Transcriptional Upregulation Research Articles

Protein disulfide isomerase-mediated transcriptional upregulation of Nox1 contributes to vascular dysfunction in hypertension.

Nox1 signaling is a causal key element in arterial hypertension. Recently, we identified protein disulfide isomerase A1 (PDI) as a novel regulatory protein that regulates Nox1 signaling in VSMCs. Spontaneously hypertensive rats (SHR) have increased levels of PDI in mesenteric resistance arteries compared with Wistar controls; however, its consequences remain unclear. Herein, we investigated the role of PDI in mediating Nox1 transcriptional upregulation and its effects on vascular dysfunction in hypertension. We demonstrate that PDI contributes to the development of hypertension via enhanced transcriptional upregulation of Nox1 in vascular smooth muscle cells (VSMCs). We show for the first time that PDI sulfenylation by hydrogen peroxide contributes to EGFR activation in hypertension via increased shedding of epidermal growth factor-like ligands. PDI also increases intracellular calcium levels, and contractile responses induced by ANG II. PDI silencing or pharmacological inhibition in VSMCs significantly decreases EGFR activation and Nox1 transcription. Overexpression of PDI in VSMCs enhances ANG II-induced EGFR activation and ATF1 translocation to the nucleus. Mechanistically, PDI increases ATF1-induced Nox1 transcription and enhances the contractile responses to ANG II. Herein we show that ATF1 binding to Nox1 transcription putative regulatory regions is augmented by PDI. Altogether, we provide evidence that HB-EGF in SHR resistance vessels promotes the nuclear translocation of ATF1, under the control of PDI, and thereby induces Nox1 gene expression and increases vascular reactivity. Thus, PDI acts as a thiol redox-dependent enhancer of vascular dysfunction in hypertension and could represent a novel therapeutic target for the treatment of this disease.

CK2 expression in cancer and associated clinical characteristics.

e15156 Background: The protein casein kinase 2 (CK2) is a ubiquitously expressed, highly pleiotropic kinase. CK2 may have incredibly varied substrate specificity and catalytic activity, as it functions as both a tetrameric and monomeric enzyme. CK2 participates in many cellular processes such as cellular proliferation, growth, and survival, and has significant potential to assist in oncogenic signaling. We and others have demonstrated CK2 gene and protein overexpression, and sometimes underexpression, in a variety of cancers. Here we investigate how the expression of CK2 subunits in cancer is associated with clinical characteristics in several distinct cancer types. Methods: Data analysis was performed on 32 non-overlapping cancer datasets obtained from The Cancer Genome Atlas (TCGA) and Broad GDAC Firehose Legacy projects, one dataset from Cancer Cell Line Encyclopedia (CCLE), and normal tissue datasets from Gene Expression Omnibus. Statistical analysis was performed using RStudio to explore the relationship between the expression levels of the three CK2 alpha transcripts (CSNK2A1, CSNK2A2, and CSNK2A3) and beta transcripts (CSNK2B) with sample clinical characteristics provided by TCGA/Broad. Relationships between CK2 expression and cancer-type specific characteristics were also explored, such as KRAS mutations in colorectal adenocarcinoma and serum AFP at sample procurement in hepatocellular carcinoma. CCLE data was used to explore correlations between cancer-type dependent CK2 gene expression and half maximal inhibitory concentration (IC50) of over 250 anticancer drugs. Tumor Immune Estimation Resource (TIMER) 2.0 was used to assess the relationship between CK2 subunit expression and tumor immune infiltration. Results: As seen in our previous analysis, CK2 transcripts were upregulated in the majority of cancer types analyzed. We also found that CK2 transcript upregulation correlated with worse survival outcomes in several previously unstudied cancers. CK2 expression differed significantly between cancer subtypes such as in ductal carcinoma and lobular carcinoma in breast cancer. CK2 expression significantly correlated with tumor grade, TNM staging, clinical staging in certain cancer types. CK2 expression significantly correlated with altered response to various anticancer drugs in certain cancers. CK2 expression was associated with both increased and decreased immune infiltration in a variety of cancers. Conclusions: Overall, this study demonstrates that CK2 transcript expression correlates with a variety of cancer-type dependent characteristics and clinical outcomes. As a result, CK2 may be useful as a prognostic biomarker and therapeutic target.

