Sensitive Transcription Factor Research Articles

Hypoxia-inducible Factor-1α Pathway in Cerebral Ischemia: From Molecular Mechanisms to Therapeutic Targets.

Ischemic injury to the brain can result in a variety of life-threatening conditions, mortality, or varying degrees of disability. Hypoxia-inducible factor 1α (HIF 1α), an oxygen- sensitive transcription factor that controls the adaptive metabolic response to hypoxia, is a critical constituent of cerebral ischemia. It participates in numerous processes, such as metabolism, proliferation, and angiogenesis, and plays a major role in cerebral ischemia. Through the use of a number of different search engines like Scopus, PubMed, Bentham, and Elsevier databases, a literature review was carried out for investigating the pharmacological modulation of HIF-1α pathways for the treatment of cerebral ischemia. Various signalling pathways, such as Mitogen-activated protein kinase (MAPK), Janus kinase/ signal transducers and activators (JAK/STAT), Phosphoinositide-3-kinase (PI3-K), and cAMPresponse element binding protein (CREB) play a vital role in modulation of HIF-1α pathway, which helps in preventing the pathogenesis of cerebral ischemia. The pharmacological modulation of the HIF-1α pathway via various molecular signalling pathways, such as PI3-K, MAPK, CREB, and JAK/STAT activators, offer a promising prospect for future interventions and treatment for cerebral ischemia.

The Aryl Hydrocarbon Receptor and Its Crosstalk: A Chemopreventive Target of Naturally Occurring and Modified Phytochemicals.

The aryl hydrocarbon receptor (AhR) is an environmentally sensitive transcription factor (TF) historically associated with carcinogenesis initiation via the activation of numerous carcinogens. Nowadays, the AhR has been attributed to multiple endogenous functions to maintain cellular homeostasis. Moreover, crosstalk, often reciprocal, has been found between the AhR and several other TFs, particularly estrogen receptors (ERs) and nuclear factor erythroid 2-related factor-2 (Nrf2). Adequate modulation of these signaling pathways seems to be an attractive strategy for cancer chemoprevention. Several naturally occurring and synthetically modified AhR or ER ligands and Nrf2 modulators have been described. Sulfur-containing derivatives of glucosinolates, such as indole-3-carbinol (I3C), and stilbene derivatives are particularly interesting in this context. I3C and its condensation product, 3,3'-diindolylmethane (DIM), are classic examples of blocking agents that increase drug-metabolizing enzyme activity through activation of the AhR. Still, they also affect multiple essential signaling pathways in preventing hormone-dependent cancer. Resveratrol is a competitive antagonist of several classic AhR ligands. Its analogs, with ortho-methoxy substituents, exert stronger antiproliferative and proapoptotic activity. In addition, they modulate AhR activity and estrogen metabolism. Their activity seems related to a number of methoxy groups introduced into the stilbene structure. This review summarizes the data on the chemopreventive potential of these classes of phytochemicals, in the context of AhR and its crosstalk modulation.

The cytoskeleton dynamics-dependent LINC complex in periodontal ligament stem cells transmits mechanical stress to the nuclear envelope and promotes YAP nuclear translocation

BackgroundPeriodontal ligament stem cells (PDLSCs) are important seed cells in tissue engineering and clinical applications. They are the priority receptor cells for sensing various mechanical stresses. Yes-associated protein (YAP) is a recognized mechanically sensitive transcription factor. However, the role of YAP in regulating the fate of PDLSCs under tension stress (TS) and its underlying mechanism is still unclear.MethodsThe effects of TS on the morphology and fate of PDLSCs were investigated using fluorescence staining, transmission electron microscopy, flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). Then qRT-PCR, western blotting, immunofluorescence staining and gene knockdown experiments were performed to investigate the expression and distribution of YAP and its correlation with PDLSCs proliferation. The effects of cytoskeleton dynamics on YAP nuclear translocation were subsequently explored by adding cytoskeleton inhibitors. The effect of cytoskeleton dynamics on the expression of the LINC complex was proved through qRT-PCR and western blotting. After destroying the LINC complex by adenovirus, the effects of the LINC complex on YAP nuclear translocation and PDLSCs proliferation were investigated. Mitochondria-related detections were then performed to explore the role of mitochondria in YAP nuclear translocation. Finally, the in vitro results were verified by constructing orthodontic tooth movement models in Sprague-Dawley rats.ResultsTS enhanced the polymerization and stretching of F-actin, which upregulated the expression of the LINC complex. This further strengthened the pull on the nuclear envelope, enlarged the nuclear pore, and facilitated YAP’s nuclear entry, thus enhancing the expression of proliferation-related genes. In this process, mitochondria were transported to the periphery of the nucleus along the reconstructed microtubules. They generated ATP to aid YAP’s nuclear translocation and drove F-actin polymerization to a certain degree. When the LINC complex was destroyed, the nuclear translocation of YAP was inhibited, which limited PDLSCs proliferation, impeded periodontal tissue remodeling, and hindered tooth movement.ConclusionsOur study confirmed that appropriate TS could promote PDLSCs proliferation and periodontal tissue remodeling through the mechanically driven F-actin/LINC complex/YAP axis, which could provide theoretical guidance for seed cell expansion and for promoting healthy and effective tooth movement in clinical practice.

