Integration Of Signaling Research Articles

Engrailed-2 and inflammation convergently and independently impinge on cerebellar Purkinje cell differentiation

Autism spectrum disorders (ASD) have a complex pathogenesis thought to include both genetic and extrinsic factors. Among the latter, inflammation of the developing brain has recently gained growing attention. However, how genetic predisposition and inflammation might converge to precipitate autistic behavior remains elusive. Cerebellar structure and function are well known to be affected in autism. We therefore used cerebellar slice cultures to probe whether inflammatory stimulation and (over)expression of the autism susceptibility gene Engrailed-2 interact in shaping differentiation of Purkinje cells, key organizers of cerebellar histogenesis and function. We show that lipopolysaccharide treatment reduces Purkinje cell dendritogenesis and that this effect is enhanced by over-expression of Engrailed-2 in these cells. The effects of lipopolysaccharide can be blocked by inhibiting microglia proliferation and also by blocking tumor necrosis factor alpha receptor signaling, suggesting microglia and tumor necrosis factor alpha are major players in this scenario. These findings identify Purkinje cells as a potential integrator of genetic and environmental signals that lead to an autism-associated morphology.

FGF21 affects the glycolysis process via mTOR-HIF1α axis in hepatocellular carcinoma.

Metabolic reprogramming, particularly glycolysis, is essential in processes like cancer and immune response. While FGF21's role in hepatocyte glucose metabolism has been linked to glucose transporters and its impact on aerobic glycolysis and cellular growth in HCC remain unclear. In this study, we investigated FGF21-mediated modulation of glucose metabolism in HCC through mTOR and HIF1α axis in HCC. The study evaluated the dysregulation of FGF21 and its prognostic impact in HCC using various datasets. The literature review was done to identify glycolysis related genes to find significant interaction with FGF21 using stringdb and their correlation in datasets. The regulation of FGF21 was validated in HepG2 cell lines by transfecting FGF21 and measuring its effects on glycolysis, including glucose uptake, lactate levels, and key glycolytic enzymes using rt-PCR. Additionally, the effect of FGF21 transfection on mTOR and HIF1α was also evaluated using rt-PCR. The insilico analysis indicates that the FGF21-mTOR-HIF1α signaling axis regulates glucose metabolism, with mTOR as a central integrator of signals from FGF21 and HIF1α. Invitro experiments showed that silencing FGF21 expression via siRNA reduced glycolytic enzyme expression, glucose uptake, lactate levels, and cell proliferation in HepG2 cells. Conversely, recombinant FGF21 treatment has a reverse effect in HepG2 cells. Additionally, FGF21 treatment also affected mTOR and HIF1α expression, highlighting its role in metabolic regulation and disease through the mTOR-HIF1α axis. The regulation of FGF21 influences glycolysis via the mTOR-HIF1α axis, highlighting its critical role in glucose metabolism and metabolic adaptation in response to energy availability.

Nitrogen at the crossroads of light: Integration of light signalling and plant nitrogen metabolism.

Plants have developed complex mechanisms to perceive, transduce, and respond to environmental signals, such as light, which are essential for acquiring and allocating resources, including nitrogen (N). This review delves into the complex interaction between light signals and N metabolism, emphasising light-mediated regulation of N uptake and assimilation. Firstly, we discuss the details of light-mediated regulation of N uptake and assimilation, focusing on the light-responsive activity of nitrate reductase (NR) and nitrate transporters. Secondly, we discuss the influence of light on N-dependent developmental plasticity, elucidating how N availability regulates crucial developmental transitions such as flowering time, shoot branching, and root growth, as well as how light modulates these processes. Additionally, we discuss the molecular interaction between light and N signalling, focusing on photoreceptors and transcription factors such as HY5, which are necessary for N uptake and assimilation under varying light conditions. A recent understanding of the nitrate signalling and perception of low N is also highlighted. The insilico transcriptome analysis suggests a reprogramming of N signalling genes by shade and identifies NLP7, bZIP1, CPK30, CBL1, LBD37, LBD38 and HRS1 as crucial molecular regulators integrating light-regulated N metabolism.

