CCAAT/enhancer-binding Protein Delta Research Articles

CCAAT/enhancer binding protein delta (CEBPD) elevating PTX3 expression inhibits macrophage-mediated phagocytosis of dying neuron cells

The CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ, NF-IL6β) is induced in many inflammation-related diseases, suggesting that CEBPD and its downstream targets may play central roles in these conditions. Neuropathological studies show that a neuroinflammatory response parallels the early stages of Alzheimer's disease (AD). However, the precise mechanistic correlation between inflammation and AD pathogenesis remains unclear. CEBPD is upregulated in the astrocytes of AD patients. Therefore, we asked if activation of astrocytic CEBPD could contribute to AD pathogenesis. In this report, a novel role of CEBPD in attenuating macrophage-mediated phagocytosis of damaged neuron cells was found. By global gene expression profiling, we identified the inflammatory marker pentraxin-3 (PTX3, TNFAIP5, TSG-14) as a CEBPD target in astrocytes. Furthermore, we demonstrate that PTX3 participates in the attenuation of macrophage-mediated phagocytosis of damaged neuron cells. This study provides the first demonstration of a role for astrocytic CEBPD and the CEBPD-regulated molecule PTX3 in the accumulation of damaged neurons, which is a hallmark of AD pathogenesis.

Minireview: Pref-1: Role in Adipogenesis and Mesenchymal Cell Fate

Preadipocyte factor-1 [Pref-1; also called Dlk1 (Delta-like protein 1)] is made as an epidermal growth factor-repeat containing transmembrane protein that produces a biologically active soluble form by TNF-alpha-converting enzyme (TACE)-mediated cleavage. Soluble Pref-1 activates the MAPK kinase/ERK pathway. In adipose tissue, Pref-1 is specifically expressed in preadipocytes but not in adipocytes and thus is used as a preadipocyte marker. Inhibition of adipogenesis by Pref-1 has been well established in vitro as well as in vivo by ablation and overexpression of Pref-1. SRY (sex determining region Y)-box 9 (Sox9), a transcription factor expressed in preadipocytes to suppress CCAAT enhancer binding protein beta and (C/EBP) delta expression, is required to be down-regulated before adipocyte differentiation can proceed. By activating MAPK kinase/ERK, Pref-1 prevents down-regulation of Sox9, resulting in inhibition of adipogenesis. Furthermore, by inducing Sox9, Pref-1 promotes chondrogenic induction of mesenchymal cells but prevents chondrocyte maturation as well as osteoblast differentiation. Thus, Pref-1 directs multipotent mesenchymal cells toward the chondrogenic lineage but inhibits differentiation into adipocytes as well as osteoblasts and chondrocytes. Pref-1, encoded by an imprinted gene, has also been detected in progenitor cells in various tissues during regeneration and therefore may have a more general role in maintaining cells in an undifferentiated state.

Acute Lymphoblastic Leukemia (ALL) with t(8;14)(q11.2;q32): B-Lineage Disease with High Proportion of Down Syndrome. A Children's Oncology Group (COG) Study.

