Activate Signal Transduction Research Articles

RTID-02. SILMET TRIAL: A RANDOMIZED, PLACEBO-CONTROLLED STUDY TO EVALUATE SILIBININ IN PREVENTING INTRACRANIAL RECURRENCE AFTER COMPLETE RESECTION OF A SINGLE BRAIN METASTASIS FROM NSCLC OR BREAST CANCER

Abstract BACKGROUND: A subpopulation of reactive astrocytes (RAs) with activated signal transducer and activator of transcription 3 (STAT3) signalling in peritumoral tissue of brain metastases (BM) has been shown to favor a pro-metastatic microenvironment. High-pSTAT3 expression in RAs of BM has been correlated with a shorter intracranial progression-free survival (i-PFS) as compared with low-pSTAT3 BM. Pharmacological inhibition of the STAT3 pathway with silibinin has been suggested to be active in preliminary cohort of patients with BM. Patients and methods. A new “prevention” trial has been initiated in June 2023 to investigate the efficacy of silibinin in comparison to placebo (PCB) to prevent a secondary intracranial relapse in a multicentric cohort of patients with gross-total resected BM from NSCLC and breast cancer. Inclusion criteria are as follows: histologically confirmed BM from NSCLC or BC; single BM who underwent a complete resection; systemic disease not requiring a change of antineoplastic therapy; pSTAT3 presence in RAs of peritumoral tissue confirmed by central assessment; ≥ 18 – 70 years of age; KPS ≥ 70. The primary endpoint is to demonstrate a superiority of silibinin in prolonging time to local recurrence as compared to PCB. The secondary endpoints are time to distant recurrence of BM in the brain, i-PFS, overall PFS (intracranial and/or systemic), overall survival, tolerability, quality of life. Collection of blood and CSF samples for the analysis of STAT3 downstream pathways is an exploratory objective. The sample size (70 patients) was calculated by assuming a median time to local recurrence of BM of 7.5 months for PCB (control arm) according to Mahajan [1] and ≥15 months for silibinin arm. Clinical trial information: NCT05689619 [1] Mahajan A et al. Post-operative stereotactic radiosurgery versus observation for completely resected brain metastases: a single-centre, randomised, controlled, phase 3 trial. Lancet Oncol. 2017

Endotoxin accelerates insulin amyloid formation and inactivates insulin signal transduction

Aims and objectivesThe aim of this study is to discuss the influence of endotoxin on insulin amyloid formation, to provide guidance for therapeutic insulin preparation and storage. Materials and methodsThe ThT and ANS binding assays were applied to characterize the dynamics curve of insulin amyloid formation with the presence or absence of endotoxin. The morphological structures of intermediate and mature insulin fibrils were observed with SEM and TEM. Secondary structural changes of insulin during fibriliation were examined with CD, FTIR and Raman spectral analysis. The cytotoxic effects of oligomeric and amyloidogenic insulin aggregates were detected using a cck-8 cell viability assay kit. The influence of endotoxin on insulin efficacy was analyzed by monitoring the activation of insulin signal transduction. Key findingsThT analysis showed that endotoxin, regardless of species, accelerated insulin fibrils formation in a dose-dependent manner, as observed with a shorter lag phase. ANS binding assay demonstrated endotoxin provoked the exposure of insulin hydrophobic patches. The results of SEM and TEM data displayed that endotoxin drove insulin to cluster into dense and viscous form, with thicker and stronger filaments. Based on CD, FTIR and Raman spectra, endotoxin promoted the transition of α-helix to random coil and β-strand secondary structures during insulin aggregation. Insulins in both oligomeric and amyloidogenic forms were cytotoxic to HepG2 cells, with the former being more severe. Finally, the efficacy of endotoxin treated insulin obviously decreased. SignificanceOur studies revealed that endotoxin disrupts the structural integrity of insulin and promotes its amyloidosis. These findings offered theoretical guidance for insulin storage and safe utilization, as well as pointing up a new direction for insulin resistance research.

