miRNA-146 negatively regulates the production of pro-inflammatory cytokines via NF-κB signalling in human gingival fibroblasts
© Xie et al.; licensee BioMed Central. 2014
Received: 23 January 2014
Accepted: 15 November 2014
Published: 6 December 2014
In human gingival fibroblasts (HGFs), TLR4 recognises Pathogen-associated molecular patterns (PAMPs), such as LPS, and subsequently activates downstream signals that lead to the production of pro-inflammatory cytokines. The aim of this study was to explore the mechanisms of LPS-induced miRNA-146 regulation of TLR4 signals in HGFs.
Materials and methods
HGFs were treated with Porphyromonas gingivalis (P.g) LPS, the cells were harvested, and kinase phosphorylation levels were detected by western blot. Selective pharmacological inhibitors and agonists were used to block or activate the relevant kinases, miRNA-146a/b expression levels were detected by real-time PCR, and IL-1, IL-6, and TNF-α production were measured by enzyme-linked immunosorbent assays (ELISA). A luciferase reporter plasmid containing miRNA-146a/b promoter was tested in terms of p50/p65 regulation.
After P.g LPS treatment, NF-κB and Erk1/2 were strongly activated in HGFs. miRNA-146a/b, IL-1, IL-6 and TNF-α levels were down-regulated when NF-κB inhibitor was used. p50/p65 strongly activated miRNA-146a/b promoter as measured with the luciferase assay.
In TLR4 signalling in HGFs, both miRNA-146a and miRNA-146b are downstream targets of NF-κB, but not of AP-1 signalling. miRNA-146a/b expression was specifically dependent on NF-κB but not Erk1/2 or JNK signalling.
Periodontal disease is one of the most common oral diseases. The third China National Oral Health Survey found that the overall prevalence of periodontal disease in the 35-44-year-old and 65-74-year-old Chinese populations exceeded 85% . Toll-like receptor 4 (TLR4) is responsible for the recognition of distinct bacterial cell-wall components, such as LPS, and signal transduction . LPS first binds to the cluster of differentiation-14 (CD14) receptor via the LPS-binding protein (LBP) and is then transferred to TLR4. Thereafter, the myeloid differentiation factor 88 (MyD88) adaptor protein links TLR4 to the interleukin-1 receptor-associated kinase-4 (IRAK4), which induces the phosphorylation of IRAK1. Tumour necrosis factor receptor-associated factor-6 (TRAF6) is also recruited to the receptor complex via association with phosphorylated IRAK1. TRAF6 transduces the signal through the TGF-β-activated kinase-1 (TAK1), TAK1-binding protein-1 (TAB1) and TAK1-binding protein-2 (TAB2) complexes, phosphorylates IκB kinase 1 (IKK1) and IκB kinase 2 (IKK2), and finally ubiquitinates inhibitor of NF-κB (IκB) and drives p65/p50 to translocate into the nucleus . Simultaneously, TLR4 activates c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (Erk), which leads to the activation of activating protein-1 (AP-1), which ultimately results in the production of inflammatory cytokines such as IL-1β, IL-6 and TNF-α. It has been reported that LPS stimulation can also induce the phosphorylation of p44 and p42 (Erk1 and Erk2, respectively)  and the expression of c-jun and c-fos  in human gingival fibroblasts (HGFs). Understanding the molecular mechanisms by which LPS-TLR4 signalling is regulated in periodontal cells will aid the design of effective strategies for the diagnosis and treatment of human periodontal diseases.
miRNAs are short (18–25 nucleotides long) non-coding RNAs that regulate gene expression by binding to the 3’-untranslated region (UTR) of the mRNAs of target genes . miRNAs were first discovered in 1993 in Caenorhabditis elegans. miRNAs are important post-transcriptional regulators of diverse biological processes, such as development, tumourigenesis, inflammation, and infection . Earlier research found that miR-146a is strongly elevated in LPS-stimulated human monocytic THP-1 cells via an NF-κB-dependent pathway, and thus miR-146 is considered as an important repressor of LPS-induced signalling via its targeting of IRAK1 and TRAF6 . miR-146 also plays an important role in regulating IL-1β-induced cytokine production in human alveolar epithelial cell . This function has also been reported in VSV (Vesicular Stomatitis Virus) -infected macrophages, and IRAK2 has been found to be a new target of miR-146a . Together, these findings suggest that miR-146 has an important role in negative regulatory loop of LPS-TLR4 signalling in diverse cell types.
