STAT3 Activation Research Articles

NF-κB and STAT3 activation in CD4 T cells in pediatric MOG antibody-associated disease

In this study, we examined CD4 T cell activation using various stimuli in pediatric MOGAD patients (n = 4, untreated remission samples) and healthy controls (n = 5), to understand how both antigen-specific and bystander mechanisms contribute to CD4 T cell activation in MOGAD. TNFα, IL6, and MOG peptide pool were found to activate NF-κB or STAT3 pathways by measuring the expression of regulators (A20, IκBα) and phosphorylated subunits (phospho-p65 and phospho-STAT3) using immunolabeling. Prednisolone reversed activation of both NF-κB and STAT3 and increased the expression of A20 and IκBα. TNFR blocking partially reversed NF-κB activation in certain CD4 T cell subsets, but did not effect STAT3 activation. We observed that activation of NF-κB and STAT3 in response to various stimuli behaves mostly same in MOGAD (remission) and HC. IL6 stimulation resulted in higher STAT3 phosphorylation in MOGAD patients at 75 min, specifically in central and effector memory CD4 T cells (with unadjusted p-values). These findings suggest the potential therapeutic targeting of NF-κB and STAT3 pathways in MOGAD. Further investigation is needed to validate the significance of extended STAT3 phosphorylation and its correlation with IL6 receptor blocker treatment response.

PGC-1α inhibits M2 macrophage polarization and alleviates liver fibrosis following hepatic ischemia reperfusion injury

Oxidative stress can induce inflammation, promoting macrophage polarization and liver fibrosis following hepatic ischemia-reperfusion (I/R). Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has anti-oxidant and anti-inflammatory activity. However, how PGC-1α regulates macrophage polarization following hepatic I/R remains largely unknown. Male C57BL/6 wild-type mice were pre-treated with vehicle or trichostatin A (TSA) for 2 days and subjected to surgical induction of I/R. Liver injury and fibrosis in individual mice were examined longitudinally and the expression levels of IL-6, STAT3, M2-type macrophage markers, Collagen I and α-SMA in the liver of mice were analyzed by immunohistochemistry, RT-qPCR and Western blot. The potential interaction of PGC-1α with phosphorylated NF-kBp65 was determined by immunoprecipitation. The impacts of PGC-1α deficiency in hepatocytes on their IL-6 production and macrophage polarization were tested in a Transwell co-culture system. Moreover, the M2-type macrophage polarization and liver fibrosis were examined in hepatocyte-specific PGC-1α knockout mice and AAV8-mediated PGC-1α over-expressing mice following liver I/R. The down-regulated PGC-1α expression by I/R was negatively correlated with IL-6 levels in the liver of I/R mice and PGC-1α deficiency enhanced IL-6 expression, STAT3 activation and M2-type macrophage polarization in the I/R mice, which were abrogated by TSA treatment. In addition, PGC-1α directly interacted with phosphorylated NF-kBp65 in I/R livers. Hepatocyte-specific PGC-1α deficiency increased IL-6 production and promoted macrophage polarization toward M2 type when co-culture. More importantly, administration with AAV8-PGC-1α rescued the I/R-induced liver fibrosis by inhibiting the IL-6/JAK2/STAT3 signaling and M2-type macrophage polarization in the liver. These results suggest that PGC-1α may alleviate the I/R-induced liver fibrosis by attenuating the IL-6/JAK2/STAT3 signaling to limit M2-type macrophage polarization. PGC-1α may be a therapeutic target for the treatment of liver fibrosis.

