STAT3 Binding Research Articles

Evaluation of Xihuang Pill in inducing pyroptosis in glioma cells through modulation of miR-21-5p.

This study aims to elucidate the mechanism by which Xihuang Pill induces pyroptosis in glioma cells via the regulation of miR-21-5p. Human glioma cell lines U-87 and LN-229 were used as experimental models to assess the effects of Xihuang Pill on glioma pyroptosis. Cells were incubated with Xihuang Pill extract at concentrations of 7.5, 15, and 30µg/mL for 24h, alongside transfection with miR-21-5p mimic, an overexpression vector for STAT3, or incubation with 50µg/mL of the STAT3 activator Colivelin for 4h. Cell viability was measured using the CCK-8 assay, apoptosis was detected by flow cytometry, and expression levels of p-STAT3/STAT3 and pyroptosis-related proteins were determined by Western Blot. Additionally, cleaved caspase-1 was assessed by immunofluorescence, miR-21-5p expression by qRT-PCR, and STAT3 binding to the miR-21-5p promoter region by ChIP and dual-luciferase reporter assays. Results showed that Xihuang Pill significantly reduced cell viability, increased apoptosis, and upregulated the expression of pyroptosis-related proteins such as NLRP3, IL-1β, cleaved caspase-1, and GSDMD-N, while reducing p-STAT3/STAT3 and miR-21-5p levels (P < 0.05). Xihuang Pill inhibited STAT3 activation, which modulated miR-21-5p expression by binding to its promoter region. Co-transfection with miR-21-5p mimic reversed the effect of Xihuang Pill on glioma pyroptosis (P < 0.05). In conclusion, Xihuang Pill promotes glioma cell pyroptosis through the STAT3/miR-21-5p pathway.

Network pharmacology and experimental verification unraveled the mechanism of Bailing Capsule against asthma.

Asthma is a serious public health challenge around the world. Recent studies into traditional Chinese medicine preparations for asthma have yielded promising findings regarding Bailing Capsule's potential in bronchial asthma prevention and treatment. This study aims to initially clarify the potential mechanism of Bailing Capsule in the treatment of asthma using network pharmacology and in vitro experimental approaches. Network pharmacology was adopted to detect the active ingredients of Bailing Capsule via Traditional Chinese Medicine Systems Pharmacology Database, and the key targets and signaling pathways in the treatment of asthma were predicted. Docking and molecular dynamics simulations were conducted to verify the most important interactions formed by these probes within different regions of the binding site. The predicted targets were validated in lipopolysaccharide-induced 16HBE cell experiment. Seven active ingredients were screened from Bailing Capsule, 294 overlapping targets matched with asthma were considered potential therapeutic targets, such as SRC, TP53, STAT3, and E1A binding protein P300. The main functional pathways involving these key targets include phosphatidylinositol 3-kinase/protein kinase B, mitogen-activated protein kinase, renin-angiotensin system and other signaling pathways, which were mainly involved in the inflammatory response, apoptosis, and xenobiotic stimulus. Moreover, molecular docking showed that Cerevisterol have higher affinity for SRC, TP53, STAT3, and E1A binding protein P300 than other main active components, which is close to the docking results of the co-crystallized ligands to proteins. Consequently, Cerevisterol was selected for molecular dynamics simulation and the results show that Cerevisterol can bind most tightly to SRC, TP53, and STAT3. Bailing Capsule can promote the growth of 16HBE cell, reduce the production of IL-4, TNF-α and IL-6, and down-regulate the levels of SRC and STAT3 mRNA. This study preliminarily reveals the potential mechanism of Bailing Capsule against asthma with the aid of network pharmacology and in vitro cell experiment, which provided reference and guidance for in-depth research and clinical application.

The aryl hydrocarbon receptor shapes monocyte transcriptional responses to interleukin-4 by prolonging STAT6 binding to promoters.

