Ligand Signaling Research Articles

Transcriptional regulation of development by SMAX1-LIKE proteins, targets of strigolactone and karrikin/KAI2 ligand signaling.

SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE (SMXL) proteins comprise a family of plant growth regulators that includes downstream targets of the karrikin (KAR)/KAI2 ligand (KL) and strigolactone (SL) signaling pathways. Following the perception of KAR/KL or SL signals by α/β hydrolases, some types of SMXL proteins are polyubiquitinated by an E3 ubiquitin ligase complex containing the F-box protein MORE AXILLARY GROWTH2 (MAX2)/DWARF3 (D3), and proteolyzed. Because SMXL proteins interact with TOPLESS (TPL) and TPL-related (TPR) transcriptional corepressors, SMXL degradation initiates changes in gene expression. This simplified model of SMXL regulation and function in plants must now be revised in light of recent discoveries. It has become apparent that SMXL abundance is not regulated by KAR/KL or SL alone, and that some SMXL proteins are not regulated by MAX2/D3 at all. Therefore, SMXL proteins should be considered signaling hubs that integrate multiple cues. Here we review the current knowledge of how SMXL proteins impose transcriptional regulation of plant development and environmental responses. SMXL proteins can bind DNA directly and interact with transcriptional regulators from several protein families. Multiple mechanisms of downstream genetic control by SMXL proteins have been identified recently that do not involve the recruitment of TPL/TPR, expanding the paradigm of SMXL function.

Cell signaling communication within papillary craniopharyngioma revealed by an integrated analysis of single-cell RNA sequencing and bulk RNA sequencing

ObjectiveThis study aims to elucidate the primary signaling communication among papillary craniopharyngioma (PCP) tumor cells.MethodsFive samples of PCP were utilized for single-cell RNA sequencing. The most relevant ligand and receptor interactions among different cells were calculated using the CellChat package in R software. Bulk RNA sequencing of 11 tumor samples and five normal controls was used to investigate the pair interactions detected by single-cell RNA sequencing.ResultsFibroblasts were not found in ACP, whereas they were detected in PCP. InferCNV revealed high CNV scores for the clusters of epithelial cells and fibroblasts using immune cells as a reference. Epithelial Mesenchymal Transition, Interferon Gamma Response, p53 Pathway, and Estrogen Response Early are pathways commonly shared by fibroblasts and epithelial cells, ranking high in priority. The Wnt signaling pathway and PI3K-Akt signaling pathway play a crucial role in facilitating communication between epithelial cells and fibroblasts. Neutrophils were recognized as the main receivers of incoming signals, with ANXA1-FPR1 and MIF-(CD74 + CXCR2) being identified as the primary signals transmitted from fibroblasts to neutrophils.ConclusionThrough analyzing the communication of essential signaling pathways, ligands, and receptors among epithelial cells, fibroblasts, and neutrophils in PCP tumor tissues, we have identified certain molecules with promising prognostic and therapeutic potential.

The Dynamics, Degradation, and Afterlives of Pectins: Influences on Cell Wall Assembly and Structure, Plant Development and Physiology, Agronomy, and Biotechnology.

Pectins underpin the assembly, molecular architecture, and physical properties of plant cell walls and through their effects on cell growth and adhesion influence many aspects of plant development. They are some of the most dynamic components of plant cell walls, and pectin remodeling and degradation by pectin-modifying enzymes can drive developmental programming via physical effects on the cell wall and the generation of oligosaccharides that can act as signaling ligands. Here, we introduce pectin structure and synthesis and discuss pectin functions in plants. We highlight recent advances in understanding the structure-function relationships of pectin-modifying enzymes and their products and how these advances point toward new approaches to bridging key knowledge gaps and manipulating pectin dynamics to control plant development. Finally, we discuss how a deeper understanding of pectin dynamics might enable innovations in agronomy and biotechnology, unlocking new benefits from these ubiquitous but complex polysaccharides.

