NFAT Inhibitor

Involvement of JNK signaling pathway in lipopolysaccharide-induced complement C3 transcriptional activation from amphioxus Branchiostoma belcheri

Abstract

Complement C3 plays a crucial role in all three pathways of complement activation. In the circulatory system, C3 is primarily produced by the hepatic cecum. During acute inflammation, hepatocytes increase both the expression and secretion of C3. However, the detailed mechanisms that regulate C3 gene transcription remain poorly understood. In this study, the 5’-flanking region of the amphioxus C3 gene was characterized to investigate its regulatory elements. A series of luciferase reporter gene constructs, including deletions and mutations of specific regulatory sites, were created to analyze the promoter region’s function. The experiments demonstrated that a C-JUN-1 binding site within the proximal promoter is essential for full activation of the C3 promoter. Other transcription factor binding sites, including those for NF-κB, AP-1, C-JUN-2, and NFAT, contribute to maintaining the promoter’s activity under normal, homeostatic conditions. Further investigation showed that treatment with SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), reduced the lipopolysaccharide (LPS)-induced activation of the C3 promoter, as well as C3 mRNA expression and protein secretion. These results indicate that the JNK signaling pathway influences C3 gene transcription by targeting C-JUN binding sites within the promoter region. This pathway mediates the LPS-induced activation of C3, suggesting a potential therapeutic target for modulating C3 expression.

Keywords: Complement C3, Promoter activity, Transcription factor, Lipopolysaccharide, c-Jun N-terminal kinase

Introduction

Amphioxus, also known as lancelet, is a basal chordate and has recently been recognized as a valuable model organism for studying the origin and evolution of vertebrates. It possesses a vertebrate-like body plan, including a circulatory system organized similarly to that of vertebrates. Despite this similarity, amphioxus is less complex due to a simpler genome, which lacks extensive genomic duplications, and the absence of lymphoid organs and free circulating blood cells. These features, along with its structural and genomic simplicity, make amphioxus an excellent organism for investigating the origins and evolutionary development of the vertebrate immune system, particularly adaptive immunity, as well as the innate immune mechanisms found in vertebrates.

The amphioxus has a hepatic cecum, a forward-protruding pouch extending from the digestive tube near the posterior part of the pharynx. This structure is widely considered a precursor to the vertebrate liver. In vertebrates, the liver is the main site of synthesis for complement components, including C3 proteins, playing an essential role in immune defense against pathogens. The acute phase response in amphioxus shows similarities to that in vertebrates, further supporting its use as a model for immune system studies.

Complement C3 is a central molecule indispensable for all complement activation pathways. Beyond its role in activating the complement system, C3 and its breakdown products facilitate phagocytosis, trigger inflammatory responses to pathogens, and regulate the differentiation and maturation of B cells, T cells, and dendritic cells. The complement system contributes significantly to both innate and adaptive immunity by mediating processes such as phagocytosis, chemotaxis, and cell lysis. As an acute-phase protein, its production rapidly increases upon inflammatory stimuli.

Immunomodulators like lipopolysaccharide (LPS) enhance complement and antibody responses, thereby increasing resistance to bacterial and parasitic infections. LPS, a component of the outer membrane of Gram-negative bacteria, activates various cell types and initiates multiple intracellular signaling pathways. These include activation of NF-κB, which promotes the production of proinflammatory mediators, and the mitogen-activated protein kinase (MAPK) pathways—ERK, JNK (also known as stress-activated protein kinase, SAPK), and p38. These MAPK pathways regulate diverse cellular responses including differentiation, apoptosis, and inflammation.