Cytokines induce functional and metabolic adaptations in immune cells, typically through transcriptional responses that can be influenced by other extracellular signals and by intracellular factors. The binding of the cytokine interleukin-4 (IL-4) to its receptor induces the phosphorylation and activation of the transcription factor STAT6. The aryl hydrocarbon receptor (AhR), a transcription factor activated by various endogenous and microbe-derived metabolites, modulates the responses of immune cells to danger signals or inflammatory mediators such as cytokines. Here, we investigated cross-talk between the AhR and signaling stimulated by IL-4 in human and mouse monocytes. AhR activation was required for a subset of IL-4-induced transcriptional responses and inhibited the IL-4-induced metabolic switch to fatty acid β-oxidation. The promoters of the genes that were induced by IL-4 in an AhR-dependent manner lacked canonical AhR binding sites, implying a nongenomic mechanism of AhR action. Mechanistically, AhR activation reduced the activity of SHP-1, a phosphatase that targets and inhibits STAT6, and prolonged STAT6 phosphorylation and binding to specific target loci, thus extending the duration of STAT6 activity. Our results identify AhR as a key player in the molecular control of responses to IL-4 in monocytes and suggest a nongenomic mechanism through which AhR ligands may influence the functional responses of cells to IL-4.

Blocking M2-Like Macrophage Polarization Using Decoy Oligodeoxynucleotide-Based Gene Therapy Prevents Immune Evasion for Pancreatic Cancer Treatment.

M2-like macrophages exhibit immunosuppressive activity and promote pancreatic cancer progression. Reactive oxygen species (ROS) affect macrophage polarization; however, the mechanism remains unclear. This study aimed to elucidate the underlying molecular basis and design a gene therapy to inhibit M2-like polarization. Microarray analysis and immunofluorescence staining were performed in M1-like and M2-like macrophages to ascertain the expression of CYBB, a major intracellular ROS source. Coculture assay and syngeneic orthotopic pancreatic cancer mice models were used to study the mechanism of M2-like skewing. Decoy oligodeoxynucleotides (ODNs) were designed to manipulate CYBB transcription to inhibit M2-like polarization and control tumor growth. Lipopolysaccharide treatment polarized U937 cells to M1-like macrophages in which CYBB expression was increased. In contrast, coculture with PANC-1 cells induced M2-like polarization in U937 cells with CYBB downregulation. High CD204 M2-like expression in combination with low CYBB expression was associated with the worst prognosis in patients with pancreatic cancer. STAT6 and HDAC2 in U937 cells were activated by cancer cell-derived IL4 after coculture and then bound to the CYBB promoter to repress CYBB expression, resulting in M2-like polarization. Diphenyleneiodonium, 8λ³-iodatricyclo[7.4.0.02,⁷]trideca-1(13),2,4,6,9,11-hexaen-8-ylium chloride that inhibits ROS production could block this action. Knockdown of STAT6 and HDAC2 also inhibited M2-like polarization and maintained the M1-like phenotype of U937 cells after coculture. Decoy ODNs interrupting the binding of STAT6 to the CYBB promoter counteracted M2-like polarization and tumor growth and triggered antitumor immunity in vivo. Gene therapy using STAT6-CYBB decoy ODNs can inhibit M2-like polarization, representing a potential therapeutic tool for pancreatic cancer.

A suboptimal OCT4-SOX2 binding site facilitates the naïve-state specific function of a Klf4 enhancer.

Enhancers have critical functions in the precise, spatiotemporal control of transcription during development. It is thought that enhancer grammar, or the characteristics and arrangements of transcription factor binding sites, underlie the specific functions of developmental enhancers. In this study, we sought to identify grammatical constraints that direct enhancer activity in the naïve state of pluripotency, focusing on the enhancers for the naïve-state specific gene, Klf4. Using a combination of biochemical tests, reporter assays, and endogenous mutations in mouse embryonic stem cells, we have studied the binding sites for the transcription factors OCT4 and SOX2. We have found that the three Klf4 enhancers contain suboptimal OCT4-SOX2 composite binding sites. Substitution with a high-affinity OCT4-SOX2 binding site in Klf4 enhancer E2 rescued enhancer function and Klf4 expression upon loss of the ESRRB and STAT3 binding sites. We also observed that the low-affinity of the OCT4-SOX2 binding site is crucial to drive the naïve-state specific activities of Klf4 enhancer E2. Altogether, our work suggests that the affinity of OCT4-SOX2 binding sites could facilitate enhancer functions in specific states of pluripotency.

