CCL2 Promoter Research Articles

The endocannabinoid anandamide mediates anti-inflammatory effects through activation of NR4A nuclear receptors.

Endocannabinoids are lipid mediators, which elicit complex biological effects that extend beyond the central nervous system. Tissue concentrations of endocannabinoids increase in atherosclerosis, and for the endocannabinoid N-arachidonoyl-ethanolamine (anandamide, AEA), this has been linked to an anti-inflammatory function. In this study, we set out to determine the anti-inflammatory mechanism of action of AEA, specifically focusing on vascular smooth muscle cells. RNA-sequencing, RT-qPCR, LC-MS/MS, NanoBit, ChIP, microscale thermophoresis, NMR structural footprinting, Gal4 reporter gene assays and loss of function approaches in cell and ex vivo organ culture were used. AEA pretreatment attenuated the cytokine-mediated induction of inflammatory gene expression such as CCL2. This effect was also observed in preparations obtained from cannabinoid receptor knockout mice and after pertussis toxin treatment. The anti-inflammatory effect of AEA required preincubation, suggesting an effect through gene induction. AEA increased the expression of the nuclear receptors NR4A1 and NR4A2. Knockdown and knockout of these receptors blocked the AEA-mediated anti-inflammatory effect in cell culture and aortic organ culture, respectively. Conversely, NR4A agonists (CsnB, C-DIM12) attenuated inflammatory gene expression. AEA binds to NR4A, and mutations in NR4A attenuated this effect. The interaction of AEA with NR4A caused recruitment of the nuclear corepressor NCoR1 to the CCL2 promoter, resulting in gene suppression. By binding to NR4A, AEA elicits an anti-inflammatory response in vascular smooth muscle cells. NR4A-binding by AEA analogues may represent novel anti-inflammatory agents.

Procaine regulates the STAT3/CCL5 axis and inhibits microglia M1 polarization to alleviate CFA rats pain behavior.

Neuropathic pain (NP) caused by sciatic nerve injury can significantly impact the quality of life of patients. The M1 phenotype of microglia has been reported to promote the progression of NP. Procaine is a lipid-soluble local anesthetic drug that exerts narcotic analgesic effects. Nevertheless, the detailed effect of procaine in NP is not clear. In order to explore the role of procaine in the polarization of NP microglia, HAPI cells were exposed to LPS to polarize into M1 type. In addition, the number of the M1 phenotype of HAPI cells was assessed using flow cytometry. The binding site between CCL5 and STAT3 was explored using the dual luciferase assay. Furthermore, in vivo experiments were applied for testing the impact of procaine on NP.LPS significantly inhibited HAPI cell viability, which was reversed by procaine. Consistently, procaine alleviated LPS-induced upregulation of inflammatory factors. Additionally, it significantly inhibited HAPI cells M1 polarization induced by LPS. Meanwhile, overexpression of STAT3 was able to promote HAPI cells M1 polarization through binding with the CCL5 promoter region and activating the PI3K/Akt signaling. Procaine could alleviate the painful behavior of complete Freund's adjuvant (CFA) rats by modulating the STAT3/CCL5 axis and inhibiting microglia M1 polarization. In conclusion, procaine alleviated the painful behavior of CFA rats via regulating the STAT3/CCL5 axis and inhibiting microglia M1 polarization. Hence, the research might provide a novel agent for NP treatment.Significance statement Neuropathic pain (NP) refers to pain caused by damage to the somatosensory system, which can be caused by brain or spinal cord injury and have a serious impact on the patient's quality of life. The M1 phenotype of microglia plays a crucial role in promoting the progression of NP. In this study, we investigated the specific mechanism of local anesthetic procaine in improving NP by inhibiting microglia polarization towards M1 type. Our findings may provide a new drug for NP treatment.

