No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
C/EBP Regulation in Lipopolysaccharide-Stimulated Macrophages
Abstract
C/EBP family members contribute to the induction of the interleukin-12 p40 gene and the genes encoding several other mediators of inflammation. Here, we show by chromatin immunoprecipitation that C/EBPbeta binds the p40 promoter following lipopolysaccharide stimulation of peritoneal macrophages. However, three modes of C/EBPbeta regulation reported in other cell types were not detected, including alternative translation initiation, nuclear translocation, and increased DNA binding following posttranslational modification. In contrast, C/EBPbeta concentrations greatly increased following stimulation via MAP kinase-dependent induction of C/EBPbeta gene transcription. Increased C/EBPbeta concentrations were unimportant for p40 induction, however, as transcription of the p40 gene initiated before C/EBPbeta concentrations increased. Furthermore, disruption of C/EBPbeta upregulation by a MAP kinase inhibitor only slightly diminished p40 induction. Phosphopeptide mapping revealed that endogenous C/EBPbeta in macrophages is phosphorylated on only a single tryptic peptide containing 14 potential phosphoacceptors. This peptide was constitutively phosphorylated in primary and transformed macrophages, in contrast to its inducible phosphorylation in other cell types in response to Ras and growth hormone signaling. Altered-specificity experiments supported the hypothesis that C/EBPbeta activity in macrophages does not require an inducible posttranslational modification. These findings suggest that, although C/EBPbeta contributes to the induction of numerous proinflammatory genes, it is fully active in unstimulated macrophages and poised to stimulate transcription in conjunction with other factors whose activities are induced.
... C/EBPβ -/mice are produced at sub-Mendelian ratios and experience high rates of perinatal death, attributed to hypoglycemia as a result of failure of the liver to release glucose (Croniger, Trus et al. 1997). In addition, mice harbouring the deletion of the C/EBPβ gene are resistant to diet-induced obesity, exhibit abnormal female reproduction and are immunocompromised (Sterneck, Tessarollo et al. 1997;Bradley, Zhou et al. 2003;Millward, Heaney et al. 2007). ...
... During the innate immune response, activated macrophages are recruited to the site of inflammation and induce the expression of pro-inflammatory cytokines, including interleukin-1 (IL-1), IL-6, IL-8, nitric oxide synthase and TNF-α (Baer, Williams et al. 1998;Bradley, Zhou et al. 2003). The expression of C/EBPβ is also induced by inflammatory stimuli, resulting in an increase in its transcriptional activity due to phosphorylation (Trautwein, Caelles et al. 1993;Poli 1998 (Bradley, Zhou et al. 2003). ...
... During the innate immune response, activated macrophages are recruited to the site of inflammation and induce the expression of pro-inflammatory cytokines, including interleukin-1 (IL-1), IL-6, IL-8, nitric oxide synthase and TNF-α (Baer, Williams et al. 1998;Bradley, Zhou et al. 2003). The expression of C/EBPβ is also induced by inflammatory stimuli, resulting in an increase in its transcriptional activity due to phosphorylation (Trautwein, Caelles et al. 1993;Poli 1998 (Bradley, Zhou et al. 2003). C/EBPβ binding sites have been identified in the regulatory regions of various genes involved in inflammation, including cytokines IL-6, IL-8 and TNF-α (Natsuka, Akira et al. 1992). ...
... It is probably not surprising that C/EBP family of transcription factors can participate in regulating cytokine expression, consistent with many previous studies (107,117,124,125,132,133,235,236). However, it is very interesting that the expression of C/EBP and C/EBP are abolished in MyD88-deficient or IRAK-4-deficient macrophages stimulated with LPS, unlike NF-B, AP-1, or IRF family of transcription factors. ...
... These results parallel with those observed in MyD88-deficient cells (Figure III-1C) (66). Furthermore, for the initial phase of TNF induction, we cannot exclude the possibility that C/EBP/ may also play a role together with other transcription factors ( Figure III-5 and 8) (66,235). ...
... C/EBP and C/EBP can be regulated by phosphorylation to modulate their DNA binding abilities and transcriptional activities (104). It has been shown that C/EBPβ can be phosphorylated by cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC), calcium dependent kinase, p90 ribosomal S kinase (RSK), glycogen synthase kinase 3- (GSK-3), and MAPK (104,106,235). Among these kinases, it is known that TLRs can activate PKC, MAPK and GSK-3. ...
... The two longer isoforms, Full and LAP, have activity as transcription factors, whereas the shorter C/EBPb isoform, LIP, lacks the transactivation domain and acts as a dominant negative form (Descombes and Schibler, 1991). Although nuclear translocation and phosphorylation have been reported, the most frequent mode of regulation of C/EBPb function is by increased de novo synthesis (see Bradley et al. (2003) for references). ...
... A band of approximately 50 kDa was also observed and probably corresponds to the so-called ''cross reactive material'' (crm; Figs. 2-5), a previously reported nonspecific band (Bradley et al., 2003;Cardinaux and Magistretti, 1996). ...
... Our finding that LPS increases C/EBPb levels also in microglial cells is a novel observation to our knowledge. Although C/EBPb activity can certainly be modulated by post-translational modifications, the most common mechanism for C/EBPb activation is increased expression (see Bradley et al. (2003) for references). Therefore, the increased C/EBPb levels in microglia induced by LPS are likely to result in increased C/EBPb activity. ...
C/EBPβ (CCAAT/enhancer binding protein beta) es un factor de transcripción de la familia b-zip cuya expresión aumenta en células microgliales activadas. Nuestro objetivo en este trabajo ha sido estudiar si C/EBPβ regula en la activación microglial la expresión de NOS2, un enzima inducible productor de NO y que se cree es clave en los efectos patogénicos de la respuesta neuroinflamatoria. El gen de NOS2 presenta secuencias consenso para C/EBP en sus regiones reguladoras. El trabajo se ha realizado en cultivos de glía mixta de corteza cerebral de embriones (E19) de ratoneswild-type y deficientes en C/EBPβ. Estos cultivos constan principalmente de astrocitos y microglía y han sido tratados con LPS o LPS+IFNγ para inducir su activación. Hemos observado que en la activación glial se induce la expresión de NOS2 (qRT-PCR), que se localiza en microglía y no en astrocitos (inmunocitoquímica) y que en estas condiciones C/EBPβ se une al promotor de NOS2 (qChIP). Por otro lado, en cultivos gliales deficientes en C/EBPβ tratados con LPS o LPS+IFNγ se produce una marcada atenuación en la inducción de NOS2 a nivel de mRNA (qRT-PCR), proteína (WB e icc) y producción de NO (Griess). Finalmente, al cocultivar neuronas con microglía wild-type o deficiente en C/EBPβ hemos observado que la neurotoxicidad inducida por células gliales activadas se ve totalmente abolida cuando la microglía es deficiente en C/EBPβ. Este trabajo demuestra que C/EBPβ regula la expresión de NOS2 en células microgliales activadas y apunta a C/EBPβ como un importante regulador de la expresión de genes con efectos neurotóxicos en la respuesta neuroinflamatoria.
... Because C/EBPβ and AEP are tightly regulated by oxidative stress and inflammation 32,34,37,38 , we investigated oxidative stress and inflammatory status in human α-SNCA mice. Iba-1 staining revealed that microglia cells were age-dependently activated in both striatum and SN in α-SNCA mice ( Supplementary Fig. 1b), and so were the levels of 4-HNE (4-hydroxynonenal), a biomarker for oxidative stress ( Supplementary Fig. 1c). ...
... In the current report, we show that C/EBPβ/AEP axis is agedependently upregulated and activated in α-SNCA transgenic mice and PD patients. Moreover, ROS and neuro-inflammation were temporally escalated in α-SNCA mice, fitting with C/EBPβ and AEP augmentation because both of them are strongly regulated by oxidative stress and inflammation 34,38 . In alignment with these findings, NQO1, an inducible antioxidative enzyme, was temporally reduced in the α-SNCA mice. ...
Parkinson’s disease (PD) is the most common neurodegenerative motor disorder, and its pathologic hallmarks include extensive dopaminergic neuronal degeneration in the Substantia nigra associated with Lewy bodies, predominantly consisting of phosphorylated and truncated α-Synuclein (α-Syn). Asparagine endopeptidase (AEP) cleaves human α-Syn at N103 residue and promotes its aggregation, contributing to PD pathogenesis. However, how AEP mediates Lewy body pathologies during aging and elicits PD onset remains incompletely understood. Knockout of AEP or C/EBPβ from α-SNCA mice, and their chronic rotenone exposure models were used, and the mechanism of α-Syn from the gut that spread to the brain was observed. Here we report that C/EBPβ/AEP pathway, aggravated by oxidative stress, is age-dependently activated and cleaves α-Syn N103 and regulates Lewy body-like pathologies spreading from the gut into the brain in human α-SNCA transgenic mice. Deletion of C/EBPβ or AEP substantially diminished the oxidative stress, neuro-inflammation, and PD pathologies, attenuating motor dysfunctions in aged α-SNCA mice. Noticeably, PD pathologies initiate in the gut and progressively spread into the brain. Chronic gastric exposure to a low dose of rotenone initiates Lewy body-like pathologies in the gut that propagate into the brain in a C/EBPβ/AEP-dependent manner. Hence, our studies demonstrate that C/EBPβ/AEP pathway is critical for mediating Lewy body pathology progression in PD.
... C/EBPβ DNA binding affinity and specificity are regulated through cooperation with other transcription factors such as the p50 subunit of NF-κB, CREB/ATF, AP-1, and SP1 [26]. It has been shown that the interaction between SP1 and C/EBPβ cooperatively activated IL-10 gene expression in LPS-stimulated macrophages [28] and that the CREB-C/EBPβ cascade was essential for induction of the type 2 macrophages anti-inflammatory cytokines such as IL10 [32][33][34][35][36][37]. CREB activity is mainly regulated through its phosphorylation at serine residue 133 by kinases p38 MAPK [38] and by GSK-3 and, at serine residue 129, by GSK-3 [39]. ...
... Results of EMSA experiments showed that both inhibitors reduced C/EBPβ DNA binding activity ( Figure 2A). The downstream targets of p38 MAPK include a wide array of cytoplasmic and nuclear factors including CREB, which is known to induce C/EBPβ transcription following its phosphorylation on serine residues [32,37]. Western blot analysis clearly showed a rapid and intense CREB phosphorylation at Ser133 and Ser129 in GM/DCs, as compared to low levels of CREB phosphorylation and expression in IL-4/DCs ( Figure 2B). ...
Dendritic cells (DCs) play a major role in innate and adaptive immunity and self-immune tolerance. Immunogenic versus tolerogenic DC functions are dictated by their levels of costimulatory molecules and their cytokine expression profile. The transcription factor C/EBPβ regulates the expression of several inflammatory genes in many cell types including macrophages. However, little is known regarding the role of C/EBPβ in tolerogenic versus immunogenic DCs functions. We have previously reported that bone marrow-derived DCs generated with GM-CSF (GM/DCs) acquire the signature of semi-mature tolerogenic IL-10-producing DCs as opposed to immunogenic DCs generated with GM-CSF and IL-4 (IL-4/DCs). Here, we show that tolerogenic GM/DCs exhibit higher levels of phosphorylation and enhanced DNA binding activity of C/EBPβ and CREB than immunogenic IL-4/DCs. We also show that the p38 MAPK/CREB axis and GSK3 play an important role in regulating C/EBPβ phosphorylation and DNA binding activity. Inhibition of p38 MAPK in GM/DCs resulted in a drastic decrease of C/EBPβ and CREB DNA binding activities, a reduction of their IL-10 production and an increase of their IL-12p70 production, a characteristic of immunogenic IL-4/DCs. We also present evidence that GSK3 inhibition in GM/DCs reduced C/EBPβ DNA binding activity and increased expression of costimulatory molecules in GM/DCs and their production of IL-10. Analysis of GM/DCs of C/EBPβ−/− mice showed that C/EBPβ was essential to maintain the semimature phenotype and the production of IL-10 as well as low CD4+ T cell proliferation. Our results highlight the importance of the p38MAPK-C/EBPβ pathway in regulating phenotype and function of tolerogenic GM/DCs.
... Furthermore, p205 has been shown to colocalize and bind directly with the c/EBPb in mouse adipose-derived stem cells (27). Furthermore, c/EBPb has also been implicated in the induction of several proinflammatory genes in macrophages (36). Therefore, we tested the effect of ectopic expression of c/EBPb or p65/RelA on Asc reporter gene expression. ...
... The transcription factor c/EBPb is important in controlling macrophage differentiation. c/EBPb remains fully active in resting macrophages and stays positioned on target genes, ready to stimulate transcription with other inducible transcription factors (36). p205 has previously been shown to interact with c/EBPb in adipocytes. ...
Members of the IFN-inducible PYHIN protein family, such as absent in melanoma-2 and IFN-γ-inducible protein (IFI)16, bind dsDNA and form caspase-1-activating inflammasomes that are important in immunity to cytosolic bacteria, DNA viruses, or HIV. IFI16 has also been shown to regulate transcription of type I IFNs during HSV infection. The role of other members of the PYHIN protein family in the regulation of immune responses is much less clear. In this study, we identified an immune-regulatory function for a member of the murine PYHIN protein family, p205 (also called Ifi205). Examination of immune responses induced by dsDNA and other microbial ligands in bone marrow-derived macrophages lacking p205 revealed that inflammasome activation by dsDNA, as well as ligands that engage the NLRP3 inflammasome, was severely compromised in these cells. Further analysis revealed that p205-knockdown cells showed reduced expression of apoptosis-associated speck-like molecule containing CARD domain (Asc) at the protein and RNA levels. p205 knockdown resulted in reduced binding of actively transcribing RNA polymerase II to the endogenous Asc gene, resulting in decreased transcription and processing of Asc pre-mRNA. Deletion of p205 in B16 melanoma cells using CRISPR/Cas9 showed a similar loss of Asc expression. Ectopic expression of p205 induced expression of an Asc promoter-luciferase reporter gene. Together, these findings suggest that p205 controls expression of Asc mRNA to regulate inflammasome responses. These findings expand on our understanding of immune-regulatory roles for the PYHIN protein family.
... Furthermore, p205 has been shown to colocalize and bind directly with the c/EBPb in mouse adipose-derived stem cells (27). Furthermore, c/EBPb has also been implicated in the induction of several proinflammatory genes in macrophages (36). Therefore, we tested the effect of ectopic expression of c/EBPb or p65/RelA on Asc reporter gene expression. ...
... The transcription factor c/EBPb is important in controlling macrophage differentiation. c/EBPb remains fully active in resting macrophages and stays positioned on target genes, ready to stimulate transcription with other inducible transcription factors (36). p205 has previously been shown to interact with c/EBPb in adipocytes. ...
... C/ EBPβ has a crucial function in inflammation 20 . Numerous proinflammatory genes contain potential consensus sequences of C/EBPβ 21 , and macrophages 22 and glial cells 23 exhibit an increase in C/EBPβ levels under the exposure to pro-inflammatory stimuli. In C/EBPβ-null brain, both pro-inflammatory genes and the neurotoxic consequences of activated microglia are diminished 24 . ...
Alzheimer’s disease (AD) is the most common dementia. It is known that women with one ApoE4 allele display greater risk and earlier onset of AD compared with men. In mice, we previously showed that follicle–stimulating hormone (FSH), a gonadotropin that rises in post–menopausal females, activates its receptor FSHR in the hippocampus, to drive AD–like pathology and cognitive impairment. Here we show in mice that ApoE4 and FSH jointly trigger AD-like pathogenesis by activating C/EBPβ/δ-secretase signaling. ApoE4 and FSH additively activate C/EBPβ/δ-secretase pathway that mediates APP and Tau proteolytic fragmentation, stimulating Aβ and neurofibrillary tangles. Ovariectomy-provoked AD-like pathologies and cognitive defects in female ApoE4-TR mice are ameliorated by anti-FSH antibody treatment. FSH administration facilitates AD-like pathologies in both young male and female ApoE4-TR mice. Furthermore, FSH stimulates AD-like pathologies and cognitive defects in ApoE4-TR mice, but not ApoE3-TR mice. Our findings suggest that in mice, augmented FSH in females with ApoE4 but not ApoE3 genotype increases vulnerability to AD-like process by activating C/EBPβ/δ-secretase signalling.