A mechanistic integration of hypoxia signaling with energy, redox and hormonal cues.

Oxygen deficiency (hypoxia) occurs naturally in many developing plant tissues but can become a major threat during acute flooding stress. Consequently, plants as aerobic organisms must rapidly acclimate to hypoxia and the associated energy crisis to ensure cellular and ultimately organismal survival. In plants, oxygen sensing is tightly linked with oxygen-controlled protein stability of group VII ETHYLENE-RESPONSE FACTORs (ERFVII) which, when stabilized under hypoxia, act as key transcriptional regulators of hypoxia-responsive genes (HRGs). Multiple signaling pathways feed into hypoxia signaling to fine-tune cellular decision making under stress. First, ATP shortage upon hypoxia directly affects the energy status and adjusts anaerobic metabolism. Secondly, altered redox homeostasis leads to reactive oxygen and nitrogen species (ROS and RNS) accumulation, evoking signaling and oxidative stress acclimation. Finally, the phytohormone ethylene promotes hypoxia signaling to improve acute stress acclimation, while hypoxia signaling in turn can alter ethylene, auxin, abscisic acid, salicylic acid and jasmonate signaling to guide development and stress responses. In this Update, we summarize the current knowledge on how energy, redox and hormone signaling pathways are induced under hypoxia and subsequently integrated at the molecular level to ensure stress-tailored cellular responses. We show that some HRGs are responsive to changes in redox, energy and ethylene independently of the oxygen status, and propose an updated HRG list that is more representative for hypoxia marker gene expression. We discuss the synergistic effects of hypoxia, energy, redox and hormone signaling and their phenotypic consequences in the context of both environmental and developmental hypoxia.

Phase separation of chimeric antigen receptor promotes immunological synapse maturation and persistent cytotoxicity.

Major challenges of chimeric antigen receptor (CAR)-T cell therapy include poor antigen sensitivity and cell persistence. Here, we report a solution to these issues by exploiting CAR phase separation. We found that incorporation of an engineered Tcell receptor CD3ε motif, EB6I, into the conventional 28Z or BBZ CAR induced self-phase separation through cation-π interactions. EB6I CAR formed a mature immunological synapse with the CD2 corolla to transduce efficient antigen and costimulatory signaling, although its tonic signaling remained low. Functionally, EB6I CAR-T cells exhibited improved signaling and cytotoxicity against low-antigen tumor cells and persistent tumor-killing function. In multiple primary and relapsed murine tumor models, EB6I CAR-T cells exerted better antitumor functions than conventional CAR-T cells against blood and solid cancers. This study thus unveils a CAR engineering strategy to improve CAR-T cell immunity by leveraging molecular condensation and signaling integration.

Another-regulin regulates cardiomyocyte calcium handling via integration of neuroendocrine signaling with SERCA2a activity

Calcium (Ca2+) dysregulation is a hallmark feature of cardiovascular disease. Intracellular Ca2+ regulation is essential for proper heart function and is controlled by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a). Another-regulin (ALN) is a newly discovered cardiomyocyte-expressed SERCA2a inhibitor, suggesting cardiomyocyte Ca2+-handling is more complex than previously appreciated. To study the role of ALN in cardiomyocytes, we generated ALN null mice (knockout, KO) and found that cardiomyocytes from these animals displayed enhanced Ca2+ cycling and contractility compared to wildtype (WT) mice, indicating enhanced SERCA2a activity. In vitro and in vivo studies show that ALN is post-translationally modified via phosphorylation on Serine 19 (S19), suggesting this contributes to its ability to regulate SERCA2a. Immunoprecipitation and FRET analysis of ALN-WT, phospho-deficient ALN (S19A), or phosphomimetic ALN (S19D) revealed that S19 phosphorylation alters the SERCA2a-ALN interaction, leading to relief of its inhibitory effects. Adeno-associated virus mediated delivery of ALN-WT or phospho-mutant ALN-S19A/D in ALN KO mice showed that cardiomyocyte-specific expression of phospho-deficient ALN-S19A resulted in increased SERCA2a inhibition characterized by reduced rates of cytoplasmic Ca2+ clearance compared to ALN-WT and ALN-S19D expressing cells, further supporting a role for this phosphorylation event in controlling SERCA2a-regulation by ALN. Levels of ALN phosphorylation were markedly increased in cardiomyocytes in response to Gαq agonists (angiotensin II, endothelin-1, phenylephrine) and Gαq-mediated phosphorylation of ALN translated to increased Ca2+ cycling in cardiomyocytes from WT but not ALN KO mice. Collectively, these results indicate that ALN uniquely regulates Ca2+ handling in cardiomyocytes via integration of neuroendocrine signaling with SERCA2a activity.

