CCAAT/enhancer binding protein β directly regulates the expression of the complement component 3 gene in neural cells: implications for the pro-inflammatory effects of this transcription factor

RESEARCH Open Access CCAAT/enhancer binding protein βdirectly regulates the expression of the complement component 3 gene in neural cells: implications for the pro-inflammatory effects of this transcription factor Elena Hernandez-Encinas 1,2 , Diana Aguilar-Morante 1,2 , Marta Cortes-Canteli 1,3 , Jose A Morales-Garcia 1,2 , Elena Gine 2,4 , Angel Santos 2,4* and Ana Perez-Castillo 1,2* Abstract Background: The CCAAT/enhancer-binding protein β(C/EBPβ) is a transcription factor, which was first identified as a regulator of differentiation and inflammatory processes mainly in adipose tissue and liver; however, its function in the brain was largely unknown for many years. Previous studies from our laboratory indicated that C/EBPβis implicated in inflammatory process and brain injury, since mice lacking this gene were less susceptible to kainic acid-induced injury. Methods: We first performed cDNA microarrays analysis using hippocampal RNA isolated from C/EBPβ +/+ and C/EBPβ −/− mice. Immunocytochemical and immunohistochemical studies were done to evaluate C/EBPβand C3 levels. Transient transfection experiments were made to analyze transcriptional regulation of C3 by C/EBPβ.To knockdown C/EBPβand C3 expression, mouse astrocytes were infected with lentiviral particles expressing an shRNA specific for C/EBPβor an siRNA specific for C3. Results: Among the genes displaying significant changes in expression was complement component 3 (C3), which showed a dramatic decrease in mRNA content in the hippocampus of C/EBPβ −/− mice. C3 is the central component of the complement and is implicated in different brain disorders. In this work we have found that C/EBPβregulates C3 levels in rodents glial in vitro and in the rat Substantia nigra pars compacta (SNpc) in vivo following an inflammatory insult. Analysis of the mouse C3 promoter showed that it is directly regulated by C/EBPβthrough a C/EBPβconsensus site located at position −616/-599 of the gene. In addition, we show that depletion of C/EBPβby a specific shRNA results in a significant decrease in the levels of C3 together with a reduction in the increased levels of pro-inflammatory agents elicited by lipopolysaccharide treatment. Conclusions: Altogether, these results indicate that C3 is a downstream target of C/EBPβ, and it could be a mediator of the pro-inflammatory effects of this transcription factor in neural cells. Keywords: C/EBPβ, C3, neuroinflammation, neural cells, transcriptional regulation * Correspondence: [email protected];[email protected] 2 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain 1 Instituto de Investigaciones Biomédicas, (CSIC-UAM), Arturo Duperier, 4, 28029 Madrid, Spain Full list of author information is available at the end of the article JOURNAL OF NEUROINFLAMMATION © 2015 Hernandez-Encinas et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hernandez-Encinas et al. Journal of Neuroinflammation (2015) 12:14 DOI 10.1186/s12974-014-0223-2 Background CCAAT/enhancer-binding protein β(C/EBPβ), is a member of a family of transcription factors consisting of six structurally related basic leucine-zipper DNA-binding proteins. All six members of this family exhibit a high sequence similarity in their C-terminal DNA binding domain and a more diverge regulatory N-terminus [1,2]. C/EBPβis encoded by a single mRNA, yet translation of this mRNA results in the synthesis of three C/EBPβisoforms based on alternative translation initiation sites [3,4]. C/EBPβis expressed in numerous tissues, including liver, adipose tissue, kidney, lung, ovary, mammary gland, and hematopoietic tissues and regulates a variety of biological processes, including metabolism, proliferation and differentiation, and immune response [1,5-7]. This regulation takes place through the induction or repression of many genes involved in these