by screening a human monophage-derived cDNA library in a yeast one-hybrid system. Electrophoretic mobility shift assay studies suggest that C/EBP-beta dynamically regulates p48 gene expression upon IFN-gamma stimulation by undergoing changes in its heterodimerization partners. Transient transfection studies demonstrate that overexpression of C/EBP-beta strongly enhanced IFN-gamma-induced transcription from the p48 promoter. However, deletion mutants of C/EBP-beta that lack the N-terminal transactivation domain were unable to stimulate the p48 promoter. Western blotting revealed that C/EBP-beta is induced by IFN-gamma stimulation in THP-1-derived macrophages. Collectively, these results suggest that C/EBP-beta plays an important role in the human IFN-gamma signaling pathway by transcriptional regulation of p48 gene expression, an essential component in the IFN signaling pathway.

Insulin suppresses transactivation by CAAT/enhancer-binding proteins beta (C/EBPbeta). Signaling to p300/CREB-binding protein by protein kinase B disrupts interaction with the major activation domain of C/EBPbeta.

CAAT/enhancer-binding proteins (C/EBPs) play an important role in the regulation of gene expression in insulin-responsive tissues. We have found that a complex containing C/EBPbeta interacts with an insulin response sequence in the insulin-like growth factor-binding protein-1 (IGFBP-1) gene and that a C/EBP-binding site can mediate effects of insulin on promoter activity. Here, we examined mechanisms mediating this effect of insulin. The ability of insulin to suppress promoter activity via a C/EBP-binding site is blocked by LY294002, a phosphatidylinositol 3-kinase inhibitor, but not by rapamycin, which blocks activation of p70(S6 kinase). Dominant negative phosphatidylinositol 3-kinase and protein kinase B (PKB) block the effect of insulin, while activated PKB suppresses promoter function via a C/EBP-binding site, mimicking the effect of insulin. Coexpression studies indicate that insulin and PKB suppress transactivation by C/EBPbeta, but not C/EBPalpha, and that N-terminal transactivation domains in C/EBPbeta are required. Studies with Gal4 fusion proteins reveal that insulin and PKB suppress transactivation by the major activation domain in C/EBPbeta (AD II), located between amino acids 31 and 83. Studies with E1A protein indicate that interaction with p300/CBP is required for transactivation by AD II and the effect of insulin and PKB. Based on a consensus sequence, we identified a PKB phosphorylation site (Ser(1834)) within the region of p300/CBP known to bind C/EBPbeta. Mammalian two-hybrid studies indicate that insulin and PKB disrupt interactions between this region of p300 and AD II and that Ser(1834) is critical for this effect. Signaling by PKB and phosphorylation of Ser(1834) may play an important role in modulating interactions between p300/CBP and transcription factors and mediate effects of insulin and related growth factors on gene expression.

C/EBPbeta, when expressed from the C/ebpalpha gene locus, can functionally replace C/EBPalpha in liver but not in adipose tissue.

Knockout of C/EBPalpha causes a severe loss of liver function and, subsequently, neonatal lethality in mice. By using a gene replacement approach, we generated a new C/EBPalpha-null mouse strain in which C/EBPbeta, in addition to its own expression, substituted for C/EBPalpha expression in tissues. The homozygous mutant mice C/ebpalpha(beta/beta) are viable and fertile and show none of the overt liver abnormalities found in the previous C/EBPalpha-null mouse line. Levels of hepatic PEPCK mRNA are not different between C/ebpalpha(beta/beta) and wild-type mice. However, despite their normal growth rate, C/ebpalpha(beta/beta) mice have markedly reduced fat storage in their white adipose tissue (WAT). Expression of two adipocyte-specific factors, adipsin and leptin, is significantly reduced in the WAT of C/ebpalpha(beta/beta) mice. In addition, expression of the non-adipocyte-specific genes for transferrin and cysteine dioxygenase is reduced in WAT but not in liver. Our study demonstrates that when expressed from the C/ebpalpha gene locus, C/EBPbeta can act for C/EBPalpha to maintain liver functions during development. Moreover, our studies with the C/ebpalpha(beta/beta) mice provide new insights into the nonredundant functions of C/EBPalpha and C/EBPbeta on gene regulation in WAT.