The c-Jun N-terminal kinases (JNK1, JNK2, and JNK3) were originally identified as stress-activated protein kinases that respond to conditions inhibiting protein synthesis. Upon activation, JNKs bind to and phosphorylate the DNA-binding protein c-Jun, increasing its transcriptional activity. C-Jun is part of the AP-1 transcription complex, a key regulator of gene expression involved in controlling cytokine genes and responding to environmental stress, radiation, and growth factors. JNKs also play critical roles in programmed cell death (apoptosis). Inhibiting JNK enhances the effects of chemotherapy on tumor cells, suggesting a role for JNK as a therapeutic target in cancer treatment. Additionally, JNK inhibitors have shown promise in animal models of rheumatoid arthritis.

This study focused on isolating the regulatory region of the C3 gene from the genomic DNA library of adult amphioxus Branchiostoma belcheri. The main objectives were to characterize the expression pattern of the C3 gene in adult amphioxus, examine its regulation in response to LPS challenge, and determine the involvement of the JNK signaling pathway in LPS-induced transcriptional activation of the C3 gene.

Materials and Methods

C3 Promoter Constructs

The regulatory regions of the amphioxus C3 gene promoters were amplified from Branchiostoma belcheri genomic DNA using polymerase chain reaction (PCR). The PCR products were cloned using a TA cloning system to generate plasmids for further analysis. The C3 promoter sequences were inserted into vectors at specific restriction enzyme sites to create various plasmid constructs containing promoter fragments of different lengths. This enabled the study of promoter activity through reporter assays.

In addition, a series of mutant promoter-luciferase constructs were engineered. These constructs included point mutations targeting specific transcription factor binding sites such as NF-κB, AP-1, C-JUN-1, C-JUN-2, and NFAT. Some constructs featured triple point mutations combining several binding sites. All single point mutations were designed based on prior research. Mutant promoters spanning nucleotides -466 to -1 relative to the transcription start site were generated by PCR using wild-type templates, while triple mutation promoters were derived from single mutation constructs. This approach allowed detailed functional analysis of the individual and combined roles of these transcription factor binding sites in regulating C3 promoter activity.

Cell Culture

HEK 293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics, including 100 units/mL penicillin G and 100 units/mL streptomycin sulfate. Cells were maintained in an incubator at 37°C with 5% CO2 and 95% humidity, and were passaged no more than 20 times to ensure consistency.

Measurement of C3 Promoter Activity

The transcriptional regulation of the C3 gene was studied by transiently transfecting HEK 293T cells with C3 reporter constructs, including enhanced green fluorescent protein (EGFP) reporter plasmids containing promoter fragments of different lengths, as well as promoter-luciferase reporter plasmids. Twenty-four hours post-transfection, EGFP fluorescence was observed using a fluorescence microscope, and luciferase activity was measured using a Dual Luciferase Reporter Assay System according to the manufacturer’s instructions.

Transient Transfection of C3 Reporter Constructs

For reporter assays, HEK 293T cells were separately transfected with mutant C3 promoter plasmids containing mutations in NF-κB, AP-1, C-JUN-1, C-JUN-2, NFAT, combined mutations such as C-JUN-1-C-JUN-2, AP-1-C-JUN-1-C-JUN-2, NF-κB-NFAT, and a wild-type C3 promoter plasmid. These transfections were conducted with or without treatment of lipopolysaccharide (LPS) and the JNK inhibitor SP600125 as specified. The wild-type and mutant C3 promoter plasmids, along with the pGL3 control plasmid, had been previously characterized.

Cells at approximately 80% confluence were transfected with 0.75 micrograms of total DNA using Lipofectamine 2000, following the manufacturer’s protocol. All plasmid DNA preparations were endotoxin-free. After transfection, cells were treated with either LPS at 10 micrograms per milliliter or SP600125 at 20 micromolar for six hours. SP600125 was dissolved in 10% dimethyl sulfoxide (DMSO) and added to culture media prior to LPS treatment. The final DMSO concentration never exceeded 0.1%.