Recurrent chromosome translocations play a critical role in the pathogenesis of ALL and many translocations have important prognostic significance. The t(8;14)(q11.2;q32) is a recurrent translocation that fuses the chromosome 8 CEBPD (CCAAT enhancer binding protein delta) gene to the IgH (immunoglobulin heavy chain) gene, leading most likely to deregulated CEPD expression. We previously reported the clinical data of 10 such patients (Leukemia , 2000; 14:238–240). Using the COG ALL cytogenetics database we expand the report to include 22 patients (13 females and 9 males) with the t(8;14)(q11.2;q32). All patients with available immunophenotyping data (n=20) had B-lineage ALL. The median age at diagnosis was 9.2 years (range: 1.6 months – 17.1 years). Median diagnostic white blood cell count (WBC) was 9,950 (range: 700–172,000). Of the 22 patients, 7 were NCI standard risk and 15 were high risk. The most common additional cytogenetic abnormality was trisomy 21. Of the ten cases with trisomy 21, seven were constitutional (Down Syndrome), thus confirming previous reports that a significant fraction of t(8;14) patients (7/22; 31.8%) have Down Syndrome, which is much higher than the 3% (80/2811) overall rate of children with Down Syndrome in a recent COG ALL trial (p<0.0001). In addition to the Down Syndrome cases, one case with phenotypic Turner syndrome had a mosaic constitutional r(Y)(p11q11.2); thus, eight cases (36%) had constitutional abnormalities. Secondary abnormalities included additional X (n=5); additional 5 (n=3); Philadelphia chromosome (n=1). Two cases had a second der(14)t(8;14) and two had loss of the der(8) t(8;14), consistent with the der(14)t(8;14) as the significant abnormality. Four cases (18%) had 9p deletions compared with 11% in the overall ALL population, and two cases (9%) had abnormalities of 13q compared with 2% in the overall population. Numerically, the cases were pseudodiploid (n=12) and hyperdiploid (n=10). Children with Down Syndrome had superior estimated 5-year event-free survival of 100%, compared to non-Down patients with 50.1±17.7% (p=0.04). Overall survival was also different but did not reach statistical significance: 100% vs. 60.9±17.0% (p=0.088). In summary, the rare t(8;14)(q11.2;q32) is associated with B-lineage phenotype and occurs with greatly increased frequency in children with Down Syndrome, who have an excellent outcome with standard COG therapy as compared to non-Down Syndrome patients with this translocation.

Temporal gene expression profiling reveals CEBPD as a candidate regulator of brain disease in prosaposin deficient mice

BackgroundProsaposin encodes, in tandem, four small acidic activator proteins (saposins) with specificities for glycosphingolipid (GSL) hydrolases in lysosomes. Extensive GSL storage occurs in various central nervous system regions in mammalian prosaposin deficiencies.ResultsOur hypomorphic prosaposin deficient mouse, PS-NA, exhibited 45% WT levels of brain saposins and showed neuropathology that included neuronal GSL storage and Purkinje cell loss. Impairment of neuronal function was observed as early as 6 wks as demonstrated by the narrow bridges tests. Temporal transcriptome microarray analyses of brain tissues were conducted with mRNA from three prosaposin deficient mouse models: PS-NA, prosaposin null (PS-/-) and a V394L/V394L glucocerebrosidase mutation combined with PS-NA (4L/PS-NA). Gene expression alterations in cerebrum and cerebellum were detectable at birth preceding the neuronal deficits. Differentially expressed genes encompassed a broad spectrum of cellular functions. The number of down-regulated genes was constant, but up-regulated gene numbers increased with age. CCAAT/enhancer-binding protein delta (CEBPD) was the only up-regulated transcription factor in these two brain regions of all three models. Network analyses revealed that CEBPD has functional relationships with genes in transcription, pro-inflammation, cell death, binding, myelin and transport.ConclusionThese results show that: 1) Regionally specific gene expression abnormalities precede the brain histological and neuronal function changes, 2) Temporal gene expression profiles provide insights into the molecular mechanism during the GSL storage disease course, and 3) CEBPD is a candidate regulator of brain disease in prosaposin deficiency to participate in modulating disease acceleration or progression.

Ultraviolet radiation (UVR) activates p38 MAP kinase and induces post‐transcriptional stabilization of the C/EBPδ mRNA in G0 growth arrested mammary epithelial cells

The G(0) growth arrest (quiescent) state is highly conserved in evolution to promote survival under adverse environmental conditions. To maintain viability, G(0) growth arrested cells limit gene expression to essential growth control and pro-survival genes. CCAAT enhancer binding protein delta (C/EBPdelta), a member of the C/EBP family of nuclear proteins, is highly expressed in G(0) growth arrested mammary epithelial cells (MECs). Although C/EBPdelta gene transcription is elevated during G(0) growth arrest, C/EBPdelta mRNA and protein are relatively short lived, suggesting tight control of the cellular C/EBPdelta content in unstressed, quiescent cells. Treatment of G(0) growth arrested MECs with ultraviolet radiation (UVR) dramatically increases the C/EBPdelta mRNA half-life (approximately 4-fold) and protein content (approximately 3-fold). The mRNA stabilizing effects of UVR treatment are mediated by the C/EBPdelta mRNA 3'untranslated region, which contains an AU rich element. UVR increased p38 MAP kinase (MAPK) activation and SB203580, a p38 MAPK inhibitor, blocked UVR-induced C/EBPdelta mRNA stabilization. UVR increased the nuclear to cytoplasmic translocation of HuR, an ARE-binding protein that functions in mRNA stabilization. Finally, HuR siRNA treatment blocked UVR-induced stabilization of the C/EBPdelta and C/EBPbeta mRNAs but had no effect on C/EBPzeta (CHOP) mRNA stability. In summary, G(0) growth arrested MECs respond to UVR treatment by activating p38 MAPK, increasing HuR translocation and HuR/C/EBPdelta mRNA binding and stabilizing the C/EBPdelta mRNA. These results identify post-transcriptional stabilization of the C/EBPdelta mRNA as a mechanism to increase C/EBPdelta levels in the stress response of quiescent cells to UVR.