DOP033 Pre-Clinical Development of SL-325, a High Affinity DR3 Blocking Antibody, for Durable Blockade of the DR3/TL1A Axis in Inflammatory Bowel Disease

Abstract Background TL1A blocking antibodies have demonstrated promising clinical remission rates in patients with inflammatory bowel disease (Sands B et al, NEJM 2024). TL1A promotes inflammation through binding to a single receptor, DR3. There are no known signaling ligands for DR3 aside from TL1A. Whereas TL1A is transiently expressed by antigen presenting and endothelial cells at sites of active inflammation in the small and large intestine, DR3 is constitutively expressed by lymphoid cells in the peripheral blood, and throughout the GI tract at both inflamed and adjacent non-inflamed tissue. Methods A human DR3 blocking antibody, SL-325, was produced from stably transfected CHO cells and purified using an affinity chromatography capture step, followed by two polishing steps. Sequence equivalents of tulisokibart and RO7790121 were produced by transient transfection and affinity purification. The safety, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of SL-325 were evaluated in a GLP toxicology study in cynomolgus macaques over a 6-week in-life period followed by a 4-week recovery at Charles River Laboratories. Results SL-325 binding affinity to human DR3 was measured at 1.3 pM. SL-325 bound to both monomeric and trimeric human DR3 prevented recombinant human TL1A binding with an approximately ~10X improvement in potency in comparison to sequence equivalents of tulisokibart or RO7790121. The DR3 epitope bound by SL-325 did not trigger receptor-mediated endocytosis, and no evidence of SL-325 mediated DR3 agonism was observed in the absence or presence of T cell receptor activation of primary human lymphocytes from healthy donors, or donors with Crohn’s Disease or Ulcerative Colitis. Naïve cynomolgus macaques received 3 doses of intravenous SL-325 (vehicle, 1 mg/kg, 10 mg/kg or 100 mg/kg dose groups), each given two weeks apart, and no evidence of toxicity, organ dysfunction or changes to complete blood counts or metabolic panels were observed. Full and durable DR3 receptor occupancy (RO) was observed in peripheral blood lymphocytes at doses of 1 mg/kg and higher within two hours of infusion and throughout the 14 day inter-dose interval. Serum exposure of free SL-325 increased in a dose proportional manner. No evidence of residual DR3 agonist activity was observed. Conclusion These results indicate SL-325 is a high-affinity DR3 blocking antibody with no evidence of toxicity or residual agonism in cynomolgus macaques, and with an RO/PK profile suggestive of extended dosing intervals that will be studied in an upcoming Phase 1 clinical trial. References Sands BE, Feagan BG, Peyrin-Biroulet L, Danese S, Rubin DT, Laurent O, Luo A, Nguyen DD, Lu J, Yen M, Leszczyszyn J, Kempinski R, McGovern DPB, Ma C, Ritter TE, Targan S. Phase 2 Trial of Anti-TL1A Monoclonal Antobidy Tulisokibart for Ulcerative Colitis. NEJM 2024;391(12):1119-1129

The effects and mechanisms of PAD2 inhibitor AFM-30a attenuates pulmonary fibrosis in silicotic mice