Pregabalin inhibits purinergic P2Y2 receptor and TRPV4 to suppress astrocyte activation and to relieve neuropathic pain

BackgroundsTransient receptor potential vanilloid 4 (TRPV4)-mediated astrocyte activation is critical to neuropathic pain. Pregabalin, a widely used drug to treat chronic pain, is reported to lower the intracellular calcium level. However, the molecular mechanism by which pregabalin decreases the intracellular calcium level remains unknown. Purinergic P2Y2 receptor—a member of the G protein-coupled receptor (GPCR) family—regulates calcium-related signal transduction in astrocyte activation. We investigated whether P2Y2 receptor is involved in the pharmacological effects of pregabalin on neuropathic pain. MethodsNeuropathic pain was induced by chronic compression of the dorsal root ganglion (CCD) in rats. Paw withdrawal mechanical threshold (PWMT) was used for behavioral testing. Intracellular calcium concentration was measured using a fluorescent calcium indicator (Fluo-4 AM). ResultsWe found that P2Y2 receptor protein was upregulated and astrocytes were activated in the experimental rats after CCD surgery. Lipopolysaccharide (LPS) increased the intracellular calcium concentration and induced astrocyte activation in cultured astrocytes but was prevented via P2Y2 receptor inhibitor AR-C118925 or pregabalin. Furthermore, plasmid-mediated P2Y2 receptor overexpression induced an elevation of the intracellular calcium levels and inflammation in astrocytes, which was abolished by the TRPV4 inhibitor HC-067047. AR-C118925, HC-067047, and pregabalin relieved neuropathic pain and inflammation in rats after CCD surgery. Finally, plasmid-mediated P2Y2 receptor overexpression induced neuropathic pain in rats, which was abolished by pregabalin administration. ConclusionsPathophysiological variables that upregulated the P2Y2 receptor/TRPV4/calcium axis contribute to astrocyte activation in neuropathic pain. Pregabalin exerts an analgesic effect by inhibiting this pathway.

Chaperon Protein GRP78 Released from MPN Cells Increase Expression of Lysyl Oxidase in Human Stroma Cell Line

Impaired function of the endoplasmic stress (ER) response causes numerous pathological conditions, including tissue fibrosis. In the present study, we aimed to determine the pathological role of ER stress response systems in myeloproliferative neoplasms (MPNs). We found an increased expression of the chaperone protein glucose-regulated protein (GRP)78, which is a central regulator of ER stress in megakaryocytes from primary myelofibrosis (PMF) (n=7) or post-essential thrombocythemia myelofibrosis(POST-ET-MF) patients (n=2) regardless of the type of driver mutation. In accord with these notions, we found that GRP78 was overexpressed in JAK2V617F-harboring cell lines (HEL and SET-2) and in UT-7/TPO cells overexpressing either type1 or type2 mutant form of calreticulin. To address the mechanism responsible for the induction of GRP78, we treated the HEL and SET-2 cells with 4-phenyl butyric acid and tauroursodeoxycholic acid both of which are well known inhibitors of ER stress, however, these inhibitors did not affect the expression levels of GRP78. These results indicated that overexpression of GRP78 shown in these cells were not a result of activation of ER stress. In contrast, ruxolitinib, an inhibitor of JAK2V617F, blocked GRP78 expression in these cells. We also found ruxolitinib blocked nuclear accumulation of the transcription factor 4 (ATF4), one of the main transcription factors which regulate GRP78. Furthermore, knockdown of ATF4 using siRNA also resulted in suppression of GRP78 levels in HEL and SET-2 cells. These results suggested that JAK2V617F induce GRP78 through activation of ATF4. We also confirmed that megakaryocytes from MF patients had statistically higher nuclear to cytoplasmic intensity of ATF4 compared to megakaryocytes found in control subjects. For the next step, we tried to find out the pathological action of GRP78 in MPN cells. Although the GRP78 usually works as chaperon protein in cytosol, some cancer cells secret GRP78 into tumor microenvironment and activates signal transduction molecules in surrounding cells. Therefore, we hypothesized that overexpressed GRP78 proteins in MPN cells may secret and modulate phenotype of bone marrow stroma cells. Initially, we confirmed the presence of GRP78 proteins in culture media from HEL and SET-2 cells. Next, we investigated whether GRP78 secreted by HEL and SET-2 cells affect phenotype of bone marrow stromal cells. For this purpose, we used human bone marrow stroma cell line, HS-5. HS-5 cells were co-cultured with HEL or SET-2 cells with trans-well system, and changes of several key molecules involved in development of fibrosis were analyzed. We found that co-culture with HEL or SET-2 cells clearly indued expression of lysyl oxidase (LOX) , which mediate cross-linking of collagen fiber and induce tissue fibrosis , in HS-5 cells. Anti-GRP78 neutralizing antibody abrogated LOX elevation, in contrast, recombinant form of GRP78 proteins induced LOX proteins in HS-5. According to the above results, we examined LOX expression in bone marrow specimens from MF patients. The areas of LOX expression cells were statistically higher in MF patients compared to control group. In addition, high power field observations detected not only in round cells but also in spindle like cells expressed LOX in MF patients, suggesting that bone marrow stromal cells in MF patients express LOX. In conclusion, we demonstrated that GRP78 was overexpressed both in MPN derived cell lines and also in primary megakaryocytes from MF patients. Our observations proposed that overexpressed GRP78 in MPN cells contribute development of myelofibrosis through secreted in microenvironment and induced expression of extracellular matrix modulating enzyme LOX in bone marrow stromal cells.