Eravacycline improves the efficacy of anti-PD1 immunotherapy via AP1/CCL5 mediated M1 macrophage polarization in melanoma

Screening approved library is a promising and safe strategy to overcome the limitation of low response rate and drug resistance in immunotherapy. Accumulating evidence showed that the application of antibiotics has been considered to reduce the effectiveness of anti-PD1 immunotherapy in tumor treatment, however, in this study, an antibiotic drug (Eravacycline, ERV) was identified to improve the efficacy of anti-PD1 immunotherapy in melanoma through screening approved library. Administration of ERV significantly attenuated melanoma cells growth as well as directly or indirectly benefited M1 macrophage polarization. Meanwhile, ERV treatment significantly induced cellular autophagy via damage of mitochondria, leading to up-regulation of ROS production, subsequently, raised CCL5 secretion through elevation AP1 binding to CCL5 promoter via p38 or JNK1/2 activation. Knockdown of Ccl5 expression attenuated ERV triggered M1 macrophage polarization in melanoma cells. Clinical analysis revealed a positive association between high expression of CCL5 and improved prognosis as well as a favorable anti-PD1 therapy in melanoma patients. As expected, application of ERV improved the efficacy of anti-PD1. Overall, our results approved that ERV enhances the efficacy of anti-PD1 immunotherapy in melanoma by promoting the polarization of M1 macrophages, which provided novel therapeutic strategy for improving the effectiveness of melanoma anti-PD1 immunotherapy.

Activation of Wnt/β-catenin signaling promotes immune evasion via the β-catenin/IKZF1/CCL5 axis in hepatocellular carcinoma

Immune checkpoint therapy (ICT) has been shown to produce durable responses in various cancer patients. However, its efficacy is notably limited in hepatocellular carcinoma (HCC), with only a small percentage of patients responding positively to treatment. The mechanism underlying resistance to ICT in HCC remains poorly understood. Here, we showed that combination treatment of ICG-001, an inhibitor of the Wnt/β-catenin signaling pathway, with anti-PD-1 antibody effectively suppresses tumor growth and promotes the infiltration of immune cells such as DCs and CD8+ T cells in the tumor microenvironment (TME). By inhibiting the activity of β-catenin and blocking its binding to the transcription factor IKAROS family zinc finger 1 (IKZF1), ICG-001 upregulated the expression of CCL5. Moreover, IKZF1 regulated the activity of the CCL5 promoter and its endogenous expression. Through inhibition of the WNT/β-catenin signaling pathway, upregulation of the expression of CCL5 was achieved, which subsequently recruited more DCs into the TME via C–C motif chemokine receptor 5 (CCR5). This, in turn, resulted in an increase in the infiltration of CD8+ T cells in the TME, thereby enhancing the antitumor immune response. Analysis of a tissue microarray derived from HCC patient samples revealed a positive correlation between survival rate and prognosis and the expression levels of CCL5/CD8. In conclusion, our findings suggest that combined application of ICG-001 and anti-PD-1 antibody exhibits significantly enhanced antitumor efficacy. Hence, combining a WNT/β-catenin signaling pathway inhibitor with anti-PD-1 therapy may be a promising treatment strategy for patients with HCC.

VDR promotes pancreatic cancer progression in vivo by activating CCL20-mediated M2 polarization of tumor associated macrophage.

Activation of VDR pathway was a promising anti-tumor therapy strategy. However, numerous clinical studies have demonstrated the effect of activating VDR is limited, which indicates that VDR plays a complex role in vivos. We analyzed the TCGA database to examine the association between VDR expression and immune cell infiltration in pancreatic adenocarcinoma (PAAD). Western blot, ELISA, ChIP, and dual-luciferase reporter assays were performed to determine the mechanism of VDR regulating CCL20. Migration assay and immunofluorescence were used to investigate the role of CCL20 in M2 macrophage polarization and recruitment. We employed multiplexed immunohistochemical staining and mouse models to validate the correlation of VDR on macrophages infiltration in PAAD. Flow cytometry analysis of M2/M1 ratio in subcutaneous graft tumors. VDR is extensively expressed in PAAD, and patients with elevated VDR levels exhibited a significantly reduced overall survival. VDR expression in PAAD tissues was associated with increased M2 macrophages infiltration. PAAD cells overexpressing VDR promote macrophages polarization towards M2 phenotype and recruitment in vitro and vivo. Mechanistically, VDR binds to the CCL20 promoter and up-regulates its transcription. The effects of polarization and recruitment on macrophages can be rescued by blocking CCL20. Finally, the relationship between VDR and M2 macrophages infiltration was evaluated using clinical cohort and subcutaneous graft tumors. A positive correlation was demonstrated between VDR/CCL20/CD163 in PAAD tissues and mouse models. High expression of VDR in PAAD promotes M2 macrophage polarization and recruitment through the secretion of CCL20, which activates tumor progression. This finding suggests that the combination of anti-macrophage therapy may improve the efficacy of VDR activation therapy in PAAD.