Exploring the in vitro and in vivo anticancer activity of lasiokaurin on nasopharyngeal carcinoma

ObjectiveTo evaluate the anticancer activity of the natural diterpenoid lasiokaurin (LAS) against nasopharyngeal carcinoma (NPC) both in vitro and in vivo, along with investigating its underlying mechanism. MethodsMTT and colony formation assays were used to assess cell viability. Flow cytometry was utilized for the analysis of cell cycle arrest and apoptosis. Additionally, wound healing and transwell invasion assays were conducted separately to investigate cell migration and invasion. To uncover potential targets and pathways of LAS within NPC, a network pharmacological study based on RNA-sequencing (RNA-seq) was performed. Immunoblotting was subsequently used to determine protein levels. Furthermore, the anti-NPC activity of LAS was evaluated in vivo using a xenograft mouse model. ResultsBased on in vitro investigations, it is evident that LAS exerted substantial inhibitory effects on NPC cells. Notably, LAS demonstrated significant reductions in cell viability, migration, and invasion capabilities, while simultaneously inducing apoptosis and G2/M cell cycle arrest. Furthermore, LAS suppressed the activation of MAPK, mTOR, STAT3, and NF-κB pathways within NPC cells. Additionally, in vivo experiments underscored LAS's capacity to attenuate NPC tumor growth without eliciting any discernible impact on body weight. ConclusionOur data clearly demonstrate the anticancer activity of LAS against NPC, both in vitro and in vivo. These findings firmly position LAS as a potential candidate for the treatment of NPC.

Renal Fibrosis Is Alleviated through Targeted Inhibition of IL-11–Induced Renal Tubular Epithelial-to-Mesenchymal Transition

Renal fibrosis is a pathologic process that leads to irreversible renal failure without effective treatment. Epithelial-to-mesenchymal transition (EMT) plays a key role in this process. The current study found that aberrant expression of IL-11 is critically involved in tubular EMT. IL-11 and its receptorsubunitalpha-1 (IL-11Rα1) were significantly induced in renal tubular epithelial cells (RTECs) in unilateral ureteral obstruction (UUO) kidneys, co-localized with transforming growth factor-β1. IL-11 knockdown ameliorated UUO-induced renal fibrosis invivo and transforming growth factor-β1-induced EMT invitro. IL-11 intervention directly induced the transdifferentiation of RTECs to the mesenchymal phenotype and increased the synthesis of profibrotic mediators. The EMT response induced by IL-11 was dependent on the sequential activation of STAT3 and extracellular signal-regulated kinase 1/2 signaling pathways and the up-regulation of metadherin in RTECs. Micheliolide (MCL) competitively inhibited the binding of IL-11 with IL-11Rα1, suppressing the activation of STAT3 and extracellular signal-regulated kinase 1/2-metadherin pathways, ultimately inhibiting renal tubular EMT and interstitial fibrosis induced by IL-11. In addition, treatment with dimethylaminomicheliolide, a pro-drug of MCL for invivo use, significantly ameliorated renal fibrosis exacerbated by IL-11 in the UUO model. These findings suggest that IL-11 is a promising target in renal fibrosis and that MCL/dimethylaminomicheliolide exerts its antifibrotic effect by suppressing IL-11/IL-11Rα1 interaction and blocking its downstream effects.

Melatonin Attenuates Sepsis-Induced Acute Lung Injury via Inhibiting Excessive Mitophagy.

Epidemiological studies have indicated that lung injury is a frequent complication of sepsis. Mitophagy is vital to multiple pathological processes and diseases; however, its influence on sepsis-induced acute lung injury remains elusive. Melatonin has multiple antioxidant action and anti-inflammatory effects, including regulating mitophagy and inflammatorycytokine expression. Whereas, little is known about the affection of melatonin and mitophagy on CLP-induced ALI. The in vivo effect of melatonin on OPTN-mediated mitophagy was studied by CLP-induced ALI in a mouse model using C57BL/6 followed by treatment with vehicle and melatonin (30 mg/kg/d, intraperitoneal injection). ALI was assayed by lung wet /dry ratio, hematoxylin and eosin staining, and immunohistochemical staining. Signaling pathway changes were subsequently determined by Western blotting and immunofluorescence staining. The effects of melatonin on STAT3 activation and TNF-α production were detected by Western blotting, PCR, and immunohistochemical staining. Our results indicated that OPTN, mitophagy adaptors were significantly repressed in CLP-induced ALI, accompanied by overactivation ofmitophagy and inflammation. At the same time, we found that melatonin treatment alleviated ALI caused by CLP, and the effect was highly correlated with OPTN-related mitophagy. Furthermore, we demonstrated that OPTN-related mitophagy, which was normalized by melatonin, blocked STAT3 involved epithelial barrier and inflammation in vivo. Overall, our results confirm that mitophagy is adjusted by melatonin in the CLP-induced ALI. Moreover, manipulation of mitophagy through melatonin could be a possible treatment to reduce sepsis-associated lung injury.