Mechanoactivation of Single Stem Cells in Microgels Using a 3D-Printed Stimulation Device.

In this study, the novel 3D-printed pressure chamber for encapsulated single-cell stimulation (3D-PRESS) platform is introduced for the mechanical stimulation of single stem cells in 3D microgels. The custom-designed 3D-PRESS, allows precise pressure application up to 400kPa at the single-cell level. Microfluidics is employed to encapsulate single mesenchymal stem cells within ionically cross-linked alginate microgels with cell adhesion RGD peptides. Rigorous testing affirms the leak-proof performance of the 3D-PRESS device up to 400kPa, which is fully biocompatible. 3D-PRESS is implemented on mesenchymal stem cells for mechanotransduction studies, by specifically targeting intracellular calcium signaling and the nuclear translocation of a mechanically sensitive transcription factor. Applying 200kPa pressure on individually encapsulated stem cells reveals heightened calcium signaling in 3D microgels compared to conventional 2D culture. Similarly, Yes-associated protein (YAP) translocation into the nucleus occurs at 200kPa in 3D microgels with cell-binding RGD peptides unveiling the involvement of integrin-mediated mechanotransduction in singly encapsulated stem cells in 3D microgels. Combining live-cell imaging with precise mechanical control, the 3D-PRESS platform emerges as a versatile tool for exploring cellular responses to pressure stimuli, applicable to various cell types, providing novel insights into single-cell mechanobiology.

Effects of Cinnamon on Cancer Prevention and Progression

Cinnamon has been used medicinally for centuries, but recently research has suggested it may have a role in cancer prevention and potentially treatment. The search for alternative and subjunctive therapies is essential due to the public demand and the increasing cost of healthcare. Here we review the biologically active components of cinnamon and discuss the methods of potential cinnamon activity against cancer including: transcription factor regulation and kinase activity. Nuclear Factor kappa B (NF-\(𝜿\)B) is a stress sensitive transcription factor that regulates transcription of genes involved in tumor progression and is inhibited by cinnamon components. Another way that cinnamon inhibits tumor growth is by suppression of transcription factor activator protein -1 (AP-1) which interacts with genes responsible for apoptosis, metastasis and inflammation. Hypoxia-inducible transcription factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) are involved in angiogenesis, especially in the tumor microenvironment. The HIF-1-VEGF pathway are targeted by cinnamaldehyde, a compound found in cinnamon. Nuclear factor erythroid related factor 2 (Nrf2) is also examined and has been indicated to both potentially prevent cancer as well as induce it; various cinnamon derivatives target Nrf2. A cinnamaldehyde derivative has been implicated in a reduction of the mitogen-activated protein kinases (MAPKs), which are a group of kinases that regulate proliferation. Additionally, cinnamon components have been tied to cancer prevention by positively affecting the gut microbiome and inhibiting inflammation. The review concludes with a discussion of the future research needed and potential risk associated with cinnamon intake.