Generation of truncated C/EBPbeta isoforms by in vitro proteolysis.

Multiple forms of the transcriptional regulator CCAAT/enhancer-binding protein beta (C/EBPbeta) with molecular masses of approximately 38, 34, 20, and 14 kDa have been observed in cell extracts. It has been proposed that these proteins arise by alternative initiation at in-frame AUG codons. The truncated C/EBPbeta isoforms (p14 and p20/LIP) lack transactivation domains but retain DNA-binding and dimerization sequences and are therefore assumed to function as competitive inhibitors of C/EBP-mediated transcription in vivo. By comparing various extraction procedures to analyze endogenous and overexpressed C/EBPbeta proteins, we determined that p20-C/EBPbeta is generated predominantly by in vitro proteolytic cleavage during isolation from cells and that p14-C/EBPbeta is produced exclusively by this mechanism. In transfected cells, the full-length (p34 and p38) isoforms but not the truncated proteins were detectable in the cytoplasm, indicating that the latter are not primary translation products. In addition, the C/EBPbeta leucine zipper dimerization domain was essential for the appearance of the truncated species, demonstrating that protein folding or dimerization are critical determinants of proteolytic sensitivity. Our findings suggest that the presence of truncated C/EBPbeta proteins in cell extracts must be interpreted with caution and that assumptions about the in vivo relevance of these isoforms should be re-evaluated.

The C/EBP bZIP domain can mediate lipopolysaccharide induction of the proinflammatory cytokines interleukin-6 and monocyte chemoattractant protein-1.

C/EBPalpha, beta, and delta are all expressed by bone marrow-derived 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/EBPalpha, beta, and delta are redundant in regard to expression of IL-6 and MCP-1. Surprisingly, the bZIP region of C/EBPbeta, 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/EBPbeta, C/EBPdelta, and, to a lesser extent, C/EBPalpha 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/EBPbeta bZIP region to activate the IL-6 promoter in transient transfections is completely dependent upon an intact NF-kappaB-binding site, supporting a model where the bZIP protein primarily functions to augment the activity of NF-kappaB. Replacement of the leucine zipper of C/EBPbeta 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.

C/EBPbeta modulates the early events of keratinocyte differentiation involving growth arrest and keratin 1 and keratin 10 expression.

The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that become postmitotic and undergo sequential changes in gene expression during terminal differentiation. The expression of the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) within mouse epidermis and primary keratinocytes has recently been described; however, the function of C/EBPbeta within the epidermal keratinocyte is unknown. We report here that transient transfection of mouse primary keratinocytes with a C/EBP-responsive promoter-reporter construct resulted in a sevenfold increase in luciferase activity when keratinocytes were switched to culture conditions that induce growth arrest and differentiation. Forced expression of C/EBPbeta in BALB/MK2 keratinocytes inhibited growth, induced morphological changes consistent with a more differentiated phenotype, and upregulated two early markers of differentiation, keratin 1 (K1) and keratin 10 (K10) but had a minimal effect on the expression of late-stage markers, loricrin and involucrin. Analysis of the epidermis of C/EBPbeta-deficient mice revealed a mild epidermal hyperplasia and decreased expression of K1 and K10 but not of involucrin and loricrin. C/EBPbeta-deficient primary keratinocytes were partially resistant to calcium-induced growth arrest. Analysis of terminally differentiated spontaneously detached keratinocytes or those induced to differentiate by suspension culture revealed that C/EBPbeta-deficient keratinocytes displayed striking decreases in K1 and K10, while expression of later-stage markers was only minimally altered. Our results demonstrate that C/EBPbeta plays an important role in the early events of stratified squamous differentiation in keratinocytes involving growth arrest and K1 and K10 expression.

CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene.