TRPM7 transactivates the FOSL1 gene through STAT3 and enhances glioma stemness

IntroductionWe previously reported that TRPM7 regulates glioma cells’ stemness through STAT3. In addition, we demonstrated that FOSL1 is a response gene for TRPM7, and the FOSL1 gene serves as an oncogene to promote glioma proliferation and invasion.MethodsIn the present study, we determined the effects of FOSL1 on glioma stem cell (GSC) markers CD133 and ALDH1 by flow cytometry, and the maintenance of stem cell activity by extreme limiting dilution assays (ELDA). To further gain insight into the mechanism by which TRPM7 activates transcription of the FOSL1 gene to contribute to glioma stemness, we constructed a FOSL1 promoter and its GAS mutants followed by luciferase reporter assays and ChIP-qPCR in a glioma cell line and glioma patient-derived xenoline. We further examined GSC markers ALDH1 and TRPM7 as well as FOSL1 by immunohistochemistry staining (IHC) in brain tissue microarray (TMA) of glioma patients.ResultsWe revealed that FOSL1 knockdown reduces the expression of GSC markers CD133 and ALDH1, and FOSL1 is required to maintain stem cell activity in glioma cells. The experiments also showed that mutations of − 328 to − 336 and − 378 to − 386 GAS elements markedly reduced FOSL1 promoter activity. Constitutively active STAT3 increased while dominant-negative STAT3 decreased FOSL1 promoter activity. Furthermore, overexpression of TRPM7 enhanced while silencing of TRPM7 reduced FOSL1 promoter activity. ChIP-qPCR assays revealed that STAT3, present in nuclear lysates of glioma cells stimulated by constitutively activated STAT3, can bind to two GAS elements, respectively. We demonstrated that deacetylation of FOSL1 at the Lys-116 residue located within its DNA binding domain led to an increase in FOSL1 transcriptional activity. We found that the expression of TRPM7, ALDH1, and FOSL1 protein is associated with grades of malignant glioma, and TRPM7 protein expression correlates to the expression of ALDH1 and FOSL1 in glioma patients.ConclusionsThese combined results demonstrated that TRPM7 induced FOSL1 transcriptional activation, which is mediated by the action of STAT3, a mechanism shown to be important in glioma stemness. These results indicated that FOSL1, similar to GSC markers ALDH1 and TRPM7, is a diagnostic marker and potential drug target for glioma patients.

Targeted and Logical Discovery of Piperazic Acid-Bearing Natural Products Based on Genomic and Spectroscopic Signatures.

A targeted and logical discovery method was devised for natural products containing piperazic acid (Piz), which is biosynthesized from ornithine by l-ornithine N-hydroxylase (KtzI) and N-N bond formation enzyme (KtzT). Genomic signature-based screening of a bacterial DNA library (2020 strains) using polymerase chain reaction (PCR) primers targeting ktzT identified 62 strains (3.1%). The PCR amplicons of KtzT-encoding genes were phylogenetically analyzed to classify the 23 clades into two monophyletic groups, I and II. Cultivating hit strains in media supplemented with 15NH4Cl and applying 1H-15N heteronuclear multiple bond correlation (HMBC) along with 1H-15N heteronuclear single quantum coherence (HSQC) and 1H-15N HSQC-total correlation spectroscopy (HSQC-TOCSY) NMR experiments detected the spectroscopic signatures of Piz and modified Piz. Chemical investigation of the hit strains prioritized by genomic and spectroscopic signatures led to the identification of a new azinothricin congener, polyoxyperuin B seco acid (1), previously reported chloptosin (2) in group I, depsidomycin D (3) incorporating two dehydropiperazic acids (Dpz), and lenziamides A and B (4 and 5), structurally novel 31-membered cyclic decapeptides in group II. By consolidating the phylogenetic and chemical analyses, clade-structure relationships were elucidated for 19 of the 23 clades. Lenziamide A (4) inhibited STAT3 activation and induced G2/M cell cycle arrest, apoptotic cell death, and tumor growth suppression in human colorectal cancer cells. Moreover, lenziamide A (4) resensitized 5-fluorouracil (5-FU) activity in both in vitro cell cultures and the in vivo 5-FU-resistant tumor xenograft mouse model. This work demonstrates that the genomic and spectroscopic signature-based searches provide an efficient and general strategy for new bioactive natural products containing specific structural motifs.