Hepatic ChREBP orchestrates intrahepatic carbohydrate metabolism to limit hepatic glucose 6-phosphate and glycogen accumulation in a mouse model for acute Glycogen Storage Disease type Ib

ObjectiveCarbohydrate Response Element Binding Protein (ChREBP) is a glucose 6-phosphate (G6P)-sensitive transcription factor that acts as a metabolic switch to maintain intracellular glucose and phosphate homeostasis. Hepatic ChREBP is well-known for its regulatory role in glycolysis, the pentose phosphate pathway, and de novo lipogenesis. The physiological role of ChREBP in hepatic glycogen metabolism and blood glucose regulation has not been assessed in detail, and ChREBP's contribution to carbohydrate flux adaptations in hepatic Glycogen Storage Disease type 1 (GSD I) requires further investigation. MethodsThe current study aimed to investigate the role of ChREBP as a regulator of glycogen metabolism in response to hepatic G6P accumulation, using a model for acute hepatic GSD type Ib. The immediate biochemical and regulatory responses to hepatic G6P accumulation were evaluated upon G6P transporter inhibition by the chlorogenic acid S4048 in mice that were either treated with a short hairpin RNA (shRNA) directed against ChREBP (shChREBP) or a scrambled shRNA (shSCR). Complementary stable isotope experiments were performed to quantify hepatic carbohydrate fluxes in vivo. ResultsShChREBP treatment normalized the S4048-mediated induction of hepatic ChREBP target genes to levels observed in vehicle- and shSCR-treated controls. In parallel, hepatic shChREBP treatment in S4048-infused mice resulted in a more pronounced accumulation of hepatic glycogen and further reduction of blood glucose levels compared to shSCR treatment. Hepatic ChREBP knockdown modestly increased glucokinase (GCK) flux in S4048-treated mice while it enhanced UDP-glucose turnover as well as glycogen synthase and phosphorylase fluxes. Hepatic GCK mRNA and protein levels were induced by shChREBP treatment in both vehicle- and S4048-treated mice, while glycogen synthase 2 (GYS2) and glycogen phosphorylase (PYGL) mRNA and protein levels were reduced. Finally, knockdown of hepatic ChREBP expression reduced starch domain binding protein 1 (STBD1) mRNA and protein levels while it inhibited acid alpha-glucosidase (GAA) activity, suggesting reduced capacity for lysosomal glycogen breakdown. ConclusionsOur data show that ChREBP activation controls hepatic glycogen and blood glucose levels in acute hepatic GSD Ib through concomitant regulation of glucose phosphorylation, glycogenesis, and glycogenolysis. ChREBP-mediated control of GCK enzyme levels aligns with corresponding adaptations in GCK flux. In contrast, ChREBP activation in response to acute hepatic GSD Ib exerts opposite effects on GYS2/PYGL enzyme levels and their corresponding fluxes, indicating that GYS2/PYGL expression levels are not limiting to their respective fluxes under these conditions.

Extracellular matrix stiffness controls cardiac fibroblast proliferation via the nuclear factor-Y (NF-Y) transcription factor

The proliferative expansion of cardiac fibroblasts (CF) contributes towards cardiac fibrosis, which results in myocardial stiffening, cardiac dysfunction, and heart failure. CF sense and respond to increased stiffness of their local extracellular matrix, modulating their phenotype towards increased collagen synthesis and higher proliferation, leading potentially to a vicious circle of positive feedback.Here we describe a novel mechanism that mediates increased CF proliferation in response to a pathologically stiff Exteracellular matrix (ECM). The mechanism we describe is independent of the well-characterised mechano-sensitive transcript factors, YAP-TEAD and MKL1-SRF, which our data indicate are only responsible for part of the genes induced by stiffened ECM. Instead, our data identify Nuclear Factor-Y (NF-Y) as a novel mechanosensitive transcription factor, which mediates enhanced CF proliferation in response to a stiff ECM. We show that levels of NF-YA protein, the major regulatory subunit of NF-Y, and NF-Y transcriptional activity, are increased by a stiff ECM. Indeed, NF-Y activity drives the expression of multiple cell-cycle genes. Furthermore, NF-YA protein levels are dependent on FAK signalling suggesting a mechanistic link to ECM composition. Consistent with its role as a mechano-sensor, inhibition of NF-Y using siRNA or dominant negative mutant blocks CF proliferation on plastic in vitro, which models a stiff ECM, whereas ectopic expression of NF-YA increases the proliferation of cells interacting under conditions that model a physiologically soft ECM.In summary, our data demonstrate that NF-Y is a biomechanically sensitive transcription factor that promotes CF proliferation in a model of pathologically stiffened ECM.

