Ectopic expression of C/EBPalpha in the lung epithelium disrupts late lung development. (65/177)

The lung develops from the endoderm through a process of branching morphogenesis. This process is highly active during the pseudoglandular stage of lung development and continues into the canalicular stage, resulting in the formation of terminal sacs. CCAAT/enhancer binding proteins (C/EBPs) are transcription factors regulating central aspects of differentiation and proliferation. We report here the developmental expression of C/EBPalpha, -beta, and -delta in the lung. C/EBPalpha exhibits a dynamic expression pattern and is first detected during the late pseudoglandular stage. At this stage, expression is observed in a subset of epithelial cells in the distal parts of the branching tubules. The expression of C/EBPalpha is confined to nonproliferating cells. To examine the role of C/EBPalpha in lung development, we generated transgenic mice ectopically expressing C/EBPalpha in the lung epithelium using the human surfactant protein C promoter. Lungs from these mice were of normal size but exhibited a phenotype characterized by fewer and larger developing epithelial tubules, indicating that the branching process was affected. No effects on overall proliferation or cellular differentiation were observed. When this phenotype was compared with that of mice carrying a targeted mutation of the Cebpa gene, the Cebpa-/- mice exhibited a similar developmental phenotype. In conclusion, our results show a role for C/EBPalpha in lung development and suggest a function in the later stages of lung branching morphogenesis.  (+info)

CCAAT/enhancer-binding protein delta activates the Runx2-mediated transcription of mouse osteocalcin II promoter. (66/177)

CCAAT/enhancer-binding proteins (C/EBPs) are involved in the regulation of cell proliferation, differentiation, and control of metabolic function. Although the roles of C/EBPs in osteoblasts are largely unknown, both C/EBPbeta and -delta have been shown to enhance rat osteocalcin promoter activity through the synergistic activation of Runx2 at the C/EBP element. Here we show that in the mouse, C/EBPdelta increases the expression of osteocalcin whereas C/EBPbeta does not. This increased expression was found to occur at the transcriptional level, as demonstrated by the increased transcriptional activity from mouse osteocalcin II (OG2) promoter by C/EBPdelta. Although we found three putative C/EBP sites in the -637/+/-34 region of the OG2 promoter, none of these sites showed binding activity with in vitro translated C/EBP proteins. Notably, we show that C/EBPdelta physically interacts with Runx2 and that C/EBPdelta overexpression increases binding between the Runx2-C/EBPdelta complex and the OSE2 element, a critical osteoblast-specific cis-acting element in the OG2 promoter. Consistent with these DNA binding data, a mutation in OSE2 abrogated the stimulatory effect of C/EBPdelta on this promoter activity. Finally, chromatin immunoprecipitation analysis in MC3T3-E1 cells showed in vivo occupancy of the OG2 promoter by Runx2 and C/EBPdelta. In conclusion, C/EBPdelta was found to regulate mouse osteocalcin OG2 promoter activity indirectly by interacting with Runx2 in the context of the OSE2 element and this subsequently resulted in the cooperative activation of the OG2 promoter.  (+info)

Decrease in circulating myeloid dendritic cell precursors in coronary artery disease. (67/177)