Socs1-knockout in skin-resident CD4+ T cells in a protracted contact-allergic reaction results in an autonomous skin inflammation with features of early-stage mycosis fungoides

Recent detailed genomic analysis of mycosis fungoides (MF) identified suppressor of cytokine signaling 1 (SOCS1), an inhibitor of JAK/STAT signaling, as one of the frequently deleted tumor suppressors in MF, and one-copy deletion of SOCS1 was confirmed in early-stage MF lesions. To better understand the functional role of SOCS1 in the genesis of MF, we used a genetically engineered mouse model emulating heterozygous SOCS1 loss in skin resident CD4+ T cells. In these mice an experimentally induced contact-allergic reaction was maintained for 20 weeks. Ten weeks after discontinuing contact-allergic challenges, only the skin with locally one-copy deletion of Socs1 in CD4+ T cells still showed high numbers of CD3+/CD4+ Socs1 k.o. cells in the dermis (p ＜ 0.0001) with prevalent Stat3 activation (p ＜0.001). And in one out of 9 mice, this had progressed to far more dramatic increases, including the thickened epidermis, and with an explosive growth of Socs1 k.o. T cells in circulation; indicative of cutaneous lymphoma. Hence, we show that Socs1 mono-allelic loss in CD4+ T cells locally in protractedly inflamed skin results in autonomous skin inflammation with features of early-stage MF.

Reduced DEFA5 Expression and STAT3 Activation Underlie the Submucosal Invasion of Early Gastric Cancers

Introduction: Submucosal invasion is a core hallmark of early gastric cancer (EGC) with poor prognosis. However, the molecular mechanism of the progression from intramucosal gastric cancer (IMGC) to early submucosal-invasive gastric cancer (SMGC) is not fully understood. The objective of this study was to identify genes and pathways involved in the submucosal invasion in EGC using comprehensive gene expression analysis. Methods: Gene expression profiling was performed for eight cases of IMGC and eight cases of early SMGC with submucosal invasion ≥500 μm. To validate the findings of gene expression analysis and to examine the gene expression pattern in tissues, immunohistochemical (IHC) staining was performed for 50 cases of IMGC and SMGC each. Results: Gene expression analysis demonstrated that the expression levels of small intestine-specific genes were significantly decreased in SMGC. Among them, defensin alpha 5 (DEFA5) was the most downregulated gene in SMGC, which was further validated in SMGC tissues by IHC staining. Gene set enrichment analysis showed a strong association between SMGC, the JAK-STAT signaling pathway, and the upregulation of STAT3-activating cytokines. The expression of phosphorylated STAT3 was significant in the nucleus of tumor cells in SMGC tissues but not in areas expressing DEFA5. Conclusion: The results of this study strongly suggest that the downregulation of DEFA5 and the activation of STAT3 play a significant role in the submucosal invasion of EGC.

The presence of blastocyst within the uteri facilitates lumenal epithelium transformation for implantation via upregulating lysosome proteostasis activity