LncRNA MEG3 Reduces the Ratio of M2/M1 Macrophages Through the HuR/CCL5 Axis in Hepatocellular Carcinoma.

Tumor-associated macrophages play a crucial role in the development of hepatocellular carcinoma (HCC). Our study aimed to investigate the relationship between long coding RNA (lncRNA) maternally expressed gene 3 (MEG3), RNA-binding protein human antigen R (HuR), and messenger RNA C-C motif chemokine 5 (CCL5) in the modulation of M1 and M2 macrophage polarization in HCC. To induce M1 or M2 polarization, LPS/IFNγ- or IL4/IL13 were used to treat bone marrow derived macrophages (BMDMs). The localization of MEG3 in M1 and M2 macrophages was assessed using fluorescence in situ hybridization assay. Expression levels of MEG3, HuR, CCL5, M1, and M2 markers were measured by RT-qPCR or immunofluorescence staining. Flow cytometry was performed to determine the proportion of F4/80+CD206+ and F4/80+CD68+ cells. RNA pulldown assay was performed to detect the binding of lncRNA MEG3 and HuR. The impacts of HuR on CCL5 stability and activity of CCL5 promoter were evaluated using actinomycin D treatment and luciferase reporter assay. Cell migration, invasiveness, and angiogenesis were assessed using transwell migration and invasion assays and a tube formation assay. A mixture of Huh-7 cells and macrophages were injected into nude mice to explore the effect of MEG3 on tumorigenesis. MEG3 promoted M1-like polarization while dampening M2-like polarization of BMDMs. MEG3 bound to HuR in M1 and M2 macrophages. HuR downregulated CCL5 by inhibiting CCL5 transcription in macrophages. In addition, overexpression of MEG3 suppressed cell metastasis, invasion, and angiogenesis by obstructing macrophage M2 polarization. MEG3 inhibited tumorigenesis in HCC via promotion of M1-like polarization and inhibition of M2-like polarization. Rescue experiments showed that depletion of CCL5 in M2 macrophages reversed MEG3-induced suppressive effect on cell migration, invasion, and tube formation. MEG3 suppresses HCC progression by promoting M1-like while inhibiting M2-like macrophage polarization via binding to HuR and thus upregulating CCL5.

SCUBE3 Exerts a Tumor-Promoting Effect in Tongue Squamous Cell Carcinoma by Promoting CEBPA Binding to the CCL2 Promoter.

Our data revealed a tumor-promoting role for SCUBE3 in TSCC via the CEBPA/CCL2/STAT3 axis, which provided new insight into novel potential therapeutic target for TSCC.

Tributyrin Mitigates Ethanol-Induced Lysine Acetylation of Histone-H3 and p65-NFκB Downregulating CCL2 Expression and Consequent Liver Inflammation and Injury.

