Nuclear Factor Κ-light Chain Enhancer Research Articles

Role of Orai-family channels in the activation and regulation of transcriptional activity.

Store operated Ca2+ entry (SOCE) is a cornerstone for the maintenance of intracellular Ca2+ homeostasis and the regulation of a variety of cellular functions. SOCE is mediated by STIM and Orai proteins following the activation of inositol 1,4,5-trisphosphate receptors. Then, a reduction of the endoplasmic reticulumintraluminal Ca2+ concentration is sensed by STIM proteins, which undergo a conformational change and activate plasma membraneCa2+ channels comprised by Orai proteins. STIM1/Orai-mediated Ca2+ signals are finely regulated and modulate the activity of different transcription factors, including certain isoforms of the nuclear factor of activated T-cells, the cAMP-response element binding protein, the nuclear factor κ-light chain-enhancer of activated B cells, c-fos, and c-myc. These transcription factors associate SOCE with a plethora of signaling events and cellular functions. Here we provide an overview of the current knowledge about the role of Orai channels in the regulation of transcription factors through Ca2+ -dependent signaling pathways.

Egg White Ovotransferrin Attenuates RANKL-Induced Osteoclastogenesis and Bone Resorption.

Ovotransferrin, a member of the transferrin family, is the second main protein found in egg white. Ovotransferrin was reported to have antimicrobial, antioxidant, and immunomodulating activities. The aim of this work was to characterize the cellular and molecular functions of egg white ovotransferrin on osteoclasts differentiation and function. Osteoclasts were prepared from mouse macrophage RAW 264.7 cells stimulated with receptor activator of nuclear factor κB ligand (RANKL). Ovotransferrin inhibited osteoclasts differentiation and the calcium–phosphate resorptive ability via the suppression of RANKL-induced nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Ovotransferrin induced apoptosis of matured osteoclasts, accompanied by increased expression of Bcl-2-like protein 11 (Bim) and Bcl-2-assoicated death promoter (Bad), but decreased expression of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra-large (Bcl-xl). We established a novel role of egg white ovotransferrin as an inhibitor of osteoclastogenesis, which may be used for the prevention of osteoporosis.

Proteomic analysis of aqueous humor in acute primary angle-closure glaucoma

Objective: To analyze the difference among expression of aqueous humor proteins in acute primary angle-closure glaucoma (APACG). Methods: Case-control study. The patients with APACG combined cataract (APACG with cataract group) and patients with cataract (cataract group), who had undertaken surgical treatment at the Tianjin Medical University Eye Hospital from October 2016 to June2017 were collected. Upon receipt of patient's consent, 50 μl of aqueous humor were collected with 1 ml syringe and No.1 needle through the surgical access during the surgery, and then injected into a sterile collection tube to be stored at -80 ℃. Those proteins extracted from aqueous humor were analyzed by quantitative proteomic mass spectrometry. The differential significance test was performed by Maxquant significances A approach. The differential proteins of the two groups were screened and determined with the conditions of P<0.05 and difference multiple>2. The functions and signal pathway of differential proteins in aqueous humor were annotated in biological big data, on the basis gene ontology (GO) and the Kyoto gene and genomic encyclopedia (KEGG) analyses. Results: There were 3 males and 7 females with an average age of (68±6) years in the APACG group. The cataract group included 2 males and 8 females with an average age of (71±8) years. There were no statistical differences in gender ratio and age between the two groups (both P>0.05). A total of 91 differential proteins were detected in this experiment, including 50 up-regulated proteins (annexinA1, vimentin, S100 calcium binding protein A8, interleukin 6, C reactive protein, laminin β2, etc.) and 41 down-regulated (keratin 85, γ-crystallin D, syntaxin-binding protein 5, semaphoring 4B, matrilin 2, cathepsin O, cadherin 4, semaphoring 3B, platelet-derived growth factor D, transforming growth factor β, etc.). On one hand, the functions of differential proteins involved in many aspects. AnnexinA1, CD163, S100 calcium-binding protein A8, C reactive protein, interleukin 6 are involved in the inflammatory reaction, cadherin 4 and laminin β2 regulate cell adhesion, matrilin 2, vimentin and laminin β2 participate in tissue fibrosis; on the other hand, KEGG analysis showed that the differential proteins participate diverse signaling pathways such as phosphatidylinositol-3-kinase-protein kinase B signaling pathway, transformation growth factor β signaling pathway, mitogen activated protein kinase signaling pathway, Toll-like receptor signaling pathway, the nuclear factor κ-light chain enhancer of the activated B cells signaling pathway, focal adhension and extracellular matrix receptor interaction pathway and so on. Conclusions: The expression of annexin A1 is significantly up-regulated in the aqueous humor in APACG, while some other factors such as transformation growth factor β, cadherin-4, and matrilin 2 are down-regulated. The change of proteins in aqueous humor is related with the outbreak of APACG. (Chin J Ophthalmol, 2019, 55: 687-694).