ABSTRACT The mammalian endometrium is covered by the lumenal epithelium (Le), which directly interacts with the blastocyst and plays an important role in the establishment of reciprocal crosstalk between the embryo and receptive uterus during implantation. However, the effect of the blastocyst on uterine differentiation during the window of receptivity is far from well understood. Through transcriptomic profiling of the uterine Le isolated by laser capture microdissection (LCM), it was demonstrated that global gene expression changes occurred in Le between pseudopregnant mice without embryos and pregnant mice with embryos. Some differentially expressed genes, including upregulated Areg (amphiregulin), Ihh (Indian hedgehog), Lifr (leukemia inhibitory factor receptor) and downregulated Msx1 (msh homeobox 1), Pgr (progesterone receptor), and Gata2 (GATA binding protein 2) in pregnant mice, have been reported to regulate the establishment of uterine receptivity. Besides, we found that blastocysts induced an increase in both the number and acidification of lysosome, consistent with enhanced lysosomal hydrolase activity in uterine Le. Further exploration uncovered that blastocyst-derived IGF2 was involved into the activation of epithelial STAT3 to induce lysosomal hydrolase expression, and inhibition of lysosomal function derails both uterine receptive maker gene expressions and embryo implantation. Finally, based on the proteomic data of both epithelia and the separated lysosome, it was revealed that CLDN1 (claudin 1) and MUC1 (mucin 1, transmembrane), two well-known downregulated molecules for successful implantation, are degraded by epithelial lysosome. In brief, our data demonstrated that blastocysts induced normal epithelium differentiation with lysosome activation to promote the uterine epithelial differentiation for embryo implantation. Abbreviations ACTB: actin beta; AREG: amphiregulin; ATP6V0A4: ATPase, H+ transporting, lysosomal V0 subunit A4; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CLDN1: claudin 1; CTSB: cathepsin B; DEGs: differentially expressed genes; E2: 17β-estradiol; ESR: estrogen receptor; GATA2: GATA binding protein 2; GLA: galactosidase, alpha; GO: gene ontology; HBEGF: heparin-binding EGF-like growth factor; IGF1R: insulin-like growth factor 1 receptor; Ihh: Indian hedgehog; ISH: in situ hybridization; LAMP1: lysosomal-associated membrane protein 1; LCM: laser capture microdissection; Le: lumenal epithelium; LGMN: legumain; LIF: leukemia inhibitory factor; LIFR: LIF receptor alpha; MSX1: msh homeobox 1; MUC1: mucin 1, transmembrane; P4: progesterone; PBS: phosphate-buffered saline; PCA: principal component analysis; PPT1: palmitoyl-protein thioesterase 1; PGR: progesterone receptor; PSP: pseudopregnancy; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; qPCR: quantitative real-time polymerase chain reaction; SP: pregnancy; TFEB: transcription factor EB.

Primary Cutaneous Anaplastic Large Cell Lymphoma-A Review of Clinical, Morphological, Immunohistochemical, and Molecular Features.

Primary cutaneous anaplastic large cell lymphoma (ALCL) is the second most common cutaneous T-cell lymphoma after mycosis fungoides and belongs to the spectrum of cutaneous CD30+ T-cell lymphoproliferative disorders. Although primary cutaneous ALCL usually presents as a localized nodule or papule with or without ulceration, multifocal lesions may occur in up to 20% of cases. Histologically, primary cutaneous ALCL consists of a diffuse dermal infiltrate of medium to large anaplastic/pleomorphic cells with abundant amphophilic-to-eosinophilic cytoplasm, horseshoe-shaped nuclei, strong and diffuse expression of CD30, and with focal or no epidermotropism. The neoplastic infiltrate may show angiocentric distribution and may extend to the subcutis. Patients with localized or multifocal disease have a similar prognosis with a 10-year overall survival rate of 90%. Approximately 30% of primary cutaneous ALCLs harbor a DUSP22 (6p25.3) gene rearrangement that results in decreased expression of this dual-specific phosphatase, decreased STAT3 activation, and decreased activity of immune and autoimmune-mediated mechanisms regulated by T-cells.

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.

Inhibition of BACE1 attenuates microglia-induced neuroinflammation after intracerebral hemorrhage by suppressing STAT3 activation.