The Genes Induced by Signal Transducer and Activators of Transcription (STAT)3 and STAT5 in Mammary Epithelial Cells Define the Roles of these STATs in Mammary Development

Prolactin and leukemia inhibitory factor (LIF) have different roles in the adult mammary gland, which are mediated in part by the signal transducers and activators of transcription (STAT)5 and STAT3. In vivo studies have shown that STAT5 contributes to prolactin-dependent lobuloalveolar development and lactation whereas STAT3 mediates LIF-dependent epithelial apoptosis during postlactational involution. To understand the molecular basis of these STAT-dependent pathways, we demonstrate the ligand-independent effects of STAT5 and STAT3 in mammary epithelial cells in vitro and also identify the genes regulated by these related transcription factors. Thus, using conditionally active STAT3- or STAT5a-GyraseB fusion proteins, we observed that enforced and specific dimerization of STAT3 induced apoptosis whereas STAT5 induced differentiation of mammary epithelial cells. Furthermore, STAT5 attenuated apoptosis mediated by LIF, the physiological inducer of STAT3. Microarray analysis of STAT3- and STAT5-induced genes using this system demonstrated a marked specificity, which reflected their different physiological effects in vitro and in vivo. STAT5-specific gene targets included the milk protein genes alpha-casein and kallikrein-8 and the survival factors prosaposin and Grb10. STAT3-specific genes included the apoptosis regulators CCAAT enhancer binding protein-delta, phosphatidylinositol 3-kinase-regulatory subunits, purine nucleoside phosphorylase, and c-fos. These data illustrate that specific activation of STAT3 and STAT5 alone is sufficient to induce and suppress apoptosis, respectively, and that these transcription factors elicit their actions by inducing distinct subsets of target genes in mammary epithelial cells.

CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: I. "Loss of function" alterations in the c/EBPdelta growth inhibitory pathway in breast cancer cell lines.

"Loss of function" alterations in growth inhibitory signal transduction pathways are common in cancer cells. In this study, we show that growth arrest (GA) treatments--serum and growth factor withdrawal and growth inhibitory IL-6 family cytokines (Interleukin-6 and Oncostatin M (OSM))--increase STAT3 phosphorylation (pSTAT3), increase CCAAT enhancer binding protein delta (C/EBPdelta) gene expression and induce GA of primary, finite-lifespan human mammary epithelial cells (HMECs), and immortalized breast cell lines (MCF-10A and MCF-12A). In contrast, serum and growth factor withdrawal from human breast cancer cell lines (MCF-7, SK-BR-3, T-47D, and MDA-MB-231) for up to 48 h induced a relatively modest increase in pSTAT3 levels and C/EBPdelta gene expression and resulted in varying levels of GA. In most breast cancer cell lines, IL-6 family cytokine treatment increased pSTAT3 levels and C/EBPdelta gene expression, however, growth inhibition was cell line dependent. In addition to "loss of function" alterations in growth inhibitory pathways, breast cancer cell lines also exhibit "gain of function" alterations in growth signaling pathways. The Akt growth/ survival pathway is constitutively activated in T-47D and MCF-7 breast cancer cells. The Akt inhibitor LY 294,002 significantly enhanced T-47D growth inhibition by serum and growth factor withdrawal or IL-6 family cytokine treatment. Finally, we show that activation of the pSTAT3/C/EBPdelta growth control pathway is independent of estrogen receptor status. These results demonstrate that "loss of function" alterations in the pSTAT3/C/EBPdelta growth inhibitory signal transduction pathway are relatively common in human breast cancer cell lines. Defective activation of the pSTAT3/ C/EBPdelta growth inhibitory signal transduction pathway, in conjunction with constitutive activation of the Akt growth stimulatory pathway, may play a synergistic role in the etiology or progression of breast cancer.

