Feed-forward Loop Research Articles

Ubiquitin-Specific Protease 15 Plays an Important Role in Controlling the Tolerance to Salt, Drought and Abscisic Acid in Arabidopsis thaliana

Ubiquitin-specific proteases (UBPs), the largest subfamily of deubiquitinating enzymes (DUBs), are critical for plant growth and development as well as abiotic-stress responses. In this study, we discovered that the expression of the ubiquitin-specific protease 15 (UBP15) gene of the gene ubiquitin-specific protease 15 (UBP15) was induced by salt, mannitol and abscisic acid (ABA) treatments. Further research revealed that UBP15 is involved in modulation of salt, drought tolerance and ABA signaling during seed germination, early seedling development, post-germination root growth or adult-plant stage. Enrichment analysis showed that many genes related to abiotic stresses and metabolic pathways were altered in the ubp15-1 mutant. Through the joint analysis of the quantitative real-time polymerase chain reaction (qRT-PCR) and differentially-expressed gene relationship network, we found that UBP15 may mainly regulate salt-stress tolerance by modulating the dwarf and delayed flowering 1 (DDF1) pathway through a cascade reaction. In the regulation of drought-stress responses, ring domain ligase1 (RGLG1) may be a direct substrate of UBP15. Moreover, we cannot exclude the possibility that UBP15 acts in a feed-forward loop mechanism in the regulation of drought-stress responses via ethylene response factor 53 (ERF53) and its ubiquitin (Ub) ligase RGLG1. In ABA signal transduction, UBP15 may play a role in at least three aspects of the ABA signaling pathway: ABA synthesis, stomatal closure regulated by ABA signaling, and transcription factors in the ABA pathway. Taken together, our results suggest that UBP15 is involved in salt, osmotic, and drought-stress tolerance and the ABA signaling pathway by directly regulating the stability of key substrates or indirectly affecting the expression of genes related to abiotic stresses in Arabidopsis thaliana. Our research provides new germplasm resources for stress-resistant crops cultivation. These results demonstrate that UBP15 is a key regulator of salt, drought and ABA tolerance in Arabidopsis.

Stress Can Induce Bovine Alpha-Herpesvirus 1 (BoHV-1) Reactivation from Latency

Bovine alpha-herpesvirus 1 (BoHV-1) is a significant problem for the cattle industry, in part because the virus establishes latency, and stressful stimuli increase the incidence of reactivation from latency. Sensory neurons in trigeminal ganglia and unknown cells in pharyngeal tonsils are importantsites for latency. Reactivation from latency can lead to reproductive problems in pregnant cows, virus transmission to young calves, suppression of immune responses, and bacterial pneumonia. BoHV-1 is also a significant cofactor in bovine respiratory disease (BRD). Stress, as mimicked by the synthetic corticosteroid dexamethasone, reproducibly initiates reactivation from latency. Stress-mediated activation of the glucocorticoid receptor (GR) stimulates viral replication and transactivation of viral promoters that drive the expression of infected cell protein 0 (bICP0) and bICP4. Notably, GR and Krüppel-like factor 15 (KLF15) form a feed-forward transcription loop that cooperatively transactivates immediate early transcription unit 1 (IEtu1 promoter). Two pioneer transcription factors, GR and KLF4, cooperatively transactivate the bICP0 early promoter. Pioneer transcription factors bind silent viral heterochromatin, remodel chromatin, and activate gene expression. Thus, wepredict that these novel transcription factors mediate early stages of BoHV-1 reactivation from latency.

Investigating the Role of Hub Calcification Proteins in Atherosclerosis via Integrated Transcriptomics and Network-Based Approach

