Nuclear STAT3 Research Articles

Spatiotemporal functions of leukemia inhibitory factor in embryo attachment and implantation chamber formation

Embryo implantation is crucial for successful pregnancy, requiring appropriate uterine responses to implantation-competent blastocysts. Molecular communication at the maternal–fetal junction governs this process. Leukemia inhibitory factor (Lif) plays a pivotal role in implantation across species. Lif is abundantly expressed in the glandular epithelium during blastocyst-receptive phase and is induced in the stroma surrounding attached blastocysts. While diminished Lif expression leads to infertility, its influence on peri-implantation uteri remains unclear. Therefore, we investigated the role of Lif in uterine physiology using its uterine-specific knockout (uKO) and uterine epithelial-specific KO (eKO) in mice. Lif eKO and uKO mice displayed infertility owing to failed embryo attachment. Recombinant Lif supplementation rescued the reproductive phenotype of Lif eKO mice, but not Lif uKO mice; however, recombinant Lif injection rescued embryo attachment in Lif uKO mice. RNA-seq analysis indicated that Lif governs uterine epithelial genes, but not embryonic genes, to facilitate embryo attachment via activating nuclear Stat3. Concordantly, three-dimensional imaging of the uterine epithelium revealed that luminal closure and crypt formation are regulated by the uterine Lif–Stat3 axis as well as the presence of blastocysts. Collectively, our findings shed light on previously unknown mechanism on how Lif influences uterine functions molecularly and physiologically during early pregnancy.

STAT3 Increases CVB3 Replication and Acute Pancreatitis and Myocarditis Pathology via Impeding Nuclear Translocation of STAT1 and Interferon-Stimulated Gene Expression.

Acute pancreatitis (AP) is an inflammatory disease initiated by the death of exocrine acinar cells, but its pathogenesis remains unclear. Signal transducer and activator of transcription 3 (STAT3) is a multifunctional factor that regulates immunity and the inflammatory response. The protective role of STAT3 is reported in Coxsackievirus B3 (CVB3)-induced cardiac fibrosis, yet the exact role of STAT3 in modulating viral-induced STAT1 activation and type I interferon (IFN)-stimulated gene (ISG) transcription in the pancreas remains unclarified. In this study, we tested whether STAT3 regulated viral-induced STAT1 translocation. We found that CVB3, particularly capsid VP1 protein, markedly upregulated the phosphorylation and nuclear import of STAT3 (p-STAT3) while it significantly impeded the nuclear translocation of p-STAT1 in the pancreases and hearts of mice on day 3 postinfection (p.i.). Immunoblotting and an immunofluorescent assay demonstrated the increased expression and nuclear translocation of p-STAT3 but a blunted p-STAT1 nuclear translocation in CVB3-infected acinar 266-6 cells. STAT3 shRNA knockdown or STAT3 inhibitors reduced viral replication via the rescue of STAT1 nuclear translocation and increasing the ISRE activity and ISG transcription in vitro. The knockdown of STAT1 blocked the antiviral effect of the STAT3 inhibitor. STAT3 inhibits STAT1 activation by virally inducing a potent inhibitor of IFN signaling, the suppressor of cytokine signaling-3 ((SOCS)-3). Sustained pSTAT1 and the elevated expression of ISGs were induced in SOCS3 knockdown cells. The in vivo administration of HJC0152, a pharmaceutical STAT3 inhibitor, mitigated the viral-induced AP and myocarditis pathology via increasing the IFNβ as well as ISG expression on day 3 p.i. and reducing the viral load in multi-organs. These findings define STAT3 as a negative regulator of the type I IFN response via impeding the nuclear STAT1 translocation that otherwise triggers ISG induction in infected pancreases and hearts. Our findings identify STAT3 as an antagonizing factor of the IFN-STAT1 signaling pathway and provide a potential therapeutic target for viral-induced AP and myocarditis.