The wall-associated receptor-like kinase CsWAKL01, positively regulated by the transcription factor CsWRKY53, confers resistance to citrus bacterial canker via regulation of phytohormone signaling.

Citrus bacterial canker (CBC) is a disease that poses a major threat to global citrus production and is caused by infection with Xanthomonas citri subsp. citri (Xcc). Wall-associated receptor-like kinase (WAKL) proteins play an important role in shaping plant resistance to various bacterial and fungal pathogens. In a previous report, CsWAKL01 was identified as a candidate Xcc-inducible gene found to be up-regulated in CBC-resistant citrus plants. However, the functional role of CsWAKL01 and the mechanisms whereby it may influence resistance to CBC have yet to be clarified. Here, CsWAKL01 was found to localize to the plasma membrane, and the overexpression of the corresponding gene in transgenic sweet oranges resulted in pronounced enhancement of CBC resistance, whereas its knockdown had the opposite effect. Mechanistically, the effect of CsWAKL01 was linked to its ability to reprogram jasmonic acid, salicylic acid, and abscisic acid signaling activity. CsWRKY53 was further identified as a transcription factor capable of directly binding to the CsWAKL01 promoter and inducing its transcriptional up-regulation. CsWRKY53 silencing conferred greater CBC susceptibility to infected plants. Overall, these data support a model wherein CsWRKY53 functions as a positive regulator of CsWAKL01 to enhance resistance to CBC via the reprogramming of phytohormone signaling. Together these results offer new insights into the mechanisms whereby WAKLs shape phytopathogen resistance while underscoring the potential value of targeting the CsWRKY53-CsWAKL01 axis when seeking to breed CBC-resistant citrus plant varieties.

Transcriptional Up-Regulation of FBXW7 by KCa1.1 K+ Channel Inhibition through the Nrf2 Signaling Pathway in Human Prostate Cancer LNCaP Cell Spheroid Model.

The tumor suppressor gene F-box and WD repeat domain-containing (FBXW) 7 reduces cancer stemness properties by promoting the protein degradation of pluripotent stem cell markers. We recently demonstrated the transcriptional repression of FBXW7 by the three-dimensional (3D) spheroid formation of several cancer cells. In the present study, we found that the transcriptional activity of FBXW7 was promoted by the inhibition of the Ca2+-activated K+ channel, KCa1.1, in a 3D spheroid model of human prostate cancer LNCaP cells through the Akt-Nrf2 signaling pathway. The transcriptional activity of FBXW7 was reduced by the siRNA-mediated inhibition of the CCAAT-enhancer-binding protein C/EBP δ (CEBPD) after the transfection of miR223 mimics in the LNCaP spheroid model, suggesting the transcriptional regulation of FBXW7 through the Akt-Nrf2-CEBPD-miR223 transcriptional axis in the LNCaP spheroid model. Furthermore, the KCa1.1 inhibition-induced activation of FBXW7 reduced (1) KCa1.1 activity and protein levels in the plasma membrane and (2) the protein level of the cancer stem cell (CSC) markers, c-Myc, which is a molecule degraded by FBXW7, in the LNCaP spheroid model, indicating that KCa1.1 inhibition-induced FBXW7 activation suppressed CSC conversion in KCa1.1-positive cancer cells.

Identification of RNAi-Related Genes and Transcriptome Assembly of Loblolly Pine (Pinus taeda, L.) Seedlings Exposed to Insect-Specific dsRNA

Considerable research has focused on gene silencing in tree-feeding insects, but how trees recognize and process double-stranded RNA (dsRNA) engineered to target plant pests is unknown. We performed transcriptomic assembly, preliminary differential expression analysis, and in silico annotation on loblolly pine (Pinus taeda, L.) seedlings exposed to southern pine beetle-specific dsRNA. This pilot study sought to elucidate the baseline response of seedlings challenged with insect-specific dsRNA. Treated and untreated seedlings were sequenced and following transcriptome assembly 20 RNAi-related proteins (RRPs) were annotated. Differential gene expression analysis conducted using DESeq2 followed by pathway enrichment revealed 7131 differentially expressed transcripts, of which 33% were upregulated and 67% were downregulated. Only two RRPs selected for analysis were upregulated in treated seedlings, showing a lack of detectable RNAi response with our methodology. Beyond RNAi-related proteins, pathway enrichment mapped to immune response systems and genetic and cellular processing. Upregulated transcripts included autophagy, amino sugar and nucleotide sugar metabolism, and plant hormone signal transduction. Downregulated transcripts included RNA degradation and fatty acid metabolism pathways. Multiple DICER-LIKE and ARGONAUTE proteins were also annotated in five other North American pines, revealing diversity among these crucial proteins. Understanding host plant response to RNAi-mediated pest control is essential to further develop this technology against tree pests.

Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.

Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.

Single nuclei RNA-seq reveals a medium spiny neuron glutamate excitotoxicity signature prior to the onset of neuronal death in an ovine Huntington's disease model.

Huntington's disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. The 73 polyglutamine repeat is somatically stable and therefore likely captures a prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. To better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. We have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in medium spiny neurons, the cell type preferentially lost early in HD. Further, we observed an upregulation of astrocytic glutamate uptake transporters and medium spiny neuron GABAA receptors, which may maintain glutamate homeostasis. Taken together, these observations support the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process but the threshold of toxicity may be regulated by several protective mechanisms. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death.

Upregulation of the AMPK-FOXO1-PDK4 pathway is a primary mechanism of pyruvate dehydrogenase activity reduction in tafazzin-deficient cells

Barth syndrome (BTHS) is a rare disorder caused by mutations in the TAFAZZIN gene. Previous studies from both patients and model systems have established metabolic dysregulation as a core component of BTHS pathology. In particular, features such as lactic acidosis, pyruvate dehydrogenase (PDH) deficiency, and aberrant fatty acid and glucose oxidation have been identified. However, the lack of a mechanistic understanding of what causes these conditions in the context of BTHS remains a significant knowledge gap, and this has hindered the development of effective therapeutic strategies for treating the associated metabolic problems. In the current study, we utilized tafazzin-knockout C2C12 mouse myoblasts (TAZ-KO) and cardiac and skeletal muscle tissue from tafazzin-knockout mice to identify an upstream mechanism underlying impaired PDH activity in BTHS. This mechanism centers around robust upregulation of pyruvate dehydrogenase kinase 4 (PDK4), resulting from hyperactivation of AMP-activated protein kinase (AMPK) and subsequent transcriptional upregulation by forkhead box protein O1 (FOXO1). Upregulation of PDK4 in tafazzin-deficient cells causes direct phospho-inhibition of PDH activity accompanied by increased glucose uptake and elevated intracellular glucose concentration. Collectively, our findings provide a novel mechanistic framework whereby impaired tafazzin function ultimately results in robust PDK4 upregulation, leading to impaired PDH activity and likely linked to dysregulated metabolic substrate utilization. This mechanism may underlie previously reported findings of BTHS-associated metabolic dysregulation.

Holistic transcriptional responses of Cordyceps militaris to different culture temperatures

Cordyceps militaris is a medicinal entomopathogenic fungus containing valuable biometabolites for pharmaceutical applications. Its genetic inheritance and environmental factors play a crucial role in the production of biomass enriched with cordycepin. While temperature is a crucial controlled parameter for fungal cultivation, its impacts on growth and metabolite biosynthesis remains poorly characterized. This study aimed to investigate the metabolic responses and cordycepin production of C. militaris strain TBRC6039 under various temperature conditions through transcriptome analysis. Among 9599 expressed genes, 576 genes were significantly differentially expressed at culture temperatures of 15 and 25 °C. The changes in the transcriptional responses induced by these temperatures were found in several metabolisms involved in nutrient assimilation and energy source, including amino acids metabolism (e.g., glycine, serine and threonine metabolism) and lipid metabolism (e.g., biosynthesis of unsaturated fatty acids and steroid biosynthesis). At the lower temperature (15 °C), the biosynthetic pathways of lipids, specifically ergosterol and squalene, were the target for maintaining membrane function by transcriptional upregulation. Our study revealed the responsive mechanisms of C. militaris in acclimatization to temperature conditions that provide an insight on physiological manipulation for the production of metabolites by C. militaris.

Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway

Oxidative stress-induced death of neurons and astrocytes contributes to the pathogenesis of numerous neurodegenerative diseases. While significant progress has been made in identifying neuroprotective molecules against neuronal oxidative damage, little is known about their counterparts for astrocytes. Prolactin (PRL), a hormone known to stimulate astroglial proliferation, viability, and cytokine expression, exhibits antioxidant effects in neurons. However, its role in protecting astrocytes from oxidative stress remains unexplored. Here, we investigated the effect of PRL against hydrogen peroxide (H2O2)-induced oxidative insult in primary cortical astrocyte cultures. Incubation of astrocytes with PRL led to increased enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX), resulting in higher total antioxidant capacity. Concomitantly, PRL prevented H2O2-induced cell death, reactive oxygen species accumulation, and protein and lipid oxidation. The protective effect of PRL upon H2O2-induced cell death can be explained by the activation of both signal transducer and activator of transcription 3 (STAT3) and NFE2 like bZIP transcription factor 2 (NRF2) transduction cascades. We demonstrated that PRL induced nuclear translocation and transcriptional upregulation of Nrf2, concurrently with the transcriptional upregulation of the NRF2-dependent genes heme oxygenase 1, Sod1, Sod2, and Gpx1. Pharmacological blockade of STAT3 suppressed PRL-induced transcriptional upregulation of Nrf2, Sod1 and Gpx1 mRNA, and SOD and GPX activities. Furthermore, genetic ablation of the PRL receptor increased astroglial susceptibility to H2O2-induced cell death and superoxide accumulation, while diminishing their intrinsic antioxidant capacity. Overall, these findings unveil PRL as a potent antioxidant hormone that protects astrocytes from oxidative insult, which may contribute to brain neuroprotection.

Resveratrol targeting NRF2 disrupts the binding between KEAP1 and NRF2-DLG motif to ameliorate oxidative stress damage in mice pulmonary infection

Ethnopharmacological relevanceThe root of Polygonum cuspidatum Sieb. et Zucc (PC), known as 'Huzhang' in the Chinese Pharmacopoeia, has been traditionally employed for its anti-inflammatory, antiviral, antimicrobial, and other biological activities. Polydatin (PD) and its aglycone, resveratrol (RES), are key pharmacologically active components responsible for exerting anti-inflammatory and antioxidant effects. However, its specific targets and action mechanisms remain unclear. Aim of the studyThe equilibrium of the KEAP1-NRF2 system serves as the primary protective response to oxidative and electrophilic stresses within the body, particularly in cases of acute lung injury caused by pathogenic microbial infection. In this study, the precise mechanisms by which RES alleviates oxidative stress damage in conjunction with NRF2 activators are discussed. Materials and methodsThe active components from PC were screened to evaluate their potential to inhibit reactive oxygen species (ROS) and activate antioxidant activity dependent on antioxidant response elements (ARE). RES was evaluated for its potential to alleviate the oxidative stress caused by pathogenic microbial infection. Functional probes were designed to study the RES distribution and identify its targets. A lipopolysaccharide (LPS)-induced oxidative injury model was used to evaluate the effects of RES on the KEAP1-NRF2/ARE pathway in RAW 264.7 cells. The interaction between RES and NRF2 was elucidated using drug-affinity responsive target stability (DARTS), cellular thermal shift assays (CETSA), co-immunoprecipitation (Co-IP), and microscale thermophoresis (MST) techniques. The key binding sites were predicted using molecular docking and validated in NRF2-knockdownand reconstructed cells. Finally, protective effects against pulmonary stress were verified in a mouse model of pathogenic infection. ResultsThe accumulation of RES in lung macrophages disrupted the binding between KEAP1 and NRF2, thereby preventing the ubiquitination degradation of NRF2 through its interaction with Ile28 on the NRF2-DLG motif. The activation of NRF2 resulted in the upregulation of nuclear transcription, enhances the expression of antioxidant genes dependent on ARE, suppresses ROS generation, and ameliorates oxidative damage both in vivo and in vitro. ConclusionThese findings shed light on the potential of RES to mitigate oxidative stress damage caused by pathogenic microorganism-induced lung infections and facilitate the discovery of novel small molecule modulators targeting the KEAP1-NRF2 DLG motif interaction.