processes, such as proliferative- or differentiation-related markers, cytokines, and many proinflammatory genes [1,8]. Also, due to its relevance in the indicated cellular processes, C/EBPβalso is involved in the pathogenesis of different diseases, for example, cancer, hyperinflammatory processes, and bacterial infections [1]. Regarding the central nervous system (CNS), it has been shown that the C/EBPβgene is expressed in different areas of adult rodents [9] and different studies, including those from our group, have indicated that C/EBPβcan have important functions in the brain. It has been shown that C/EBPβplays an important role in the consolidation of long-term memory, suggesting a very important role for this protein in the hippocampus [10,11] and Menard et al. have defined a MEK-C/EBP pathway as being essential for the differentiation of cortical progenitor cells into postmitotic neurons [12]. In this regard, we have demonstrated that C/EBPβserves as a critical factor in neuronal differentiation [13]. More recently we have demonstrated that C/EBPβregulates the expression of several genes involved in inflammatory processes and brain injury [14] and mice lacking C/EBPβshowed a reduced inflammatory response after kainic acid injection and exhibited a dramatic reduction in pyramidal cell loss in the CA1 and CA3 subfields of the hippocampus [15]. These data reveal an essential function for C/EBPβin the pathways leading to brain damage and suggest that C/EBPβshould be considered as a therapeutic target in brain injury and neurodegenerative disorders where neuronal cell death and inflammation are involved. Finally, we have also shown that C/EBPβis expressed in the dentate gyrus of the hippocampus and has a key role in regulating the proliferation and differentiation of the stem/progenitor cell population [16]. The complement system plays important roles in the immune response, including chemotaxis, phagocytosis, cell adhesion, and B and T cell differentiation [17,18], and it is a very important line of defense against infections through the elimination of invading pathogens and regulation of the adaptive immune response [19,20]. More recent works show that complement components can be produced by astrocytes, microglia and neurons and that the complement system also plays important roles in the CNS that extend far beyond host defense and inflammatory processes [21-27]. Complement component 3 (C3) is a 185-kDa glycoprotein, which is the central player in the complement cascade and is involved in opsonization of pathogens, inflammatory processes, and immune response [28]. Regarding the brain, C3 has been shown to be essential for the elimination, or pruning, of incorrect synapsis during development [26,29,30]. In fact, mice deficient in C3 exhibit deficits in synaptic remodeling, increased synaptic connectivity, and enhanced epileptiform activity due to failed synaptic pruning [31]. Also, it has been shown that C3 regulates hippocampal neurogenesis in adult mammalian brain [32] and C3-deficient mice present impaired neurogenesis following cerebral ischemia [33,34]. These results point to C3 as a positive regulator of both basal and ischemia-induced neurogenesis. Besides its role in the CNS in physiological conditions, uncontrolled complement activation in the brain also has been shown to be involved in the neuroimmunological and inflammatory processes that underlie the pathophysiology of many acute and neurodegenerative disorders, including Alzheimer’s, Parkinson’s, dementia, meningoencephalitis, and multiple sclerosis [25,35-42]. Increased levels of C3 have been found in the cerebrospinal fluid (CSF) of patients with Parkinson’sandAlzheimer’sdiseasesandtheselevels augment with the progression of the disease [42]. Zanjani et al. showed that C3 localized surrounding β-amyloid plaques in early stages of Alzheimer disease, when an important loss of synapsis takes place [43]. Also, enhanced synthesis of different complement components has been reported in animal models of neurodegeneration