Malignant solitary fibrous tumor of the kidney with IGF2 secretion and without hypoglycemia

BackgroundSolitary fibrous tumor (SFT) is a rare fibroblastic mesenchymal tumor that mostly involves the pleura and infrequently involves extra-pleural sites. De novo SFT of the kidney is uncommon, and malignant SFT is extremely rare.Case presentationWe report a case of a 51-year-old man with a large malignant SFT in the left kidney. Pathological examination confirmed the diagnosis of SFT based on typical morphology, nuclear STAT6 expression, and NAB2-STAT6 gene fusion. The malignant subtype was determined by a large tumor size (≥ 15 cm) and high mitotic counts (8/10 high-power fields). KRAS mutation was identified by DNA sequencing. Insulin-like growth factor 2 (IGF2) was diffusely and strongly expressed in tumor cells, however, hypoglycemia was not observed. Hyperglycemia and high adrenocorticotropic hormone (ACTH) concentration were observed one month after surgery. Hormone measurements revealed normal blood cortisol and aldosterone levels, and increased urinary free cortisol level. A pituitary microadenoma was identified using brain magnetic resonance imaging, which may be responsible for the promotion of hyperglycemia.ConclusionsWe report a case of renal malignant SFT with a KRAS mutation, which was previously unreported in SFT and may be associated with its malignant behavior. Additionally, we emphasize that malignant SFT commonly causes severe hypoglycemia due to the production of IGF2. However, this effect may be masked by the presence of other lesions that promote hyperglycemia. Therefore, when encountering a malignant SFT with diffuse and strong IGF2 expression and without hypoglycemia, other lesions promoting hyperglycemia need to be ruled out.

Single Living Cell "Observation-Analysis" Integrated Platform Decodes Cell Migration Plasticity Orchestrated by Nucleocytoplasmic STAT3.

Cell migration requires the interplay among diverse migration patterns. The molecular basis of distinct migration programs is undoubtedly vital but not fully explored. Meanwhile, the lack of tools for investigating spontaneous migratory plasticity in a single living cell also adds to the hindrance. Here, we developed a micro/nanotechnology-enabled single-cell analytical platform to achieve coherent monitoring of spontaneous migratory pattern and signaling molecules. Via the platform, we unveiled a previously unappreciated STAT3 regionalization on the multifunctional regulations of migration. Specifically, nuclear STAT3 is associated with amoeboid migration, while cytoplasmic STAT3 promotes mesenchymal movement. Opposing effects of JAK2 multisite phosphorylation shape its response to STAT3 distribution in a dynamic and antagonistic manner, eventually triggering a reversible amoeboid-mesenchymal transition. Based on the above results, bioinformatics further revealed a possible downstream regulator of nucleocytoplasmic STAT3. Thus, our platform, as an exciting technological advance in single-cell migration research, can provide in-depth mechanism interpretations of tumor metastasis and progression.

GGT1 is a SNP eQTL gene involved in STAT3 activation and associated with the development of Post-ERCP pancreatitis

Post-ERCP pancreatitis (PEP) is an acute pancreatitis caused by endoscopic-retrograde-cholangiopancreatography (ERCP). About 10% of patients develop PEP after ERCP. Here we show that gamma-glutamyltransferase 1 (GGT1)-SNP rs5751901 is an eQTL in pancreatic cells associated with PEP and a positive regulator of the IL-6 amplifier. More PEP patients had the GGT1 SNP rs5751901 risk allele (C) than that of non-PEP patients at Hokkaido University Hospital. Additionally, GGT1 expression and IL-6 amplifier activation were increased in PEP pancreas samples with the risk allele. A mechanistic analysis showed that IL-6-mediated STAT3 nuclear translocation and STAT3 phosphorylation were suppressed in GGT1-deficient cells. Furthermore, GGT1 directly associated with gp130, the signal-transducer of IL-6. Importantly, GGT1-deficiency suppressed inflammation development in a STAT3/NF-κB-dependent disease model. Thus, the risk allele of GGT1-SNP rs5751901 is involved in the pathogenesis of PEP via IL-6 amplifier activation. Therefore, the GGT1-STAT3 axis in pancreas may be a prognosis marker and therapeutic target for PEP.

Giant cell-rich solitary fibrous tumour of the urinary bladder: case report of an unusual histological variant and literature review