The pro-inflammatory cytokines IL-1β and IL-6 promote upregulation of the ST6GAL1 sialyltransferase in pancreatic cancer cells

The ST6GAL1 sialyltransferase is overexpressed in multiple cancers including pancreatic ductal adenocarcinoma (PDAC). ST6GAL1 adds an α2-6-linked sialic acid to N-glycosylated membrane receptors, which consequently modulates receptor structure and function. While many studies have investigated the effects of ST6GAL1 on cell phenotype, there is a dearth of knowledge regarding mechanisms that regulate ST6GAL1 expression. In the current study, we evaluated the regulation of ST6GAL1 by two pro-inflammatory cytokines, IL-1β and IL-6, that are abundant within the PDAC tumor microenvironment. Cytokine activity was monitored using the Suit-2 PDAC cell line and two Suit-2-derived metastatic subclones, S2-013 and S2-LM7AA. For all three cell models, treatment with IL-1β or IL-6 increased the expression of ST6GAL1 protein and mRNA. Specifically, IL-1β and IL-6 induced expression of the ST6GAL1 YZ mRNA isoform, which is driven by the P3 promoter. The ST6GAL1 H and X isoforms were not detected. Promoter reporter assays confirmed that IL-1β and IL-6 activated transcription from the P3 promoter. We then examined downstream signaling mechanisms. IL-1β is known to signal through the NFκB transcription factor, whereas IL-6 signals through the STAT3 transcription factor. CUT&RUN experiments revealed that IL-1β promoted the binding of NFκB to the ST6GAL1 P3 promoter, and IL-6 induced the binding of STAT3 to the P3 promoter. Finally, we determined that inhibitors of NFκB and STAT3 blocked the upregulation of ST6GAL1 stimulated by IL-1β and IL-6, respectively. Together, these results highlight a novel molecular pathway by which cytokines within the tumor microenvironment stimulate the upregulation of ST6GAL1 in PDAC cells.

Stat3-mediated Atg7 expression regulates anti-tumor immunity in mouse melanoma

Epigenetic modifications to DNA and chromatin control oncogenic and tumor-suppressive mechanisms in melanoma. Ezh2, the catalytic component of the Polycomb Repressive Complex 2 (PRC2), which mediates methylation of lysine 27 on histone 3 (H3K27me3), can regulate both melanoma initiation and progression. We previously found that mutant Ezh2Y641F interacts with the immune regulator Stat3 and together they affect anti-tumor immunity. However, given the numerous downstream targets and pathways affected by Ezh2, many mechanisms that determine its oncogenic activity remain largely unexplored. Using genetically engineered mouse models, we further investigated the role of pathways downstream of Ezh2 in melanoma carcinogenesis and identified significant enrichment in several autophagy signatures, along with increased expression of autophagy regulators, such as Atg7. In this study, we investigated the effect of Atg7 on melanoma growth and tumor immunity within the context of a wild-type or Ezh2Y641F epigenetic state. We found that the Atg7 locus is controlled by multiple Ezh2 and Stat3 binding sites, Atg7 expression is dependent on Stat3 expression, and that deletion of Atg7 slows down melanoma cell growth in vivo, but not in vitro. Atg7 deletion also results in increased CD8 + T cells in Ezh2Y641F melanomas and reduced myelosuppressive cell infiltration in the tumor microenvironment, particularly in Ezh2WT melanomas, suggesting a strong immune system contribution in the role of Atg7 in melanoma progression. These findings highlight the complex interplay between genetic mutations, epigenetic regulators, and autophagy in shaping tumor immunity in melanoma.

Small Molecule Induces Time-Dependent Inhibition of Stat3 Dimerization and DNA-Binding Activity and Regresses Human Breast Tumor Xenografts.

Aberrantly-active signal transducer and activator of transcription (Stat)3 has a causal role in many human cancers and represents a validated anticancer drug target, though it has posed significant challenge to drug development. A new small molecule, JKB887, was identified through library screening and is predicted to interact with Lys591, Arg609 and Pro63 in the phospho-tyrosine (pTyr)-binding pocket of the Stat3 SH2 domain. JKB887 inhibited Stat3 DNA-binding activity in vitro in a time-dependent manner, with IC50 of 2.2-4.5 μM at 30-60-min incubation. It directly disrupted both the Stat3 binding to the cognate, high-affinity pTyr (pY) peptide, GpYLPQTV-NH2 in fluorescent polarization assay with IC50 of 3.5-5.5 μM at 60-90-min incubation, and to the IL-6 receptor/gp130 or Src in treated malignant cells. Treatment with JKB887 selectively blocked constitutive Stat3 phosphorylation, nuclear translocation and transcriptional activity, and Stat3-regulated gene expression, and decreased viable cell numbers, cell growth, colony formation, migration, and survival in human or mouse tumor cells. By contrast, JKB887 had minimal effects on Stat1, pErk1/2MAPK, pShc, pJAK2, or pSrc induction, or on cells that do not harbor aberrantly-active Stat3. Additionally, JKB887 inhibited growth of human breast cancer xenografts in mice. JKB887 is a Stat3-selective inhibitor with demonstrable antitumor effects against Stat3-dependent human cancers.