Atherosclerosis (AS) is a chronic inflammatory condition of the arteries, characterized by plaque formation that can restrict blood flow and lead to potentially fatal cardiovascular events. Given that AS is responsible for a quarter of global deaths, this study aimed to develop a systematic bioinformatics approach to identify biomarkers and regulatory targets involved in plaque development, with the goal of reducing cardiovascular disease risk. AS-specific mRNA expression profiles were retrieved from a publicly accessible database, followed by differentially expressed genes (DEGs) identification and AS-specific weighted gene co-expression network (WGCN) construction. Thereafter, calcification and atherosclerosis-specific (CASS) DEGs were utilized for protein–protein interaction network (PPIN) formation, followed by gene ontology (GO) term and pathway enrichment analyses. Lastly, AS-specific 3-node miRNA feed-forward loop (FFL) construction and analysis was performed. Microarray datasets GSE43292 and GSE28829 were obtained from gene expression omnibus (GEO). A total of 3785 and 6176 DEGs were obtained in case of GSE28829 and GSE43292; 3256 and 5962 module DEGs corresponding to GSE28829 and GSE43292 were obtained from WGCN. From a total of 54 vascular calcification (VC) genes, 20 and 29 CASS-DEGs corresponding to GSE28829 and GSE43292 were overlapped. As observed from FFL centrality measures, the highest-order subnetwork motif comprised one TF (SOX7), one miRNA (miR-484), and one mRNA (SPARC) in the case of GSE28829. Also, in the case of GSE43292, the highest-order subnetwork motif comprised one TF (ESR2), one miRNA (miR-214-3p), and one mRNA (MEF2C). These findings have important implications for developing new therapeutic strategies for AS. The identified TFs and miRNAs may serve as potential therapeutic targets for treating atherosclerotic plaques, offering insights into the molecular mechanisms underlying the pathogenesis and highlighting new avenues for research and treatment.

Biochemical characterization of the feedforward loop between CDK1 and FOXM1 in epidermal stem cells

The complex network governing self-renewal in epidermal stem cells (EPSCs) is only partially defined. FOXM1 is one of the main players in this network, but the upstream signals regulating its activity remain to be elucidated. In this study, we identify cyclin-dependent kinase 1 (CDK1) as the principal kinase controlling FOXM1 activity in human primary keratinocytes. Mass spectrometry identified CDK1 as a key hub in a stem cell-associated protein network, showing its upregulation and interaction with essential self renewal-related markers. CDK1 phosphorylates FOXM1 at specific residues, stabilizing the protein and enhancing its nuclear localization and transcriptional activity, promoting self-renewal. Additionally, FOXM1 binds to the CDK1 promoter, inducing its expression.We identify the CDK1-FOXM1 feedforward loop as a critical axis sustaining EPSCs during in vitro cultivation. Understanding the upstream regulators of FOXM1 activity offers new insights into the biochemical mechanisms underlying self-renewal and differentiation in human primary keratinocytes.

A coherent feed-forward loop in the Arabidopsis root stem cell organizer regulates auxin biosynthesis and columella stem cell maintenance.

Stem cells in plant meristems are kept undifferentiated by signals from surrounding cells and provide the basis for continuous organ formation. In the stem cell organizer of the Arabidopsis thaliana root, the quiescent centre (QC), the WOX5 transcription factor, functions as a central hub in regulating columella stem cell (CSC) homoeostasis. However, the processes mediating WOX5 function are only poorly understood. Here we identify the transcription factor HAN as a central mediator of WOX5-regulated stem cell maintenance. HAN is required for mitotic quiescence of QC and CSC maintenance and is sufficient to induce ectopic stem cells. WOX5 and HAN repress transcription of the differentiation factor gene CDF4 in a coherent feed-forward loop (cFFL), one output of which is the expression of the auxin biosynthesis gene TAA1 and maintenance of auxin response maxima in the organizer. These findings and mathematical modelling provide a mechanistic framework for WOX5 function in the root stem cell niche.

CREB Is Critically Implicated in Skin Mast Cell Degranulation Elicited via FcεRI and MRGPRX2.

Skin mast cells (MCs) mediate acute allergic reactions in the cutaneous environment and contribute to chronic dermatoses, including urticaria, and atopic or contact dermatitis. The cAMP response element binding protein (CREB), an evolutionarily well conserved transcription factor (TF) with over 4,000 binding sites in the genome, was recently found to form a feedforward loop with KIT, maintaining MC survival. The most selective MC function is degranulation with its acute release of prestored mediators. Herein, we asked whether CREB contributes to the expression and function of the degranulation-competent receptors FcεRI and MRGPRX2. Interference with CREB by pharmacological inhibition (CREBi, 666-15) or RNA interference only slightly affected the expression of these receptors, while KIT was strongly attenuated. Interestingly, MRGPRX2 surface expression moderately increased following CREB-knockdown, whereas MRGPRX2-dependent exocytosis simultaneously decreased. FcεRI expression and function were regulated consistently, although the effect was stronger at the functional level. Preformed MC mediators (tryptase, histamine, β-hexosaminidase) remained comparable following CREB attenuation, suggesting that granule synthesis did not rely on CREB function. Collectively, in contrast to KIT, FcεRI and MRGPRX2 moderately depend on unperturbed CREB function. Nevertheless, CREB is required to maintain MC releasability irrespective of stimulus, insinuating that CREB may operate by safeguarding the degranulation machinery. To our knowledge, CREB is the first factor identified to regulate MRGPRX2 expression and function in opposite direction. Overall, the ancient TF is an indispensable component of skin MCs, orchestrating not only survival and proliferation but also their secretory competence.