Given that different cell types have different cellular environments, the behaviours of miRNAs are widely diverse across distinct cellular environments. Although the LPS-TLR4 signal is important in HGFs, whether miRNAs, and if so which miRNAs, play key regulating roles remains obscure. In our previous studies , we found that miR-146a and miR-146b are highly expressed in inflammatory gingival tissues compared to healthy tissues. We also confirmed that miR-146 plays a critical role in down-regulating inflammatory cytokines in HGFs by targeting IRAK1 but not TRAF6, which implies that the behaviour of miR-146 in HGFs is unique. Based on these findings, we further identified a precise method for controlling miR-146 expression in HGFs. This approach employs pharmacological methods to block the activities of up- (IRAK1/4) and down-stream (IκB, JNK, and Erk) regulators of miR-146 with the aim of completely mapping the molecular regulation miR-146 to provide a drug design strategy based on miR-146 as a microRNA therapeutics for clinical trials.
Materials and methods
HGF cell culture
HGFs were prepared from explants of the gingiva of 10 periodontitis patients who were acquired during periodontal flap surgery after receiving the informed consent of the patients. The epithelial tissues were torn from the gingiva after 24 h of soaking in 2 U/ml dispase II (Takara, Japan). Gingival connective tissues were cut into pieces and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) with 20% foetal bovine serum (FBS) (Hyclone, USA). The medium was changed every 3 days for total 10–20 days until confluent cell monolayers were formed . After four or five subcultures, homogeneous, slim, spindle-shaped cells were obtained and cultured in DMEM with 10% FBS, penicillin (100 U/ml) and streptomycin sulphate (50 μg/ml). TPCA-1 (an IKK-2 inhibitor), PD98059 (a MEK-1/2 inhibitor), SP600125 (a JNK-1/2 inhibitor) or an IRAK1/4 inhibitor was added to the culture at the concentrations and times indicated below, with or without simulation of 1 μg/ml of P.g LPS (Ultrapure, activates TLR4 only, InvivoGen, USA). All inhibitors were purchased from Sigma (USA). Betulinic acid was purchased from R&D (USA).
Total RNA was extracted from cultured cells with TRIzol (Invitrogen, USA) according to the manufacturer’s instructions. miRNA was polyadenylated and reverse transcribed using poly(A) polymerase and MMLV reverse transcriptase (Clontech, USA). miR-146a and miR-146b expressions in the total RNA extracts were measured with SYBR Advantage qPCR Premix (Clontech, USA), and the reactions were run on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, USA). The U6 small nuclear RNA (NR_003027) was used as an internal control. Each sample was amplified in triplicate. Data were analysed with SDS software (ABI, USA). The sequences of the target mature miRNAs and specific forward PCR primers were follows: hsa-miR-146a, GGGTGAGAACTGAATTCCA; hsa-miR-146b-5p, GGGTGAGAACTGAATTCCA; universal reverse PCR primer, CAGTGCGTGTCGTGGAGT; and U6 small nuclear RNA primers, GCTTCGGCAGCACATATACTAAAAT (U6-forward) and CGCTTCACGAATTTGCGTGTCAT (U6-reverse).