Biological Ce/Fe-metal organic framework multifunctional nanozyme-based colorimetric sensor array for multi-analyte detection in human sweat

Diabetes, a widespread metabolic disorder, is a major public health concern. To address the ongoing need for effective diagnosis, a novel approach has been developed that allows the rapid, low-cost, and ultra-sensitive detection of disease-specific biomarkers in various body fluids using a single platform. Notably, this method focuses on biomarkers found in human sweat—glucose, L-Tryptophan, uric acid, and acetone—that are indicative of diabetes. The new strategy involves a nanozyme-based colorimetric sensor array made from a Ce/Fe fumarate biological metal–organic framework (Ce/Fe-Fum Bio-MOF) doped with Cu and Ni, and further enhanced with Ag nanoparticles. This nanozyme displays multiple enzyme-like activities, which result in distinct color changes when tested with specific chromogenic substrates. When these color changes are reduced by the introduction of biomarkers, the results can be easily captured using a smartphone. This method offers a wide linear detection range (20–1000 µM) and low detection limits for each biomarker, showing strong selectivity, reproducibility, and practical performance in detecting biomarkers in human sweat samples. The integration of smartphone signaling with this sensor array creates potential for a portable, handheld device for point-of-care applications, and could also be adapted for other sensing and biosensing needs. This colorimetric assay marks a significant advancement in the detection of diabetes biomarkers and the development of point-of-care diagnostic systems.

The axis of tumor-associated macrophages, extracellular matrix proteins, and cancer-associated fibroblasts in oncogenesis.

The extracellular matrix (ECM) is a complex, dynamic network of multiple macromolecules that serve as a crucial structural and physical scaffold for neighboring cells. In the tumor microenvironment (TME), ECM proteins play a significant role in mediating cellular communication between cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Revealing the ECM modification of the TME necessitates the intricate signaling cascades that transpire among diverse cell populations and ECM proteins. The advent of single-cell sequencing has enabled the identification and refinement of specific cellular subpopulations, which has substantially enhanced our comprehension of the intricate milieu and given us a high-resolution perspective on the diversity of ECM proteins. However, it is essential to integrate single-cell data and establish a coherent framework. In this regard, we present a comprehensive review of the relationships among ECM, TAMs, and CAFs. This encompasses insights into the ECM proteins released by TAMs and CAFs, signaling integration in the TAM-ECM-CAF axis, and the potential applications and limitations of targeted therapies for CAFs. This review serves as a reliable resource for focused therapeutic strategies while highlighting the crucial role of ECM proteins as intermediates in the TME.

Network Analysis of Brain and Bone Tissue Transcripts Reveals Shared Molecular Mechanisms Underlying Alzheimer's Disease and Related Dementias and Osteoporosis.

