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Hexavalent chromium (Cr(VI)) exposure has been linked with gastrointestinal toxicity, whereas the molecular pathways and key targets remain elusive. Computational toxicology analysis predicted the correlation between protein phosphatase 2A (PP2A) and genes regarding Cr(VI)-induced intestinal injury. Here, we generated a mouse model with intestinal epithelium-specific knock out of Ppp2r1a (encoding PP2A Aα subunit) to investigate the mechanisms underlying Cr(VI)-induced small intestinal toxicity. Heterozygous (HE) mice and matched WT littermates were administrated with Cr(VI) at 0, 5, 20, and 80 mg/l for 28 successive days. Cr(VI) treatment led to crypt hyperplasia, epithelial cell apoptosis, and intestinal barrier dysfunction, accompanied by the decline of goblet cell counts and Occludin expression in WT mice. Notably, these effects were aggravated in HE mice, indicating that PP2A Aα deficiency conferred mice with susceptibility to Cr(VI)-induced intestinal injury. The combination of data analysis and biological experiments revealed Cr(VI) exposure could decrease YAP1 phosphorylation at Ser127 but increase protein expression and activity, together with elevated transcriptional coactivator with PDZ-binding motif protein driving epithelial crypt cells proliferation following damage, suggesting the involvement of Hippo/YAP1 signaling pathway in Cr(VI)-induced intestinal toxicity. Nevertheless, the enhanced phosphorylation of YAP1 in HE mice resulted in proliferation/repair defects in intestinal epithelium, thereby exacerbating Cr(VI)-induced gut barrier dysfunction. Notably, by molecular docking and further studies, we identified urolithin A, a microbial metabolite, attenuated Cr(VI)-induced disruption of intestinal barrier function, partly by modulating YAP1 expression and activity. Our findings reveal the novel molecular pathways participated in Cr(VI)-caused small intestinal injury and urolithin A could potentially protect against environmental hazards-induced intestinal diseases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Chemotherapy is still the main therapeutic strategy for gastric cancer (GC). However, most patients eventually acquire multidrug resistance (MDR). Hyperactivation of the EGFR signaling pathway contributes to MDR by promoting cancer cell proliferation and inhibiting apoptosis. We previously identified the secreted protein CGA as a novel ligand of EGFR and revealed a CGA/EGFR/GATA2 positive feedback circuit that confers MDR in GC. Herein, we outline a microRNA-based treatment approach for MDR reversal that targets both CGA and GATA2. We observed increased expression of CGA and GATA2 and increased activation of EGFR in GC samples. Bioinformatic analysis revealed that miR-107 could simultaneously target CGA and GATA2, and the low expression of miR-107 was correlated with poor prognosis in GC patients. The direct interactions between miR-107 and CGA or GATA2 were validated by luciferase reporter assays and Western blot analysis. Overexpression of miR-107 in MDR GC cells increased their susceptibility to chemotherapeutic agents, including fluorouracil, adriamycin, and vincristine, in vitro. Notably, intratumor injection of the miR-107 prodrug enhanced MDR xenograft sensitivity to chemotherapies in vivo. Molecularly, targeting CGA and GATA2 with miR-107 inhibited EGFR downstream signaling, as evidenced by the reduced phosphorylation of ERK and AKT. These results suggest that miR-107 may contribute to the development of a promising therapeutic approach for the treatment of MDR in GC., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetic nephropathy (DN) is one of the most important comorbidities for diabetic patients, which is the main factor leading to end-stage renal disease. Heparin analogs can delay the progression of DN, but the mechanism is not fully understood. In this study, we found that low molecular weight heparin therapy significantly upregulated some downstream proteins of the peroxisome proliferator-activated receptor (PPAR) signaling pathway by label-free quantification of the mouse kidney proteome. Through cell model verification, low molecular weight heparin can protect the heparan sulfate of renal tubular epithelial cells from being degraded by heparanase that is highly expressed in a high-glucose environment, enhance the endocytic recruitment of fatty acid-binding protein 1, a coactivator of the PPAR pathway, and then regulate the activation level of intracellular PPAR. In addition, we have elucidated for the first time the molecular mechanism of heparan sulfate and fatty acid-binding protein 1 interaction. These findings provide new insights into understanding the role of heparin in the pathogenesis of DN and developing corresponding treatments., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aberrant regulation of signal transduction pathways can adversely derail biological processes for tissue development. One such process is the embryonic eyelid closure that is dependent on the mitogen-activated protein kinase kinase kinase 1 (MAP3K1). Map3k1 KO in mice results in defective eyelid closure and an autosomal recessive eye-open at birth phenotype. We have shown that in utero exposure to dioxin, a persistent environmental toxicant, induces the same eye defect in Map3k1 +/- heterozygous but not WT pups. Here, we explore the mechanisms of the Map3k1 (gene) and dioxin (environment) interactions (GxE) underlying defective eyelid closure. We show that, acting through the aryl hydrocarbon receptor, dioxin activates epidermal growth factor receptor signaling, which in turn depresses MAP3K1-dependent Jun N-terminal kinase (JNK) activity. The dioxin-mediated JNK repression is moderate but is exacerbated by Map3k1 heterozygosity. Therefore, dioxin exposed Map3k1 +/- embryonic eyelids have a marked reduction of JNK activity, accelerated differentiation and impeded polarization in the epithelial cells. Knocking out Ahr or Egfr in eyelid epithelium attenuates the open-eye defects in dioxin-treated Map3k1 +/- pups, whereas knockout of Jnk1 and S1pr that encodes the sphigosin-1-phosphate (S1P) receptors upstream of the MAP3K1-JNK pathway potentiates the dioxin toxicity. Our novel findings show that the crosstalk of aryl hydrocarbon receptor, epidermal growth factor receptor, and S1P-MAP3K1-JNK pathways determines the outcome of dioxin exposure. Thus, gene mutations targeting these pathways are potential risk factors for the toxicity of environmental chemicals., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Antimicrobial resistance poses a serious threat to human health worldwide and its incidence continues to increase owing to the overuse of antibiotics and other factors. Macrolide antibiotics such as erythromycin (EM) have immunomodulatory effects in addition to their antibacterial activity. Long-term, low-dose administration of macrolides has shown clinical benefits in treating non-infectious inflammatory respiratory diseases. However, this practice may also increase the emergence of drug-resistant bacteria. In this study, we synthesized a series of EM derivatives, and screened them for two criteria: (i) lack of antibacterial activity and (ii) ability to suppress tumor necrosis factor-α (TNF-α) production in THP-1 cells stimulated with lipopolysaccharide. Among the 37 synthesized derivatives, we identified a novel 12-membered ring macrolide EM982 that lacked antibacterial activity against Staphylococcus aureus and suppressed the production of TNF-α and other cytokines. The effects of EM982 on Toll-like receptor 4 (TLR4) signaling were analyzed using a reporter assay and Western blotting. The reporter assay showed that EM982 suppressed the activation of transcription factors, NF-κB and/or activator protein 1 (AP-1), in HEK293 cells expressing human TLR4. Western blotting showed that EM982 inhibited the phosphorylation of both IκB kinase (IKK) β and IκBα, which function upstream of NF-κB, whereas it did not affect the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal kinase, which act upstream of AP-1. These results suggest that EM982 suppresses cytokine production by inhibiting phosphorylation of IKKβ and IκBα, resulting in the inactivation of NF-κB., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Periprosthetic osteolysis and subsequent aseptic loosening are the primary causes of failure following total joint arthroplasty. Wear particle-induced osteogenic impairment is recognized as an important contributing factor in the development of osteolysis, with endoplasmic reticulum (ER) stress emerging as a pivotal underlying mechanism. Hence, searching for potential therapeutic targets and agents capable of modulating ER stress in osteoblasts is crucial for preventing aseptic loosening. Kaempferol (KAE), a natural flavonol compound, has shown promising osteoprotective effects and anti-ER stress properties in diverse diseases. However, the influence of KAE on ER stress-mediated osteogenic impairment induced by wear particles remains unclear. In this study, we observed that KAE effectively relieved TiAl 6 V 4 particles-induced osteolysis by improving osteogenesis in a mouse calvarial model. Furthermore, we demonstrated that KAE could attenuate ER stress-mediated apoptosis in osteoblasts exposed to TiAl 6 V 4 particles, both in vitro and in vivo. Mechanistically, our results revealed that KAE mitigated ER stress-mediated apoptosis by upregulating the IRE1α-XBP1s pathway while concurrently partially inhibiting the IRE1α-regulated RIDD and JNK activation. Collectively, our findings suggest that KAE is a prospective therapeutic agent for treating wear particle-induced osteolysis and highlight the IRE1α-XBP1s pathway as a potential therapeutic target for preventing aseptic loosening., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Homologous recombination (HR) plays a key role in maintaining genomic stability, and the efficiency of the HR system is closely associated with tumor response to chemotherapy. Our previous work reported that CK2 kinase phosphorylates HIV Tat-specific factor 1 (HTATSF1) Ser748 to facilitate HTATSF1 interaction with TOPBP1, which in turn, promotes RAD51 recruitment and HR repair. However, the clinical implication of the CK2-HTATSF1-TOPBP1 pathway in tumorigenesis and chemotherapeutic response remains to be elucidated. Here, we report that the CK2-HTATSF1-TOPBP1 axis is generally hyperactivated in multiple malignancies and renders breast tumors less responsive to chemotherapy. In contrast, deletion mutations of each gene in this axis, which also occur in breast and lung tumor samples, predict higher HR deficiency scores, and tumor cells bearing a loss-of-function mutation of HTATSF1 are vulnerable to poly(ADP-ribose) polymerase inhibitors or platinum drugs. Taken together, our study suggests that the integrity of the CK2-HTATSF1-TOPBP1 axis is closely linked to tumorigenesis and serves as an indicator of tumor HR status and modulates chemotherapy response., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The Hippo signaling pathway plays an essential role in organ size control and tumorigenesis. Loss of Hippo signal and hyper-activation of the downstream oncogenic YAP signaling are commonly observed in various types of cancers. We previously identified STRN3-containing PP2A phosphatase as a negative regulator of MST1/2 kinases (i.e., Hippo) in gastric cancer (GC), opening the possibility of selectively targeting the PP2Aa-STRN3-MST1/2 axis to recover Hippo signaling against cancer. Here, we further discovered 1) disulfiram (DSF), an FDA-approved drug, which can similarly block the binding of STRN3 to PP2A core enzyme and 2) CX-6258 (CX), a chemical inhibitor, that can disrupt the interaction between STRN3 and MST1/2, both allowing reactivation of Hippo activity to inhibit GC. More importantly, we found these two compounds, via an MST1/2 kinase-dependent manner, inhibit DNA repair to sensitize GC towards chemotherapy. In addition, we identified thiram, a structural analog of DSF, can function similarly to inhibit cancer cell proliferation or enhance chemotherapy sensitivity. Interestingly, inclusion of copper ion enhanced such effects of DSF and thiram on GC treatment. Overall, this work demonstrated that pharmacological targeting of the PP2Aa-STRN3-MST1/2 axis by drug compounds can potently recover Hippo signal for tumor treatment., Competing Interests: Conflict of interest The authors have filed a patent (202110534762.X) for the thiram compound in China., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF-related protein family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE and that larger species are dispensable for BMP inhibition. Additionally, we used an in silico approach to identify a helix, termed the ligand-binding domain, that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the ligand-binding domain is crucial for activity through luciferase assays and surface plasmon resonance analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands., Competing Interests: Conflict of interest T. B. T. is a consultant/advisor for Keros Therapeutics and Oviva Therapeutics. All other authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Oligodendrocyte precursor cells are present in the adult central nervous system, and their impaired ability to differentiate into myelinating oligodendrocytes can lead to demyelination in patients with multiple sclerosis, accompanied by neurological deficits and cognitive impairment. Exosomes, small vesicles released by cells, are known to facilitate intercellular communication by carrying bioactive molecules. In this study, we utilized exosomes derived from human umbilical cord mesenchymal stem cells (HUMSCs-Exos). We performed sequencing and bioinformatics analysis of exosome-treated cells to demonstrate that HUMSCs-Exos can stimulate myelin gene expression in oigodendrocyte precursor cells. Functional investigations revealed that HUMSCs-Exos activate the Pi3k/Akt pathway and regulate the Tbr1/Wnt signaling molecules through the transfer of miR-23a-3p, promoting oligodendrocytes differentiation and enhancing the expression of myelin-related proteins. In an experimental autoimmune encephalomyelitis model, treatment with HUMSCs-Exos significantly improved neurological function and facilitated remyelination. This study provides cellular and molecular insights into the use of cell-free exosome therapy for central nervous system demyelination associated with multiple sclerosis, demonstrating its great potential for treating demyelinating and neurodegenerative diseases., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Notch regulates the immune and inflammatory response and has been associated with the pathogenesis of osteoarthritis in humans and preclinical models of the disease. Notch2 tm1.1Ecan mice harbor a NOTCH2 gain-of-function and are sensitized to osteoarthritis, but the mechanisms have not been explored. We examined the effects of tumor necrosis factor α (TNFα) in chondrocytes from Notch2 tm1.1Ecan mice and found that NOTCH2 enhanced the effect of TNFα on Il6 and Il1b expression. Similar results were obtained in cells from a conditional model of NOTCH2 gain-of-function, Notch2 2.1Ecan mice, and following the expression of the NOTCH2 intracellular domain in vitro. Recombination signal-binding protein for immunoglobulin Kappa J region partners with the NOTCH2 intracellular domain to activate transcription; in the absence of Notch signaling it inhibits transcription, and Rbpj inactivation in chondrocytes resulted in Il6 induction. Although TNFα induced IL6 to a greater extent in the context of NOTCH2 activation, there was a concomitant inhibition of Notch target genes Hes1, Hey1, Hey2, and Heyl. Electrophoretic mobility shift assay demonstrated displacement of recombination signal-binding protein for immunoglobulin Kappa J region from DNA binding sites by TNFα explaining the increased Il6 expression and the concomitant decrease in Notch target genes. NOTCH2 enhanced the effect of TNFα on NF-κB signaling, and RNA-Seq revealed increased expression of pathways associated with inflammation and the phagosome in NOTCH2 overexpressing cells in the absence and presence of TNFα. Collectively, NOTCH2 has important interactions with TNFα resulting in the enhanced expression of Il6 and inflammatory pathways in chondrocytes., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential inhibition. We examined the prenylation of carboxy-terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to prenylation and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide, statin sensitivity, and extent of prenylation. Our results also show a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings indicate a plausible mechanism allowing for statins to differentially perturb heterotrimeric G protein signaling in cells depending on their Gγ-subtype composition. Our results may also provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest concerning the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Pharmacological inhibition of mitochondrial fatty acid oxidation (FAO) has been clinically used to alleviate certain metabolic diseases by remodeling cellular metabolism. However, mitochondrial FAO inhibition also leads to mechanistic target of rapamycin complex 1 (mTORC1) activation-related protein synthesis and tissue hypertrophy, but the mechanism remains unclear. Here, by using a mitochondrial FAO inhibitor (mildronate or etomoxir) or knocking out carnitine palmitoyltransferase-1, we revealed that mitochondrial FAO inhibition activated the mTORC1 pathway through general control nondepressible 5-dependent Raptor acetylation. Mitochondrial FAO inhibition significantly promoted glucose catabolism and increased intracellular acetyl-CoA levels. In response to the increased intracellular acetyl-CoA, acetyltransferase general control nondepressible 5 activated mTORC1 by catalyzing Raptor acetylation through direct interaction. Further investigation also screened Raptor deacetylase histone deacetylase class II and identified histone deacetylase 7 as a potential regulator of Raptor. These results provide a possible mechanistic explanation for the mTORC1 activation after mitochondrial FAO inhibition and also bring light to reveal the roles of nutrient metabolic remodeling in regulating protein acetylation by affecting acetyl-CoA production., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Bivalent epigenomic regulatory domains containing both activating histone 3 lysine 4 (H3K4me3) and repressive lysine 27 (H3K27me3) trimethylation are associated with key developmental genes. These bivalent domains repress transcription in the absence of differentiation signals but maintain regulatory genes in a poised state to allow for timely activation. Previous studies demonstrated that enhancer of zeste homolog 2 (Ezh2), a histone 3 lysine 27 (H3K27) methyltransferase, suppresses osteogenic differentiation and that inhibition of Ezh2 enhances commitment of osteoblast progenitors in vitro and bone formation in vivo. Here, we examined the mechanistic effects of Tazemetostat (EPZ6438), an Food and Drug Administration approved Ezh2 inhibitor for epithelioid sarcoma treatment, because this drug could potentially be repurposed to stimulate osteogenesis for clinical indications. We find that Tazemetostat reduces H3K27me3 marks in bivalent domains in enhancers required for bone formation and stimulates maturation of MC3T3 preosteoblasts. Furthermore, Tazemetostat activates bivalent genes associated with the Wingless/integrated (WNT), adenylyl cyclase (cAMP), and Hedgehog (Hh) signaling pathways based on transcriptomic (RNA-seq) and epigenomic (chromatin immunoprecipitation [ChIP]-seq) data. Functional analyses using selective pathway inhibitors and silencing RNAs demonstrate that the WNT and Hh pathways modulate osteogenic differentiation after Ezh2 inhibition. Strikingly, we show that loss of the Hh-responsive transcriptional regulator Gli1, but not Gli2, synergizes with Tazemetostat to accelerate osteoblast differentiation. These studies establish epigenetic cooperativity of Ezh2, Hh-Gli1 signaling, and bivalent regulatory genes in suppressing osteogenesis. Our findings may have important translational ramifications for anabolic applications requiring bone mass accrual and/or reversal of bone loss., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Substance abuse is on the rise, and while many people may use illicit drugs mainly due to their rewarding effects, their societal impact can range from severe, as is the case for opioids, to promising, as is the case for psychedelics. Common with all these drugs' mechanisms of action are G protein-coupled receptors (GPCRs), which lie at the center of how these drugs mediate inebriation, lethality, and therapeutic effects. Opioids like fentanyl, cannabinoids like tetrahydrocannabinol, and psychedelics like lysergic acid diethylamide all directly bind to GPCRs to initiate signaling which elicits their physiological actions. We herein review recent structural studies and provide insights into the molecular mechanisms of opioids, cannabinoids, and psychedelics at their respective GPCR subtypes. We further discuss how such mechanistic insights facilitate drug discovery, either toward the development of novel therapies to combat drug abuse or toward harnessing therapeutic potential., Competing Interests: Conflict of interest D. W. has consulted for Otsuka Pharmaceutical, Longboard Pharmaceuticals and Ocean Bio Ltd on the design of psychedelic-based therapeutics. None of the companies listed herein contributed to the funding or narrative of the manuscript. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