Chemokine-driven leukocyte infiltration and sustained inflammation contribute to alcohol-associated liver disease (ALD). Elevated hepatic CCL2 expression, seen in ALD, is associated with disease severity. However, mechanisms of CCL2 regulation are not completely elucidated. Post-translational modifications (PTMs) of proteins, particularly acetylation, modulate gene expression. This study examined the acetylation changes of promoter-associated histone-H3 and key transcription factor-NFκB in regulating hepatic CCL2 expression and subsequent inflammation and injury. Further, the effect of therapeutic modulation of the acetylation state by tributyrin (TB), a butyrate prodrug, was assessed. Hepatic CCL2 expression was assessed in mice fed control (PF) or an ethanol-containing Lieber-DeCarli (5% v/v, EF) diet for 7 weeks with or without oral administration of tributyrin (TB, 2 g/kg, 5 days/week). A chromatin immunoprecipitation (ChIP) assay evaluated promoter-associated modifications. Nuclear association between SIRT1, p300, and NFκB-p65 and acetylation changes of p65 were determined using immunoprecipitation and Western blot analyses. A Student's t-test and one-way ANOVA determined the significance. Ethanol significantly increased promoter-associated histone-H3-lysine-9 acetylation (H3K9Ac), reflecting a transcriptionally permissive state with a resultant increase in hepatic CCL2 mRNA and protein expression. Moreover, increased lysine-310-acetylation of nuclear RelA/p65 decreased its association with SIRT1, a class III HDAC, but concomitantly increased with p300, a histone acetyltransferase. This further led to enhanced recruitment of NF-κB/p65 and RNA polymerase-II to the CCL2 promoter. Oral TB administration prevented ethanol-associated acetylation changes, thus downregulating CCL2 expression, hepatic neutrophil infiltration, and inflammation/ injury. The modulation of a protein acetylation state via ethanol or TB mechanistically regulates hepatic CCL2 upregulation in ALD.

ZNF451 favors triple-negative breast cancer progression by enhancing SLUG-mediated CCL5 transcriptional expression

Triple-negative breast cancer (TNBC) is the most aggressive subtype with limited effective therapies because of the absence of definitive targets. Here, we demonstrate that the expression of ZNF451, a poorly characterized vertebrate zinc-finger protein, is upregulated in TNBC and associated with a poor prognosis. Elevated ZNF451 expression facilitates TNBC progression by interacting with and enhancing the activity of the transcriptional activator snail family transcriptional repressor 2 (SLUG). Mechanistically, the ZNF451-SLUG complex preferentially recruits the acetyltransferase p300/CBP-associated factor (PCAF) to the CCL5 promoter, selectively facilitating CCL5 transcription by enhancing the acetylation of SLUG and local chromatin, leading to recruitment and activation of tumor-associated macrophages (TAMs). Disturbing the ZNF451-SLUG interaction using a peptide suppresses TNBC progression by reducing CCL5 expression and counteracting the migration and activation of TAMs. Collectively, our work provides mechanistic insights into the oncogene-like functions of ZNF451 and suggests that ZNF451 is a potential target for development of effective therapies against TNBC.

Hydrogel Encapsulation of Genome-Engineered Stem Cells for Long-Term Self-Regulating Anti-Cytokine Therapy.

Biologic therapies have revolutionized treatment options for rheumatoid arthritis (RA) but their continuous administration at high doses may lead to adverse events. Thus, the development of improved drug delivery systems that can sense and respond commensurately to disease flares represents an unmet medical need. Toward this end, we generated induced pluripotent stem cells (iPSCs) that express interleukin-1 receptor antagonist (IL-1Ra, an inhibitor of IL-1) in a feedback-controlled manner driven by the macrophage chemoattractant protein-1 (Ccl2) promoter. Cells were seeded in agarose hydrogel constructs made from 3D printed molds that can be injected subcutaneously via a blunt needle, thus simplifying implantation of the constructs, and the translational potential. We demonstrated that the subcutaneously injected agarose hydrogels containing genome-edited Ccl2-IL1Ra iPSCs showed significant therapeutic efficacy in the K/BxN model of inflammatory arthritis, with nearly complete abolishment of disease severity in the front paws. These implants also exhibited improved implant longevity as compared to the previous studies using 3D woven scaffolds, which require surgical implantation. This minimally invasive cell-based drug delivery strategy may be adapted for the treatment of other autoimmune or chronic diseases, potentially accelerating translation to the clinic.

Notch-mediated hepatocyte MCP-1 secretion causes liver fibrosis.

