Yes-associated Protein Research Articles

UBR1 promotes anaplastic thyroid carcinoma progression via stabilizing YAP through monoubiquitylation

Anaplastic thyroid carcinoma (ATC) is a highly aggressive human malignancy without effective treatment. Yes-associated protein (YAP) is a critical effector of the Hippo pathway, which is essential in thyroid carcinogenesis. However, the underlying mechanisms of aberrant YAP expression in ATC are not completely understood. Ubiquitylation-related enzyme siRNA screening identified the ubiquitin protein ligase E3 component n-recognin 1 (UBR1) as a stabilizer of YAP in ATC cells. UBR1 deficiency reduced YAP protein levels and its target gene expression. UBR1 directly interacted with YAP and promoted its monoubiquitylation, competitively suppressing its polyubiquitylation and resulting in extended protein half-life. UBR1 depletion reduced ATC cell proliferation and migration in vitro. Xenograft tumor studies also suggested that UBR1 knockdown suppressed ATC cell growth in vivo. Furthermore, exogenous YAP expression partially reversed the inhibitive effects of UBR1 depletion on ATC cell proliferation and migration. Our studies demonstrated that UBR1 directly interacts with YAP and stabilized it in a monoubiquitylation-dependent manner, consequently promoting ATC tumorigenesis, suggesting that UBR1 might be a potentially therapeutic target for ATC treatment.

Angiotensin II depends on hippo/YAP signaling to reprogram angiogenesis and promote liver fibrosis

Liver fibrosis is a chronic pathological process in which the abnormal proliferation of connective tissue is induced by various pathogenic factors. During the process of fibrosis, excessive angiogenesis is observed. Physiological angiogenesis has the potential to impede the progression of liver fibrosis through augmenting matrix metalloenzyme activity; however, pathological angiogenesis can exacerbate liver fibrosis by promoting collagen accumulation. Therefore, a key scientific research focus in the treatment of liver diseases is to search for the “on-off” mechanism that regulates angiogenesis from normal proliferation to pathological proliferation. In this study, we found that excessive angiogenesis appeared during the initial phase of hepatic fibrosis without mesenchymal characteristics. In addition, angiogenesis accompanied by significant endothelial-to-mesenchymal transition (EndMT) was observed in mice after the intraperitoneal injection of angiotensin II (Ang II). Interestingly, the changes in Yes-associated protein (YAP) activity in endothelial cells (ECs) can affect the regulation of angiogenesis by Ang II. The results of in vitro experiments revealed that the regulatory influence of Ang II on ECs was significantly attenuated upon suppression of YAP activity. Furthermore, the function of Ang II in regulating angiogenesis during fibrosis was investigated in liver-specific transgenic mice. The results revealed that Ang II gene deletion could restrain liver fibrosis and EndMT. Meanwhile, Ang II deletion downregulated the profibrotic YAP signaling pathway in ECs. The small molecule AT1R agonist olmesartan targeting Ang II-YAP signaling could also alleviate liver fibrosis. In conclusion, this study identified Ang II as a pivotal regulator of EndMT during the progression of liver fibrosis and evaluated the therapeutic effect of the Ang II-targeted drug olmesartan on liver fibrosis.

IL-32 aggravates metabolic disturbance in human nucleus pulposus cells by activating FAT4-mediated Hippo/YAP signaling