CD40 signaling has long been a target in autoimmunity. Attempts to block signaling between CD40 and CD154 during clinical trials using monoclonal antibodies suffered severe adverse events. Previously, we developed a peptide, KGYY 15 , that targets CD40 and, in preclinical trials, prevents type 1 diabetes in >90% of cases and reverses new-onset hyperglycemia in 56% of cases. It did so by establishing normal effector T-cell levels rather than ablating the cells and causing immunosuppression. However, the relationship between KGYY 15 and other elements of the complex signaling network of CD40 is not clear. Studying interactions between proteins from autoimmune and nonautoimmune mice, we demonstrate interactions between CD40 and integrin CD11a/CD18, which complicates the understanding of the inflammatory nexus and how to prevent autoinflammation. In addition to interacting with CD40, KGYY 15 interacts with the integrins CD11a/CD18 and CD11b/CD18. We argue that modulation of CD40-CD154 signaling may be more advantageous than complete inhibition because it may preserve normal immunity to pathogens., Competing Interests: Conflict of interest D. H. W. Jr is the founder and chief scientific officer of Op-T LLC., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Fibroblast growth factor (FGF) is a multifunctional protein that exhibits a wide range of biological effects. Most commonly, it acts as a mitogen, but it also has regulatory, morphological, and endocrine effects. The four receptor subtypes of FGF are activated by more than 20 different FGF ligands. FGF2, one of the FGF ligands, is an essential factor for cell culture in stem cells for regenerative medicine; however, recombinant FGF2 is extremely unstable. Here, we successfully generated homobivalent agonistic single-domain antibodies (variable domain of heavy chain of heavy chain antibodies referred to as VHHs) that bind to domain III and induce activation of the FGF receptor 1 and thus transduce intracellular signaling. This agonistic VHH has similar biological activity (EC 50 ) as the natural FGF2 ligand. Furthermore, we determined that the agonistic VHH could support the proliferation of human-induced pluripotent stem cells (PSCs) and human mesenchymal stem cells, which are PSCs for regenerative medicine. In addition, the agonistic VHH could maintain the ability of mesenchymal stem cells to differentiate into adipocytes or osteocytes, indicating that it could maintain the properties of PSCs. These results suggest that the VHH agonist may function as an FGF2 mimetic in cell preparation of stem cells for regenerative medicine with better cost effectiveness., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein-coupled receptor involved in raising cytosolic cAMP levels, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. Our previous work suggested that dissociation of autoproteolytically generated N-terminal and C-terminal fragments of GPR133 at the plasma membrane correlates with receptor activation and signaling. To promote the goal of developing biologics that modulate GPR133 function, we investigated the effects of antibodies against the N-terminus of GPR133 on receptor signaling. Here, we show that treatment of HEK293T cells overexpressing GPR133 with these antibodies increased cAMP levels in a concentration-dependent manner. Analysis of culture medium following antibody treatment further indicated the presence of complexes of these antibodies with the autoproteolytically cleaved N-terminal fragments of GPR133. In addition, cells expressing a cleavage-deficient mutant of GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage dependent. Finally, we demonstrate the antibody-mediated stimulation of WT GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that the intramolecular cleavage in the N-terminus modulates receptor activation and signaling., Competing Interests: Conflict of interest D. G. P. and NYU Grossman School of Medicine own an EU and Hong Kong patent titled “Method for treating high-grade gliomas” on the use of GPR133 as a treatment target in glioma. D. G. P. has received consultant fees from Tocagen, Synaptive Medical, Monteris and Robeaute. The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

