Thrombopoietic effects of CCAAT/enhancer-binding protein ß on the early-stage differentiation of megakaryocytes.

CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor that is involved in adipocytic and monocytic differentiation. However, the physiological role of C/EBPß in megakaryocytes (MKs) is not clear. In this study, we investigated the effects of C/EBPß on the early-stage differentiation of MKs, and explored the potential mechanisms of action. We established a cytosine arabinoside-induced thrombocytopenia mouse model using C57BL/6 mice. In the thrombocytopenia mice, the platelet count was found to be decreased, and the mRNA and protein expression levels of C/EBPß in MKs were also reduced. Furthermore, the maturation of Dami (MKs cell line) cells was induced by phorbol 12-myristate 13-acetate. When C/EBPß was silenced in Dami cells by transfection using C/EBPß-small interfering RNA, the expression of MKs-specific markers CD41 and CD62P, was dramatically decreased, resulting in morphological changes and differentiation retardation in low ploidy, which were evaluated using flow cytometry, real-time polymerase chain reaction, western blot, and confocal microscopy. The mitogen activated protein kinase-extracellular signal-regulated kinase signaling pathway was found to be required for the differentiation of MKs; knockdown of C/EBPß in MEK/ERK1/2 pathway attenuated MKs differentiation. Overexpression of C/EBPß in MEK/ERK1/2 pathway inhibited by U0126 did not promote MKs differentiation. To the best of our knowledge, C/EBPß plays an important role in MKs differentiation and polyploidy cell cycle control. Taken together, C/EBPß may have thrombopoietic effects in the differentiation of MKs, and may assist in the development of treatments for various disorders.

Myeloid transformation by MLL-ENL depends strictly on C/EBP.

Chromosomal rearrangements of the mixed-lineage leukemia gene MLL1 are the hallmark of infant acute leukemia. The granulocyte-macrophage progenitor state forms the epigenetic basis for myelomonocytic leukemia stemness and transformation by MLL-type oncoproteins. Previously, it was shown that the establishment of murine myelomonocytic MLL-ENL transformation, but not its maintenance, depends on the transcription factor C/EBPα, suggesting an epigenetic hit-and-run mechanism of MLL-driven oncogenesis. Here, we demonstrate that compound deletion of Cebpa/Cebpb almost entirely abrogated the growth and survival of MLL-ENL-transformed cells. Rare, slow-growing, and apoptosis-prone MLL-ENL-transformed escapees were recovered from compound Cebpa/Cebpb deletions. The escapees were uniformly characterized by high expression of the resident Cebpe gene, suggesting inferior functional compensation of C/EBPα/C/EBPß deficiency by C/EBPε. Complementation was augmented by ectopic C/EBPß expression and downstream activation of IGF1 that enhanced growth. Cebpe gene inactivation was accomplished only in the presence of complementing C/EBPß, but not in its absence, confirming the Cebpe dependency of the Cebpa/Cebpb double knockouts. Our data show that MLL-transformed myeloid cells are dependent on C/EBPs during the initiation and maintenance of transformation.

C/EBPß deletion in oncogenic Ras skin tumors is a synthetic lethal event.

Therapeutic targeting of specific genetic changes in cancer has proven to be an effective therapy and the concept of synthetic lethality has emerged. CCAAT/enhancer-binding protein-ß (C/EBPß), a basic leucine zipper transcription factor, has important roles in cellular processes including differentiation, inflammation, survival, and energy metabolism. Using a genetically engineered mouse model, we report that the deletion C/EBPß in pre-existing oncogenic Ha-Ras mouse skin tumors in vivo resulted in rapid tumor regression. Regressing tumors exhibited elevated levels of apoptosis and p53 protein/activity, while adjacent C/EBPß-deleted skin did not. These results indicate that the deletion of C/EBPß de-represses p53 in oncogenic Ras tumors but not in normal wild-type Ras keratinocytes, and that C/EBPß is essential for survival of oncogenic Ras tumors. Co-deletion of C/EBPß and p53 in oncogenic Ras tumors showed p53 is required for tumor regression and elevated apoptosis. In tumors, loss of a pathway that confers adaptability to a stress phenotype of cancer/tumorigenesis, such as DNA damage, could result in selective tumor cell killing. Our results show that oncogenic Ras tumors display a significant DNA damage/replicative stress phenotype and these tumors have acquired a dependence on C/EBPß for their survival. RNAseq data analysis of regressing tumors deleted of C/EBPß indicates a novel interface between p53, type-1 interferon response, and death receptor pathways, which function in concert to produce activation of extrinsic apoptosis pathways. In summary, the deletion of C/EBPß in oncogenic Ras skin tumors is a synthetic lethal event, making it a promising target for future potential anticancer therapies.