Effects of molting on the expression of ecdysteroid responsive genes in the crustacean molting gland (Y-organ)

Ecdysteroid molting hormones coordinate arthropod growth and development. Binding of 20-hydroxyecdysone (20E) to ecdysteroid receptor EcR/RXR activates a cascade of nuclear receptor transcription factors that mediate tissue responses to hormone. Insect ecdysteroid responsive and Forkhead box class O (FOXO) transcription factor gene sequences were used to extract orthologs from blackback land crab (Gecarcinus lateralis) Y-organ (YO) transcriptome: Gl-Ecdysone Receptor (EcR), Gl-Broad Complex (Br-C), Gl-E74, Gl-Hormone Receptor 3 (HR3), Gl-Hormone Receptor 4 (HR4), Gl-FOXO, and Gl-Fushi tarazu factor-1 (Ftz-f1). Quantitative polymerase chain reaction quantified mRNA levels in tissues from intermolt animals and in YO of animals induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). Gl-EcR, Gl-Retinoid X Receptor (RXR), Gl-Br-C, Gl-HR3, Gl-HR4, Gl-E74, Gl-E75, Gl-Ftz-f1, and Gl-FOXO were expressed in all 10 tissues, with Gl-Br-C, Gl-E74, Gl-E75, and Gl-HR4 mRNA levels in the YO lower than those in most of the other tissues. In MLA animals, molting had no effect on Gl-Br-C, Gl-E74, and Gl-Ftz-f1 mRNA levels and little effect on Gl-EcR, Gl-E75, and Gl-HR4 mRNA levels. Gl-HR3 and Gl-FOXO mRNA levels were increased during premolt stages, while Gl-RXR mRNA level was highest during intermolt and premolt stages and lowest at postmolt stage. In ESA animals, YO mRNA levels were not correlated with hemolymph ecdysteroid titers. ESA had no effect on Gl-EcR, Gl-E74, Gl-HR3, Gl-HR4, Gl-Ftz-f1, and Gl-FOXO mRNA levels, while Gl-RXR, Gl-Br-C, and Gl-E75 mRNA levels were decreased at 3 days post-ESA. These data suggest that transcriptional up-regulation of Gl-FOXO and Gl-HR3 contributes to increased YO ecdysteroidogenesis during premolt. By contrast, transcriptional regulation of ecdysteroid responsive genes and ecdysteroidogenesis were uncoupled in the YO of ESA animals.

The temperature sensor TWA1 is required for thermotolerance in Arabidopsis

Plants exposed to incidences of excessive temperatures activate heat-stress responses to cope with the physiological challenge and stimulate long-term acclimation1,2. The mechanism that senses cellular temperature for inducing thermotolerance is still unclear3. Here we show that TWA1 is a temperature-sensing transcriptional co-regulator that is needed for basal and acquired thermotolerance in Arabidopsis thaliana. At elevated temperatures, TWA1 changes its conformation and allows physical interaction with JASMONATE-ASSOCIATED MYC-LIKE (JAM) transcription factors and TOPLESS (TPL) and TOPLESS-RELATED (TPR) proteins for repressor complex assembly. TWA1 is a predicted intrinsically disordered protein that has a key thermosensory role functioning through an amino-terminal highly variable region. At elevated temperatures, TWA1 accumulates in nuclear subdomains, and physical interactions with JAM2 and TPL appear to be restricted to these nuclear subdomains. The transcriptional upregulation of the heat shock transcription factor A2 (HSFA2) and heat shock proteins depended on TWA1, and TWA1 orthologues provided different temperature thresholds, consistent with the sensor function in early signalling of heat stress. The identification of the plant thermosensors offers a molecular tool for adjusting thermal acclimation responses of crops by breeding and biotechnology, and a sensitive temperature switch for thermogenetics.

GPR160 regulates the self-renewal and pluripotency of mouse embryonic stem cells via JAK1/STAT3 signaling pathway

G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and regulate various physiological and pathological processes. Despite extensive studies, the roles of GPCRs in mouse embryonic stem cells (mESCs) remain poorly understood. Here, we show that GPR160, a class A member of GPCRs, is dramatically downregulated concurrent with mESC differentiation into embryoid bodies in vitro. Knockdown of Gpr160 leads to downregulation of the expression of pluripotency-associated transcription factors and upregulation of the expression of lineage markers, accompanying with the arrest of the mESC cell-cycle in the G0/G1 phase. RNA-seq analysis shows that GPR160 participates in the JAK/STAT signaling pathway crucial for maintaining ESC stemness, and the knockdown of Gpr160 results in the downregulation of STAT3 phosphorylation level, which in turn is partially rescued by colivelin, a STAT3 activator. Consistent with these observations, GPR160 physically interacts with JAK1, and cooperates with leukemia inhibitory factor receptor (LIFR) and gp130 to activate the STAT3 pathway. In summary, our results suggest that GPR160 regulates mESC self-renewal and pluripotency by interacting with the JAK1–LIFR–gp130 complex to mediate the JAK1/STAT3 signaling pathway.

Ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation mediates female reproductive dysfunction induced by cold exposure

Ambient air temperature is a key factor affecting human health. Female reproductive disorders are representative health risk events under low temperature. However, the mechanism involving in cold-induced female reproductive disorders remains largely unknown. Female mice were intermittently exposed to cold conditions (4 °C) to address the health risk of low temperature on female reproductive system. Primary granulosa cells (GCs) were prepared and cultured under low temperature (35 °C) or exposed to β3-adrenoreceptor agonist, isoproterenol, to mimic the condition of cold exposure. Western-blot, RT-PCR, co-IP, ELISA, pharmacological inhibition or siRNA-mediated knockdown of target gene were performed to investigate the possible role of hormones, gap conjunction proteins, and ER stress sensor protein in regulating female reproductive disorders under cold exposure. Cold exposure induced estrous cycle disorder and follicular dysplasia in female mice, accompanying with abnormal upregulation of progesterone and its synthetic rate-limiting enzyme, StAR, in the ovarian granulosa cells. Under the same conditions, an increase in connexin 43 (CX43) expressions in the GCs was also observed, which contributed to elevated progesterone levels in the ovary. Moreover, ER stress sensor protein, PERK, was activated in the ovarian GCs after cold exposure, leading to the upregulation of downstream NRF2-dependent CX43 transcription and aberrant increase in progesterone synthesis. Most importantly, blocking PERK expression in vivo significantly inhibited NRF2/CX43/StAR/progesterone pathway activation in the ovary and efficiently rescued the prolongation of estrous cycle and the increase in follicular atresia of the female mice induced by cold stress. We have elucidated the mechanism of ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation in mediating female reproductive disorder under cold exposure. Targeting PERK might be helpful for maintaining female reproductive health under cold conditions.

Evolutionary conservation of receptor compensation for stem cell homeostasis in Solanaceae plants.

Stem cell homeostasis is pivotal for continuous and programmed formation of organs in plants. The precise control of meristem proliferation is mediated by the evolutionarily conserved signaling that encompasses complex interactions among multiple peptide ligands and their receptor-like kinases. Here, we identified compensation mechanisms involving the CLAVATA1 (CLV1) receptor and its paralogs, BARELY ANY MERISTEMs (BAMs), for stem cell proliferation in two Solanaceae species, tomato and groundcherry. Genetic analyses of higher-order mutants deficient in multiple receptor genes, generated via CRISPR-Cas9 genome editing, reveal that tomato SlBAM1 and SlBAM2 compensate for slclv1 mutations. Unlike the compensatory responses between orthologous receptors observed in Arabidopsis, tomato slclv1 mutations do not trigger transcriptional upregulation of four SlBAM genes. The compensation mechanisms within receptors are also conserved in groundcherry, and critical amino acid residues of the receptors associated with the physical interaction with peptide ligands are highly conserved in Solanaceae plants. Our findings demonstrate that the evolutionary conservation of both compensation mechanisms and critical coding sequences between receptor-like kinases provides a strong buffering capacity during stem cell homeostasis in tomato and groundcherry.

Effect of Phenol and Alkylamide Interaction on α-Glucosidase Inhibition and Cellular Antioxidant Activity during In Vitro Digestion: Using Szechuan Pepper (Zanthoxylum genus) as a Model.

Although recent evidence indicated significant phenol and alkylamide interaction in aqueous solutions, the gastrointestinal digestion influence of the combination remains unclear. This study aims to investigate phenol and alkylamide interaction during in vitro digestion, focusing on bioaccessibility and bioactivity, including α-glucosidase inhibition and cellular antioxidant activity. Additionally, the structural mechanism of phenol and alkylamide interaction during in vitro digestion was explored. The results indicated that the presence of phenols and alkylamides significantly increased or decreased their respective bioaccessibility, depending on the Zanthoxylum varieties. Furthermore, although antagonistic phenol/alkylamide interaction was evident during α-glucosidase inhibition, cellular oxidative stress alleviation, and antioxidant gene transcription upregulation, this effect weakened gradually as digestion progressed. Glycoside bond cleavage and the methylation of phenols as well as alkylamide isomerization and addition were observed during digestion, modifying the hydrogen bonding sites and interaction behavior. This study provided insights into the phenol/alkylamide interaction in the gastrointestinal tract.