NAMPT mediates PDGF-induced pulmonary arterial smooth muscle cell proliferation by TLR4/NF-κB/PLK4 signaling pathway

Nicotinamide phosphoribosyltransferase (NAMPT), a pleiotropic protein, promotes the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), which is associated with the genesis and progression of pulmonary arterial hypertension (PAH). NAMPT is highly increased in PAH patient's plasma and highly relevant to PAH severity. The mRNA and protein levels of NAMPT are elevated in PAH animal models. However, the underlying molecular mechanisms how NAMPT mediated platelet-derived growth factor (PDGF)-induced PASMCs proliferation are still unclear. The present study aimed to address these issues. Primary cultured PASMCs were attained from male Sprague-Dawley (SD) rats. Western blotting, RT-PCR, ELISA, cell transfection, Cell Counting Kit-8 (CCK-8) and EdU incorporation assays were used in the experiments. We showed that PDGF upregulated NAMPT expression through the activation of signal transducers and activators of transcription 5 (STAT5), and elevated extracellular NAMPT further promoted the activation of NF-κB through Toll-like receptor 4 (TLR4), which ultimately upregulated polo-like kinase 4 (PLK4) expression leading to PASMCs proliferation. Knockdown of STAT5, NAMPT or PLK4, and inhibition of TLR4 or NF-κB suppressed PDGF-induced PASMCs proliferation. Our study suggests that NAMPT plays an essential role in PDGF-induced PASMCs proliferation via TLR4/NF-κB/PLK4 pathway, suggesting that targeting NAMPT might be valuable in ameliorating pulmonary arterial hypertension.

Analysis of Skin Regeneration and Barrier-Improvement Efficacy of Polydeoxyribonucleotide Isolated from Panax Ginseng (C.A. Mey.) Adventitious Root.

Polydeoxyribonucleotide (PDRN) has the ability to regenerate skin cells and improve the skin barrier and wound healing. This study investigated the possibility of replacing animal-derived PDRN with plant-derived PDRN. To test this, the adventitious roots of Korean ginseng (Panax ginseng C.A. Meyer), which is commonly used to treat various diseases, were suspension-cultivated through tissue culture; subsequently, PDRN was purified using microfluidization, an ultra-high-pressure physical grinding method. The results showed that purified Panax PDRN was effective in healing skin wounds and enhancing the skin barrier. Panax PDRN promoted the proliferation of keratinocytes and fibroblasts by increasing the expression of fibronectin, filaggrin, Ki-67, Bcl-2, inhibin beta A, and Cyclin D1. It also acted as an agonist of the adenosine A2A receptor and induced the phosphorylation of focal adhesion kinase, adenosine triphosphate-dependent tyrosine kinase, and mitogen-activated protein kinase. This activated signal transduction, thereby regenerating skin cells and strengthening the barrier. These results were not only observed in skin cells but also in an artificial skin model (KeraSkinTM). The use of plant-derived PDRN instead of animal-derived PDRN can promote animal welfare and environmental sustainability. Furthermore, Panax PDRN can potentially be a new plant-derived PDRN (PhytoPDRN) that may be utilized in the treatment of various skin diseases.

The RNA-binding protein CSDE1 promotes hematopoietic stem and progenitor cell generation via translational control of Wnt signaling.

In vertebrates, the earliest hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of specialized endothelial cells, hemogenic endothelial cells, in the aorta-gonad-mesonephros region through endothelial-to-hematopoietic transition. HSPC generation is efficiently and accurately regulated by a variety of factors and signals; however, the precise control of these signals remains incompletely understood. Post-transcriptional regulation is crucial for gene expression, as the transcripts are usually bound by RNA-binding proteins (RBPs) to regulate RNA metabolism. Here, we report that the RBP protein Csde1-mediated translational control is essential for HSPC generation during zebrafish early development. Genetic mutants and morphants demonstrated that depletion of csde1 impaired HSPC production in zebrafish embryos. Mechanistically, Csde1 regulates HSPC generation through modulating Wnt/β-catenin signaling activity. We demonstrate that Csde1 binds to ctnnb1 mRNAs (encoding β-catenin, an effector of Wnt signaling) and regulates translation but not stability of ctnnb1 mRNA, which further enhances β-catenin protein level and Wnt signal transduction activities. Together, we identify Csde1 as an important post-transcriptional regulator and provide new insights into how Wnt/β-catenin signaling is precisely regulated at the post-transcriptional level.