Dual Luciferase Reporter Assays

Luciferase reporter assays were performed using a Dual Luciferase Reporter Assay System following the manufacturer’s guidelines. Renilla luciferase activity was used for normalization. After 24 hours of culture, cells were washed with phosphate-buffered saline (PBS) and lysed with 100 microliters of passive lysis buffer. Lysates were subjected to freeze-thaw cycles at -80°C for 12 hours. Subsequently, 20 microliters of cell lysate were mixed with 50 microliters of luciferase substrate solution and luminescence was measured using a spectrofluorimeter according to the kit’s protocol.

In Vivo Infection

Adult amphioxus Branchiostoma belcheri, averaging about 4 centimeters in length, were obtained and acclimatized in sterilized, filtered seawater for two days. They were divided into groups and exposed to either control conditions, 20 micrograms per milliliter of LPS, or 40 micromolar of the JNK inhibitor SP600125 in filtered seawater at room temperature. Samples of eight animals per group were collected at 6 and 12-hour intervals. The hepatic cecum tissues were harvested separately for RNA extraction and western blot analysis.

Western Blotting Analysis

After treatment with LPS and SP600125, amphioxus tissues were homogenized using RIPA buffer containing 1% Triton X-100, 1% deoxycholate, and 0.1% SDS. Protein concentrations were determined through the BCA Protein Assay Kit. The tissue homogenates were then combined with SDS sample loading buffer, which included components such as Tris-HCl, SDS, glycerol, dithiothreitol, and bromophenol blue. This mixture was subjected to ultrasonication followed by boiling for 2 minutes. For each sample, 40 micrograms of protein were loaded and separated by SDS-PAGE, running for two hours at 120 volts.

Proteins were subsequently transferred onto PVDF membranes, which were washed three times with TBST buffer. The membranes were blocked with 5% bovine serum albumin (BSA) and then incubated overnight at 4°C with primary antibodies. Following this incubation, the membranes were washed and treated with peroxidase-conjugated secondary antibodies for two hours at room temperature. After additional washes, the antigen-antibody complexes were detected using enhanced chemiluminescence and analyzed with image analysis software.

RNA Extraction and cDNA Synthesis

To assess mRNA expression levels of the C3 gene, total RNA was extracted from various adult amphioxus tissues, including gonad, notochord, intestine, gill, muscle, and hepatic cecum. Post-stimulation expression patterns were evaluated using RNA isolated from the hepatic cecum. Samples from 24 individuals were pooled to create three biological replicates, each containing RNA from eight animals. Total RNA was extracted from these replicates at each sampling point using Trizol reagent. Reverse transcription was performed to synthesize cDNA employing an oligo(dT) primer and Superscript II enzyme. The concentration and quality of the RNA samples were measured by spectrophotometry, and their integrity was verified by electrophoresis on a 1% agarose gel. Both RNA and cDNA samples were stored at -80°C for subsequent analyses.

Quantitative Real-Time PCR Analysis

After validating the quality of the cDNA templates and primers, mRNA levels of target genes and the internal reference gene β-actin were quantified using real-time PCR in triplicate. This was conducted on an ABI StepOnePlus real-time PCR system. SYBR Premix Ex Taq was used following the manufacturer’s protocol, with primer concentrations set at 200 nM. The total reaction volume was 20 µL, containing 2 µL of diluted cDNA template, 0.4 µM of each primer, 0.4 µL of ROX Reference Dye, and 10 µL of 2× SYBR Premix Ex Taq II. The PCR cycling program included an initial denaturation step at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. A dissociation step was carried out at the end of the amplification cycles to confirm specificity of the PCR products.

Fluorescence was measured at the end of each extension phase to determine threshold cycle (Ct) values. Data analysis was performed using StepOnePlus SDS software, with relative gene expression quantified by the comparative Ct (2^-ΔΔCt) method. The expression levels of complement genes were normalized against β-actin to calculate relative mRNA levels.

Statistics

Results are presented as means ± standard deviation (SD) based on at least three replicates for each time point and three independent experiments. Differences between group means were assessed using one-way ANOVA followed by Dunn’s multiple range test. Statistical analyses were conducted with SPSS version 20.0 for Windows. Statistical significance was accepted at a two-sided p-value less than 0.05. Densitometric analysis of visible protein bands was performed using Image J software.