BackgroundGiant cell-rich solitary fibrous tumour (GCR-SFT), previously referred to as giant cell angiofibroma, is an uncommon soft tissue tumour that classically occurs in the orbit but very rarely presents in deep organs. Here, we present a case of GCR-SFT occurring in the urinary bladder, which is one of the unusual histological subtypes of SFT.Case presentationA 56-year-old man was incidentally found to have a mass measuring 4.5 × 4.3 × 4.0 cm located in the left posterior wall of the bladder by computed tomography during a physical examination. The lesion was confirmed as GCR-SFT by pathological examination after laparoscopic radical surgery. Histopathologically, the tumour was a well-circumscribed, nonencapsulated lesion that was composed of bland spindle-ovoid tumour cells alternating with hypocellular and hypercellular areas, staghorn-like vasculatures and scattered large dark-stained multinucleate giant cells lining pseudovascular spaces. The spindle-ovoid cells and multinucleate giant cells showed strong and diffuse expression of CD34 and nuclear STAT6. In addition, the hallmark of the NAB2ex4-STAT6ex5 fusion gene was detected by RT‒PCR. The patient was classified as having a low risk of recurrence or metastasis according to the risk stratification criteria. The patient underwent regular follow-up for 34 months after surgery, and there was no evidence of local recurrence or metastasis.ConclusionThis is the first reported case of GCR-SFT occurring in the urinary bladder with underlying NAB2ex4-STAT6ex5 fusion. Complete surgical excision of the tumour and long-term follow-up are recommended to ensure no local recurrence or metastasis.

Sinonasal Glomangiopericytoma with Skull Base Involvement: A Diagnostic Dilemma.

Introduction: Glomangiopericytoma (GPC) is a rare type of neoplasm with hemangiopericytoma-like vasculature and perivascular hyalinization of capillary-sized veins. CD34 and S100 protein staining might be positive in a small percentage of GPC. Solitary fibrous tumors (SFTs) present clinically like GPC. However, challenges remain when differentiating GPC from SFT. Case Presentation: A 37-year-old male, smoker, presented with 3 years history of right-sided epistaxis and nasal congestion. He was also complaining of hyposmia but no headaches or visual complaints. On nasal endoscopy, he was found to have a right-sided nasal mass occupying the ethmoid cavity. Computed tomography showed a right sinonasal mass abutting the anterior skull base and magnetic resonance imaging demonstrated a hyperintense, enhancing mass within the right ethmoid cavity, superior septum, and anterior skull base. The patient underwent endoscopic resection with gross total resection and skull base reconstruction. Postoperative pathology initially was thought to be an SFT; however, subsequent STAT6 expression was negative, and therefore GPC diagnosis was confirmed. At the most recent follow-up (6 months), the patient remained free of local disease. Discussion: SFT, unlike GPC, stains strongly for CD34, in this case, due to strong CD34, the diagnosis of SFT was initially made. Nuclear STAT6 expression is highly specific and sensitive for SFT. This later returned as negative; therefore, GPC was confirmed. Conclusion: We present a case of sinonasal GPC with skull base involvement that was treated with endoscopic resection. At the most recent follow-up (6 months), the patient remained free of local disease.

ROLE OF GATA3 AND STAT6 IMMUNOHISTOCHEMISTRY IN CLASSIC HODGKIN LYMPHOMA (CHL) AND NODULAR LYMPHOCYTE PREDOMINANT HODGKIN LYMPHOMA (NLPHL)

Objective: The purpose of our study is to investigate the utility of immunohistochemical expression of GATA3 and STAT6 in differentiating Classic Hodgkin Lymphoma (CHL) from Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL). Material and Methods: We approached database of Shaukat Khanum Memorial Cancer Hospital and Research Centre and selected a total of 79 CHLs and 15 NLPHLs diagnosed either on Trucut biopsy or excision biopsy diagnosed during the time 2020-2021. One slide from each of these 94 cases was stained with GATA3 and STAT6 independently and pattern of staining (either nuclear or cytoplasmic or dual nuclear and cytoplasmic) was accessed. Results: 75/79(95%) cases of CHL were positive for GATA3 staining [60/79(75.9%) cases showed nuclear GATA3 staining, 15/79(18.9%) cases showed both nuclear and cytoplasmic staining] and 4/79(5%) CHL cases were negative for GATA3. 15/15(100%) cases of NLPHL were negative for GATA3[12/15(80%) cases showed no staining and 3/15(20%) cases showed only cytoplasmic blush]. 65/79(82%) cases of CHL were positive for STAT6[17/79(21.5%) cases showed nuclear STAT6 expression, 48/79(60.7%) cases showed both nuclear and cytoplasmic staining], and 14/79(18%) cases of CHL were negative for STAT6 expression [8/79(10%) cases showed only cytoplasmic staining and 6/79(8%) showed neither nuclear nor cytoplasmic staining]. 14/15(93.3%) cases of NLPHL were negative for STAT6[5/79(33.3%) cases showed only cytoplasmic staining and 9/15(60%) cases showed no staining] and 1/15(6.7%) case of NLPHL was positive for STAT6[showed nuclear STAT6 staining]. Conclusion: Nuclear staining of GATA3 and STAT6 either alone or in combination with cytoplasmic staining can be used to differentiate CHL from NLPHL. GATA3 effectively excludes NLPHL with 100% negative predictive value. Keywords: GATA3, STAT6, Classic Hodgkin lymphoma, Nodular lymphocyte predominant Hodgkin lymphoma