Objective: To observe the effects of peptidylarginine deiminase 2 (PAD2) inhibitor AFM-30a on silicotic mice and its possible mechanisms. Methods: In May 2022, 40 SPF male C57BL/6J mice were randomly divided into control group, AFM-30a group, silicosis model group and AFM-30a treatment group, with 10 mice in each group. Silicosis model group and AFM-30a treatment group were perfused with silicon dioxide (SiO(2)) suspension (10 mg/piece, 50 μl), and the other groups were perfused with an equal amount of sodium chloride solution. After 2 weeks, AFM-30a group and AFM-30a treatment group were intraperitoneally injected AFM-30a (20 mg/kg, 100 μl) daily, and mice of other groups were injected with equal amounts of sodium chloride solution for 4 weeks. Mouse RAW264.7 monocytes/macrophages were cultured in vitro and divided into blank control group, AFM-30a group (5 μmol/L), SiO(2) group (200 μg/ml), and SiO(2)+AFM-30a group (200 μg/ml SiO(2) induction for 12 h, followed by 5 μmol/L AFM-30a treatment for 12 h). As well as blank control group, vimentin (Vim) group (2 μg/ml), citrullinated vimentin (Cit-Vim) group (2 μg/ml), and Cit-Vim+TLR4-C34 group (10 μmol/L TLR4-C34 treatment for 1 h, followed by 2 μg/ml Cit-Vim induction for 24 h). Hematoxylin Eosin (HE) and Masson staining were used to observe the pathological morphology of lung. The lung fieldclarity and lung texture of each group was observed by micro-CT. The number of positive cells was detected by tartrate resistant acid phosphatase (TRAP) staining. The localization and expression levels of PAD2, Cit-Vim, toll-like receptor 4 (TLR4) signaling and receptor activator of nuclear factor-κB ligand (RANKL) signaling proteins were measured by Immunofluorescence staining and Western blotting in vitro and in vivo. The experimental data were all presented as Mean±SD. A completely random design of one-way analysis of variance was used among the groups. The pduo comparison was performed using LSD test for homogeneity of variance and Tamhane's test for inconsistency. Results: Compared with the control group, the silicosis model group showed the formation of silicon nodules accompanied by collagen deposition, the silicosis model group showed thickened, and several high-density shadows of varying sizes in the lung field, and the number of TRAP positive cells in silicosis model group were increased significantly, the expression levels of PAD2, Cit-Vim, TLR4 and RANKL signal-related proteins were also significantly increased in silicosis groupmodel (P<0.05). Compared with the silicosis model group, the AFM-30a treatment group reduced deposition of collagen in lung, and the number of TRAP positive cells was decreased in AFM-30a treatment group. The expression levels of PAD2, Cit-Vim, TLR4 and RANKL signaling related proteins were significantly decreased in AFM-30a treatment group (P<0.05). In vitro, compared with the blank control group, the number of TRAP positive cells and the expression levels of PAD2, Cit-Vim, TLR4 and RANKL signaling related proteins in the SiO(2) group were significantly increased (P<0.05). Compared with the SiO(2) group, the number of TRAP positive cells and the expression levels of PAD2, Cit-Vim, TLR4 and RANKL signaling related proteins in the SiO(2)+AFM-30a group were significantly decreased (P<0.05). Compared with the blank control group, the expression levels of TLR4 and RANKL signaling related proteins in the Cit-Vim group were significantly increased (P<0.05). Compared with the Cit-Vim group, the expression levels of TLR4 and RANKL signaling related proteins in the Cit-Vim+TLR4-C34 group were significantly decreased (P<0.05) . Conclusion: PAD2 inhibitor AFM-30a may play an antagonisticrole in silicotic fibrosis in mice by potentialregulating TLR4 and RANKL signaling pathways.

Langerhans Cells Drive Tfh and B Cell Responses Independent of Canonical Cytokine Signals.

Dendritic cells (DCs) are key regulators of adaptive immunity, guiding T helper (Th) cell differentiation through antigen presentation, co-stimulation, and cytokine production. However, in steady-state conditions, certain DC subsets, such as Langerhans cells (LCs), induce T follicular helper (Tfh) cells and B cell responses without inflammatory stimuli. Using multiple mouse models and in vitro systems, we investigated the mechanisms underlying steady-state LC-induced adaptive immune responses. We found that LCs drive germinal center Tfh and B cell differentiation and antibody production independently of interleukin-6 (IL-6), type-I interferons, and ICOS ligand (ICOS-L) signaling, which are critical in inflammatory settings. Instead, these responses relied on CD80/CD86-mediated co-stimulation. Our findings challenge the conventional three-signal paradigm by demonstrating that cytokine signaling is dispensable for LC-mediated Tfh and B cell responses in steady-state. These insights provide a framework for understanding homeostatic immunity and the immune system's role in maintaining tolerance or developing autoimmunity under non-inflammatory conditions.

Molecular basis of interchain disulfide bond formation in BMP-9 and BMP-10.

BMP-9 and BMP-10 are TGF-β family signaling ligands naturally secreted into blood. They act on endothelial cells and are required for proper development and maintenance of the vasculature. In hereditary hemorrhagic telangiectasia, regulation is disrupted due to mutations in the BMP-9/10 pathway, namely in the type I receptor ALK1 or the co-receptor endoglin. It has been demonstrated that BMP-9/10 heterodimers are the most abundant signaling species in the blood, but it is unclear how they form. Unlike other ligands of the TGF-β family, BMP-9 and -10 are secreted as a mixture of disulfide-linked dimers and monomers in which the interchain cysteine (Cys-392) remains either unpaired or paired. Here, we show that the monomers are secreted in a cysteinylated form that crystallizes as a non-covalent dimer. Despite this, monomers do not self-associate at micromolar or lower concentrations and have reduced signaling potency compared to disulfide-linked dimers. We further show using protein crystallography that the interchain disulfide of the BMP-9 homodimer adopts a highly strained syn-periplanar conformation. Hence, geometric strain across the interchain disulfide is responsible for infrequent interchain disulfide bond formation, not the cysteinylation. Additionally, we show that interchain disulfide bond formation occurs less in BMP-9 than BMP-10 and these frequencies can be reversed by swapping residues near the interchain disulfide that form attractive interactions with the opposing protomer. Finally, we discuss the implications of these observations on BMP-9/10 heterodimer formation.