An antioxidant response element regulates the HIF1α axis in breast cancer cells.

The redox sensitive transcription factor NRF2 is a central regulator of the transcriptional response to reactive oxygen species (ROS). NRF2 is widely recognized for its ROS-responsive upregulation of antioxidant genes that are essential for mitigating the damaging effects of oxidative stress. However, multiple genome-wide approaches have suggested that NRF2's regulatory reach extends well beyond the canonical antioxidant genes, with the potential to regulate many noncanonical target genes. Recent work from our lab and others suggests HIF1A, which encodes the hypoxia-responsive transcription factor HIF1α, is one such noncanonical NRF2 target. These studies found that NRF2 activity is associated with high HIF1A expression in multiple cellular contexts, HIF1A expression is partially dependent on NRF2, and there is a putative NRF2 binding site (antioxidant response element, or ARE) approximately 30 kilobases upstream of HIF1A. These findings all support a model in which HIF1A is a direct target of NRF2, but did not confirm the functional importance of the upstream ARE in HIF1A expression. Here we use CRISPR/Cas9 genome editing to mutate this ARE in its genomic context and test the impact on HIF1A expression. We find that mutation of this ARE in a breast cancer cell line (MDA-MB-231) eliminates NRF2 binding and decreases HIF1A expression at the transcript and protein levels, and disrupts HIF1α target genes as well as phenotypes driven by these HIF1α targets. Taken together, these results indicate that this NRF2 targeted ARE plays an important role in the expression of HIF1A and activity of the HIF1α axis in MDA-MB-231cells.

Regulation by Nrf2 of IL-1β-induced inflammatory and oxidative response in VSMC and its relationship with TLR4.

Introduction: Vascular oxidative stress and inflammation play an important role in the pathogenesis of cardiovascular diseases (CVDs). The proinflammatory cytokine Interleukin-1β (IL-1β) participates in the vascular inflammatory and oxidative responses and influences vascular smooth muscle cells (VSMC) phenotype and function, as well as vascular remodelling in cardiovascular diseases. The Toll-like receptor 4 (TLR4) is also involved in the inflammatory response in cardiovascular diseases. A relationship between Interleukin-1β and Toll-like receptor 4 pathway has been described, although the exact mechanism of this interaction remains still unknown. Moreover, the oxidative stress sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) promotes the transcription of several antioxidant and anti-inflammatory genes. Nuclear factor-erythroid 2-related factor 2 activators have shown to possess beneficial effects in cardiovascular diseases in which oxidative stress and inflammation are involved, such as hypertension and atherosclerosis; however, the molecular mechanisms are not fully understood. Here, we analysed the role of Toll-like receptor 4 in the oxidative and inflammatory effects of Interleukin-1β as well as whether nuclear factor-erythroid 2-related factor 2 activation contributes to vascular alterations by modulating these effects. Materials: For this purpose, vascular smooth muscle cells and mice aortic segments stimulated with Interleukin-1β were used. Results: Interleukin-1β induces MyD88 expression while the Toll-like receptor 4 inhibitor CLI-095 reduces the Interleukin-1β-elicited COX-2 protein expression, reactive oxygen species (ROS) production, vascular smooth muscle cells migration and endothelial dysfunction. Additionally, Interleukin-1β increases nuclear factor-erythroid 2-related factor 2 nuclear translocation and expression of its downstream proteins heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and superoxide dismutase-2, by an oxidative stress-dependent mechanism; moreover, Interleukin-1β reduces the expression of the nuclear factor-erythroid 2-related factor 2 inhibitor Keap1. The nuclear factor-erythroid 2-related factor 2 activator tert-butylhydroquinone (tBHQ) reduces the effects of Interleukin-1β on the increased reactive oxygen species production and the expression of the proinflammatory markers (p-p38, p-JNK, p-c-Jun, COX-2), the increased cell proliferation and migration and prevents the Interleukin-1β-induced endothelial dysfunction in mice aortas. Additionally, tert-butylhydroquinone also reduces the increased MyD88 expression, NADPHoxidase activity and cell migration induced by lipopolysaccharide. Conclusions: In summary, this study reveals that Toll-like receptor 4 pathway contributes to the prooxidant and proinflammatory Interleukin-1β-induced effects. Moreover, activation of nuclear factor-erythroid 2-related factor 2 prevents the deleterious effects of Interleukin-1β, likely by reducing Toll-like receptor 4-dependent pathway. Although further research is needed, the results are promising as they suggest that nuclear factor-erythroid 2-related factor 2 activators might protect against the oxidative stress and inflammation characteristic of cardiovascular diseases.