Transcriptome Analysis of Peritoneal Cells Reveals the Early Immune Response of Flounder (Paralichthys olivaceus) to Inactivated Vibrio anguillarum Immunization.

Vibrio anguillarum (V. anguillarum) is a bacterium that seriously harms flounder and other aquaculture species. Vaccination is an effective means of preventing vibriosis and is mainly administered by intraperitoneal injection. Effective antigen processing at the initial stage of immunization is essential to elicit adaptive immune responses and improve vaccine efficacy. To understand the early immune response of flounder caused by inactivated V. anguillarum, we detected the transcriptome profiles of the cells in the peritoneal cavity (PoPerCs) after inactivated V. anguillarum immunization. More than 10 billion high-quality reads were obtained, of which about 89.33% were successfully mapped to the reference genome of flounder. A total of 1985, 3072, 4001, and 5476 differentially expressed genes were captured at 6, 12, 24, and 48 h post immunization, respectively. The hub module correlated with the immunization time was identified by WGCNA. GO and KEGG analysis showed that hub module genes were abundantly expressed in various immune-related aspects, including the response to stimuli, the immune system process, signal transducer activity, autophagy, the NOD-like receptor signaling pathway, the toll-like receptor signaling pathway, the T cell receptor signaling pathway, and Th17 cell differentiation. Additionally, genes related to Th cell differentiation are presented as heatmaps. These genes constitute a complex immune regulatory network, mainly involved in pathogen recognition, antigen processing and presentation, and Th cell differentiation. The results of this study provide the first transcriptome profile of PoPerCs associated with inactivated V. anguillarum immunity and lay a solid foundation for further studies on effective V. anguillarum vaccines.

Phytochemical Investigation of Equisetum arvense and Evaluation of Their Anti-Inflammatory Potential in TNFα/INFγ-Stimulated Keratinocytes.

Equisetum arvense L. (Equisetaceae), widely known as 'horsetail', is a perennial plant found extensively across Asia. Extracts of E. arvense have been used in traditional medicine, particularly for the treatment of inflammatory disorders. This study aimed to determine the phytochemical compounds in E. arvense ethanolic extract and their anti-inflammatory properties. Subsequently, we isolated and identified nine secondary metabolites, including kaempferol 3,7-di-O-β-D-glucopyranoside (1), icariside B2 (2), (Z)-3-hexenyl β-D-glucopyranoside (3), luteolin 5-O-β-D-glucopyranoside (4), 4-O-β-D-glucopyranosyl caffeic acid (5), clemastanin B (6), 4-O-caffeoylshikimic acid (7), (7S,8S)-threo-7,9,9'-trihydroxy-3,3'-dimethoxy-8-O-4'-neolignan-4-O-β-D-glucopyranoside (8), and 3-O-caffeoylshikimic acid (9). The chemical structures of the isolated compounds (1-9) were elucidated using HR-ESI-MS data, NMR spectra, and ECD data. Next, the anti-inflammatory effects of the isolates were evaluated in tumor necrosis factor (TNF)α/interferon (IFN)γ-induced HaCaT, a human keratinocyte cell line. Among the isolates, compound 3 showed the highest inhibitory effect on the expression of pro-inflammatory chemokines, followed by compounds 6 and 8. Correspondingly, the preceding isolates inhibited TNFα/IFNγ-induced activation of pro-inflammatory transcription factors, signal transducer and activator of transcription 1, and nuclear factor-κB. Collectively, E. arvense could be employed for the development of prophylactic or therapeutic agents for improving dermatitis.

Integrative Proteomic Analysis Reveals the Cytoskeleton Regulation and Mitophagy Difference Between Ischemic Cardiomyopathy and Dilated Cardiomyopathy

Ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM) are the two primary etiologies of end-stage heart failure. However, there remains a dearth of comprehensive understanding the global perspective and the dynamics of the proteome and phosphoproteome in ICM and DCM, which hinders the profound comprehension of pivotal biological characteristics as well as differences in signal transduction activation mechanisms between these two major types of heart failure. We conducted high-throughput quantification proteomics and phosphoproteomics analysis of clinical heart tissues with ICM or DCM, which provided us the system-wide molecular insights into pathogenesis of clinical heart failure in both ICM and DCM. Both protein and phosphorylation expression levels exhibit distinct separation between heart failure and normal control heart tissues, highlighting the prominent characteristics of ICM and DCM. By integrating with omics results, Western blots, phosphosite-specific mutation, chemical intervention, and immunofluorescence validation, we found a significant activation of the PRKACA–GSK3β signaling pathway in ICM. This signaling pathway influenced remolding of the microtubule network and regulated the critical actin filaments in cardiac construction. Additionally, DCM exhibited significantly elevated mitochondria energy supply injury compared to ICM, which induced the ROCK1–vimentin signaling pathway activation and promoted mitophagy. Our study not only delineated the major distinguishing features between ICM and DCM but also revealed the crucial discrepancy in the mechanisms between ICM and DCM. This study facilitates a more profound comprehension of pathophysiologic heterogeneity between ICM and DCM and provides a novel perspective to assist in the discovery of potential therapeutic targets for different types of heart failure.

Ube2L6 Promotes M1 Macrophage Polarization in High-Fat Diet-Fed Obese Mice via ISGylation of STAT1 to Trigger STAT1 Activation

Introduction: In obesity-related type 2 diabetes mellitus (T2DM), M1 macrophages aggravate chronic inflammation and insulin resistance. ISG15-conjugation enzyme E2L6 (Ube2L6) has been demonstrated as a promoter of obesity and insulin resistance. This study investigated the function and mechanism of Ube2L6 in M1 macrophage polarization in obesity. Methods: Obesity was induced in Ube2L6^AKO mice and age-matched Ube2L6^flox/flox control mice by high-fat diet (HFD). Stromal vascular cells were isolated from the epididymal white adipose tissue of mice. Polarization induction was performed in mouse bone marrow-derived macrophages (BMDMs) by exposure to IFN-γ, lipopolysaccharide, or IL-4. F4/80 expression was assessed by immunohistochemistry staining. Expressions of M1/M2 macrophage markers and target molecules were determined by flow cytometry, RT-qPCR, and Western blotting, respectively. Protein interaction was validated by co-immunoprecipitation (Co-IP) assay. The release of TNF-α and IL-10 was detected by ELISA. Results: The polarization of pro-inflammatory M1 macrophages together with an increase in macrophage infiltration was observed in HFD-fed mice, which could be restrained by Ube2L6 knockdown. Additionally, Ube2L6 deficiency triggered the repolarization of BMDMs from M1 to M2 phenotypes. Mechanistically, Ube2L6 promoted the expression and activation of signal transducer and activator of transcription 1 (STAT1) through interferon-stimulated gene 15 (ISG15)-mediated ISGlylation, resulting in M1 macrophage polarization. Conclusion: Ube2L6 exerts as an activator of STAT1 via post-translational modification of STAT1 by ISG15, thereby triggering M1 macrophage polarization in HFD-fed obese mice. Overall, targeting Ube2L6 may represent an effective therapeutic strategy for ameliorating obesity-related T2DM.

Complement receptor 3-mediated neurotoxic glial activation contributes to rotenone-induced cognitive decline in mice

Microglia-mediated chronic neuroinflammation has been associated with cognitive decline induced by rotenone, a well-known neurotoxic pesticide used in agriculture. However, the mechanisms remain unclear. This work aimed to elucidate the role of complement receptor 3 (CR3), a highly expressed receptor in microglia, in cognitive deficits induced by rotenone. Rotenone up-regulated the expression of CR3 in the hippocampus and cortex area of mice. CR3 deficiency markedly ameliorated rotenone-induced cognitive impairments, neurodegeneration and phosphorylation (Ser129) of α-synuclein in mice. CR3 deficiency also attenuated rotenone-stimulated microglial M1 activation. In microglial cells, siRNA-mediated knockdown of CR3 impeded, while CR3 activation induced by LL-37 exacerbated, rotenone-induced microglial M1 activation. Mechanistically, CR3 deficiency blocked rotenone-induced activation of nuclear factor κB (NF-κB), signal transducer and activator of transcription 1 (STAT1) and STAT3 signaling pathways. Pharmacological inhibition of NF-κB or STAT3 but not STAT1 was confirmed to suppress microglial M1 activation elicited by rotenone. Further study revealed that CR3 deficiency or knockdown also reduced rotenone-induced expression of C3, an A1 astrocyte marker, and production of microglial C1q, TNFα and IL-1α, a cocktail for activated microglia to induce neurotoxic A1 astrocytes, via NF-κB and STAT3 pathways. Finally, a small molecule modulator of CR3 efficiently mitigated rotenone-elicited cognitive deficits in mice even administered after the establishment of cognitive dysfunction. Taken together, our findings demonstrated that CR3 is a key factor in mediating neurotoxic glial activation and subsequent cognitive impairments in rotenone-treated mice, giving novel insights into the immunopathogenesis of cognitive impairments in pesticide-related Parkinsonism.