Danlian-Tongmai formula improves diabetic vascular calcification by regulating CCN3/NOTCH signal axis to inhibit inflammatory reaction.

Vascular calcification (VC) commonly occurs in diabetes and is associated with cardiovascular disease incidence and mortality. Currently, there is no drug treatment for VC. The Danlian-Tongmai formula (DLTM) is a traditional Chinese medicine (TCM) prescription used for diabetic VC (DVC), but its mechanisms of action remain unclear. This study aims to elucidate the effects of DLTM on DVC and explore the underlying mechanisms of action. Ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) was used to identify the metabolites of DLTM. A DVC rat model was established using streptozotocin (STZ) combined with vitamin D3 (VitD3). The effects of DLTM on DVC were evaluated through alizarin red staining, calcium deposition, and changes in osteogenic and contractile markers. The specific molecular mechanism of DLTM in treating diabetic VC was comprehensively analyzed by transcriptomics, molecular docking and in vivo experimental verification. We identified 108 major metabolites of DLTM. In vivo, high-dose DLTM significantly alleviated VC in diabetic rats. Transcriptomic analysis showed that DLTM treatment markedly altered the transcriptomic profile of rat aortas, which was associated with regulating the CCN3/NOTCH signaling pathway, promoting vascular smooth muscle contraction, and inhibiting the inflammatory responses. Molecular docking and molecular dynamics simulation demonstrated strong binding interactions between DLTM metabolites and key molecules within the CCN3/NOTCH pathway, including NOTCH1, DLL1, DLL4, hes1, and hey1. In vivo experiments confirmed that DLTM could upregulate CCN3, inhibit the activation of NOTCH signaling ligands DLL1 and downstream transcription factors hes1 and hey1, and reduce the release of inflammatory cytokines IL6, IL1β, and TNFα. DLTM alleviates DVC by regulating the CCN3/NOTCH signaling axis to inhibit inflammatory responses. Our research provides experimental basis for clinical treatment and drug transformation of diabetic VC.

Structural insights into the distinct ligand recognition and signaling of the chemerin receptors CMKLR1 and GPR1

Structural insights into the distinct ligand recognition and signaling of the chemerin receptors CMKLR1 and GPR1

Identification of CENPM as a key gene driving adrenocortical carcinoma metastasis via physical interaction with immune checkpoint ligand FGL1.

Distant metastasis occurs in the majority of adrenocortical carcinoma (ACC), leading to an extremely poor prognosis. However, the key genes driving ACC metastasis remain unclear. Weighted gene co-expression network analysis (WGCNA) and functional enrichment analysis were conducted to identify ACC metastasis-related genes. Data from RNA-seq and microarray were analyzed to reveal correlations of the CENPM gene with cancer, metastasis, and survival in ACC. Immunohistochemistry was used to assess CENPM protein expression. The impact of CENPM on metastasis behaviour was verified in ACC (H295R and SW-13) cells and xenograft NPG mice. DIA quantitative proteomics analysis, western blot, immunofluorescence, and co-immunoprecipitation assay were performed to identify the downstream target of CENPM. Among the 12035 analyzed genes, 363 genes were related to ACC metastasis and CENPM was identified as the hub gene. CENPM was upregulated in ACC samples and associated with metastasis and poor prognosis. Knockdown of CENPM inhibited proliferation, invasion, and migration of ACC cells and suppressed liver metastasis in xenograft NPG mice. Collagen-containing extracellular matrix signalling was primarily downregulated when CENPM was knocked down. FGL1, important components of ECM signalling and immune checkpoint ligand of LAG3, were downregulated following CENPM silence, overexpressed in human advanced ACC samples, and colocalized with CENPM. Physical interaction between CENPM and FGL1 was identified. Overexpression of FGL1 rescued migration and invasion of CENPM knockdown ACC cells. CENPM is a key gene in driving ACC metastasis. CENPM promotes ACC metastasis through physical interaction with the immune checkpoint ligand FGL1. CENPM can be used as a new prognostic biomarker and therapeutic target for metastatic ACC. CENPM is the key gene that drives ACC metastasis, and a robust biomarker for ACC prognosis. Silencing CENPM impedes ACC metastasis in vitro and in vivo by physical interaction with immune checkpoint ligand FGL1. FGL1 is overexpressed in ACC and promotes ACC metastasis.

