Downstream Activation Research Articles

Broad-spectrum inflammasome inhibition by thiomuscimol.

Inflammasome formation, arising from pathogen or internal activating signals, is a key step in canonical pyroptosis, a gasdermin-mediated inflammatory cell death. Inhibition of pyroptosis has great clinical relevance due to its involvement in many different disease states. Current inhibitors of pyroptosis either only inhibit the final lytic step, which still allows inflammatory signal release, or only inhibit a single inflammasome, which does not account for inherent redundancy in activation of other inflammatory pathways. Here, we show that thiomuscimol, a structural analog of the lysis inhibitor muscimol, exhibits unique inhibitory activity upstream of plasma membrane rupture. We find that thiomuscimol inhibits inflammasome formation, as well as downstream caspase-1 activation, initiated by multiple pyroptotic signals, regardless of whether NLR recruitment of caspase-1 to the inflammasome relies on the ASC adapter protein. The ability of thiomuscimol to block multiple different inflammasomes opens the door for development of therapeutics with increased applications to broadly inhibit pyroptosis in multiple pathological settings.

Porcine reproductive and respiratory syndrome virus degrades TANK-binding kinase 1 via chaperon-mediated autophagy to suppress type I interferon production and facilitate viral proliferation.

Porcine reproductive and respiratory syndrome virus (PRRSV) has led to significant economic losses in the global swine industry. Type I interferon (IFN-I) plays a crucial role in the host's resistance to PRRSV infection. Despite extensive research showing that PRRSV employs multiple strategies to antagonise IFN-I induction, the underlying mechanisms remain to be fully elucidated. In this study, we have discovered that PRRSV inhibits the production of IFN-I by degrading TANK-binding kinase 1 (TBK1) through chaperon-mediated autophagy (CMA). From a mechanistic standpoint, PRRSV nonstructural protein 2 (Nsp2) increases the interaction between the heat shock protein member 8 (HSPA8) and TBK1. This interaction leads to the translocation of TBK1 into lysosomes for degradation, mediated by lysosomal-associated membrane protein 2A (LAMP2A). As a result, the downstream activation of IFN regulatory factor 3 (IRF3) and the production of IFN-I are hindered. Together, these results reveal a new mechanism by which PRRSV suppresses host innate immunity and contribute to the development of new antiviral strategies against the virus.

Abstract 4136316: BMP10 as novel marker for right atrial dilatation and pressure in precapillary pulmonary hypertension

Introduction: Precapillary pulmonary hypertension (precPH) leads to increased right atrial (RA) stretch and pressure. The right atrium is the major source of bone morphogenetic protein 10 (BMP10) in adults. Aim: This study aims to investigate BMP10 in relation to RA dilatation and pressure overload. Methods: We studied BMP10 mRNA in fresh RA tissue of N=5 Chronic Thromboembolic Pulmonary Hypertension (CTEPH) patients and N=9 controls and protein expression in paraffin-embedded RA tissue of N=4 Pulmonary Arterial Hypertension (PAH) patients and N=6 controls. We prospectively included 48 precPH patients (N=22 idiopathic PAH, N=14 hereditary PAH and N=12 CTEPH, and N=16 controls. To study BMP10-induced transcriptional activity, we assayed serum samples in human microvascular endothelial cells (HMEC) with a BMP-responsive transcriptional luciferase assay. BMP10 activity was calculated by subtracting BMP activity after incubation with antibodies for Alk1Fc and bone morphogenetic protein 9 (BMP9). We assessed correlations between BMP10 activity and RA dilatation (RA max volume>79 ml/m 2 for male or >69 ml/m 2 for female) and right ventricular (RV) dilatation (RV end-diastolic volume>108 ml/m 2 for male and >96 ml/m 2 for female). In a cohort of CTEPH patients that underwent pulmonary endarterectomy (PEA) we assessed the effect of pressure unloading on BMP10 activity. Normality of data was checked and statistical differences between groups were tested using an independent sample t-test or Wilcoxon rank-sum test. Results: BMP10 mRNA, protein and downstream activity were higher in the precPH RA tissue (Figure 1A-F). High systemic BMP10 activity was associated with RA dilatation and high RA pressure, but not RV dilatation in precPH (Figure 2A-C). Finally, pressure unloading after PEA in CTEPH patients results in a reduction in BMP10 activity (figure 2D). Conclusions: Local RA BMP10 expression and activity is increased in precPH. Increased systemic BMP10 activity was related to RA dilatation and pressure. Pressure unloading of the right heart leads to a reduction of systemic BMP10 activity. Future studies are needed to determine whether increased BMP10 release is an adaptative mechanism or can be used as therapeutic target.