Methoxyhispolon Methyl Ether, a Hispolon Analog, Thwarts the SRC/STAT3/BCL-2 Axis to Provoke Human Triple-Negative Breast Cancer Cell Apoptosis In Vitro.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with few treatment options. A promising TNBC treatment approach is targeting the oncogenic signaling pathways pivotal to TNBC initiation and progression. Deregulated activation of signal transducer and activator of transcription 3 (STAT3) is fundamental to driving TNBC malignant transformation, highlighting STAT3 as a promising TNBC therapeutic target. Methoxyhispolon Methyl Ether (MHME) is an analog of Hispolon, an anti-cancer polyphenol found in the medicinal mushroom Phellinus linteus. Still, MHME's anti-cancer effects and mechanisms remain unknown. Herein, we present the first report about MHME's anti-TNBC effect and its action mechanism. We first revealed that MHME is proapoptotic and cytotoxic against human TNBC cell lines HS578T, MDA-MB-231, and MDA-MB-463 and displayed a more potent cytotoxicity than Hispolon's. Mechanistically, MHME suppressed both constitutive and interleukin 6 (IL-6)-induced activation of STAT3 represented by the extent of tyrosine 705-phosphorylated STAT3 (p-STAT3). Notably, MHME-evoked apoptosis and clonogenicity impairment were abrogated in TNBC cells overexpressing a dominant-active mutant of STAT3 (STAT3-C); supporting the blockade of STAT3 activation is an integral mechanism of MHME's cytotoxic action on TNBC cells. Moreover, MHME downregulated BCL-2 in a STAT3-dependent manner, and TNBC cells overexpressing BCL-2 were refractory to MHME-induced apoptosis, indicating that BCL-2 downregulation is responsible for MHME's proapoptotic effect on TNBC cells. Finally, MHME suppressed SRC activation, while v-src overexpression rescued p-STAT3 levels and downregulated apoptosis in MHME-treated TNBC cells. Collectively, we conclude that MHME provokes TNBC cell apoptosis through the blockade of the SRC/STAT3/BCL-2 pro-survival axis. Our findings suggest the potential of applying MHME as a TNBC chemotherapy agent.

Suppression of IRF9 Promotes Osteoclast Differentiation by Decreased Ferroptosis via STAT3 Activation.

Osteoporosis is a chronic disease that endangers the health of the elderly. Inhibiting osteoclast hyperactivity is a key aspect of osteoporosis prevention and treatment. Several studies have shown that interferon regulatory factor 9 (IRF9) not only regulates innate and adaptive immune responses but also plays an important role in inflammation, antiviral response, and cell development. However, the exact role of IRF9 in osteoclasts has not been reported. To elucidate the role of IRF9 in osteoclast differentiation, we established the ovariectomized mouse model of postmenopausal osteoporosis and found that IRF9 expression was reduced in ovariectomized mice with overactive osteoclasts. Furthermore, knockdown of IRF9 expression enhanced osteoclast differentiation in vitro. Using RNA sequencing, we identified that the differentially expressed genes enriched by IRF9 knockdown were related to ferroptosis. We observed that IRF9 knockdown promoted osteoclast differentiation via decreased ferroptosis in vitro and further verified that IRF9 knockdown reduced ferroptosis by activating signal transducer and activator of transcription 3 (STAT3) to promote osteoclastogenesis. In conclusion, we identified an essential role of IRF9 in the regulation of osteoclastogenesis in osteoporosis and its underlying mechanism.

FRI286 Stimulation Of Egr1 Expression Is Mediated By A Gαq/11-signaling Pathway In A Pattern Distinct From That Of MEK/ERK At High And Low GnRH Pulse Frequencies