CDK2-activated TRIM32 phosphorylation and nuclear translocation promotes radioresistance in triple-negative breast cancer

IntroductionDespite radiotherapy being one of the major treatments for triple-negative breast cancer (TNBC), new molecular targets for its treatment are still required due to radioresistance. CDK2 plays a critical role in TNBC. However, the mechanism by which CDK2 promotes TNBC radioresistance remains to be clearly elucidated. ObjectivesWe aimed to elucidate the relationship between CDK2 and TRIM32 and the regulation mechanism in TNBC. MethodsWe performed immunohistochemical staining to detect nuclear TRIM32, CDK2 and STAT3 on TNBC tissues. Western blot assays and PCR were used to detect the protein and mRNA level changes. CRISPR/Cas9 used to knock out CDK2. shRNA-knockdown and transfection assays also used to knock out target genes. GST pull-down analysis, immunoprecipitation (IP) assay and in vitro isomerization analysis also used. Tumorigenesis studies also used to verify the results in vitro. ResultsHerein, tripartite motif-containing protein 32 (TRIM32) is revealed as a substrate of CDK2. Radiotherapy promotes the binding of CDK2 and TRIM32, thus leading to increased CDK2-dependent phosphorylation of TRIM32 at serines 328 and 339. This causes the recruitment of PIN1, involved in cis–trans isomerization of TRIM32, resulting in importin α3 binding to TRIM32 and contributing to its nuclear translocation. Nuclear TRIM32 inhibits TC45-dephosphorylated STAT3, Leading to increased transcription of STAT3 and radioresistance in TNBC. These results were validated by clinical prognosis confirmed by the correlative expressions of the critical components of the CDK2/TRIM32/STAT3 signaling pathway. ConclusionsOur findings demonstrate that regulating the CDK2/TRIM32/STAT3 pathway is a promising strategy for reducing radioresistance in TNBC.

Solitary Fibrous Tumor of the Pancreas: Analysis of 9 Cases With Literature Review.

Solitary fibrous tumor (SFT) has been increasingly reported in various anatomic sites. However, it is still extremely rare in the pancreas. Herein, we present the first series of primary pancreatic SFTs. Nine cases of primary pancreatic SFTs were analyzed. The mean age was 60 years (36 to 76y) with no sex predilection. Six tumors were in the head, 3 were in the tail. On imaging studies, tumors were described as a hypervascular mass, 2 revealed cystic areas, and 3 were favored to be neuroendocrine tumors. On biopsy, 2 cases were diagnosed as atypical spindle cell tumor; one was misdiagnosed as suspicious for sarcoma, and another case as metastatic renal cell carcinoma. Two were diagnosed as low-grade sarcoma and low-grade stromal tumor on frozen sections. Grossly, tumors were well-demarcated with a median size of 4cm (0.9 to 15cm). Microscopically, they were composed of ovoid to spindle tumor cells with no significant mitotic activity and were arranged in alternating hypercellular and hypocellular areas. Staghorn-like vessels and entrapped pancreatic parenchyma were also detected within all tumors. Tumor cells revealed diffuse/strong nuclear STAT6 expression in 7 of 8, CD34 in 7 of 9, and bcl-2 in 4 of 4 tested cases. One tested tumor harbored NAB2 - STAT6 fusion. Eight patients with available follow-up data were free of disease at a mean follow-up of 76 months (3 to 189mo). SFT should be considered in the differential diagnoses of mesenchymal neoplasms of the pancreas. Immunohistochemical nuclear STAT6 expression is a characteristic feature of SFT. Primary pancreatic SFTs seem to have favorable biological behavior in our series.

Hovenia dulcis Thunb. Fruit Extract Attenuates Psoriatic Skin Inflammation in Tumor Necrosis Factor-α-Stimulated Human Keratinocyte HaCaT Cells In Vitro.