As a crucial metabolic intermediate, l-lactate is involved in redox balance, energy balance, and acid-base balance in organisms. Moderate exercise training transiently elevates plasma l-lactate levels and ameliorates obesity-associated type 2 diabetes. However, whether moderate l-lactate administration improves obesity-associated insulin resistance remains unclear. In this study, we defined 800 mg/kg/day as the dose of moderate l-lactate administration. In mice fed with a high-fat diet (HFD), moderate l-lactate administration for 12 weeks was shown to alleviate weight gain, fat accumulation, and insulin resistance. Along with the phenotype alterations, white adipose tissue thermogenesis was also found to be elevated in HFD-fed mice. Meanwhile, moderate l-lactate administration suppressed the infiltration and proinflammatory M1 polarization of adipose tissue macrophages (ATMs) in HFD-fed mice. Furthermore, l-lactate treatment suppressed the lipopolysaccharide-induced M1 polarization of bone marrow-derived macrophages (BMDMs). l-lactate can bind to the surface receptor GPR132, which typically drives the downstream cAMP-PKA signaling. As a nutrient sensor, AMP-activated protein kinase (AMPK) critically controls macrophage inflammatory signaling and phenotype. Thus, utilizing inhibitors of the kinases PKA and AMPK as well as siRNA against GPR132, we demonstrated that GPR132-PKA-AMPKα1 signaling mediated the suppression caused by l-lactate treatment on BMDM M1 polarization. Finally, l-lactate addition remarkably resisted the impairment of lipopolysaccharide-treated BMDM conditional media on adipocyte insulin sensitivity. In summary, moderate l-lactate administration suppresses ATM proinflammatory M1 polarization through activation of the GPR132-PKA-AMPKα1 signaling pathway to improve insulin resistance in HFD-fed mice, suggesting a new therapeutic and interventional approach to obesity-associated type 2 diabetes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera. It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found that WFA dose-dependently (within the concentration range of 0.3-1.25 μM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhesion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome analysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, suggesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and impedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologies., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the content of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Bisphenol A and its derivatives are recognized as endocrine disruptors based on their complex effects on estrogen receptor (ER) signaling. While the effects of bisphenol derivatives on ERα have been thoroughly evaluated, how these chemicals affect ERβ signaling is less well understood. Herein, we sought to identify novel ERβ ligands using a radioligand competitive binding assay to screen a chemical library of bisphenol derivatives. Many of the compounds identified showed intriguing dual activities as both ERα agonists and ERβ antagonists. Docking simulations of these compounds and ERβ suggested that they bound not only to the canonical binding site of ERβ but also to the coactivator binding site located on the surface of the receptor, suggesting that they act as coactivator-binding inhibitors (CBIs). Receptor-ligand binding experiments using WT and mutated ERβ support the presence of a second ligand-interaction position at the coactivator-binding site in ERβ, and direct binding experiments of ERβ and a coactivator peptide confirmed that these compounds act as CBIs. Our study is the first to propose that bisphenol derivatives act as CBIs, presenting critical insight for the future development of ER signaling-based drugs and their potential to function as endocrine disruptors., Competing Interests: Conflict of interest R. M. E. holds the March of Dimes Chair in Molecular and Developmental Biology at the Salk Institute. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The orphan nuclear receptor Nur77 is an immediate-early response gene that based on tissue and cell context is implicated in a plethora of cellular processes, including proliferation, differentiation, apoptosis, metabolism, and inflammation. Nur77 has a ligand-binding pocket that is obstructed by hydrophobic side groups. Naturally occurring, cell-endogenous ligands have not been identified, and Nur77 transcriptional activity is thought to be regulated through posttranslational modification and modulation of protein levels. To determine whether Nur77 is transcriptionally active in hematopoietic cells in vivo, we used an upstream activating sequence (UAS)-GFP transgenic reporter. We found that Nur77 is transcriptionally inactive in vivo in hematopoietic cells under basal conditions, but that activation occurs following cytokine exposure by G-CSF or IL-3. We also identified a series of serine residues required for cytokine-dependent transactivation of Nur77. Moreover, a kinase inhibitor library screen and proximity labeling-based mass spectrometry identified overlapping kinase pathways that physically interacted with Nur77 and whose inhibition abrogated cytokine-induced activation of Nur77. We determined that transcriptional activation of Nur77 by G-CSF or IL-3 requires functional JAK and mTor signaling since their inhibition leads to Nur77 transcriptional inactivation. Thus, intracellular cytokine signaling networks appear to regulate Nur77 transcriptional activity in mouse hematopoietic cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Intraneuronal neurofibrillary tangles composed of Tau aggregates have been widely accepted as an important pathological hallmark of Alzheimer's disease. A current therapeutic avenue for treating Alzheimer's disease is aimed at inhibiting Tau accumulation with small molecules such as natural flavonoids. Liquid-liquid phase separation (LLPS) of Tau can lead to its aggregation, and Tau aggregates can then be degraded by autophagy. However, it is unclear whether natural flavonoids modulate the formation of phase-separated Tau droplets or promote autophagy and Tau clearance. Here, using confocal microscopy and fluorescence recovery after photobleaching assays, we report that a natural antioxidant flavonoid compound myricetin slows LLPS of full-length human Tau, shifting the equilibrium phase boundary to a higher protein concentration. This natural flavonoid also significantly inhibits pathological phosphorylation and abnormal aggregation of Tau in neuronal cells and blocks mitochondrial damage and apoptosis induced by Tau aggregation. Importantly, using coimmunoprecipitation and Western blotting, we show that treatment of cells with myricetin stabilizes the interaction between Tau and autophagy-related protein 5 (ATG5) to promote clearance of phosphorylated Tau to indirectly limit its aggregation. Consistently, this natural flavonoid inhibits mTOR pathway, activates ATG5-dependent Tau autophagy, and almost completely suppresses Tau toxicity in neuronal cells. Collectively, these results demonstrate how LLPS and abnormal aggregation of Tau are inhibited by natural flavonoids, bridging the gap between Tau LLPS and aggregation in neuronal cells, and also establish that myricetin could act as an ATG5-dependent autophagic activator to ameliorate the pathogenesis of Alzheimer's disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cyclin-dependent kinase 7 (CDK7) is a master regulatory kinase that drives cell cycle progression and stimulates expression of oncogenes in a myriad of cancers. Inhibitors of CDK7 (CDK7i) are currently in clinical trials; however, as with many cancer therapies, patients will most likely experience recurrent disease due to acquired resistance. Identifying targets underlying CDK7i resistance will facilitate prospective development of new therapies that can circumvent such resistance. Here we utilized triple-negative breast cancer as a model to discern mechanisms of resistance as it has been previously shown to be highly responsive to CDK7 inhibitors. After generating cell lines with acquired resistance, high-throughput RNA sequencing revealed significant upregulation of genes associated with efflux pumps and transforming growth factor-beta (TGF-β) signaling pathways. Genetic silencing or pharmacological inhibition of ABCG2, an efflux pump associated with multidrug resistance, resensitized resistant cells to CDK7i, indicating a reliance on these transporters. Expression of activin A (INHBA), a member of the TGF-β family of ligands, was also induced, whereas its intrinsic inhibitor, follistatin (FST), was repressed. In resistant cells, increased phosphorylation of SMAD3, a downstream mediator, confirmed an increase in activin signaling, and phosphorylated SMAD3 directly bound the ABCG2 promoter regulatory region. Finally, pharmacological inhibition of TGF-β/activin receptors or genetic silencing of SMAD4, a transcriptional partner of SMAD3, reversed the upregulation of ABCG2 in resistant cells and phenocopied ABCG2 inhibition. This study reveals that inhibiting the TGF-β/Activin-ABCG2 pathway is a potential avenue for preventing or overcoming resistance to CDK7 inhibitors., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell migration is an essential physiological process, and aberrant migration of epithelial cells underlies many pathological conditions. However, the molecular mechanisms governing cell migration are not fully understood. We report here that growth factor-induced epithelial cell migration is critically dependent on the crosstalk of two molecular switches, namely phosphorylation switch (P-switch) and transcriptional switch (T-switch). P-switch refers to dynamic interactions of deleted in liver cancer 1 (DLC1) and PI3K with tensin-3 (TNS3), phosphatase and tensin homolog (PTEN), C-terminal tension, and vav guanine nucleotide exchange factor 2 (VAV2) that are dictated by mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated protein kinase 1/2-dependent phosphorylation of TNS3, PTEN, and VAV2. Phosphorylation of TNS3 and PTEN on specific Thr residues led to the switch of DLC1-TNS3 and PI3K-PTEN complexes to DLC1-PTEN and PI3K-TNS3 complexes, whereas Ser phosphorylation of VAV2 promotes the transition of the PI3K-TNS3/PTEN complexes to PI3K-VAV2 complex. T-switch denotes an increase in C-terminal tension transcription/expression regulated by both extracellular signal-regulated protein kinase 1/2 and signal transducer and activator of transcription 3 (STAT3) via interleukin-6-Janus kinase-STAT3 signaling pathway. We have found that, the P-switch is indispensable for both the initiation and continuation of cell migration induced by growth factors, whereas the T-switch is only required to sustain cell migration. The interplay of the two switches facilitated by the interleukin-6-Janus kinase-STAT3 pathway governs a sequence of dynamic protein-protein interactions for sustained cell migration. That a similar mechanism is employed by both normal and tumorigenic epithelial cells to drive their respective migration suggests that the P-switch and T-switch are general regulators of epithelial cell migration and potential therapeutic targets., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Pulmonary endothelial barrier dysfunction is a major pathophysiology observed in acute respiratory distress syndrome (ARDS). Ghrelin, a key regulator of metabolism, has been shown to play protective roles in the respiratory system. However, its effects on lipopolysaccharide (LPS)-induced pulmonary endothelial barrier injury are unknown. In this study, the effects of ghrelin on LPS-induced ARDS and endothelial cell injury were evaluated in vivo and in vitro. In vivo, mice treated with LPS (3 mg/kg intranasal application) were used to establish the ARDS model. Annexin V/propidium iodide apoptosis assay, scratch-wound assay, tube formation assay, transwell permeability assay, and Western blotting experiment were performed to reveal in vitro effects and underlying mechanisms of ghrelin on endothelial barrier function. Our results showed that ghrelin had protective effects on LPS-induced ARDS and endothelial barrier disruption by inhibiting apoptosis, promoting cell migration and tube formation, and activating the PI3K/AKT signaling pathway. Furthermore, ghrelin stabilized LPS-induced endothelial barrier function by decreasing endothelial permeability and increasing the expression of the intercellular junction protein vascular endothelial cadherin. LY294002, a specific inhibitor of the PI3K pathway, reversed the protective effects of ghrelin on the endothelial cell barrier. In conclusion, our findings indicated that ghrelin protected against LPS-induced ARDS by impairing the pulmonary endothelial barrier partly through activating the PI3K/AKT pathway. Thus, ghrelin may be a valuable therapeutic strategy for the prevention or treatment of ARDS., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The neuron-specific tyrosine phosphatase striatal-enriched phosphatase (STEP) is emerging as a key regulator of excitotoxicity, which is involved in the pathogenesis of both acute and chronic neurological diseases. However, the intracellular mechanisms that are regulated by STEP to confer neuroprotection against excitotoxic insults are not well understood. The present study investigates the role of STEP in regulating neuronal release of the proinflammatory prostanoid prostaglandin E 2 (PGE 2 ), which is associated with a wide range of pathological conditions. The findings show that glutamate-mediated activation of the N-methyl-D-aspartic acid receptor in STEP-deficient neurons leads to rapid and sustained increase in the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), a signaling molecule involved in the production of inflammatory mediators. Such sustained p38 MAPK activation increases the activity of cytosolic phospholipase A 2 , which catalyzes the release of arachidonic acid, the initial substrate for PGE 2 biosynthesis. Sustained p38 MAPK activation also induces nuclear factor-κB-mediated increase in expression of cyclooxygenase-2 that is involved in the conversion of arachidonic acid to prostanoids, resulting in enhanced biosynthesis and release of PGE 2 from neurons. Restoration of STEP function with a STEP mimetic (TAT-STEP-myc peptide) significantly decreases the activation of p38 MAPK-mediated cytosolic phospholipase A 2 /cyclooxygenase-2/PGE 2 signaling cascade. This study identifies an important mechanism involved in the neuronal release of the proinflammatory mediator PGE 2 after excitotoxic insult and highlights for the first time the immunomodulatory ability of a neuronal tyrosine phosphatase., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Glucose-mediated signaling regulates the expression of a limited number of genes in human pancreatic β-cells at the transcriptional level. However, it is unclear whether glucose plays a role in posttranscriptional RNA processing or translational control of gene expression. Here, we asked whether glucose affects posttranscriptional steps and regulates protein synthesis in human β-cell lines. We first showed the involvement of the mTOR pathway in glucose-related signaling. We also used the surface sensing of translation technique, based on puromycin incorporation into newly translated proteins, to demonstrate that glucose treatment increased protein translation. Among the list of glucose-induced proteins, we identified the proconvertase PCSK1, an enzyme involved in the proteolytic conversion of proinsulin to insulin, whose translation was induced within minutes following glucose treatment. We finally performed global proteomic analysis by mass spectrometry to characterize newly translated proteins upon glucose treatment. We found enrichment in proteins involved in translation, glycolysis, TCA metabolism, and insulin secretion. Taken together, our study demonstrates that, although glucose minorly affects gene transcription in human β-cells, it plays a major role at the translational level., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Reperfusion therapy, the standard treatment for acute myocardial infarction, can trigger necrotic death of cardiomyocytes and provoke ischemia/reperfusion (I/R) injury. However, signaling pathways that regulate cardiomyocyte necrosis remain largely unknown. Our recent genome-wide RNAi screen has identified a potential necrosis suppressor gene PRKAR1A, which encodes PKA regulatory subunit 1α (R1α). R1α is primarily known for regulating PKA activity by sequestering PKA catalytic subunits in the absence of cAMP. Here, we showed that depletion of R1α augmented cardiomyocyte necrosis in vitro and in vivo, resulting in exaggerated myocardial I/R injury and contractile dysfunction. Mechanistically, R1α loss downregulated the Nrf2 antioxidant transcription factor and aggravated oxidative stress following I/R. Degradation of the endogenous Nrf2 inhibitor Keap1 through p62-dependent selective autophagy was blocked by R1α depletion. Phosphorylation of p62 at Ser349 by mammalian target of rapamycin complex 1 (mTORC1), a critical step in p62-Keap1 interaction, was induced by I/R, but diminished by R1α loss. Activation of PKA by forskolin or isoproterenol almost completely abolished hydrogen-peroxide-induced p62 phosphorylation. In conclusion, R1α loss induces unrestrained PKA activation and impairs the mTORC1-p62-Keap1-Nrf2 antioxidant defense system, leading to aggravated oxidative stress, necrosis, and myocardial I/R injury. Our findings uncover a novel role of PKA in oxidative stress and necrosis, which may be exploited to develop new cardioprotective therapies., Competing Interests: Conflict of interest Dr Stratakis' laboratory at the NIH holds patents on PRKAR1A and related genes and/or their function and has received funding from Pfizer Inc. on research projects unrelated to the subject of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Bone fractures are common impact injuries typically resolved through natural processes of osteogenic regeneration and bone remodeling, restoring the biological and mechanical function. However, dysfunctionality in bone healing and repair often arises in the context of aging-related chronic disorders, such as Alzheimer's disease (AD). There is unmet need for effective pharmacological modulators of osteogenic differentiation and an opportunity to probe the complex links between bone biology and cognitive disorders. We previously discovered the small molecule DIPQUO, which promotes osteoblast differentiation and bone mineralization in mouse and human cell culture models, and in zebrafish developmental and regenerative models. Here, we examined the detailed function of this molecule. First, we used kinase profiling, cellular thermal shift assays, and functional studies to identify glycogen synthase kinase 3-beta (GSK3-β) inhibition as a mechanism of DIPQUO action. Treatment of mouse C2C12 myoblasts with DIPQUO promoted alkaline phosphatase expression and activity, which could be enhanced synergistically by treatment with other GSK3-β inhibitors. Suppression of the expression or function of GSK3-β attenuated DIPQUO-dependent osteogenic differentiation. In addition, DIPQUO synergized with GSK3-β inhibitors to stimulate expression of osteoblast genes in human multipotent progenitors. Accordingly, DIPQUO promoted accumulation and activation of β-catenin. Moreover, DIPQUO suppressed activation of tau microtubule-associated protein, an AD-related effector of GSK3-β signaling. Therefore, DIPQUO has potential as both a lead candidate for bone therapeutic development and a pharmacological modulator of GSK3-β signaling in cell culture and animal models of disorders including AD., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Olfactory receptors (ORs), the largest family of G protein-coupled receptors, are expressed in the nasal epithelium where they mediate the sense of smell. However, ORs are also found in other non-nasal tissues, but the role of these ectopic ORs in cell signaling, proliferation, and survival is not well understood. Here, using an inducible expression system in the lymph node carcinoma of the prostate (LNCaP) cell line, we investigated two ectopic ORs, OR51E1 and OR51E2, which have been shown to be upregulated in prostate cancer. We found that, consistent with previous studies, OR51E1 stimulated adenylyl cyclase in response to treatment by short-chain to medium-chain organic acids (C3-C9) but not by acetate. OR51E2 responded to acetate and propionate but not to the longer chain organic acids. Stimulation of LNCaP cells with butyrate inhibited their growth, and the knockdown of the endogenous OR51E1 negated this cytostatic effect. Most significantly, overexpression of OR51E1 or OR51E2 suppressed LNCaP cell proliferation. Overexpression of another ectopic OR OR2AT4, β2-adrenergic receptor, or treatment of cells with forskolin did not suppress cell proliferation, indicating that a rise in cAMP is not sufficient to induce cytostasis. Overexpression of OR51E1 caused an upregulation of cytostatic and cell death markers including p27, p21, and p53, strongly increased annexin V staining, and stimulated extracellular signal-regulated protein kinases 1 and 2. Overexpression and/or activation of OR51E1 did not affect human embryonic kidney 293 cell proliferation, indicating that cytotoxicity of OR51E1/OR51E2 is specific for LNCaP cells. Together, our results further our understanding of prostate cancer etiology and suggest that ectopic ORs may be useful therapeutic targets., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Heterotrimeric G protein subunits Gαq and Gα11 are inhibited by two cyclic depsipeptides, FR900359 (FR) and YM-254890 (YM), both of which are being used widely to implicate Gq/11 proteins in the regulation of diverse biological processes. An emerging major research question therefore is whether the cellular effects of both inhibitors are on-target, that is, mediated via specific inhibition of Gq/11 proteins, or off-target, that is, the result of nonspecific interactions with other proteins. Here we introduce a versatile experimental strategy to discriminate between these possibilities. We developed a Gαq variant with preserved catalytic activity, but refractory to FR/YM inhibition. A minimum of two amino acid changes were required and sufficient to achieve complete inhibitor resistance. We characterized the novel mutant in HEK293 cells depleted by CRISPR-Cas9 of endogenous Gαq and Gα11 to ensure precise control over the Gα-dependent cellular signaling route. Using a battery of cellular outcomes with known and concealed Gq contribution, we found that FR/YM specifically inhibited cellular signals after Gαq introduction via transient transfection. Conversely, both inhibitors were inert across all assays in cells expressing the drug-resistant variant. These findings eliminate the possibility that inhibition of non-Gq proteins contributes to the cellular effects of the two depsipeptides. We conclude that combined application of FR or YM along with the drug-resistant Gαq variant is a powerful in vitro strategy to discern on-target Gq against off-target non-Gq action. Consequently, it should be of high value for uncovering Gq input to complex biological processes with high accuracy and the requisite specificity., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyperinflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 receptor 5 (IL-1R5, also known as IL-18 receptor alpha chain), leading to the recruitment of the coreceptor, IL-1 receptor 7 (IL-1R7, also known as IL-18 receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs) and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6, and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans-induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19., Competing Interests: Conflict of interest K. B. and U. P. were employed by MAB Discovery GmbH, Neuried, Germany. S. F. is the CEO of MAB Discovery GmbH. J. F. H. has received consulting fees from MAB Discovery GmbH. Other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Adult progenitor cell populations typically exist in a quiescent state within a controlled niche environment. However, various stresses or forms of damage can disrupt this state, which often leads to dysfunction and aging. We built a glucocorticoid (GC)-induced liver damage model of mice, found that GC stress induced liver damage, leading to consequences for progenitor cells expansion. However, the mechanisms by which niche factors cause progenitor cells proliferation are largely unknown. We demonstrate that, within the liver progenitor cells niche, Galectin-3 (Gal-3) is responsible for driving a subset of progenitor cells to break quiescence. We show that GC stress causes aging of the niche, which induces the up-regulation of Gal-3. The increased Gal-3 population increasingly interacts with the progenitor cell marker CD133, which triggers focal adhesion kinase (FAK)/AMP-activated kinase (AMPK) signaling. This results in the loss of quiescence and leads to the eventual stemness exhaustion of progenitor cells. Conversely, blocking Gal-3 with the inhibitor TD139 prevents the loss of stemness and improves liver function. These experiments identify a stress-dependent change in progenitor cell niche that directly influence liver progenitor cell quiescence and function., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Yang et al.)