Patients with nonalcoholic steatohepatitis (NASH) have increased expression of liver monocyte chemoattractant protein-1 (MCP-1), but its cellular source and contribution to various aspects of NASH pathophysiology remain debated. We demonstrated increased liver CCL2 (which encodes MCP-1) expression in patients with NASH, and commensurately, a 100-fold increase in hepatocyte Ccl2 expression in a mouse model of NASH, accompanied by increased liver monocyte-derived macrophage (MoMF) infiltrate and liver fibrosis. To test repercussions of increased hepatocyte-derived MCP-1, we generated hepatocyte-specific Ccl2-knockout mice, which showed reduced liver MoMF infiltrate as well as decreased liver fibrosis. Forced hepatocyte MCP-1 expression provoked the opposite phenotype in chow-fed wild-type mice. Consistent with increased hepatocyte Notch signaling in NASH, we observed a close correlation between markers of Notch activation and CCL2 expression in patients with NASH. We found that an evolutionarily conserved Notch/recombination signal binding protein for immunoglobulin kappa J region binding site in the Ccl2 promoter mediated transactivation of the Ccl2 promoter in NASH diet-fed mice. Increased liver MoMF infiltrate and liver fibrosis seen in opposite gain-of-function mice was ameliorated with concomitant hepatocyte Ccl2 knockout or CCR2 inhibitor treatment. Hepatocyte Notch activation prompts MCP-1-dependent increase in liver MoMF infiltration and fibrosis.

Regulatory role and translational potential of CCL11 in liver fibrosis.

Myofibroblasts are considered the major effector cell type of liver fibrosis and primarily derived from hepatic stellate cells (HSCs). In the present study, we investigated the contribution of C-C motif chemokine (CCL11) to HSC-myofibroblast trans -differentiation and its implication in liver fibrosis. We report that CCL11 levels were elevated in HSCs, but not in hepatocytes or Kupffer cells, isolated from mice with liver fibrosis compared with the control mice. CCL11 levels were also up-regulated by 2 pro-fibrogenic growth factors TGF-β and platelet derived growth factor in cultured HSCs. Mechanistically, zinc finger factor 281 bound to the CCL11 promoter and mediated CCL11 trans -activation in HSCs. Depletion of CCL11 attenuated whereas treatment with recombinant CCL11 promoted HSC activation. Further, global CCL11 deletion ( CCL11-/- ) or HSC/myofibroblast-specific CCL11 knockdown mitigated fibrogenesis in mice. RNA-sequencing revealed that CCL11 might regulate HSC activation by stimulating the transcription of Jagged 1. Reconstitution of Jagged 1 restored the fibrogenic response in CCL11-/- mice. Finally, several targeting strategies that aimed at blockading CCL11 signaling, either by administration of an antagonist to its receptor C-C motif chemokine receptor 3 or neutralizing antibodies against CCL11/C-C motif chemokine receptor 3, ameliorated liver fibrosis in mice. Our data unveil a previously unrecognized role for CCL11 in liver fibrosis and provide proof-of-concept evidence that targeting CCL11 can be considered as an effective therapeutic approach.

A nerve injury-specific long noncoding RNA promotes neuropathic pain by increasing Ccl2 expression.

Maladaptive changes of nerve injury–associated genes in dorsal root ganglia (DRGs) are critical for neuropathic pain genesis. Emerging evidence supports the role of long noncoding RNAs (lncRNAs) in regulating gene transcription. Here we identified a conserved lncRNA, named nerve injury–specific lncRNA (NIS-lncRNA) for its upregulation in injured DRGs exclusively in response to nerve injury. This upregulation was triggered by nerve injury–induced increase in DRG ELF1, a transcription factor that bound to the NIS-lncRNA promoter. Blocking this upregulation attenuated nerve injury–induced CCL2 increase in injured DRGs and nociceptive hypersensitivity during the development and maintenance periods of neuropathic pain. Mimicking NIS-lncRNA upregulation elevated CCL2 expression, increased CCL2-mediated excitability in DRG neurons, and produced neuropathic pain symptoms. Mechanistically, NIS-lncRNA recruited more binding of the RNA-interacting protein FUS to the Ccl2 promoter and augmented Ccl2 transcription in injured DRGs. Thus, NIS-lncRNA participates in neuropathic pain likely by promoting FUS-triggered DRG Ccl2 expression and may be a potential target in neuropathic pain management.