Periodontitis and Diabetes Differentially Affect Inflammation in Obesity.

Periodontitis (PD) potentiates systemic inflammatory diseases and fuels a feed-forward loop of pathogenic inflammation in obesity and type 2 diabetes (T2D). Published work in this area often conflates obesity with obesity-associated T2D; thus, it remains unclear whether PD similarly affects the inflammatory profiles of these 2 distinct systemic diseases. We collected peripheral blood mononuclear cells (PBMCs) from cross-sectionally recruited subjects to estimate the ability of PD to affect cytokine production in human obesity and/or T2D. We analyzed 2 major sources of systemic inflammation: T cells and myeloid cells. Bioplex quantitated cytokines secreted by PBMCs stimulated with T cell- or myeloid-targeting activators, and we combinatorially analyzed outcomes using partial least squares discriminant analysis. Our data show that PD significantly shifts peripheral T cell- and myeloid-generated inflammation in obesity. PD also changed myeloid- but not T cell-generated inflammation in T2D. T2D changed inflammation in samples from subjects with PD, and PD changed inflammation in samples from subjects with T2D, consistent with the bidirectional relationship of inflammation between these 2 conditions. PBMCs from T2D subjects with stage IV PD produced lower amounts of T cell and myeloid cytokines compared with PBMCs from T2D subjects with stage II to III PD. We conclude that PD and T2D affect systemic inflammation through overlapping but nonidentical mechanisms in obesity, indicating that characterizing both oral and metabolic status (beyond obesity) is critical for identifying mechanisms linking PD to systemic diseases such as obesity and T2D. The finding that stage IV PD cells generate fewer cytokines in T2D provides an explanation for the paradoxical findings that the immune system can appear activated or suppressed in PD, given that many studies do not report PD stage. Finally, our data indicate that a focus on multiple cellular sources of cytokines will be imperative to clinically address the systemic effects of PD in people with obesity.

Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems.

Gene therapy holds great therapeutic potential. Yet, controlling cargo expression in single cells is limited due to the variability of delivery methods. We implement an incoherent feedforward loop based on proteolytic cleavage of CRISPR-Cas activation or inhibition systems to reduce gene expression variability against the variability of vector delivery. We demonstrate dosage control for activation and inhibition, post-delivery tuning, and RNA-based delivery, for a genome-integrated marker. We then target the RAI1 gene, the haploinsufficiency and triplosensitivity of which cause two autism-related syndromes, Smith-Magenis-Syndrome (SMS) and Potocki-Lupski-Syndrome, respectively. We demonstrate dosage control for RAI1 activation in HEK293s, Neuro-2As, and mouse cortical neurons via AAVs and lentiviruses. Finally, we activate the intact RAI1 copy in SMS patient-derived cells to an estimated two-copy healthy range, avoiding the harmful three-copy regime. Our circuit paves the way for viable therapy in dosage-sensitive disorders, creating precise and tunable gene regulation systems for basic and translational research.

A regulatory module mediating temperature control of cell-cell communication facilitates tree bud dormancy release.

The control of cell-cell communication via plasmodesmata (PD) plays a key role in plant development. In tree buds, low-temperature conditions (LT) induce a switch in plasmodesmata from a closed to an open state, which restores cell-to-cell communication in the shoot apex and releases dormancy. Using genetic and cell-biological approaches, we have identified a previously uncharacterized transcription factor, Low-temperature-Induced MADS-box 1 (LIM1), as an LT-induced, direct upstream activator of the gibberellic acid (GA) pathway. The LIM1-GA module mediates low temperature-induced plasmodesmata opening, by negatively regulating callose accumulation to promote dormancy release. LIM1 also activates expression of FT1 (FLOWERING LOCUS T), another LT-induced factor, with LIM1-FT1 forming a coherent feedforward loop converging on low-temperature regulation of gibberellin signaling in dormancy release. Mathematical modeling and experimental validation suggest that negative feedback regulation of LIM1 by gibberellin could play a crucial role in maintaining the robust temporal regulation of bud responses to low temperature. These results reveal genetic factors linking temperature control of cell-cell communication with regulation of seasonally-aligned growth crucial for adaptation of trees.