OBJECTIVES: We analyzed the frequency of myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC) precursors in blood of patients with coronary artery disease (CAD) and in atherosclerotic carotid plaques of patients with cerebrovascular disease (CVD). BACKGROUND: Circulating DC precursors are reduced in several autoimmune diseases. Atherosclerosis has features of an autoimmune disease, such as the presence of autoantibodies or autoreactive T cells. Tissue-resident DCs were previously described in atheromata, and it is assumed that they are important for the activation of T cells against autoantigens there. METHODS: Circulating mDC and pDC precursors were flow cytometrically detected in healthy controls (n = 19), CAD patients with stable (n = 20) and unstable angina pectoris (n = 19), and acute myocardial infarction (n = 17). In human carotid plaques (n = 65), mDC and pDC precursors were identified immunohistochemically. RESULTS: Circulating mDC precursors were significantly reduced in patients with stable angina pectoris (0.19%, p = 0.04), unstable angina pectoris (0.16%, p = 0.004), and acute myocardial infarction (0.08%, p < 0.001) compared with control patients (0.22% of peripheral blood mononuclear cells). In contrast, pDC numbers were not significantly altered. Circulating mDC precursors inversely correlated with high-sensitivity C-reactive protein (r = -0.38, p = 0.001) or interleukin-6 (r = -0.42, p < 0.001). In contrast to pDC, significantly more mDC precursors were observed in vulnerable carotid plaques (24, 0.25 mm2; n = 31; p = 0.003) than in stable ones (6.4, 0.25 mm2; n = 34). CONCLUSIONS: Similar to autoimmune diseases, circulating mDC precursors were significantly reduced in patients with CAD. The emergence of mDC precursors in vulnerable plaques suggests their recruitment into atheromata as a possible reason for their decrease in blood. In contrast, no significant association of circulating pDC precursors with atherosclerosis was observed.  (+info)

c/ebpdelta Null mouse as a model for the double knock-out of slc5a8 and slc5a12 in kidney. (68/177)

slc5a8 and slc5a12 represent the high affinity and low affinity Na+/lactate co-transporters, respectively, in the kidney. Here we show that these transporters are expressed in the apical membrane of the proximal tubular cells in mouse kidney, indicating that these transporters are likely to mediate the first step in the renal reabsorption of lactate. Interestingly, the renal expression of both transporters is almost completely ablated in mice homozygous for the deletion of the transcription factor c/ebpdelta. This effect is tissue-specific since the expression of the transporters is not affected in non-renal tissues. The functional role of C/EBPdelta in the expression of SLC5A8 and SLC5A12 is demonstrable in HEK293 cells in reporter assays using gene-specific promoters. The ablation of the transporters in the kidney is accompanied by a marked increase in urinary excretion of lactate as well as a decrease in blood levels of lactate in c/ebpdelta-/- mice. These data provide evidence for an obligatory role for slc5a8 and slc5a12 in the renal absorption of lactate. In addition, we show that urinary excretion of urate is significantly elevated in c/ebpdelta-/- mice even though the expression of URAT1, the transporter responsible for the apical membrane uptake of urate in renal proximal tubule, is not altered. These data provide in vivo evidence for the functional coupling between lactate reabsorption and urate reabsorption in the kidney. Thus, the fortuitous double knock-out of slc5a8 and slc5a12 in kidney in c/ebpdelta-/- mice reveals the physiologic role of these transporters in the renal handling of lactate and urate.  (+info)

IL-1 and IL-6 induce hepatocyte plasminogen activator inhibitor-1 expression through independent signaling pathways converging on C/EBPdelta. (69/177)

To elucidate signaling pathways activated by IL-1 and IL-6 that contribute to increased expression of plasminogen activator inhibitor-1 (PAI-1), we studied human hepatoma (HepG2) cells and primary mouse hepatocytes. HepG2 cell PAI-1 mRNA increased in response to IL-1beta, IL-6, and IL-1beta plus IL-6 as shown by real-time PCR. Activity of the transiently transfected PAI-1 promoter (-829 to +36 bp) increased as well. Systematic promoter deletion assays showed that the region from -239 to -210 bp containing a putative CCAAT-enhancer binding protein (C/EBP) binding site was critical. Point mutations in this region abolished the IL-1beta and IL-6 responses. Antibody interference electrophoretic mobility shift assays showed that C/EBPdelta (but not C/EBPalpha or C/EBPbeta) binding and protein were increased by IL-1beta, IL-6, and IL-1beta plus IL-6 in HepG2 cells. IL-1beta and IL-6 increased expression of both PAI-1 mRNA and C/EBPdelta mRNA in mouse primary hepatocytes as well. Downregulation of C/EBPdelta induced with small interfering RNA (siRNA) decreased secretion of PAI-1. As judged from results obtained with inhibitors, signal transduction in all three of the mitogen-activated protein kinase pathways was involved in IL-1-inducible PAI-1 expression. By contrast, JAK signaling was responsible for the IL-6-induced inducible expression. Thus IL-1 and IL-6 exert directionally similar effects on PAI-1 expression, but the induction involves distinct signaling pathways with a final common mediator, C/EBPdelta.  (+info)