IFN-γ and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation.

BackgroundOverexpression of CCL2 (MCP-1) has been implicated in pathogenesis of metabolic conditions, such as obesity and T2D. However, the mechanisms leading to increased CCL2 expression in obesity are not fully understood. Since both IFN-γ and LPS levels are found to be elevated in obesity and shown to be involved in the regulation of metabolic inflammation and insulin resistance, we investigated whether these two agents could synergistically trigger the expression of CCL2 in obesity.MethodsMonocytes (Human monocytic THP-1 cells) were stimulated with IFN-γ and LPS. CCL2 gene expression was determined by real-time RT-PCR. CCL2 protein was determined by ELISA. Signaling pathways were identified by using epigenetic inhibitors and STAT1 siRNA. Acetylation of H3K27 was analyzed by Western blotting. The acetylation level of histone H3K27 in the transcriptional initiation region of CCL2 gene was determined by ChIP-qPCR.ResultsOur results show that the co-incubation of THP-1 monocytes with IFN-γ and LPS significantly enhanced the expression of CCL2, compared to treatment with IFN-γ or LPS alone. Similar results were obtained using primary monocytes and macrophages. Interestingly, IFN-γ priming was found to be more effective than LPS priming in inducing synergistic expression of CCL2. Moreover, STAT1 deficiency significantly suppressed this synergy for CCL2 expression. Mechanistically, we showed that IFN-γ priming induced acetylation of lysine 27 on histone 3 (H3K27ac) in THP-1 cells. Chromatin immunoprecipitation (ChIP) assay followed by qRT-PCR revealed increased H3K27ac at the CCL2 promoter proximal region, resulting in stabilized gene expression. Furthermore, inhibition of histone acetylation with anacardic acid suppressed this synergistic response, whereas trichostatin A (TSA) could substitute IFN-γ in this synergy.ConclusionOur findings suggest that IFN-γ, in combination with LPS, has the potential to augment inflammation via the H3K27ac-mediated induction of CCL2 in monocytic cells in the setting of obesity.

Upregulated lncARAT in Schwann cells promotes axonal regeneration by recruiting and activating proregenerative macrophages

BackgroundAxonal regeneration following peripheral nerve injury (PNI) depends on the complex interaction between Schwann cells (SCs) and macrophages, but the mechanisms underlying macrophage recruitment and activation in axonal regeneration remain unclear.MethodsRNA sequencing (RNA-seq) was conducted to identify differentially expressed long noncoding RNAs (DElncRNAs) between crushed sciatic nerves and intact contralateral nerves. The putative role of lncRNAs in nerve regeneration was analyzed in vitro and in vivo.ResultsAn lncRNA, called axon regeneration-associated transcript (lncARAT), was upregulated in SCs and SC-derived exosomes (SCs-Exo) after sciatic nerve injury. LncARAT contributed to axonal regeneration and improved motor function recovery. Mechanistically, lncARAT epigenetically activated C–C motif ligand 2 (CCL2) expression by recruiting KMT2A to CCL2 promoter, resulting in increased histone 3 lysine 4 trimethylation (H3K4me3) and CCL2 transcription in SCs. CCL2 facilitated the infiltration of macrophages into the injured nerves. Meanwhile, lncARAT-enriched exosomes were released from SCs and incorporated into macrophages. LncARAT functioned as an endogenous sponge to adsorb miRNA-329-5p in macrophages, resulting in increased suppressor of cytokine signaling (SOCS) 2 expression, which induced a proregenerative function of macrophages through a signal transducer and activator of transcription (STAT) 1/6-dependent pathway.ConclusionsLncARAT may represent a promising therapeutic avenue for peripheral nerve repair.