The PI3K-AKT-mTOR axis persists as a therapeutic dependency in KRASG12D-driven non-small cell lung cancer.

KRASG12C and KRASG12D inhibitors represent a major translational breakthrough for non-small cell lung cancer (NSCLC) and cancer in general by directly targeting its most mutated oncoprotein. However, resistance to these small molecules has highlighted the need for rational combination partners necessitating a critical understanding of signaling downstream of KRAS mutant isoforms. We contrasted tumor development between KrasG12C and KrasG12D genetically engineered mouse models (GEMMs). To corroborate findings and determine mutant subtype-specific dependencies, isogenic models of KrasG12C and KrasG12D initiation and adaptation were profiled by RNA sequencing. We also employed cell line models of established KRAS mutant NSCLC and determined therapeutic vulnerabilities through pharmacological inhibition. We analysed differences in survival outcomes for patients affected by advanced KRASG12C or KRASG12D-mutant NSCLC. KRASG12D exhibited higher potency in vivo, manifesting as more rapid lung tumor formation and reduced survival of KRASG12D GEMMs compared to KRASG12C. This increased potency, recapitulated in an isogenic initiation model, was associated with enhanced PI3K-AKT-mTOR signaling. However, KRASG12C oncogenicity and downstream pathway activation were comparable with KRASG12D at later stages of tumorigenesis in vitro and in vivo, consistent with similar clinical outcomes in patients. Despite this, established KRASG12D NSCLC models depended more on the PI3K-AKT-mTOR pathway, while KRASG12C models on the MAPK pathway. Specifically, KRASG12D inhibition was enhanced by AKT inhibition in vitro and in vivo. Our data highlight a unique combination treatment vulnerability and suggest that patient selection strategies for combination approaches using direct KRAS inhibitors should be i) contextualised to individual RAS mutants, and ii) tailored to their downstream signaling.

Dengue Virus Infection: Immune Response and Therapeutic Targets.

Flavivirus infection, especially dengue virus infection caused by DENV, is known to be a significant health concern globally owing to the high incidence and mortality rate. The expanding and increasing disease burden calls for the need to develop an effective treatment and prevent the event of fatal complications, including dengue hemorrhagic fever/dengue shock syndrome. The DENV-induced immune response has been described as paradoxical because it has a protective role in viral clearance but, at the same time, causes more severe infection through viral-specific immunity. This is further complicated by high homology and cross-reactivity between different serotypes of DENV, causing a more severe disease presentation during secondary infection by a heterologous serotype. This serotype complexity poses a challenge for the development of a universal flavivirus vaccine. This review highlights the significance of high motility group box 1 (HMGB1) and nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) inflammasome activation pathways in initiating an inflammatory response through the downstream activation of nuclear factor κB and proinflammatory cytokine Interleukin (IL)-1B, IL-18 release in DENV infection. It also discusses the role of NLRP3 in activating cellular apoptosis and pyroptosis leading to systemic failure, especially in peripheral tissues. Over the decades, there has been much progress in understanding the immunopathogenesis of DENV infection. Researchers have been studying key pathogenic molecules for potential therapeutic targets including HMGB1 and NLRP3 inflammasome inhibitors, which is explored in this review. Ultimately, although there is not yet an effective antiviral or vaccine for DENV, immunomodulators continue to pave the way to decrease disease severity in infected individuals.