Characterization of the transcriptional and functional effects of fibroblast growth factor-1 on human preadipocyte differentiation. (70/177)

We recently established that fibroblast growth factor (FGF)-1 promotes adipogenesis of primary human preadipocytes (phPA). In the current report, we have characterized the adipogenic effects of FGF-1 in phPA and also in a human PA strain derived from an individual with Simpson-Golabi-Behmel syndrome (SGBS PA), which exhibit an intrinsic capacity to differentiate with high efficiency. In further studies, we compared these models with the well-characterized murine 3T3-L1 preadipocyte cell line (3T3-L1 PA). FGF-1 up-regulated the adipogenic program in phPA, with increased expression of peroxisome proliferator-activated receptor-gamma in confluent PA prior to induction of differentiation and increased expression of adipocyte markers during differentiation. Moreover, phPA differentiated in the presence of FGF-1 were more insulin responsive and secreted increased levels of adiponectin. FGF-1 treatment of SGBS PA further enhanced differentiation. For the most part, the adipogenic program in phPA paralleled that observed in 3T3-L1 PA; however, we found no evidence of mitotic clonal expansion in the phPA. Finally, we investigated a role for extracellular regulated kinase 1/2 (ERK1/2) in adipogenesis of phPA. FGF-1 induced robust phosphorylation of ERK1/2 in early differentiation and inhibition of ERK1/2 activity significantly reduced phPA differentiation. These data suggest that FGF-1 treated phPA represent a valuable in vitro model for the study of adipogenesis and insulin action and indicate that ERK1/2 activation is necessary for human adipogenesis in the absence of mitotic clonal expansion.  (+info)

Pneumococcal peptidoglycan-polysaccharides induce the expression of interleukin-8 in airway epithelial cells by way of nuclear factor-kappaB, nuclear factor interleukin-6, or activation protein-1 dependent mechanisms. (71/177)

Cell envelope compounds of bacteria trigger immune and inflammatory reactions by way of chemokines/cytokines. In this study, we demonstrated that pneumococcal peptidoglycan-polysaccharides (PGPS) induced the production of interleukin (IL)-8 by way of nuclear factor (NF)-kappaB, nuclear factor interleukin (NF-IL)6, and activation protein (AP)-1 dependent mechanisms in the human bronchial epithelial cells (NL-20) in a dose- and time-dependent manner in vitro, and the mutation of either the NF-kappaB, NF-IL6, or AP-1 binding sites in the promoter of IL-8 abrogated the IL-8 transcriptional activity. In a similar way, lipopolysaccharides induced the promoter activation of IL-8 in NL-20. However, the PGPS-induced IL-8 promoter activation in rodent middle ear epithelial cells required NF-kappaB and NF-IL6 but not AP-1.  (+info)

Molecular characterization of transcription factors that bind to the cAMP responsive region of the substance P precursor gene. cDNA cloning of a novel C/EBP-related factor. (72/177)

A cAMP response element (CRE) plays an important role in the cAMP-mediated gene regulation. Several factors that recognize a CRE have been characterized, and it has been shown that they need either covalent modification by protein kinase A or a cofactor such as the adenovirus Ela to function as an activator. In this study we show that the substance P precursor gene expression is regulated by protein kinase A and identify the CRE sequence in its promoter region. We find that a novel factor and ATF2 bind to the region containing the CRE of the substance P precursor gene. The sequence analysis indicates that the novel protein, designated CELF, has a significant homology to C/EBP gene family proteins in the carboxyl-terminal part containing the basic region and the leucine zipper motif. Ubiquitous expression of CELF suggests that this factor is utilized by various genes. Cell-free transcription analyses indicate that CELF is a constitutive transcriptional activator without apparent phosphorylation by protein kinase A. These results demonstrate that multiple factors are responsible for transcriptional control of the substance P precursor gene through the CRE region.  (+info)