Results

Characterization of the 5’-Flanking Region of the Amphioxus C3 Gene Promoter

To explore the regulatory mechanisms underlying LPS-induced expression of the C3 gene, the promoter region of the amphioxus C3 gene was analyzed. Computational analysis indicated that the core promoter is located within approximately 2000 nucleotides upstream of the transcription start site. To assess transcriptional activity, three distinct upstream regions of the gene—ranging from -2160 to +1 nt, -963 to +1 nt, and -466 to +1 nt—were cloned into a plasmid vector and introduced into HEK 293T cells. The expression of reporter protein was detected in all three constructs, demonstrating that each region possesses promoter activity. The most critical regulatory elements required for basal transcription were found to reside between -466 and +1 nucleotides relative to the transcription initiation site.

Further functional analyses using luciferase-based reporter constructs revealed that removing the segment between -963 and -466 had little impact on overall promoter activity. This result supports the conclusion that the primary transcriptional control elements are contained within the proximal -466 nucleotide region upstream of the gene’s transcription start site.

LPS Significantly Enhances C3 Gene Expression in HEK 293T Cells and Amphioxus

The effect of LPS stimulation on C3 gene expression was first examined in HEK 293T cells. Cells were treated with either 10 µg/mL of LPS or PBS as a control for 24 hours. Following treatment, the cells were harvested and subjected to luciferase assays to quantify promoter activity. LPS treatment resulted in an approximate tenfold increase in C3 expression when compared to the PBS control, indicating a strong activation of C3 transcription in response to LPS.

The in vivo response to LPS was also evaluated in amphioxus, particularly focusing on tissue-specific expression. Transcripts of the C3 gene were highly expressed in the notochord, muscle, gill, and hepatic cecum, with lower expression observed in the gonad and intestine. Based on this expression pattern, the hepatic cecum was selected for further analysis. Amphioxus specimens were treated with LPS for 12 hours, after which hepatic cells were isolated and analyzed using quantitative real-time PCR. The results showed that LPS stimulation led to an approximately threefold increase in C3 mRNA levels, confirming that LPS enhances C3 gene expression in both in vitro and in vivo systems.

Identification of Critical Regulatory Motifs in the Amphioxus C3 Promoter

To pinpoint specific transcription factor binding sites involved in basal promoter activity, a detailed bioinformatic analysis was performed on the -466 to +1 region of the C3 promoter. This analysis revealed several putative binding motifs for various transcription factors.

Targeted mutagenesis was conducted to alter individual and combined binding sites for transcription factors such as NF-κB, AP-1, C-JUN-1, C-JUN-2, and NFAT. These mutations were incorporated into luciferase reporter constructs. Assays indicated that mutations in NF-κB, AP-1, C-JUN-2, and NFAT had minimal effect on promoter activity. However, mutations targeting the C-JUN-1 site alone or in combination with adjacent sites significantly reduced promoter-driven transcription. These results highlight the importance of the C-JUN-1 binding site as a key element maintaining the basal transcriptional activity of the C3 gene.

LPS-Induced Activation of C3 Gene Through C-JUN Binding Motifs

Additional experiments were conducted to determine how LPS influences the activity of specific motifs within the C3 promoter. HEK 293T cells were transfected with various constructs containing full-length or truncated promoter sequences and then treated with LPS or PBS for 24 hours. The luciferase assays revealed that constructs containing regions up to -963 or -466 nucleotides upstream of the transcription start site exhibited approximately a tenfold increase in activity after LPS treatment.