... Therefore enrichment of SPi1 binding in exercised BMDMs may explain the attenuated responses to LPS in these macrophages. cEBPb, a member of the C/EBP family of proteins, is transcriptionally induced by the CREB transcriptional activator (102) and plays a crucial role in the antibacterial activity of macrophages (103). When macrophages are activated, CREB-mediated induction of cEBPb is required for certain M2-associated genes, like ARG1 (82). ...
Despite its importance in protecting the host from infections and injury, excessive inflammation may lead to serious human diseases including autoimmune disorders, cardiovascular diseases, diabetes, and cancer. Exercise is a known immunomodulator; however, whether exercise causes long term changes in inflammatory responses and how these changes occur are lacking. Here, we show that chronic moderate intensity training of mice leads to persistent metabolic rewiring and changes to chromatin accessibility in bone marrow derived macrophages (BMDMs), which, in turn, tempers their inflammatory responses. We show that BMDMs from exercised mice exhibited a decrease in lipopolysaccharide (LPS) induced NF-kB activation and pro-inflammatory gene expression along with an increase in M2-like associated genes when compared to BMDMs from sedentary mice. This was associated with improved mitochondrial quality and increased reliance on oxidative phosphorylation accompanied with reduced mitochondrial ROS production. Mechanistically, ATAC-seq analysis showed changes in chromatin accessibility of genes associated with inflammatory and metabolic pathways. Overall, our data suggest that chronic moderate exercise can influence the inflammatory responses of macrophages by reprogramming their metabolic and epigenetic landscape.
... Furthermore, in the presence of SB239063, NPB had no effect on the expression of C/EBPβ and PPARγ. Furthermore, it was found that in various cell types, p38 could induce the transcription and transcriptional activity of C/EBPβ [45][46][47], which is critically involved in early adipogenesis [15]. Overall, these results provide strong evidence that NPB stimulates adipogenesis in porcine preadipocytes in a p38-dependend manner. ...
Neuropeptide B (NPB) affects energy homeostasis and metabolism by binding and activating NPBWR1 and NPBWR2 in humans and pigs. Recently, we reported that NPB promotes the adipogenesis of rat white and brown preadipocytes as well as 3T3-L1 cells. In the present study, we evaluated the effects of NPB on the proliferation and differentiation of white porcine preadipocytes into mature adipocytes. We identified the presence of NPB, NPBWR1, and NPBWR2 on the mRNA and protein levels in porcine white preadipocytes. During the differentiation process, NPB increased the mRNA expression of PPARγ, C/EBPβ, C/EBPα, PPARγ, and C/EBPβ protein production in porcine preadipocytes. Furthermore, NPB stimulated lipid accumulation in porcine preadipocytes. Moreover, NPB promoted the phosphorylation of the p38 kinase in porcine preadipocytes, but failed to induce ERK1/2 phosphorylation. NPB failed to stimulate the expression of C/EBPβ in the presence of the p38 inhibitor. Taken together, we report that NPB promotes the differentiation of porcine preadipocytes via a p38-dependent mechanism.
... We observed that differentiation of hTSCs (Figs. 4 and 7) and freshly isolated CytT from mid-gestation human placenta to SynT (20), resulted in marked induction of NRF2, C/EBPβ, and PPARγ. Moreover, C/EBPβ and PPARγ have been found to act as signaling partners of NRF2 and are implicated in the development of an anti-inflammatory M2 Mϕ phenotype (93,94). Further, C/ EBPβ was reported to be important for B-cell development, whereas C/EBPβ-deficient mice were highly susceptible to Listeria monocytogenes infection (95,96). ...
Using cultured human trophoblast stem cells (hTSCs), mid-gestation human trophoblasts in primary culture, and gene-targeted mice, we tested the hypothesis that the multinucleated syncytiotrophoblast (SynT) serves a critical role in pregnancy maintenance through production of key immune modulators/checkpoint proteins (ICPs) under control of the O2-regulated transcription factor, NRF2/NFE2L2. These ICPs potentially act at the maternal-fetal interface to protect the hemi-allogeneic fetus from rejection by the maternal immune system. Using cultured hTSCs, we observed that several ICPs involved in the induction and maintenance of immune tolerance were markedly upregulated during differentiation of cytotrophoblasts (CytT) to SynT. These included HMOX1, kynurenine receptor, AhR, PD-L1, and GDF15. Intriguingly, NRF2, C/EBPβand PPARγ, were markedly induced when CytT fused to form SynT in a 20% O2 environment. Notably, when hTSCs were cultured in a hypoxic (2% O2) environment, SynT fusion and the differentiation-associated induction of NRF2, C/EBPβ, aromatase (CYP19A1; SynT differentiation marker), and ICPs were blocked. NRF2 knockdown also prevented induction of aromatase, C/EBPβand the above-mentioned ICPs. ChIP-qPCR revealed that temporal induction of the ICPs in hTSCs and mid-gestation human trophoblasts cultured in 20% O2 was associated with increased binding of endogenous NRF2 to putative response elements within their promoters. Moreover, placentas of 12.5 days post-coitum (dpc) mice with a global Nrf2 knockout manifested decreased mRNA expression of C/ebpβ, Pparγ, Hmox1, Ahr, and Nqo1, another direct downstream target of Nrf2, compared wild-type mice. Collectively, these compelling findings suggest that O2-regulated NRF2 serves as a key regulator of ICP expression during SynT differentiation.
... 33,34 Putative C/EBPβ consensus sequences are present in the promoters of many pro-inflammatory genes, which upregulates the response to pro-inflammatory stimuli. [35][36][37][38] Previous studies have revealed that multiple genes related to cell growth and differentiation are regulated by EGR proteins [39][40][41] and that knockout of EGR2 leads to a lethal autoimmune syndrome related to excessive systemic pro-inflammatory cytokines. 42 Moreover, Tatyana showed that EGR2 is involved in the polarization and plasticity of macrophages and high levels of C/EBPβ negatively regulate EGR2 in M1 macrophages. ...
Prolongation of postsurgical pain caused by pre-operative stress is a clinically significant problem, although the mechanisms are not fully understood. Stress can promote the pro-inflammatory activation of microglia, and the transcription factor CCAAT/enhancer-binding protein (C/EBP) β regulates pro-inflammatory gene expression in microglia. Therefore, we speculated that C/EBPβ in spinal microglia may have critical roles in the development of chronic postsurgical pain. Accordingly, in this study, we used a single prolonged stress (SPS) procedure and plantar incisions to evaluate the roles of C/EBPβ in postsurgical pain. Our experiments showed that SPS exposure prolonged mechanical allodynia, increased the expression of C/EBPβ and pro-inflammatory cytokines, and potentiated the activation of spinal microglia. Subsequently, microinjection of C/EBPβ siRNA attenuated the duration of SPS-prolonged postoperative mechanical allodynia and inhibited microglial activation in the spinal cord. Conversely, mimicking this increase in C/EBPβ promoted microglial activation via pretreatment with a pre-injection of AAV5-C/EBPβ, leading to prolongation of postsurgical pain. Overall, these results suggested that spinal microglia may play key roles in prolongation of postsurgical pain induced by pre-operative stress and that C/EBPβ may be a potential target for disease treatment.
... Hypomethylated DMRs and DARs in Scnn1b-Tg AMs were significantly enriched for TFs known to regulate inflammatory responses and macrophage polarization. For example, we identified highly enriched motifs for C/EBP, IRF2, IRF3, and STAT6 (Fig. 1j) in DMRs and motifs for the inflammation associated TFs JUNB, FRA1, NFkB-p65 (RELA), and the M2 polarizing TF ATF3 in DARs (Fig. 1k) [34][35][36][37][38] . Prediction of potential master regulators of changes in chromatin accessibility revealed cytokines such as TGF-β, TNF, IFN-γ, IL-4, as well as bacterial-derived LPS as activators in Scnn1b-Tg AMs (Fig. 1l). ...
Lung diseases, such as cystic fibrosis and COPD, are characterized by mucus obstruction and chronic airway inflammation, but their mechanistic link remains poorly understood. Here, we focus on the function of the mucostatic airway microenvironment on epigenetic reprogramming of airway macrophages (AM) and resulting transcriptomic and phenotypical changes. Using a mouse model of muco-obstructive lung disease ( Scnn1b -transgenic), we identify epigenetically controlled, differentially regulated pathways and transcription factors involved in inflammatory responses and macrophage polarization. Functionally, AMs from Scnn1b -transgenic mice have reduced efferocytosis and phagocytosis, and excessive inflammatory responses upon lipopolysaccharide challenge, mediated through enhanced Irf1 function and expression. Ex vivo stimulation of wild-type AMs with native mucus impairs efferocytosis and phagocytosis capacities. In addition, mucus induces gene expression changes, comparable with those observed in AMs from Scnn1b -transgenic mice. Our data show that mucostasis induces epigenetic reprogramming of AMs, leading to changes favoring tissue damage and disease progression. Targeting these altered AMs may support therapeutic approaches in patients with muco-obstructive lung diseases.
... It was first shown to directly activate the expression of the anti-inflammatory associated genes Il10 [47] and Arg1 [48] in activated macrophages in vitro. In macrophages activated by LPS in vitro, Cebpb was transcriptionally activated by the cAMP responsive element binding protein (CREB) transcription factor through its binding to 2 cAMP response elements (CREs) in the Cebpb promoter [49,50]. To study the role of C/EBPβ during skeletal muscle regeneration, Ruffell and colleagues generated a mouse model where the 2 CREs in the Cbepb promoter were mutated [51]. ...
Skeletal muscle is a tissue able to fully regenerate after an acute injury. Macrophages play an essential role during skeletal muscle regeneration. Resolution of inflammation is a crucial step during the regeneration process, allowing to contain the inflammatory response to avoid damage of the healthy surrounding muscle and triggers the recovery phase during which the muscle regenerates. Resolution of inflammation is mainly mediated by macrophage phenotypic shift that is the transition from a pro-inflammatory damage associated profile towards an anti-inflammatory restorative phenotype, which is characterized by a major transcriptional rewiring. Failure of the resolution of inflammation is observed in chronic diseases such as degenerative myopathies where permanent asynchronous muscle injuries trigger contradictory inflammatory cues, leading to fibrosis and alteration of muscle function. This review will focus on the described molecular pathways that control macrophage inflammatory shift during skeletal muscle regeneration. First, we will highlight the transcriptional changes that characterize macrophage inflammatory shift during skeletal muscle regeneration. Then, we will describe how the signaling pathways and the metabolic changes associated with this shift are controlled. Finally, we will emphasize the transcription factors involved.
... Other candidate TFs, which can shape macrophage behavior, include cAMP response element-binding protein (СREB) and CCAAT-enhancer-binding proteins (C/EBPs) TF family [235][236][237][238]. The phosphorylated CREB inhibits NF-kB activation, thus limiting proinflammatory responses [239,240]. ...
Tumor-associated macrophages (TAMs) are the essential components of the tumor microenvironment. TAMs originate from blood monocytes and undergo pro- or anti-inflammatory polarization during their life span within the tumor. The balance between macrophage functional populations and the efficacy of their antitumor activities rely on the transcription factors such as STAT1, NF-κB, IRF, and others. These molecular tools are of primary importance, as they contribute to the tumor adaptations and resistance to radio- and chemotherapy and can become important biomarkers for theranostics. Herein, we describe the major transcriptional mechanisms specific for TAM, as well as how radio- and chemotherapy can impact gene transcription and functionality of macrophages, and what are the consequences of the TAM-tumor cooperation.
... C/EBPβ also plays a critical role in inflammation [25]. Promoters of many pro-inflammatory genes contain putative C/EBPβ consensus sequences [26], and C/EBPβ levels are upregulated in response to proinflammatory stimuli in macrophages [27] and glial cells [28]. Upon lipopolysaccharides treatment, C/EBPβ is highly upregulated both in astrocytes and in microglia [29]. ...
The apolipoprotein E ε4 (APOE4) allele is a major genetic risk factor for Alzheimer’s disease (AD), and its protein product, ApoE4, exerts its deleterious effects mainly by influencing amyloid-β (Aβ) and Tau (neurofibrillary tangles, NFTs) deposition in the brain. However, the molecular mechanism dictating its expression during ageing and in AD remains incompletely clear. Here we show that C/EBPβ acts as a pivotal transcription factor for APOE and mediates its mRNA levels in an age-dependent manner. C/EBPβ binds the promoter of APOE and escalates its expression in the brain. Knockout of C/EBPβ in AD mouse models diminishes ApoE expression and Aβ pathologies, whereas overexpression of C/EBPβ accelerates AD pathologies, which can be attenuated by anti-ApoE monoclonal antibody or deletion of ApoE via its specific shRNA. Remarkably, C/EBPβ selectively promotes more ApoE4 expression versus ApoE3 in human neurons, correlating with higher activation of C/EBPβ in human AD brains with ApoE4/4 compared to ApoE3/3. Therefore, our data support that C/EBPβ is a crucial transcription factor for temporally regulating APOE gene expression, modulating ApoE4’s role in AD pathogenesis.
... The antiadipogenic effect of 5 µM is in line with reports showing that a knockdown of C/EBPβ inhibited adipocyte differentiation in 3T3-L1 preadipocytes [45]; however, in our work, CCT did not affect C/EBPδ expression. It is possible that an unaltered C/EBPδ expression can compensate for the decrease in C/EBPβ at the early stages of differentiation [46], thus playing a role in the induction of PPARγ expression. Most carotenoids inhibit the adipogenic process via the repression of PPARγ [47,48]; however, CCT did not alter PPARγ mRNA levels at the FD time-point. ...
Saffron, as a food colorant, has been displaced by low-cost synthetic dyes. These have unhealthy properties; thus, their replacement with natural food colorants is an emerging trend. Obesity is a worldwide health problem due to its associated comorbidities. Crocetin esters (crocins) are responsible for the red saffron color. Crocetin (CCT) exhibits healthful properties. We aimed to broaden the existing knowledge on the health properties of CCT isolated from saffron, to facilitate its consideration as a healthy natural food colorant in the future. We evaluated the ability of CCT (1 and 5 μM) to reduce lipid accumulation during the differentiation of 3T3-L1 preadipocytes. Intracellular fat was quantified by Oil Red O staining. CTT cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The number and size of lipid droplets were analyzed using WimLipid software. The expression of adipogenic genes (CCAAT/enhancer-binding protein (C/EBPβ, C/EBPδ, C/EBPα), and peroxisome proliferator-activated receptor γ (PPARγ)) was analyzed using quantitative real-time PCR (qRT-PCR). CCT 5 μM decreased intracellular fat by 22.6%, without affecting viability or lipid droplet generation, via a decrease in C/EBPα expression, implicated in lipid accumulation. Thus, CCT is a potential candidate to be included in dietary therapies aimed at reversing adipose tissue accumulation in obesity.
... C/EBPβ is implicated in regulating proinflammatory gene expression in glial activation [23]. Promoters of many pro-inflammatory genes contain putative C/EBPβ consensus sequences [36,37] and C/EBPβ levels are upregulated in response to pro-inflammatory stimuli in macrophages [38] and glial cells [39][40][41][42]. Interestingly, C/EBPβ deficiency provides neuroprotection following ischemic [43] or excitotoxic injuries [44]. ...