Hovenia dulcis Thunb. fruit (HDF) is traditionally used for treating liver diseases and alcohol poisoning. The purpose of this study was to explore the effects of HDF on hyperproliferation, levels of inflammatory cytokines, and signaling mechanisms in human psoriatic keratinocyte HaCaT cells. HDF showed a preventive effect on tumor necrosis factor-α (TNF-α)-induced abnormal proliferation of psoriatic keratinocytes. Furthermore, real-time reverse transcription-PCR analysis showed that HDF suppressed the expressions of inflammatory cytokines; interleukin (IL)-1α and IL-1β and chemokines; CCL-20 and CXCL-8 in TNF-α-induced HaCaT cells. Western blotting revealed that HDF suppressed the levels of phosphorylated IκB and STAT3 together with a decline in the levels of phosphorylated mitogen-activated protein kinases (MAPKs). These outcomes indicate that HDF prevents the abnormal proliferation of keratinocytes and modulates inflammatory responses by suppressing nuclear factor-κB (NF-κB) and STAT3 activation through downregulation of the MAPK signaling pathway in TNF-α-induced psoriatic keratinocytes. Our study demonstrates that HDF is prospective and beneficial for psoriatic skin inflammation.

Subsets of interferon signaling and response to immune checkpoint blockade.

9522 Background: Interferon (IFN) signaling in tumors is critical for both response and resistance to immune checkpoint inhibitors (ICI). While PD-L1 is an approved biomarker for ICI response in some cancers, most have no approved biomarkers for ICI response. Further, there are no clinical strategies to predict IFN sensitive vs resistant states. We hypothesized that distinct patterns of IFN signaling in melanoma, including STAT1 (precursor to IFN signaling), phosphorylated STAT1 (pSTAT1, marker of active IFN signaling), and PD-L1 (canonical inhibitory output), are associated with response or resistance to ICI. Methods: Samples from 2 tissue microarrays of 97 patients total with metastatic melanoma who received ICI from 2011-2017 were randomized into discovery [D] or validation [V] cohorts. Multiplexed immunofluorescent microscopy was used to assess STAT1, pSTAT1, and PD-L1. 54 tumors were assessed for CD3. Signals were quantified via AQUA (Automated Quantitative Analysis). High vs low signals were defined around the discovery cohort median AQUA scores. Treatment response was assessed using RECIST, and 3-year survival was analyzed. For in vitro studies, 4 human melanoma cell lines were stimulated with IFNγ/β, and Western blots were performed for STAT1, pSTAT1, and PD-L1. Results: Median follow up time was 20.2 mos (range 0.9-95.4 mos). Higher overall histospot, tumor, and nuclear STAT1 were in responders (CR/PR/SD > 6 mos) compared to non-responders (SD < 6 mos/PD, histospot p = 0.029 [D] and 0.040 [V]) and were associated with improved survival (histospot HR 0.25 [95% CI 0.08-0.76] p = 0.015 [D]; HR 0.32 [95% CI 0.12-0.84] p = 0.021 [V]). PD-L1 and pSTAT1 did not validate as associated with ICI response/survival. While many tumors had concordant high/low STAT1, pSTAT1, and PD-L1, many also had a discordant STAT1 or PD-L1 predominance. To assess the basis of this discordance, we performed IFN stimulation and Western blots of melanoma cell lines. IFNβ upregulated STAT1 greater than PD-L1 in both acute and chronic settings (p < 0.001 and < 0.001), while chronic IFNγ upregulated PD-L1 compared to the acute setting (p = 0.039) but not STAT1 (p = 0.105). Therefore, we hypothesized patients with discordant STAT1 and PD-L1 expression may have differential responses to ICI. In the overall cohort, STAT1highPD-L1high tumors (IFN engaged) were associated with improved survival compared to STAT1lowPD-L1low tumors (IFN low, HR 0.28 [95% CI 0.11-0.70] p = 0.007). Interestingly, STAT1highPD-L1low tumors were associated with improved survival compared to STAT1lowPD-L1high tumors (HR 0.28 [95% CI 0.08-0.94] p = 0.04). Accordingly, STAT1highPD-L1high and STAT1highPD-L1low tumors had greater CD3 AQUA scores compared to STAT1lowPD-L1low tumors (p < 0.001 and p = 0.018 respectively). Conclusions: Combined STAT1 and PD-L1 expression patterns may better predict melanoma response to ICI and provide insight into IFN sensitive versus resistant states.