Dynamics of two feed forward genetic motifs in the presence of molecular noise

Understanding the function of common motifs in gene regulatory networks is an important goal of systems biology. Feed forward loops (FFLs) are an example of such a motif. In FFLs, a gene (X) regulates another gene (Z) both directly and via an intermediary gene (Y). Previous theoretical studies have suggested several possible functions for FFLs, based on their transient responses to changes in input signals (using deterministic models) and their fluctuations around steady state (using stochastic models). In this paper we study stochastic models of the two most common FFLs, “coherent type 1” and “incoherent type 1”. We incorporate molecular noise by treating DNA binding, transcription, translation, and decay as stochastic processes. By comparing the dynamics of these loops with models of alternative networks (in which X does not regulate Y), we explore how FFLs act to process information in the presence of noise. This work highlights the importance of incorporating realistic molecular noise in studying both the transient and steady-state behavior of gene regulatory networks.

Knockdown of microglial iron import gene, Slc11a2, worsens cognitive function and alters microglial transcriptional landscape in a sex-specific manner in the APP/PS1 model of Alzheimer’s disease

BackgroundMicroglial cell iron load and inflammatory activation are significant hallmarks of late-stage Alzheimer’s disease (AD). In vitro, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and excess iron can augment cellular inflammation, suggesting a feed-forward loop between iron import mechanisms and inflammatory signaling. However, it is not understood whether microglial iron import mechanisms directly contribute to inflammatory signaling and chronic disease in vivo. These studies determined the effects of microglial-specific knockdown of Slc11a2 on AD-related cognitive decline and microglial transcriptional phenotype.MethodsIn vitro experiments and RT-qPCR were used to assess a role for DMT1 in amyloid-β-associated inflammation. To determine the effects of microglial Slc11a2 knockdown on AD-related phenotypes in vivo, triple-transgenic Cx3cr1Cre−ERT2;Slc11a2flfl;APP/PS1+or – mice were generated and administered corn oil or tamoxifen to induce knockdown at 5–6 months of age. Both sexes underwent behavioral analyses to assess cognition and memory (12–15 months of age). Hippocampal CD11b+ microglia were magnetically isolated from female mice (15–17 months) and bulk RNA-sequencing analysis was conducted.ResultsDMT1 inhibition in vitro robustly decreased Aβ-induced inflammatory gene expression and cellular iron levels in conditions of excess iron. In vivo, Slc11a2KDAPP/PS1 female, but not male, mice displayed a significant worsening of memory function in Morris water maze and a fear conditioning assay, along with significant hyperactivity compared to control WT and APP/PS1 mice. Hippocampal microglia from Slc11a2KDAPP/PS1 females displayed significant increases in Enpp2, Ttr, and the iron-export gene, Slc40a1, compared to control APP/PS1 cells. Slc11a2KD cells from APP/PS1 females also exhibited decreased expression of markers associated with subsets of disease-associated microglia (DAMs), such as Apoe, Ctsb, Ly9, Csf1, and Hif1α.ConclusionsThis work suggests a sex-specific role for microglial iron import gene Slc11a2 in propagating behavioral and cognitive phenotypes in the APP/PS1 model of AD. These data also highlight an association between loss of a DAM-like phenotype in microglia and cognitive deficits in Slc11a2KDAPP/PS1 female mice. Overall, this work illuminates an iron-related pathway in microglia that may serve a protective role during disease and offers insight into mechanisms behind disease-related sex differences.

The human disease-associated gene ZNFX1 controls inflammation through inhibition of the NLRP3 inflammasome.