PI3K-AKT-mTOR and NFκB Pathways in Ovarian Cancer: Implications for Targeted Therapeutics.

Ovarian cancer is the most lethal gynecologic malignancy in the United States, with an estimated 22,530 new cases and 13,980 deaths in 2019. Recent studies have indicated that the phosphoinositol 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), as well as the nuclear factor-κ light chain enhancer of activated B cells (NFκB) pathways are highly mutated and/or hyper-activated in a majority of ovarian cancer patients, and are associated with advanced grade and stage disease and poor prognosis. In this review, we will investigate PI3K/AKT/mTOR and their interconnection with NFκB pathway in ovarian cancer cells.

FKBP8 inhibits virus-induced RLR-VISA signaling.

The mitochondrial antiviral signal protein mitochondrial antiviral signaling protein, also known as virus-induced signaling adaptor (VISA), plays a key role in regulating host innate immune signaling pathways. This study identifies FK506 binding protein 8 (FKBP8) as a candidate interacting protein of VISA through the yeast two-hybrid technique. The interaction of FKBP8 with VISA, retinoic acid inducible protein 1 (RIG-I), and IFN regulatory factor 3 (IRF3) was confirmed during viral infection in mammalian cells by coimmunoprecipitation. Overexpression of FKBP8 using a eukaryotic expression plasmid significantly attenuated Sendai virus-induced activation of the promoter interferons β (IFN-β), and transcription factors nuclear factor κ-light chain enhancer of activated B cells (NF-κB) and IFN-stimulated response element (ISRE). Overexpression of FKBP8 also decreased dimer-IRF3 activity, but enhanced virus replication. Conversely, knockdown of FKBP8 expression by RNA interference showed opposite effects. Further studies indicated that FKBP8 acts as a negative interacting partner to regulate RLR-VISA signaling by acting on VISA and TANK binding kinase 1 (TBK1). Additionally, FKBP8 played a negative role on virus-induced signaling by inhibiting the formation of TBK1-IRF3 and VISA-TRAF3 complexes. Notably, FKBP8 also promoted the degradation of TBK1, RIG-I, and TRAF3 resulting from FKBP8 reinforced Sendai virus-induced endogenous polyubiquitination of RIG-I, TBK1, and TNF receptor-associated factor 3 (TRAF3). Therefore, a novel function of FKBP8 in innate immunity antiviral signaling regulation was revealed in this study.

Co-factor-independent phosphoglycerate mutase of Leishmania donovani modulates macrophage signalling and promotes T-cell repertoires bearing epitopes for both MHC-I and MHC-II.

Immunoactivation depends upon the antigen potential to modulate T-cell repertoires. The present study has enumerated the effect of 61 kDa recombinant Leishmania donovani co-factor-independent phosphoglycerate mutase (rLd-iPGAM) on mononuclear cells of healthy and treated visceral leishmaniasis subjects as well as on THP-1 cell line. rLd-iPGAM stimulation induced higher expression of interleukin-1β (IL-1β) in the phagocytic cell, its receptor and CD69 on T-cell subsets. These cellular activations resulted in upregulation of host-protective cytokines IL-2, IL-12, IL-17, tumour necrosis factor-α and interferon-γ, and downregulation of IL-4, IL-10 and tumour growth factor-β. This immune polarization was also evidenced by upregulation of nuclear factor-κ light-chain enhancer of activated B cells p50 and regulated expression of suppressor of mother against decapentaplegic protein-4. rLd-iPGAM stimulation also promoted lymphocyte proliferation and boosted the leishmaniacidal activity of macrophages by upregulating reactive oxygen species. It also induced 1·8-fold higher release of nitric oxide (NO) by promoting the transcription of inducible nitric oxide synthase gene. Besides, in silico analysis suggested the presence of major histocompatibility complex class I and II restricted epitopes, which can proficiently trigger CD8+ and CD4+ cells, respectively. This study reports rLd-iPGAM as an effective immunoprophylactic agent, which can be used in future vaccine design.