Elevated levels of fasting insulin release and insufficient glucose-stimulated insulin secretion (GSIS) are hallmarks of diabetes. Studies have established cross-talk between integrin signaling and insulin activity, but more details of how integrin-dependent signaling impacts the pathophysiology of diabetes are needed. Here, we dissected integrin-dependent signaling pathways involved in the regulation of insulin secretion in β-cells and studied their link to the still debated autocrine regulation of insulin secretion by insulin/insulin-like growth factor (IGF) 2-AKT signaling. We observed for the first time a cooperation between different AKT isoforms and focal adhesion kinase (FAK)-dependent adhesion signaling, which either controlled GSIS or prevented insulin secretion under fasting conditions. Indeed, β-cells form integrin-containing adhesions, which provide anchorage to the pancreatic extracellular matrix and are the origin of intracellular signaling via FAK and paxillin. Under low-glucose conditions, β-cells adopt a starved adhesion phenotype consisting of actin stress fibers and large peripheral focal adhesion. In contrast, glucose stimulation induces cell spreading, actin remodeling, and point-like adhesions that contain phospho-FAK and phosphopaxillin, located in small protrusions. Rat primary β-cells and mouse insulinomas showed an adhesion remodeling during GSIS resulting from autocrine insulin/IGF2 and AKT1 signaling. However, under starving conditions, the maintenance of stress fibers and the large adhesion phenotype required autocrine IGF2-IGF1 receptor signaling mediated by AKT2 and elevated FAK-kinase activity and ROCK-RhoA levels but low levels of paxillin phosphorylation. This starved adhesion phenotype prevented excessive insulin granule release to maintain low insulin secretion during fasting. Thus, deregulation of the IGF2 and adhesion-mediated signaling may explain dysfunctions observed in diabetes., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Arous et al.)