Alzheimer's disease and related dementias (ADRD) and osteoporosis (OP) are 2 prevalent diseases of aging with demonstrated epidemiological association, but the underlying molecular mechanisms contributing to this association are unknown. We used network analysis of bone and brain transcriptomes to discover common molecular mechanisms underlying these 2 diseases. Our study included RNA-sequencing data from the dorsolateral prefrontal cortex tissue of autopsied brains in 629 participants from ROSMAP (Religious Orders Study and the Rush Memory and Aging Project), with a subgroup of 298 meeting criteria for inclusion in 5 ADRD categories, and RNA array data from transiliac bone biopsies in 84 participants from the Oslo study of postmenopausal women. After developing each network within each tissue, we analyzed associations between modules (groups of coexpressed genes) with multiple bone and neurological traits, examined overlap in modules between networks, and performed pathway enrichment analysis to discover conserved mechanisms. We discovered 3 modules in ROSMAP that showed significant associations with ADRD and bone-related traits and 4 modules in Oslo that showed significant associations with multiple bone outcomes. We found significant module overlap between the 2 networks in modules linked to signaling, tissue homeostasis, and development, and Wingless-related integration site (Wnt) signaling was found to be highly enriched in OP and ADRD modules of interest. These results provide translational opportunities in the development of treatments and biomarkers for ADRD and OP.

Synergistic activation by Glass and Pointed promotes neuronal identity in the Drosophila eye disc

The integration of extrinsic signaling with cell-intrinsic transcription factors can direct progenitor cells to differentiate into distinct cell fates. In the developing Drosophila eye, differentiation of photoreceptors R1–R7 requires EGFR signaling mediated by the transcription factor Pointed, and our single-cell RNA-Seq analysis shows that the same photoreceptors require the eye-specific transcription factor Glass. We find that ectopic expression of Glass and activation of EGFR signaling synergistically induce neuronal gene expression in the wing disc in a Pointed-dependent manner. Targeted DamID reveals that Glass and Pointed share many binding sites in the genome of developing photoreceptors. Comparison with transcriptomic data shows that Pointed and Glass induce photoreceptor differentiation through intermediate transcription factors, including the redundant homologs Scratch and Scrape, as well as directly activating neuronal effector genes. Our data reveal synergistic activation of a multi-layered transcriptional network as the mechanism by which EGFR signaling induces neuronal identity in Glass-expressing cells.

Control of Dopamine Signal in High-Order Receptor Complex on Striatal Astrocytes.

The receptor-receptor interaction (RRI) of G protein-coupled receptors (GPCRs) leads to new functional entities that are conceptually distinct from the simple addition of signals mediated by the activation of the receptors that form the heteromers. Focusing on astrocytes, there is evidence for the existence of inhibitory and facilitatory RRIs, including the heteromers formed by the adenosine A2A and the dopamine D2 receptors, by A2A and the oxytocin receptor (OTR), and the D2-OTR heteromers. The possible involvement of these receptors in mosaicism has never been investigated in striatal astrocytes. By biophysical and functional approaches, we focused our attention on the existence of an A2A-D2-OTR high-order receptor complex and its role in modulating cytosolic calcium levels and endogenous glutamate release, when striatal astrocyte processes were stimulated with 4-aminopyridine. Functional data indicate a permissive role of OTR on dopamine signaling in the regulation of the glutamatergic transmission, and an inhibitory control mediated by A2A on both the D2-mediated signaling and on the OTR-facilitating effect on D2. Imaging biochemical and bioinformatic evidence confirmed the existence of the A2A-D2-OTR complex and its ternary structure in the membrane. In conclusion, the D2 receptor appears to be a hotspot in the control of the glutamate release from the astrocytic processes and may contribute to the regulation and integration of different neurotransmitter-mediated signaling in the striatum by the A2A-D2-OTR heterotrimers. Considering the possible selectivity of allosteric interventions on GPCRs organized as receptor mosaics, A2A-D2-OTR heterotrimers may offer selective pharmacological targets in neuropsychiatric disorders and neurodegenerative diseases.

Genome-wide identification, phylogenetic, structural and functional evolution of thecore components of ABA signaling in plant species: a focus on rice.