Fine-Tuned Deep Transfer Learning Models for Large Screenings of Safer Drugs Targeting Class A GPCRs.

G protein-coupled receptors (GPCRs) remain a focal point of research due to their critical roles in cell signaling and their prominence as drug targets. However, directly linking drug efficacy to receptor-mediated activation of specific intracellular transducers and the resulting physiological outcomes remains challenging. It is unclear whether the enhanced therapeutic window of certain drugs - defined as the dose range that provides effective therapy with minimal side effects - stems from their low intrinsic efficacy across all signaling pathways or ligand bias, wherein specific transducer subtypes are preferentially activated in a given cellular system compared to a reference ligand. Accurately predicting safer compounds, whether through low intrinsic efficacy or ligand bias, would greatly advance drug development. While AI models hold promise for such predictions, the development of deep learning models capable of reliably forecasting GPCR ligands with defined bioactivities remains challenging, largely due to the limited availability of high-quality data. To address this, we pre-trained a model on receptor sequences and ligand datasets across all class A GPCRs, and then refined it to predict low-efficacy compounds or biased agonists for individual class A GPCRs. This was achieved using transfer learning and a neural network incorporating natural language processing of target sequences and receptor mutation effects on signaling. These two fine-tuned models-one for low-efficacy agonists and one for biased agonists-are available on demand for each class A GPCR and enable virtual screening of large chemical libraries, thereby facilitating the discovery of compounds with potentially improved safety profiles.

FMT Restores Colonic Protein Biosynthesis and Cell Proliferation in Patients with Recurrent Clostridioides difficile Disease.

Recurrent C. difficile infection (CDI) is a major health threat with significant mortality and financial costs. Fecal Microbiota Transplantation (FMT) is an effective therapy, however the mechanisms by which it acts, particularly on the host, are poorly understood. Here we enrolled a prospective cohort of human patients with recurrent CDI (n=16) undergoing FMT therapy. Colonic biopsies were collected and bulk RNA sequencing was performed to compare changes in host gene expression pre- and two months post-FMT. Transcriptional profiles were significantly altered after FMT therapy, with many differentially expressed genes (∼15% of annotated genes detected). Enrichment analysis determined that these changes were reflective of increased protein production post-FMT, with enrichment of pathways such as Ribosome Biogenesis, Protein Processing, and signaling pathways (Myc, mTORc1, E2F) associated with cell proliferation and protein biosynthesis. Histology of H&E-stained biopsies identified a significant increase in colonic crypt length post-FMT, suggesting that this treatment promotes cell proliferation. Crypt length was significantly correlated with enriched Myc and mTOR signaling pathways as well as genes associated with polyamine biosynthesis, providing a potential mechanism through which this may occur. Finally, signaling pathways upstream of Myc and mTOR, notably IL-33 Signaling and EGFR ligands, were significantly upregulated, suggesting that FMT may utilize these signals to promote cell proliferation and restoration of the intestine.

A collection of split-Gal4 drivers targeting conserved signaling ligands in Drosophila.

Communication between cells in metazoan organisms is mediated by a remarkably small number of highly conserved signaling pathways. Given this small number of signaling pathways, the existence of multiple related ligands for many of these pathways represents a key evolutionary innovation for encoding complexity into cell-cell signaling. Relatedly, crosstalk between pathways is another critical feature which allows a modest number pathways to ultimately generate an enormously diverse range of outcomes. It would thus be useful to have genetic tools to identify and manipulate not only those cells which express a given signaling ligand, but also those cells that specifically co-express pairs of signaling ligands. We present a collection of split-Gal4 knock-in lines targeting many of the ligands for highly conserved signaling pathways in Drosophila (Notch, Hedgehog, FGF, EGF, TGFβ, JAK/STAT, JNK, and PVR). We demonstrate that these lines faithfully recapitulate the endogenous expression pattern of their targets, and that they can be used to identify cells and tissues that co-express pairs of ligands. As a proof of principle, we demonstrate that the 4th chromosome TGFβ ligands myoglianin and maverick are broadly co-expressed in muscles and other tissues of both larva and adults, and that the JAK/STAT ligands upd2 and upd3 are partially co-expressed from cells of the midgut following gut damage. Together with our previously collection of split-Gal4 lines targeting the seven Wnt ligands, this resource allows Drosophila researchers to identify and genetically manipulate cells that specifically express pairs of conserved ligands from nearly all the major intercellular signaling pathways.