Selective Lesioning of Nuclear Factor-κB Activated Cells in the Nucleus Accumbens Shell Attenuates Alcohol Place Preference.

Nuclear factor κ-light chain enhancer of activated B cells (NF-κB) is a transcription factor commonly associated with innate immunity and is activated by infection and inflammation. NF-κB has recently gained attention as a mediator of complex psychiatric phenomena such as stress and addiction. In regards to alcohol, most research on NF-κB has focused on neurotoxicity and few studies have explored the role of NF-κB in alcohol reward, reinforcement, or consumption. In these studies, we used conditioned place preference to assess the activity of NF-κB in response to rewarding doses of alcohol. To measure NF-κB activity we used a line of transgenic mice that express the LacZ gene under the control of an NF-κB-regulated promoter. In these animals, staining for β-galactosidase (β-gal) identifies cells in which NF-κB has been activated. We then used the Daun02 inactivation method to specifically silence NF-κB-expressing cells during place preference conditioning. Daun02 is an inactive prodrug that is converted to the inhibitory molecule daunorubicin by β-gal. After alcohol place conditioning, we observed increased β-gal staining in the nucleus accumbens (NAC) shell and dorsal raphe nucleus, and found that disruption of NF-κB-expressing cells using Daun02 attenuated the development of alcohol place preference when infused into the NAC shell following conditioning sessions. We found this effect to be regionally and temporally specific. These results suggest that, in addition to its role in alcohol-induced neurotoxicity, NF-κB mediates the development of alcohol place preference via its actions in the NAC shell.

The Roles of Reactive Oxygen Species and Nitric Oxide in Perfluorooctanoic Acid-Induced Developmental Cardiotoxicity and l-Carnitine Mediated Protection.

Perfluorooctanoic acid (PFOA) is an environmental contaminant that could induce developmental cardiotoxicity in a chicken embryo, which may be alleviated by l-carnitine. To explore the roles of reactive oxygen species (ROS) and nitric oxide (NO) in such changes and the potential effects of l-carnitine, fertile chicken eggs were exposed to PFOA via an air cell injection, with or without l-carnitine co-treatment. The ROS and NO levels in chicken embryo hearts were determined with electron spin resonance (ESR), and the protein levels of the nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) p65 and inducible nitric oxide synthase (iNOS) in chicken embryo hearts were assessed with western blotting. The results of ESR indicated that PFOA exposure induced an elevation in the ROS levels in ED19 chicken embryo hearts and hatchling chicken hearts, while l-carnitine could alleviate such changes. Meanwhile, increased NO levels were observed in ED19 embryo hearts and hatchling hearts following PFOA exposure, while l-carnitine co-treatment exerted modulatory effects. Western blotting revealed that p65 translocation in ED19 embryo hearts and hatchling hearts was enhanced by PFOA, while l-carnitine co-treatment alleviated such changes. iNOS expression levels in ED19 embryo hearts followed the same pattern as NO levels, while a suppression of expression was observed in hatchling hearts exposed to PFOA. ROS/NF-κB p65 and iNOS/NO seem to be involved in the late stage (ED19 and post hatch) of PFOA-induced developmental cardiotoxicity in a chicken embryo. l-carnitine could exert anti-oxidant and NO modulatory effects in the developing chicken embryo hearts, which likely contribute to its cardioprotective effects.

Rapidly cycling Lgr5+ stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk.