and neuroinflammation, such as exposure to neurotoxins, virus-induced encephalomyelitis, and excitotoxic kainic acid lesion [44-47]. In this work, we have analyzed the regulation of C3 by C/EBPβand its possible involvement as a mediator of the effects of C/EBPβin inflammatory processes. First, we have shown, by microarray and quantitative RT-PCR analysis, a dramatic decrease in C3 mRNA content in the hippocampus of C/EBPβknockout mice. The analysis of the mouse C3 promoter showed that C3 is directly regulated by C/EBPβ, primarily through a C/EBPβ consensus site located at position −616/-599 of the gene. Expression studies showed a parallel induction of C/EBPβand C3 proteins by lipopolysaccharide (LPS) in vitro in primary cultures of astrocytes and microglial cells and in vivo in the Substantia nigra pars compacta (SNpc) of adult rats. Ectopic expression of C/EBPβin mouse cultured astrocytes induced the expression of the endogenous C3 gene. On the other hand, depletion of Hernandez-Encinas et al. Journal of Neuroinflammation (2015) 12:14 Page 2 of 16 C/EBPβby a specific shRNA diminished C3 expression levels together with a reduction in interleukin-1β(IL-1β) and cyclooxygenase type 2 (COX-2), after LPS treatment. Altogether, these results suggest a direct role of C3 in the pro-inflammatory effects of C/EBPβ. Methods Animals Adult male Wistar rats (8 to 12-weeks old) and neonatal Wistar rats (2-days-old) were used throughout the study. MicroarraysanalysiswasperformedinadultC/EBPβ +/+ and C/EBPβ −/− mice. C/EBPβ +/+ and C/EBPβ −/− mice were generated from heterozygous breeding pairs, kindly provided by C. M. Croniger and R. W. Hanson (Case Western Reserve University, Cleveland, OH) [48]. Genotypes were identified using genomic PCR, with DNA prepared from tail using the REDExtract-N-AmpTM tissue PCR kit (XNAT kit, Sigma- Aldrich, Madrid, Spain). All procedures with animals were specifically approved by the Ethics Committee for Animal Experimentation of the Instituto de Investigaciones Biomedicas and carried out in accordance with the European Communities Council, directive 86/609/EEC. Special care was taken to minimize pain or discomfort of animals. Microarray analysis Total RNA was extracted from hippocampus of C/EBPβ +/+ and C/EBPβ −/− mice using Trizol (Agilent Technologies, Madrid, Spain) and the integrity of RNA confirmed in an Agilent 2100 Bioanalyzer. The microarray analysis (seven wild type and seven knockouts samples) was performed using the G4122F Whole Mouse Genome Microarray Kit (Agilent Technologies, Madrid, Spain), at the CNIC Genomic Department. The chips were scanned using the Agilent G2567AA Microarray Scanner System (Agilent Technologies, Madrid, Spain). Image analysis and data collection were carried out using the Agilent Feature Extraction 9.1.3.1. (AFE, Agilent Technologies, Madrid, Spain). The AFE algorithm yields a background-adjusted signal by subtracting the background signal from the mean signal. Real-time PCR Total hippocampal RNA samples (2 μg) were used for the synthesis of cDNA by reverse transcription using the Reverse Transcription System (Promega Corporation, Madrid, Spain) with a pd(N)6 random hexamer. Real-time PCR was performed in an ABI Prism machine using the SYBR Green PCR Master Mix (Applied Biosystems, Madrid, Spain) and 300 nM concentrations of specific primer. The primers used for the determination of the concentration of C3 mRNA were: 5’-ACC TTA CCT CGG CAA GTT TCT-3’(forward sequence) and 5’-TTG TAG AGC TGC TGG TCA GG- 3’(reverse sequence), which synthesized a fragment of 140 bp. In all runs, melting curves were performed to ensure that only one DNA fragment was amplified. Cycle threshold for wild type mice (dilution 1:10) was around 25. Amplification of the 18S rRNA was used for normalization of cDNA loading in the PCR as previously described [49]. The relative mRNA content was determined with the 2 -ΔΔCt method [50]. Cells culture, gene silencing and lentiviral infection GL261 murine glioblastoma cells were obtained from