Single-cell data revealed the regulatory mechanism of TNK cell heterogeneity in liver metastasis from gastric cancer

AimThe present work set out to classify cell subpopulations related to liver metastasis from gastric cancer (GC) and the mechanisms of their interactions with other immune cell subpopulations.BackgroundGC is characterized by a high degree of heterogeneity and liver metastasis. Exploring the mechanism of liver metastasis of GC from the perspective of heterogeneity of the tumor microenvironment (TME) might help improve the efficacy of GC treatment.ObjectiveBased on the cellular subpopulation characteristics of GC with liver metastasis, the regulatory mechanisms contributing to GC progression were analyzed, with special focuses on the roles of signaling pathways, transcription factors (TFs) and ligand–receptor pairs.MethodsThe GSE163558 dataset was downloaded from the Gene Expression Omnibus (GEO) database to collect single-cell transcriptomic data of GC patients and their metastasis groups for cell clustering and relevant analyses. Differentially expressed genes (DEGs) in the GC and GC liver metastasis groups were screened and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. SCENIC analysis was used to mine TFs that affected cellular subpopulations during liver metastasis from GC. The relative expression levels of TFs in GC were determined using qRT-PCR. Transwell and wound healing assays were utilized to verify the regulation of the TFs on the migration and invasion of GC cells. Interaction network between the cellular subpopulations was developed applying CellChat.ResultsSingle-cell clustering was performed to group six major cell subpopulations, namely, Myeloid cells, B cells, Mast cells, Epithelial cells, Fibroblasts, and TNK cells, among which the number of TNK cells was significantly increased in the GC liver metastasis group. Differentially enriched pathways of TNK cells between GC and GC liver metastasis groups mainly included IL-17 and Pi3k–Akt signaling pathways. TNK cell subsets could be further categorized into CD8 T cells, Exhausted T cells, NK cells, NKT cells, and Treg cells, with the GC liver metastasis group showing significantly more CD8 T cells and NKT cells. FOS and JUNB were the TFs of TNK cell marker genes that contributed to liver metastasis from GC and the invasion and migration of GC cell lines. Significant differences in immune cell communication ligand–receptor pairs existed between the GC and GC liver metastasis groups.ConclusionThis study revealed the critical role of TNK cell subsets in GC with liver metastasis applying single-cell transcriptomics analysis. The findings provided an important theoretical basis for developing novel therapies to inhibit liver metastasis from GC.

Abstract 4120290: Sex-specific Impact of Early to Mid-gestational Testosterone Excess on Sheep Fetal and Adult Offspring Lungs