Abstract Disclosure: G.A. Stamatiades: None. H.K. Kim: None. R.S. Carroll: None. U.B. Kaiser: None. Introduction: The pulsatile release of the hypothalamic decapeptide, GnRH, activates signal transduction cascades in pituitary gonadotropes to control the synthesis and secretion of LH and FSH, hormones critical for normal reproductive function and fertility. LH and FSH release are differentially regulated by varying GnRH pulse patterns, with LH secretion being preferentially stimulated at high (e.g., every 30 min) GnRH pulse frequencies, while FSH secretion is preferentially induced at low (e.g., every 2 hour) GnRH pulses. LH is a heterodimer comprised of a common α subunit and a distinct LHβ subunit, with Lhb gene (encoding LHβ) transcription being preferentially stimulated at the higher GnRH pulse frequencies. It is well established that Egr1 is a key inducer of Lhb expression upon pulsatile GnRH stimulation. In addition, it has been shown that MEKI/II inhibition abrogates the induction of Lhb mRNA levels at both high and low GnRH pulse frequencies. Hypothesis:Egr1 expression is mediated by a Gαq/11-signaling pathway that includes MEK/ERK at both high and low GnRH pulse frequencies. Methods: To determine whether a Gαq/11-mediated pathway regulates Egr1 expression upon GnRH stimulation, LβT2 cells transduced with lentiviruses encoding scrambled or Gnaq/11 shRNA were perifused and treated with pulsatile GnRH at either high (q30min) or low (q2h) pulse frequencies, or with medium only, for 20 hours. Following the same perifusion paradigm, ERK1/2 phosphorylation was also assessed. Results: LβT2 cells transduced with a scrambled control shRNA showed a GnRH pulse frequency-dependent pattern of induction of Egr1 expression, with significantly greater induction at high than at low GnRH pulse frequency, compared to cells perifused with media only. In addition, Gαq/11 depletion reduced Egr1 mRNA levels at both high and low GnRH pulse frequencies, but the frequency dependence was maintained. A GnRH pulse frequency-dependent pattern of induction of ERK1/2 phosphorylation was observed, with significantly greater induction at low than at high GnRH pulse frequency in control cells transduced with scrambled control shRNA, consistent with prior studies from our group. Following Gnaq/11 KD, the induction of ERK1/2 phosphorylation was reduced at both GnRH pulse frequencies, with loss of the pulse frequency dependence. Conclusion: Taken together, these findings suggest that a Gαq/11-signaling pathway involving Egr1 induces Lhb expression at both pulse frequencies. However, the link between MEK/ERK and Egr1 needs to be further investigated, given the differences in the GnRH pulse frequency dependent patterns of induction. Presentation: Friday, June 16, 2023

TCR ligand potency differentially impacts PD-1 inhibitory effects on diverse signaling pathways.

Checkpoint blockade revolutionized cancer therapy, but we still lack a quantitative, mechanistic understanding of how inhibitory receptors affect diverse signaling pathways. To address this issue, we developed and applied a fluorescent intracellular live multiplex signal transduction activity reporter (FILMSTAR) system to analyze PD-1-induced suppressive effects. These studies identified pathways triggered solely by TCR or requiring both TCR and CD28 inputs. Using presenting cells differing in PD-L1 and CD80 expression while displaying TCR ligands of distinct potency, we found that PD-1-mediated inhibition primarily targets TCR-linked signals in a manner highly sensitive to peptide ligand quality. These findings help resolve discrepancies in existing data about the site(s) of PD-1 inhibition in T cells while emphasizing the importance of neoantigen potency in controlling the effects of checkpoint therapy.

Honokiol prevents lung metastasis of triple-negative breast cancer by regulating polarization and recruitment of macrophages

Metastasis is the leading cause of breast cancer-associated death. Lung metastasis commonly occurs in triple-negative breast cancer (TNBC) metastasis, worsening the TNBC prognosis. Considering their role in tumor progression and metastasis, tumor-associated macrophages (TAMs) are essential therapeutic targets in cancer therapy. Previous studies have demonstrated that honokiol inhibits tumor growth and progression. Here we assessed how honokiol inhibits lung metastasis of TNBC by regulating the polarization of macrophages. We found that honokiol decreased the expression of IL-13-triggered M2 markers like CD206, Arg1, and CCL2, preventing the invasion and migration ability of TNBC cells. The activation of signal transducer and activator of transcription STAT6 and STAT3 was significantly suppressed by honokiol in M2 polarized macrophages. Meanwhile, honokiol increased the expression of LPS/IFNγ-induced M1 markers such as CD11c, iNOS, and IL12 by promoting STAT1 phosphorylation. Besides, honokiol decreased both the ratio of M2/M1 macrophages and the expression of the IL-10/IL-12 gene in lung tissues, thereby inhibiting the proliferation and metastasis of murine breast cancer. Moreover, honokiol reduced the infiltration of macrophages to the lung tissue through the CCL2/CCR2 pathways. These results highlight the potential of honokiol in suppressing TNBC tumor progression and lung metastasis by regulating the polarization and recruitment of macrophages.