IL-27 Induces CCL5 Production by T Lymphocytes, Which Contributes to Antitumor Activity.

IL-27 is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells including T lymphocytes. In this study, we demonstrate that IL-27 directly induces CCL5 production by T lymphocytes, particularly CD8+ T cells in vitro and in vivo. IL-27-induced CCL5 production is IL-27R-dependent. In CD4+ T cells, IL-27-induced CCL5 production was primarily dependent on Stat1 activation, whereas in CD8+ T cells, Stat1 deficiency does not abrogate CCL5 induction. A chromatin immunoprecipitation assay revealed that in the CCL5 promoter region, both putative Stat3 binding sites exhibit significant binding to Stat3, whereas only one out of four Stat1 binding sites displays moderate binding to Stat1. In tumor-bearing mice, IL-27 induced dramatic production of CCL5 in tumor-infiltrating T cells. IL-27-induced CCL5 appears to contribute to an IL-27-mediated antitumor effect. This is signified by diminished tumor inhibition in anti-CCL5- and IL-27-treated mice. Additionally, intratumor delivery of CCL5 mRNA using lipid nanoparticles significantly inhibited tumor growth. Thus, IL-27 induces robust CCL5 production by T cells, which contributes to antitumor activity.

LncRNA Tug1 maintains blood-testis barrier integrity by modulating Ccl2 expression in high-fat diet mice.

Sertoli cells are essential for spermatogenesis in the testicular seminiferous tubules by forming blood-testis barrier (BTB) and creating a unique microenvironment for spermatogenesis. Many lncRNAs have been reported to participate in spermatogenesis. However, the role of long noncoding RNAs (lncRNAs) in Sertoli cells has rarely been examined. Herein, we found that a high-fat diet (HFD) decreased sperm quality, impaired BTB integrity and resulted in accumulation of saturated fatty acids (SFAs), especially palmitic acid (PA), in mouse testes. PA decreased the expression of tight junction (TJ)-related proteins, increased permeability and decreased transepithelial electrical resistance (TER) in primary Sertoli cells and TM4 cells. Moreover, lncRNA Tug1 was found to be involved in PA-induced BTB disruption by RNA-seq. Tug1 depletion distinctly impaired the TJs of Sertoli cells and overexpression of Tug1 alleviated the disruption of BTB integrity induced by PA. Moreover, Ccl2 was found to be a downstream target of Tug1, and decreased TJ-related protein levels and TER and increased FITC-dextran permeability in vitro. Furthermore, the addition of Ccl2 damaged BTB integrity after overexpression of Tug1 in the presence of PA. Mechanistically, we found that Tug1 could directly bind to EZH2 and regulate H3K27me3 occupancy in the Ccl2 promoter region by RNA immunoprecipitation and chromatin immunoprecipitation assays. Our study revealed an important role of Tug1 in the BTB integrity of Sertoli cells and provided a new view of the role of lncRNAs in male infertility.

STING inhibitor ameliorates LPS-induced ALI by preventing vascular endothelial cells-mediated immune cells chemotaxis and adhesion.

Acute lung injury (ALI) is a common and devastating clinical disorder featured by excessive inflammatory responses. Stimulator of interferon genes (STING) is an indispensable molecule for regulating inflammation and immune response in multiple diseases, but the role of STING in the ALI pathogenesis is not well elucidated. In this study, we explored the molecular mechanisms of STING in regulating lipopolysaccharide (LPS)-induced lung injury. Mice were pretreated with a STING inhibitor C-176 (15, 30 mg/kg, i.p.) before LPS inhalation to induce ALI. We showed that LPS inhalation significantly increased STING expression in the lung tissues, whereas C-176 pretreatment dose-dependently suppressed the expression of STING, decreased the production of inflammatory cytokines including TNF-α, IL-6, IL-12, and IL-1β, and restrained the expression of chemokines and adhesion molecule vascular cell adhesion protein-1 (VCAM-1) in the lung tissues. Consistently, in vitro experiments conducted in TNF-α-stimulated HMEC-1cells (common and classic vascular endothelial cells) revealed that human STING inhibitor H-151 or STING siRNA downregulated the expression levels of adhesion molecule and chemokines in HMEC-1cells, accompanied by decreased adhesive ability and chemotaxis of immunocytes upon TNF-α stimulation. We further revealed that STING inhibitor H-151 or STING knockdown significantly decreased the phosphorylation of transcription factor STAT1, which subsequently influenced its binding to chemokine CCL2 and adhesive molecule VCAM-1 gene promoter. Collectively, STING inhibitor can alleviate LPS-induced ALI in mice by preventing vascular endothelial cells-mediated immune cell chemotaxis and adhesion, suggesting that STING may be a promising therapeutic target for the treatment of ALI.