Gene expression profiling identifies a unique androgen-mediated inflammatory/immune signature and a PTEN (phosphatase and tensin homolog deleted on chromosome 10)-mediated apoptotic response specific to the rat ventral prostate.

Understanding androgen regulation of gene expression is critical for deciphering mechanisms responsible for the transition from androgen-responsive (AR) to androgen-independent (AI) prostate cancer (PCa). To identify genes differentially regulated by androgens in each prostate lobe, the rat castration model was used. Microarray analysis was performed to compare dorsolateral (DLP) and ventral prostate (VP) samples from sham-castrated, castrated, and testosterone-replenished castrated rats. Our data demonstrate that, after castration, the VP and the DLP differed in the number of genes with altered expression (1496 in VP vs. 256 in DLP) and the nature of pathways modulated. Gene signatures related to apoptosis and immune response specific to the ventral prostate were identified. Microarray and RT-PCR analyses demonstrated the androgen repression of IGF binding protein-3 and -5, CCAAT-enhancer binding protein-delta, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) genes, previously implicated in apoptosis. We show that PTEN protein was increased only in the luminal epithelial cells of the VP, suggesting that it may be a key mediator of VP apoptosis in the absence of androgens. The castration-induced immune/inflammatory gene cluster observed specifically in the VP included IL-15 and IL-18. Immunostaining of the VP, but not the DLP, showed an influx of T cells, macrophages, and mast cells, suggesting that these cells may be the source of the immune signature genes. Interestingly, IL-18 was localized mainly to the basal epithelial cells and the infiltrating macrophages in the regressing VP, whereas IL-15 was induced in the luminal epithelium. The VP castration model exhibits immune cell infiltration and loss of PTEN that is often observed in progressive PCa, thereby making this model useful for further delineation of androgen-regulated gene expression with relevance to PCa.

Therapeutic effect of all-trans retinoic acid on rats with anti-GBM antibody glomerulonephritis

All-trans retinoic acid (ATRA) has antiproliferative and anti-inflammatory effects and is currently used in the treatment of leukemia and dermatologic diseases. We tested the therapeutic potential of ATRA on anti-glomerular basement membrane (GBM) glomerulonephritis rats. Glomerulonephritis was induced in male Wistar-Kyoto rats on day 0 by an intravenous injection of antirat GBM antibody. On day 14 after the induction of anti-GBM glomerulonephritis, some rats were sacrificed (N = 5). Another 10 rats were divided into two groups: the vehicle group (N = 5) and the ATRA treated group (N = 5). ATRA was orally administrated from day 14 to day 27 after disease induction. Blood pressure, body weight, urinary protein excretion, and blood chemistry was determined on days 1, 14, 21, and 27. Kidney samples were obtained on day 28. The kidneys were examined with periodic acid-Schiff staining (PAS) and immunohistochemistry using antibodies against the proliferative cell nuclear antigen (PCNA), rat monocyte and macrophage (ED-1), and alpha-smooth muscle actin (alpha-SMA). Glomerular RNA was extracted from isolated glomeruli, and reverse transcription (RT) followed by polymerase chain reaction (PCR) was performed. ATRA administration produced a 55% reduction of proteinuria in glomerulonephritis rats. Light microscopic analysis revealed severe necrosis/crescent formation (>50% of the glomerulus) affecting 34% of glomeruli in vehicle rats, whereas ATRA treatment reduced the glomeruli showing severe change to 14%. ATRA also significantly reduced PCNA-positive cells, ED-1-positive cells and alpha-SMA-positive area in the glomeruli. RT-PCR analyses revealed that a wide variety of genes including inflammation related [tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and CCAAT enhancer-binding protein delta (C/EBPdelta)], cell proliferation-related [platelet-derived growth factor (PDGF)] and fibrosis-related [transforming growth factor-beta1 (TGF-beta1), type I collagen, and alpha-SMA) genes were suppressed in the glomeruli of ATRA-treated rats. ATRA administration significantly reduced severe necrosis/crescent formation and urinary protein excretion in glomerulonephritis rats. Suppression of a wide variety of gene expression may partly explain the mechanism of ATRA's antiproliferative and anti-inflammatory effects. These data suggest a novel therapeutic application of ATRA toward glomerulonephritis.