Extracellular matrix (ECM) metabolism disorders in the inflammatory microenvironment play a key role in the pathogenesis of intervertebral disc degeneration (IDD). Interleukin-32 (IL-32) has been reported to be involved in the progression of various inflammatory diseases; however, it remains unclear whether it participates in the matrix metabolism of nucleus pulposus (NP) cells. Therefore, this study aimed to investigate the mechanism of IL-32 on regulating the ECM metabolism in the inflammatory microenvironment. RNA-seq was used to identify aberrantly expressed genes in NP cells in the inflammatory microenvironment. Western blotting, real-time quantitative PCR, immunohistochemistry and immunofluorescence analysis were performed to measure the expression of IL-32 and metabolic markers in human NP tissues or NP cells treated with or without tumor necrosis factor-α (TNF-α). In vivo, an adeno-associated virus overexpressing IL-32 was injected into the caudal intervertebral discs of rats to assess its effect on IDD. Proteins interacting with IL-32 were identified via immunoprecipitation and mass spectrometry. Lentivirus overexpressing IL-32 or knocking down Fat atypical cadherin 4 (FAT4), yes-associated protein (YAP) inhibitor-Verteporfin (VP) were used to treat human NP cells, to explore the pathogenesis of IL-32. Hippo/YAP signaling activity was verified in human NP tissues. IL-32 expression was significantly upregulated in degenerative NP tissues, as indicated in the clinical samples. Furthermore, IL-32 was remarkably overexpressed in TNF-α-induced degenerative NP cells. IL-32 overexpression induced IDD progression in the rat model. Mechanistically, the elevation of IL-32 in the inflammatory microenvironment enhanced its interactions with FAT4 and mammalian sterile 20-like kinase1/2 (MST1/2) proteins, prompting MST1/2 phosphorylation, and activating the Hippo/YAP signaling pathway, causing matrix metabolism disorder in NP cells. Our results suggest that IL-32 mediates matrix metabolism disorders in NP cells in the inflammatory micro-environment via the FAT4/MST/YAP axis, providing a theoretical basis for the precise treatment of IDD.

Trilobatin, a Novel Naturally Occurring Food Additive, Ameliorates Alcoholic Liver Disease in Mice: Involvement of Microbiota-Gut-Liver Axis and Yap/Nrf2 Signaling Pathway.

Trilobatin, a novel natural food additive, exerts a protective effect on acute liver injury. However, whether Trilobatin can protect against alcoholic liver disease (ALD) has not been elucidated. This research is intended to ascertain the impact of Trilobatin on ALD in mice and decipher the potential underlying mechanisms. Lieber-DeCarli liquid alcohol diet was used to induce ALD in mice, followed by administration of Trilobatin (10, 20, 40 mg·kg-1·d-1) for 15 days. The results suggested that Trilobatin significantly alleviated ethanol-induced hepatic injury in mice. Furthermore, RNA-Seq analysis revealed that yes-associated protein (YAP) downregulation occurred in the liver after Trilobatin treatment. Mechanistically, Trilobatin directly bound to YAP and hindered its nuclear translocation, which activated the Nrf2 pathway to reduce pro-inflammatory cytokines and oxidative stress. Intriguingly, 16S rDNA analysis results revealed that Trilobatin reshaped the gut microbiota, reducing harmful bacteria and increasing beneficial bacteria. It also enhanced tight junction proteins, defending against damage to the intestinal barrier. These findings not only highlight the microbiota-gut-liver axis and YAP/Nrf2 pathway as crucial potential targets to treat ALD but also reveal that Trilobatin effectively protects against ALD, at least partly, through modulating the microbiota-gut-liver axis and YAP/Nrf2 pathway.

Downregulation of SSR2 Enhances Hepatocellular Carcinoma Cisplatin Sensitivity via the Hippo Pathway.

Chemotherapy resistance is an obstacle to promoting the survival of patients with hepatocellular carcinoma (HCC). Thus, finding promising therapeutic targets to enhance HCC chemotherapy is necessary. Signal sequence receptor subunit (SSR2) expression analysis was performed using quantitative real time polymerase chain reaction (qPCR) and Western blotting assays. Colony formation, apoptosis, anchorage-independent growth assay, and in vivo animal models were used to investigate the effect of SSR2 expression on the resistance of HCC cells to Cisplatin (DDP). Western blotting and luciferase reporter gene techniques were used to explore the molecular mechanism of SSR2 on the resistance of HCC cells to DDP. We found that the SSR2 is upregulated in HCC and associated with poor survival. Further analysis showed that the downregulation of SSR2 increased the sensitivity of HCC to DDP. Mechanically, SSR2 inhibited the Yes-associated protein (YAP) phosphorylation and promoted the transcription of Hippo signaling downstream genes. Finally, the Hippo pathway inhibitor can suppress colony formation and tumorigenesis arising from SSR2 upregulation. Our study shows that SSR2 is important in HCC progression via the Hippo pathway. Thus, targeting the SSR2/Hippo axis might be a potential strategy for overcoming HCC resistance to DDP.