Unraveling the unphosphorylated STAT3-unphosphorylated NF-κB pathway in loss of function STAT3 Hyper IgE syndrome.

Patients with loss of function signal transducer and activator of transcription 3-related Hyper IgE Syndrome (LOF STAT3 HIES) present with recurrent staphylococcal skin and pulmonary infections along with the elevated serum IgE levels, eczematous rashes, and skeletal and facial abnormalities. Defective STAT3 signaling results in reduced Th17 cells and an impaired IL-17/IL-22 response primarily due to a compromised canonical Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway that involves STAT3 phosphorylation, dimerization, nuclear translocation, and gene transcription. The non-canonical pathway involving unphosphorylated STAT3 and its role in disease pathogenesis, however, is unexplored in HIES. This study aims to elucidate the role of unphosphorylated STAT3-unphosphorylated NF-κB (uSTAT3-uNF-κB) activation pathway in LOF STAT3 HIES patients. The mRNA expression of downstream molecules of unphosphorylated STAT3-unphosphorylated NF-κB pathway was studied in five LOF STAT3 HIES patients and transfected STAT3 mutants post-IL-6 stimulation. Immunoprecipitation assays were performed to assess the binding of STAT3 and NF-κB to RANTES promoter. A reduced expression of the downstream signaling molecules of the uSTAT3-uNF-κB complex pathway, viz., RANTES, STAT3, IL-6, IL-8, ICAM1, IL-8, ZFP36L2, CSF1, MRAS, and SOCS3, in LOF STAT3 HIES patients as well as the different STAT3 mutant plasmids was observed. Immunoprecipitation studies showed a reduced interaction of STAT3 and NF-κB to RANTES in HIES patients. The reduced expression of downstream signaling molecules, specially RANTES and STAT3, confirmed the impaired uSTAT3-uNF-κB pathway in STAT3 LOF HIES. Decreased levels of RANTES and STAT3 could be a significant component in the disease pathogenesis of Hyper IgE Syndrome.

A New Renieramycin T Right-Half Analog as a Small Molecule Degrader of STAT3.

Constitutive activation of STAT3 contributes to tumor development and metastasis, making it a promising target for cancer therapy. (1R,4R,5S)-10-hydroxy-9-methoxy-8,11-dimethyl-3-(naphthalen-2-ylmethyl)-1,2,3,4,5,6-hexahydro-1,5-epiminobenzo[d]azocine-4-carbonitrile, DH_31, a new derivative of the marine natural product Renieramycin T, showed potent activity against H292 and H460 cells, with IC50 values of 5.54 ± 1.04 µM and 2.9 ± 0.58 µM, respectively. Structure-activity relationship (SAR) analysis suggests that adding a naphthalene ring with methyl linkers to ring C and a hydroxyl group to ring E enhances the cytotoxic effect of DH_31. At 1-2.5 µM, DH_31 significantly inhibited EMT phenotypes such as migration, and sensitized cells to anoikis. Consistent with the upregulation of ZO1 and the downregulation of Snail, Slug, N-cadherin, and Vimentin at both mRNA and protein levels, in silico prediction identified STAT3 as a target, validated by protein analysis showing that DH_31 significantly decreases STAT3 levels through ubiquitin-proteasomal degradation. Immunofluorescence and Western blot analysis confirmed that DH_31 significantly decreased STAT3 and EMT markers. Additionally, molecular docking suggests a covalent interaction between the cyano group of DH_31 and Cys-468 in the DNA-binding domain of STAT3 (binding affinity = -7.630 kcal/mol), leading to destabilization thereafter. In conclusion, DH_31, a novel RT derivative, demonstrates potential as a STAT3-targeting drug that significantly contribute to understanding of the development of new targeted therapy.