O-GlcNAcylation of RPA2 at S4/S8 antagonizes phosphorylation and regulates checkpoint activation during replication stress

O-linked N-acetylglucosamine (O-GlcNAc) is the most abundant mono-saccharide modification occurring in the cytoplasm, nucleus and mitochondria. Recent advent of the mass spectrometry technology has enabled identification of abundant O-GlcNAc transferase (OGT) substrates in diverse biological processes, such as cell cycle progression, replication and DNA damage response. Herein we report the O-GlcNAcylation of Replication Protein A2 (RPA2), a component of the heterotrimeric RPA complex pivotal for DNA metabolism. We found that RPA2 interacts with OGT, and a topoisomerase II inhibitor, etoposide, diminishes the association. Using higher-energy collisional dissociation mass spectrometry, we mapped RPA2 O-GlcNAc sites to be Ser-4/Ser-8, which are well-known PIKK-dependent RPA2 phosphorylation sites involved in checkpoint activation upon replication stress. We further demonstrated that Ser-4/Ser-8 O-GlcNAcylation antagonizes phosphorylation and impairs downstream Chk1 activation. Moreover, RPA2 O-GlcNAcylation sustains H2AX phosphorylation upon etoposide treatment, and promotes inappropriate cell cycle progression, indicative of checkpoint defects. Our work not only unveils a new OGT substrate, but also underscores the distinct roles of OGT in replication versus replication stress.

Growth hormone and bone: a basic perspective.

Growth hormone is fundamental for growth during childhood and for maintaining bone mass and homeostasis in the adults. GH deficiency causes decreased bone growth and osteopenia, whereas GH excess causes increased bone fragility and decreased bone quality. In the past, it was common knowledge that GH effects on the skeletal system were due to the production of IGF1 from the liver, which has a huge bone anabolic effect per se. However, with the progress of basic research techniques new light has been shed on the mechanisms underlying GH effect in bone, and it is now clear that GH has effects that go beyond the downstream activation of liver IGFs. Therefore, the purpose of this review is to summarize the milestones in basic research that led to the discovery of GH local activity on bone.

CD24 regulates liver immune response and ameliorates acute hepatic injury through controlling hepatic macrophages.

Liver injury releases danger-associated molecular patterns, which trigger the immune response. CD24 negatively regulates the immune response by binding with danger-associated molecular patterns, but the specific role of CD24 in modulating macrophage-related inflammation during liver injury remains largely unexplored. Here, we aimed to investigate the mechanisms of macrophage CD24 in the development of liver injury. Our results show that CD24 expression is upregulated primarily in hepatic macrophages (HMs) during acute liver injury. CD24-deficient mice exhibited more severe liver injury and showed a significantly higher frequency and number of HMs, particularly Ly6Chi monocyte-derived macrophages. Mechanistically, the CD24-Siglec-G interaction plays a vital role in mitigating acute liver injury. CD24-mediated inhibitory signaling in HMs primarily limits downstream NF-κB and p38 MAPK activation through the recruitment of SHP1. Our work unveils the critical role of macrophage CD24 in negatively regulating innate immune responses and protecting against acute liver injury, thus providing potential therapeutic targets for liver-associated diseases.

Leveraging Covalency to Stabilize Ternary Complex Formation For Cell-Cell "Induced Proximity".