Abstract PO1-24-05: EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis

Abstract Background: Metaplastic breast carcinomas (mBrCAs) are a highly aggressive subtype of triple negative breast cancer with histological evidence of deregulated differentiation towards non-glandular components. Previous studies have demonstrated that human mBrCA often exhibit activation of differentiation pathways, including canonical Wnt/b-catenin and EZH2-mediated transcriptional repression. Our lab has identified CCN6 as a tumor suppressor in mBrCA. MMTV-Cre;Ccn6fl/fl (CCN6KO) mice develop spindle mBrCAs and CCN6 is reduced/lost in 68% of human mBrCAs. Recently, we have demonstrated that the tumor suppressor function of CCN6 in spindle mBrCA requires activation of Wnt/b-catenin signaling pathway. Here, we tested the hypothesis that CCN6 KO leads to the upregulation of EZH2 histone methyltransferase promoting mBrCA. Furthermore, we investigated the requirement for the activation of the Wnt/b-catenin pathway in this mechanism. Methods: To test the effect of CCN6/b-catenin on EZH2 gene and protein expression we performed IHC, IF, qRT-PCR, ChIP-Seq, RNA-seq, invasion and adhesion assays, EZH2 reporter assay and immunoblots in mBrCA cell lines and MMTV-Cre;Ccn6fl/fl tumors. To investigate the role of CCN6KO-induced b-catenin activation on EZH2 activity and neoplastic functions we employed three independent approaches: i) Expression of a dominant-negative Tcf4 (dnTcf4) rescued with EZH2-WT, dSET and dNLS domain mutants versus vector in MMTV-Cre;Ccn6fl/fl tumor-derived cells; ii) Expression of a constitutively active mutant (S33Y) b-catenin in concert with treatment with recombinant human CCN6 (rhCCN6; 500 ug/ml) versus control; iii) Syngeneic orthotopic mammary tumor transplants of MMTV-Cre;Ccn6fl/fl were used for in vivo rescue experiments with rhCCN6 or BSA. To assess the therapeutic benefit of inhibiting EZH2 methyltransferase activity, MMTV-Cre;Ccn6fl/fl tumor cells (CCN6KO cells) were implanted orthotopically or intracardially in FVB mice, followed by treatment with EPZ-6438 (a selective EZH2 methyltransferase inhibitor) or vehicle. We tested CCN6, b-catenin, and EZH2 expression by IHC in a cohort of 27 human mBrCA tumor samples. Results: CCN6KO-induced b-catenin/TCF activation mediates EZH2 transcriptional upregulation and the deposition of repressive H3K27me3 in spindle mBrCAs. We found that the invasion program triggered by CCN6KO-induced Wnt/b-catenin requires EZH2 catalytic activity as WT-EZH2 (but not dSET-EZH2) rescued the reduced invasion of CCN6KO-dnTcf4 cells. RNA-seq and 3H3K27 ChIP-seq of CCN6KO-dnTcf4 cells transduced with EZH2-WT or dSET-EZH2 identify specific CCN6KO-b-catenin/Tcf targets that require EZH2 transcriptional repressor function. In vivo, administration of CCN6 protein to MMTV-Cre;Ccn6fl/fl tumor transplants reduces tumor growth and nuclear b-catenin, EZH2 and 3H3K27 in the tumors. Pharmacologic inhibition of EZH2 reduces the growth and metastasis of CCN6KO mBrCA tumors and improves survival. We identify a subset of human spindle mBrCA (54%) that display a CCN6Low/nuclear b-cat/EZH2High phenotype. Conclusion: We found a critical role for EZH2 activation in CCN6-deficient mBrCA tumor phenotypes via b-catenin/TCF Wnt canonical signaling. We demonstrate the effectiveness of pharmacological inhibition of EZH2 methyltransferase activity in reducing primary tumor growth and distant metastasis in mouse models of spindle mBrCA. In clinical samples, low CCN6 is significantly associated with activated b-catenin and high EZH2 in spindle mBrCAs compared to other subtypes. These data reveal a novel tumor suppressor mechanism of CCN6 and provide compelling evidence supporting the potential therapeutic value of CCN6 restoration, b-catenin or EZH2 inhibition as promising approaches for the treatment of spindle mBrCAs. Citation Format: Maria Gonzalez, Ahmad Eido, GIUSEPPINA AUGIMERI, Celina Kleer. EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-05.