Inherited deficiency of zinc finger NFX1-type containing 1 (ZNFX1), a dsRNA virus sensor, is associated with severe familial immunodeficiency, multisystem inflammatory disease, increased susceptibility to viruses, and early mortality. However, limited treatments for patients with pathological variants of ZNFX1 exist due to an incomplete understanding of the diseases resulting from ZNFX1 mutations. Here, we demonstrate that ZNFX1 specifically inhibits the activation of the NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome in response to NLRP3 activators both in vitro and in vivo. ZNFX1 retains NLRP3 in the cytoplasm and prevents its accumulation in the TGN38 + /TGN46+ vesicles in the resting state. Upon NLRP3 inflammasome activation, ZNFX1 is cleaved by caspase-1, establishing a feed-forward loop that promotes NLRP3 accumulation in the trans-Golgi network (TGN) and amplifies the activity of the downstream cascade. Expression of wild-type ZNFX1, but not of ZNFX1 with human pathogenic mutations, rescues the impairment of NLRP3 inflammasome inhibition. Our findings reveal a dual role of ZNFX1 in virus sensing and suppression of inflammation, which may become valuable for the development of treatments for ZNFX1 mutation-related diseases.

Selective Vulnerability of GABAergic Inhibitory Interneurons to Bilirubin Neurotoxicity in the Neonatal Brain.

Hyperbilirubinemia (HB) is a key risk factor for hearing loss in neonates, particularly premature infants. Here we report that bilirubin (BIL)-dependent cell death in auditory brainstem of neonatal mice of both sexes is significantly attenuated by ZD7288, a blocker for hyperpolarization-activated cyclic nucleotide-gated (HCN) channel mediated current (Ih), or by genetic deletion of HCN1. GABAergic inhibitory interneurons predominantly express HCN1, on which BIL selectively acts to increase their intrinsic excitability and mortality by enhancing HCN1 activity and Ca2+-dependent membrane targeting. Chronic BIL elevation in neonatal mice in vivo increases the fraction of spontaneously active interneurons and their firing frequency, Ih and death, compromising audition at young adult stage in HCN1+/+, but not in HCN1-/- genotype. We conclude that HB preferentially targets HCN1 to injure inhibitory interneurons, fueling a feedforward loop in which lessening inhibition cascades hyperexcitability, Ca2+ overload, neuronal death and auditory impairments. These findings rationalize HCN1 as a potential target for managing HB encephalopathy.Significance Statement This study demonstrated that bilirubin preferentially targets GABAergic interneurons where it facilitates not only gating of HCN1 channels but also targeting of intracellular HCN1 to plasma membrane in calcium-dependent manner, resulting in neuronal hyperexcitability, injury and sensory dysfunction. These findings implicate HCN1 channel not only as a potential driver for auditory abnormalities in neonatal patients with bilirubin encephalopathy, but also potential intervention target for clinical management of neurological impairments associated with severe jaundice. Selective vulnerability of interneurons to neurotoxicity may be of general significance for understanding other forms of brain injury.

PITAR, a DNA damage-inducible cancer/testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA.

In tumors with WT p53, alternate mechanisms of p53 inactivation are reported. Here, we have identified a long noncoding RNA, PITAR (p53 Inactivating TRIM28 Associated RNA), as an inhibitor of p53. PITAR is an oncogenic Cancer/testis lncRNA and is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSC). We establish that TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase, is a direct target of PITAR. PITAR interaction with TRIM28 RNA stabilized TRIM28 mRNA, which resulted in increased TRIM28 protein levels and reduced p53 steady-state levels due to enhanced p53 ubiquitination. DNA damage activated PITAR, in addition to p53, in a p53-independent manner, thus creating an incoherent feedforward loop to inhibit the DNA damage response by p53. While PITAR silencing inhibited the growth of WT p53 containing GSCs in vitro and reduced glioma tumor growth in vivo, its overexpression enhanced the tumor growth in a TRIM28-dependent manner and promoted resistance to Temozolomide. Thus, we establish an alternate way of p53 inactivation by PITAR, which maintains low p53 levels in normal cells and attenuates the DNA damage response by p53. Finally, we propose PITAR as a potential GBM therapeutic target.

Abstract B014: NF-kB Regulates Innate Immunity in the Muscle Microenvironment to Control Distinct States of Wasting in Pancreatic Cancer-Induced Cachexia