Evaluation of HIF-1 Involvement in the BDNF and ProBDNF Signaling Pathways among Obstructive Sleep Apnea Patients.

Obstructive Sleep Apnea (OSA) is a chronic condition characterized by intermittent hypoxia associated with multiple comorbidities, including psychiatric disorders, such as depression, insomnia, and cognitive impairment. The brain-derived neurotrophic factor (BDNF) and proBDNF singling pathways have been shown to be involved in this group of diseases. Furthermore, their expression might be affected by hypoxia-inducible factor 1 (HIF-1), which is an oxygen sensitive transcription factor due to its alpha subunit. Therefore, this study aimed to evaluate the association between HIF-1α, BDNF, and proBDNF protein levels among OSA patients. This study included 40 individuals who underwent polysomnography (PSG) and were divided into the OSA group (n = 20; AHI ≥ 30) and healthy control (n = 20; AHI < 5) based on the apnea−hypopnea index (AHI). All participants had their peripheral blood collected in the evening before and the morning after the PSG. BDNF, proBDNF, and HIF-1α protein concertation measurements were performed using ELISA. No differences were found in BDNF, proBDNF, and HIF-1α protein levels between OSA and the control group, both in the evening and in the morning. In the OSA group, i.e., the linear regression model, the morning BDNF protein level was predicted by age (ß = −0.389, p = 0.023) and the mean SpO2 of desaturations during sleep (ß = −0.577, p = 0.002). This model accounted for 63.3% of the variability in the morning BDNF protein level (F = 14.639, p < 0.001). The morning proBDNF protein level was predicted by age (ß = −0.395, p = 0.033) and HIF-1α morning protein level (ß = −3.192, p = 0.005). This model accounted for 52.4% of the variability in the morning BDNF protein level (F = 9.355, p = 0.002). The obtained results suggest that the HIF-1 transcription factor might be involved in the pathway activated by proBDNF, which may have protective properties from hypoxia in OSA patients.

Mechanisms underlying Nrf2 nuclear translocation by non-lethal levels of hydrogen peroxide: p38 MAPK-dependent neutral sphingomyelinase2 membrane trafficking and ceramide/PKCζ/CK2 signaling.

Hydrogen peroxide is an aerobic metabolite playing a central role in redox signaling and oxidative stress. H2O2 could activate redox sensitive transcription factors, such as Nrf2, AP-1 and NF-κB by different manners. In some cells, treatment with non-lethal levels of H2O2 induces rapid activation of Nrf2, which upregulates expression of a set of genes involved in glutathione (GSH) synthesis and defenses against oxidative damage. It depends on two steps, the rapid translational activation of Nrf2 and facilitation of Nrf2 nuclear translocation. We review the molecular mechanisms by which H2O2 induces nuclear translocation of Nrf2 in cultured cells by highlighting the role of neutral sphingomyelinase 2 (nSMase2), a GSH sensor. H2O2 enters cells through aquaporin channels in the plasma membrane and is rapidly reduced to H2O by GSH peroxidases to consume cellular GSH, resulting in nSMase2 activation to generate ceramide. H2O2 also activates p38 MAP kinase, which enhances transfer of nSMase2 from perinuclear regions to plasma membrane lipid rafts to accelerate ceramide generation. Low levels of ceramide activate PKCζ, which then activates casein kinase 2 (CK2). These protein kinases are able to phosphorylate Nrf2 to stabilize and activate it. Notably, Nrf2 also binds to caveolin-1 (Cav1), which protects Nrf2 from Keap1-mediated degradation and limits Nrf2 nuclear translocation. We propose that Cav1serves as a signaling hub for the control of H2O2-mediated phosphorylation of Nrf2 by kinases, which results in release of Nrf2 from Cav1 to facilitate nuclear translocation. In summary, H2O2 induces GSH depletion which is recovered by Nrf2 activation dependent on p38/nSMase2/ceramide signaling.