Abstract Tu134: Oncostatin M favors cardiomyocyte survival by promoting glycolysis

Introduction: Cardiomyocytes is in high demand of energy and is susceptible to low oxygen environment. Timely switch of energy metabolism after cardiac ischaemia is helpful for cardiomyocyte preservation. We previously identified a critical role of Oncostatin M (OSM) in cardiomyocyte proliferation. Since the level of OSM quickly rises in myocardium after myocardial injury, OSM may play a protective role in early phase after myocardial injury through metabolic regulation. Hypothesis: We hypothesized that Oncostatin M regulates cardiomyocyte energy metabolism to promote its resistance to hypoxia. Methods and Results: We cultivated cardiomyocytes under hypoxic condition observed that in OSM-treated group, the percentage of TUNEL positive cardiomyocytes significantly decreased compared with that in vehicle-treated group. Published RNA sequencing data from OSM-treated neonatal cardiomyocytes revealed alterations in genes related to energy metabolism. Our Seahorse metabolic stress assays demonstrated that OSM-treated cardiomyocytes exhibited higher glycolytic metabolism and lower mitochondrial respiration than vehicle-treated cells. Pyruvate kinase M2 (PKM2), among the regulatory enzymes, was significantly upregulated in OSM-treated cardiomyocytes as was detected via western-blotting. Interestingly, PKM2 has lower catalytic activity compared to its isotype PKM1, suggesting that the augmented glycolysis might be mediated by PKM2. The mutually exclusive splicing of the gene Pkm between exons 9 and 10 led to the switch between PKM1 and PKM2. qPCR results indicated a 1.5-fold increase in Pkm transcripts and a 2-fold increase in the ratio of Pkm2 to Pkm1 , suggesting that OSM concomitantly promoted Pkm transcription as well as alternative splicing of Pkm2 . Furthermore, we performed RNA interference to knock down Stat3 , the canonical downstream signal of OSM, in cardiomyocytesand observed that OSM failed to upregulate Pkm transcription. ChIP-qPCR analysis on cardiomyocyte cell line HL-1 confirmed an increased STAT3 binding signal upstream of Pkm after OSM treatment. Additionally, RIP-qPCR assays revealed a 3-fold increase in PTBP1-binding signal to Pkm transcripts in cardiomyocytes following OSM treatment. Conclusions: Our results suggest that OSM promotes glycolysis and suppresses mitochondrial respiration in cardiomyocytes by upregulates PKM2 through STAT3 mediated transcription of Pkm and PTBP1-mediated alternative splicing of Pkm transcripts.

Regulation of the expression of casein alpha S1 and S2 genes in the bovine mammary epithelial cells by STAT5A

Cow milk is rich in protein. Major cow milk proteins include casein α S1 (CSN1S1), casein α S2 (CSN1S2), casein β (CSN2), casein kappa (CSN3), lactalbumin α (LALBA), and β-lactoglobulin (LGB). These milk proteins are produced through gene expression in the mammary epithelial cells. Little is known about the molecular mechanism that mediates the expression of milk protein genes in cows. In this study, we tested the hypothesis that the expression of milk protein genes in cows is mediated by STAT5A, a transcription factor that is induced to bind and activate the transcription of target genes by extracellular signals such as prolactin. To circumvent the need of prolactin-responsive bovine mammary epithelial cells, we generated a plasmid that expresses a constitutively active bovine STAT5A variant, bSTAT5ACA. Transfection of the bovine mammary epithelial cell line MAC-T cells with the bSTAT5ACA expression plasmid caused a more than 100,000-fold and 600-fold increase in the expression of CSN1S1 and CSN1S2 mRNAs, respectively, compared with transfection of the wild-type bovine STAT5A (bSTAT5A) expression plasmid. Transfection of bSTAT5ACA, however, had no significant effect on the expression of CSN2, CSN3, LALBA, or LGB mRNA in MAC-T cells. Transfection of bSTAT5ACA caused a more than 260-fold and 120-fold increase in the expression of a luciferase reporter gene linked to the bovine CSN1S1 and CSN1S2 promoters in MAC-T cells, respectively, compared with that of bSTAT5A. The bovine CSN1S1 and CSN1S2 promoters each contain a putative STAT5 binding site, and gel-shift and super-shift assays confirmed bSTAT5ACA binding to both sites. These results together suggest that STAT5A plays a major role in regulating the expression of CSN1S1 and CSN1S2 genes in the bovine mammary epithelial cells and that STAT5A regulates the expression of these genes at least in part by binding to the STAT5 binding sites in their promoter regions. These results also suggest that STAT5A does not play a major role in regulating the expression of other major milk protein genes.