Two putative CCAAT/enhancer-binding protein (C/EBP) response elements were identified in the proximal promoter of the human steroidogenic acute regulatory protein (StAR) gene, which encodes a key protein-regulating steroid hormone synthesis. Expression of C/EBPalpha and -beta increased StAR promoter activity in COS-1 and HepG2 cells. Cotransfection of C/EBPalpha or -beta and steroidogenic factor 1, a transcription factor required for cAMP regulation of StAR expression, into COS-1 augmented 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP)-stimulated promoter activity. When the putative C/EBP response elements were mutated, individually or together, a pronounced decline in basal StAR promoter activity in human granulosa-lutein cells resulted, but the fold stimulation of promoter activity by 8-Br-cAMP was unaffected. Recombinant C/EBPalpha and -beta bound to the two identified sequences but not the mutated elements. Human granulosa-lutein cell nuclear extracts also bound these elements but not the mutated sequences. An antibody to C/EBPbeta, but not C/EBPalpha, supershifted the nuclear protein complex associated with the more distal element. The complex formed by nuclear extracts with the proximal element was not supershifted by either antibody. Western blot analysis revealed the presence of C/EBPalpha and C/EBPbeta in human granulosa-lutein cell nuclear extracts. C/EBPbeta levels were up-regulated 3-fold by 8-Br-cAMP treatment. Our studies demonstrate a role for C/EBPbeta as well as yet to be identified proteins, which can bind to C/EBP response elements, in the regulation of StAR gene expression and suggest a mechanism by which C/EBPbeta participates in the cAMP regulation of StAR gene transcription.

Autocrine signals control CCAAT/enhancer binding protein beta expression, localization, and activity in macrophages.

The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta, or NF-IL6) is expressed in macrophages, where it participates in lipopolysaccharide (LPS)-mediated induction of proinflammatory cytokine genes such as interleukin-6 (IL-6) and IL-1beta. We have identified activities in conditioned medium from a macrophage tumor cell line that regulates the expression, localization, and transcriptional activity of C/EBPbeta. One factor was shown to be tumor necrosis factor- (TNF-), which increased C/EBPbeta expression by a posttranscriptional mechanism. A second activity, designated autocrine macrophage factor (AMF), elicited a change in C/EBPbeta localization from a punctate nuclear staining pattern to diffuse nuclear distribution. The punctate form of C/EBPbeta correlated with increased susceptibility of this protein to cleavage by an endogenous protease during nuclear extract preparation. Conditioned medium stimulated the ability of C/EBPbeta to transactivate a reporter gene and activated the expression of two cytokine genes that are putative targets of C/EBPbeta. These observations suggest that diffuse distribution of C/EBPbeta in the nucleus corresponds to an activated form of this protein. AMF activity could not be mimicked by an extensive set of recombinant cytokines and growth factors and therefore may represent a novel extracellular factor.

Tumor necrosis factor alpha transcription in macrophages is attenuated by an autocrine factor that preferentially induces NF-kappaB p50.

Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), which are expressed during conditions of inflammation, infection, or injury. We identified an activity secreted by a macrophage tumor cell line that negatively regulates bacterial lipopolysaccharide (LPS)-induced expression of TNF-alpha. This activity, termed TNF-alpha-inhibiting factor (TIF), suppressed the induction of TNF-alpha expression in macrophages, whereas induction of three other proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-6, and monocyte chemoattractant protein 1) was accelerated or enhanced. A similar or identical inhibitory activity was secreted by IC-21 macrophages following LPS stimulation. Inhibition of TNF-alpha expression by macrophage conditioned medium was associated with selective induction of the NF-kappaB p50 subunit. Hyperinduction of p50 occurred with delayed kinetics in LPS-stimulated macrophages but not in fibroblasts. Overexpression of p50 blocked LPS-induced transcription from a TNF-alpha promoter reporter construct, showing that this transcription factor is an inhibitor of the TNF-alpha gene. Repression of the TNF-alpha promoter by TIF required a distal region that includes three NF-kappaB binding sites with preferential affinity for p50 homodimers. Thus, the selective repression of the TNF-alpha promoter by TIF may be explained by the specific binding of inhibitory p50 homodimers. We propose that TIF serves as a negative autocrine signal to attenuate TNF-alpha expression in activated macrophages. TIF is distinct from the known TNF-alpha-inhibiting factors IL-4, IL-10, and transforming growth factor beta and may represent a novel cytokine.