Red Palm Oil Ameliorates Oxidative Challenge and Inflammatory Responses Associated with Lipopolysaccharide-Induced Hepatic Injury by Modulating NF-κβ and Nrf2/GCL/HO-1 Signaling Pathways in Rats.

Lipopolysaccharide (LPS), a well-conserved cell wall component of Gram positive bacteria, exerts its toxic effects via inducing oxidative and pro-inflammatory responses. Red palm oil (RPO) is a unique natural product with a balanced ratio of saturated and unsaturated fatty acids, with reported antioxidant and anti-inflammatory effects. In this study, we assess the protective effect and mechanistic action of RPO using a lipopolysaccharide (LPS)-induced hepatic injury model. Male Wistar rats were assigned into four groups (10 animals/group): normal control (NC), RPO, LPS and RPO + LPS. Animals in the RPO and RPO + LPS groups were administered RPO (200 μL/day) for 28 days. On the 27th day of experiment, animals in LPS and RPO + LPS groups were injected with LPS (0.5 mg/kg body weight). Animals were sacrificed 24 h later, and blood and liver tissues harvested for biochemical and molecular analysis. RPO resolved hepatic histological dysfunction induced by LPS, and lowered alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and γ-glutamyl transferase activities in the serum. Hepatic malondialdehyde and conjugated dienes, as well as pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6 and TNFα were significantly diminished (p < 0.05) by RPO pre-treatment. Activity of hepatic antioxidant enzymes including superoxide dismutase, glutathione reductase, glutathione peroxidase, as well as glutathione redox status (GSH:GSSG), and markers of antioxidant capacity that decreased as a result of LPS injection were improved by RPO pre-treatment. Mechanistically, RPO up-regulated mRNA expression of redox sensitive transcription factor Nrf2 and its downstream targets GCL and HO-1, while also suppressing the expression of NFκβ and associated inflammatory protein, Iκβ kinase (IκKβ). In conclusion, this study highlights the ameliorating effects of RPO against LPS-induced hepatic injury and revealed the Nrf2/GCL/HO-1 and NFκβ signaling axis as potential contributing mechanisms.

Glutathione peroxidase 2 is a metabolic driver of the tumor immune microenvironment and immune checkpoint inhibitor response

BackgroundThe existence of immunologically ‘cold tumors’ frequently found across a wide spectrum of tumor types represents a significant challenge for cancer immunotherapy. Cold tumors have poor baseline pan-leukocyte infiltration, including...

Nano-titanium dioxide inhalation exposure during gestation drives redox dysregulation and vascular dysfunction across generations

BackgroundPregnancy is associated with many rapid biological adaptations that support healthy development of the growing fetus. One of which is critical to fetal health and development is the coordination between maternal liver derived substrates and vascular delivery. This crucial adaptation can be potentially derailed by inhalation of toxicants. Engineered nanomaterials (ENM) are commonly used in household and industrial products as well as in medicinal applications. As such, the potential risk of exposure remains a concern, especially during pregnancy. We have previously reported that ENM inhalation leads to upregulation in the production of oxidative species. Therefore, we aimed to determine if F0 dam maternal nano-TiO2 inhalation exposure (exclusively) resulted in altered H2O2 production capacity and changes in downstream redox pathways in the F0 dams and subsequent F1 pups. Additionally, we investigated whether this persisted into adulthood within the F1 generation and how this impacted F1 gestational outcomes and F2 fetal health and development. We hypothesized that maternal nano-TiO2 inhalation exposure during gestation in the F0 dams would result in upregulated H2O2 production in the F0 dams as well as her F1 offspring. Additionally, this toxicological insult would result in gestational vascular dysfunction in the F1 dams yielding smaller F2 generation pups.ResultsOur results indicate upregulation of hepatic H2O2 production capacity in F0 dams, F1 offspring at 8 weeks and F1 females at gestational day 20. H2O2 production capacity was accompanied by a twofold increase in phosphorylation of the redox sensitive transcription factor NF-κB. In cell culture, naïve hepatocytes exposed to F1-nano-TiO2 plasma increased H2O2 production. Overnight exposure of these hepatocytes to F1 plasma increased H2O2 production capacity in a partially NF-κB dependent manner. Pregnant F1- nano-TiO2 females exhibited estrogen disruption (12.12 ± 3.1 pg/ml vs. 29.81 ± 8.8 pg/ml sham-control) and vascular dysfunction similar to their directly exposed mothers. F1-nano-TiO2 uterine artery H2O2 production capacity was also elevated twofold. Dysfunctional gestational outcomes in the F1-nano-TiO2 dams resulted in smaller F1 (10.22 ± 0.6 pups vs. sham-controls 12.71 ± 0.96 pups) and F2 pups (4.93 ± 0.47 g vs. 5.78 ± 0.09 g sham-control pups), and fewer F1 male pups (4.38 ± 0.3 pups vs. 6.83 ± 0.84 sham-control pups).ConclusionIn conclusion, this manuscript provides critical evidence of redox dysregulation across generations following maternal ENM inhalation. Furthermore, dysfunctional gestational outcomes are observed in the F1-nano-TiO2 generation and impact the development of F2 offspring. In total, this data provides strong initial evidence that maternal ENM exposure has robust biological impacts that persists in at least two generations.