MODERN MARKERS OF OSTEODYSMETABOLIC SYNDROME

Osteoporosis is a prevalent systemic osteodysmetabolic disease affecting bone tissue, characterized by a loss of bone mass, microstructure disturbances, and an increased susceptibility to low-traumatic fractures. Global statistical data from 2019 indicate that 32 million people worldwide were diagnosed with osteoporosis, with 25.5 million being women and 6.5 million men. Hypoestrogeny, considered one of the key mechanisms in the development of osteometabolic syndrome, disrupts the RANK/RANKL/OTG signalling system by activating nuclear factor-κB (NF-KB) or STAT-3. This activation triggers osteoclastogenesis and subsequently leads to the development of osteoporosis, which is a significant global health concern.
 The aim of this study is to investigate the specific features of osteometabolic changes in bone tissue and assess the 10-year risks of osteoporotic fractures and hip fractures. An examination was conducted on 130 individuals (116 women and 14 men) with an average age of 55.3±15.4 years. The participants were further divided into three groups: Group I (main group, n=85), Group II (control group, n=31), and Group III (comparative group, n=14). Anthropometric parameters were evaluated, revealing deviations in body mass index (BMI) from the norm. The average BMI values were as follows: Group I - 27.2±5.2 kg/m2, Group II - 23.4±4.3 kg/m2, and Group III - 25.8±3.5 kg/m2 (p = 0.0013).
 Estimating the 10-year probability of developing osteoporotic fractures using the FRAX model indicated a higher likelihood in the main group compared to the control group (7.4% [4.0–15.0%] vs. 2.7% [2.4–3.3%], p = 0.0001). When analyzing the 10-year risk of hip fracture, the results were as follows: Group I - 1.1% [0.2–5.1%], Group II - 0.1% [0–0.3%], and Group III - 0.15% [0.1–0.4%] (p = 0.0001). These findings suggest the activation of systemic inflammatory pathways as a consequence of hypoestrogenism in women from the main group.
 This study clearly demonstrates a higher likelihood of 10 different osteoporotic fractures and hip fractures, as indicated by the FRAX model, in the main group compared to Groups II and III. Therefore, utilizing ultrasound densitometry in conjunction with the FRAX model can help prevent the occurrence of osteoporotic fractures and hip fractures. Furthermore, when changes in tissue mineral density and markers of systemic inflammation are detected, it enables the development of gender-specific approaches for further diagnosis and treatment

Abstract 4991: Phosphorylation of MERTK is required for nuclear localization in non-small cell lung cancer (NSCLC)

Abstract MERTK is a transmembrane receptor that belongs to the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases. The role of MERTK in cancer progression and resistance to therapies are reported in many cancer types, including non-small cell lung cancer (NSCLC). In canonical signaling, MERTK is localized at the cell membrane and mediates intracellular signal transduction pathways. Non-canonical signaling of receptor tyrosine kinases (RTKs) has been reported, including signaling which occurs when RTKs translocate to the nucleus. Our group previously reported nuclear localization of MERTK in leukemia cells and we now extend these findings to non-small cell lung cancer. We found exogenous MERTK localized to the nucleus in cells transfected with a plasmid expressing wild-type MERTK. Additionally, endogenous nuclear MERTK was phosphorylated, suggesting that MERTK may have a functional role in the nucleus. MERTK kinase inhibitor MRX-2843 modulates its nuclear translocation. Introducing point mutations to MERTK autophosphorylation sites diminished nuclear translocation in comparison to cells transfected with wild-type MERTK. Our analysis of proteins that may interact with MERTK to regulate nuclear localization and function demonstrated co-immunoprecipitation of nuclear MERTK and STAT1. Current studies are underway to further explore nuclear MERTK functions and how these roles may be modulated upon interaction with STAT1. Citation Format: K.M. Tanim, Deborah DeRyckere, Douglas K. Graham. Phosphorylation of MERTK is required for nuclear localization in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4991.

NO-producing Polymorphonuclear Neutrophils Confer Protection against Chlamydia psittaci in Mouse Lung Infection.

Whether polymorphonuclear neutrophils (PMN) exert a protective role upon chlamydial infection by expressing inducible nitric oxide (NO) synthase (iNOS) and producing NO remains unclear. This issue was addressed using BALB/c mice infected with C. psittaci (Cps) 6BC strain. Methods mainly included flow cytometry, immunofluorescence, qRT-PCR, and Western blot. The number of PMN was significantly increased during Cps infection, which was accompanied by the increased iNOS expression and NO production in the mouse lungs. PMN were the major source of NO during pulmonary Cps infection and inhibited Cps multiplication in an iNOS/NO-dependent manner. Depletion of PMN aggravated Cps-induced disease and increased Cps burden. Mechanically, NF-κB and STAT1 signaling pathways, but not MAPK signaling pathways, were required for the induction of iNOS expression and NO production in PMN by Cps infection. Thus, our findings highlight the protective role of NO-producing PMN in Cps infection. NO-producing PMN confer a protective role during pulmonary Cps infection in mice, and thus our study sheds new light on PMN function during Chlamydia infection.