Myeloid cell deficiency of the inflammatory transcription factor Stat4 protects long-term synaptic plasticity from the effects of a high-fat, high-cholesterol diet

Neuroinflammation is associated with neurodegenerative diseases, including Alzheimer’s and Parkinson’s. The cytokine interleukin-12 activates signal transducer and activator of transcription 4 (Stat4), and consumption of a high-fat, high-cholesterol diet (HFD-C) and Stat4 activity are associated with inflammation, atherosclerosis, and a diabetic metabolic phenotype. In studies of in vitro hippocampal slices from control Stat4fl/flLdlr−/− mice fed a HFD-C diabetogenic diet, we show that Schaffer collateral-CA1 synapses exhibited larger reductions in activity-dependent, long-term potentiation (LTP) of synaptic transmission, compared to mice fed a standard diet. Glucose tolerance and insulin sensitivity shifts produced by HFD-C diet were reduced in Stat4ΔLysMLdlr−/− mice compared to Stat4fl/flLdlr−/− controls. Stat4ΔLysMLdlr−/− mice, which lack Stat4 under control of the LysMCre promoter, were resistant to HFD-C induced impairments in LTP. In contrast, Schaffer collateral-CA1 synapses in Stat4ΔLysMLdlr−/− mice fed the HFD-C diet showed larger LTP than control Stat4fl/flLdlr−/− mice. Expression of a number of neuroinflammatory and synaptic plasticity genes was reduced by HFD-C diet in control mice, and less affected by HFD-C diet in Stat4ΔLysMLdlr−/− mice. These data suggest that suppression of Stat4 activation may protect against effects of Western diet on cognition, type 2 diabetes, and reduce risk of Alzheimer’s disease and other neurodegenerative disorders associated with neuroinflammation.

Ubiquitination and cell-autonomous immunity.

Cell-autonomous immunity is the first line of defense by which cells recognize and contribute to eliminating invasive pathogens. It is composed of immune signaling networks that sense microbial pathogens, promote pathogen restriction, and stimulate their elimination, including host cell death. Ubiquitination is a pivotal orchestrator of these pathways, by changing the activity of signal transducers and effector proteins in an efficient way. In this review, we will focus on how ubiquitin connects the pathways that sense pathogens to the cellular responses to invaders and shed light on how ubiquitination impacts the microenvironment around the infected cell, stimulating the appropriate immune response. Finally, we discuss therapeutic options directed at favoring cell-autonomous immune responses to infection.

Combined transcriptomic and metabolomic analysis of alginate oligosaccharides alleviating salt stress in rice seedlings

BackgroundSalt stress is one of the key factors limiting rice production. Alginate oligosaccharides (AOS) enhance plant stress resistance. However, the molecular mechanism underlying salt tolerance in rice induced by AOS remains unclear. FL478, which is a salt-tolerant indica recombinant inbred line and IR29, a salt-sensitive rice cultivar, were used to comprehensively analyze the effects of AOS sprayed on leaves in terms of transcriptomic and metabolite profiles of rice seedlings under salt stress.ResultsIn this experiment, exogenous application of AOS increased SOD, CAT and APX activities, as well as GSH and ASA levels to reduce the damage to leaf membrane, increased rice stem diameter, the number of root tips, aboveground and subterranean biomass, and improved rice salt tolerance. Comparative transcriptomic analyses showed that the regulation of AOS combined with salt treatment induced the differential expression of 305 and 1030 genes in FL478 and IR29. The expressed genes enriched in KEGG pathway analysis were associated with antioxidant levels, photosynthesis, cell wall synthesis, and signal transduction. The genes associated with light-trapping proteins and RLCK receptor cytoplasmic kinases, including CBA, LHCB, and Lhcp genes, were fregulated in response to salt stress. Treatment with AOS combined with salt induced the differential expression of 22 and 50 metabolites in FL478 and IR29. These metabolites were mainly related to the metabolism of amino and nucleotide sugars, tryptophan, histidine, and β -alanine. The abundance of metabolites associated with antioxidant activity, such as 6-hydroxymelatonin, wedelolactone and L-histidine increased significantly. Combined transcriptomic and metabolomic analyses revealed that dehydroascorbic acid in the glutathione and ascorbic acid cycles plays a vital role in salt tolerance mediated by AOS.ConclusionAOS activate signal transduction, regulate photosynthesis, cell wall formation, and multiple antioxidant pathways in response to salt stress. This study provides a molecular basis for the alleviation of salt stress-induced damage by AOS in rice.