A genome-wide analysis had identified 642 ABA core component genes from 20 plant species, which were further categorized into three distinct subfamilies. The gene structures and evolutionary relationships of these genes had been characterized. PP2C_1, PP2C_2, and SnRK2_1 had emerged as key players in mediating the ABA signaling transduction pathway, specifically in rice, in response to abiotic stresses. The plant hormone abscisic acid (ABA) is essential for growth, development, and stress response, relying on its core components, pyrabactin resistance, pyrabactin resistance-like, and the regulatory component of ABA receptor (PYR/PYL/RCAR), 2C protein phosphatase (PP2C), sucrose non-fermenting-1-related protein kinase 2 (SnRK2). However, there's a lack of research on their structural evolution and functional differentiation across plants. Our study analyzed the phylogenetic, gene structure, homology, and duplication evolution of this complex in 20 plant species. We found conserved patterns in copy number and homology across subfamilies. Segmental and tandem duplications drove the evolution of these genes, while whole-genome duplication (WGD) expanded PYR/PYL/RCAR and PP2C subfamilies, enhancing environmental adaptation. In rice and Arabidopsis, the PYR/PYL/RCAR, PP2C, and SnRK2 genes showed distinct tissue-specific expression and responded to various stresses. Notably, PP2C_1 and PP2C_2 interacted with SnRK2_1 and were crucial for ABA signaling in rice. These findings offered new insights into ABA signaling evolution, interactions, and integration in green plants, benefiting future research in agriculture, evolutionary biology, ecology, and environmental science.

Molecular advances in bud dormancy in trees.

Seasonal bud dormancy in perennial woody plants is a crucial and intricate process that is vital for the survival and development of plants. Over the past few decades, significant advancements have been made in understanding many features of bud dormancy, particularly in model species, where certain molecular mechanisms underlying this process have been elucidated. We provide an overview of recent molecular progress in understanding bud dormancy in trees, with a specific emphasis on the integration of common signaling and molecular mechanisms identified across different tree species. Additionally, we address some challenges that have emerged from our current understanding of bud dormancy and offer insights for future studies.

Mapping proteomic composition of excitatory postsynaptic sites in the cerebellar cortex.

Functions of the cerebellar cortex, from motor learning to emotion and cognition, depend on the appropriate molecular composition at diverse synapse types. Glutamate receptor distributions have been partially mapped using immunogold electron microscopy. However, information is lacking on the distribution of many other components, such as Shank2, a postsynaptic scaffolding protein whose cerebellar dysfunction is associated with autism spectrum disorders. Here, we used an adapted Magnified Analysis of the Proteome, an expansion microscopy approach, to map multiple glutamate receptors, scaffolding and signaling proteins at single synapse resolution in the cerebellar cortex. Multiple distinct synapse-selective distribution patterns were observed. For example, AMPA receptors were most concentrated at synapses on molecular layer interneurons and at climbing fiber synapses, Shank1 was most concentrated at parallel fiber synapses on Purkinje cells, and Shank2 at both climbing fiber and parallel fiber synapses on Purkinje cells but little on molecular layer interneurons. Our results are consistent with gene expression data but also reveal input-selective targeting within Purkinje cells. In specialized glomerular structures of the granule cell layer, AMPA receptors as well as most other synaptic components preferentially targeted to synapses. However, NMDA receptors and the synaptic GTPase activating protein SynGAP preferentially targeted to extrasynaptic sites. Thus, glomeruli may be considered integrative signaling units through which mossy fibers differentially activate synaptic AMPA and extrasynaptic NMDA receptor complexes. Furthermore, we observed NMDA receptors and SynGAP at adherens junctions, suggesting a role in structural plasticity of glomeruli. Altogether, these data contribute to mapping the cerebellar 'synaptome'.

The Arabidopsis SGN3/GSO1 receptor kinase integrates soil nitrogen status into shoot development

The Casparian strip is a barrier in the endodermal cell walls of plants that allows the selective uptake of nutrients and water. In the model plant Arabidopsis thaliana, its development and establishment are under the control of a receptor-ligand mechanism termed the Schengen pathway. This pathway facilitates barrier formation and activates downstream compensatory responses in case of dysfunction. However, due to a very tight functional association with the Casparian strip, other potential signaling functions of the Schengen pathway remain obscure. In this work, we created a MYB36-dependent synthetic positive feedback loop that drives Casparian strip formation independently of Schengen-induced signaling. We evaluated this by subjecting plants in which the Schengen pathway has been uncoupled from barrier formation, as well as a number of established barrier-mutant plants, to agar-based and soil conditions that mimic agricultural settings. Under the latter conditions, the Schengen pathway is necessary for the establishment of nitrogen-deficiency responses in shoots. These data highlight Schengen signaling as an essential hub for the adaptive integration of signaling from the rhizosphere to aboveground tissues.