Parkinson’s disease (PD) is characterized by dopaminergic neuronal loss and the presence of intra-neuronal Lewy body (LB) inclusions with aggregated α-synuclein (α-Syn) as the major component. MAOB, a crucial monoamine oxidase for dopamine metabolism, triggers oxidative stress in dopaminergic neurons and α-Syn aggregation. However, the key molecular mechanism that mediates PD pathogenesis remains elusive. Here we show that C/EBPβ acts as an age-dependent transcription factor for both α-Syn and MAOB, and initiates the PD pathologies by upregulating these two pivotal players, in addition to escalating δ-secretase activity to cleave α-Syn and promotes its neurotoxicity. Overexpression of C/EBPβ in human wild-type α-Syn transgenic mice facilitates PD pathologies and elicits motor disorders associated with augmentation of δ-secretase, α-Syn, and MAOB. In contrast, depletion of C/EBPβ from human α-Syn Tg mice abolishes rotenone-elicited PD pathologies and motor impairments via downregulating the expression of these key factors. Hence, our study supports that C/EBPβ/δ-secretase signaling mediates PD pathogenesis via regulating the expression and cleavage of α-Syn and MAOB.
... 45 Production of these cytokines is controlled by distinct transcription factors (NF-κB and CCAAT/enhancer-binding protein b, respectively). 116,117 Moreover, mitogen-activated protein/extracellular signal-regulated kinase 3 is implicated in production of IL-6 (and not TNF-α) by lipopolysaccharide-activated macrophages. 118 Taking all this into account, assessing the effects of citrulline supplementation on these transcription factors is suggested for further research. ...
Objective:
Diabetes mellitus is a prevalent endocrine disorder worldwide. Citrulline is an α-amino acid, which is abundant in watermelon, and a precursor of arginine and nitric oxide. Decreased bioavailability of nitric oxide is associated with insulin resistance. The present systematic review focused on the existing evidence of citrulline and watermelon extract effects on metabolic and inflammatory parameters in diabetes mellitus.
Methods:
A systematic search of the databases PubMed, Scopus, EMBASE, ProQuest and Google Scholar was conducted for relevant papers published from inception until October 2018. All clinical trials, animal and in vitro studies published in English-language that assessed the role of citrulline and watermelon extract on diabetes mellitus, were eligible. Studies providing inadequate information were excluded.
Results:
Out of 1262 articles we found, only 8 articles met the inclusion criteria for analysis. In three studies an increase in the synthesis of nitric oxide was reported with citrulline and watermelon extract supplementation. Four studies showed a significant reduction in blood glucose after supplementation with watermelon extract, and two studies reported a decrease in a number of inflammatory biomarkers following citrulline supplementation. Although citrulline intake caused a significant reduction in HOMA-IR in one study, inconsistent results were revealed on the effects of citrulline and watermelon extract on insulin levels and lipid profile.
Conclusion:
Citrulline and watermelon extract could improve nitric oxide synthesis, glycemic status and inflammation in diabetes mellitus. However, further studies are required to shed light on the underlying mechanisms.
... In the brain, these changes are sugges-tive of age-related induction of neuro-inflammation and activation of glia. C/EBPb (coded by the Cebpb gene) is a basicleucine zipper DNA-binding protein that controls expression of cytokines and other pro-inflammatory genes and induces pathways that are critical for glia activation (Bradley et al., 2003;Wang et al., 2018). Apart from being implicated in neuro-inflammation, C/EBPb controls expression of delta-secretase enzyme, which is thought to contribute to the pathology in Alzheimer's disease by cleaving both Tau and beta-amyloid precursor protein . ...
To understand the changes in gene expression thatoccur as a result of age, which might create a permis-sive or causal environment for age-related diseases,we produce a multi-time point age-related geneexpression signature (AGES) from liver, kidney, skel-etal muscle, and hippocampus of rats, comparing6-, 9-, 12-, 18-, 21-, 24-, and 27-month-old animals.We focus on genes that changed in one directionthroughout the lifespan of the animal, either early inlife (early logistic changes), at mid-age (mid-logistic),late in life (late-logistic), or linearly, throughout thelifespan of the animal. The pathways perturbedbecause of chronological age demonstrate organ-specific and more-global effects of aging and pointto mechanisms that could potentially be counter-regulated pharmacologically to treat age-associateddiseases. A small number of genes are regulated byaging in the same manner in every tissue, suggestingthey may be more-universal markers of aging.
... CCAAT-enhancer binding protein (C/EBP-β) is a member of the C/EBP family of bZIP transcription factors that bind DNA as dimers. Through the alternative use of three translation initiation codons, three C/EBP isoforms (called LAP * , LAP, and LIP) are translated from a single mRNA (Bradley et al., 2003). LAP * and LAP function as transcriptional activators, while the LIP function as a transcriptional repressor in a dominant negative fashion because of lacking a transactivation domain. ...
Methamphetamine (MA) is a widely abused psychoactive drug that primarily damages the nervous system. However, the involvement of MA in the survival of microglia remains poorly understood. CCAAT-enhancer binding protein (C/EBP-β) is a transcription factor and an important regulator of cell apoptosis. Lipocalin2 (lcn2) is a known apoptosis inducer and is involved in many cell death processes. We hypothesized that C/EBP-β is involved in MA-induced lcn2-mediated microglial apoptosis. To test this hypothesis, we measured the protein expression of C/EBP-β after MA treatment and evaluated the effects of silencing C/EBP-β or lcn2 on MA-induced apoptosis in BV-2 cells and the mouse striatum after intrastriatal MA injection. MA exposure increased the expression of C/EBP-β and stimulated the lcn2-mediated modulation of apoptosis. Moreover, silencing the C/EBP-β-dependent lcn2 upregulation reversed the MA-induced microglial apoptosis. The in vivo relevance of these findings was confirmed in mouse models, which demonstrated that the microinjection of anti-C/EBP-β into the striatum ameliorated the MA-induced decrease survival of microglia. These findings provide a new insight regarding the specific contributions of C/EBP-β-lcn2 to microglial survival in the context of MA abuse.
... We have previously shown that c/EBPβ is a central regulator of anti-inflammatory macrophages [18] that controls the expression of IRAK-M and several miRNAs involved in macrophage activation [2,19,20]. Elevated levels of c/EBPβ in naive macrophages may indicate a state of reduced M1 activation capacity and a poised state to induce higher levels of anti-inflammatory genes following an appropriate stimulus [21]. ...
Macrophages are central mediators of inflammation, orchestrating the inflammatory response through the production of cytokines and nitric oxide. Macrophages obtain pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, which can be modulated by soluble factors, including natural products. Despite the crucial protective role of inflammation, chronic or deregulated inflammation can lead to pathological states, such as autoimmune diseases, metabolic disorders, cardiovascular diseases, and cancer. In this case, we studied the anti-inflammatory activity of neorogioltriol (1) in depth and identified two structurally related diterpenes, neorogioldiol (2), and O11,15-cyclo-14-bromo-14,15-dihydrorogiol-3,11-diol (3), with equally potent activity. We investigated the mechanism of action of metabolites 1⁻3 and found that all three suppressed macrophage activation and promoted an M2-like anti-inflammatory phenotype by inducing expression of Arginase1, MRC1, IRAK-M, the transcription factor C/EBPβ, and the miRNA miR-146a. In addition, they suppressed iNOS induction and nitric oxide production. Importantly, treatment of mice with 2 or 3 suppressed DSS-induced colitis by reducing tissue damage and pro-inflammatory cytokine production. Thus, all these three diterpenes are promising lead molecules for the development of anti-inflammatory agents targeting macrophage polarization mechanisms.
... In the network model, we included only the ones which seem to be most important. F. Network definition and reference for molecular interactions Myc E2F [6], MAPK [6,7], Notch [8], SHH [9], Wnt [10], pRb [6] AP2 [11], TGF-β [12,13], p53 [6] p21 AP2 [14], GR [15], Gata1/2 [16], HNF4α [17], Runx1 [18], TGF-β [13], TNF-α [19], p53 [6,19] Akt [4], Hes1 [20], Myc [19,21] p27 E-Cadherin [22], Gata1/2 [23], NR4A [24], PTEN [25], TGF-β [26] Akt [25], Hes1 [27], Myc [6] pRb Cyclin D/Cdk4,6 [1,28], Cyclin E/Cdk2 [1,28] Retinoblastoma protein phosphorylation Ras EGFR [29], FGFR2 [30], HGF [31], IL-6 [32,33], IR [34], VEGF [35,36] GR [37], p53 [38] Apoptosis Bad TNF-α [39], p53 [40] Akt [41], MAPK [41], NF-κB [42], p21 [43] Bax Bim [44], p53 [45,46] Akt [47], Bcl-2 [48], Sox4 [49] Bcl-2 Integrin [50], NF-κB [51], VEGF [52] Caspase 3 [52], TGF-β [53], p53 [54] Bcl-xL EGFR [55], NF-κB [56], Stat5 [57] Caspase 3 [58] Bim Akt [41], MAPK [41] Caspase HIF [83], IL-1 [84], MAPK [85], NR2F2 [86], Notch [87], SHH [88], Stat5 [89] C/EBPβ AP2 [90], IL-1 [84], MAPK [91] Hoxa3 [92], TGF-β [93] Continued on next page AP2 [107], Foxa2 [108], RARs [109,110] Sox4 [111] Foxa2 C/EBPα [112], RARs [109], SHH [113] Akt [114], PRDM14 [115] Gata1/2 Cdx2 [116], Notch [117], Stat3 [118] Hes1 [119], Hoxa10 [120], Pu.1 [121,122], VEGF [123], c-Jun [124] Gata4/6 PKA [125], Sox7 [126], Wnt [127] Hey2 [128], PRDM14 [115], Sox2 [100,126,129], c-Jun [130] Hes1 ...
... C/EBPβ has been found to induce the transcriptional activation of XBP1 (61,62) and, conversely, C/EBPβ is induced by XBP1 via an X-box present in human cells that is absent in rats and mice (63), as well as through the PERK route (64). In addition, C/EBPβ is fully active in unstimulated macrophages and poised to be recruited into enhanceosomes to team up with other factors to stimulate transcription, given its well-known function as a pioneer factor that promotes the opening of silent chromatin (49,65,66). The present results are of clinical relevance because DCs in tumor milieus show an activation of XBP1 that blunts the immune response and favors tumor cell progression (13). ...
Human monocyte-derived dendritic cells (DCs) exposed to pathogen-associated molecular patterns (PAMPs) undergo bioenergetic changes that influence the immune response. We found that stimulation with PAMPs enhanced glycolysis in DCs, whereas oxidative phosphorylation remained unaltered. Glucose starvation and the hexokinase inhibitor 2-deoxy-d-glucose (2-DG) modulated cytokine expression in stimulated DCs. Strikingly, IL23A was markedly induced upon 2-DG treatment, but not during glucose deprivation. Since 2-DG can also rapidly inhibit protein N-glycosylation, we postulated that this compound could induce IL-23 in DCs via activation of the endoplasmic reticulum (ER) stress response. Indeed, stimulation of DCs with PAMPs in the presence of 2-DG robustly activated inositol-requiring protein 1α (IRE1α) signaling and to a lesser extent the PERK arm of the unfolded protein response. Additional ER stressors such as tunicamycin and thapsigargin also promoted IL-23 expression by PAMP-stimulated DCs. Pharmacological, biochemical, and genetic analyses using conditional knockout mice revealed that IL-23 induction in ER stressed DCs stimulated with PAMPs was IRE1α/X-box binding protein 1-dependent upon zymosan stimulation. Interestingly, we further evidenced PERK-mediated and CAAT/enhancer-binding protein β-dependent trans-activation of IL23A upon lipopolysaccharide treatment. Our findings uncover that the ER stress response can potently modulate cytokine expression in PAMP-stimulated human DCs.
... The second TF that we focused on, C/EBPβ, is a regulator of M1/M2 polarization of macrophages. It is expressed at basal levels in M1 macrophages but upregulated for M2 activation[53]. Deletion of two CREB-binding sites from the CEBPβ promoter abrogates its induction upon macrophage activation[16], linking the TF CREB with CEBPβ. CREB-C/EBPβ signaling has been linked to induction of the M2-specific genes Msr1, Il10, II13ra, and Arg1. ...
For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 β (IL-1β) and tumor necrosis factor α (TNF-α). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPβ signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPβsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.
... MAPK signaling is also necessary for LPS-dependent IL-10 induction through C/EBPβ [21]. In murine macrophages, C/EBPβ transcription, DNA binding [55] and cytokine production [62][63][64] is induced through p-38 kinase. In our model, zinc-mediated inhibition of C/EBPβ accumulation and phosphorylation in human macrophages did not decrease p38 phosphorylation (Fig 5E), which indicates that zinc modulates the activity of a yet to be identified kinase(s) or phosphatase(s) upstream of C/EBPβ in a TLR4-dependent signaling pathway. ...
Two vital functions of the innate immune system are to initiate inflammation and redistribute micronutrients in favor of the host. Zinc is an essential micronutrient used in host defense. The zinc importer ZIP8 is uniquely induced through stimulation of the NF-κB pathway by LPS in monocytes and functions to regulate inflammation in a zinc-dependent manner. Herein we determined the impact of zinc metabolism following LPS-induced inflammation in human macrophages. We observed that ZIP8 is constitutively expressed in resting macrophages and strikingly elevated following LPS exposure, a response that is unique compared to the 13 other known zinc import proteins. During LPS exposure, extracellular zinc concentrations within the physiological range markedly reduced IL-10 mRNA expression and protein release but increased mRNA expression of TNFα, IL-8, and IL-6. ZIP8 knockdown inhibited LPS-driven cellular accumulation of zinc and prevented zinc-dependent reduction of IL-10 release. Further, zinc supplementation reduced nuclear localization and activity of C/EBPβ, a transcription factor known to drive IL-10 expression. These studies demonstrate for the first time that zinc regulates LPS-mediated immune activation of human macrophages in a ZIP8-dependent manner, reducing IL-10. Based on these findings we predict that macrophage zinc metabolism is important in host defense against pathogens.
... Thus, having a population of TFs searching simultaneously is critical. In yeast, the number of TF molecules per cell can range from 4 × 10 3 -2.5 × 10 6 -where increasing concentration reduces the effective search time [15,[35][36][37][38]. However, even at high concentrations not all gene targets will be saturated by TFbinding; there will still be many instances when target loci remain vacant [15,36]. ...
Dynamic reprogramming of transcriptional networks enables cells to adapt to a changing environment. Thus, it is crucial not only to understand what gene targets are regulated by a transcription factor (TF) but also when. This review explores the way TFs function with respect to time, paying particular attention to discoveries made in plants - where coordinated, genome-wide responses to environmental change is crucial to the survival of these sessile organisms. We investigate the molecular mechanisms that mediate transient TF-DNA binding, and assess how these rapid and dynamic interactions translate to long-term temporal regulation of genomes. We also discuss how current molecular techniques can catch, and sometimes miss, transient TF-target interactions that underlie dynamic cellular responses.
... Thus, our data show that poly(I:C) induces IFN-γ and IL-12 in BAL, and IFNα and IL-12 production by alveolar macrophages 22,44 . Also while blocking IL-12 in our model diminishes the Th1 response induced by poly(I:C), treating mice with IL-10 both blocks IL-12 production and prevents the Th1 differentiation 45,46 . Interestingly two different cell types are mainly involved: whilst we show that alveolar macrophages are the major cell type producing Th1-polarizing cytokines, small macrophages are restricted to make Th17-inducing cytokines. ...
Infections caused by bacteria in the airway preferentially induce a Th17 response. However, the mechanisms involved in the regulation of CD4 T-cell responses in the lungs are incompletely understood. Here, we have investigated the mechanisms involved in the regulation of Th17 differentiation in the lungs in response to immunization with lipopolysaccharide (LPS) as an adjuvant. Our data show that both Myd88 and TRIF are necessary for Th17 induction. This distinctive fate determination can be accounted for by the pattern of inflammatory cytokines induced by airway administration of LPS. We identified the production of interleukin (IL)-1β and IL-6 by small macrophages and IL-23 by alveolar dendritic cells (DCs), favoring Th17 responses, and IL-10 repressing interferon (IFN)-γ production. Furthermore, we show that exogenous IL-1β can drastically alter Th1 responses driven by influenza and lymphocytic choriomeningitis virus infection models and induce IL-17 production. Thus, the precision of the lung immune responses to potential threats is orchestrated by the cytokine microenvironment, can be repolarized and targeted therapeutically by altering the cytokine milieu. These results indicate that how the development of Th17 responses in the lung is regulated by the cytokines produced by lung DCs and macrophages in response to intranasal immunization with LPS adjuvant.Mucosal Immunology advance online publication, 22 June 2016; doi:10.1038/mi.2016.54.