Recent advances in the field of translational chemical biology use diverse "proximity-inducing" synthetic modalities to elicit new modes of "event driven" pharmacology. These include mechanisms of targeted protein degradation and immune clearance of pathogenic cells. Heterobifunctional "chimeric" compounds like Proteolysis TArgeting Chimeras (PROTACs) and Antibody Recruiting Molecules (ARMs) leverage these mechanisms, respectively. Both systems function through the formation of reversible "ternary" or higher-order biomolecular complexes. Critical to function are key parameters, such as bifunctional molecule affinity for endogenous proteins, target residence time, and turnover. To probe the mechanism and enhance function, covalent chemical approaches have been developed to kinetically stabilize ternary complexes. These include electrophilic PROTACs and Covalent Immune Recruiters (CIRs), the latter designed to uniquely enforce cell-cell induced proximity. Inducing cell-cell proximity is associated with key challenges arising from a combination of steric and/or mechanical based destabilizing forces on the ternary complex. These factors can attenuate the formation of ternary complexes driven by high affinity bifunctional/proximity inducing molecules. This Account describes initial efforts in our lab to address these challenges using the CIR strategy in antibody recruitment or receptor engineered T cell model systems of cell-cell induced proximity. ARMs form ternary complexes with serum antibodies and surface protein antigens on tumor cells that subsequently engage immune cells via Fc receptors. Binding and clustering of Fc receptors trigger immune cell killing of the tumor cell. We applied the CIR strategy to convert ARMs to covalent chimeras, which "irreversibly" recruit serum antibodies to tumor cells. These covalent chimeras leverage electrophile preorganization and kinetic effective molarity to achieve fast and selective covalent engagement of the target ternary complex protein, e.g., serum antibody. Importantly, covalent engagement can proceed via diverse binding site amino acids beyond cysteine. Covalent chimeras demonstrated striking functional enhancements compared to noncovalent ARM analogs in functional immune assays. We revealed this enhancement was in fact due to the increased kinetic stability and not concentration, of ternary complexes. This finding was recapitulated using analogous CIR modalities that integrate peptidic or carbohydrate binding ligands with Sulfur(VI) Fluoride Exchange (SuFEx) electrophiles to induce cell-cell proximity. Mechanistic studies in a distinct model system that uses T cells engineered with receptors that recognize covalent chimeras or ARMs, revealed covalent receptor engagement uniquely enforces downstream activation signaling. Finally, this Account discusses potential challenges and future directions for adapting and optimizing covalent chimeric/bifunctional molecules for diverse applications in cell-cell induced proximity.

A new-disease-causing dominant-negative variant in CARD11 gene in a Chinese case with recurrent fever

Immunodeficiency 11B with atopic dermatitis (IMD11B, OMIM:617638) is rare primary immunodeficiency disease caused by germline dominant negative (DN) mutations in the CARD11 gene. Affected patients present with immune dysfunction, recurrent infections and atopic dermatitis. In this study, we sought to identify and characterize the genetic variant in one patient with periodic fever, recurrent infections, and eczema. Trio whole-exome sequencing (WES) was employed in this patient and her parents, and Sanger sequencing validated the potential pathogenic variant. In vitro functional study was performed to evaluate the pathogenicity of genetic variant identified. A very rare missense mutation (c.2324C > T, p.S775L) in CARD11 gene (NM_032415) was identified by WES in the patient but not her parents. Luciferase reporter assays and co-immunoprecipitation demonstrated mutation exerts a dominant-interfering effect on wild-type CARD11, inhibiting the activity of NF-κB. RNA sequencing analysis also confirmed that mutant CARD11 inhibited down-stream transcriptional activity of NF-κB. A review of literature doesn’t found significant genotype–phenotype correlation. We identified a vary rare DN CARD11 mutation, expanding the genetic and phenotypic spectrum of CARD11.