The unfolded protein response (UPR) plays a central role in regulating endoplasmic reticulum (ER) and global cellular physiology in response to pathologic ER stress. The UPR is comprised of three signaling pathways activated downstream of the ER membrane proteins IRE1, ATF6, and PERK. Once activated, these proteins initiate transcriptional and translational signaling that functions to alleviate ER stress, adapt cellular physiology, and dictate cell fate. Imbalances in UPR signaling are implicated in the pathogenesis of numerous, etiologically-diverse diseases, including many neurodegenerative diseases, protein misfolding diseases, diabetes, ischemic disorders, and cancer. This has led to significant interest in establishing pharmacologic strategies to selectively modulate IRE1, ATF6, or PERK signaling to both ameliorate pathologic imbalances in UPR signaling implicated in these different diseases and define the importance of the UPR in diverse cellular and organismal contexts. Recently, there has been significant progress in the identification and characterization of UPR modulating compounds, providing new opportunities to probe the pathologic and potentially therapeutic implications of UPR signaling in human disease. Here, we describe currently available UPR modulating compounds, specifically highlighting the strategies used for their discovery and specific advantages and disadvantages in their application for probing UPR function. Furthermore, we discuss lessons learned from the application of these compounds in cellular and in vivo models to identify favorable compound properties that can help drive the further translational development of selective UPR modulators for human disease., Competing Interests: Conflict of interest—R. L. W. is an inventor on patents filed for the ATF6 activators AA147 and AA263 described in this review. Both R. L. W. and J. M. D. G. are inventors on a patent filed for the IRE1/XBP1s activator IXA4 described in this review., (© 2020 Grandjean and Wiseman.)