Selectively enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/enhancer binding protein delta.

CCAAT/enhancer binding protein delta (C/EBPdelta) is a transcriptional regulator implicated in the hepatic acute phase response and in adipogenic and myeloid cell differentiation. We found that C/EBPdelta is widely expressed in the peripheral and central nervous systems, including neurons of the hippocampal formation, indicating a role in neural functions. To examine the role of C/EBPdelta in vivo, we generated mice with a targeted deletion of the C/EBPdelta gene. This mutation does not interfere with normal embryonic and postnatal development. Performance in a battery of behavioral tests indicates that basic neurological functions are normal. Furthermore, performance in a Morris water maze task suggests that C/EBPdelta mutant mice have normal spatial learning. However, in the contextual and auditory-cue-conditioned fear task, C/EBPdelta null mice displayed significantly more conditioned freezing to the test context than did wild-type controls, but equivalent conditioning to the auditory cue. These data demonstrate a selectively enhanced contextual fear response in mice carrying a targeted genomic mutation and implicate C/EBPdelta in the regulation of a specific type of learning and memory.

beta-adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein delta.

Stimulation of beta-adrenergic receptors (BAR) by clenbuterol (CLE) increases nerve growth factor (NGF) biosynthesis in the rat cerebral cortex but not in other regions of the brain. We have explored the transcription mechanisms that may account for the cortex-specific activation of the NGF gene. Although the NGF promoter contains an AP-1 element, AP-1-binding activity in the cerebral cortex was not induced by CLE, suggesting that other transcription factors govern the brain area-specific induction of NGF. Because BAR activation increases cAMP levels, we examined the role of CCAAT/enhancer-binding proteins (C/EBP), some of which are known to be cAMP-inducible. In C6-2B glioma cells, whose NGF expression is induced by BAR agonists, (i) CLE increased C/EBPdelta-binding activity, (ii) NGF mRNA levels were increased by overexpressing C/EBPdelta, and (iii) C/EBPdelta increased the activity of an NGF promoter-reporter construct. Moreover, DNase footprinting and deletion analyses identified a C/EBPdelta site in the proximal region of the NGF promoter. C/EBPdelta appears to be responsible for the BAR-mediated activation of the NGF gene in vivo, since CLE elicited a time-dependent increase in C/EBPdelta-binding activity in the cerebral cortex only. Our data suggest that, while AP-1 may regulate basal levels of NGF expression, C/EBPdelta is a critical component determining the area-specific expression of NGF in response to BAR stimulation.

C/EBPepsilon is a myeloid-specific activator of cytokine, chemokine, and macrophage-colony-stimulating factor receptor genes.

C/EBPepsilon is a member of the CCAAT/enhancer binding protein family of basic region/leucine zipper transcriptional activators. The C/EBPepsilon protein is highly conserved between rodents and humans, and its domain structure is very similar to C/EBPalpha. In mice C/EBPepsilon mRNA is only detected in hematopoietic tissues, including embryonic liver and adult bone marrow and spleen. Within the hematopoietic system, C/EBPepsilon is expressed primarily in myeloid cells, including promyelocytes, myelomonocytes, and their differentiated progeny. To identify potential functions of C/EBPepsilon, cell lines over-expressing the C/EBPepsilon protein were generated in the P388 lymphoblastic cell line. In contrast to the parental cell line, C/EBPepsilon-expressing cell lines displayed lipopolysaccharide-inducible expression of the interleukin-6 and monocyte chemoattractant protein 1 (MCP-1) genes as well as elevated basal expression of the MIP-1alpha and MIP-1beta chemokine genes. In the EML-C1 hematopoietic stem cell line, C/EBPepsilon mRNA levels increased as the cells progressed along the myeloid lineage, just preceding activation of the gene encoding the receptor for macrophage-colony-stimulating factor (M-CSFR). M-CSFR expression was stimulated in C/EBPepsilon-expressing P388 cell lines, when compared with either the parental P388 cells or P388 cell lines expressing either C/EBPalpha or C/EBPbeta. These results suggest that C/EBPepsilon may be an important regulator of differentiation of a subset of myeloid cell types and may also participate in the regulation of cytokine gene expression in mature cells.