The C/EBPß LIP isoform rescues loss of C/EBPß function in the mouse.

The transcription factor C/EBPß regulates hematopoiesis, bone, liver, fat, and skin homeostasis, and female reproduction. C/EBPß protein expression from its single transcript occurs by alternative in-frame translation initiation at consecutive start sites to generate three isoforms, two long (LAP*, LAP) and one truncated (LIP), with the same C-terminal bZip dimerization domain. The long C/EBPß isoforms are considered gene activators, whereas the LIP isoform reportedly acts as a dominant-negative repressor. Here, we tested the putative repressor functions of the C/EBPß LIP isoform in mice by comparing monoallelic WT or LIP knockin mice with Cebpb knockout mice, in combination with monoallelic Cebpa mice. The C/EBPß LIP isoform was sufficient to function in coordination with C/EBPα in murine development, adipose tissue and sebocyte differentiation, and female fertility. Thus, the C/EBPß LIP isoform likely has more physiological functions than its currently known role as a dominant-negative inhibitor, which are more complex than anticipated.

Absence of myeloid Klf4 reduces prostate cancer growth with pro-atherosclerotic activation of tumor myeloid cells and infiltration of CD8 T cells.

The microenvironment of prostate cancer often includes abundant tumor-associated macrophages (TAMs), with their acquisition of an M2 phenotype correlating with local aggressiveness and metastasis. Tumor-derived M-CSF contributes to TAM M2 polarization, and M-CSF receptor inhibition slows prostate cancer growth in model systems. As additional cytokines can direct TAM M2 polarization, targeting downstream transcription factors could avoid resistance. Klf4 and C/EBPß each contribute to monocyte development, and reduced expression of macrophage Klf4 or C/EBPß favors their adoption of a pro-inflammatory M1 state. We find that a Hi-Myc C57BL/6 prostate cancer line grows more slowly in syngeneic Klf4(f/f);Lys-Cre compared with Klf4(f/f) mice when inoculated subcutaneously, but grows equally rapidly in C/EBPß(f/f);Lys-Cre and C/EBPß(f/f) hosts. In the absence of myeloid Klf4, TAMs have reduced expression of surface mannose receptor and Fizz1 mRNA, both M2 markers. Global gene expression analysis further revealed activation of pro-inflammatory, pro-atherosclerotic pathways. Analysis of tumor-infiltrating lymphocytes (TILs) demonstrated markedly increased activated CD8 T cell numbers, and CD8 T cell depletion obviated the inhibitory effect of myeloid Klf4 deletion on prostate cancer growth. These findings suggest that reducing expression or activity of the Klf4 transcription factor in tumor myeloid cells may contribute to prostate cancer therapy.

The use of siRNA as a pharmacological tool to assess a role for the transcription factor NF-IL6 in the brain under in vitro and in vivo conditions during LPS-induced inflammatory stimulation.

BACKGROUND: Studies with NF-IL6-deficient mice indicate that this transcription factor plays a dual role during systemic inflammation with pro- and anti-inflammatory capacities. Here, we aimed to characterize the role of NF-IL6 specifically within the brain. METHODS: In this study, we tested the capacity of short interfering (si) RNA to silence the inflammatory transcription factor nuclear factor-interleukin 6 (NF-IL6) in brain cells under in vitro and in vivo conditions. RESULTS: In cells of a mixed neuronal and glial primary culture from the rat area postrema (AP), short interfering RNA (siRNA) directed against NF-IL6 strongly reduced basal and lipopolysaccharide (LPS)-induced nuclear immunoreactivity of this transcription factor, with the strongest effect on astrocytes. The siRNA did not exert inflammatory effects in the primary culture as confirmed by unaltered levels of IL-6 in supernatants. In vivo, intracerebroventricular (i.c.v.) injections of fluorochrome labelled siRNA caused its appearance in relevant brain structures for fever induction pathways such as the vascular organ of lamina terminalis, the subfornical organ, the median preoptic nucleus (MnPO) and the AP in several cell types, including microglial cells. However, i.c.v. injections of siRNA per se caused signs of fever, anorexia and reduced locomotor activity, i.e. sickness behavior. CONCLUSIONS: This approach was, thus, not suitable to characterize the role NF-IL6 in the brain in vivo, namely during experimentally induced systemic inflammation.