Oxidative Stress and FOXO-1 Relationship in Stage III Periodontitis

Objectives8-Hydroxideoxyguanosine (8-OHdG) is a marker of oxidative stress, and Forkhead Box-O1 (FOXO1) is a transcription factor and signaling integrator in cell and tissue homeostasis. This study aims to determine FOXO1 and 8-OHdG levels in serum and saliva samples of periodontitis patients and to evaluate their relationship with clinical periodontal parameters.Materials and MethodsTwenty healthy individuals, twenty generalized Stage III Grade B periodontitis patients, and nineteen generalized Stage III Grade C periodontitis patients were included in the study. Clinical periodontal parameters (plaque index (PI), probing depth (PD), bleeding on probing (BOP), and clinical attachment level (CAL)) were recorded. Salivary and serum 8-OHdG and FOX-O1 levels were analyzed by enzyme-linked immunosorbent assay (ELISA).ResultsClinical periodontal parameters showed a statistically significant increase in periodontitis groups compared to the control group (p < 0.05). 8-OHdG salivary levels were significantly higher in both periodontitis groups compared to the control group. The salivary FOXO1 levels were significantly lower in both periodontitis groups compared to the control group. Salivary FOXO1 level had a low-grade negative correlation with BOP and salivary 8-OHdG level.ConclusionsWhile reactive oxygen species increase in periodontal inflammation, low expression of FOXO1, an important transcription factor for antioxidant enzymes, supports that this molecule plays a vital role in tissue destruction, and FOXO1 can be seen as a potential immune modulator.Clinical relevanceThe role of FOXO1 in supporting antioxidant defense may suggest that FOXO1 is a candidate target for periodontitis treatment.

Integration of nitrate and abscisic acid signaling in plants.

To meet the demands of the new Green Revolution and sustainable agriculture, it is important to develop crop varieties with improved yield, nitrogen use efficiency, and stress resistance. Nitrate is the major form of inorganic nitrogen available for plant growth in many well-aerated agricultural soils, and acts as a signaling molecule regulating plant development, growth, and stress responses. Abscisic acid (ABA), an important phytohormone, plays vital roles in integrating extrinsic and intrinsic responses and mediating plant growth and development in response to biotic and abiotic stresses. Therefore, elucidating the interplay between nitrate and ABA can contribute to crop breeding and sustainable agriculture. Here, we review studies that have investigated the interplay between nitrate and ABA in root growth modulation, nitrate and ABA transport processes, seed germination regulation, and drought responses. We also focus on nitrate and ABA interplay in several reported omics analyses with some important nodes in the crosstalk between nitrate and ABA. Through these insights, we proposed some research perspectives that could help to develop crop varieties adapted to a changing environment and to improve crop yield with high nitrogen use efficiency and strong stress resistance.

Feed restriction of lactating cows triggers acute downregulation of mammary mammalian target of rapamycin signaling and chronic reduction of mammary epithelial mass