... The CCAAT enhancer binding proteins C/EBP␣ and C/EBP are critical transcription factors for myelomonocytic lineage commitment, granulocyte differentiation and macrophage function (18)(19)(20). Expression of C/EBP␣ and - proteins are strictly controlled at the mRNA-translation initiation level (21)(22)(23). From consecutive initiation codons in the C/EBPα mRNA three different protein isoforms are synthesised. ...
Mutations in the Shwachman–Bodian–Diamond Syndrome (SBDS) gene cause Shwachman–Diamond Syndrome (SDS), a rare congenital disease
characterized by bone marrow failure with neutropenia, exocrine pancreatic dysfunction and skeletal abnormalities. The SBDS
protein is important for ribosome maturation and therefore SDS belongs to the ribosomopathies. It is unknown, however, if
loss of SBDS functionality affects the translation of specific mRNAs and whether this could play a role in the development of the clinical features of SDS. Here, we report that translation of
the C/EBPα and -β mRNAs, that are indispensible regulators of granulocytic differentiation, is altered by SBDS mutations or knockdown. We show that SBDS function is specifically required for efficient translation re-initiation into
the protein isoforms C/EBPα-p30 and C/EBPβ-LIP, which is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5′ untranslated regions (5′ UTRs) of both mRNAs. Furthermore, we show
that as a consequence of the C/EBPα and -β deregulation the expression of MYC is decreased with associated reduction in proliferation, suggesting that failure of progenitor proliferation contributes
to the haematological phenotype of SDS. Therefore, our study provides the first indication that disturbance of specific translation
by loss of SBDS function may contribute to the development of the SDS phenotype.
... L'expression de C/EBPβ est régulée positivement par IL-1, IL-6 et TNF dans un cadre inflammatoire (Labialle et al., 2002). D'autre part, C/EBPβ contrôle la régulation de diverses cytokines et chimiokines dont IL-6, IL-1β, IL-8, IL-12, TNFα et MCP-1 (Bradley et al., 2003). Des travaux montrent que le TNF augmente la quantité d'ARNm de MDR1 ainsi que l'expression de la Pgp dans des cellules endothéliales cérébrales microvasculaires (Yu et al., 2007). ...
The central nervous system is a complex and privileged organ protected by, among others, the blood-brain barrier (BBB). On this barrier are localised efflux pumps such as P-glycoprotein. The BBB is involved in neuro-inflammation associated to many cerebral diseases.
The endothelin 1 (ET-1) peptide seems to play a key role in the process. We have used an in vitro human autologous adult BBB model and an in vitro human foetal BBB model (co-culture of glial cells and brain microvascular endothelial cells) to evaluate ET-1's influence on the P-glycoprotein.
A difference was shown between the two models concerning Pgp's activity modulation by ET-1. We have also been looking at ET-1 induced Monocyte chemoattractant protein 1 and interleukin 8 secretions. The results show that ET-1 reduces Pgp's activity and induces MCP-1 and IL-8 secretion.
... More is known about their role in macrophages and given the ontogenic and functional relationship between both cell types we think it is important to summarize the literature on the role of C/EBPβ and C/EBPδ in macrophages since it may offer clues about their roles in microglia. The upregulation of C/EBPβ and C/EBPδ expression by proinflammatory stimuli in macrophages has been well characterized (Bradley et al. 2003;Litvak et al. 2009). In activated macrophages C/EBPβ promotes the expression of proinflammatory cytokines and NO-and PGE2-producing enzymes (Gorgoni et al. 2001;Rahman et al. 2012;Screpanti et al. 1995;Tanaka et al. 1995;Uematsu et al. 2002;Yan et al. 2012b). ...
CCAAT/enhancer binding protein (C/EBP) β and C/EBPδ are transcription factors of the basic-leucine zipper class which share phylogenetic, structural and functional features. In this review we first describe in depth their basic molecular biology which includes fascinating aspects such as the regulated use of alternative initiation codons in the C/EBPβ mRNA. The physical interactions with multiple transcription factors which greatly opens the number of potentially regulated genes or the presence of at least five different types of post-translational modifications are also remarkable molecular mechanisms that modulate C/EBPβ and C/EBPδ function. In the second part, we review the present knowledge on the localization, expression changes and physiological roles of C/EBPβ and C/EBPδ in neurons, astrocytes and microglia. We conclude that C/EBPβ and C/EBPδ share two unique features related to their role in the CNS: whereas in neurons they participate in memory formation and synaptic plasticity, in glial cells they regulate the pro-inflammatory program. Because of their role in neuroinflammation, C/EBPβ and C/EBPδ in microglia are potential targets for treatment of neurodegenerative disorders. Any strategy to reduce C/EBPβ and C/EBPδ activity in neuroinflammation needs to take into account its potential side-effects in neurons. Therefore, cell-specific treatments will be required for the successful application of this strategy.
Copyright © 2015. Published by Elsevier Ltd.
... (1) The expression and function of C ⁄ EBPb are regulated in a complex way during transcription, translation, post-translational modification, and protein-protein interactions. (4)(5)(6)(7)(8) Notably, alternative translation through the use of different initiation codons generates three different isoforms of C ⁄ EBPb: liverenriched activating protein* (LAP* or full-length), liverenriched activating protein (LAP), and liver-enriched inhibitory protein (LIP) (Fig. 1). (6) Both LAP* and LAP are transcriptional activators, whereas LIP (which is the shortest isoform and lacks transactivation domains but retains DNA binding and dimerization domains) acts as a repressor or a dominant negative inhibitor of other C ⁄ EBP family transcription factors. ...
Steady-state hematopoiesis responds to extracellular stimuli to meet changing demands and also to pathologically-altered intracellular signaling. Granulocyte production increases upon infection or in response to cytokine stimulation, and activation of the C/EBPβ transcription factor is required for such stress-induced granulopoiesis, whereas C/EBPα plays a critical role in maintaining steady-state granulopoiesis. Such different roles of these C/EBP transcription factors in different modes of hematopoiesis are evolutionally conserved from zebrafish to humans. In addition to reactions against infections, C/EBPβ is responsible for cancer-driven myelopoiesis, which promotes cancer progression at least in part by abrogating the immune response in the cancer microenvironment. The BCR-ABL fusion protein activates emergency-specific pathway of granulopoiesis by up-regulating C/EBPβ. This in turn causes chronic phase chronic myeloid leukemia, which is characterized by myeloid expansion. C/EBPβ also plays a role in other hematological malignancies of both myeloid- and lymphoid lineage-origin. Thus, elucidation of the upstream and downstream networks surrounding C/EBPβ will lead to the development of novel therapeutic strategies for diseases mediated by non-steady-state hematopoiesis. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... To test for methodological error, we measured the number of molecules of the transcription factor Ikaros in total thymocytes using identical methods and a recombinant Ikaros protein standard (see "Experimental Procedures"). Two determinations yielded values of ϳ150,000 and ϳ250,000 Ikaros molecules per thymocyte (Western blots not shown), similar to the previously determined value of ϳ250,000 (46). ...
The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pull down assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (KD ~0.4 μM). Using the Hermes transposase as a guide, we constructed a 36 kD "mini" RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and residues N-terminal to it, but lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (aa 997-1008) near the RAG1 C-terminus and a region of RAG1 from aa 479-559. Two adjacent acidic amino acids in this region (D546 and E547) are important for both the RAG1-RAG2 interaction and recombination activity, with D546 of particular importance. Structural modeling of Mini-RAG1 suggests that D546/E547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative western blotting allowed us to estimate that mouse thymocytes contain on average ~1,800 monomers of RAG1 and ~15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the KD for their interaction, which could help limit off-target RAG activity.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... The increase in mRNA expression levels of C/EBPβ and C/EBPδ correlates with similar results in other studies showing a differential regulation of C/EBP isoforms in response to LPS and different cytokines. This has been shown by RT-PCR analysis of macrophages, where in general, the expression of C/EBPα was reduced and that of C/EBPβ and C/EBPδ was induced in the presence of LPS (Tengku-Muhammad et al., 2000;Bradley et al., 2003). An additional RT-PCR study indicates that C/EBPα, C/EBPβ and C/EBPδ mRNA expression is upregulated in rat glomerular mesangial cells after LPS treatment (Granger et al., 2000a). ...
... Notably, among the putative mechanisms of this effect, the production of these cytokines is controlled by different transcription factors, e.g. CCAAT/enhancer-binding protein b for IL-6 (26) or NF-kB for TNF-a (27) . Moreover, mitogen-activated protein/extracellular signal-regulated kinase kinase 3 has been considered to be essential for IL-6 production by lipopolysaccharide-activated macrophages, unlike TNF-a (28) . ...
In type 2 diabetes (T2D) macrophage dysfunction increases susceptibility to infection and mortality. This may result from the associated decreased plasma concentration of arginine, an amino acid that plays an important role in immunity.
In vitro
, increasing arginine availability leads to an improvement in macrophage function; however, arginine supplementation in diabetic obese patients may be detrimental. The aim of the present study was to assess
in vitro
whether citrulline, an arginine precursor, could replace arginine in the regulation of macrophage function under a condition of diabetes and obesity. Peritoneal macrophages from diabetic obese or lean rats were incubated for 6 h in an arginine-free medium, in the presence of increasing citrulline concentrations (0·1, 0·5, 1 or 2 mmol/l). Cytokine and NO production was determined. Peritoneal macrophages from either lean or diabetic obese rats produced NO, and at higher levels in the cells from lean rats. In diabetic obese rats, TNF-α production decreased with increasing citrulline concentrations, but was higher than that in the cells from lean rats. In contrast, IL-6 production increased with increasing citrulline concentrations. The present experiment shows that citrulline is effectively used for NO production and regulates cytokine production in macrophages from diabetic obese rats. This effect warrants
in vivo
evaluation in T2D-related inflammation.
... We first investigated whether TGR5 could regulate C/EBPβ expression. In accordance with the proinflammatory role of C/EBPβ (56), stimulation of primary macrophages with LPS induced C/EBPβ expression, at both the mRNA and the protein level (Figure 7, A-C). Notably, activation of TGR5 by INT-777 in LPS-treated BMDMs did not affect the levels of Cebpb mRNA, but reversed the ratio of C/EBPβ isoforms, marked by an increase in LIP protein (Figure 7, A-C). ...
The bile acid-responsive G protein-coupled receptor TGR5 is involved in several metabolic processes, and recent studies suggest that TGR5 activation may promote pathways that are protective against diet-induced diabetes. Here, we investigated the role of macrophage-specific TGR5 signaling in protecting adipose tissue from inflammation and associated insulin resistance. Examination of adipose tissue from obese mice lacking macrophage Tgr5 revealed enhanced inflammation, increased chemokine expression, and higher macrophage numbers compared with control obese animals. Moreover, macrophage-specific deletion of Tgr5 exacerbated insulin resistance in obese animals. Conversely, pharmacological activation of TGR5 markedly decreased LPS-induced chemokine expression in primary macrophages. This reduction was mediated by AKT-dependent activation of mTOR complex 1, which in turn induced the differential translation of the dominant-negative C/EBPβ isoform, liver inhibitory protein (LIP). Overall, these studies reveal a signaling pathway downstream of TGR5 that modulates chemokine expression in response to high-fat diet and suggest that targeting this pathway has the potential to be therapeutically exploited for prevention of chronic inflammatory diseases and type 2 diabetes mellitus.
... 30 It is quite possible that different cell types differ in their activation of C/EBPb in response to inflammatory stimuli. 44 Alternatively, the regulation mechanism of the chicken 5-HTT gene in response to LPS might be different from that of the human 5-HTT gene. Indeed, a careful sequence analysis of the 1.9-kb 5 0 -flanking region of the chicken 5-HTT gene and the human 5-HTT gene revealed that the sequences considerably differ from each other. ...
Serotonin (5-hydroxytryptamine; 5-HT) transporter (5-HTT) is involved in inflammation and the stress response. In this study, we examined the regulation of 5-HTT expression in macrophage HD11 cells in response to bacterial LPS. Long-term exposure of cells to LPS (6-18 h) produced a decrease in 5-HTT mRNA expression. Accordingly, reduced 5-HTT activity measured by 5-HT uptake was also observed in LPS-treated HD11 cells. Moreover, LPS treatment, as well as co-transfection with an expression vector encoding the chicken CCAAT/enhancer binding protein beta (C/EBPβ), resulted in inhibition of 5-HTT promoter activity. Indeed, sequence analysis revealed several C/EBPβ binding motifs in the upstream region of the 5-HTT gene, which specifically interacted with C/EBPβ both in an in vitro band shift assay and in living HD11 cells. The C/EBPβ binding was activated in cells treated with LPS. The role of C/EBPβ in LPS inhibition of 5-HTT expression was further confirmed by small interfering RNA interference, which demonstrated that knockdown of endogenous C/EBPβ attenuated the inhibition of 5-HTT expression in LPS-treated cells. Taken together, the results suggest that C/EBPβ plays a critical role in regulating the 5-HTT gene in macrophages in response to pro-inflammatory stimuli.
... In fact, activation of ATF2 by tunicamycin and the cooperation of ATF2 with ATF4 to induce CHOP transcription under amino acid deprivation have been reported (39). The enhancement of C/EBP binding to CHOP-C/EBP and C/EBP sites by LPS can also explain the strong ability of LPS to activate il12/23b expression, which is regulated by the combined activation of NF-B and C/EBP (40). The path- ways triggered by zymosan revolve around the -glucan receptor dectin-1, which, unlike the TLR4/LPS route, activates the tyrosine kinase SYK, phospholipase C␥2, and the Ca 2ϩ and diacylglycerol/PKC routes. ...
Current views on the control of IL-23 production focus on the regulation of il23a, the gene encoding IL-23 p19, by NF-κB in combination with other transcription factors. C/EBP homologous protein (CHOP),
X2-Box-binding protein 1 (XBP1), activator protein 1 (AP1), SMAD, CCAAT/enhancer-binding protein (C/EBPβ), and cAMP-response
element-binding protein (CREB) have been involved in response to LPS, but no data are available regarding the mechanism triggered
by the fungal mimic and β-glucan-containing stimulus zymosan, which produces IL-23 and to a low extent the related cytokine
IL-12 p70. Zymosan induced the mobilization of CHOP from the nuclear fractions to phagocytic vesicles. Hypha-forming Candida also induced the nuclear disappearance of CHOP. Assay of transcription factor binding to the il23a promoter showed an increase of Thr(P)-71–Thr(P)-69-activating transcription factor 2 (ATF2) binding in response to zymosan.
PKC and PKA/mitogen- and stress-activated kinase inhibitors down-regulated Thr(P)-71–ATF2 binding to the il23a promoter and il23a mRNA expression. Consistent with the current concept of complementary phosphorylations on N-terminal Thr-71 and Thr-69 of
ATF2 by ERK and p38 MAPK, MEK, and p38 MAPK inhibitors blunted Thr(P)-69–ATF2 binding. Knockdown of atf2 mRNA with siRNA correlated with inhibition of il23a mRNA, but it did not affect the expression of il12/23b and il10 mRNA. These data indicate the following: (i) zymosan decreases nuclear proapoptotic CHOP, most likely by promoting its accumulation
in phagocytic vesicles; (ii) zymosan-induced il23a mRNA expression is best explained through coordinated κB- and ATF2-dependent transcription; and (iii) il23a expression relies on complementary phosphorylation of ATF2 on Thr-69 and Thr-71 dependent on PKC and MAPK activities.