Myeloid C/EBPß deficiency reshapes microglial gene expression and is protective in experimental autoimmune encephalomyelitis.

BACKGROUND: CCAAT/enhancer binding protein ß (C/EBPß) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia. Mice deficient for C/EBPß show protection against excitotoxic and ischemic CNS damage, but the involvement in this neuroprotective effect of the various C/EBPß-expressing cell types is not solved. Since C/EBPß-deficient microglia show attenuated neurotoxicity in culture, we hypothesized that specific C/EBPß deficiency in microglia could be neuroprotective in vivo. In this study, we have tested this hypothesis by generating mice with myeloid C/EBPß deficiency. METHODS: Mice with myeloid C/EBPß deficiency were generated by crossing LysMCre and C/EBPßfl/fl mice. Primary microglial cultures from C/EBPßfl/fl and LysMCre-C/EBPßfl/fl mice were treated with lipopolysaccharide ± interferon γ (IFNγ) for 6 h, and gene expression was analyzed by RNA sequencing. Gene expression and C/EBPß deletion were analyzed in vivo in microglia isolated from the brains of C/EBPßfl/fl and LysMCre-C/EBPßfl/fl mice treated systemically with lipolysaccharide or vehicle. Mice of LysMCre-C/EBPßfl/fl or control genotypes were subjected to experimental autoimmune encephalitis and analyzed for clinical signs for 52 days. One- or two-way ANOVA or Kruskal-Wallis with their appropriate post hoc tests were used. RESULTS: LysMCre-C/EBPßfl/fl mice showed an efficiency of C/EBPß deletion in microglia of 100 and 90% in vitro and in vivo, respectively. These mice were devoid of female infertility, perinatal mortality and reduced lifespan that are associated to full C/EBPß deficiency. Transcriptomic analysis of C/EBPß-deficient primary microglia revealed C/EBPß-dependent expression of 1068 genes, significantly enriched in inflammatory and innate immune responses GO terms. In vivo, microglial expression of the pro-inflammatory genes Cybb, Ptges, Il23a, Tnf and Csf3 induced by systemic lipopolysaccharide injection was also blunted by C/EBPß deletion. CNS expression of C/EBPß was upregulated in experimental autoimmune encephalitis and in multiple sclerosis samples. Finally, LysMCre-C/EBPßfl/fl mice showed robust attenuation of clinical signs in experimental autoimmune encephalitis. CONCLUSION: This study provides new data that support a central role for C/EBPß in the biology of activated microglia, and it offers proof of concept for the therapeutic potential of microglial C/EBPß inhibition in multiple sclerosis.

Identification of an atypical monocyte and committed progenitor involved in fibrosis.

Monocytes and macrophages comprise a variety of subsets with diverse functions. It is thought that these cells play a crucial role in homeostasis of peripheral organs, key immunological processes and development of various diseases. Among these diseases, fibrosis is a life-threatening disease of unknown aetiology. Its pathogenesis is poorly understood, and there are few effective therapies. The development of fibrosis is associated with activation of monocytes and macrophages. However, the specific subtypes of monocytes and macrophages that are involved in fibrosis have not yet been identified. Here we show that Ceacam1+Msr1+Ly6C-F4/80-Mac1+ monocytes, which we term segregated-nucleus-containing atypical monocytes (SatM), share granulocyte characteristics, are regulated by CCAAT/enhancer binding protein ß (C/EBPß), and are critical for fibrosis. Cebpb deficiency results in a complete lack of SatM. Furthermore, the development of bleomycin-induced fibrosis, but not inflammation, was prevented in chimaeric mice with Cebpb-/- haematopoietic cells. Adoptive transfer of SatM into Cebpb-/- mice resulted in fibrosis. Notably, SatM are derived from Ly6C-FcεRI+ granulocyte/macrophage progenitors, and a newly identified SatM progenitor downstream of Ly6C-FcεRI+ granulocyte/macrophage progenitors, but not from macrophage/dendritic-cell progenitors. Our results show that SatM are critical for fibrosis and that C/EBPß licenses differentiation of SatM from their committed progenitor.

C/EBPß regulates homeostatic and oncogenic gastric cell proliferation.