Droplet-based microfluidics for engineering shape-controlled hydrogels with stiffness gradient

Current biofabrication strategies are limited in their ability to replicate native shape-to-function relationships, that are dependent on adequate biomimicry of shape, size and spatial heterogeneity, within cell-laden hydrogels. In this study, a diffusion-based microfluidics platform is presented that meets these needs in a two-step process. In the first step, a hydrogel-precursor solution is dispersed into a continuous oil phase within the microfluidics tubing. By adjusting the dispersed and oil phase flow rates, the physical architecture of hydrogel-precursor phases can be adjusted to generate spherical and plug-like structures, as well as continuous meter-long hydrogel-precursor phases (up to 1.75 m). The second step involves the controlled introduction a small molecule-containing aqueous phase through a T-shaped tube connector to enable controlled small molecule diffusion across the interface of the aqueous phase and hydrogel-precursor. Application of this system is demonstrated by diffusing co-initiator sodium persulfate (SPS) into hydrogel-precursor solutions, where the controlled SPS diffusion into the hydrogel-precursor and subsequent photo-polymerization allows for the formation of unique radial stiffness patterns across the shape- and size-controlled hydrogels, as well as allowing the formation of hollow hydrogels with controllable internal architectures. Mesenchymal stromal cells are successfully encapsulated within hollow hydrogels and hydrogels containing radial stiffness gradient. The cells are observed to respond to the microscale spatial heterogeneity as evidenced by increased cell elongation in softer core regions of the hydrogel as compared to the peripheral stiffer hydrogel regions, as well as stiffness-dependent nuclear accumulation of the yes-associated protein mechano-regulator. Finally, breast cancer cells are found to phenotypically switch in response to stiffness gradients, causing a shift in their ability to aggregate, which may have implications for metastasis. The diffusion-based microfluidics will mimic native shape-to-function relationship and provides a platform to further study the roles of micro- and macroscale architectural features that exist within native tissues.

Stepwise-targeting and hypoxia-responsive liposome AMVY@NPs carrying siYAP and verteporfin for glioblastoma therapy

BackgroundThe Hippo pathway is a conserved tumour suppressor signalling pathway, and its dysregulation is often associated with abnormal cell growth and tumorigenesis. We previously revealed that the transcriptional coactivator Yes-associated protein (YAP), the key effector of the Hippo pathway, is a molecular target for glioblastoma (GBM), the most common malignant brain tumour. Inhibiting YAP with small interfering RNA (siYAP) or the specific inhibitor verteporfin (VP) can diminish GBM growth to a certain degree.ResultsIn this study, to enhance the anti-GBM effect of siYAP and VP, we designed stepwise-targeting and hypoxia-responsive liposomes (AMVY@NPs), which encapsulate hypoxia-responsive polymetronidazole-coated VP and DOTAP adsorbed siYAP, with angiopep-2 (A2) modification on the surface. AMVY@NPs exhibited excellent blood‒brain barrier crossing, GBM targeting, and hypoxia-responsive and efficient siYAP and VP release properties. By inhibiting the expression and function of YAP, AMVY@NPs synergistically inhibited both the growth and stemness of GBM in vitro. Moreover, AMVY@NPs strongly inhibited the growth of orthotopic U87 xenografts and improved the survival of tumour-bearing mice without adverse effects.ConclusionSpecific targeting of YAP with stepwise-targeting and hypoxia-responsive liposome AMVY@NPs carrying siYAP and VP efficiently inhibited GBM progression. This study provides a valuable drug delivery platform and creative insights for molecular targeted treatment of GBM in the future.

Inhibition of MRTF-A Ameliorates Pathological Remodeling of the Pressure-loaded Right Ventricle.