the NCI-Frederick Cancer Research Tumor Repository (Frederick, MD) and propagated in RPMI medium with 10% fetal bovine serum as described [51]. Rat and mouse primary astrocyte and microglial cultures were prepared as previously described [52]. Cultures were stimulated with LPS (10 μg/ml) and cells harvested 24 hours later for evaluation of C/EBPβand C3 content. For C3 silencing, mouse astrocytes were grown in 60-mm plates and transiently transfected with 150 pmoles of an siRNA specific for C3 (Santa Cruz Biotechnology, Heidelberg, Germany) using Lipofectamine (as indicated by the manufacturer). The control siRNA also was provided by Santa Cruz Biotechnology. After 24 hours of transfection, plates were treated with LPS at a concentration of 10 μg/ml, and 48 hours later plates were washed twice with cold PBS 1× and processed for protein extraction. To knockdown C/EBPβexpression, mouse astrocytes were infected with lentiviral particles expressing a shRNA specific for C/EBPβ. The interfering sequence was: GAG CGACGAGTACAAGATG [53]. Annealed oligonucleotides were cloned into pGreenPuroTM shRNA lentiviral vector (Cloning and Expression Lentivector; SBI, System Biosciences, Mountain View, CA, USA), according to the manufacter’s protocol, in which shRNA was expressed under the control of the H1 promoter. The control pGreenPuro™construct with the luciferase shRNA Template provided by System Biosciences was used as control in all the experiments. The lentiviral particles were obtained in HEK293T cells, which were transfected with the appropriate lentiviral expression vectors and the third generation packaging vectors pMD2-G, pMDLg/ pRRE, and pRSV-Rev [54]. The medium containing the lentiviruses was recovered, filtered through a 0.45-μm filter and used to infect astrocytes. The infection was repeated 12 h and 24 h later. After 14 h from the last infection, half of the plates of each of the groups were treated with LPS (10 μg/ml) for 24 h after which all plates were processed for extraction of protein. Promoter cloning and luciferase assay A fragment of the mouse C3 promoter, from −1624/+87, was PCR-amplified from mouse genomic DNA using specific primers and high-fidelity Eppendorf Triple Master PCR System (Eppendorf, Hamburg, Germany). The primers used were: 5’-GGA GAA CAA TAC AGA GAG GAG G-3’ (forward sequence) and 5’-AGT GAA GGA AAA AGG Hernandez-Encinas et al. Journal of Neuroinflammation (2015) 12:14 Page 3 of 16 TGG AAG-3’(reverse sequence). The entire promoter fragment, P.C3/1711 was sequenced and subcloned in the promoterless luciferase reporter vector pGL4.10. Also, two shorter fragments (−255/+42; P.C3/297) and −79/+42; P.C3/121) were PCR amplified from the P.C3/1711 fragment. Point mutations were generated by PCR using specific mutated primers and the Quick Change Site-Directed Mutagenesis Kit (Agilent Technologies, Madrid, Spain). This system uses the Pfu Turbo DNA Polymerase and subsequent digestion with DpnI, to eliminate the methylated parental band. Mutations were confirmed by sequencing. Mutated primers were: P.C3/MutA: 5’-AAG TAG GGC CCG GGT CCT GCC CAG CAA CTG A-3 (for the C/EBPβbinding site located at −616/-599) and P.C3/ MutB: 5’-CTT AGG AAA CGC ACA ATG TCT CAA GTG GGC AGT CCC-3’(for the C/EBPβbinding site at positions −108/-94). For transient transfection experiments, semiconfluent GL261 cells were grown in 24- well plates and transfected with lipofectamine 2000 (Invitrogen, Madrid, Spain) using the different constructs described above (1.5 μg DNA/well) in the presence or absence of the C/EBPβoverexpression plasmid pcDNA3-C/ EBPβ(2 μg DNA/well). Forty-eight hours after transfection, cells were harvested and the luciferase activity determined using a reporter assay system (Promega Corporation, Madrid, Spain). β-galactosidase was used to determine transfection efficiency. Chromatin immunoprecipitation Primary cultures of mouse astrocytes were treated with LPS (10 μg/ml) and 24 hr later chromatin immunoprecipitation (ChIP) assay was performed, following the