... IL-10 promoter contained several binding sites for C/EBPb, which were required for LPS-induced transactivation of IL-10 (Liu et al. 2003). It is also reported that C/EBPb was induced by LPS with or without IFN-c stimulation (Bradley et al. 2003;Ruffell et al. 2009), which was shown to be necessary for the production of IL-10 in macrophages (Ruffell et al. 2009). In the present study, the role of C/EBPb in the increased expression of IL-10 in Siglec-9-expressing cells was studied. ...
Interleukin-10 (IL-10) expression was significantly elevated upon stimulation with lipopolysaccharide (LPS) when the sialic acid-recognizing Ig-superfamily lectin Siglec-5 or -9 was overexpressed in RAW264 cells. During the course to clarify the mechanism for this activation, we found that IL-10 promoter proximal region up to -500 bp led to transactivation similar to that up to -1,500 bp. Among the transcription factors that activate the mouse IL-10 promoter so far reported, the level of C/EBPβ was increased in Siglec-9-expressing cells. Transient expression of the C/EBPβ major isoform LAP led to an increase in the expression of IL-10 in Siglec-9-expressing cells, but not in mock-transfected control RAW264 cells upon stimulation with LPS, as assessed by either a luciferase assay or the production of IL-10. Without LPS, the IL-10 promoter was activated by transiently expressed LAP in Siglec-9-expressing cells, however, the magnitude of transactivation was less than that with the LPS stimulation. The knockdown of C/EBPβ down-regulated the production of IL-10. Taken together, these results suggest that one of the reasons for the stimulation of IL-10 expression in Siglec-9-expressing cells may be an increase in intracellular C/EBPβ level.
Cerebral ischemic stroke and glioma are the two leading causes of patient mortality globally. Despite physiological variations, 1 in 10 people who have an ischemic stroke go on to develop brain cancer, most notably gliomas. In addition, glioma treatments have also been shown to increase the risk of ischemic strokes. Stroke occurs more frequently in cancer patients than in the general population, according to traditional literature. Unbelievably, these events share multiple pathways, but the precise mechanism underlying their co-occurrence remains unknown. Transcription factors (TFs), the main components of gene expression programmes, finally determine the fate of cells and homeostasis. Both ischemic stroke and glioma exhibit aberrant expression of a large number of TFs, which are strongly linked to the pathophysiology and progression of both diseases. The precise genomic binding locations of TFs and how TF binding ultimately relates to transcriptional regulation remain elusive despite a strong interest in understanding how TFs regulate gene expression in both stroke and glioma. As a result, the importance of continuing efforts to understand TF-mediated gene regulation is highlighted in this review, along with some of the primary shared events in stroke and glioma.
Hepatoblastoma (HB) is the predominant hepatic neoplasm in infants and young children. Sorafenib has been used to treat adult and pediatric hepatocellular carcinoma. However, efficacy of monotherapy of sorafenib in HB is not sustained. In this study, we tested a possible combinatory therapy of sorafenib with the CCAAT/enhancer‐binding proteins (C/EBP) overexpression in HB cell line. Firstly, we evaluated the expression level of C/EBPβ in the patients with HB by analyzing TCGA data. Lower level of C/EBPβ was observed in tumor tissues in comparison with matched normal tissues. Next, we observed that combination of sorafenib and C/EBPβ overexpression led to dramatic growth and migration inhibition of live tumor cells which implied promising probability for clinical trial. Mechanistically, C/EBPβ which can be downregulated by Ras v12, augmented mRNA and protein levels of p53. These data suggested that a combination of sorafenib and C/EBPβ overexpression inhibited tumor growth synergistically and provided a promising approach to treat HB.
This article is protected by copyright. All rights reserved.
Interactions between pattern recognition receptors (PRRs) shape innate immune responses to particular classes of pathogens. Here, we review interactions between TLRs and nucleotide‐binding oligomerization domain 1 and 2 (NOD1 and NOD2) receptors, two major groups of PRRs involved in innate recognition of bacteria. Most of experimental data both in vitro and in vivo suggest that NODs and TLRs synergize with each other at inducing the production of cytokines and antimicrobial peptides. Molecular mechanisms of this synergy remain poorly understood, although several scenarios can be proposed: (i) direct interactions of signaling pathways downstream of NODs and TLRs; (ii) mutual transcriptional regulation of unique components of NOD‐dependent and TLR‐dependent signaling pathways; and (iii) interactions at the post‐transcriptional level. Potential practical implications of NOD‐TLR synergy are dual. In sepsis, where synergistic effects probably contribute to excessive proinflammatory cytokine production, blockade of NOD1, and/or NOD2 in addition to TLR4 blockade may be required to achieve therapeutic benefit. On the other hand, synergistic combinations of relatively small doses of NOD and TLR agonists administered before infection could be used to boost innate resistance against bacterial pathogens. NOD1/NOD2 and TLRs synergize at induction of innate immune responses, which can be beneficial (boosting protective immunity) or deleterious (in sepsis)
An inducible program of inflammatory gene expression is a hallmark of antimicrobial defenses. Recently, cellular nucleic acid–binding protein (CNBP) was identified as a regulator of nuclear factor-kappaB (NF-κB)–dependent proinflammatory cytokine gene expression. Here, we generated mice lacking CNBP and found that CNBP regulates a very restricted gene signature that includes IL-12β. CNBP resides in the cytosol of macrophages and translocates to the nucleus in response to diverse microbial pathogens and pathogen-derived products. Cnbp -deficient macrophages induced canonical NF-κB/Rel signaling normally but were impaired in their ability to control the activation of c-Rel, a key driver of IL-12β gene transcription. The nuclear translocation and DNA-binding activity of c-Rel required CNBP. Lastly, Cnbp -deficient mice were more susceptible to acute toxoplasmosis associated with reduced production of IL-12β, as well as a reduced T helper type 1 (Th1) cell IFN-γ response essential to controlling parasite replication. Collectively, these findings identify CNBP as important regulator of c-Rel–dependent IL-12β gene transcription and Th1 immunity.
Interferon-inducible PYHIN protein family includes the DNA-binding proteins, AIM2 and IFI16, which form ASC-caspase 1 dependent inflammasomes, important in immunity against cytosolic bacteria, DNA viruses and HIV. The role of other members of this family in the recognition of DNA and/or regulation of immune responses is unclear. We identified an immune regulatory function of p205, another member of the PYHIN family, in the transcriptional control of immune genes. Knockdown of p205 in macrophages revealed that inflammasome activation due to dsDNA and ligands that engage the NLRP3 inflammasome were severely compromised. Detailed mechanistic analysis showed that loss of p205 was associated with a decrease in Asc mRNA and protein levels. p205 knockdown resulted in reduced RNA Polymerase II-mediated endogenous Asc gene transcription and mRNA processing, suggesting a co-transcriptional control of Asc gene expression. Ectopically expressed p205 induced expression of an Asc gene-luciferase reporter and collaborated with other transcription factors, such as c/EBPβ, p65/RelA, to further enhance expression. p205 knockdown also affected the expression of the immune genes Cd86, Cox2, Cxcl2, Il1α, Il10, Il12α, Il6 and Ifnα in LPS-stimulated macrophages. Together these findings suggest that p205 regulates inflammation through control of Asc gene expression, and other immune genes. Fungal infections activate both caspase 1-dependent and -independent inflammasomes. In an independent study, we show that Paracoccidioides brasiliensis fungal infection also induces caspase 8-dependent non-canonical inflammasome. Caspase 8-dependent IL-1β processing required dectin-1, Syk and Asc. Rip3-/- Casp8-/- mice infected with P. brasiliensis displayed increased fungal load and showed worse disease progression compared to wild type and Rip3-/- mice. These results revealed the importance of caspase 8 in activating and regulating inflammasome responses during fungal infection in vivo.
Neuroinflammation, a neuroprotective process, which facilitates recovery is “driven” by activated resident glial cells (astrocytes and microglia). The activation of glial cells is accompanied by increase in production of TNF-α, IL-1β, and IL-6, chemokines, NO, prostaglandin E2, PAF, and ROS along with invasion of circulating immune cells, which secrete more proinflammatory cytokines. Increase in cytokines is promoted by increase in inflammatory signaling pathways, which involve the participation of Jun N-terminal kinase (JNK), IκB kinase (IKK)-β, nuclear factor (NF)-κB, and redox-sensitive transcription factor. Microglial activation may not only increase oxidative stress through the generation of proinflammatory cytokines, but also through the production of nitric oxide. Proinflammatory cytokines and high nityric oxide levels may promote ROS formation, which in turn accelerates lipid peroxidation, damaging membrane phospholipids. Collectively, these processes initiate and establish uncontrolled brain damage pathway for the pathogenesis of neurodegenerative diseases.
Las citocinas IL-12 p70 e IL-23 polarizan la respuesta inmune a los tipos Th1 y Th17. Estas citocinas comparten una subunidad, la IL-12 p40 (gen IL12/23B) y difieren en una subunidad específica, la IL-12 p35 (gen IL12A) en la IL-12 p70, y la IL-23 p19 (gen IL23A) en la IL-23. La regulación del balance IL-12 p70/IL-23 es relevante en infecciones fúngicas invasivas y enfermedades autoinmunes. La transcripción de IL23A se regula por una combinación de c-Rel con coactivadores como ATF2, que se activa por fosforilación complementaria, dependiente de PKC y MAPKs, de las Thr-71 y Thr-69. Los receptores de patrones fúngicos Dectin-1 y Dectin-2 inducen la producción de LTB4, cisteinil-leucotrienos y PAF que contribuyen a la fosforilación de ATF2. La transcripción de IL12A se reprime por el estímulo fúngico zimosano mediante la desacetilación dependiente de NAD+ de las histonas del promotor de IL12A por SIRT1, reduciendo la accesibilidad de c-Rel.
El libro impreso se puede adquirir en ProQuest (ISBN 978-1339531007), en CreateSpace (https://www.createspace.com/5953836), en Amazon (https://www.amazon.es/dp/1339531003) y en Book Depository (https://www.bookdepository.com/Activacion-de-Il23-y-Represion-de-Il12-Por-Metabolitos-En-L-Respuest-Inmune-Th17-Mario-Rodriguez-Pen/9781339531007)
Enlace en Teseo: https://www.educacion.gob.es/teseo/mostrarRef.do?ref=1191189
Macrophage activation by cytokines or microbial products such as LPS results in the induction and release of several key immune effector molecules including NO and IL-12. These have been shown to play crucial roles in the development of immunity to intracellular pathogens such as Leishmania. The molecular mechanisms underlying the induction of these effector molecules are not fully understood. We now show that the extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases play differential roles in the regulation of LPS-stimulated inducible NO synthase and IL-12 gene expression. In macrophages, LPS stimulates the simultaneous activation of all three classes of MAP kinases, ERK, c-jun N-terminal kinase, and p38, albeit with differential activation kinetics. However, studies using inhibitors selective for ERK (PD98059) and p38 (SB203580) show that while p38 plays an essential role in the induction of inducible NO synthase, ERK MAP kinases play only a minor role in promoting NO generation. In contrast, while p38 promotes induction of IL-12 (p40) mRNA, ERK activation suppresses LPS-mediated IL-12 transcription. The biological relevance of these regulatory signals is demonstrated by our finding that Leishmania lipophosphoglycans, which promote parasite survival, act by stimulating ERK MAP kinase to inhibit macrophage IL-12 production. Thus, as ERK and p38 MAP kinases differentially regulate the induction of the macrophage effector molecules, inducible NO synthase and IL-12, these kinases are potential targets not only for the development of novel strategies to combat intracellular pathogens but also for therapeutic immunomodulation.
The mechanisms by which growth factor-induced signals are propagated to the nucleus, leading to the activation of the transcription factor CREB, have been characterized. Nerve growth factor (NGF) was found to activate multiple signaling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine 133 (Ser-133). NGF activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs), which in turn activate the pp90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases, all three members of which were found to catalyze CREB Ser-133 phosphorylation in vitro and in vivo. In addition to the ERK/RSK pathway, we found that NGF activated the p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in phosphorylation of CREB at Ser-133. Inhibition of either the ERK/RSK or the p38/MAPKAP kinase 2 pathway only partially blocked NGF-induced CREB Ser-133 phosphorylation, suggesting that either pathway alone is sufficient for coupling the NGF signal to CREB activation. However, inhibition of both the ERK/RSK and the p38/MAPKAP kinase 2 pathways completely abolished NGF-induced CREB Ser-133 phosphorylation. These findings indicate that NGF activates two distinct MAPK pathways, both of which contribute to the phosphorylation of the transcription factor CREB and the activation of immediate-early genes.
The nuclear oncogenes v-myc or v-myb specifically transform avian myeloid cells. In both cases, the transformed cells remain dependent on chicken myelomonocytic growth factor (cMGF). This factor dependence can be relieved by expression of kinase-type oncogenes such as v-mil or v-erbB, leading to expression of cMGF and autocrine growth stimulation. In erythroid cells the same kinase-type oncogenes cause transformation but do not induce cMGF expression. Here we investigated the molecular mechanisms of the observed lineage specific oncogene collaboration. We found that kinase-type oncogenes and TPA activate the cMGF promoter via AP-1 like transcription factors. The activation of the cMGF promoter is, however, strictly dependent on the binding of nuclear proteins to both halves of an inverted repeat adjacent to the AP-1 binding site. These proteins are related to C/EBP. They are expressed exclusively in myeloid cells and were therefore termed NF-M. Our results indicate that the lineage specific cooperation of kinase type oncogenes with v-myb or v-myc in leukemia formation is based on the concerted action of AP-1 and NF-M on the cMGF promoter.
Eucaryotic organisms possess natural defense processes triggered by stress factors such as injury, infection, and inflammation. The acute phase response is an early defense mechanism during which striking changes in protein synthesis occur in the liver and other tissues. The altered expression of many acute phase protein genes is at the transcriptional level. Some of these genes have DNA binding sites for the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. We report here that in vivo expression of three isoforms of C/EBP is dramatically changed during the acute phase response. The steady-state mRNA levels of C/EBP alpha decreased significantly in the liver, lung, and fat tissues of lipopolysaccharide (LPS)-treated mice; moreover, nuclear run-off transcription assays indicated a decrease in the rate of C/EBP alpha gene transcription in isolated liver nuclei. The steady-state levels of C/EBP beta and a new isoform, C/EBP delta, were dramatically increased in many tissues within 4 h following LPS treatment. The rates of transcription of these two genes were only minimally altered in liver but significantly increased in kidney nuclei isolated from stimulated animals. Thus, the C/EBP isoforms exhibit differential mechanisms in their responses to LPS in various tissues and are likely to play an important role in mediating the transcriptional activation of genes involved in the acute phase response.
The c-fos serum response element (SRE) is a multifunctional regulatory region of the c-fos promoter that responds to a variety of inducers. Recently, we have demonstrated that the SRE binds the C/EBP-related transcription factor rat NFIL-6 (rNFIL-6). In this study we show that rNFIL-6 is regulated by the cAMP second messenger pathway in the rat pheochromocytoma PC12 cell line. Following forskolin treatment, rNFIL-6 binding to the SRE is increased, and the factor becomes phosphorylated and undergoes a trans-location to the nucleus. In transient cotransfection assays, rNFIL-6 is capable of trans-activating the c-fos promoter in a manner dependent on the SRE. These data show that rNFIL-6 undergoes a novel activation in which cAMP-induced nuclear trans-location allows rNFIL-6 to bind to the SRE and contribute to c-fos activation. We propose that rNFIL-6 is an additional regulatory component of the c-fos gene, which provides cAMP responsiveness to the multifunctional SRE.
A C/EBP-like transcription factor, AGP/EBP, that binds to three distinct motifs in the 5'-flanking region of alpha 1-acid
glycoprotein gene (AGP) has been identified. Here we report the cloning and properties of cDNA corresponding to mouse AGP/EBP.