Right ventricular (RV) fibrosis is associated with RV dysfunction in a variety of RV pressure-loading conditions where RV mechanical stress is increased, but the underlying mechanisms driving RV fibrosis are incompletely understood. In pulmonary and cardiovascular diseases characterized by elevated mechanical stress and transforming growth factor - beta-1 (TGF-β1) signaling, myocardin-related transcription factor A (MRTF-A) is a mechanosensitive protein critical to driving myofibroblast transition and fibrosis. Here we investigated whether MRTF-A inhibition improves RV pro-fibrotic remodeling and function in response to a pulmonary artery banding (PAB) model of RV pressure-loading. Rats were assigned into either 1) sham or 2) PAB groups. MRTF-A inhibitor CCG-1423 was administered daily at 0.75mg/kg in a subset of PAB animals. Echocardiography and pressure-volume hemodynamics were obtained at a terminal experiment 6-weeks later. RV myocardial samples were analyzed for fibrosis, cardiomyocyte hypertrophy, and pro-fibrotic signaling. MRTF-A inhibition slightly reduced systolic dysfunction in PAB rats reflected by increased lateral tricuspid annulus peak systolic velocity, while diastolic function parameters were not significantly improved. RV remodeling was attenuated in PAB rats with MRTF-A inhibition, displaying reduced fibrosis. This was accompanied with a reduction in PAB-induced upregulation of yes-associated protein (YAP) and its paralog transcriptional co-activator with PDZ-binding motif (TAZ). We also confirmed using a second-generation MRTF-A inhibitor CCG-203971 that MRTF-A is critical in driving RV fibroblast expression of TAZ and markers of myofibroblast transition in response to TGF-β1 stress and RhoA activation. These studies identify RhoA, MRTF-A, and YAP/TAZ as interconnected regulators of pro-fibrotic signaling in RV pressure-loading, and as potential targets to improve RV pro-fibrotic remodeling.

Hydrogel biomaterials that stiffen and soften on demand reveal that skeletal muscle stem cells harbor a mechanical memory

Muscle stem cells (MuSCs) are specialized cells that reside in adult skeletal muscle poised to repair muscle tissue. The ability of MuSCs to regenerate damaged tissues declines markedly with aging and in diseases such as Duchenne muscular dystrophy, but the underlying causes of MuSC dysfunction remain poorly understood. Both aging and disease result in dramatic increases in the stiffness of the muscle tissue microenvironment from fibrosis. MuSCs are known to lose their regenerative potential if cultured on stiff plastic substrates. We sought to determine whether MuSCs harbor a memory of their past microenvironment and if it can be overcome. We tested MuSCs in situ using dynamic hydrogel biomaterials that soften or stiffen on demand in response to light and found that freshly isolated MuSCs develop a persistent memory of substrate stiffness characterized by loss of proliferative progenitors within the first three days of culture on stiff substrates. MuSCs cultured on soft hydrogels had altered cytoskeletal organization and activity of Rho and Rac guanosine triphosphate hydrolase (GTPase) and Yes-associated protein mechanotransduction pathways compared to those on stiff hydrogels. Pharmacologic inhibition identified RhoA activation as responsible for the mechanical memory phenotype, and single-cell RNA sequencing revealed a molecular signature of the mechanical memory. These studies highlight that microenvironmental stiffness regulates MuSC fate and leads to MuSC dysfunction that is not readily reversed by changing stiffness. Our results suggest that stiffness can be circumvented by targeting downstream signaling pathways to overcome stem cell dysfunction in aged and disease states with aberrant fibrotic tissue mechanics.

Retinoic acid-induced protein 14 links mechanical forces to Hippo signaling

Cells sense and respond to various mechanical forces from the extracellular matrix primarily by modulating the actin cytoskeleton. Mechanical forces can be translated into biochemical signals in a process called mechanotransduction. Yes-associated protein (YAP) is an effector of Hippo signaling and a mediator of mechanotransduction, but how mechanical forces regulate Hippo signaling is still an open question. We propose that retinoic acid-induced protein 14 (RAI14) responds to mechanical forces and regulates Hippo signaling. RAI14 positively regulates the activity of YAP. RAI14 interacts with NF2, a key component of the Hippo pathway, and the interaction occurs on filamentous actin. When mechanical forces are kept low in cells, NF2 dissociates from RAI14 and filamentous actin, resulting in increased interactions with LATS1 and activation of the Hippo pathway. Clinical data show that tissue stiffness and expression of RAI14 and YAP are upregulated in tumor tissues and that RAI14 is strongly associated with adverse outcome in patients with gastric cancer. Our data suggest that RAI14 links mechanotransduction with Hippo signaling and mediates Hippo-related biological functions such as cancer progression.

A highly sensitive reporter system to monitor endogenous YAP1/TAZ activity and its application in various human cells.