Notch 1 impairs osteoblastic cell differentiation.

Notch receptors are single pass transmembrane receptors activated by membrane-bound ligands with a role in cell proliferation and differentiation. As Notch 1 and 2 mRNAs are expressed by osteoblasts and induced by cortisol, we postulated that Notch could regulate osteoblastogenesis. We investigated the effects of retroviral vectors directing the constitutive expression of the Notch 1 intracellular domain (NotchIC) in murine ST-2 stromal and in MC3T3 cells. NotchIC overexpression was documented by increased Notch 1 transcripts and activity of the Notch-dependent Hairy Enhancer of Split promoter. In the presence of bone morphogenetic protein-2 (BMP-2), ST-2 cells differentiated toward osteoblasts forming mineralized nodules, and Notch 1 opposed this effect and decreased the expression of osteocalcin, type I collagen, and alkaline phosphatase transcripts and Delta2Delta FosB protein. Further, NotchIC decreased Wnt/beta-catenin signaling. As cells differentiated in the presence of BMP-2, they underwent apoptosis, and Notch opposed this event. In the presence of cortisol, NotchIC induced the formation of mature adipocytes and enhanced the effect of cortisol on adipsin, peroxisome proliferator-activated receptor-gamma2 and CCAAT enhancer binding protein alpha and delta mRNA levels. NotchIC also opposed MC3T3 cell differentiation and the expression of a mature osteoblastic phenotype. In conclusion, NotchIC impairs osteoblast differentiation and enhances adipogenesis in stromal cell cultures.

Profound molecular changes following hippocampal slice preparation: loss of AMPA receptor subunits and uncoupled mRNA/protein expression.

The acute hippocampal slice preparation is a convenient, in vitro model widely used to study the biological basis of synaptic plasticity. Although slices may preserve their electrophysiological properties for several hours, profound molecular changes in response to the injury caused by the slicing procedure are likely to occur. To determine the magnitude and duration of these changes we examined the post-slicing expression kinetics of three classes of genes known to be implicated in long-term synaptic plasticity: glutamate AMPA receptors (GluR), transcription factors and neurotrophins. Slicing resulted in a striking loss of GluR1 and GluR3, but not of GluR2 proteins suggesting that rapid changes in the composition of major neurotransmitter receptors may occur. Slicing caused a significant induction of the transcription factors c-fos, zif268, CCAAT enhancer binding protein (C/EBP ) beta and delta mRNAs and of the neurotrophin brain-derived neurothophic factor (BDNF ) mRNA. In contrast, there was no augmentation, and sometimes a decline, in the levels of the corresponding proteins. These data reveal that significant discrepancies exist between the slice preparation and the intact hippocampus in terms of the metabolism of molecular components known to be involved in synaptic plasticity.

Differential expression of CCAAT enhancer binding protein family in rat alveolar epithelial cell proliferation and in acute lung injury

Although alveolar reorganization after acute lung injury depends on regeneration of alveolar epithelial cells, there is little knowledge of regulation of pulmonary healing process. Transcription factors may play key roles in this regulation. To investigate whether the CCAAT enhancer binding protein (C/EBP) family, alpha, beta, and delta, were involved in alveolar reorganization after injury, we examined expression of C/EBP proteins and mRNAs in lung injuries induced by lipopolysaccharide (LPS) or bleomycin (Bleo) and in cell proliferation by keratinocyte growth factor (KGF). By immunohistochemistry, we demonstrated that C/EBP alpha and C/EBP beta were expressed in alveolar type II cells and alveolar macrophages, but C/EBP delta was expressed restrictedly in some of alveolar type II cells in a spatial pattern in the control lungs. Further, these three C/EBP family members were differentially expressed in alveolar cell proliferation and in acute lung injury, in which, interestingly, C/EBP alpha and C/EBP delta were reciprocally expressed in alveolar type II cell proliferation and in pulmonary fibrosis. However, expressions of their mRNAs by in situ hybridization were dramatically increased in the affected lesions of the lungs by LPS and Bleo, and Northern blot analysis showed an increased abundance of the mRNA for C/EBP beta in LPS-treated lungs and for C/EBP delta in Bleo-treated lungs, compared with those in the control lungs. Thus, differential expression of the C/EBP family may be required to maintain and reorganize the basic integrity of alveolar structure during pathological states, which suggests an important role for the C/EBP family in maintaining normal alveolar architecture and function and in repairing the damaged epithelium after injury.