AGP/EBP and C/EBP share 87% amino acid sequence homology in the "leucine zipper" and its associated DNA-binding domains, while
their sequences outside these domains and the sizes of their mRNAs are different. Unlike the limited expression of C/EBP in
tissues and cells, AGP/EBP appears to be ubiquitously expressed in tissues like lung, spleen, kidney, heart, testis, and liver
and cell lines like p388D1, 129P (hepatoma cell line of C3H/HeJ), FO (mouse myeloma), and L929. Antibody against cloned and
expressed AGP/EBP which was raised in rabbits could recognize AGP/EBP from nuclear extract of a number of cells and tissues.
On the basis of our findings about the structural relationship and the similarity of motif recognition, we propose that a
family of C/EBP-like transcription factors exists.
Mouse and rat genomic DNA libraries were screened by reduced stringency hybridization with the DNA-binding domain of the c/ebp gene as a probe. Three genes were isolated that encode bZIP DNA-binding proteins (designated CRP1, CRP2, and CRP3) with strong amino acid sequence similarities to the C/EBP-binding domain. CRP2 is identical to the protein described recently by other groups as NF-IL6, LAP, IL-6DBP, and AGP/EBP, whereas CRP1 and CRP3 represent novel proteins. Several lines of evidence indicate that these three proteins, along with C/EBP, comprise a functional family. Each bacterially expressed polypeptide binds to DNA as a dimer with recognition properties that are virtually identical to those of C/EBP. Every member also bears a conserved cysteine residue at or near the carboxyl terminus, immediately following the leucine zipper, that at least in vitro allows efficient disulfide cross-linking between paired zipper helices. We developed a gel assay for covalent dimers to assess leucine zipper specificities among the family members. The results demonstrate that all pairwise combinations of dimer interactions are possible. To the extent that we have examined them, the same heterodimeric complexes can be detected intracellularly following cotransfection of the appropriate pair of genes into recipient cells. All members are also capable of activating in vivo transcription from promoters that contain a C/EBP-binding site. Our findings indicate that a set of potentially interacting C/EBP-like proteins exists, whose complexity is comparable to that of other bZIP protein subfamilies such as Jun, Fos, and ATF/CREB.
This chapter discusses the phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Peptide mapping is a powerful technique used to help determine peptide structure and composition of proteins. Peptide maps or fingerprints of proteolyzed proteins are usually obtained by resolution on either one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high-performance liquid chromatography (HPLC), or by two-dimensional separation on thin-layer cellulose (TLC) plates. The most common applications of peptide mapping are (1) to compare proteins encoded by the same or related genes, (2) to prepare individual peptides for determining amino acid composition and sequence, and (3) to determine the precise location of amino acid residues that are posttranslationally modified by fatty acid acylation, glycosylation, methylation, acetylation, or phosphorylation.
NF-IL6 is a nuclear factor that specifically binds to an IL1-responsive element in the IL-6 gene. In this study the gene encoding NF-IL6 has been cloned by direct screening of a lambda gt11 library using NF-IL6 binding sequence as a ligand. The full-length cDNA encoded a 345 amino acid protein with a potential leucine zipper structure and revealed a high degree of homology to a liver-specific transcriptional factor, C/EBP, at the C-terminal portion. The bacterial fusion protein bound to the CCAAT homology as well as the viral enhancer core sequences as in the case of C/EBP. Recombinant NF-IL6 activated the human IL-6 promoter in a sequence-specific manner. Southern blot analysis demonstrated the high-degree conservation of the NF-IL6 gene through evolution and the existence of several other related genes sharing the DNA-binding domain. NF-IL6 mRNA was normally not expressed, but induced by the stimulation with either LPS, IL-1 or IL-6. Interestingly, NF-IL6 was shown to bind to the regulatory regions for various acute-phase protein genes and several other cytokine genes such as TNF, IL-8 and G-CSF, implying that NF-IL6 has a role in regulation not only for the IL-6 gene but also for several other genes involved in acute-phase reaction, inflammation and hemopoiesis.
A gene, encoding a liver-enriched transcriptional activator protein (LAP) has been isolated. LAP is a 32-kD protein that stimulates the transcription of chimeric genes containing albumin D-promoter elements both in vivo and in vitro. LAP shares extensive sequence homology (71%) in its DNA-binding and leucine zipper domains with C/EBP. As a consequence, these two proteins show an indistinguishable DNA-binding specificity and readily heterodimerize. In addition, both genes, lap and cebp, are devoid of intervening sequences. Although correctly initiated transcripts from the LAP gene accumulate in the six examined tissues--liver, lung, spleen, kidney, brain, and testis--LAP protein is highly enriched in liver nuclei. Thus, the preferential accumulation of LAP protein in liver appears to be regulated post-transcriptionally.
We have used the technique of reverse transcription coupled to the polymerase chain reaction to detect mRNA precursors [heterogeneous nuclear RNA (hnRNA)] transcribed from the thymidine kinase (TK) gene of human diploid fibroblasts. With this method, the amplification products of both hnRNA (containing the introns) and mature mRNA can be detected on Southern blots with appropriate hybridization probes. With the experimental conditions used, the sensitivity of the technique is such that TK mRNA can be detected in as few as 20 S-phase cells. TK hnRNA is maximally expressed early in the S phase of the cell cycle after quiescent human fibroblasts are stimulated to proliferate. At this point, the ratio of TK hnRNA to TK mRNA is 1:155. A small amount of TK hnRNA can be detected in populations of cells that appear to be quiescent. However, the presence of the precursor in these populations correlates with the number of cells still cycling. No TK hnRNA can be detected in truly quiescent human diploid fibroblasts, suggesting that in these cells, the TK gene is not transcribed in G0.
We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription
initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using
the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus
promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA
and Ad 2 VA).
Two members of the C/EBP family of basic region-leucine zipper proteins enriched in the liver, C/EBP (C/EBP alpha) and CRP2 (C/EBP beta), were previously shown to transactivate the albumin promoter in a cell type-dependent manner. These proteins function efficiently in HepG2 hepatoma cells, but inefficiently in HeLa (epithelial) and L (fibroblastic) cells. Here we have investigated the mechanism for cell-specific control of CRP2 activity. We show that CRP2 contains a negative regulatory region composed of two elements, RD1 and RD2. Deletions of RD2 relieve the inhibition of CRP2 activity in L cells, but do not affect CRP2 function in HepG2 cells. These deletions also increase the DNA binding activity of CRP2 approximately 3-fold, suggesting that RD2-mediated repression of DNA binding activity is responsible for CRP2 inhibition in L cells. The adjacent RD1 element functions independently of RD2 and modulates the CRP2 activation domain, which we show to be composed of three subdomains that are conserved within the C/EBP protein family. RD1 does not affect cell type specificity, but inhibits the transactivation potential of GAL4-CRP2 hybrid proteins by 50-fold. These findings suggest that CRP2 assumes a tightly folded conformation in which the DNA binding and activation domains are masked by interactions with the regulatory domain and that to function efficiently in HepG2 cells the protein must undergo an activation step. We propose that relief of inhibition conferred by the regulatory domains also accounts for CRP2 activation in response to extracellular signals.
C/EBP beta is considered a key element of interleukin-6 (IL-6) signalling as well as an important transcriptional regulator of the IL-6 gene itself. We describe here how mice lacking C/EBP beta develop a pathology similar to mice overexpressing IL-6 and nearly identical to multicentric Castleman's disease in human patients, with marked splenomegaly, peripheral lymphadenopathy and enhanced haemopoiesis. Humoral, innate and cellular immunity are also profoundly distorted, as shown by the defective activation of splenic macrophages, the strong impairement of IL-12 production, the increased susceptibility to Candida albicans infection and the altered T-helper function. Our data show that C/EBP beta is crucial for the correct functional regulation and homeostatic control of haemopoietic and lymphoid compartments.
The effect of insulin on expression of CCAAT/enhancer binding protein (C/EBP) α, β, and was investigated in fully-differentiated 3T3-L1 adipocytes. Treatment of adipocytes with insulin stimulated rapid dephosphorylation
of C/EBPα, and repressed the expression of C/EBPα within 2-4 h, with >90% suppression occurring at 24 h. While insulin induced
expression of C/EBPβ and C/EBP within 1 h and caused a >20-fold increase by 4 h, expression returned to nearly pretreatment levels by 24 h. The insulin
concentration dependence of these effects was consistent with involvement of the insulin receptor. Gel shift analysis revealed
that 6 h of insulin treatment decreased the binding of nuclear C/EBPα while increasing binding of nuclear C/EBPβ and C/EBP. The reciprocal effects of insulin on the steady-state levels of C/EBP transcription factors can be accounted for kinetically
and quantitatively by changes in their mRNA levels, which can be accounted for by effects on gene transcription. The effects
of insulin on adipocyte gene transcription (e.g. GLUT4) may be mediated, at least in part, by down-regulation of C/EBPα and/or its dephosphorylation.
Protein-protein interactions between the CCAAT box enhancer-binding proteins (C/EBP) and the Rel family of transcription factors
have been implicated in the regulation of cytokine gene expression. We have used sequence-specific DNA affinity chromatography
to purify a complex from avian T cells that binds to a consensus C/EBP motif. Our results provide evidence that Rel-related
proteins are components of the C/EBP-DNA complex as a result of protein-protein interactions with the C/EBP proteins. A polyclonal
antiserum raised against the Rel homology domain of v-Rel and antisera raised against two human RelA-derived peptides specifically
induced a supershift of the C/EBP-DNA complex in mobility shift assays using the affinity-purified C/EBP. In addition, several
kappa B-binding proteins copurified with the avian C/EBP complex through two rounds of sequence-specific DNA affinity chromatography.
The kappa B-binding proteins are distinct from the C/EBP proteins that directly contact DNA containing the C/EBP binding site.
The identification of a protein complex that binds specifically to a consensus C/EBP site and contains both C/EBP and Rel
family members suggests a novel mechanism for regulation of gene expression by Rel family proteins.
Phosphorylation of transcription factors is regarded as a major mechanism to control their activity in regulation of gene expression. C/EBP beta is a transcription factor that becomes activated after phosphorylation to induce genes involved in inflammation, acute-phase response, cytokine expression, cell growth, and differentiation. The chicken homolog NF-M collaborates with Myb and various kinase oncogenes in normal myeloid differentiation as well as in the leukemic transformation of myelomonocytic cells. Here, we examined the structure of NF-M and its mechanism of activation. We show that NF-M is a repressed transcription factor with concealed activation potential. Derepressed NF-M exhibits enhanced transcriptional efficacy in reporter assays. More importantly, NF-M activates resident chromatin-embedded, myelomonocyte-specific target genes, even in heterologous cell types such as fibroblasts or erythroblasts. We identified two regions within NF-M that act to repress trans-activation. Repression is abolished by deletion of these regions, activation of signal transduction kinases including v-erbB, polyoma middle T, ras and mil/raf, or point mutation of a critical phosphorylation site for MAP kinases. We provide evidence that phosphorylation plays a unique role to derepress rather than to enhance the trans-activation domain as a novel mechanism to regulate gene expression by NF-M/C/EBP beta.
LAP (NF-IL6 or C/EBP beta), is a liver transcriptional activator protein that confers liver-specific gene expression. Because LAP has a characteristic phosphoacceptor sequence for cAMP-dependent protein kinase A (PKA), we tested if in vitro phosphorylation of LAP by PKA modulates its interaction with specific DNA sequences. The major PKA phosphorylation site of LAP was identified as Ser105, which is a predicted PKA site. As expected, this PKA phosphorylation site disappears after mutation of Ser105 to Ala. Kinetic studies with LAP and LAP Asp105 (which mimics a phosphoserine residue) demonstrated that phosphorylation of Ser105 itself has no effect on DNA binding. Phosphorylation of other sites by PKA, identified in the region between Ser173 and Ser223 and at Ser240, by analysis of truncated and mutated LAP peptides, resulted in an inhibition of DNA binding. LAP was also phosphorylated by purified protein kinase C in vitro, and the major phosphoacceptor was shown to be Ser240 within the DNA-binding domain of LAP. Phosphorylation of LAP at this residue or introduction of a Ser240 to Asp mutation resulted in marked decrease in its binding to DNA. These results suggest that site-specific phosphorylations of LAP modulate transactivation of its target genes.
Single binding sites for transcription factors NF-IL6 and NF-kappa B are present in the promoter of the interleukin (IL) 6 gene. Previous studies of internally deleted promoter mutants demonstrated that these two sites are important for the transcriptional regulation of this gene. In this report, we describe the synergistic activation of the IL-6 promoter by transcription factors NF-IL6 and NF-kappa B. Cotransfection of NF-IL6 with the NF-kappa B p65 subunit resulted in strong synergistic activation of an IL-6 promoter-reporter construct. Both the NF-IL6 and NF-kappa B binding sites in the IL-6 promoter were required for synergistic activation. Similar synergistic activation was observed in the IL-8 promoter, which also contains both NF-IL6 and NF-kappa B binding sites. Furthermore, we demonstrated that NF-IL6 and the NF-kappa B p65 subunit directly associated via the basic leucine-zipper domain of NF-IL6 and the Rel homology domain of p65. Since the promoters of many other genes involved in the inflammatory and acute-phase responses also contain binding sites for NF-IL6 and NF-kappa B, the cooperation between these two factors may have an important role in these responses. We also discuss the possible interplay between various viral gene products and these two factors in the process of viral infection and constitutive cytokine production.
NF-IL6, a member of the basic leucine zipper (bZIP) family transcription factors, is involved in expression of inducible genes involved in immune and inflammatory responses. We observed that coexpression of oncogenic p21ras stimulated the transactivating activity of NF-IL6 and induced phosphorylation of Thr-235 located just N-terminal to the DNA binding domain of NF-IL6. Recently, mitogen-activated protein (MAP) kinases have been shown to be implicated in the cellular response to activated ras. Purified MAP kinases specifically phosphorylated Thr-235 of NF-IL6 in vitro. Mutation of Thr-235 abolished the ras-dependent activation of NF-IL6. From these results, we conclude that NF-IL6 is regulated through phosphorylation by MAP kinases in response to activated ras.
Retroviral oncogenes encode nuclear regulators of gene expression or signal transduction molecules, such as protein kinases, which stimulate the activity of cellular transcription factors. Here we describe the cloning of NF-M, a myeloid-specific transcription factor related to C/EBP beta, which is a target of activated protein kinases. NF-M stimulates the expression of the gene encoding cMGF, a myeloid cell-specific growth factor, creating an autocrine growth loop crucial to oncogene transformation of myeloid cells. The NF-M protein bound directly to the cMGF gene promoter and activated its transcription, even in erythroid cells where the promoter is usually inactive. In addition, a truncated, dominant-negative form of NF-M inhibited cMGF expression in macrophages, indicating that NF-M is required for the normal activation of the gene. When multipotent hematopoietic progenitor cells were stimulated to differentiate, NF-M expression was induced at a very early stage, suggesting that the transcription factor plays a role in lineage commitment. The stimulation of transformed myelomonocytic cells or of normal peripheral blood macrophages with kinases or LPS or TPA respectively, led to the rapid redistribution of NF-M protein from the cell bodies to the nucleus, consistent with the notion that NF-M was directly affected by such treatments. Our data indicate that NF-M plays a key role in myelomonocytic differentiation, in signal transduction during macrophage activation and in the development of myelogenous leukemia.
Tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) activate transcription of the TSG-6 gene in normal human fibroblasts through a promoter region (-165 to -58) that encompasses an AP-1 and a NF-IL6 site. We show by deletion analysis and substitution mutagenesis that both sites are necessary for activation by TNF-alpha. Activation by IL-1 requires the NF-IL6 site and is enhanced by the AP-1 site. These results suggest that the NF-IL6 and AP-1 family transcription factors functionally cooperate to mediate TNF-alpha and IL-1 signals. Consistent with this possibility, IL-1 and TNF-alpha markedly increase the binding of Fos and Jun to the AP-1 site, and NF-IL6 activates the native TSG-6 promoter. Activation by NF-IL6 requires an intact NF-IL6 site and is modulated by the ratio of activator to inhibitor NF-IL6 isoforms that are translated from different in-frame AUGs. However, the inhibitor isoform can also bind to the AP-1 site and repress AP-1 site-mediated transcription. The finding that the inhibitor isoform antagonizes activation of the native TSG-6 promoter by IL-1 and TNF-alpha suggests that NF-IL6 has a physiologic role in these cytokine responses. Thus, the functionally distinct NF-IL6 isoforms cooperate with Fos and Jun to positively and negatively regulate the native TSG-6 promoter by TNF-alpha and IL-1.
Protein-protein interactions between the CCAAT box enhancer-binding proteins (C/EBP) and the Rel family of transcription factors have been implicated in the regulation of cytokine gene expression. We have used sequence-specific DNA affinity chromatography to purify a complex from avian T cells that binds to a consensus C/EBP motif. Our results provide evidence that Rel-related proteins are components of the C/EBP-DNA complex as a result of protein-protein interactions with the C/EBP proteins. A polyclonal antiserum raised against the Rel homology domain of v-Rel and antisera raised against two human RelA-derived peptides specifically induced a supershift of the C/EBP-DNA complex in mobility shift assays using the affinity-purified C/EBP. In addition, several kappa B-binding proteins copurified with the avian C/EBP complex through two rounds of sequence-specific DNA affinity chromatography. The kappa B-binding proteins are distinct from the C/EBP proteins that directly contact DNA containing the C/EBP binding site. The identification of a protein complex that binds specifically to a consensus C/EBP site and contains both C/EBP and Rel family members suggests a novel mechanism for regulation of gene expression by Rel family proteins.
The interleukin-8 promoter is transcriptionally activated by interleukin-1, tumor necrosis factor alpha, phorbol myristate acetate, or hepatitis B virus X protein through a sequence located between positions -91 and -71. This region contains an NF-kappa B-like and a C/EBP-like binding site. We show here that several members of the NF-kappa B family, including p65, p50, p52, and c-Rel, can bind to this region, confirming an authentic NF-kappa B binding site in the interleukin-8 promoter. Further, C/EBP binds only weakly to the interleukin-8 promoter site. Electrophoretic mobility shift assays with proteins overexpressed in COS cells and with nuclear extracts from tumor necrosis factor alpha-stimulated HeLa cells demonstrated a strong cooperative binding of C/EBP to its site when NF-kappa B is bound to its adjacent binding site. Transfection studies lead to a model that suggests a highly complex regulation of interleukin-8 gene expression at multiple levels: independent binding of C/EBP and NF-kappa B to their respective sites, cooperative binding of C/EBP and NF-kappa B to DNA, and positive synergistic activation through the C/EBP binding site and inhibition through the NF-kappa B binding site by combinations of C/EBP and NF-kappa B. Thus, the ultimate regulation of interleukin-8 gene expression depends on the ratio of cellular C/EBP and NF-kappa B.
A C/EBP-like transcription factor, AGP/EBP, that binds to three distinct motifs in the 5'-flanking region of alpha 1-acid glycoprotein gene (AGP) has been identified. Here we report the cloning and properties of cDNA corresponding to mouse AGP/EBP. AGP/EBP and C/EBP share 87% amino acid sequence homology in the "leucine zipper" and its associated DNA-binding domains, while their sequences outside these domains and the sizes of their mRNAs are different. Unlike the limited expression of C/EBP in tissues and cells, AGP/EBP appears to be ubiquitously expressed in tissues like lung, spleen, kidney, heart, testis, and liver and cell lines like p388D1, 129P (hepatoma cell line of C3H/HeJ), FO (mouse myeloma), and L929. Antibody against cloned and expressed AGP/EBP which was raised in rabbits could recognize AGP/EBP from nuclear extract of a number of cells and tissues. On the basis of our findings about the structural relationship and the similarity of motif recognition, we propose that a family of C/EBP-like transcription factors exists.
Interleukin-12 (IL-12) is a heterodimeric cytokine produced mostly by phagocytic cells in response to bacteria, bacterial products, and intracellular parasites, and to some degree by B lymphocytes. IL-12 induces cytokine production, primarily of IFN-gamma from NK and T cells, acts as a growth factor for activated NK and T cells, enhances the cytotoxic activity of NK cells, and favors cytotoxic T lymphocyte generation. In vivo IL-12 acts primarily at three stages during the innate resistance/adaptive immune response to infection: 1. Early in the infection, IL-12 is produced and induces production from NK and T cells of IFN-gamma, which contributes to phagocytic cell activation and inflammation; 2. IL-12 and IL-12-induced IFN-gamma favor Th1 cell differentiation by priming CD4(+) T cells for high IFN-gamma production; and 3. IL-12 contributes to optimal IFN-gamma production and to proliferation of differentiated Th1 cells in response to antigen. The early preference expressed in the immune response depends on the balance between IL-12, which favors Th1 responses, and IL-4, which favors Th2 responses. Thus, IL-12 represents a functional bridge between the early nonspecific innate resistance and the subsequent antigen-specific adaptive immunity.
We report that TGFα induces activation of the p90 ribosomal S kinase (RSK), which results in the phosphorylation of rat C/EBPβ on Ser-105 and of mouse C/EBPβ on Thr-217 and concomitantly stimulates proliferation in differentiated hepatocytes. Moreover, C/EBPβ−/− mouse hepatocytes respond to TGFα when wild-type C/EBPβ is reexpressed, whereas they remain refractory to the growth effect of TGFα when expressing phosphoacceptor mutants rat C/EBPβ Ala-105 or mouse C/EBPβ Ala-217. In contrast, C/EBPβ−/− hepatocytes expressing the phosphorylation mimic mutants, rat C/EBPβ Asp-105 or mouse C/EBPβ Glu-217, exhibited marked proliferation in the absence of TGFα. Thus, a site-specific phosphorylation of the transcription factor C/EBPβ is critical for hepatocyte proliferation induced by TGFα and other stimuli that activate RSK.
Alterations in intracellular oxidative status activate several signal transduction pathways resulting in distinct patterns
of gene expression. Treatment of colorectal cancer cells with antioxidants can lead to apoptosis by induction of p21 through
a mechanism involving CCAAT/enhancer-binding protein β (C/EBPβ). Herein, we demonstrate that the antioxidant pyrrolidinedithiocarbamate
activates cAMP-dependent protein kinase (PKA) in a colorectal cancer cell line DKO-1. Activation of PKA phosphorylates Ser299 within C/EBPβ, which is essential for protein translocation to the nucleus. Pharmacological inhibition of PKA and mutation
of Ser299 to alanine blocks C/EBPβ nuclear translocation and induction of p21. Our results indicate that a cAMP-dependent phosphorylation
of C/EBPβ at Ser299 is critical for nuclear translocation of this protein and its subsequent transactivation of genes in response to antioxidant
treatment.
The transcription factors CCAAT/enhancer binding protein (C/EBP)-β and -δ are key regulators for the expression of the acute phase genes in the liver, such as complement component C3 and antichymotrypsin. In the brain, these acute phase proteins are produced in response to pro-inflammatory cytokines by the reactive astrocytes, in particular those surrounding the amyloid plaques of Alzheimer's disease brains. Here we show that lipopolysaccharides (LPS), IL-1β, and TNFα induce the expression of the c/ebpβ and -δ genes in mouse primary astrocytes. This induction precedes the expression of the acute phase genes coding for the complement component C3 and the mouse homologue of antichymotrypsin. The induction of these two acute phase genes by LPS is blocked by cycloheximide, whereas this protein synthesis inhibitor does not affect the expression of the c/ebp genes. Altogether, our data support a role as immediate-early genes for c/ebpβ and -δ, whose expression is induced by pro-inflammatory cytokines in mouse cortical astrocytes. In the liver, these transcription factors are known to play an important role in inflammation and energy metabolism regulation. Therefore, C/EBPβ and -δ could be pivotal transcription factors involved in brain inflammation, in addition to their previously demonstrated role in brain glycogen metabolism regulation (Cardinaux and Magistretti. J Neurosci 16:919–929, 1996). GLIA 29:91–97, 2000. © 2000 Wiley-Liss, Inc.
Using the 3T3-F442A preadipocyte line as a model of on-dependent differentiation, early changes in the DNA-binding affinity of transcription factors in response to on addition were investigated. Addition of 50 ng/ml human on to cells in chemically defined medium led to a rapid increase in binding activity of activator protein 1 (AP-1) and CCAAT enhancer-binding protein (C/EBP), which was significant at 30 min and reached maximal induction by 2 h (3-fold for AP-1, 2.5-fold for C/EBP). Induction in AP-1 DNA binding correlates with a concomitant on trans-activation of c-jun and c-fos genes described previously. Using specific antibodies in electrophoretic mobility shift assays and Western blots, it was shown that the increase in activity of C/EBP is the result of an increase in synthesis of two alternatively translated forms of C/EBP beta: 40-C/EBP beta and 23-C/EBP beta. This increase in protein was not accompanied by alteration in mRNA level and could be blocked by a Janus kinase 2 tyrosine kinase inhibitor and a C kinase inhibitor at concentrations shown to inhibit on-dependent activation of microtubule-associated protein (MAP) kinases. Concomitant with the translationally activated increase in C/EBP beta, a on-dependent increase was observed in C/EBP delta transcription. This was accompanied by an increase in mRNA for C/EBP delta, which was superinduced by cycloheximide and, unlike the increase in C/EBP beta protein, was not observed with insulin. Thus on exerts its effects on C/EBP isoforms at two levels: transcriptional activation of C/EBP delta and translational activation of C/EBP beta. It is proposed that on-dependent phosphorylation results in the efficient translation of 40-C/EBP beta and 23-C/EBP beta (the mouse homolog of the inhibitor liver-enriched inhibitory protein), and that together with the induction of C/EBP delta, these may be involved in initiating the adipocyte differentiation program.
Describes how MCB University Press has started to come to terms
with the metamorphosis which electronic publishing offers. Sees a future
where interactive multimedia products and services are the norm and are
quite differently distributed, based on new alliances from within but
also from outside the traditional players. Explores how MCB's strengths
might be used to succeed in the new frameworks and concludes that
double-loop action learning is the only viable way ahead. Identifies
that authors will be a constant point of reference and that networked
desktop PCs and networked homes will open vast new markets to those who
can re-present knowledge and information to gain and hold their
attention. Outlines what MCB has done so far.
Alterations in intracellular calcium levels activate several signal transduction pathways resulting in distinct patterns of gene expression. Here, a pathway for calcium-mediated signals is demonstrated that involves C/EBP beta, a member of the bZip family of transcription factors. In pituitary cells C/EBP beta was phosphorylated in response to increased intracellular calcium concentrations as a consequence of the activation of a calcium-calmodulin-dependent protein kinase. Phosphorylation of serine at position 276 within the leucine zipper of C/EBP beta appeared to confer calcium-regulated transcriptional stimulation of a promoter that contained binding sites for C/EBP beta.
The NF-kappa B-p50 polypeptide, a member of the Rel family of transcription factors, was produced as a fusion protein containing amino-terminal peptide additions that facilitate purification and detection with a monoclonal antibody and specific radiolabeling by phosphorylation in vitro. The 32P-labeled NK-kappa B-p50 fusion polypeptide was used as the probe in Western blotting experiments and in screenings of a bacteriophage expression library to isolate cDNAs encoding interacting protein domains. As expected, cDNAs encoding proteins of the Rel family were identified. Surprisingly, the 32P-labeled NF-kappa B protein also specifically bound to proteins encoded by cDNAs for the human NF-IL6 transcription factor. The NF-kappa B-p50 and NF-IL6 proteins directly interact, and the Rel homology domain and leucine-zipper motif, respectively, are important for this interaction. Since induction of the NF-kappa B and NF-IL6 factors are important events in immune and acute-phase responses, this interaction could permit coregulation of genes.
Using a DNA probe from the DNA-binding portion of the NF-IL6 gene and an antibody against the DNA-binding domain of NF-IL6, we isolated a gene homologous to NF-IL6 in the DNA-binding and leucine zipper domains. This intronless gene, termed NF-IL6 beta encodes a 269-amino acid protein with a potential leucine zipper structure, and the gene product can bind to the CCAAT homology as well as the viral enhancer core sequence, as in the cases of NF-IL6 and C/EBP. This gene is expressed at an undetectable or a minor level in normal tissues but is induced by lipopolysaccharide or inflammatory cytokines, as in the case of NF-IL6. NF-IL6 beta easily forms a heterodimer with NF-IL6 in vitro and the heterodimeric complex binds to the same DNA sequence as the respective homodimers. When examined by transient luciferase assays, NF-IL6 beta is consistently a stronger transactivator than NF-IL6. Furthermore, NF-IL6 beta shows a synergistic transcriptional effect with NF-IL6. These data suggest that NF-IL6 beta is an important transcriptional activator in addition to NF-IL6 in regulation of the genes involved in the immune and inflammatory responses.
In an effort to identify protein factors that play a regulatory role in the differentiation of adipocytes, we have isolated two genes that encode polypeptides related to CCAAT/enhancer-binding protein (C/EBP; hereafter termed C/EBP alpha). The proteins encoded by these C/EBP-related genes, termed C/EBP beta and C/EBP delta, exhibit similar DNA-binding specificities and affinities compared with C/EBP alpha. Furthermore, C/EBP beta and C/EBP delta readily form heterodimers with one another as well as with C/EBP alpha. The transcriptional activating capacity of these two newly identified C/EBP isoforms was demonstrated by transient transfection experiments in which expression vectors encoding C/EBP beta and C/EBP delta were observed to induce transcription from the promoter of the serum albumin gene in cultured hepatoma cells. The mRNAs encoding C/EBP beta and C/EBP delta were detected in a number of tissues, most of which corresponded to sites of expression of C/EBP alpha. The expression pattern of C/EBP beta and C/EBP delta during adipose conversion of 3T3-L1 cells was examined by Western and Northern blotting assays. In contrast to the expression profile of the gene encoding C/EBP alpha, whose product is not detectable until the late phase of adipocyte differentiation, the c/ebp beta and c/ebp delta genes were actively expressed very early during adipocyte differentiation. Moreover, transcription of the c/ebp beta and c/ebp delta genes was observed to be induced directly by adipogenic hormones. The accumulation of C/EBP beta and C/EBP delta reached a maximal level during the first 2 days of differentiation and declined sharply before the onset of C/EBP alpha accumulation. The temporal pattern of expression of these three C/EBP isoforms during adipocyte differentiation may reflect the underpinnings of a regulatory cascade that controls the process of terminal cell differentiation.
LAP, a transcriptional activator, and LIP, a transcriptional repressor, are translated from a single mRNA species by using two AUGs within the same reading frame. These two proteins share the 145 C-terminal amino acids that contain the basic DNA-binding domain and the leucine zipper dimerization helix. Probably owing to its higher affinity for its DNA cognate sequences, LIP can attenuate the transcriptional stimulation by LAP in substoichiometric amounts. As revealed by transient transfection experiments, a moderate increase in the LAP/LIP ratio results in a significantly higher transcriptional activation of an appropriate target gene. The LAP/LIP ratio increases about 5-fold during terminal rat liver differentiation and is thus likely to modulate the activity of LAP in the intact animal.
We analyzed a family of proteins from hepatoma cell nuclei that bind to interleukin-6 responsive elements (IL-6REs) of several acute-phase genes. This family is characterized by leucine zipper domains compatible with that of the CCAAT/enhancer binding protein (C/EBP). A cDNA clone coding for a member of the family, IL-6DBP, was isolated; it is strongly homologous to C/EBP in the region of the basic domain and in the leucine zipper sequence. IL-6DBP and C/EBP can interact in vitro to form heterodimers that bind to DNA with the same specificity as the respective homodimers, and they can interact functionally in vivo. Both the DNA binding activity and the trans-activating capacity of IL-6DBP are induced in hepatoma cells by treatment with IL-6 through a posttranslational mechanism, implicating it as a nuclear target of IL-6 and as a mediator of the IL-6-dependent transcriptional activation of liver genes during the acute-phase response.