Hematoma-induced neuroinflammation is the cause of poor prognosis in intracerebral hemorrhage (ICH); therefore, promoting blood clearance and blocking overactivated inflammation are rational approaches for ICH treatment. β-site amyloid precursor protein (APP) lyase-1 (BACE1) is a key molecule regulating the microglial phenotype transition in neurodegenerative diseases. Therefore, the aim of this study was to investigate the role of BACE1 in microglial phagocytosis and inflammatory features in ICH. Here, we demonstrated the unique advantages of targeting BACE1 in microglia using an autologous blood model and primary microglia hemoglobin stimulation. When BACE1 was inhibited early in ICH, fewer residual hematomas remained, consistent with an increase in genetic features that favor phagocytosis and anti-inflammation. In addition, inhibition of BACE1 enhanced the secretion of anti-inflammatory cytokines and substantially reduced the expression of proinflammatory genes, which was regulated by signal transduction and phosphorylation of activator of transcription 3 (STAT3). Further pharmacological inhibition of STAT3 phosphorylation effectively blocked the proinflammatory and weak phagocytic phenotype of microglia due to BACE1 induction. In summary, BACE1 is the critical molecule regulating the inflammatory and phagocytic phenotypes of microglia after ICH, and targeted inhibition of the BACE1/STAT3 pathway is an important strategy for the future treatment of ICH-induced neurological injury.

BSBM-03 A NOVEL COMBINED IMMUNOTHERAPY TO IMPROVE THERAPEUTIC RESPONSES IN SYMPTOMATIC BRAIN METASTASIS BASED ON TARGETING A SUBPOPULATION OF IMMUNOSUPPRESSIVE ASTROCYTES

Abstract The diagnosis of brain metastasis involves high morbidity and mortality. Recently, immune checkpoint blockade antibodies (ICB) have shown clinical benefits mainly when applied to asymptomatic brain metastasis patients. However, variability is broad and responses to this therapy drop considerably when treating clinically relevant disease. Although potentially involved in the lack of response to immunotherapy, corticoids do not seem to fully explain this situation. Thus, it is currently unknown how to effectively target symptomatic brain metastases with immunotherapy. We previously reported a clinically relevant protumoral program driven by STAT3 activation in a subpopulation of reactive astrocytes during advanced stages of the disease. By further exploiting astrocyte heterogeneity, we have found a potential strategy to improve the number of responders to immunotherapy in symptomatic brain metastasis. Specifically, we have developed a comprehensive strategy including genetic and pharmacologic approaches to define a novel immunosuppressive axis involving astrocyte-secreted TIMP1 signaling on CD63+ CD8+ T cells. Based on these findings, we developed a combined immunotherapy to boost the systemic activation of T cells with ICB while blocking local TIMP1-dependent immunosuppression in various preclinical models. Furthermore, the detection of TIMP1 in the CSF provides a biomarker to select patients who would benefit the most from the combined immunotherapy. Even more, our data using Patient Derived Organotypic Cultures from fresh brain metastasis neurosurgeries confirmed that our therapeutic strategy is valid for symptomatic brain metastases from any primary source. In conclusion, our study has identified a novel combined immunotherapy that could be especially relevant for symptomatic brain metastases. Our findings have emerged from a strong scientific rationale whereby a specific subpopulation of astrocytes is shown to activate the local immunosuppressive environment for brain-infiltrating CD8+ T cells.

Bromodomain protein 4 mediates the roles of TGFβ1-induced Stat3 signaling in mouse liver fibrogenesis

Epigenetic reader Bromodomain protein 4 (BrD4) functions as a global genomic regulator to direct hepatic stellate cell (HSC) activation (a key step in liver fibrogenesis) and liver fibrosis. The pivotal pro-fibrotic cytokine transforming growth factor-β1 (TGFβ1) signals through both Smad and Stat3 to elicit a wide array of biological effects. Stat3 is widely acknowledged as a regulator of gene transcription and is involved in fibrosis of multiple tissues. The present study focused on BrD4 function implication in the roles of TGFβ1-induced Stat3 signaling in HSC activation and liver fibrosis by using heterozygous TGFβ1 knockout mice and HSC culture. Results showed that Stat3 was required for TGFβ1-induced BrD4 expression in HSCs. BrD4 expression paralleled Stat3 activation in activated HSCs in human cirrhotic livers. BrD4 was involved in the roles of TGFβ1-induced Stat3 in HSC activation and liver fibrogenesis. Smad3 bound to phosphorylated-Stat3 and contributed to TGFβ1-induced Stat3 signaling. BrD4 expression induced by Stat3 signaling required the early-immediate gene Egr1. Egr1 had a positive feedback on Stat3 activation. In conclusion, a network consisting of Stat3 signaling, Smad3 signaling, Egr1, and BrD4 was involved in the effects of TGFβ1 on liver fibrosis, providing new toxicological mechanisms for TGFβ1 in liver fibrogenesis.