Panax notoginseng saponins (PNS) attenuate Th17 cell differentiation in CIA mice via inhibition of nuclear PKM2-mediated STAT3 phosphorylation

Context Rheumatoid arthritis (RA) is an autoimmune disease with aberrant Th17 cell differentiation. Panax notoginseng (Burk.) F. H. Chen (Araliaceae) saponins (PNS) have an anti-inflammatory effect and can suppress Th17 cell differentiation. Objective To investigate mechanisms of PNS on Th17 cell differentiation in RA, and the role of pyruvate kinase M2 (PKM2). Materials and methods Naive CD4+T cells were treated with IL-6, IL-23 and TGF-β to induce Th17 cell differentiation. Apart from the Control group, other cells were treated with PNS (5, 10, 20 μg/mL). After the treatment, Th17 cell differentiation, PKM2 expression, and STAT3 phosphorylation were measured via flow cytometry, western blots, or immunofluorescence. PKM2-specific allosteric activator (Tepp-46, 50, 100, 150 μM) and inhibitor (SAICAR, 2, 4, 8 μM) were used to verify the mechanisms. A CIA mouse model was established and divided into control, model, and PNS (100 mg/kg) groups to assess an anti-arthritis effect, Th17 cell differentiation, and PKM2/STAT3 expression. Results PKM2 expression, dimerization, and nuclear accumulation were upregulated upon Th17 cell differentiation. PNS inhibited the Th17 cells, RORγt expression, IL-17A levels, PKM2 dimerization, and nuclear accumulation and Y705-STAT3 phosphorylation in Th17 cells. Using Tepp-46 (100 μM) and SAICAR (4 μM), we demonstrated that PNS (10 μg/mL) inhibited STAT3 phosphorylation and Th17 cell differentiation by suppressing nuclear PKM2 accumulation. In CIA mice, PNS attenuated CIA symptoms, reduced the number of splenic Th17 cells and nuclear PKM2/STAT3 signaling. Discussion and conclusions PNS inhibited Th17 cell differentiation through the inhibition of nuclear PKM2-mediated STAT3 phosphorylation. PNS may be useful for treating RA.

Alleviation of imiquimod-induced psoriasis-like symptoms in Rorα-deficient mouse skin

Retinoic acid receptor-related orphan receptor α (RORα) plays a vital role in various physiological processes, including metabolism, cancer, circadian rhythm, cerebellar development, and inflammation. Although RORα is expressed in the skin, its role in skin physiology remains poorly elucidated. Herein, Rorα was expressed in the basal and suprabasal layers of the epidermis; however, keratinocyte-specific Rorα deletion did not impact normal epidermal formation. Under pathophysiological conditions, Rorα-deficient mice exhibited alleviated psoriasis-like symptoms, including relatively intact epidermal stratification, reduced keratinocyte hyperproliferation, and low-level expression of inflammatory cytokines in keratinocytes. Unexpectedly, the splenic population of Th17 cells was significantly lower in keratinocyte-specific RORα deficient mice than in the control. Additionally, Rorα-deficiency reduced imiquimod-induced activation of nuclear factor-κB and STAT3 in keratinocytes. Therefore, we expect that RORα inhibitors act on immune cells and keratinocytes to suppress the onset and progression of psoriasis.

Leptin plays a role in the multiplication of and inflammation in ovarian granulosa cells in polycystic ovary syndrome through the JAK1/STAT3 pathway

ObjectivesThe authors determined the level of Expression of Leptin (LEP) in Polycystic Ovary Syndrome (PCOS) patients with or without obesity and in GCs treated with insulin. MethodsLEP expression was first assessed in ovary cortex specimens collected from women with PCOS with or without obesity as well as from healthy controls. Ovarian Granulosa Cells (OGCs) induced by insulin extracted from a mouse model were used in further functional research. ResultsReal-time PCR and western blotting indicated that LEP expression was upregulated in GCs induced by insulin, in comparison with that in GCs not induced by insulin. Furthermore, the knockdown of LEP resulted in a reduction in growth and multiplication and an increase in apoptosis and inflammation in GCs induced by insulin. Next, the authors evaluated the effect of LEP on three key pathways of inflammation (MAPK, NF-kB, and JAK1/STAT3); results showed that the JAK1/STAT3 pathway was induced by LEP knockdown, as evidenced by the upregulation of phosphor-JAK1, phosphor-STAT3, and nuclear STAT3 expression. Administration of curcumin, a specific inhibitor of STAT3, counteracted the effect of LEP knockdown on cell inflammation and apoptosis. ConclusionThe present data suggest that upregulation of LEP expression in the PCOS granulosa cell model is essential for reducing apoptosis and inflammation by modulating the JAK1/STAT3 pathway axis.