Using the translationally relevant sheep model of prenatal testosterone (T) excess, we previously reported intrauterine growth retardation (IUGR) in both sexes, adverse sex specific programming of fetal heart and cardiometabolic dysfunction in adult female offspring. Because of the intricate reciprocal relationship between the heart and lungs, we hypothesized that gestational T excess will have detrimental sex-specific effects on offspring lungs beginning as early as fetal life and extending into adulthood. Pregnant ewes were intramuscularly injected with 100 mg of testosterone propionate twice weekly from gestational days (GD) 30-90 (term 147 days) and offspring lungs were harvested at GD120, 30 days after cessation of T treatment; (n=4-7/group) and in adulthood (n=4-6/group) and processed for molecular and histological analyses. Data was analyzed by student T test within each sex and Cohen (d) effect size was calculated (Cohens d&gt;0.8 represents large effect size. Lung collagen was significantly increased in T-male fetuses (TM) vs control males (CM) (p=0.01, d=1.99) that persisted into adulthood (TM vs CM: p=0.01, d=1.83). The effects of gestational T excess was evidenced as an increase in collagen only in the lungs of adult T-female fetuses (TF) relative to control females (CF) (p=0.003, d=2.38), but not during fetal life. Gestational T excess induced significant parallel increases in TUNEL positive nuclei/frame in TM fetuses relative to CM (n=3/group, p=0.01, d=3.35) persisting as a large magnitude increase of TUNEL positive nuclei/frame in adult TM (p=0.12, d=0.96). In TF females, apoptotic changes were not noted in fetal lungs but an increase of TUNEL positive nuclei/frame was evidenced in adult TF (p=0.08, d=1.14). At the molecular level prenatal T excess induced an increase in histone deacetylase 1 (HDAC1) expression in fetal TM vs CM (p=0.04, d=1.50), and decrease in HDAC1 in TF vs CF (p=0.10, d=1.09) suggestive of T-induced epigenetic alteration. Transcriptomic analysis of fetal lungs revealed upregulation of pathways associated with estrogen signaling and neuroactive ligand receptor interaction, and downregulation of pathways associated with protein processing in endoplasmic reticulum and antigen processing&amp;presentation (unadjusted p&lt; 0.05, log FC&gt;1.5) in both sexes. Overall, early to mid-gestational exposure to testosterone adversely reprograms the lungs in sex specific fashion as early as fetal life extending into postnatal adult life.

Single-cell profiling of trabecular meshwork identifies mitochondrial dysfunction in a glaucoma model that is protected by vitamin B3 treatment.

Since the trabecular meshwork (TM) is central to intraocular pressure (IOP) regulation and glaucoma, a deeper understanding of its genomic landscape is needed. We present a multimodal, single-cell resolution analysis of mouse limbal cells (includes TM). In total, we sequenced 9,394 wild-type TM cell transcriptomes. We discovered three TM cell subtypes with characteristic signature genes validated by immunofluorescence on tissue sections and whole-mounts. The subtypes are robust, being detected in datasets for two diverse mouse strains and in independent data from two institutions. Results show compartmentalized enrichment of critical pathways in specific TM cell subtypes. Distinctive signatures include increased expression of genes responsible for 1) extracellular matrix structure and metabolism (TM1 subtype), 2) secreted ligand signaling to support Schlemm's canal cells (TM2), and 3) contractile and mitochondrial/metabolic activity (TM3). ATAC-sequencing data identified active transcription factors in TM cells, including LMX1B. Mutations in LMX1B cause high IOP and glaucoma. LMX1B is emerging as a key transcription factor for normal mitochondrial function and its expression is much higher in TM3 cells than other limbal cells. To understand the role of LMX1B in TM function and glaucoma, we single-cell sequenced limbal cells from Lmx1b V265D/+ mutant mice. In V265D/+ mice, TM3 cells were uniquely affected by pronounced mitochondrial pathway changes. This supports a primary role of mitochondrial dysfunction within TM3 cells in initiating the IOP elevation that causes glaucoma in these mice. Importantly, treatment with vitamin B 3 (nicotinamide), to enhance mitochondrial function and metabolic resilience, significantly protected Lmx1b mutant mice from IOP elevation.

Molecular basis of interchain disulfide-bond formation in BMP-9 and BMP-10.

BMP-9 and BMP-10 are TGF-β family signaling ligands naturally secreted into blood. They act on endothelial cells and are required for proper development and maintenance of the vasculature. In hereditary hemorrhagic telangiectasia, regulation is disrupted due to mutations in the BMP-9/10 pathway, namely in the type I receptor ALK1 or the co-receptor endoglin. It has been demonstrated that BMP-9/10 heterodimers are the most abundant signaling species in the blood, but it is unclear how they form. Unlike other ligands of the TGF-β family, BMP-9 and -10 are secreted as a mixture of monomers and disulfide-linked dimers. Here, we show that the monomers are secreted in a cysteinylated form that crystallizes as a noncovalent dimer. Despite this, monomers do not self-associate at micromolar or lower concentrations and have reduced signaling potency compared to dimers. We further show using protein crystallography that the interchain disulfide of the BMP-9 homodimer adopts a highly strained syn-periplanar conformation. Hence, geometric strain across the interchain disulfide is responsible for the reduced propensity to dimerize, not the cysteinylation. Additionally, we show that the dimerization propensity of BMP-9 is lower than BMP-10 and these propensities can be reversed by swapping residues near the interchain disulfide that form attractive interactions with the opposing monomer. Finally, we discuss the implications of these observations on BMP-9/10 heterodimer formation.