Branched-chain α-keto acids (BCKAs) are catabolites of branched-chain amino acids (BCAAs). Intracellular BCKAs are cleared by branched-chain ketoacid dehydrogenase (BCKDH), which is sensitive to inhibitory phosphorylation by BCKD kinase (BCKDK). Accumulation of BCKAs is an indicator of defective BCAA catabolism and has been correlated with glucose intolerance and cardiac dysfunction. However, it is unclear whether BCKAs directly alter insulin signaling and function in the skeletal and cardiac muscle cell. Furthermore, the role of excess fatty acids (FAs) in perturbing BCAA catabolism and BCKA availability merits investigation. By using immunoblotting and ultra-performance liquid chromatography MS/MS to analyze the hearts of fasted mice, we observed decreased BCAA-catabolizing enzyme expression and increased circulating BCKAs, but not BCAAs. In mice subjected to diet-induced obesity (DIO), we observed similar increases in circulating BCKAs with concomitant changes in BCAA-catabolizing enzyme expression only in the skeletal muscle. Effects of DIO were recapitulated by simulating lipotoxicity in skeletal muscle cells treated with saturated FA, palmitate. Exposure of muscle cells to high concentrations of BCKAs resulted in inhibition of insulin-induced AKT phosphorylation, decreased glucose uptake, and mitochondrial oxygen consumption. Altering intracellular clearance of BCKAs by genetic modulation of BCKDK and BCKDHA expression showed similar effects on AKT phosphorylation. BCKAs increased protein translation and mTORC1 activation. Pretreating cells with mTORC1 inhibitor rapamycin restored BCKA's effect on insulin-induced AKT phosphorylation. This study provides evidence for FA-mediated regulation of BCAA-catabolizing enzymes and BCKA content and highlights the biological role of BCKAs in regulating muscle insulin signaling and function., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Biswas et al.)