EGR1 and RXRA transcription factors link TGF-\u03b2 pathway and CCL2 expression in triple negative breast cancer cells

Transforming growth factor beta (TGF-β) is the main cytokine responsible for the induction of the epithelial-mesenchymal transition of breast cancer cells, which is a hallmark of tumor transformation to the metastatic phenotype. Recently, research demonstrated that the chemokine CCL2 gene expression level directly correlates with the TGF-β activity in breast cancer patients. CCL2 attracts tumor-associated macrophages and is, therefore, considered as an important inductor of breast cancer progression; however, the precise mechanisms underlying its regulation by TGF-β are unknown. Here, we studied the behavior of the CCL2 gene in MDA-MB-231 and HCC1937 breast cancer cells representing mesenchymal-like phenotype activated by TGF-β. Using bioinformatics, deletion screening and point mutagenesis, we identified binding sites in the CCL2 promoter and candidate transcription factors responsible for its regulation by TGF-β. Among these factors, only the knock-down of EGR1 and RXRA made CCL2 promoter activity independent of TGF-β. These factors also demonstrated binding to the CCL2 promoter in a TGF-β-dependent manner in a chromatin immunoprecipitation assay, and point mutations in the EGR1 and RXRA binding sites totally abolished the effect of TGF-β. Our results highlight the key role of EGR1 and RXRA transcription factors in the regulation of CCL2 gene in response to TGF-β pathway.

MLL3 Inhibits Apoptosis of Rheumatoid Arthritis Fibroblast-Like Synoviocytes and Promotes Secretion of Inflammatory Factors by Activating CCL2 and the NF-κB Pathway

Rheumatoid arthritis (RA) remains the most common inflammatory arthritis and a major cause of disability. This study investigated the mechanism of MLL3 in fibroblast-like synoviocyte (FLS) apoptosis and inflammatory factor secretion in RA. Expression of MLL3 in synovial tissue of RA patients and patients with bone trauma was detected. FLS was isolated and identified by flow cytometry. Expressions of TNF-α, IL-1β, IL-8, and IL-10 and apoptosis were measured by MTT, flow cytometry, and ELISA. Western blot and qRT-PCR were performed to detect MLL3 and CCL2 expressions, H3K4me3 level, and NF-κB pathway-related proteins in rat joints. MLL3 was highly expressed in the synovial tissue of RA patients, and silencing MLL3 in FLS-RA promoted apoptosis, inhibited pro-inflammatory factors TNF-α, IL-1β, and IL-8 secretion, and promoted anti-inflammatory factor IL-10 secretion. Inhibition of MLL3 suppressed intracellular H3K4me3 and CCL2 expressions. CCL2 activated the NF-κB pathway to promote pro-inflammatory factors TNF-α, IL-1β, and IL-8, inhibit anti-inflammatory factor IL-10, and inhibit apoptosis in FLS-RA. Inhibition of MLL3 expression in RA rats reduced joint redness, swelling, and intra-articular inflammation, but increasing H3K4me3 level reversed the ameliorative effects of sh-MLL3 on RA rats. Collectively, MLL3 activated the NF-κB pathway by increasing H3K4me3 modification in the CCL2 promoter region in FLS-RA, thereby inhibiting apoptosis and promoting pro-inflammatory factors of FLS-RA.