The C/EBPbeta transcription factor regulates epithelial cell proliferation and differentiation in the mammary gland.

Studies of C/EBPbeta-deficient mice have demonstrated a pivotal role for this transcription factor in hematopoiesis, adipogenesis, and ovarian function. Here we show that C/EBPbeta is also essential for normal development and function of the mammary gland. Ductal morphogenesis in virgin C/EBPbeta-deficient mice was disrupted, with ducts displaying reduced growth and branching. To distinguish whether the effect of C/EBPbeta deficiency on mammary epithelium is indirect or cell autonomous, we performed ovarian and mammary gland transplants. Transplants of wild-type ovaries into mutant females partially restored ductal morphogenesis during puberty but failed to support mammopoiesis during pregnancy. At term, mutant mice harboring wild-type ovaries exhibited reduced alveolar proliferation and impaired epithelial cell differentiation, including a complete absence of milk protein expression. Mammary gland transplant experiments demonstrated that development of C/EBPbeta-deficient epithelium was defective within a wild-type stroma and host background. Cell proliferation during pregnancy was reduced and differentiation, as measured by the activity of milk protein genes, was inhibited. However, wild-type epithelium developed in a C/EBPbeta-deficient stroma. Thus, C/EBPbeta plays an essential, cell autonomous role in the proliferation and differentiation of mammary secretory epithelial cells and is required for the activation of milk protein genes.

Inhibition of CCAAT/enhancer-binding protein alpha and beta translation by upstream open reading frames.

CCAAT/enhancer-binding protein (C/EBP) alpha is a bZIP transcription factor whose expression is restricted to specific cell types. Analysis of C/EBPalpha mRNA and protein levels in various mammalian cells indicates that expression of this gene is controlled both transcriptionally and post-transcriptionally. We report here that C/EBPalpha translation is repressed in several cell lines by an evolutionarily conserved upstream open reading frame (uORF), which acts in cis to inhibit C/EBPalpha translation. Mutations that disrupt the uORF completely abolished translational repression of C/EBPalpha. The related c/ebpbeta gene also contains an uORF that suppresses translation. The length of the spacer sequence between the uORF terminator and the ORF initiator codon (7 bases in all c/ebpalpha genes and 4 bases in c/ebpbeta homologs) is precisely conserved. The effects of insertions, deletions, and base substitutions in the C/EBPalpha spacer showed that both the length and nucleotide sequence of the spacer are important for efficient translational repression. Our data indicate that the uORFs regulate translation of full-length C/EBPalpha and C/EBPbeta and do not play a role in generating truncated forms of these proteins, as has been suggested by start site multiplicity models.

CCAAT/enhancer binding protein beta is a neuronal transcriptional regulator activated by nerve growth factor receptor signaling.

CCAAT/enhancer binding protein beta (C/EBPbeta) is a transcriptional regulator of the basic leucine zipper family. By in situ hybridization analysis, we found that C/EBPbeta is widely expressed in the CNS of adult mice, including cells of the hippocampus and dentate gyrus and cerebellar Purkinje and granule cells. Expression of C/EBPbeta had also been reported in the PC12 cell line, which undergoes differentiation to neuron-like cells in response to nerve growth factor (NGF). We show that C/EBPbeta mRNA expression increases while protein levels decrease during differentiation of PC12 cells. In transactivation assays, C/EBPbeta activity was stimulated by NGF receptor signaling. Mutations of a phosphorylation site for mitogen-activated protein kinase in C/EBPbeta affected its capacity to transactivate in a promoter-specific manner. Our data identify the C/EBPbeta protein and gene as direct downstream targets of the NGF receptor and suggest a role for C/EBPbeta in neurotrophin signaling in the brain.