Glucagon induces the hepatic expression of inflammatory markers in vitro and in vivo.

Glucagon exerts multiple hepatic actions, including stimulation of glycogenolysis/gluconeogenesis. The liver plays a crucial role in chronic inflammation by synthesizing proinflammatory molecules, which are thought to contribute to insulin resistance and hyperglycaemia. Whether glucagon affects hepatic expression of proinflammatory cytokines and acute-phase reactants is unknown. Herein, we report a positive relationship between fasting glucagon levels and circulating interleukin (IL)-1β (r=0.252, p=.042), IL-6 (r=0.230, p=.026), fibrinogen (r=0.193, p=.031), complement component 3 (r=0.227, p=.024) and high sensitivity C-reactive protein (r=0.230, p=.012) in individuals without diabetes. In CD1 mice, 4-week continuous treatment with glucagon induced a significant increase in circulating IL-1β (p=.02), and IL-6 (p=.001), which was countered by the contingent administration of the glucagon receptor antagonist, GRA-II. Consistent with these results, we detected a significant increase in the hepatic activation of inflammatory pathways, such as expression of NLRP3 (p < .02), and the phosphorylation of nuclear factor kappaB (NF-κB; p < .02) and STAT3 (p < .01). In HepG2 cells, we found that glucagon dose-dependently stimulated the expression of IL-1β (p < .002), IL-6 (p < .002), fibrinogen (p < .01), complement component 3 (p < .01) and C-reactive protein (p < .01), stimulated the activation of NLRP3 inflammasome (p < .01) and caspase-1 (p < .05), induced the phosphorylation of TRAF2 (p < .01), NF-κB (p < .01) and STAT3 (p < .01). Preincubating cells with GRA-II inhibited the ability of glucagon to induce an inflammatory response. Using HepaRG cells, we confirmed the dose-dependent ability of glucagon to stimulate the expression of NLRP3, the phosphorylation of NF-κB and STAT3, in the absence of GRA-II. These results suggest that glucagon has proinflammatory effects that may participate in the pathogenesis of hyperglycaemia and unfavourable cardiometabolic risk profile.

New Mechanistic Insight into the Protective Effects of Ganoderma lucidum Polysaccharides Against Palmitic Acid-Induced Cell Damage in Porcine Intestinal Epithelial Cell Line IPEC-J2

Ganoderma lucidum ( G. lucidum) is one of the well-known mushrooms in China, which has G. lucidum polysaccharides (GLP) that have been widely studied for various biological activities, such as antioxidant, antitumor, antiinflammatory, antiviral, antidiabetes, and immunomodulatory activities. A signal transducer and activator of transcription (STAT) signaling pathway is related to cell proliferation and apoptosis. The relationship between STAT and intestinal protection of GLP is still unknown. We studied the inhibitors AG490 in the STAT pathway and its downstream molecules to analyze the unique effects in the protection of GLP against palmitic acid (PA)-induced porcine intestinal epithelial cells (IPEC-J2) injury. Compared to PA treatment, GLP + PA obviously decreased Ca2+ concentration, H2O2 production, NF-E2-related factor 2 (Nrf2) nuclear translocation, STAT1 and STAT2 protein levels, and increased nuclear factor kappa-B (NF-κB) nuclear translocation and p-STAT3/STAT3 ratio in IPEC-J2 cells. After inhibition of STAT3 signaling, p-STAT3/STAT3 ratio, NF-κB nuclear translocation obviously decreased and Nrf2 nuclear translocation significantly increased in the GLP + PA group. The protection of GLP on proliferation and apoptosis of PA-induced IPEC-J2 cells was suppressed by inhibiting STAT3. The STAT3 pathway regulated the enterocyte-protective effects of GLP by modulating the nuclear translocation of Nrf2 and NF-κB. We provide new insights into the mechanism of STAT signaling for the protection of GLP on PA-induced intestinal epithelial cell injury.