Both C/EBP and GCN4 are sequence-specific DNA binding proteins that control gene expression. Recent evidence implicates C/EBP as a transcriptional regulator of genes involved in lipid and carbohydrate metabolism. The C/EBP protein binds avidly to the dyad symmetric sequence 5'-ATTGCGCAAT-3'; GCN4 regulates the transcription of genes that control amino acid biosynthesis in yeast, and binds avidly to the dyad symmetric sequence 5'-ATGA(G/C)TCAT-3'. Both C/EBP and GCN4 bind DNA via the same structural motif. This motif has been predicted to be bipartite, consisting of a dimerization interface termed the "leucine zipper" and a DNA contact surface termed the "basic region." Specificity of DNA binding has been predicted to be imparted by the basic region. As a test of this hypothesis, recombinant proteins were created wherein the basic regions and leucine zippers of GCN4 and C/EBP were reciprocally exchanged. In both of the recombinant polypeptides, DNA binding specificity is shown to track with the basic region.
To investigate the role of NF-IL6 in vivo, we have generated NF-IL6 (-/-) mice by gene targeting. NF-IL6 (-/-) mice were highly susceptible to infection by Listeria monocytogenes. Electron microscopic observation revealed the escape of a larger number of pathogens from the phagosome to the cytoplasm in activated macrophages from NF-IL6 (-/-) mice. Furthermore, the tumor cytotoxicity of macrophages from NF-IL6 (-/-) mice was severely impaired. However, cytokines involved in macrophage activation, such as TNF and IFN gamma, were induced normally in NF-IL6 (-/-) mice. Nitric oxide (NO) formation was induced to a similar extent in macrophages from both wild-type and NF-IL6 (-/-) mice. These results demonstrate the crucial role of NF-IL6 in macrophage bactericidal and tumoricidal activities as well as the existence of a NO-independent mechanism of these activities. We also demonstrate that NF-IL6 is essential for the induction of G-CSF in macrophages and fibroblasts.
Terminal differentiation of cultured 3T3-L1 fibroblasts to the adipogenic phenotype is potently stimulated by dexamethasone (DEX) and methylisobutylxanthine (MIX). Previous studies have shown that these hormones induce the expression of genes encoding two members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. In the absence of new protein synthesis DEX activates the gene encoding C/EBPδ. Likewise, MIX is a direct inducer of C/EBPβ gene expression. Optimal conditions for differentiation entail a 2-day period wherein confluent fibroblasts are exposed to DEX and MIX, followed by removal of the hormones and subsequent culture in the presence of insulin and fetal bovine serum. During the early phase of differentiation, high levels of C/EBPδ and C/EBPβ accumulate. These transcription factors diminish during the terminal phase of differentiation and come to be replaced by a third member of the C/EBP family, C/EBPα. Conclusive evidence has already shown that C/EBPα regulates terminal adipocyte differentiation, turning on the battery of fat-specific genes required for the synthesis, uptake, and storage of long chain fatty acids. Here we provide evidence that C/EBPδ and C/EBPβ play early catalytic roles in the differentiation pathway, relaying the effects of the hormonal stimulants DEX and MIX in a cascade-like fashion, leading to the activation of the gene encoding C/EBPα. Conditions facilitating the precocious expression of either C/EBPδ or C/EBPβ were observed to accelerate adipogenesis and, in the case of C/EBPβ, relieve dependence on the early hormonal stimulants. Likewise, conditions that prevented the expression of functional C/EBPβ effectively blocked terminal differentiation. Finally, we have discovered that ectopic expression of C/EBPβ in multipotential NIH-3T3 cells results in their conversion into committed adipoblasts capable, upon hormonal stimulation, of synchronous and uniform differentiation into fat- laden adipocytes.
Activation of adipogenesis in 3T3 preadipocytes by exposure to the adipogenic inducers dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum is accompanied by a transient burst of C/EBP beta protein expression that precedes the induction of the fat gene program. In this study we have investigated the role of C/EBP beta in initiating the adipogenic program by overexpressing C/EBP beta in multipotential NIH-3T3 fibroblasts. Conditional ectopic expression of C/EBP beta was accomplished by using an artificial transcriptional regulatory system based on the Escherichia coli tetracycline repressor to generate a stable cell line, beta 2, that expresses C/EBP beta mRNA and protein in a tightly controlled tetracycline dose-dependent manner. Induction of C/EBP beta DNA-binding activity in NIH-3T3 beta 2 cells exposed to dexamethasone in the presence of insulin and fetal bovine serum activates the expression of an adipocyte-specific nuclear hormone receptor, PPAR gamma, that stimulates the conversion of these fibroblasts into committed preadipocytes. Either ectopic expression of C/EBP beta or treatment with dexamethasone alone is incapable of inducing PPAR gamma expression, but when present together, they have a synergistic effect on the adipogenic program. Exposure of these stimulated cells to a PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumulation of fat droplets and expression of the adipocyte-enriched genes aP2 and glycerol phosphate dehydrogenase (GPD). The number of beta 2 cells that can differentiate into adipocytes is related to the concentration of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed. C/EBP beta can induce PPAR gamma mRNA in the absence of ETYA; however, expression of aP2 mRNA and maximum fat deposition is dependent on the PPAR activator. Our results suggest that enhanced expression of C/EBP beta converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators to differentiate into adipocytes.
Interleukin 12 (IL-12) is an inducible cytokine composed of 35- and 40-kDa subunits that is critical for promoting T helper type 1 development and cell-mediated immunity against pathogens. The 40-kDa subunit, expressed by activated macrophages and B cells, is induced by several pathogens in vivo and in vitro and is augmented or inhibited by gamma interferon (IFN-gamma) or IL-10, respectively. Control of IL-12 p40 expression is therefore important for understanding resistance and susceptibility to a variety of pathogens, including Leishmania major and perhaps human immunodeficiency virus. In this report, we provide the first characterization of IL-12 p40 gene regulation in macrophages. We localize inducible activity of the promoter to the sequence -122GGGGAATTTTA-132 not previously recognized to bind Rel family transcription factors. We demonstrate binding of this sequence to NF-kappa B (p50/p65 and p50/c-Rel) complexes in macrophages activated by several p40-inducing pathogens and provide functional data to support a role for NF-kappa B family members in IL-12 p40 activation. Finally, we find that IFN-gamma treatment of cells enhances this binding interaction, thus potentially providing a mechanism for IFN-gamma augmentation of IL-12 production by macrophages.
Interleukin-12 (IL-12) is a heterodimeric cytokine produced mostly by phagocytic cells in response to bacteria, bacterial products, and intracellular parasites, and to some degree by B lymphocytes. IL-12 induces cytokine production, primarily of IFN-gamma, from NK and T cells, acts as a growth factor for activated NK and T cells, enhances the cytotoxic activity of NK cells, and favors cytotoxic T lymphocyte generation. In vivo IL-12 acts primarily at three stages during the innate resistance/adaptive immune response to infection: 1. Early in the infection, IL-12 is produced and induces production from NK and T cells of IFN-gamma, which contributes to phagocytic cell activation and inflammation; 2. IL-12 and IL-12-induced IFN-gamma favor Th1 cell differentiation by priming CD4+ T cells for high IFN-gamma production; and 3. IL-12 contributes to optimal IFN-gamma production and to proliferation of differentiated Th1 cells in response to antigen. The early preference expressed in the immune response depends on the balance between IL-12, which favors Th1 responses, and IL-4, which favors Th2 responses. Thus, IL-12 represents a functional bridge between the early nonspecific innate resistance and the subsequent antigen-specific adaptive immunity.
Using the 3T3-F442A preadipocyte line as a model of GH-dependent differentiation, early changes in the DNA-binding affinity of transcription factors in response to GH addition were investigated. Addition of 50 ng/ml human GH to cells in chemically defined medium led to a rapid increase in binding activity of activator protein 1 (AP-1) and CCAAT enhancer-binding protein (C/EBP), which was significant at 30 min and reached maximal induction by 2 h (3-fold for AP-1, 2.5-fold for C/EBP). Induction in AP-1 DNA binding correlates with a concomitant GH trans-activation of c-jun and c-fos genes described previously. Using specific antibodies in electrophoretic mobility shift assays and Western blots, it was shown that the increase in activity of C/EBP is the result of an increase in synthesis of two alternatively translated forms of C/EBP beta: 40-C/EBP beta and 23-C/EBP beta. This increase in protein was not accompanied by alteration in mRNA level and could be blocked by a Janus kinase 2 tyrosine kinase inhibitor and a C kinase inhibitor at concentrations shown to inhibit GH-dependent activation of microtubule-associated protein (MAP) kinases. Concomitant with the translationally activated increase in C/EBP beta, a GH-dependent increase was observed in C/EBP delta transcription. This was accompanied by an increase in mRNA for C/EBP delta, which was superinduced by cycloheximide and, unlike the increase in C/EBP beta protein, was not observed with insulin. Thus GH exerts its effects on C/EBP isoforms at two levels: transcriptional activation of C/EBP delta and translational activation of C/EBP beta. It is proposed that GH-dependent phosphorylation results in the efficient translation of 40-C/EBP beta and 23-C/EBP beta (the mouse homolog of the inhibitor liver-enriched inhibitory protein), and that together with the induction of C/EBP delta, these may be involved in initiating the adipocyte differentiation program.
Interleukin-12 (IL-12) is necessary for the production of IFN-gamma by NK cells during the generation of innate immunity and by T cells for the development of the Th1 response during specific cell-mediated immunity. Here we demonstrate that the endogenous production of IL-12 is critical to the survival of both immunocompromised SCID mice and normal C.B-17 control mice during a primary infection with Listeria monocytogenes. When IL-12 is neutralized in vivo, both strains of mice die at a normally sublethal dose of Listeria. Anti-IL-12 antibody-treated mice showed a decrease in macrophage I-Ad expression and an increase Listeria burden in the spleen. Furthermore, as has been demonstrated in vitro, these effects of IL-12 in vivo were predominantly regulated through the production of IFN-gamma. Administration of IFN-gamma simultaneously with neutralizing antibodies to IL-12 restored macrophage I-Ad expression, limited the spread of the infection, and resulted in the survival of SCID mice. Thus, IL-12 is critical for resistance to infection with Listeria monocytogenes, and this resistance is mediated through stimulation by IL-12 of IFN-gamma production. Concomitant experiments confirmed that anti-TNF antibodies also resulted in uncontrolled infection and a decrease in macrophage I-Ad expression. However, administration of IFN-gamma restored the levels of I-Ad in macrophages but did not limit Listeria growth.
Tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) activate transcription of the TSG-6 gene in normal human
fibroblasts through a promoter region (-165 to -58) that encompasses an AP-1 and a NF-IL6 site. We show by deletion analysis
and substitution mutagenesis that both sites are necessary for activation by TNF-alpha. Activation by IL-1 requires the NF-IL6
site and is enhanced by the AP-1 site. These results suggest that the NF-IL6 and AP-1 family transcription factors functionally
cooperate to mediate TNF-alpha and IL-1 signals. Consistent with this possibility, IL-1 and TNF-alpha markedly increase the
binding of Fos and Jun to the AP-1 site, and NF-IL6 activates the native TSG-6 promoter. Activation by NF-IL6 requires an
intact NF-IL6 site and is modulated by the ratio of activator to inhibitor NF-IL6 isoforms that are translated from different
in-frame AUGs. However, the inhibitor isoform can also bind to the AP-1 site and repress AP-1 site-mediated transcription.
The finding that the inhibitor isoform antagonizes activation of the native TSG-6 promoter by IL-1 and TNF-alpha suggests
that NF-IL6 has a physiologic role in these cytokine responses. Thus, the functionally distinct NF-IL6 isoforms cooperate
with Fos and Jun to positively and negatively regulate the native TSG-6 promoter by TNF-alpha and IL-1.
Activated macrophages produce substantial quantities of paracrine mediators, including cytokines, nitric oxide, and prostaglandins. Transforming growth factor beta 1 (TGF-beta) is a potent modulator of immune function. TGF-beta inhibits the cytotoxic activity of endotoxin/lipopolysaccharide (LPS)-activated macrophage cell lines and primary macrophage cultures, reducing their expression of cytokines and nitric oxide. In this report we demonstrate that TGF-beta also attenuates the LPS-induced synthesis and secretion of prostaglandin E2 in murine RAW 264.7 macrophage cells. Macrophage activation also induces accumulation of the recently described ligand-responsive prostaglandin synthase (PGS) TIS10/PGS-2. While TGF-beta alone has no effect on expression from the TIS10/PGS-2 gene, this cytokine inhibits LPS-induced TIS10/PGS-2 protein accumulation and synthesis, as well as LPS-induced TIS10/PGS-2 message accumulation in RAW 264.7 cells. TGF-beta concentrations in the range of 0.1-1.0 ng/ml (4-40 pM) maximally inhibit LPS-induced TIS10/PGS-2 message accumulation. In contrast, neither LPS nor TGF-beta has any effect on the level of PGS-1 (EC 1.14.99.1) message. TGF-beta also attenuates LPS-induced accumulation of unspliced TIS10/PGS-2 transcripts in RAW 264.7 cells, suggesting that this cytokine exerts its effects on TIS10/PGS-2 expression at the transcriptional level. TGF-beta inhibits the LPS-induced accumulation of TIS10/PGS-2 protein and message in cultured murine peritoneal macrophages, as well as in macrophage cell lines.
One of the members of the bZIP family of transcriptional activators is NF-IL6/LAP (IL-6 DBP, C/EBP beta, CRP2). NF-IL6/LAP protein is highly expressed in liver nuclei, where it has been implicated as a master regulator of the acute-phase response, induced by interleukin-6 (IL-6) and other inflammatory mediators. Also, NF-IL6/LAP is involved in the activation of the IL-6 promoter in response to IL-1 and bacterial lipopolysaccharide. The control of NF-IL6/LAP expression and activity is complex and poorly understood. Under some conditions the NF-IL6/LAP gene is transcriptionally activated by IL-1 and lipopolysaccharide, whereas in other instances, its binding to cognate DNA sequences is enhanced by cytokines. Additionally, the ability of constitutively expressed NF-IL6/LAP to activate transcription is strongly augmented by IL-6, through an unknown signalling pathway. We now show that stimulation of the protein kinase C pathway increases the phosphorylation of Ser 105 within the activation domain of NF-IL6/LAP, and enhances its transcriptional efficacy.
The interleukin-8 promoter is transcriptionally activated by interleukin-1, tumor necrosis factor alpha, phorbol myristate
acetate, or hepatitis B virus X protein through a sequence located between positions -91 and -71. This region contains an
NF-kappa B-like and a C/EBP-like binding site. We show here that several members of the NF-kappa B family, including p65,
p50, p52, and c-Rel, can bind to this region, confirming an authentic NF-kappa B binding site in the interleukin-8 promoter.
Further, C/EBP binds only weakly to the interleukin-8 promoter site. Electrophoretic mobility shift assays with proteins overexpressed
in COS cells and with nuclear extracts from tumor necrosis factor alpha-stimulated HeLa cells demonstrated a strong cooperative
binding of C/EBP to its site when NF-kappa B is bound to its adjacent binding site. Transfection studies lead to a model that
suggests a highly complex regulation of interleukin-8 gene expression at multiple levels: independent binding of C/EBP and
NF-kappa B to their respective sites, cooperative binding of C/EBP and NF-kappa B to DNA, and positive synergistic activation
through the C/EBP binding site and inhibition through the NF-kappa B binding site by combinations of C/EBP and NF-kappa B.
Thus, the ultimate regulation of interleukin-8 gene expression depends on the ratio of cellular C/EBP and NF-kappa B.