Context-dependent T-cell Receptor Gene Repertoire Profiles in Proliferations of T Large Granular Lymphocytes.

T cell large granular lymphocyte (T-LGL) lymphoproliferations constitute a disease spectrum ranging from poly/oligo to monoclonal. Boundaries within this spectrum of proliferations are not well established. T-LGL lymphoproliferations co-occur with a wide variety of other diseases ranging from autoimmune disorders, solid tumors, hematological malignancies, post solid organ, and hematopoietic stem cell transplantation, and can therefore arise as a consequence of a wide variety of antigenic triggers. Persistence of a dominant malignant T-LGL clone is established through continuous STAT3 activation. Using next-generation sequencing, we profiled a cohort of 27 well-established patients with T-LGL lymphoproliferations, aiming to identify the subclonal architecture of the T-cell receptor beta (TRB) chain gene repertoire. Moreover, we searched for associations between TRB gene repertoire patterns and clinical manifestations, with the ultimate objective of discriminating between T-LGL lymphoproliferations developing in different clinical contexts and/or displaying distinct clinical presentation. Altogether, our data demonstrates that the TRB gene repertoire of patients with T-LGL lymphoproliferations is context-dependent, displaying distinct clonal architectures in different settings. Our results also highlight that there are monoclonal T-LGL cells with or without STAT3 mutations that cause symptoms such as neutropenia on one end of a spectrum and reactive oligoclonal T-LGL lymphoproliferations on the other. Longitudinal analysis revealed temporal clonal dynamics and showed that T-LGL cells might arise as an epiphenomenon when co-occurring with other malignancies, possibly reactive toward tumor antigens.

Lysozyme promotes renal fibrosis through the JAK/STAT3 signal pathway in diabetic nephropathy.

Diabetic nephropathy (DN) is a leading cause of kidney failure. Lysozyme (LYZ) is an essential component of innate immunity and exhibits antibacterial properties. However, LYZ has been reported to induce nephropathy, implying a possible association between impaired renal function and lysozyme expression. Bioinformatics analysis was used to predict the hub gene associated with DN, and the differential expression of the hub gene was confirmed using a mouse model. A mouse model of streptozotocin (STZ)-induced diabetic nephropathy was established to investigate the correlation between DN and LYZ expression, and the functionality of LYZ was verified through knockdown and overexpression experiments conducted in vivo. Immunohistochemistry (IHC) was utilized to assess fibrosis-related markers and cytokines, while Masson staining was performed to assess renal fibrosis. Fibroblast proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. The role of the JAK pathway was confirmed using the JAK inhibitor AG490, and Western blot was used to investigate the underlying mechanisms. Mechanistically, 25 mM glucose promotes the expression of LYZ in fibroblastic cells, and LYZ may in turn promote the proliferation of renal interstitial fibroblasts. Western blot shows that glucose can activate STAT3 in an LYZ-dependent manner, and the JAK inhibitor AG490 can partially suppress LYZ-induced STAT3 activation. Furthermore, in vivo observations have revealed that overexpression of LYZ is associated with the senescent phenotype of renal tubular epithelial cells (RTECs). Lysozyme promotes kidney fibrosis via the JAK/STAT3 signaling pathway in diabetic nephropathy, and glucose may promote fibroblast proliferation by promoting LYZ auto-secretion.