The activation of yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) has been implicated in both regeneration and tumorigenesis, thus representing a double-edged sword in tissue homeostasis. However, how the activity of YAP1/TAZ is regulated or what leads to its dysregulation in these processes remains unknown. To explore the upstream stimuli modulating the cellular activity of YAP1/TAZ, we developed a highly sensitive YAP1/TAZ/TEAD-responsive DNA element (YRE) and incorporated it into a lentivirus-based reporter cell system to allow for sensitive and specific monitoring of the endogenous activity of YAP1/TAZ in terms of luciferase activity invitro and Venus fluorescence invivo. Furthermore, by replacing YRE with TCF- and NF-κB-binding DNA elements, we demonstrated the applicability of this reporter system to other pathways such as Wnt/β-catenin/TCF- and IL-1β/NF-κB-mediated signaling, respectively. The practicality of this system was evaluated by performing cell-based reporter screening of a chemical compound library consisting of 364 known inhibitors, using reporter-introduced cells capable of quantifying YAP1/TAZ- and β-catenin-mediated transcription activities, which led to the identification of multiple inhibitors, including previously known as well as novel modulators of these signaling pathways. We further confirmed that novel YAP1/TAZ modulators, such as potassium ionophores, Janus kinase inhibitors, platelet-derived growth factor receptor inhibitors, and genotoxic stress inducers, alter the protein level or phosphorylation of endogenous YAP1/TAZ and the expression of their target genes. Thus, this reporter system provides a powerful tool to monitor endogenous signaling activities of interest (even in living cells) and search for modulators in various cellular contexts.

A novel mechanism of ferroptosis inhibition-enhanced atherosclerotic plaque stability: YAP1 suppresses vascular smooth muscle cell ferroptosis through GLS1.

Atherosclerosis is a leading cause of cardiovascular diseases (CVDs), often resulting in major adverse cardiovascular events (MACEs), such as myocardial infarction and stroke due to the rupture or erosion of vulnerable plaques. Ferroptosis, an iron-dependent form of cell death, has been implicated in the development of atherosclerosis. Despite its involvement in CVDs, the specific role of ferroptosis in atherosclerotic plaque stability remains unclear. In this study, we confirmed the presence of ferroptosis in unstable atherosclerotic plaques and demonstrated that the ferroptosis inhibitor ferrostatin-1 (Fer-1) stabilizes atherosclerotic plaques in apolipoprotein E knockout (Apoe-/-) mice. Using bioinformatic analysis combining RNA sequencing (RNA-seq) with single-cell RNA sequencing (scRNA-seq), we identified Yes-associated protein 1 (YAP1) as a potential key regulator of ferroptosis in vascular smooth muscle cells (VSMCs) of unstable plaques. Invitro, we found that YAP1 protects against oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in VSMCs. Mechanistically, YAP1 exerts its anti-ferroptosis effects by regulating the expression of glutaminase 1 (GLS1) to promote the synthesis of glutamate (Glu) and glutathione (GSH). These findings establish a novel mechanism where the inhibition of ferroptosis promotes the stabilization of atherosclerotic plaques through the YAP1/GLS1 axis, attenuating VSMC ferroptosis. Thus, targeting the YAP1/GLS1 axis to suppress VSMC ferroptosis may represent a novel strategy for preventing and treating unstable atherosclerotic plaques.

Involvement of lysophosphatidic acid-LPA1-YAP signaling in healthy and pathological FAPs migration

Skeletal muscle fibrosis is defined as the excessive accumulation of extracellular matrix (ECM) components and is a hallmark of muscular dystrophies. Fibro-adipogenic progenitors (FAPs) are the main source of ECM, and thus have been strongly implicated in fibrogenesis. In skeletal muscle fibrotic models, including muscular dystrophies, FAPs undergo dysregulations in terms of proliferation, differentiation, and apoptosis, however few studies have explored the impact of FAPs migration.Here, we studied fibroblast and FAPs migration and identified lysophosphatidic acid (LPA), a signaling lipid central to skeletal muscle fibrogenesis, as a significant migration inductor. We identified LPA receptor 1 (LPA1) mediated signaling as crucial for this effect through a mechanism dependent on the Hippo pathway, another pathway implicated in fibrosis across diverse tissues. This cross-talk favors the activation of the Yes-associated protein 1 (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ), leading to increased expression of fibrosis-associated genes. This study reveals the role of YAP in LPA-mediated fibrotic responses as inhibition of YAP transcriptional coactivator activity hinders LPA-induced migration in fibroblasts and FAPs.Moreover, we found that FAPs derived from the mdx4cv mice, a murine model of Duchenne muscular dystrophy, display a heightened migratory phenotype due to enhanced LPA signaling compared to wild-type FAPs. Remarkably, we found that the inhibition of LPA1 or YAP transcriptional coactivator activity in mdx4cv FAPs reverts this phenotype. In summary, the identified LPA-LPA1-YAP pathway emerges as a critical driver of skeletal muscle FAPs migration and provides insights into potential novel targets to mitigate fibrosis in muscular dystrophies.