While there is generally no consensus about how nutrients determine milk synthesis in the mammary gland, it is likely that the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) plays a role as a key integrator of nutritional and mitogenic signals that can influence a multitude of catabolic and anabolic pathways. The objectives of this study were to evaluate acute changes (<24 h) in translational signaling, in addition to chronic changes (14 d) in mammary gland structure and composition, in response to a severe feed restriction. Fourteen lactating Holstein dairy cows were assigned to either ad libitum feeding (n = 7) or a restricted feeding program (n = 7). Feed-restricted cows had feed removed after the evening milking on d 0. Mammary biopsies and blood samples were collected 16 h after feed removal, after which cows in the restricted group were fed 60% of their previously observed ad libitum intake for the remainder of the study. On d 14, animals were slaughtered and their mammary glands dissected. In response to feed removal, an acute increase in plasma nonesterified fatty acid concentration was observed, concurrent to a decrease in milk yield. In mammary tissue, we observed downregulation of the mTORC1-S6K1 signaling cascade, in addition to reductions in mRNA expression of markers of protein synthesis, endoplasmic reticulum biogenesis, and cell turnover (i.e., transcripts associated with apoptosis or cell proliferation). During the 14 d of restricted feeding, animals underwent homeorhetic adaptation to 40% lower nutrient intake, achieving a new setpoint of 14% reduced milk yield with 18% and 29% smaller mammary secretory tissue DM and CP masses, respectively. On d 14, no treatment differences were observed in markers of protein synthesis or mammary cell turnover evaluated using gene transcripts and immunohistochemical staining. These findings implicate mTORC1-S6K1 in the early phase of the adaptation of the mammary gland's capacity for milk synthesis in response to changes in nutrient supply. Additionally, changes in rates of mammary cell turnover may be transient in nature, returning to basal levels following brief alterations that have sustained effects.

Interplay of RAP2 GTPase and the cytoskeleton in Hippo pathway regulation

The Hippo signaling is instrumental in regulating organ size, regeneration, and carcinogenesis. The cytoskeleton emerges as a primary Hippo signaling modulator. Its structural alterations in response to environmental and intrinsic stimuli control Hippo signaling pathway activity. However, the precise mechanisms underlying the cytoskeleton regulation of Hippo signaling are not fully understood. RAP2 GTPase is known to mediate the mechanoresponses of Hippo signaling via activating the core Hippo kinases LATS1/2 through MAP4Ks and MST1/2. Here we show the pivotal role of the reciprocal regulation between RAP2 GTPase and the cytoskeleton in Hippo signaling. RAP2 deletion undermines the responses of the Hippo pathway to external cues tied to RhoA GTPase inhibition and actin cytoskeleton remodeling, such as energy stress and serum deprivation. Notably, RhoA inhibitors and actin disruptors fail to activate LATS1/2 effectively in RAP2-deficient cells. RNA sequencing highlighted differential regulation of both actin and microtubule networks by RAP2 gene deletion. Consistently, Taxol, a microtubule-stabilizing agent, was less effective in activating LATS1/2 and inhibiting cell growth in RAP2 and MAP4K4/6/7 knockout cells. In summary, our findings position RAP2 as a central integrator of cytoskeletal signals for Hippo signaling, which offers new avenues for understanding Hippo regulation and therapeutic interventions in Hippo-impaired cancers.

Osteosarcoma cells exhibit functional interactions with stromal cells, fostering a lung microenvironment conducive to the establishment of metastatic tumor cells.

Osteosarcoma (OS) stands out as the most common bone tumor, with approximately 20% of the patients receiving a diagnosis of metastatic OS at their initial assessment. A significant challenge lies in the frequent existence of undetected metastases during the initial diagnosis. Mesenchymal stem cells (MSCs) possess unique abilities that facilitate tumor growth, and their interaction with OS cells is crucial for metastatic spread. We demonstrated that, in vitro, MSCs exhibited a heightened migration response toward the secretome of non-metastatic OS cells. When challenged to a secretome derived from lungs preloaded with OS cells, MSCs exhibited greater migration toward lungs colonized with metastatic OS cells. Moreover, in vivo, MSCs displayed preferential migratory and homing behavior toward lungs colonized by metastatic OS cells. Metastatic OS cells, in turn, demonstrated an increased migratory response to the MSCs' secretome. This behavior was associated with heightened cathepsin D (CTSD) expression and the release of active metalloproteinase 2 (MMP2) by metastatic OS cells. Our assessment focused on two complementary tumor capabilities crucial to metastatic spread, emphasizing the significance of inherent cell features. The findings underscore the pivotal role of signaling integration within the niche, with a complex interplay of migratory responses among established OS cells in the lungs, prometastatic OS cells in the primary tumor, and circulating MSCs. Pulmonary metastases continue to be a significant factor contributing to OS mortality. Understanding these mechanisms and identifying differentially expressed genes is essential for pinpointing markers and targets to manage metastatic spread and improve outcomes for patients with OS.