Delivery of a Jagged1-PEG-MAL hydrogel with pediatric human bone cells regenerates critically sized craniofacial bone defects

Current treatments for congenital and acquired craniofacial (CF) bone abnormalities are limited and costly. Conventional methods involve surgical correction, short-term stabilization, and long-term bone grafting, which may include problematic allografts and limited autografts. While bone morphogenetic protein 2 (BMP2) has been used for bone regeneration, it can cause bone overgrowth and life-threatening inflammation. Bone marrow-derived mesenchymal stem cell therapies, though promising, are not Food and Drug Administration approved and are resource intensive. Thus, there is a need for effective, affordable, and less side-effect-prone bone regenerative therapies. Previous research demonstrated that JAGGED1 induces osteoblast commitment in murine cranial neural crest cells through a NOTCH-dependent non-canonical pathway involving JAK2–STAT5. We hypothesize that delivery of JAGGED1 and induction of its downstream NOTCH non-canonical signaling in pediatric human osteoblasts constitutes an effective bone regenerative treatment. Delivering pediatric human bone-derived osteoblast-like cells to an in vivo murine bone loss model of a critically sized cranial defect, we identified that JAGGED1 promotes human pediatric osteoblast commitment and bone formation through p70 S6K phosphorylation. This approach highlights the potential of JAGGED1 and its downstream activators as innovative treatments for pediatric CF bone loss.

Delivery of a Jagged1-PEG-MAL hydrogel with pediatric human bone cells regenerates critically sized craniofacial bone defects.

Current treatments for congenital and acquired craniofacial (CF) bone abnormalities are limited and costly. Conventional methods involve surgical correction, short-term stabilization, and long-term bone grafting, which may include problematic allografts and limited autografts. While bone morphogenetic protein 2 (BMP2) has been used for bone regeneration, it can cause bone overgrowth and life-threatening inflammation. Bone marrow-derived mesenchymal stem cell therapies, though promising, are not Food and Drug Administration approved and are resource intensive. Thus, there is a need for effective, affordable, and less side-effect-prone bone regenerative therapies. Previous research demonstrated that JAGGED1 induces osteoblast commitment in murine cranial neural crest cells through a NOTCH-dependent non-canonical pathway involving JAK2-STAT5. We hypothesize that delivery of JAGGED1 and induction of its downstream NOTCH non-canonical signaling in pediatric human osteoblasts constitutes an effective bone regenerative treatment. Delivering pediatric human bone-derived osteoblast-like cells to an in vivo murine bone loss model of a critically sized cranial defect, we identified that JAGGED1 promotes human pediatric osteoblast commitment and bone formation through p70 S6K phosphorylation. This approach highlights the potential of JAGGED1 and its downstream activators as innovative treatments for pediatric CF bone loss.

β-Mangostin Alleviates Renal Tubulointerstitial Fibrosis via the TGF-β1/JNK Signaling Pathway.

The epithelial-to-mesenchymal transition (EMT) plays a key role in the pathogenesis of kidney fibrosis, and kidney fibrosis is associated with an adverse renal prognosis. Beta-mangostin (β-Mag) is a xanthone derivative obtained from mangosteens that is involved in the generation of antifibrotic and anti-oxidation effects. The purpose of this study was to examine the effects of β-Mag on renal tubulointerstitial fibrosis both in vivo and in vitro and the corresponding mechanisms involved. As shown through an in vivo study conducted on a unilateral ureteral obstruction mouse model, oral β-Mag administration, in a dose-dependent manner, caused a lesser degree of tubulointerstitial damage, diminished collagen I fiber deposition, and the depressed expression of fibrotic markers (collagen I, α-SMA) and EMT markers (N-cadherin, Vimentin, Snail, and Slug) in the UUO kidney tissues. The in vitro part of this research revealed that β-Mag, when co-treated with transforming growth factor-β1 (TGF-β1), decreased cell motility and downregulated the EMT (in relation to Vimentin, Snail, and N-cadherin) and phosphoryl-JNK1/2/Smad2/Smad3 expression. Furthermore, β-Mag co-treated with SB (Smad2/3 kinase inhibitor) or SP600125 (JNK kinase inhibitor) significantly inhibited the TGF-β1-associated downstream phosphorylation and activation of JNK1/2-mediated Smad2 targeting the Snail/Vimentin axis. To conclude, β-Mag protects against EMT and kidney fibrotic processes by mediating the TGF-β1/JNK/Smad2 targeting Snail-mediated Vimentin expression and may have therapeutic implications for renal tubulointerstitial fibrosis.