Endocannabinoid signaling plays a regulatory role in various (neuro)biological functions. 2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid, and although its canonical biosynthetic pathway involving phosphoinositide-specific phospholipase C and diacylglycerol lipase α is known, alternative pathways remain unsettled. Here, we characterize a noncanonical pathway implicating glycerophosphodiesterase 3 (GDE3, from GDPD2 gene). Human GDE3 expressed in HEK293T cell membranes catalyzed the conversion of lysophosphatidylinositol (LPI) into monoacylglycerol and inositol-1-phosphate. The enzyme was equally active against 1-acyl and 2-acyl LPI. When using 2-acyl LPI, where arachidonic acid is the predominant fatty acid, LC-MS analysis identified 2-AG as the main product of LPI hydrolysis by GDE3. Furthermore, inositol-1-phosphate release into the medium occurred upon addition of LPI to intact cells, suggesting that GDE3 is actually an ecto-lysophospholipase C. In cells expressing G-protein-coupled receptor GPR55, GDE3 abolished 1-acyl LPI-induced signaling. In contrast, upon simultaneous ex-pression of GDE3 and cannabinoid receptor CB2, 2-acyl LPI evoked the same signal as that induced by 2-AG. These data strongly suggest that, in addition to degrading the GPR55 LPI ligand, GDE3 can act as a switch between GPR55 and CB2 signaling. Coincident with a major expression of both GDE3 and CB2 in the spleen, spleens from transgenic mice lacking GDE3 displayed doubling of LPI content compared with WT mice. Decreased production of 2-AG in whole spleen was also observed, supporting the in vivo relevance of our findings. These data thus open a new research avenue in the field of endocannabinoid generation and reinforce the view of GPR55 and LPI being genuine actors of the endocannabinoid system., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Briand-Mésange et al.)

Muscle atrophy is regulated by the balance between protein degradation and synthesis. FOXO1, a transcription factor, helps to determine this balance by activating pro-atrophic gene transcription when present in muscle fiber nuclei. Foxo1 nuclear efflux is promoted by AKT-mediated Foxo1 phosphorylation, eliminating FOXO1's atrophy-promoting effect. AKT activation can be promoted by insulin-like growth factor 1 (IGF1) or insulin via a pathway including IGF1 or insulin, phosphatidylinositol 3-kinase, and AKT. We used confocal fluorescence time-lapse imaging of FOXO1-GFP in adult isolated living muscle fibers maintained in culture to explore the effects of IGF1 and insulin on FOXO1-GFP nuclear efflux with and without pharmacological inhibitors. We observed that although AKT inhibitor blocks the IGF1- or insulin-induced effect on FOXO1 nuclear efflux, phosphatidylinositol 3-kinase inhibitors, which we show to be effective in these fibers, do not. We also found that inhibition of the protein kinase ACK1 or ATM contributes to the suppression of FOXO1 nuclear efflux after IGF1. These results indicate a novel pathway that has been unexplored in the IGF1- or insulin-induced regulation of FOXO1 and present information useful both for therapeutic interventions for muscle atrophy and for further investigative areas into insulin insensitivity and type 2 diabetes., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Russell and Schneider.)

The contraction and relaxation of the heart is controlled by stimulation of the β1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of β 1 -AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance., Competing Interests: Conflict of interest—The authors declare that they have no conflict of interest with the content of this article., (© 2020 Diering et al.)

Interaction of transforming growth factor-β (TGFβ)-induced canonical signaling with the noncanonical kinase cascades regulates glomerular hypertrophy and matrix protein deposition, which are early features of glomerulosclerosis. However, the specific target downstream of the TGFβ receptor involved in the noncanonical signaling is unknown. Here, we show that TGFβ increased the catalytic loop phosphorylation of platelet-derived growth factor receptor β (PDGFRβ), a receptor tyrosine kinase expressed abundantly in glomerular mesangial cells. TGFβ increased phosphorylation of the PI 3-kinase-interacting Tyr-751 residue of PDGFRβ, thus activating Akt. Inhibition of PDGFRβ using a pharmacological inhibitor and siRNAs blocked TGFβ-stimulated phosphorylation of proline-rich Akt substrate of 40 kDa (PRAS40), an intrinsic inhibitory component of mTORC1, and prevented activation of mTORC1 in the absence of any effect on Smad 2/3 phosphorylation. Expression of constitutively active myristoylated Akt reversed the siPDGFRβ-mediated inhibition of mTORC1 activity; however, co-expression of the phospho-deficient mutant of PRAS40 inhibited the effect of myristoylated Akt, suggesting a definitive role of PRAS40 phosphorylation in mTORC1 activation downstream of PDGFRβ in mesangial cells. Additionally, we demonstrate that PDGFRβ-initiated phosphorylation of PRAS40 is required for TGFβ-induced mesangial cell hypertrophy and fibronectin and collagen I (α2) production. Increased activating phosphorylation of PDGFRβ is also associated with enhanced TGFβ expression and mTORC1 activation in the kidney cortex and glomeruli of diabetic mice and rats, respectively. Thus, pursuing TGFβ noncanonical signaling, we identified how TGFβ receptor I achieves mTORC1 activation through PDGFRβ-mediated Akt/PRAS40 phosphorylation to spur mesangial cell hypertrophy and matrix protein accumulation. These findings provide support for targeting PDGFRβ in TGFβ-driven renal fibrosis., Competing Interests: Conflict of interest—The authors declare that they do not have any conflicts of interest with the contents of the article.

Surface-exposed Toll-like receptors (TLRs) such as TLR2 and TLR4 survey the extracellular environment for pathogens. TLR activation initiates the production of various cytokines and chemokines, including type I interferons (IFN-I). Downstream of TLR4, IFNβ secretion is only vigorously triggered in macrophages when the receptor undergoes endocytosis and switches signaling adaptor; surface TLR4 engagement predominantly induces proinflammatory cytokines via the signaling adaptor MyD88. It is unclear whether this dichotomy is generally applicable to other TLRs, cell types, or differentiation states. Here, we report that diverse TLR2 ligands induce an IFN-I response in human monocyte-like cells, but not in differentiated macrophages. This TLR2-dependent IFN-I signaling originates from the cell surface and depends on MyD88; it involves combined activation of the transcription factors IRF3 and NF-κB, driven by the kinases TBK1 and TAK1-IKKβ, respectively. TLR2-stimulated monocytes produced modest IFNβ levels that caused productive downstream signaling, reflected by STAT1 phosphorylation and expression of numerous interferon-stimulated genes. Our findings reveal that the outcome of TLR2 signaling includes an IFN-I response in human monocytes, which is lost upon macrophage differentiation, and differs mechanistically from IFN-I-induction through TLR4. These findings point to molecular mechanisms tailored to the differentiation state of a cell and the nature of receptors activated to control and limit TLR-triggered IFN-I responses., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Oosenbrug et al.)

The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the devastating COVID-19 lung disease pandemic. Here, we tested the inhibitory activities of the antiviral interferons of type I (IFN-α) and type III (IFN-λ) against SARS-CoV-2 and compared them with those against SARS-CoV-1, which emerged in 2003. Using two mammalian epithelial cell lines (human Calu-3 and simian Vero E6), we found that both IFNs dose-dependently inhibit SARS-CoV-2. In contrast, SARS-CoV-1 was restricted only by IFN-α in these cell lines. SARS-CoV-2 generally exhibited a broader IFN sensitivity than SARS-CoV-1. Moreover, ruxolitinib, an inhibitor of IFN-triggered Janus kinase/signal transducer and activator of transcription signaling, boosted SARS-CoV-2 replication in the IFN-competent Calu-3 cells. We conclude that SARS-CoV-2 is sensitive to exogenously added IFNs. This finding suggests that type I and especially the less adverse effect-prone type III IFN are good candidates for the management of COVID-19., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Felgenhauer et al.)