A collection of split-Gal4 drivers targeting conserved signaling ligands in Drosophila.

Communication between cells in metazoan organisms is mediated by a remarkably small number of highly conserved signaling pathways. Given the relatively small number of signaling pathways, the existence of multiple related ligands for many of these pathways is thought to represent a key evolutionary innovation for encoding complexity into cell-cell signaling. Relatedly, crosstalk and other interactions between pathways is another critical feature which allows a modest number pathways to ultimately generate an enormously diverse range of outcomes. It would thus be useful to have genetic tools to identify and manipulate not only those cells which express a given signaling ligand, but also those cells that specifically co-express pairs of signaling ligands. Here, we present a collection of split-Gal4 knock-in lines targeting many of the ligands for highly conserved signaling pathways in Drosophila (Notch, Hedgehog, FGF, EGF, TGFβ, JAK/STAT, JNK, and PVR). We demonstrate that these lines faithfully recapitulate the endogenous expression pattern of their targets, and that they can be used to specifically identify the cells and tissues that co-express pairs of signaling ligands. As a proof of principle, we demonstrate that the 4th chromosome TGFβ ligands myoglianin and maverick are broadly co-expressed in muscles and other tissues of both larva and adults, and that the JAK/STAT ligands upd2 and upd3 are partially co-expressed from cells of the midgut following gut damage. Together with our previously collection of split-Gal4 lines targeting the seven Wnt ligands, this resource allows Drosophila researchers to identify and genetically manipulate cells that specifically express pairs of conserved ligands from nearly all the major intercellular signaling pathways.

Secretion of endoplasmic reticulum protein VAPB/ALS8 requires topological inversion

VAMP-associated protein (VAP) is a type IV integral transmembrane protein at the endoplasmic reticulum (ER). Mutations in human VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS). The N-terminal major sperm protein (MSP) domain of VAPB (Drosophila Vap33) is cleaved, secreted, and acts as a signaling ligand for several cell-surface receptors. Although extracellular functions of VAPB are beginning to be understood, it is unknown how the VAPB/Vap33 MSP domain facing the cytosol is secreted to the extracellular space. Here we show that Vap33 is transported to the plasma membrane, where the MSP domain is exposed extracellularly by topological inversion. The externalized MSP domain is cleaved by Matrix metalloproteinase 1/2 (Mmp1/2). Overexpression of Mmp1 restores decreased levels of extracellular MSP domain derived from ALS8-associated Vap33 mutants. We propose an unprecedented secretion mechanism for an ER-resident membrane protein, which may contribute to ALS8 pathogenesis.

Regulatory role of Heparan sulfate in leptin signaling

Leptin, a hormone mainly secreted by adipocytes, has attracted significant attention since its discovery in 1994. Initially known for its role in appetite suppression and energy regulation, leptin is now recognized for its influence on various physiological processes, including immune response, bone formation, and reproduction. It exerts its effects by binding to receptors and initiating an intracellular signaling cascade. Heparan sulfate (HS) is known to regulate the intracellular signaling of various ligands. HS is present as the glycan portion of HSPGs on cell surfaces and in intercellular spaces, with diverse structures due to extensive sulfation and epimerization. Although HS chains on HSPGs are involved in many physiological processes, the detailed effects of HS chains on leptin signaling are not well understood.This study examined the role of HS chains on HSPGs in leptin signaling using Neuro2A cells expressing the full-length leptin receptor (LepR). We showed that cell surface HS was essential for efficient leptin signaling. Enzymatic degradation of HS significantly reduced leptin-induced phosphorylation of downstream molecules, such as signal transducer and activator of transcription 3 and p44/p42 Mitogen-activated protein kinase. In addition, HS regulated LepR expression and internalization, as treatment with HS-degrading enzymes decreased cell surface LepR. HS was also found to exhibit a weak interaction with LepR. Enzymatic removal of HS enhanced the interaction between LepR and low-density lipoprotein receptor-related protein 1, suggesting that HS negatively regulates this interaction. In conclusion, HS plays a significant role in modulating LepR availability on the cell surface, thereby influencing leptin signaling. These findings provide new insights into the complex regulation of leptin signaling and highlight potential therapeutic targets for metabolic disorders and obesity.