Cancer of the stomach is among the leading causes of death from cancer worldwide. The transcription factor C/EBPß is frequently overexpressed in gastric cancer and associated with the suppression of the differentiation marker TFF1. We show that the murine C/EBPß knockout stomach displays unbalanced homeostasis and reduced cell proliferation and that tumorigenesis of human gastric cancer xenograft is inhibited by knockdown of C/EBPß. Cross-species comparison of gene expression profiles between C/EBPß-deficient murine stomach and human gastric cancer revealed a subset of tumors with a C/EBPß signature. Within this signature, the RUNX1t1 tumor suppressor transcript was down-regulated in 38 % of gastric tumor samples. The RUNX1t1 promoter was frequently hypermethylated and ectopic expression of RUNX1t1 in gastric cancer cells inhibited proliferation and enhanced TFF1 expression. These data suggest that the tumor suppressor activity of both RUNX1t1 and TFF1 are mechanistically connected to C/EBPß and that cross-regulation between C/EBPß-RUNX1t1-TFF1 plays an important role in gastric carcinogenesis. KEY MESSAGE: C/EBPß controls proliferation and differentiation balance in the stomach. Homeostatic differentiation/proliferation balance is altered in gastric cancer. RUNX1t1 is a C/EBPß-associated tumor suppressor. RUNX1t1 negatively regulates C/EBPß pro-oncogenic functions.

CCAAT/enhancer binding protein beta protects muscle satellite cells from apoptosis after injury and in cancer cachexia.

CCAAT/enhancer binding protein beta (C/EBPß), a transcription factor expressed in muscle satellite cells (SCs), inhibits the myogenic program and is downregulated early in differentiation. In a conditional null model in which C/EBPß expression is knocked down in paired box protein 7+ (Pax7+) SCs, cardiotoxin (CTX) injury is poorly repaired, although muscle regeneration is efficient in control littermates. While myoblasts lacking C/EBPß can differentiate efficiently in culture, after CTX injury poor regeneration was attributed to a smaller than normal Pax7+ population, which was not due to a failure of SCs to proliferate. Rather, the percentage of apoptotic SCs was increased in muscle lacking C/EBPß. Given that an injury induced by BaCl2 is repaired with greater efficiency than controls in the absence of C/EBPß, we investigated the inflammatory response following BaCl2 and CTX injury and found that the levels of interleukin-1ß (IL-1ß), a proinflammatory cytokine, were robustly elevated following CTX injury and could induce C/EBPß expression in myoblasts. High levels of C/EBPß expression in myoblasts correlated with resistance to apoptotic stimuli, while its loss increased sensitivity to thapsigargin-induced cell death. Using cancer cachexia as a model for chronic inflammation, we found that C/EBPß expression was increased in SCs and myoblasts of tumor-bearing cachectic animals. Further, in cachectic conditional knockout animals lacking C/EBPß in Pax7+ cells, the SC compartment was reduced because of increased apoptosis, and regeneration was impaired. Our findings indicate that the stimulation of C/EBPß expression by IL-1ß following muscle injury and in cancer cachexia acts to promote SC survival, and is therefore a protective mechanism for SCs and myoblasts in the face of inflammation.

Deficiency in mTORC1-controlled C/EBPß-mRNA translation improves metabolic health in mice.

The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of physiological adaptations in response to changes in nutrient supply. Major downstream targets of mTORC1 signalling are the mRNA translation regulators p70 ribosomal protein S6 kinase 1 (S6K1p70) and the 4E-binding proteins (4E-BPs). However, little is known about vertebrate mRNAs that are specifically controlled by mTORC1 signalling and are engaged in regulating mTORC1-associated physiology. Here, we show that translation of the CCAAT/enhancer binding protein beta (C/EBPß) mRNA into the C/EBPß-LIP isoform is suppressed in response to mTORC1 inhibition either through pharmacological treatment or through calorie restriction. Our data indicate that the function of 4E-BPs is required for suppression of LIP. Intriguingly, mice lacking the cis-regulatory upstream open reading frame (uORF) in the C/EBPß-mRNA, which is required for mTORC1-stimulated translation into C/EBPß-LIP, display an improved metabolic phenotype with features also found under calorie restriction. Thus, our data suggest that translational adjustment of C/EBPß-isoform expression is one of the key processes that direct metabolic adaptation in response to changes in mTORC1 activity.

Prospective signs of cleidocranial dysplasia in Cebpb deficiency.