manufacturer’s recommendations (Santa Cruz Biotechnology). For immunoprecipitation, the following antibodies were used: 5 μg of rabbit polyclonal anti-C/EBPβ(Santa Cruz Biotechnology) and 2 μg of normal rabbit IgG (Santa Cruz Biotechnology, Mountain View, CA, USA). The precipitated DNA was analyzed by PCR using the following primers: C/EBPβ,sense:5’-TGC CCT CAC CCC TTA TCC TCC TC-3’;antisense:5’-GGC AGG TTC AGG AAG GTG GAG AC-3’(which amplified the C3 promoter from −669 to −525) and GAPDH, sense: 5’-GAG GTC CAC CAC CCT GTT GCT GTA GC -3’;antisense5’- GCT GAA CGG GAA GCT ACA TGG CAT GG -3’ (which amplified a fragment from +208 to +712). Thermal cycling conditions: one cycle at 95°C for 1 min; 38 cicles at 95°C for 30 s, 60°C for 30 s and 72°C for 30 s; and one cycle at 72°C for 7 min. Western blot analysis Proteins were isolated from cell cultures by standard methods. A total amount of 30 μgofproteinwasloaded on a 6% or 12% SDS-PAGE gel. After electrophoresis, proteins were transferred to protran nitrocellulose membranes (Sigma-Aldrich, Madrid, Spain), and blots were probed with the indicated primary antibodies, as previously described [14]. The antibodies used were the following: rabbit polyclonal anti-C3 (Abcam, Cambridge, UK), mouse monoclonal anti-C/EBPβ(Abcam, Cambridge, UK), goat polyclonal anti-COX2 (Santa Cruz Biotechnologies, Mountain View, CA, USA), rabbit polyclonal anti-IL1β (Abcam, Cambridge, UK), rabbit polyclonal anti-α-tubulin (Sigma-Aldrich, Madrid, Spain) and secondary peroxidase conjugated antibodies (Jackson Immunoresearch, West Grove, PA, USA). Quantification was performed using Scion Image software (Scion Corporation, http://scionimage.software.informer.com/). Nitrites measurement Accumulation of nitrites in media was assayed by the standard Griess reaction. After 24 hours of transfection with 150 pmoles of an siRNA specific for C3 or a nontargeting siRNA, plates were treated with LPS at a concentration of 10 μg/ml, and 48 hours later supernatants were collected and mixed with an equal volume of Griess reagent (Sigma-Aldrich, Madrid, Spain). Samples were then incubated at room temperature for 15 minutes and absorbance read using a plate reader at 492/ 540 nm. Immunocytochemistry Cells were processed for immunocytochemistry at the end of the treatment period as previously described [55]. Briefly, primary glial cultures grown on glass cover slips were washed with PBS, fixed for 30 min with 4% paraformaldehyde at 25°C and permeabilized for 30 min with 0.1% Triton X-100 at 37°C. After 1 h incubation with the corresponding primary antibody, cells were washed with PBS and incubated with Alexa-488 or Alexa-647 secondary antibodies for 45 min at 37°C. Then, 6- diaminidine-2-phenylindole (DAPI) was used to stain nuclei. Images were acquired using a Nikon eclipse 90i microscope, equipped with a DS-Fi1 digital camera (Nikon, Amsterdam, Holland). Microscope settings were adjusted to optimize signal-to-noise ratios. To compare fluorescence signals from different preparations, settings were fixed for all samples withinthesameanalysis.Thefollowing primary antibodies were used: rabbit polyclonal anti-C3 (Abcam, Cambridge, UK), mouse monoclonal anti-C/EBPβ(Abcam, Cambridge, UK). Lipopolysaccharide injection in vivo Adult male rats were anesthetized by intraperitoneal injection of ketamine (60 mg/kg) and medetomidine (0.125 mg/kg) and positioned in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA, USA). LPS (10 μgin2.5μl PBS) was injected into the right side of the SNpc (coordinates from Bregma: posterior −4.8 mm; lateral +2.0 mm; Hernandez-Encinas et al. Journal of Neuroinflammation (2015) 12:14 Page 4 of 16 ventral +8.2 mm, according to the atlas of Paxinos and Watson) with a 10-μl Hamilton syringe. Flow rate (1 μl/min) was kept constant with a motorized syringe pump (BASi, West Lafayette, IN, USA) and the needle was kept in place for 2 min postinjection before being slowly withdrawn. Control animals of the same age were injected with vehicle. Rats were then housed