Activation of kappa opioid receptor suppresses post-traumatic osteoarthritis via sequestering STAT3 on the plasma membrane

ObjectiveKappa opioid receptor (KOR) signaling is involved in joint development and inflammation in Osteoarthritis (OA), while the biochemical mechanism remains unclarified. This study aims to investigate downstream molecular events of KOR activation, to provide novel perspectives in OA pathology.MethodsU50,488H, a selective KOR agonist, was intra-articularly injected in mice upon destabilization of the medial meniscus (DMM) as OA models, with PBS injection as control. The behavioral and histological evaluation was assessed by hot plate test and red solid green staining, respectively. Alterations in mRNA and protein expression were assessed by RNA-seq, RT-qPCR, immunohistochemistry and western blotting (WB) in chondrocytes treated with TNF-α or TNF-α + U50,488H. Proteins interacted with KOR were explored using proximity labeling followed by mass spectrometry and then testified by co-immunoprecipitation (Co-IP) assay and immunofluorescence (IF).ResultsOA-induced pain was reduced and cartilage degeneration was alleviated upon KOR activation in DMM mice. In chondrocytes, activation of KOR reversed the upregulation of MMPs, IL-6, IL-1β and phosphorylated(p-) STAT3, stimulated by TNF-α, while the expression of NF-κB, MAPKs and AKT signaling weren't reversed. RNA-seq and IF results presented that KOR activation evidently reduced STAT3 nuclear translocation in chondrocytes upon TNF-α stimuli. The reduction may be resulted from the binding of KOR and STAT3 in the plasma membrane, revealed by proximity labeling and Co-IP results.ConclusionsKOR activation protects cartilage from OA, and this protective effect is mainly exerted via sequestering STAT3 on the plasma membrane, resulting in inactivation of STAT3-dependent immune responses which otherwise contributes to OA.

LncRNA CDKN2B-AS1 regulates collagen expression

The long noncoding RNA CDKN2B-AS1 harbors a major coronary artery disease risk haplotype, which is also associated with progressive forms of the oral inflammatory disease periodontitis as well as myocardial infarction (MI). Despite extensive research, there is currently no broad consensus on the function of CDKN2B-AS1 that would explain a common molecular role of this lncRNA in these diseases. Our aim was to investigate the role of CDKN2B-AS1 in gingival cells to better understand the molecular mechanisms underlying the increased risk of progressive periodontitis. We downregulated CDKN2B-AS1 transcript levels in primary gingival fibroblasts with LNA GapmeRs. Following RNA-sequencing, we performed differential expression, gene set enrichment analyses and Western Blotting. Putative causal alleles were searched by analyzing associated DNA sequence variants for changes of predicted transcription factor binding sites. We functionally characterized putative functional alleles using luciferase-reporter and antibody electrophoretic mobility shift assays in gingival fibroblasts and HeLa cells. Of all gene sets analysed, collagen biosynthesis was most significantly upregulated (Padj=9.7 × 10− 5 (AUC > 0.65) with the CAD and MI risk gene COL4A1 showing strongest upregulation of the enriched gene sets (Fold change = 12.13, Padj = 4.9 × 10− 25). The inflammatory “TNFA signaling via NFKB” gene set was downregulated the most (Padj=1 × 10− 5 (AUC = 0.60). On the single gene level, CAPNS2, involved in extracellular matrix organization, was the top upregulated protein coding gene (Fold change = 48.5, P < 9 × 10− 24). The risk variant rs10757278 altered a binding site of the pathogen responsive transcription factor STAT1 (P = 5.8 × 10− 6). rs10757278-G allele reduced STAT1 binding 14.4% and rs10757278-A decreased luciferase activity in gingival fibroblasts 41.2% (P = 0.0056), corresponding with GTEx data. CDKN2B-AS1 represses collagen gene expression in gingival fibroblasts. Dysregulated collagen biosynthesis through allele-specific CDKN2B-AS1 expression in response to inflammatory factors may affect collagen synthesis, and in consequence tissue barrier and atherosclerotic plaque stability.