BACKGROUND: Although runt-related transcription factor 2 (RUNX2) has been considered a determinant of cleidocranial dysplasia (CCD), some CCD patients were free of RUNX2 mutations. CCAAT/enhancer-binding protein beta (Cebpb) is a key factor of Runx2 expression and our previous study has reported two CCD signs including hyperdontia and elongated coronoid process of the mandible in Cebpb deficient mice. Following that, this work aimed to conduct a case-control study of thoracic, zygomatic and masticatory muscular morphology to propose an association between musculoskeletal phenotypes and deficiency of Cebpb, using a sample of Cebpb-/-, Cebpb+/- and Cebpb+/+ adult mice. Somatic skeletons and skulls of mice were inspected with soft x-rays and micro-computed tomography (µCT), respectively. Zygomatic inclination was assessed using methods of coordinate geometry and trigonometric function on anatomic landmarks identified with µCT. Masseter and temporal muscles were collected and weighed. Expression of Cebpb was examined with a reverse transcriptase polymerase chain reaction (RT-PCR) technique. RESULTS: Cebpb-/- mice displayed hypoplastic clavicles, a narrow thoracic cage, and a downward tilted zygomatic arch (p < 0.001). Although Cebpb+/- mice did not show the phenotypes above (p = 0.357), a larger mass percentage of temporal muscles over masseter muscles was seen in Cebpb+/- littermates (p = 0.012). The mRNA expression of Cebpb was detected in the clavicle, the zygoma, the temporal muscle and the masseter muscle, respectively. CONCLUSIONS: Prospective signs of CCD were identified in mice with Cebpb deficiency. These could provide an additional aetiological factor of CCD. Succeeding investigation into interactions among Cebpb, Runx2 and musculoskeletal development is indicated.

CCAAT/enhancer-binding protein ß expressed by bone marrow mesenchymal stromal cells regulates early B-cell lymphopoiesis.

The transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) regulates the differentiation of a variety of cell types. Here, the role of C/EBPß expressed by bone marrow mesenchymal stromal cells (BMMSCs) in B-cell lymphopoiesis was examined. The size of the precursor B-cell population in bone marrow was reduced in C/EBPß-knockout (KO) mice. When bone marrow cells from C/EBPß-KO mice were transplanted into lethally irradiated wild-type (WT) mice, which provide a normal bone marrow microenvironment, the size of the precursor B-cell population was restored to a level equivalent to that generated by WT bone marrow cells. In coculture experiments, BMMSCs from C/EBPß-KO mice did not support the differentiation of WT c-Kit(+) Sca-1(+) Lineage(-) hematopoietic stem cells (KSL cells) into precursor B cells, whereas BMMSCs from WT mice did. The impaired differentiation of KSL cells correlated with the reduced production of CXCL12/stromal cell-derived factor-1 by the cocultured C/EBPß-deficient BMMSCs. The ability of C/EBPß-deficient BMMSCs to undergo osteogenic and adipogenic differentiation was also defective. The survival of leukemic precursor B cells was poorer when they were cocultured with C/EBPß-deficient BMMSCs than when they were cocultured with WT BMMSCs. These results indicate that C/EBPß expressed by BMMSCs plays a crucial role in early B-cell lymphopoiesis.

Phenotypes of CCAAT/enhancer-binding protein beta deficiency: hyperdontia and elongated coronoid process.

OBJECTIVES: This investigation aimed to conduct a case-control study of mandibular morphology and dental anomalies to propose a relationship between mandibular/dental phenotypes and deficiency of CCAAT/enhancer-binding protein beta (CEBPB). MATERIALS AND METHODS: Skulls of CEBPB(-/-), CEBPB(+/-) and CEBPB(+/+) mice were inspected with micro-computed tomography. Mandibular morphology was assessed with a method of Euclidean distance matrix analysis. RESULTS: Elongation of the coronoid process was identified in CEBPB(+/-) (P ≤ 0.046) and CEBPB(-/-) 12-month-olds (P ≤ 0.028) but not in 14-day-olds (P ≥ 0.217) and 0-day-olds (P ≥ 0.189) of either genotype. Formation of supernumerary teeth in CEBPB(-/-) adult mice was demonstrated (χ(2) = 6.00, df = 1, P = 0.014). CONCLUSIONS: CEBPB deficiency was related to elongation of the coronoid process and formation of supernumerary teeth. The mandibular and dental phenotypes of CEBPB deficiency were unseen by the 14th day after birth. Future investigations into the influence of CEBPB on mandibular and dental development are needed.

C/EBPß is involved in the amplification of early granulocyte precursors during candidemia-induced "emergency" granulopoiesis.