individually to recover and sacrificed 72 h after lesioning with LPS. Immunohistochemistry Rats previously anesthetized were perfused transcardially with 4paraformaldehyde, and brains removed, postfixed and processed for immunohistochemistry as previously described [56]. The following primary antibodies were used: rabbit polyclonal anti-C3 (Abcam, Cambridge, UK), mouse monoclonal anti-C/EBPβ(Abcam, Cambridge, UK), rabbit anti-tyrosine hydroxylase (TH, Chemicon International Inc., Madrid, Spain) and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP, Dako, Glostrup, Denmark). Neurotrace fluorescent Nissl stain (Molecular Probes, Madrid, Spain) and tomato lectin (Vector Labs, Peterborough, UK) were used to stain neurons and microglial cells, respectively. After being mounted with Vectashield (Vector labs, Peterborough, UK), samples were examined with a spectral LSM710 confocal microscope (Zeiss, Madrid, Spain) equipped with a Nikon DS- Qi1Mc digital camera. To compare fluorescence signals from different preparations, confocal microscope settings were fixed for all samples within the same analysis and adjusted to produce the optimum signal-to-noise ratio. Activated glial cells (microglia and astrocytes) expressing C/EBPβwere counted in the SNpc on coronal sections doubled immunostained with tomato lectin (microglia) or GFAP (astrocytes) in five well-defined high magnification (×400) fields in five animals/experimental group, using a computer-assisted image analysis software (Soft Imaging System Corp, Lakewood, CO, USA). Double C/EBPβ/tomato-lectin or C/EBPβ/GFAP positive cells counting were expressed as a percentage over the total number of tomato-lectin + or GFAP + cells, respectively. Table 1 Genes regulated by C/EBPβinvolved in immune/inflammatory response Upregulated genes in C/EBPβ +/+ Accession no. Symbol Name Biological processes NM_009778 C3 Complement component 3 Complement activation, immune and inflammatory response, response to stress. NM_013590 Lzp-s Plysozyme structural Cell wall macromolecule catabolic processes, defence response to bacterium. NM_016972 S1c7a8 Solute carrier family (cationic amino acid transporter, y + system), member 8 Integral component of membrane, amino acid transmembrane transporter activity, toxin transporter activity. NM_009662 Alox 5 Arachidonate 5-lipoxygenase Defence response, response to stress, immune response, oxidoreductose and dioxygenase activity. NM_130452 Bbox1 Butyrobetaine (gamma), 2-oxoglutarate dioxygenase 1 (gamma-butyrobetaine hydroxylase) Carnitine biosynthesis, adrenal gland development, metabolism. NM_027961.1 Wfdc3 WAP four-disulfide core domain 3 Inflammation, bacterial opsonization, immune responses. NM_026331 S1c25a37 Solute carrier family 25, member 37 Mitochondrial biogenesis, neurotransmitter transporter. NM_053262 Dhrs8 Dehydrogenase/reductase (SDR family) member 8 Steroid dehydrogenase activity, androgen catabolic process, lipid metabolic process, oxidation-reduction process. Downregulated genes in C/EPBβ +/+ Accession no. Symbol Name Biological process NM_009628.2 ADNP Activity-dependent neuroprotective protein Neuropeptide signalling pathway, neurological disorders, inflammation. NM_008596.1 Sypl2 Synaptophysin-like 2 transporter activity, cellular calcium ion homeostasis. NM_008380 Inhba Inhibin beta-A Cytokine-cytokine receptor interaction, growth factor activity, cell cycle arrest, cellular response to cholesterol. NM_001002927 Penk1 Preproenkephalin 1 Response to stress, neuropeptide signalling pathway, signal transduction. NM_172479 S1c38a5 Solute carrier family 38, member 5 Amino acid transport across plasma membrane, oxidative stress, antioxidant defence. NM_010796 Mg11 Macrophage galactose N-acetyl-galactosamine specific lectin 1 Inflammation, macrophage polarization. NM_008198 Cfb Complement factor B Response to stress, complement activation, immune response. NM_026954 Tusc1 Tum or suppressor candidate 1 Chronic inflammation, cell cycle regulation. NM_145575 Cald1 Caldesmon 1 Immune