The majority of colon tumors are driven by aberrant Wnt signaling in intestinal stem cells, which mediates an efficient route toward initiating intestinal cancer. Natural lipophilic polyphenols and long-chain polyunsaturated fatty acids (PUFAs) generally suppress Wnt- and NF-κB- (nuclear factor-κ light-chain enhancer of activated B-cell) related pathways. However, the effects of these extrinsic agents on colonic leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5+) stem cells, the cells of origin of colon cancer, have not been documented to date. Therefore, we examined the effect of n-3 PUFA and polyphenol (curcumin) combination on Lgr5+ stem cells during tumor initiation and progression in the colon compared with an n-6 PUFA-enriched control diet. Lgr5-EGFP-IRES-creERT2 knock-in mice were fed diets containing n-6 PUFA (control), n-3 PUFA, n-6 PUFA+curcumin or n-3 PUFA+curcumin for 3 weeks, followed by 6 azoxymethane (AOM) injections, and terminated 17 weeks after the last injection. To further elucidate the effects of the dietary bioactives at the tumor initiation stage, Lgr5+ stem cells were also assessed at 12 and 24 h post AOM injection. Only n-3 PUFA+curcumin feeding reduced nuclear β-catenin in aberrant crypt foci (by threefold) compared with control at the progression time point. n-3 PUFA+curcumin synergistically increased targeted apoptosis in DNA-damaged Lgr5+ stem cells by 4.5-fold compared with control at 12 h and maximally reduced damaged Lgr5+ stem cells at 24 h, down to the level observed in saline-treated mice. Finally, RNAseq analysis indicated that p53 signaling in Lgr5+ stem cells from mice exposed to AOM was uniquely upregulated only following n-3 PUFA+curcumin cotreatment. These novel findings demonstrate that Lgr5+ stem cells are uniquely responsive to external dietary cues following the induction of DNA damage, providing a therapeutic strategy for eliminating damaged Lgr5+ stem cells to reduce colon cancer initiation.

Heavy metal mediated innate immune responses of the Indian green frog, Euphlyctis hexadactylus (Anura: Ranidae): Cellular profiles and associated Th1 skewed cytokine response

Immune cell and cytokine profiles in relation to metal exposure though much studied in mammals has not been adequately investigated in amphibians, due mainly to lack of suitable reagents for cytokine profiling in non-model species. However, interspecies cross reactivity of cytokines permitted us to assay levels of IFNγ, TNFα, IL6 and IL10in a common anuran, the Indian green frog (Euphlyctis hexadactylus), exposed to heavy metals (Cd, Cr, Cu, Zn and Pb, at ~5ppm each) under field and laboratory settings in Sri Lanka. Enumeration of immune cells in blood and melanomacrophages in the liver, assay of serum and hepatic cytokines, and Th1/Th2 cytokine polarisation were investigated.Immune cell counts indicated overall immunosuppression with decreasing total WBC and splenocyte counts while neutrophil/lymphocyte ratio increased with metal exposure, indicating metal mediated stress. Serum IL6 levels of metal exposed frogs reported the highest (~9360pg/mL) of all cytokines tested. Significantly elevated IFNγ production (P<0.05) was evident in heavy metal exposed frogs. Th1/Th2 cytokine ratio in both serum and liver tissue homogenates was Th1 skewed due to significantly higher production of pro-inflammatory cytokines, IFNγ in serum and TNFα in the liver (P<0.01).Metal mediated aggregations of melanomacrophages in the liver were positively and significantly (P<0.05) correlated with the hepatic expression of TNFα, IL6 and IL10 activity. Overall, Th1 skewed response may well be due to oxidative stress mediated nuclear factor κ-light chain enhancer of activated B cells (NFκB) which enhances the transcription of pro-inflammatory cytokines. Xenobiotic stress has recently imposed an unprecedented level of threat to wildlife, particularly to sensitive species such as amphibians. Therefore, understanding the interactions between physiological stress and related immune responses is fundamental to conserve these environmental sentinels in the face of emerging eco-challenges.

Chronic Fluoxetine Treatment Upregulates the Activity of the ERK1/2-NF-κB Signaling Pathway in the Hippocampus and Prefrontal Cortex of Rats Exposed to Forced-Swimming Stress