Granulopoiesis is tightly regulated to meet host demands during both "steady-state" and "emergency" situations, such as infections. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) plays critical roles in emergency granulopoiesis, but the precise developmental stages in which C/EBPß is required are unknown. In this study, a novel flow cytometric method was developed that successfully dissected mouse bone marrow cells undergoing granulopoiesis into five distinct subpopulations (#1-5) according to their levels of c-Kit and Ly-6G expression. After the induction of candidemia, rapid mobilization of mature granulocytes and an increase in early granulocyte precursors accompanied by cell cycle acceleration was followed by a gradual increase in granulocytes originating from the immature populations. Upon infection, C/EBPß was upregulated at the protein level in all the granulopoietic subpopulations. The rapid increase in immature subpopulations #1 and #2 observed in C/EBPß knockout mice at 1 d postinfection was attenuated. Candidemia-induced cell cycle acceleration and proliferation of hematopoietic stem/progenitors were also impaired. Taken together, these data suggest that C/EBPß is involved in the efficient amplification of early granulocyte precursors during candidemia-induced emergency granulopoiesis.

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

C/EBPs, particularly C/EBPß 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/EBPß and C/EBPδ in acute lung inflammation and injury. In this study, we show that both C/EBPß 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/EBPß 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/EBPß-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/EBPß in a murine alveolar macrophage cell line. These findings implicate C/EBPß as a critical regulator of IgG immune complex-induced inflammatory responses and injury in the lung.

Mesenchymal-specific deletion of C/EBPß suppresses pulmonary fibrosis.

The CCAAT/enhancer-binding protein ß (C/EBPß) regulates a variety of factors and cellular responses associated with pulmonary fibrosis. To distinguish its role in the mesenchyme from that in other compartments, the effects of mesenchymal-specific deletion of C/EBPß on pulmonary fibrosis was examined. Crossing of mice with the floxed C/EBPß gene with α2(I) collagen enhancer-CreER(T)-bearing mice successfully generated progeny with a conditional knockout (CKO) of C/EBPß in collagen I-expressing ("mesenchymal") cells only on treatment with tamoxifen (C/EBPß CKO). When treated with an endotracheal bleomycin injection, C/EBPß CKO mice showed significant attenuation of pulmonary fibrosis relative to control C/EBPß-intact mice. C/EBPß CKO mice also had reduced myofibroblasts in the lung. However, no significant differences in inflammatory/immune cell influx were noted in the mutant mice relative to the control mice. DNA microarray and real-time PCR analyses identified a series of myofibroblast differentiation regulators as novel target genes of C/EBPß. Interestingly, C/EBPß deficiency caused a marked induction of matrix metalloproteinase 12 expression, suggesting its potential role as a repressor, which could account for the noted reduction in fibrosis in the C/EBPß-deficient mice. Thus, these findings indicate an essential role for C/EBPß in the mesenchymal compartment in pulmonary fibrosis that is independent of its effects on inflammation or immune cell infiltration.

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

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-1ß (interleukin 1ß) 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/EBPß (CCAAT/enhancer-binding protein ß) 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/EBPß binding to the promoter and enhancer respectively. Knockout/knockdown studies established a functional role for C/EBPß in mPGES-1 gene regulation and the documented interaction between Egr-1 and C/EBPß highlights the proximal promoter co-operation with a novel distal enhancer element in regulating inducible mPGES-1 expression.

FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors.

The transcription factors that control lineage specification of haematopoietic stem cells (HSCs) have been well described for the myeloid and lymphoid lineages, whereas transcriptional control of erythroid (E) and megakaryocytic (Mk) fate is less understood. We here use conditional removal of the GATA-1 and FOG-1 transcription factors to identify FOG-1 as required for the formation of all committed Mk- and E-lineage progenitors, whereas GATA-1 was observed to be specifically required for E-lineage commitment. FOG-1-deficient HSCs and preMegEs, the latter normally bipotent for the Mk and E lineages, underwent myeloid transcriptional reprogramming, and formed myeloid, but not erythroid and megakaryocytic cells in vitro. These results identify FOG-1 and GATA-1 as required for formation of bipotent Mk/E progenitors and their E-lineage commitment, respectively, and show that FOG-1 mediates transcriptional Mk/E programming of HSCs as well as their subsequent Mk/E-lineage commitment. Finally, C/EBPs and FOG-1 exhibited transcriptional cross-regulation in early myelo-erythroid progenitors making their functional antagonism a potential mechanism for separation of the myeloid and Mk/E lineages.