Inhibition of primary cilia-hedgehog signaling axis triggers autophagic cell death and suppresses malignant progression of VHL wild-type ccRCC

Primary cilia are present on renal tubules and are implicated to play a pivotal role in transducing signals during development; however, the oncogenic role of cilia in clear cell renal cell carcinoma (ccRCC) has not been examined. Here we show that VHL wild-type ccRCC cell lines have a high incidence of primary cilia, and a high frequency of primary cilia is positively correlated with VHL expression and poor prognosis. Besides, the depletion of KIF3A and IFT88, genes required for ciliogenesis, significantly inhibited tumor proliferation and metastasis in vitro and in vivo. Further analysis found that mutations of key genes in hedgehog signaling are enriched in VHL wild ccRCC, its downstream signaling activation depends on ciliogenesis. Moreover, depletion of primary cilia or suppression of hedgehog pathway activation with inhibitor-induced robust autophagic cell death. Collectively, our findings revealed that primary cilia could serve as a diagnostic tool and provide new insights into the mechanism of VHL wild-type ccRCC progression. Targeting the primary cilia-hedgehog pathway may represent an effective therapeutic strategy for VHL wild-type ccRCC.

Aromative Dephosphinidenation of a Bisphosphirane‐Fused Anthracene toward E−H (E=H, Si, N and P) Bond Activation

AbstractA bisphosphirane‐fused anthracene (5) was prepared by treatment of a sterically encumbered amino phosphorus dichloride (3) with MgA ⋅ THF3 (A=anthracene). X‐ray diffraction analysis revealed a pentacyclic framework consisting of 5 with two phosphirane rings fused to the anthracene in a trans‐fashion. Compound 5 has been shown to be an efficient phosphinidene synthon, readily liberating two transient phosphinidene units for subsequent downstream bond activation via the reductive elimination of anthracene under mild conditions. The formal oxidative addition of H2 and E−H (E=Si, N, P) bonds by the liberated phosphinidene provided diphosphine and substituted phosphines. Furthermore, phosphinidene transfer to alkenes and alkynes smoothly yielded the corresponding phosphiranes and phosphirenes. The mechanism of the H2 activation by 5 was investigated by density functional theory (DFT) calculations.

HHIP's Dynamic Role in Epithelial Wound Healing Reveals a Potential Mechanism of COPD Susceptibility.

A genetic variant near HHIP has been consistently identified as associated with increased risk for Chronic Obstructive Pulmonary Disease (COPD), the third leading cause of death worldwide. However HHIP's role in COPD pathogenesis remains elusive. Canonically, HHIP is a negative regulator of the hedgehog pathway and downstream GLI1 and GLI2 activation. The hedgehog pathway plays an important role in wound healing, specifically in activating transcription factors that drive the epithelial mesenchymal transition (EMT), which in its intermediate state (partial EMT) is necessary for the collective movement of cells closing the wound. Herein, we propose a mechanism to explain HHIP's role in faulty epithelial wound healing, which could contribute to the development of emphysema, a key feature of COPD. Using two different Boolean models compiled from the literature, we show dysfunctional HHIP results in a lack of negative feedback on GLI, triggering a full EMT, where cells become mesenchymal and do not properly close the wound. We validate these Boolean models with experimental evidence gathered from published scientific literature. We also experimentally test if low HHIP expression is associated with EMT at the edge of wounds by using a scratch assay in a human lung epithelial cell line. Finally, we show evidence supporting our hypothesis in bulk and single cell RNA-Seq data from different COPD cohorts. Overall, our analyses suggest that aberrant wound healing due to dysfunctional HHIP, combined with chronic epithelial damage through cigarette smoke exposure, may be a primary cause of COPD-associated emphysema.