Objectives: The aim of this study was to explore whether or not the antidepressant actions of fluoxetine (FLX) are correlated with extracellular signal-regulated kinase 1 and 2 (ERK1/2) and nuclear factor κ-light chain enhancer of activated B cells (NF-κB) in the hippocampus (HC) and prefrontal cortex (PFC) of rats. Materials and Methods: A total of 108 male Sprague-Dawley rats were randomly divided into 6 groups of 18 rats each. Group 1 was the control group, while group 2 comprised the depressed model in which rats were subjected to 28 days of forced-swimming stress (FST); groups 3-6 were also subjected to 28 days of FST and treated with FLX once a day for 1 day (group 3; F1d), 1 week (group 4; F1w), 2 weeks (group 5; F2w), or 4 weeks (group 6; F4w). The control group was not subjected to FST or treated with FLX. Behavior tests that included the Morris water maze (MWM) and saccharin preference were performed, and ERK1/2 and NF-κB proteins were assayed using Western blot. Results: The rats in the control group and in groups 5 and 6 (F2w and F4w, respectively) had a significantly shorter average escape latency, needed more attempts in order to successfully cross the platform, and had a greater saccharin preference than those in the depressed group (p < 0.05). In the depressed group, the phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated NF-κB (p-NF-κB) expression in the HC and PFC were lower than in the control group (p < 0.05). Treatment with FLX reversed the changes in the expression of p-ERK1/2 and p-NF-κB in rats in the F2w and F4w groups. Conclusions: In this study, FLX treatment for 2 weeks or longer reversed the impaired spatial learning, memory, and anhedonia observed in the depressed model rats and upregulated the activities of the ERK1/2-NF-κB signaling pathway.

Knockdown of Eag1 Expression by RNA Interference Increases Chemosensitivity to Cisplatin in Ovarian Cancer Cells

Ether á go-go 1 (Eag1) is frequently highly expressed in various malignant cancers and its excessive expression is correlated with poor prognosis in various cancers. However, the relationship of Eag1 expression with the clinical outcome of patients having ovarian cancer treated with cisplatin-based adjuvant chemotherapy is still unknown. In this study, we measured the expression of Eag1 in ovarian cancer and investigated the association between cisplatin chemosensitivity of ovarian cancer cells and Eag1 expression level. We demonstrate that decreased expression of Eag1 correlates with favorable prognosis in patients treated with cisplatin-based adjuvant chemotherapy and predicts higher cisplatin sensitivity in ovarian cancer cells. In vitro, knockdown of Eag1 by small interfering RNA facilitated the sensitivity of ovarian cancer cells (SKOV3 and TYK) to cisplatin-induced apoptosis via nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) pathway. Furthermore, knockdown of Eag1 expression was associated with decreased expression of the P-glycoprotein without affecting multidrug resistance-associated protein 1 expression. Taken together, Eag1 may serve as a potential indicator to predict Eag1 chemosensitivity, and silencing Eag1 may represent a potential therapeutic strategy for ovarian cancer.

BAX inhibitor-1 is a Ca(2+) channel critically important for immune cell function and survival.

The endoplasmic reticulum (ER) serves as the major intracellular Ca(2+) store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca(2+) leak channel also implicated in the response against protein misfolding, thereby connecting the Ca(2+) store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca(2+) levels. When put into culture, purified knockout T cells and even more so B cells die spontaneously. This is preceded by increased activity of the mitochondrial initiator caspase-9 and correlated with a significant surge in mitochondrial Ca(2+) levels, suggesting an exhausted mitochondrial Ca(2+) buffer capacity as the underlying cause for cell death in vitro. In vivo, T-cell-dependent experimental autoimmune encephalomyelitis and B-cell-dependent antibody production are attenuated, corroborating the ex vivo results. These results suggest that BI-1 has a major role in the functioning of the adaptive immune system by regulating intracellular Ca(2+) homeostasis in lymphocytes.

Apurinic/Apyrimidinic Endonuclease/Redox Factor-1 (APE1/Ref-1) Redox Function Negatively Regulates NRF2

Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) (henceforth referred to as Ref-1) is a multifunctional protein that in addition to its base excision DNA repair activity exerts redox control of multiple transcription factors, including nuclear factor κ-light chain enhancer of activated B cells (NF-κB), STAT3, activator protein-1 (AP-1), hypoxia-inducible factor-1 (HIF-1), and tumor protein 53 (p53). In recent years, Ref-1 has emerged as a promising therapeutic target in cancer, particularly in pancreatic ductal carcinoma. Although a significant amount of research has centered on Ref-1, no wide-ranging approach had been performed on the effects of Ref-1 inhibition and transcription factor activity perturbation. Starting with a broader approach, we identified a previously unsuspected effect on the nuclear factor erythroid-related factor 2 (NRF2), a critical regulator of cellular defenses against oxidative stress. Based on genetic and small molecule inhibitor-based methodologies, we demonstrated that repression of Ref-1 potently activates NRF2 and its downstream targets in a dose-dependent fashion, and that the redox, rather than the DNA repair function of Ref-1 is critical for this effect. Intriguingly, our results also indicate that this pathway does not involve reactive oxygen species. The link between Ref-1 and NRF2 appears to be present in all cells tested in vitro, noncancerous and cancerous, including patient-derived tumor samples. In particular, we focused on understanding the implications of the novel interaction between these two pathways in primary pancreatic ductal adenocarcinoma tumor cells and provide the first evidence that this mechanism has implications for overcoming the resistance against experimental drugs targeting Ref-1 activity, with clear translational implications.

Oxyresveratrol suppresses lipopolysaccharide-induced inflammatory responses in murine macrophages.

Excessive inflammation is considered a critical factor in many human diseases. Oxyresveratrol(trans-2,3',4,5'-tetrahydroxystilbene), a natural hydroxystilbene, has been shown to possess antioxidant and free radical-scavenging activity. In this study, we investigated the effects of oxyresveratrol (OxyR) on the lipopolysaccharide (LPS)-induced production of inflammatory cytokines and mediators and further explored the mechanism of action in RAW264.7 murine macrophage cell line. Production of nitric oxide (NO), prostaglandin E2 (PGE2), messenger RNA (mRNA) and protein expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and granulocyte macrophage colony-stimulating factor (GM-CSF), phosphorylation of mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38), and the activation of nuclear factor κ-light chain enhancer of activated B cells (NFκB) with OxyR were assayed in LPS-stimulated RAW264.7 cells. OxyR inhibited the productions of NO, PGE2, IL-6, and GM-CSF significantly in LPS-stimulated RAW264.7 cells. OxyR suppressed mRNA and protein expressions of iNOS, COX-2, IL-6, and GM-CSF in LPS-stimulated RAW264.7 cells. OxyR suppressed the phosphorylation of Akt and JNK and p38 MAPKs and the translocation of NFκB p65 subunit into the nucleus. These results indicate that OxyR inhibits LPS-stimulated inflammatory responses though the blocking of MAPK and NFκB signaling pathway in macrophages, and suggest that OxyR possesses anti-inflammatory effects.

Effect of prostaglandin E2 on multidrug resistance transporters in human placental cells.

Prostaglandin (PG) E2, a major product of cyclooxygenase (COX)-2, acts as an immunomodulator at the maternal-fetal interface during pregnancy. It exerts biologic function through interaction with E-prostanoid (EP) receptors localized to the placenta. The activation of the COX-2/PGE2/EP signal pathway can alter the expression of the ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 [P-glycoprotein (Pgp); gene: ABCB1], and breast cancer resistance protein (BCRP; gene: ABCG2), which function to extrude drugs and xenobiotics from cells. In the placenta, PGE2-mediated changes in ABC transporter expression could impact fetal drug exposure. Furthermore, understanding the signaling cascades involved could lead to strategies for the control of Pgp and BCRP expression levels. We sought to determine the impact of PGE2 signaling mechanisms on Pgp and BCRP in human placental cells. The treatment of placental cells with PGE2 up-regulated BCRP expression and resulted in decreased cellular accumulation of the fluorescent substrate Hoechst 33342. Inhibiting the EP1 and EP3 receptors with specific antagonists attenuated the increase in BCRP. EP receptor signaling results in activation of transcription factors, which can affect BCRP expression. Although PGE2 decreased nuclear factor κ-light chain-enhancer of activated B activation and increased activator protein 1, chemical inhibition of these inflammatory transcription factors did not blunt BCRP up-regulation by PGE2. Though PGE2 decreased Pgp mRNA, Pgp expression and function were not significantly altered. Overall, these findings suggest a possible role for PGE2 in the up-regulation of placental BCRP expression via EP1 and EP3 receptor signaling cascades.