Abstract Cancer cachexia is a debilitating syndrome characterized by unintentional weight loss largely due to the depletion of adipose and skeletal muscle mass. Cachexia occurs in at least half of all patients with cancer, with increasing incidences in more advanced cases, and is estimated to be responsible for greater than 20% of all cancer related deaths. In pancreatic ductal adenocarcinoma (PDAC), which has a 5-year survival rate of 13%, the incidence of cachexia can be as high as 80% and is associated with poor disease outcomes. Mitigating the effects of cachexia has the potential to increase both quality of life and survival for patients with PDAC. However, there are currently no effective means of treating cachexia, thereby underscoring the need to further understand the pathology of this disease. Although cancer cachexia is classically characterized as a systemic inflammatory disorder, emerging evidence indicates that weight loss also associates with local tissue inflammation. We queried the regulation of this inflammation and its causality to cachexia by exploring skeletal muscle, whose atrophy strongly associates with poor outcomes. Using both a genetically engineered mouse model of PDAC- induced cachexia named KPP and patient samples, we show that cachectic muscle is marked by enhanced innate immunity. Accumulation of macrophages is regulated by an NF-kB activity that localizes to multiple cells within the muscle microenvironment. In addition, this accumulation of macrophages derives from an equal contribution of infiltrating monocytes and resident cells. Moreover, NF-kB activated cells and macrophages undergo a crosstalk; whereas NF-kB+ cells signal and recruit macrophages to inhibit muscle regeneration and promote atrophy, but interestingly at the same time can also protect myofibers, macrophages signal back to NF-kB+ cells to sustain an innate immune response in a feed-forward loop. Together, we propose that NF-kB functions in multiple cells in the muscle microenvironment to regulate innate immunity that both promotes and protects against muscle wasting in cancer. Citation Format: Denis C Guttridge, Benjamin R Pryce, Alexander Oles, Erin E Talbert, Katherine A Morgan, David A Mahvi, Michael C Ostrowski, Teresa A Zimmers, James G Tidball, David J Wang. NF-kB Regulates Innate Immunity in the Muscle Microenvironment to Control Distinct States of Wasting in Pancreatic Cancer-Induced Cachexia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B014.

Homoharringtonine promotes non-small-cell lung cancer cell death via modulating HIF-1α/ERβ/E2F1 feedforward loop.

Hypoxia conditions promote the adaptation and progression of non-small-cell lung cancer (NSCLC) via hypoxia-inducible factors (HIF). HIF-1α may regulate estrogen receptor β (ERβ) and promote the progression of NSCLC. The phytochemical homoharringtonine (HHT) exerts strong inhibitory potency on NSCLC, with molecular mechanism under hypoxia being elusive. The effects of HHT on NSCLC growth were determined by cell viability assay, colony formation, flow cytometry, and H460 xenograft models. Western blotting, molecular docking program, site-directed mutagenesis assay, immunohistochemical assay, and immunofluorescence assay were performed to explore the underlying mechanisms of HHT-induced growth inhibition in NSCLC. HIF-1α/ERβ signaling-related E2F1 is highly expressed and contributes to unfavorable survival and tumor growth. The findings in hypoxic cells, HIF-1α overexpressing cells, as well as ERβ- or E2F1-overexpressed and knockdown cells suggest that the HIF-1α/ERβ/E2F1 feedforward loop promotes NSCLC cell growth. HHT suppresses HIF-1α/ERβ/E2F1 signaling via the ubiquitin-proteasome pathway, which is dependent on the inhibition of the protein expression of HIF-1α and ERβ. Molecular docking and site-directed mutagenesis revealed that HHT binds to the GLU305 site of ERβ. HHT inhibits cell proliferation and colony formation and promotes apoptosis in both NSCLC cells and xenograft models. The formation of the HIF-1α/ERβ/E2F1 feedforward loop promotes NSCLC growth and reveals a novel molecular mechanism by which HHT induces cell death in NSCLC.

Advantage looping: Gene regulatory circuits between microRNAs and their target transcription factors in plants.

The 20-24 nucleotide microRNAs (miRNAs) and their target transcription factors (TF) have emerged as key regulators of diverse processes in plants, including organ development and environmental resilience. In several instances, the mature miRNAs degrade the TF-encoding transcripts, while their protein products in turn bind to the promoters of the respective miRNA-encoding genes and regulate their expression, thus forming feedback loops (FBLs) or feedforward loops (FFLs). Computational analysis suggested that such miRNA-TF loops are recurrent motifs in gene regulatory networks (GRNs) in plants as well as animals. In recent years, modeling and experimental studies have suggested that plant miRNA-TF loops in GRNs play critical roles in driving organ development and abiotic stress responses. Here, we discuss the miRNA-TF FBLs and FFLs that have been identified and studied in plants over the past decade. We then provide some insights into the possible roles of such motifs within GRNs. Lastly, we provide perspectives on future directions for dissecting the functions of miRNA-centric GRNs in plants.