APE1/Ref-1 Role in Inflammation and Immune Response.

Apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional enzyme that is essential for maintaining cellular homeostasis. APE1 is the major apurinic/apyrimidinic endonuclease in the base excision repair pathway and acts as a redox-dependent regulator of several transcription factors, including NF-κB, AP-1, HIF-1α, and STAT3. These functions render APE1 vital to regulating cell signaling, senescence, and inflammatory pathways. In addition to regulating cytokine and chemokine expression through activation of redox sensitive transcription factors, APE1 participates in other critical processes in the immune response, including production of reactive oxygen species and class switch recombination. Furthermore, through participation in active chromatin demethylation, the repair function of APE1 also regulates transcription of some genes, including cytokines such as TNFα. The multiple functions of APE1 make it an essential regulator of the pathogenesis of several diseases, including cancer and neurological disorders. Therefore, APE1 inhibitors have therapeutic potential. APE1 is highly expressed in the central nervous system (CNS) and participates in tissue homeostasis, and its roles in neurodegenerative and neuroinflammatory diseases have been elucidated. This review discusses known roles of APE1 in innate and adaptive immunity, especially in the CNS, recent evidence of a role in the extracellular environment, and the therapeutic potential of APE1 inhibitors in infectious/immune diseases.

REV-ERBα regulates age-related and oxidative stress-induced degeneration in retinal pigment epithelium via NRF2

Retinal pigment epithelium (RPE) dysfunction and atrophy occur in dry age-related macular degeneration (AMD), often leading to photoreceptor degeneration and vision loss. Accumulated oxidative stress during aging contributes to RPE dysfunction and degeneration. Here we show that the nuclear receptor REV-ERBα, a redox sensitive transcription factor, protects RPE from age-related degeneration and oxidative stress-induced damage. Genetic deficiency of REV-ERBα leads to accumulated oxidative stress, dysfunction and degeneration of RPE, and AMD-like ocular pathologies in aging mice. Loss of REV-ERBα exacerbates chemical-induced RPE damage, and pharmacological activation of REV-ERBα protects RPE from oxidative damage both in vivo and in vitro. REV-ERBα directly regulates transcription of nuclear factor erythroid 2-related factor 2 (NRF2) and its downstream antioxidant enzymes superoxide dismutase 1 (SOD1) and catalase to counter oxidative damage. Moreover, aged mice with RPE specific knockout of REV-ERBα also exhibit accumulated oxidative stress and fundus and RPE pathologies. Together, our results suggest that REV-ERBα is a novel intrinsic protector of the RPE against age-dependent oxidative stress and a new molecular target for developing potential therapies to treat age-related retinal degeneration.

Highly Reversible Tunable Thermal-Repressible Split-T7 RNA Polymerases (Thermal-T7RNAPs) for Dynamic Gene Regulation.