Phenotyping the complex human peripheral blood (PB) B cell compartment using multimodal Cellular Indexing of Transcriptomes and Epitopes (CITE-seq) analysis.

Abstract Classifying the heterogeneous human B cell compartment into discrete subsets based on surface phenotype alone remains a major challenge in the field of B cell biology. Our lab has discovered a novel mouse IgM+IgDlow/- B cell subset (BDL) that induces Treg proliferation, making it a potentially significant therapeutic target for autoimmune diseases. But due to their rarity and lack of sufficient cell surface identifiers, a more definitive phenotype is required to track this B cell subset in humans. The objective of these studies was to characterize human IgDlow/- B cells from PB using multiomic CITE-seq analysis. Here, we adapted a standardized gating scheme (CD27xIgD → CD24xCD38) that captures all the major canonical human PB B cell subsets. This scheme allowed us to analyze FACS-purified CD19+IgM+IgDlow/- PB B cells using CITE-seq by targeting these four protein markers and recreate biaxial plots similar to our flow cytometry analyses. It is increasingly understood that mRNA levels within a cell do not correlate well with cell surface protein expression. This represents a major drawback to using a transcriptome-only scRNA-seq approach because any ‘novel’ cell populations discovered based on the upregulation of surface protein transcripts may prove impossible to validate. These studies have established a workflow for a multiomic approach to identify discrete human B cell subsets based on cell surface expression that can be analyzed for transcriptional differences.

Understanding molecular differences in endocrine and exocrine pancreatic tissue in healthy and unhealthy metabolic states

Type II diabetes (T2D) is a metabolic disorder characterized by hyperglycemia largely due the development of insulin-resistance, and in some cases, due to insuffcient insulin secretion. The endocrine portion of the pancreas, the islets of Langerhans, is composed of a diverse population of cells (beta, alpha, delta) that play vital roles in glucose homeostasis. Intercellular signaling events that occur within this diverse cell population play an active role in T2D progression. Therefore, to properly understand the progression of T2D it is critical to understand the how both the cellular neighborhoods and cell-cell communications within the islet of Langerhans and the surrounding exocrine (non-Islet) tissue contribute to the development of T2D. As the pancreas is a heterogenous organ, techniques allowing for the identification of cellular subpopulations within the tissue, such as single-cell RNA sequencing (scRNAseq), have been critical to answering questions about the pathogenesis of T2D. However, these techniques provide no insight into the anatomical and spatial context of cells, and therefore lack the ability to determine the influence of cellular neighborhoods in T2D pathogenesis. To begin to fill the gap in our understanding of how cellular neighborhoods and cell-cell communication impacts the progression of T2D, we utilized cutting edge technology that allows for the identification of RNA and protein within tissue at cellular and subcellular level, the Nanostring CosMx Spatial Molecular Imager. In this study, we aimed to determine if the location of individual cells within the islet (exocrine adjacent versus endocrine adjacent) contributes to gene expression changes that direct disease progression. To answer this question, we collected 200 human donor pancreases and analyzed them for 1000 RNA targets using the CosMX Spatial Molecular Imager. Ig. We then performed an analysis of RNA expression of exocrine cells bordering the islets of Langerhans and endocrine cells surrounding pancreatic vasculature was compared between healthy, metabolically heathy obese (MHO), and obese T2D groups. Preliminary analysis of these data yield novel insights into the transcriptomic profile of metabolically unhealthy tissues that may play a role in the progression of T2D. In particular, we observed an upregulation of mRNA transcripts associated with pro-inflammatory IL-21 and IL-22 signaling in the alpha cells from MHO donor pancreases. Furthermore, this upregulation is primarily observed in alpha cells in close proximity to acinar cells. Overall, these data will help pave the way to addressing previously unanswered questions in the context of T2D pathogenesis and unexplored cell-cell interactions that occur between the endocrine and exocrine spaces of the pancreas. Funding: Saint Louis University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.