To assess the role of C-JUN binding elements in this response, constructs with targeted point mutations at specific transcription factor binding sites were used. Mutation of the C-JUN-1 site, as well as combined mutations of C-JUN-1 with C-JUN-2 and AP-1, resulted in a significant suppression of LPS-induced luciferase activity. In contrast, mutations in NF-κB, AP-1, C-JUN-2, and NFAT Inhibitor sites did not inhibit promoter activity under LPS stimulation. These findings suggest that LPS primarily exerts its effect on the C3 gene by interacting with the C-JUN motif.

C-JUN Regulates C3 Promoter Activity via the JNK Signaling Pathway

C-JUN is a downstream target of the JNK pathway, which is activated in response to a variety of stimuli. To investigate whether JNK signaling mediates LPS-induced activation of the C3 promoter, luciferase assays and western blotting were performed using the JNK inhibitor SP600125. Cells transfected with the -466/+1 promoter construct and treated with LPS showed strong luciferase activity. However, the addition of SP600125 significantly reduced this activity to basal levels, indicating that JNK signaling is essential for C3 promoter activation by LPS.

Western blotting further showed that LPS increased the phosphorylation of JNK, while treatment with SP600125 partially suppressed this phosphorylation. In hepatic cecum tissues from amphioxus, a 12-hour SP600125 treatment markedly reduced phosphorylated JNK protein levels and suppressed LPS-induced C3 expression. These findings demonstrate that the JNK pathway is activated in response to LPS and facilitates C3 gene transcription through C-JUN motifs in both in vitro and in vivo systems.

Discussion and Conclusion

Amphioxus is considered the closest invertebrate relative to vertebrates and serves as a valuable model for studying the evolutionary origins of the vertebrate immune system. The complement system, an essential component of innate immunity, is present in both invertebrates and vertebrates and functions by tagging pathogens for destruction. C3 is a central molecule in this system, responsible for pathogen opsonization and immune activation. However, the molecular mechanisms governing C3 gene expression have remained unclear.

In this study, we characterized the promoter region of the amphioxus C3 gene and identified a core promoter within the -466 to +1 nucleotide region. Functional assays using EGFP and dual-luciferase reporters demonstrated strong promoter activity across all fragments tested. In response to LPS, C3 transcription was significantly enhanced in both HEK 293T cells and amphioxus, particularly in hepatic cecum tissues. Further analysis of C3 promoter mutants revealed that a 179-bp region near the transcription start site is essential for promoter activity and is regulated via the JNK pathway.

The hepatic cecum of amphioxus, which shares functional similarities with the vertebrate liver, is a primary site for the expression of acute phase proteins. Our findings support the hypothesis that this tissue plays a central role in the immune response of amphioxus, similar to liver function in vertebrates.

Importantly, we demonstrated that the C-JUN motif located at -179 bp is a functional binding site critical for the transcriptional regulation of the C3 gene. Mutation of this motif substantially decreased promoter activity. Furthermore, the use of a JNK inhibitor revealed that the JNK pathway is involved in mediating LPS-induced C3 expression. Collectively, these results suggest that LPS activates JNK, which in turn phosphorylates C-JUN, leading to enhanced C3 gene transcription.

In summary, this study offers novel insights into the regulatory mechanisms of C3 gene expression in amphioxus. Our data highlight the pivotal role of the JNK-C-JUN signaling axis in mediating immune responses and suggest that this pathway may be a key component in the evolution of innate immunity. These findings also provide a foundation for future research into the transcriptional regulation of complement genes in early chordates and their evolutionary significance.

Acknowledgements

We thank Professor Hong Shi at the Third Institute of Oceanography, State Oceanic Administration for providing anti-p-JNK antibodies, and Professors Yi-quan Wang and Guang Li at Xiamen University for supplying adult amphioxus (Branchiostoma belcheri). This work was supported by the National Key R&D Program of China (Grant 2017YFC1404500), the Beihai Pilot City Program for National Innovative Marine Economic Development, the International Science & Technology Cooperation Program of China (2015DFA20500), and the Scientific Research Foundation of the Third Institute of Oceanography, SOA (2017005).