Temperature is a physical cue that is easy to apply, allowing cellular behaviors to be controlled in a contactless and dynamic manner via heat-inducible/repressible systems. However, existing heat-repressible systems are limited in number, rely on thermal sensitive mRNA or transcription factors that function at low temperatures, lack tunability, suffer delays, and are overly complex. To provide an alternative mode of thermal regulation, we developed a library of compact, reversible, and tunable thermal-repressible split-T7 RNA polymerase systems (Thermal-T7RNAPs), which fused temperature-sensitive domains of Tlpa protein with split-T7RNAP to enable direct thermal control of the T7RNAP activity between 30 and 42 °C. We generated a large mutant library with varying thermal performances via an automated screening framework to extend temperature tunability. Lastly, using the mutants, novel thermal logic circuitry was implemented to regulate cell growth and achieve active thermal control of the cell proportions within co-cultures. Overall, this technology expanded avenues for thermal control in biotechnology applications.

Systematic analysis of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs.

Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq (chromatin immunoprecipitation sequencing). We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPβ binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPβ at these locations tolerated changes in spacing ranging from 5 bp increase to >30 bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation.

BAP1 Downregulates NRF2 Target Genes and Exerts Anti-Tumorigenic Effects by Deubiquitinating KEAP1 in Lung Adenocarcinoma.

KELCH-ECH-associated protein 1 (KEAP1) is an adaptor protein of Cullin 3 (CUL3) E3 ubiquitin ligase that targets a redox sensitive transcription factor, NF-E2-related factor 2 (NRF2). BRCA1-associated protein 1 (BAP1) is a tumor suppressor and deubiquitinase whose mutations increase the risk of several types of familial cancers. In the present study, we have identified that BAP1 deubiquitinates KEAP1 by binding to the BTB domain. Lentiviral transduction of BAP1 decreased the expression of NRF2 target genes, suppressed the migration and invasion, and sensitized cisplatin-induced apoptosis in human lung adenocarcinoma (LUAD) A549 cells. Examination of the lung tissues in KrasG12D/+ mice demonstrated that the level of Bap1 and Keap1 mRNAs progressively decreases during lung tumor progression, and it is correlated with NRF2 activation and the inhibition of oxidative stress. Supporting this observation, lentiviral transduction of BAP1 decreased the growth of A549 xenografts in athymic nude mice. Transcriptome analysis of human lung tissues showed that the levels of Bap1 mRNA are significantly higher in normal samples than LUAD samples. Moreover, the expression of Bap1 mRNA is associated with a better survival of LUAD patients. Together, our study demonstrates that KEAP1 deubiquitination by BAP1 is novel tumor suppressive mechanism of LUAD.

Estimating the regulatory potential of DNA methylation in Alzheimer's disease

Epigenome-wide association studies (EWAS) often detect a large number of differentially methylated CpGs, many are located far from genes, complicating the interpretation of their functionalities. Therefore, there is a critical need to better understand the functional impact of these CpGs. Recent studies demonstrated methylated CpGs can affect gene transcription by either increase or decrease transcription factor binding strengths. To prioritize significant CpGs from EWAS, an integrative analysis that assesses the impact of CpG methylation on TF regulatory activities is proposed. We developed a new method and software, MethReg, that analyzes matched DNA-methylation and gene-expression data, along with external transcription factor (TF) binding information, to evaluate, prioritize, and annotate CpG sites with high regulatory potential. By simultaneous modeling three key elements that contribute to gene transcription (CpG methylation, target gene expression and TF activity), MethReg identifies TF-target gene associations that are present only in a subset of samples with high (or low) methylation levels at the CpG that influences TF activities, which can be missed in analyses that use all samples. We performed a MethReg analysis for the ROSMAP Alzheimer's Disease (AD) dataset with DNA methylation and RNA-seq data for 529 independent subjects. MethReg identified 60 methylation sensitive transcription factors, many of which are well-known regulators for AD such as TCF12, SPI1, NR3C1, CEBPB, GABPA, and others. Although many of these significant TFs have been previously implicated in AD pathology, their specific roles in transcription regulation and the identification of their targets in AD remain to be investigated. Currently available tools only identify the TFs but do not consider CpGs or provide detailed information on the relevant target genes. In contrast, MethReg fills this critical gap by nominating plausible TF-target associations that are mediated by DNA methylation. Therefore, MethReg analysis, which leverages additional gene expression data and provides more comprehensive information on transcription regulation for the TFs, complements existing approaches.