C5a-regulated CCAAT/enhancer-binding proteins β and δ are essential in Fcγ receptor-mediated inflammatory cytokine and chemokine production in macrophages

CCAAT/enhancer-binding protein β (C/EBPβ) and C/EBPδ are known to participate in the regulation of many genes associated with inflammation. However, little is known about the activation and function of C/EBPβ and -δ in inflammatory responses elicited by Fcγ receptor (FcγR) activation. Here we show that C/EBPβ and -δ activation are induced in IgG immune complex (IC)-treated macrophages. The increased expression of C/EBPβ and -δ occurred at both mRNA and protein levels. Furthermore, induction of C/EBPβ and -δ was mediated, to a large extent, by activating FcγRs. Using siRNA-mediated knockdown as well as macrophages deficient for C/EBPβ and/or -δ, we demonstrate that C/EBPβ and -δ play a critical role in the production of TNF-α, MIP-2, and MIP-1α in IgG IC-stimulated macrophages. Moreover, both ERK1/2 and p38 MAPK are involved in C/EBP induction and TNF-α, MIP-2, and MIP-1α production induced by IgG IC. We provide the evidence that C5a regulates IgG IC-induced inflammatory responses by enhancing ERK1/2 and p38 MAPK activities as well as C/EBPβ and -δ activities. Collectively, these data suggest that C/EBPβ and -δ are key regulators for FcγR-mediated induction of cytokines and chemokines in macrophages. Furthermore, C/EBPs may play an important regulatory role in IC-associated inflammatory responses.     

Role of C/EBPβ-LAP and C/EBPβ-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes

We investigated the role of the major isoforms of CCAAT enhancer binding protein β (C/EBPβ), C/EBPβ-LAP and C/EBPβ-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPβ gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPβ mRNA and protein levels declined. The C/EBPβ-LIP protein steady-state level decreased considerably stronger than the C/EBPβ-LAP level and the C/EBPβ-LIP half-life was significantly shorter than the C/EBPβ-LAP half-life. The turn-over of both C/EBPβ-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPβ-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPβ-LIP had antiadipogenic activity in human ASC. C/EBPβ-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPβ-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPβ-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPβ gene expression and C/EBPβ-LIP and C/EBPβ-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of human ASC and at later stages in human immature adipocytes.     

Inhibition of LPS‐induced C/EBPδ by trichostatin a has a positive effect on LPS‐induced cyclooxygenase 2 expression in RAW264.7 cells

Cyclooxygenase 2 (COX‐2) is an important inflammatory factor. Previous studies have indicated that COX‐2 is induced with lipopolysaccharide (LPS) treatment. Here, we found that an inhibitor of histone deacetylase (HDAC), trichostatin A (TSA), cannot repress LPS‐induced COX‐2 but it increased the COX‐2 level in RAW264.7 cells. We found no significant difference in NF‐κB activation and ERK1/2 phosphorylation, but LPS‐induced C/EBPδ expression was completely abolished after TSA treatment of LPS‐treated cells. Interesting, reporter assay of C/EBPδ promoter revealed that Sp1‐binding site is important. Although there was no alteration in c‐Jun levels, but the phosphorylation of c‐Jun at its C‐terminus was increased dramatically. A DNA‐associated protein assay (DAPA) and chromatin immunoprecipitation assay (ChIP) indicated that c‐Jun was recruited via Sp1 to the promoter of C/EBPδ after LPS treatment; this recruitment of c‐Jun was repressed by TSA. C/EBPδ inhibition by TSA resulted in increased binding of C/EBPα and C/EBPβ to the COX‐2 promoter. Therefore, TSA has a positive effect on LPS‐induced COX‐2 since it decreases the C/EBPδ level by reducing c‐Jun recruitment by Sp1 to the C/EBPδ promoter, resulting in increased the recruitment of C/EBPα and C/EBPβ to the COX‐2 promoter. J. Cell. Biochem. 110: 1430–1438, 2010. © 2010 Wiley‐Liss, Inc.     