Targeting the STAT3/IL-36G signaling pathway can be a promising approach to treat rosacea

BackgroundRosacea is an inflammatory skin disorder characterized by the release of inflammatory mediators from keratinocytes, which are thought to play a crucial role in its pathogenesis. Despite an incidence of approximately 5.5%, rosacea is associated with a poor quality of life. However, as the pathogenesis of rosacea remains enigmatic, treatment options are limited. ObjectivesTo investigate the pathogenesis of rosacea and explore new therapeutic strategies. MethodsTranscriptome data from rosacea patients combined with immunohistochemical staining were used to investigate the activation of STAT3 in rosacea. The role of STAT3 activation in rosacea was subsequently explored by inhibiting STAT3 activation both in vivo and in vitro. The key molecules downstream of STAT3 activation were identified through data analysis and experiments. Dual-luciferase assay and ChIP-qPCR analysis were used to validate the direct binding of STAT3 to the IL-36G promoter. DARTS, in combination with experimental screening, was employed to identify effective drugs targeting STAT3 for rosacea treatment. ResultsSTAT3 signaling was hyperactivated in rosacea and served as a promoter of the keratinocyte-driven inflammatory response. Mechanistically, activated STAT3 directly bind to the IL-36G promoter region to amplify downstream inflammatory signals by promoting IL-36G transcription, and treatment with a neutralizing antibody (α-IL36γ) could mitigate rosacea-like inflammation. Notably, a natural plant extract (pogostone), which can interact with STAT3 directly to inhibit its activation and affect the STAT3/IL36G signaling pathway, was screened as a promising topical medication for rosacea treatment. ConclusionsOur study revealed a pivotal role for STAT3/IL36G signaling in the development of rosacea, suggesting that targeting this pathway might be a potential strategy for rosacea treatment.

Blocking the MIR155HG/miR-155 axis reduces CTGF-induced inflammatory cytokine production and α-SMA expression via upregulating AZGP1 in hypertrophic scar fibroblasts

Hypertrophic scarring (HS) is a pathological condition characterized by excessive fibrosis and inflammation, resulting in excessive extracellular matrix formation in the skin. MIR155HG, a long non-coding RNA, is abnormally upregulated in fibrotic tissues; however, its underlying mechanism is poorly understood. Using single-cell sequencing data, we analyzed connective tissue growth factor (CTGF) expression in various cell types in HS and normal skin tissues and MIR155HG expression in clinical samples. To investigate the mechanism of fibrosis, an in vitro model using CTGF-treated hypertrophic scar fibroblasts (HSFBs) was established and qRT-PCR, western blotting and ELISA assays were performed to investigate the expression of interleukin (IL)-1β, IL-6, and mesenchymal markers α-smooth muscle actin (α-SMA). CTGF stimulates MIR155HG level through phosphorylated STAT3 binding to the MIR155HG promoter. We analyzed the methylation of MIR155HG, assessed the levels of miR-155-5p/−3p in CTGF-treated HSFBs and identified differentially expressed genes among HS and NS samples using the Gene Expression Omnibus RNA sequencing data. The binding between miR-155-5p/−3p and AZGP1 was confirmed using a dual-luciferase assay and inflammatory cytokine production and α-SMA expression were investigated in rescue experiments. The findings revealed that CTGF elevated inflammatory cytokine production, α-SMA and MIR155HG expression in HSFBs. MIR155HG is upregulated in HS tissues due to low DNA methylation. Mechanistically, miR-155-5p/−3p was directly bound to MIR155HG 3’UTR. MIR155HG silencing inhibited cytokine production and α-SMA expression by repressing the generation of miR-155-5p/−3p in CTGF-treated HSFBs. Bioinformatics analysis and luciferase reporter assays revealed that miR-155-5p/−3p targets AZGP1. In addition, transfection with plasmids carrying AZGP1 cDNA significantly inhibited the signaling activity of miR-155-5p/−3 p-overexpressing HSFBs. Our findings highlight the importance of the MIR155HG/miR-155/AZGP1 axis in regulating cytokine production and α-SMA in HS.

An engineered viral protein rewires signaling pathways to overcome T cell suppression.