response. Hernandez-Encinas et al. Journal of Neuroinflammation (2015) 12:14 Page 5 of 16 Figure 1 Regulation by C/EBPβof the activity of the mouse C3 promoter. (A) RT-PCR analysis of C3 mRNA content in the hippocampus of C/EBPβ +/+ and C/EBPβ −/− mice. The graphic shows the mean ± SD of three different experiments. **P<0.01. (B, C) Analysis, by transient transfection in GL261 cells, of the effect of different deletions (B) and point mutations (C) on the activity of C3 promoter. Data represent the mean ± SD of luciferase activity determined in triplicate in at least three independent experiments. ***P<0.001 versus whole (B) or non-mutated (C) C3 promoter; #P<0.05, ###P<0.001 versus C3 basal promoter activity. (D) Representative image of ChIP analysis of C/EBPβbinding to the endogenous C3 promoter in primary astrocytes. DNA before immunoprecipitation was used as positive control (Input). Hernandez-Encinas et al. Journal of Neuroinflammation (2015) 12:14 Page 6 of 16 Figure 2 Induction of C/EBPβand C3 gene expression in mouse astrocytes and microglial cells treated with lipopolysaccharide (LPS). Astrocytes and microglial cells were cultured and treated with vehicle or LPS (10 μg/ml) and the content of C/EBPβand C3 proteins determined 24 h later using specific antibodies, as indicated in Methods. (A) Representative western blot showing the levels of C/EBPβand C3. Quantification analysis is shown. Values represent the mean ± SD of five different experiments. *P<0.05, **P<0.01. (B, C) Primary astrocytes (B) or microglial (C) cells were treated as commented above and the expression of C/EBPβ(red) and C3 (green) was evaluated using immunofluorescence techniques, as described in Methods. Nuclei were stained with DAPI. Hernandez-Encinas et al. Journal of Neuroinflammation (2015) 12:14 Page 7 of 16 Figure 3 (See legend on next page.) Hernandez-Encinas et al. Journal of Neuroinflammation (2015) 12:14 Page 8 of 16 Results Microarray analysis The comparison of the hippocampal transcriptoma between wild type and C/EBPβknockout mice rendered 33 genes differentially expressed. Eighteen of these genes are genes that have been related with inflammation and cellular damage (Table 1). Among those genes, we found C3, which is a factor that plays a central role in the function of the complement system. The content of C3 transcripts was markedly reduced in the hippocampus of C/EBPβ −/− mice compared with control ones, and subsequently in this work, we pursued the study of the possible role of this gene in the pro-inflammatory and neural damage effects of C/EBPβ. C/EBPβregulates the activity of the mouse C3 promoter We initially confirmed, by real time PCR, the decrease in C3 mRNA content in the hippocampus of C/EBPβ −/− mice indicated by the microarray analysis. Our results showed an 80% decrease in the content of C3 transcripts in the hippocampus of knockout mice compared with controls (Figure 1A). To further substantiate the role of C/EBPβin regulating C3 gene expression, we studied whether C/EBPβregulates C3 promoter activity. First, we performed an in silico analysis to search for putative C/EBPβbinding sites in the mouse C3 promoter. We identified two putative consensus binding sites for this transcription factor located at positions −616/-599 (site A) and −108/-94 (site B). Transient transfection experiments in GL261 cells showed that the cotransfection of P.C3/1711 with C/EBPβsignificantly (14-fold) induced the activity of the promoter (Figure 1B), clearly indicating that C3 promoter responds to C/EBPβ,assuggestedbythe in silico analysis. In order to localize the responsive site we first analyzed the response to C/EBPβoverexpression of different deletions of C3 promoter. When site A was deleted (P.C3/297), the induction by C/EBPβwas drastically reduced. No effect of C/EBPβwas observed when both sites, A and B, were deleted (P.C3/121) (Figure 1B). A more detailed analysis, by point mutation, showed that site A is essential for the regulation by C/EBPβof the mouse C3 promoter. When site A was