Critical role for CCAAT/enhancer-binding protein β in immune complex-induced acute lung injury

C/EBPs, particularly C/EBPβ and C/EBPδ, are known to participate in the regulation of many genes associated with inflammation. However, very little is known regarding the activation and functions of C/EBPβ and C/EBPδ in acute lung inflammation and injury. In this study, we show that both C/EBPβ and C/EBPδ activation are triggered in lungs and in alveolar macrophages following intrapulmonary deposition of IgG immune complexes. We further show that mice carrying a targeted deletion of the C/EBPβ gene displayed significant attenuation of the permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), total number of WBCs, and neutrophils in bronchoalveolar lavage fluids compared with wild-type mice. Moreover, the mutant mice expressed considerably less TNF-α, IL-6, and CXC/CC chemokine and soluble ICAM-1 proteins in bronchoalveolar lavage fluids, and corresponding mRNAs in the IgG immune complex-injured lung, compared with wild-type mice. These phenotypes were associated with a significant reduction in morphological lung injury. In contrast, C/EBPδ deficiency had no effect on IgG immune complex-induced lung injury. IgG immune complex-stimulated C/EBPβ-deficient alveolar macrophages released significantly less TNF-α, IL-6, MIP-2, keratinocyte cell-derived chemokine, and MIP-1α compared with wild-type cells. Similar decreases in IgG immune complex-induced inflammatory mediator production were observed following small interfering RNA ablation of C/EBPβ in a murine alveolar macrophage cell line. These findings implicate C/EBPβ as a critical regulator of IgG immune complex-induced inflammatory responses and injury in the lung.     

CCAAT/enhancer binding protein β-deficiency enhances type 1 diabetic bone phenotype by increasing marrow adiposity and bone resorption

Bone loss in type 1 diabetes is accompanied by increased marrow fat, which could directly reduce osteoblast activity or result from altered bone marrow mesenchymal cell lineage selection (adipocyte vs. osteoblast). CCAAT/enhancer binding protein beta (C/EBPβ) is an important regulator of both adipocyte and osteoblast differentiation. C/EBPβ-null mice have delayed bone formation and defective lipid accumulation in brown adipose tissue. To examine the balance of C/EBPβ functions in the diabetic context, we induced type 1 diabetes in C/EBPβ-null (knockout, KO) mice. We found that C/EBPβ deficiency actually enhanced the diabetic bone phenotype. While KO mice had reduced peripheral fat mass compared with wild-type mice, they had 5-fold more marrow adipocytes than diabetic wild-type mice. The enhanced marrow adiposity may be attributed to compensation by C/EBPδ, peroxisome proliferator-activated receptor-γ2, and C/EBPα. Concurrently, we observed reduced bone density. Relative to genotype controls, trabecular bone volume fraction loss was escalated in diabetic KO mice (-48%) compared with changes in diabetic wild-type mice (-22%). Despite greater bone loss, osteoblast markers were not further suppressed in diabetic KO mice. Instead, osteoclast markers were increased in the KO diabetic mice. Thus, C/EBPβ deficiency increases diabetes-induced bone marrow (not peripheral) adipose depot mass, and promotes additional bone loss through stimulating bone resorption. C/EBPβ-deficiency also reduced bone stiffness and diabetes exacerbated this (two-way ANOVA P < 0.02). We conclude that C/EBPβ alone is not responsible for the bone vs. fat phenotype switch observed in T1 diabetes and that suppression of CEBPβ levels may further bone loss and decrease bone stiffness by increasing bone resorption.     

Regulation of human microsomal prostaglandin E synthase-1 by IL-1β requires a distal enhancer element with a unique role for C/EBPβ

The studies of PGE2 (prostaglandin E2) biosynthesis have focused primarily on the role of cyclo-oxygenases. Efforts have shifted towards the specific PGE2 terminal synthases, particularly mPGES-1 (microsomal PGE synthase 1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by pro-inflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1 (early growth-response factor 1). Numerous studies demonstrate that the mPGES-1 promoter (PTGES) alone cannot account for the level of IL-1β (interleukin 1β) induction. We identified two DNase I-hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near -8.6 kb. Functional analysis of the distal site revealed two elements that co-operate with basal promoter expression and a stimulus-dependent enhancer. A specific binding site for C/EBPβ (CCAAT/enhancer-binding protein β) in the enhancer was directly responsible for inducible enhancer activity. ChIP (chromatin immunoprecipitation) analysis demonstrated constitutive Egr-1 binding to the promoter and induced RNA polymerase II and C/EBPβ binding to the promoter and enhancer respectively. Knockout/knockdown studies established a functional role for C/EBPβ in mPGES-1 gene regulation and the documented interaction between Egr-1 and C/EBPβ highlights the proximal promoter co-operation with a novel distal enhancer element in regulating inducible mPGES-1 expression.