Two kilobases promoter sequence of miR-146a/b primary transcripts were cloned into the 5’ site of the luc2 reporter gene in pGL4.10 plasmid (Promega, USA). We co-transfected 200 ng of reporter plasmid, 20 ng of pRL-TK-Renillaluciferase and 100 ng of p50/p65 pcDNA3.0 plasmid into the HGFs using Lipofectamine 2000 (Invitrogen, USA). After 24 h of transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer’s instructions. The luciferase data were normalised to transfection efficiency by dividing the firefly luciferase activity by the activity of Renilla luciferase.
Western blot assay
A total of 105 HGFs per sample were harvested and lysed with 100 μl lysis buffer (50 mM HEPES, pH 7.0), 1% Nonidet P-40, 5 mM EDTA, 450 mM NaCl, 10 mM Na pyrophosphate, and 50 mM NaF and freshly supplemented with inhibitors (1 mM Na orthovanadate, 1 mM PMSF, 10 μg/ml aprotinin, leupeptin, pepstatin) at room temperature for 20 min. Aliquots of the whole-cell extracts were prepared and subjected to 10% SDS-PAGE and then electroblotted onto nitrocellulose membranes. The membranes were then probed with Abs as indicated. Antibodies against p-JNK (Thr183/Tyr185), total JNK (56G8), p-Erk1/2 (Thr202/Tyr204, D13.14.4E), total Erk1/2 (137 F5), p-IRAK4 (Thr345/Ser346), total IRAK4, total IκB-α and HRP-labelled secondary antibody were purchased from Cell Signaling. Membranes were visualised with Super Signal West Pico Chemiluminescent Substrate (Pierce, USA)
For analysis of cytokine production in the supernatant, human IL-1β, IL-6, and TNF-α ELISA Duoset kits were purchased from R&D and used according to the manufacturer’s protocol.
The results are presented as the mean ± SD where applicable. Student’s t tests were used to compare pairs of independent groups. For all tests, values of p < 0.05 were considered statistically significant.
P.g LPS induces the activation of multiple kinases in HGFs
To further test whether selective pharmacological inhibitors were functional in HGFs, we stimulated the cells with separate inhibitors and analysed the phosphorylation levels and total amounts of their targets. As expected, based on reports of other cell lines, PD98059 (an inhibitor of Erk kinase-1/2 and MEK-1/2, which is an upstream activator of Erk-1/2) , SP600125 (a JNK-1/2 inhibitor) , and TPCA-1 (which inhibits IKK-2 and prevents the degradation of the IκB-α proteins and NF-κB signals) , worked well in HGFs (Figure 1 B-E).
miRNA-146 level was consistent with the activation states of the NF-κB signals
P. g LPS-induced inflammatory cytokine expression is affected by IRAK1/4 and NF-κB inhibitor
Direct targeting of the promoters of miR-146 by NF-κB
Toll-like receptors are broadly distributed on divergent immune cells, function as primary sensors of alien PAMPs (such as LPS), and trigger the activation of signal cascades and the production of inflammatory cytokines . However, excessive activation of the TLR signalling pathway leads to immune disorders such as autoimmune or chronic inflammatory disorders . Therefore, TLR signalling and its consequences must be under tight and precise negative regulation to prevent over-response during efficiently clearing the pathogens. Thus far, multiple negative responses have been found in the TLR signalling network, and these negative regulators reduce TLR signals via multiple mechanisms, such as degradation by ubiquitination, competitive binding in the form of alternative splicing , and, most importantly and recently discovered, the down-regulation of gene expression via miRNA targeting .