B2 Molecular structure and function of rat CCAAT-enhancer binding protein-delta gene promoter

B2 Molecular structure and function of rat CCAAT-enhancer binding protein-delta gene promoter

Molecular Structure and Function of Rat CCAAT-Enhancer Binding Protein-Delta Gene Promoter

CCAAT-enhancer binding protein-delta (C/EBPδ) is a transcriptional nuclear factor, and belongs to basic region-leucine zipper class DNA binding proteins. One genomic clone containing a 12-kb sequence of the C/EBPδ gene was isolated from a rat genomic library, and a 2,056-bp fragment containing the 5′-flanking region was characterized. Sequence analysis of this fragment revealed that there were a TATA-like sequence (TAGAAAA) and many transcriptional regulatory elements. The transcription start site of the gene was determined by both primer extension analysis and riboprobe mapping. Both analyses indicated that the transcription start site was located at 31-bp downstream of the TATA-like sequence. Transient transfection experiments showed that the fragment cloned in this study was able to act as a functional promoter in rat vascular smooth muscle cells. The 5′-deletion analysis of this fragment revealed that the sequence spanning −235 through −82, which was designated as an upstream control element (UCE), remarkably increased a basal promoter activity of the C/EBPδ gene, and was also able to act as a promoter by itself. In addition, we also studied effects of the UCE on the heterologous gene promoter including rat α-actin gene promoter or SV40 virus promoter. Interestingly, the UCE specifically increased the promoter activity of the rat α-actin gene suggesting that the C/EBPδ gene may be positively controlled by the UCE via a cell-type or promoter-type specific manner.

Transcriptional Regulation of Human Prostaglandin-endoperoxide Synthase-2 Gene by Lipopolysaccharide and Phorbol Ester in Vascular Endothelial Cells: INVOLVEMENT OF BOTH NUCLEAR FACTOR FOR INTERLEUKIN-6 EXPRESSION SITE AND cAMP RESPONSE ELEMENT

There exist two distinct isozymes of prostaglandin-endoperoxide synthase (PES). PES-2 mRNA is synergistically induced by lipopolysaccharide (LPS) and 12-O-tetradecanoylphorbol-13-acetate (TPA) in bovine arterial endothelial cells. On the other hand, PES-1 mRNA is constitutively expressed under these conditions. Therefore, the promoter activities of the human genes for PES-1 and -2 in bovine arterial endothelial cells were examined. The 5'-flanking region of the human PES-2 gene (nucleotides -327 to +59) showed promoter activity inducible by LPS and TPA using transient transfection analysis, whereas that of the PES-1 gene (nucleotides -1010 to +69) showed constitutive promoter activity. Destruction of both consensus sequences for the nuclear factor responsible for the interleukin-6 expression (NF-IL6) site (nucleotides -132 to -124) and the cyclic AMP response element (CRE) (nucleotides -59 to -53) of the human PES-2 gene markedly reduced the promoter activity (25%) of the PES-2 gene after combined treatment with LPS and TPA, although single destruction of the NF-IL6 site or the CRE slightly reduced the promoter activity (60 or 90%, respectively). Moreover, cotransfection experiments showed that a trans-acting factor, CCAAT enhancer binding protein delta (C/EBP delta), which binds to both the NF-IL6 site and the CRE, increased the promoter activity of the PES-2 gene mainly through the CRE. C/EBP delta mRNA was rapidly induced by LPS. Collectively, these results suggest that transcription of the PES-2 gene in vascular endothelial cells is regulated through combination of the NF-IL6 site and the CRE and that C/EBP delta functions as one of the trans-acting factors.