Bile acids (BAs) comprise heterogenous amphipathic cholesterol-derived molecules that carry out physicochemical and signaling functions. A major site of BA action is the terminal ileum, where enterocytes actively reuptake BAs and express high levels of BA-sensitive nuclear receptors. BA pool size and composition are affected by changes in metabolic health, and vice versa. One of several factors that differentiate BAs is the presence of a hydroxyl group on C12 of the steroid ring. 12α-Hydroxylated BAs (12HBAs) are altered in multiple disease settings, but the consequences of 12HBA abundance are incompletely understood. We employed mouse primary ileum organoids to investigate the transcriptional effects of varying 12HBA abundance in BA pools. We identified Slc30a10 as one of the top genes differentially induced by BA pools with varying 12HBA abundance. SLC30A10 is a manganese efflux transporter critical for whole-body manganese excretion. We found that BA pools, especially those low in 12HBAs, induce cellular manganese efflux and that Slc30a10 induction by BA pools is driven primarily by lithocholic acid signaling via the vitamin D receptor. Administration of lithocholic acid or a vitamin D receptor agonist resulted in increased Slc30a10 expression in mouse ileum epithelia. These data demonstrate a previously unknown role for BAs in intestinal control of manganese homeostasis., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Ahmad et al.)

The supplementation of royal jelly (RJ) is known to provide a variety of health benefits, including anti-inflammatory and anti-obesity effects. RJ treatment also reportedly protects against bone loss, but no single factor in RJ has yet been identified as an anti-osteoporosis agent. Here we fractionated RJ and identified 10-hydroxy-2-decenoic acid (10H2DA) as a key component involved in inhibiting osteoclastogenesis based on mass spectrometric analysis. We further demonstrated free fatty acid receptor 4 (FFAR4) as directly interacting with 10H2DA; binding of 10H2DA to FFAR4 on osteoclasts inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced activation of NF-κB signaling, thereby attenuating the induction of nuclear factor of activated T cells (NFAT) c1, a key transcription factor for osteoclastogenesis. Oral administration of 10H2DA attenuated bone resorption in ovariectomized mice. These results suggest a potential therapeutic approach of targeting osteoclast differentiation by the supplementation of RJ, and specifically 10H2DA, in cases of pathological bone loss such as occur in postmenopausal osteoporosis., Competing Interests: Conflict of interest—T. N. received joint research funds from MORIKAWA KENKODO CO., LTD., (© 2020 Tsuchiya et al.)

Mediator complex subunit 16 (MED16) is a component of the mediator complex and functions as a coactivator in transcriptional events at almost all RNA polymerase II-dependent genes. In this study, we report that the expression of MED16 is markedly decreased in papillary thyroid cancer (PTC) tumors compared with normal thyroid tissues. In vitro , MED16 overexpression in PTC cells significantly inhibited cell migration, enhanced sodium/iodide symporter expression and iodine uptake, and decreased resistance to radioactive 131 I (RAI). Conversely, PTC cells in which MED16 had been further knocked down (MED16 KD ) exhibited enhanced cell migration, epithelial-mesenchymal transition, and RAI resistance, accompanied by decreased sodium/iodide symporter levels. Moreover, cell signaling through transforming growth factor β (TGF-β) was highly activated after the MED16 knockdown. Similar results were obtained in MED12 KD PTC cells, and a co-immunoprecipitation experiment verified interactions between MED16 and MED12 and between MED16 and TGF-βR2. Of note, the application of LY2157299, a potent inhibitor of TGF-β signaling, significantly attenuated MED16 KD -induced RAI resistance both in vitro and in vivo In conclusion, our findings indicate that MED16 reduction in PTC contributes to tumor progression and RAI resistance via the activation of the TGF-β pathway., Competing Interests: Conflict of interest—No conflict of interest exists in the submission of this manuscript from all authors., (© 2020 Gao et al.)

The transcriptional coactivator YAP1 (yes-associated protein 1) regulates cell proliferation, cell-cell interactions, organ size, and tumorigenesis. Post-transcriptional modifications and nuclear translocation of YAP1 are crucial for its nuclear activity. The objective of this study was to elucidate the mechanism by which the steroid hormone androgen regulates YAP1 nuclear entry and functions in several human prostate cancer cell lines. We demonstrate that androgen exposure suppresses the inactivating post-translational modification phospho-Ser-127 in YAP1, coinciding with increased YAP1 nuclear accumulation and activity. Pharmacological and genetic experiments revealed that intact androgen receptor signaling is necessary for androgen's inactivating effect on phospho-Ser-127 levels and increased YAP1 nuclear entry. We also found that androgen exposure antagonizes Ser/Thr kinase 4 (STK4/MST1) signaling, stimulates the activity of protein phosphatase 2A, and thereby attenuates the phospho-Ser-127 modification and promotes YAP1 nuclear localization. Results from quantitative RT-PCR and CRISPR/Cas9-aided gene knockout experiments indicated that androgen differentially regulates YAP1-dependent gene expression. Furthermore, an unbiased computational analysis of the prostate cancer data from The Cancer Genome Atlas revealed that YAP1 and androgen receptor transcript levels correlate with each other in prostate cancer tissues. These findings indicate that androgen regulates YAP1 nuclear localization and its transcriptional activity through the androgen receptor-STK4/MST1-protein phosphatase 2A axis, which may have important implications for human diseases such as prostate cancer., Competing Interests: Conflict of interest—The authors declare no competing conflicts of interest with the content of this article., (© 2020 Cinar et al.)

Pancreatic cancer (PC) remains a therapeutic challenge because of its intrinsic and extrinsic chemoresistance mechanisms. Here, we report that C- X -C motif chemokine receptor 4 (CXCR4) and hedgehog pathways cooperate in PC chemoresistance via bidirectional tumor-stromal crosstalk. We show that when PC cells are co-cultured with pancreatic stellate cells (PSCs) they are significantly more resistant to gemcitabine toxicity than those grown in monoculture. We also demonstrate that this co-culture-induced chemoresistance is abrogated by inhibition of the CXCR4 and hedgehog pathways. Similarly, the co-culture-induced altered expression of genes in PC cells associated with gemcitabine metabolism, antioxidant defense, and cancer stemness is also reversed upon CXCR4 and hedgehog inhibition. We have confirmed the functional impact of these genetic alterations by measuring gemcitabine metabolites, reactive oxygen species production, and sphere formation in vehicle- or gemcitabine-treated monocultures and co-cultured PC cells. Treatment of orthotopic pancreatic tumor-bearing mice with gemcitabine alone or in combination with a CXCR4 antagonist (AMD3100) or hedgehog inhibitor (GDC-0449) displays reduced tumor growth. Notably, we show that the triple combination treatment is the most effective, resulting in nearly complete suppression of tumor growth. Immunohistochemical analysis of Ki67 and cleaved caspase-3 confirm these findings from in vivo imaging and tumor measurements. Our findings provide preclinical and mechanistic evidence that a combination of gemcitabine treatment with targeted inhibition of both the CXCR4 and hedgehog pathways improves outcomes in a PC mouse model., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Khan et al.)

Lytic replication of Epstein-Barr virus (EBV) is not only essential for its cell-to-cell spread and host-to-host transmission, but it also contributes to EBV-induced oncogenesis. Thus, blocking EBV lytic replication could be a strategy for managing EBV-associated diseases. Previously, we identified a series of natural lignans isolated from the roots of Saururus chinensis (Asian lizard's tail) that efficiently block EBV lytic replication and virion production with low cytotoxicity. In this study, we attempted to elucidate the molecular mechanism by which these lignans inhibit EBV lytic replication. We found that a representative compound, CSC27 (manassantin B), inhibits EBV lytic replication by suppressing the expression of EBV immediate-early gene BZLF1 via disruption of AP-1 signal transduction. Further analysis revealed that manassantin B specifically blocks the mammalian target of rapamycin complex 2 (mTORC2)-mediated phosphorylation of AKT Ser/Thr protein kinase at Ser-473 and protein kinase Cα (PKCα) at Ser-657. Using phosphoinositide 3-kinase-AKT-specific inhibitors for kinase mapping and shRNA-mediated gene silencing, we validated that manassantin B abrogates EBV lytic replication by inhibiting mTORC2 activity and thereby blocking the mTORC2-PKC/AKT-signaling pathway. These results suggest that mTORC2 may have utility as an antiviral drug target against EBV infections and also reveal that manassantin B has potential therapeutic value for managing cancers that depend on mTORC2 signaling for survival., (© 2020 Wang et al.)