Selective activation of STAT3 and STAT5 dictates the fate of myeloid progenitor cells

The molecular programs that govern the directed differentiation of myeloid progenitor cells are still poorly defined. Using a previously established immortalized, phenotypically normal myeloid progenitor cell model mEB8-ER, we unveil a new mechanism mediated by STAT5 and STAT3 at a bifurcation point of myeloid progenitor cell-fate specification. We find that myeloid progenitor cells can spontaneously differentiate into neutrophils with a basal level of STAT3 phosphorylation, which is enhanced by G-CSF treatment or STAT3 over-expression, leading to elevated neutrophil differentiation. Reduced STAT3 phosphorylation caused by GM-CSF treatment, STAT3 specific inhibitor, or STAT3 depletion leads to attenuated myeloid differentiation into neutrophils, while elevating differentiation into monocytes/macrophages. In contrast, STAT5 appears to have an antagonistic function to STAT3. When activated by GM-CSF, STAT5 promotes myeloid differentiation into monocytes/macrophages but inhibits neutrophil differentiation. At the mechanistic level, GM-CSF activates STAT5 to up-regulate SOCS3, which attenuates STAT3 phosphorylation and consequently neutrophil differentiation, while enhancing monocyte/macrophage differentiation. Furthermore, inhibition of STAT5 and STAT3 in primary myeloid progenitors recapitulates the results from the mEB8-ER model. Together, our findings provide new mechanistic insights into myeloid differentiation and may prove useful for the diagnosis and treatment of diseases related to abnormal myeloid differentiation.

The ATR inhibitor VE-821 increases the sensitivity of gastric cancer cells to cisplatin

BackgroundChemoresistance is a common event after cancer chemotherapy, including gastric cancer (GC). Cisplatin has been reported to induce the DNA damage response (DDR), thus leading to chemoresistance. VE-821, a specific inhibitor of ATR, has been proven to suppress a variety of solid malignancies effectively. Our study aimed to explore the effect of VE-821 on enhancing the chemical sensitivity to cisplatin and clarify the potential molecular mechanisms. MethodsCell viability and apoptosis of MKN-45 and AGS were measured by CCK8 and flow cytometry assay respectively. Western blotting was used to detect the expression of target proteins. TCGA database was used to analyze the correlation between the ATR expression with the prognosis of GC patients. The viability of GC organoids was detected by Cell Titer Glo (CTG) through luminescence. ResultsCisplatin inhibited the proliferation and induced apoptosis of GC cells with a relatively high IC50 value, and increased the phosphorylation levels of ATR-CHK1 and H2AX. VE-821 achieved the same effects but by downregulating the phosphorylation levels of the ATR-CHK1 pathway. Besides, higher ATR expression in GC tissues was positively correlated with higher pathological stage in GC patients. Interestingly, ATR inhibition reversed cisplatin-induced STAT3 activation and enhanced H2AX levels. Moreover, VE-821 significantly sensitized GC cells to cisplatin, and these two drugs had synergistic effects in GC cell lines, organoids, and in vivo. ConclusionOur results suggested VE-821 sensitized GC cells to cisplatin via reversing DDR activation. And VE-821 treatment may be a promising therapeutic strategy for GC patients with cisplatin resistance.

Positive regulatory loop of platelet-derived growth factor DD-induced STAT3 activation is associated with poor prognosis in advanced urothelial carcinoma

Immune checkpoint inhibitor (ICI) therapy has been established for patients with advanced urothelial cancer (UC). The necessity of overcoming resistance to ICIs and identifying a predictive factor for the same has been highlighted, such as the assessment of combination therapy with other targeted drugs and the characterization of molecular signatures in the tumor microenvironment. Recently, we reported that low hemoglobin (Hb) levels and a high platelet-to-lymphocyte ratio (PLR) were significantly associated with overall survival in patients with UC who did not benefit from pembrolizumab treatment. In the present study, we identified a possible link between these unfavorable prognostic indicators and PDGF-DD-induced STAT3 activation in UC. Overlapping patients between the high STAT3- or phosphorylated STAT3-positive score group (as assessed by immunohistochemistry) and low Hb levels or high PLR group (as assessed by blood tests) showed significantly worse outcomes after pembrolizumab treatment. Additionally, using the bladder cancer JMSU1 cell line, we demonstrated a possible positive regulatory loop between autocrine/paracrine PDGF-DD and STAT3 signaling. Therefore, we suggest that STAT3 inhibition and PDGF-DD detection in the tumor microenvironment might represent a potential therapeutic strategy to overcome resistance to pembrolizumab. Moreover, this can help identify patients with UC who could benefit from combination treatment.