Acetylation at Lysine 183 of Progesterone Receptor by p300 Accelerates DNA Binding Kinetics and Transactivation of Direct Target Genes

The identification of lysine acetylation of steroid hormone receptors has previously been based on the presence of consensus motif (K/R)XKK. This study reports the discovery by mass spectrometry of a novel progesterone receptor acetylation site at Lys-183 that is not in the consensus motif. In vivo acetylation and mutagenesis experiments revealed that Lys-183 is a primary site of progesterone receptor (PR) acetylation. Lys-183 acetylation is enhanced by p300 overexpression and abrogated by p300 gene silencing, suggesting that p300 is the major acetyltransferase for Lys-183 acetylation. Furthermore, p300-mediated Lys-183 acetylation is associated with heightened PR activity. Accordingly, the acetylation-mimicking mutant PRB-K183Q exhibited accelerated DNA binding kinetics and greater activity compared with the wild-type PRB on genes containing progesterone response element. In contrast, Lys-183 acetylation had no influence on PR tethering effect on the nuclear factor κ-light chain enhancer of activated B cells (NFκB). Additionally, increases of Lys-183 acetylation by p300 overexpression or inhibition of deacetylation resulted in increases of Ser-294 phosphorylation levels. In conclusion, PR acetylation at Lys-183 by p300 potentiates PR activity through accelerated binding of its direct target genes without affecting PR tethering on other transcription factors. The effect may be mediated by enhancing Ser-294 phosphorylation.

Structural Basis and Targeting of the Interaction between Fibroblast Growth Factor-inducible 14 and Tumor Necrosis Factor-like Weak Inducer of Apoptosis

Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr(176), but not Trp(231), resulted in the loss of TWEAK binding to Fn14 substantiating Tyr(176) as the anchoring residue. Importantly, mutation of TWEAK at Tyr(176) did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor κ-light chain enhancer of activated B cell (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers.

Role of inflammation in oral carcinogenesis (Part II): CD8, FOXP3, TNF-α, TGF-β and NF-κB expression

Due to the frequent presence of inflammation in cases of carcinoma and its use as a parameter for the assessment of tumor aggressiveness, the role of inflammation in oral carcinogenesis was investigated. This was performed by evaluating the expression of cellular markers, cytokines and nuclear transcription factors that identify the cells that participate in the antitumor defense in cases of oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). A semi-quantitative immunohistochemical analysis was performed for the transcription factors cluster of differentiation 8 (CD8), forkhead box P3 (FOXP3), transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α and nuclear factor κ-light chain enhancer of activated B-cells (NF-κB), in cases of OED and OSCC. CD8, TGF-β, TNF-α and NF-κB participated in the processes of tumor transformation and progression. The presence of inflammatory infiltrate in cases of OED favors the transformation and invasion process when stromal TNF-α and NF-kB are overexpressed, as NF-kB activated by TNF-α during inflammation predisposes the lesion to transformation, functioning as a link between inflammation and cancer. The control of these inflammatory mediators may prevent malignant transformation in the oral cavity.

The c-Jun N-terminal Kinase (JNK)-binding Protein (JNKBP1) Acts as a Negative Regulator of NOD2 Protein Signaling by Inhibiting Its Oligomerization Process

NOD2 is one of the best characterized members of the cytosolic NOD-like receptor family. NOD2 is able to sense muramyl dipeptide, a specific bacterial cell wall component, and to subsequently induce various signaling pathways leading to NF-κB activation and autophagy, both events contributing to an efficient innate and adaptive immune response. Interestingly, loss-of-function NOD2 variants were associated with a higher susceptibility for Crohn disease, which highlights the physiological importance of proper regulation of NOD2 activity. We performed a biochemical screen to search for new NOD2 regulators. We identified a new NOD2 partner, c-Jun N-terminal kinase-binding protein 1 (JNKBP1), a scaffold protein characterized by an N-terminal WD-40 domain. JNKBP1, through its WD-40 domain, binds to NOD2 following muramyl dipeptide activation. This interaction attenuates NOD2-mediated NF-κB activation and IL-8 secretion as well as NOD2 antibacterial activity. JNKBP1 exerts its repressor effect by disturbing NOD2 oligomerization and RIP2 tyrosine phosphorylation, both steps required for downstream NOD2 signaling. We furthermore showed that JNKBP1 and NOD2 are co-expressed in the human intestinal epithelium and in immune cells recruited in the lamina propria, which suggests that JNKBP1 contributes to maintain NOD2-mediated intestinal immune homeostasis.