Abstract Cancer can evade destruction by depriving T cells of cytokines, such as IL-2, that cause JAK-STAT signaling required for T cell functionality. We hypothesized that the direct activation of STAT proteins in a cytokine-independent manner could restore the signals needed by T cells. Because STATs are regulated by phosphorylation, we predicted that enforced recruitment of kinase activity to STATs could activate them directly in the absence of cytokine. Tyrosine-protein kinase-interacting protein (TIP), expressed by Herpesvirus saimiri, co-opts LCK kinase by recruiting LCK to non-canonical substrates, including STATs. We evaluated whether a minimal region of TIP could be isolated from the full-length viral protein and combined with STAT binding sites derived from cytokine receptors. Using this platform, we demonstrate that LCK kinase activity can be recruited to targeted STAT proteins to activate them. We determined that a STAT5 activator (aSTAT5) allowed CD8+ T cells to remain viable in the absence of IL-2 and retained their capacity to produce IFNg and kill cancer cells ex vivo. We therefore incorporated aSTAT5 into tumor-specific T cells (TST) and transferred them into tumor-bearing mice. We determined that aSTAT5 TST caused potent tumor regression and reduced tumor outgrowth due to improved T cell persistence and functionality. Our findings highlight that T cells can be improved to resist the miscommunication that cancer causes by targeting intracellular signaling pathways.

Sialic Acid-Siglec-E Interactions Regulate the Response of Neonatal Macrophages to Group B Streptococcus.

The mammalian Siglec receptor sialoadhesin (Siglec1, CD169) confers innate immunity against the encapsulated pathogen group B Streptococcus (GBS). Newborn lung macrophages have lower expression levels of sialoadhesin at birth compared with the postnatal period, increasing their susceptibility to GBS infection. In this study, we investigate the mechanisms regulating sialoadhesin expression in the newborn mouse lung. In both neonatal and adult mice, GBS lung infection reduced Siglec1 expression, potentially delaying acquisition of immunity in neonates. Suppression of Siglec1 expression required interactions between sialic acid on the GBS capsule and the inhibitory host receptor Siglec-E. The Siglec1 gene contains multiple STAT binding motifs, which could regulate expression of sialoadhesin downstream of innate immune signals. Although GBS infection reduced STAT1 expression in the lungs of wild-type newborn mice, we observed increased numbers of STAT1+ cells in Siglece-/- lungs. To test if innate immune activation could increase sialoadhesin at birth, we first demonstrated that treatment of neonatal lung macrophages ex vivo with inflammatory activators increased sialoadhesin expression. However, overcoming the low sialoadhesin expression at birth using invivo prenatal exposures or treatments with inflammatory stimuli were not successful. The suppression of sialoadhesin expression by GBS-Siglec-E engagement may therefore contribute to disease pathogenesis in newborns and represent a challenging but potentially appealing therapeutic opportunity to augment immunity at birth.

The Myc-associated zinc finger protein epigenetically controls expression of interferon-γ-stimulated genes by recruiting STAT1 to chromatin

The MYC-Associated Zinc Finger Protein (MAZ) plays important roles in chromatin organization and gene transcription regulation. Dysregulated expression of MAZ causes diseases, such as glioblastoma, breast cancer, prostate cancer, and liposarcoma. Previously, it has been reported that MAZ controls the proinflammatory response in colitis and colon cancer via STAT3 signaling, suggesting that MAZ is involved in regulating immunity-related pathways. However, the molecular mechanism underlying this regulation remains elusive. Here, we investigate the regulatory effect of MAZ on interferon-gamma (IFN-γ)-stimulated genes via STAT1, a protein that plays an essential role in immune responses to viral, fungal, and mycobacterial pathogens. We demonstrate that about 80% of occupied STAT1-binding sites colocalize with occupied MAZ-binding sites in HAP1/K562 cells after IFN-γ stimulation. MAZ depletion significantly reduces STAT1 binding in the genome. By analyzing genome-wide gene expression profiles in the RNA-Seq data, we show that MAZ depletion significantly suppresses a subset of the immune response genes, which include the IFN-stimulated genes IRF8 and Absent in Melanoma 2. Furthermore, we find that MAZ controls expression of the immunity-related genes by changing the epigenetic landscape in chromatin. Our study reveals an important role for MAZ in regulating immune-related gene expression.

Hyperactive STAT5 hijacks T cell receptor signaling and drives immature T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.