Role of oxytocin in bone.

Oxytocin (OT) is a posterior pituitary hormone that, in addition to its role in regulating childbirth and lactation, also exerts direct regulatory effects on the skeleton through peripheral OT and oxytocin receptor (OTR). Bone marrow mesenchymal stem cells (BMSCs), osteoblasts (OB), osteoclasts (OC), chondrocytes, and adipocytes all express OT and OTR. OT upregulates RUNX2, BMP2, ALP, and OCN, thereby enhancing the activity of BMSCs and promoting their differentiation towards OB rather than adipocytes. OT also directly regulates OPG/RANKL to inhibit adipocyte generation, increase the expression of SOX9 and COMP, and enhance chondrocyte differentiation. OB can secrete OT, exerting influence on the surrounding environment through autocrine and paracrine mechanisms. OT directly increases OC formation through the NκB/MAP kinase signaling pathway, inhibits osteoclast proliferation by triggering cytoplasmic Ca2+ release and nitric oxide synthesis, and has a dual regulatory effect on OCs. Under the stimulation of estrogen, OB synthesizes OT, amplifying the biological effects of estrogen and OT. Mediated by estrogen, the OT/OTR forms a feedforward loop with OB. Apart from estrogen, OT also interacts with arginine vasopressin (AVP), prostaglandins (PGE2), leptin, and adiponectin to regulate bone metabolism. This review summarizes recent research on the regulation of bone metabolism by OT and OTR, aiming to provide insights into their clinical applications and further research.

Combinatorial targeting of glutamine metabolism and lysosomal-based lipid metabolism effectively suppresses glioblastoma

Antipsychotic drugs have been shown to have antitumor effects but have had limited potency in the clinic. Here, we unveil that pimozide inhibits lysosome hydrolytic function to suppress fatty acid and cholesterol release in glioblastoma (GBM), the most lethal brain tumor. Unexpectedly, GBM develops resistance to pimozide by boosting glutamine consumption and lipogenesis. These elevations are driven by SREBP-1, which we find upregulates the expression of ASCT2, a key glutamine transporter. Glutamine, in turn, intensifies SREBP-1 activation through the release of ammonia, creating a feedforward loop that amplifies both glutamine metabolism and lipid synthesis, leading to drug resistance. Disrupting this loop via pharmacological targeting of ASCT2 or glutaminase, in combination with pimozide, induces remarkable mitochondrial damage and oxidative stress, leading to GBM cell death invitro and invivo. Our findings underscore the promising therapeutic potential of effectively targeting GBM by combining glutamine metabolism inhibition with lysosome suppression.

Deficiency in epithelium RAD50 aggravates UC via IL-6-mediated JAK1/2-STAT3 signaling and promotes development of colitis-associated cancer in mice.

Ulcerative colitis (UC) is one of the most important risk factors for developing colitis-associated cancer (CAC). Persistent DNA damage increases CAC risk and has been observed in patients with UC. We aimed to identify the regulatory role of RAD50, a DNA double-strand breaks (DSBs) sensor, in UC progression to CAC. DSBs and RAD50 expression in IBD and CAC cell and mouse models were assessed. Mice with intestinal epithelial RAD50 deletion (RAD50IEC-KO) were used to examine the role of RAD50 in colitis and CAC. Along with the increased γ-H2AX expression in colitis and CAC models, RAD50 expression reduced in human IBD and CAC as well as in mouse models. Furthermore, RAD50IEC-KO sensitizes mice to dextran sulfate sodium (DSS)-induced acute and chronic experimental colitis. RNA-seq analyses revealed that RAD50 activated the cytokine-cytokine receptor response, which was amplified through the JAK-STAT pathway. RAD50 directly interacts with STAT3 and subsequently inhibits its phosphorylation, which may disrupt the IL-6-JAK1/2-STAT3-IL-6 feed-forward loop. Pharmacological STAT3 inhibition relieves colitis in RAD50IEC-KO mice. Severe DSBs, increased cell proliferation, and extended inflammatory response were identified in RAD50-deficienct cells, which promoted azoxymethane (AOM)-DSS-induced colon tumor development in RAD50IEC-KO mice. RAD50 exerts anti-IL-6-related inflammatory effects in colitis and suppresses CAC. Increasing RAD50 level in colon tissues may be promising for treating patients with UC and CAC.