Some miRNAs play key roles in regulating inflammatory responses or tolerance. miRNA-21, which is triggered by LPS-induced NF-κB signals in human peripheral blood mononuclear cells, targets peripheral blood CD4+ cells and thus positively influences IL-10 production. Decreases in miR-21 due to transfection with miR-21 antisense oligonucleotides lead to strong increases in NF-κB signals and IL-10 production; these findings prove that miR-21 is the fundamental molecule in the NF-κB negative regulation loop . However, in epithelial cell lines, miR-21 is activated by IL-6 and STAT3 and targets PTEN, which leads to increased NF-κB activity and tumour growth . These results imply that the activation and function of miRNAs vary across cell conditions and that the precise mechanisms controlled by miRNAs are complicated.
miR-146a expression was found to be an NF-κB-dependent via in vitro reporter assays in the breast cancer cell line . In macrophages, the inhibition of NF-κB by pyrrolidinecarbodithoic acid (PDTC), which is a chemically synthesised inhibitor of NF-κB, also impairs VSV-induced miR-146a expression . However, there are no reports regarding whether NF-κB is responsible for the expression of miR-146b. In HGFs, Erk1/2 activation and the subsequent activation of AP-1 have been reported to occur after LPS stimulation ,, but the overall relationship between the miRNA negative feedback loop and the LPS signalling cascades remains to be explored.
Here, we found that TLR4 signalling initiated a rapid and continuous activation of NF-κB activation and a much shorter-termed Erk1/2 phosphorylation in HGFs. We also observed that both miR-146a and miR-146b levels were dramatically reduced by blockade of the NF-κB signal but not by inhibition of JNK or Erk1/2. As expected, cytokine production was primarily dependent on NF-κB, and thus NF-κB signalling requires further downstream negative regulations to prevent over-responding to antigens. Our results show that both miR-146a and miR-146b are downstream targets of NF-κB but not AP-1. Thus, in HGFs, NF-κB has a greater role in the production of inflammatory cytokines than AP-1 does, and miR-146 may be one of the key downstream negative regulators of NF-κB.
miR-146a and miR-146b are highly expressed in the metastatic human breast cancer cell line MDA-MB-231 and negatively regulate NF-κB activity by targeting the 3’ UTRs of IRAK1 and TRAF6. miR-146a/b overexpression in MDA-MB-231 cells leads to markedly impaired invasion and migration capacity compared to controls, and this finding proves that miR-146 suppresses NF-κB activity via a reduction of metastatic potential . Furthermore, miR-146 levels are significantly up-regulated by breast cancer metastasis-suppressor 1 (BRMS1), a gene that affects multiple steps in the metastatic cascade, which leads to a suppression of metastasis of ~90% in breast carcinomas . In HGFs, we found that TNF-α is more sensitive to pharmacological inhibitors. These results imply that miR-146 has a fundamental function in controlling cancer-related signals and in inhibiting inflammatory cytokines.
Although only BetA or P.g LPS stimulation can up-regulate miR-146a and miR146b in HGFs, our data showed different express patterns of miR-146a and miR-146b under both BetA and P.g LPS stimulation. miR-146a and miR146b share similar sequence and function, the genomic loci of miR-146a and miR146b is on human chromosomes 5 and 10 respectively, far away from each other, which implies their transcription may under different regulation. This gave us hint that there may be other miR-146 regulation mechanisms under LPS stimulation, other than NF-κB, which we will study in our future work.
miRNAs are regulated by precise mechanisms and are thought to target multiple mRNAs to regulate gene expression. As the number of miRNAs is much smaller than the number of coding genes, single miRNAs may regulate different genes in different conditions. Our research showed that miR-146 is highly regulated in P. g LPS-treated HGFs and negatively controls TLR4 signalling by targeting IRAK1 but not TRAF6 . We also found that NF-κB but not AP-1 is responsible for the production of both miR-146a and miR-146b. Moreover, miR-146 inhibits NF-κB signals and limits immune responses to appropriate levels. It is probable that other activators in the NF-κB signalling pathway are targets of miR-146. This presumption will lead us to work of seeking to reveal the entire set of functions of miR-146 in the periodontal innate immune response.
This work was supported by the National Natural Science Foundation of China (Grant No. 81100754) and the Research Foundation for Selecting and Training Excellent Youth Scholars of Shanghai Universities (Project No. Shjdy11031).
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