UV-induced reactive oxygen species and transcriptional control of 3-deoxyanthocyanidin biosynthesis in black sorghum pericarp.

Black pericarp sorghum has notable value due to the biosynthesis of 3-deoxyanthocyanidins (3-DOAs), a rare class of bioactive polyphenols valued as antioxidant food additives and as bioactive compounds with cytotoxicity to human cancer cells. A metabolic and transcriptomic study was conducted to ascertain the cellular events leading to the activation of 3-DOA biosynthesis in black sorghum pericarp. Prolonged exposure of pericarp during grain maturation to high-fluence ultraviolet (UV) light resulted in elevated levels of reactive oxygen species (ROS) and the activation of 3-DOA biosynthesis in pericarp tissues. In conjunction with 3-DOA biosynthesis was the transcriptional activation of specific family members of early and late flavonoid biosynthesis pathway genes as well as the downstream activation of defense-related pathways. Promoter analysis of genes highly correlated with 3-DOA biosynthesis in black pericarp were enriched in MYB and HHO5/ARR-B motifs. Light microscopy studies of black pericarp tissues suggest that 3-DOAs are predominantly localized in the epicarp and are associated with the cell wall. A working model of UV-induced 3-DOA biosynthesis in black pericarp is proposed that shares features of plant immunity associated with pathogen attack or mechanical wounding. The present model depicts ROS accumulation, the transcriptional activation of receptor kinases and transcription factors (TFs) including NAC, WRKY, bHLH, AP2, and C2H2 Zinc finger domain. This study identified key biosynthetic and regulatory genes of 3-DOA accumulation in black pericarp and provided a deeper understanding of the gene networks and cellular events controlling this tissue-and genotype-specific trait.

Combined inhibition of MET and VEGF enhances therapeutic efficacy of EGFR TKIs in EGFR-mutant non-small cell lung cancer with concomitant aberrant MET activation

Abstract Background Aberrant activation of mesenchymal epithelial transition (MET) has been considered to mediate primary and acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). However, mechanisms underlying this process are not wholly clear and the effective therapeutic strategy remains to be determined. Methods The gefitinib-resistant NSCLC cell lines were induced by concentration increase method in vitro. Western blot and qPCR were used to investigate the relationship between MET and vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway. Double luciferase reporter gene and co-immunoprecipitation were used to further reveal the regulation mechanism between MET and VEGF/VEGFR2. The effect of combined inhibition of MET and VEGF/VEGFR2 signaling pathway on the therapeutic sensitivity of EGFR-TKI in gefitinib resistant cell lines with MET aberration was verified ex vivo and in vivo. Results We successfully obtained two gefitinib-resistant NSCLC cell lines with EGFR mutation and abnormal activation of MET. We observed that MET formed a positive feedback loop with the VEGF/VEGFR2 signaling, leading to persistent downstream signaling activation. Specifically, MET up-regulated VEGFR2 expression in a MAPK/ERK/ETS1-dependent manner, while VEGF promoted physical interaction between VEGFR2 and MET, thereby facilitating MET phosphorylation. A MET inhibitor, crizotinib, combined with an anti-VEGF antibody, bevacizumab, enhanced the sensitivity of NSCLC cells to gefitinib and synergistically inhibited the activation of downstream signaling in vitro. Dual inhibition of MET and VEGF combined with EGFR TKIs markedly restrained tumor growth in both human NSCLC xenograft models and in an EGFR/MET co-altered case. Conclusions Our work reveals a positive feedback loop between MET and VEGF/VEGFR2, resulting in continuous downstream signal activation. Combined inhibition of MET and VEGF/VEGFR2 signaling pathway may be beneficial for reversing EGFR TKIs resistance.