mutated, the induction by C/EBPβof the promoter activity was reduced from a 14-fold increment in P.C3/1711 to only a 4-fold increase in P.C3/MutA (Figure 1C). In contrast, no effect was observed when site B was mutated (Figure 1C). All these results suggest that the C/EBPβbinding site A is the most important for the regulation by C/EBPβof the mouse C3 promoter. To provide direct evidence that C/EBPβis recruited to the endogenous C3 promoter during transcription in vivo, we performed standard ChIP assays, which allow the detection of proteins bound to specific regions of DNA in vivo. For these assays we used primary cultures of astrocytes treated with LPS and a specific antibody against C/EBPβto precipitate the complex formed by DNA and this transcription factor. ChIP analysis with C/EBPβ antibody showed binding of this transcription factor to the C3 promoter at the C/EBPβconsensus binding site A (Figure 1D). As a control of specificity of the assay, binding of C/EBPβto the housekeeping GAPDH locus was almost undetectable. These data indicate that C/EBPβ interacts with the A region of the C3 promoter. Lipopolysaccharide simultaneously induces the expression of C/EBPβand C3 genes in primary cultures of mouse astrocytes and microglial cells We have previously shown that pro-inflammatory and cell injury factors induce the expression of C/EBPβin glial cells [15]. Here, we used the known pro-inflammatory factor LPS to induce the expression of C/EBPβand simultaneously analyzed the expression of C3 in primary cultures of mouse astrocytes and microglial cells. Western blot analysis showed that in astrocytes LPS induced the expression of both C/EBPβand C3. As can be seen in Figure 2A, LPS elicited a significant increase in the content of C/EBPβand C3 (six-fold and four-fold, respectively, compared with non-treated cultures). Immunocytochemistry analysis showed similar results with an increase of C/EBPβin the nuclei and C3 in the cytosol of astrocytes (Figure 2B). Interestingly, our results clearly show that this induction of C/EBPβand C3 protein levels by LPS occurs in the same cells (Figure 2B). In the case of microglial (See figure on previous page.) Figure 3 Effect of C/EBPβand C3 on IL-1βand COX-2 gene expression in mouse astrocytes. Astrocytes were transiently transfected with the indicated vectors or oligonucleotides, treated or not with lipopolysaccharide (LPS), and 24 h later the content of C/EBPβ, C3, IL-1βand COX2 proteins analyzed as indicated in Methods. (A) Representative western blot showing expression of C/EBPβand C3 in astrocytes overexpressing C/EBPβ(pcDNA3β). Quantification analysis is shown. Data represent the mean ± SD of five different experiments. *P<0.05, ***P<0.001 versus basal values. (B) Representative western blot showing expression of C/EBPβ, C3, IL-1β, and COX-2 in astrocytes transfected with a C/EBP shRNA and treated or not with LPS. Quantification analysis is shown. Values represent the mean ± SD of five different experiments. *P<0.05, **P<0,01, ***P<0.001 versus shRNA Ctrl. (C) Representative Western blot showing expression of C3, IL-1β, and COX-2 in astrocytes transfected with C3 siRNA and treated or not with LPS. Quantification analysis is shown. Values represent the mean ± SD of five different experiments. (D) Astrocytes were transfected with C3 siRNA or a non-targeting siRNA (siRNA Ctrl), treated or not with LPS and nitrite production was measured as indicated in Methods. ***P<0.001 versus siRNA Ctrl + LPS. Hernandez-Encinas et al. Journal of Neuroinflammation (2015) 12:14 Page 9 of 16 67. Vegeto E, Belcredito S, Etteri S, Ghisletti S, Brusadelli A, Meda C, et al. Estrogen receptor-alpha mediates the brain antiinflammatory activity of estradiol. Proc Natl Acad Sci U S A. 2003;100:9614–9. 68. Huber R, Pietsch D, Panterodt T, Brand K. Regulation of C/EBPbeta and resulting functions in cells of the monocytic lineage. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Hernandez-Encinas et al. Journal of Neuroinflammation (2015) 12:14 Page 16 of 16