The nitration of SIRT6 aggravates neuronal damage during cerebral ischemia-reperfusion in rat

Ischemic stroke is a major cause of death and disability. The activation of neuronal nitric oxide synthase (nNOS) and the resulting production of nitric oxide (NO) via NMDA receptor-mediated calcium influx play an exacerbating role in cerebral ischemia reperfusion injury. The NO rapidly reacts with superoxide (O2-) to form peroxynitrite (ONOO-), a toxic molecule may modify proteins through tyrosine residue nitration, ultimately worsening neuronal damage. SIRT6 has been proven to be crucial in regulating cell proliferation, death, and aging in various pathological settings. We have previous reported that human SIRT6 tyrosine nitration decreased its intrinsic catalytic activity in vitro. However, the exact role of SIRT6 function in the process of cerebral ischemia reperfusion injury is not yet fully elucidated. Herein, we demonstrated that an increase in the nitration of SIRT6 led to reduce its enzymatic activity and aggravated hippocampal neuronal damage in a rat model of four-artery cerebral ischemia reperfusion. In addition, reducing SIRT6 nitration resulted in increase the activity of SIRT6, alleviating hippocampal neuronal damage. Moreover, SIRT6 nitration affected its downstream molecule activity such as PARP1 and GCN5, promoting the process of neuronal ischemic injury in rat hippocampus. Additionally, treatment with NMDA receptor antagonist MK801, or nNOS inhibitor 7-NI, and resveratrol (an antioxidant) diminished SIRT6 nitration and the catalytic activity of downstream molecules like PARP1 and GCN5, thereby reducing neuronal damage. Finally, in the biochemical regulation of SIRT6 activity, tyrosine 257 was essential for its activity and susceptibility to nitration. Replacing tyrosine 257 with phenylalanine in rat SIRT6 attenuated the death of SH-SY5Y neurocytes under oxygen-glucose deprivation (OGD) conditions. These results may offer further understanding of SIRT6 function in the pathogenesis of cerebral ischemic diseases.

ZnR/GPR39 regulates hepatic insulin signaling, tunes liver bioenergetics and ROS production, and mitigates liver fibrosis and injury

Adequate supply of zinc is essential for hepatic function and its deficiency is associated with acute liver injury (ALI) and chronic nonalcoholic fatty liver disease (NAFLD). However, how zinc controls hepatic function is unknown. We found that the zinc sensitive ZnR/GPR39, a mediator of zinc signaling, enhances hepatic phosphorylation of ERK1/2, which is reduced in ZnR/GPR39 deficient livers. Surprisingly, livers from ZnR/GPR39 knockout (KO) mice exhibited elevated insulin receptor expression and downstream AKT activation. Moreover, ZnR/GPR39 KO mice had higher blood fasting glucose level, pronounced hepatic lipid accumulation, increased hepatocyte oxygen consumption rate (OCR) and reactive oxygen species (ROS) levels. These data suggest that ZnR/GPR39 modulates insulin receptor signaling, a major pathway in hepatic metabolism. Associated with the impaired signaling, ZnR/GPR39 KO livers exhibited increased tissue fibrosis, manifested by marked elevation of collagen expression, compared to wildtype (WT). Additionally, we found alteration of hepatocyte junctional proteins that was accompanied by increased macrophage infiltration and higher liver inflammation in ZnR/GPR39 KO mice. To determine the role of ZnR/GPR39 in ALI, we applied a mild LPS challenge that induced profound decrease in hepatic OCR, also leading to higher ROS generation in ZnR/GPR39 KO hepatocytes, but not in WT. We further found increased serum IL-2 and AST/ALT ratio only in ZnR/GPR39 KO mice. Our findings reveal a role of ZnR/GPR39 in controlling hepatic insulin receptor signaling and mitigating liver fibrosis and inflammation, thus underscoring the important role of ZnR/GPR39 in liver signaling and function.