Targeting Neuraminidase 4 Attenuates Kidney Fibrosis in Mice.

Despite significant progress in therapy, there remains a lack of substantial evidence regarding the molecular factors that lead to renal fibrosis. Neuraminidase 4 (NEU4), an enzyme that removes sialic acids from glycoconjugates, has an unclear role in chronic progressive fibrosis. Here, this study finds that NEU4 expression is markedly upregulated in mouse fibrotic kidneys induced by folic acid or unilateral ureter obstruction, and this elevation is observed in patients with renal fibrosis. NEU4 knockdown specifically in the kidney attenuates the epithelial-to-mesenchymal transition, reduces the production of pro-fibrotic cytokines, and decreases cellular senescence in male mice. Conversely, NEU4 overexpression exacerbates the progression of renal fibrosis. Mechanistically, NEU4254-388aa interacts with Yes-associated protein (YAP) at WW2 domain (231-263aa), promoting its nucleus translocation and activation of target genes, thereby contributing to renal fibrosis. 3,5,6,7,8,3',4'-Heptamethoxyflavone, a natural compound, is identified as a novel NEU4 inhibitor, effectively protecting mice from renal fibrosis in a NEU4-dependent manner. Collectively, the findings suggest that NEU4 may represent a promising therapeutic target for kidney fibrosis.

Neuro-mesenchymal interaction mediated by a β2 adrenergic-nerve growth factor feedforward loop promotes colorectal cancer progression.

Cancer-associated fibroblasts (CAFs) and nerves, components of the tumor microenvironment, have each been shown to directly promote gastrointestinal cancers. However, it remains unknown whether these cells interact with each other to regulate cancer progression. We found that in colorectal cancer (CRC) norepinephrine induces ADRB2-dependent nerve growth factor (NGF) secretion from CAFs, which in turn increases intra-tumor sympathetic innervation and norepinephrine accumulation. Adrenergic stimulation accelerates CRC growth through ADRA2A/Gi-mediated activation of Yes-Associated Protein (YAP). NGF from CAFs directly enhances CRC cell growth via the PI3K/AKT pathway. Treatment with a tropomyosin receptor kinase (Trk) inhibitor decreased YAP and AKT activation and CRC progression in mice. In human CRC, high NGF expression is associated with the mesenchymal-like tumor subtype and poor patient survival. These findings suggest a central role for reciprocal CAF-nerve crosstalk in promoting CRC progression. Blocking this feedforward loop with a Trk inhibitor may represent a potential therapeutic approach for CRC.

Colorectal cancer treatment strategy: Targeting O-GlcNAcylation of Yes-associated protein utilizing diselenide-bridged nanovesicles

The limited treatment options for colorectal cancer (CRC) and its adverse effects of chemotherapy underscore the urgent need for innovative therapeutic strategies. Focusing on early cellular glycosylation, particularly through the targeting of O-GlcNAcylation with small-molecule inhibitors, emerges as a promising direction. In this study, we developed artificial cell-like nanovesicles with a bilayer membrane structure to encapsulate OSMI-1, a specific inhibitor of O-GlcNAc transferase, aimed at inhibiting CRC progression. To enhance the effectiveness and specificity of the treatment, these self-targeted nanovesicles (T–Se–Lip–OSMI) were reinforced using internal diselenide bridges enabling redox-responsive release within the tumor microenvironment. In vitro assays revealed that T–Se–Lip–OSMI substantially diminished CRC cell proliferation, migration, and invasion. This was further validated using patient-derived organoid models, which highlighted the superior therapeutic efficacy of T–Se–Lip–OSMI over nanovesicles without the diselenide bridge. Subsequent in vivo studies confirmed its targeted delivery and pronounced suppression of tumor growth. Further mechanistic analysis illustrated that the therapeutic effect was mediated through the inhibition of O-GlcNAcylation of the Yes-associated protein (YAP), ultimately preventing the nuclear translocation of YAP and the activation of oncogenic downstream targets. This study leverages the unique properties of the diselenide bridge to enhance stability and functionality of the nanovesicles, offering a novel avenue for targeted CRC treatment.

Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms.

Metabolic rewiring during the proliferation-to-quiescence transition is poorly understood. Here, using a model of contact inhibition-induced quiescence, we conducted 13C-metabolic flux analysis in proliferating (P) and quiescent (Q) mouse embryonic fibroblasts (MEFs) to investigate this process. Q cells exhibit reduced glycolysis but increased TCA cycle flux and mitochondrial respiration. Reduced glycolytic flux in Q cells correlates with reduced glycolytic enzyme expression mediated by yes-associated protein (YAP) inhibition. The increased TCA cycle activity and respiration in Q cells is mediated by induced mitochondrial pyruvate carrier (MPC) expression, rendering them vulnerable to MPC inhibition. The malate-to-pyruvate flux, which generates NADPH, is markedly reduced by modulating malic enzyme 1 (ME1) dimerization in Q cells. Conversely, the malate dehydrogenase 1 (MDH1)-mediated oxaloacetate-to-malate flux is reversed and elevated in Q cells, driven by high mitochondrial-derived malate levels, reduced cytosolic oxaloacetate, elevated MDH1 levels, and a high cytoplasmic NAD+/NADH ratio. Transcriptomic analysis revealed large number of genes are induced in Q cells, many of which are associated with the extracellular matrix (ECM), while YAP-dependent and cell cycle-related genes are repressed. The results suggest that high TCA cycle flux and respiration in Q cells are required to generate ATP and amino acids to maintain de-novo ECM protein synthesis and secretion.

YAP activation in Müller cells protects against NMDA-induced retinal ganglion cell injury by regulating Bcl-xL expression.

Retinal neurodegeneration, characterized by retinal ganglion cell (RGC) death, is a leading cause of vision impairment and loss in blind diseases, such as glaucoma. Müller cells play crucial roles in maintaining retinal homeostasis. Thus, dysfunction of Müller cells has been implicated as one of the causes of retinal diseases. Yes-associated protein 1 (YAP), a nuclear effector of the Hippo pathway, regulates mammalian cell survival. In this study, we investigated the role of YAP in Müller cells during N-methyl-D-aspartic acid (NMDA)-induced excitotoxic RGC injury in rats. We found that YAP expression increased and was activated in Müller cells after NMDA-induced RGC injury. This YAP response was partly due to an increase in Yap mRNA levels, although it may be independent of the Hippo pathway and β-TrCP-mediated YAP degradation. Morphological analysis revealed that verteporfin, a selective YAP inhibitor, exacerbated NMDA-induced RGC degeneration, suggesting that YAP activation in Müller cells contributes to RGC survival in NMDA-treated retinas. Studies in the rat Müller cell line (rMC-1) demonstrated that overexpression of YAP increased the levels of Bcl-xL, while verteporfin decreased the levels of Bcl-xL and cell viability and increased the levels of cytochrome c released from mitochondria and cleaved caspase-3. Finally, we found that Bcl-xL expression increased slightly in NMDA-treated retinas, whereas intravitreal injection of verteporfin suppressed this increase. Our findings suggest that activated YAP in Müller cells protects against NMDA-induced RGC injury by upregulating Bcl-xL expression.

CRKL Enhances YAP Signaling through Binding and JNK/JUN Pathway Activation in Liver Cancer.

The Hippo pathway transducers yes-associated protein (YAP) and WW-domain containing transcription regulator 1 (WWTR1/TAZ) are key regulators of liver tumorigenesis, promoting tumor formation and progression. Although the first inhibitors are in clinical trials, targeting the relevant upstream regulators of YAP/TAZ activity could prove equally beneficial. To identify regulators of YAP/TAZ activity in hepatocarcinoma (HCC) cells, we carried out a proximity labelling approach (BioID) coupled with mass spectrometry. We verified CRK-like proto-oncogene adaptor protein (CRKL) as a new YAP-exclusive interaction partner. CRKL is highly expressed in HCC patients, and its expression is associated with YAP activity as well as poor survival prognosis. In vitro experiments demonstrated CRKL-dependent cell survival and the loss of YAP binding induced through actin disruption. Moreover, we delineated the activation of the JNK/JUN pathway by CRKL, which promoted YAP transcription. Our data illustrate that CRKL not only promoted YAP activity through its binding but also through the induction of YAP transcription by JNK/JUN activation. This emphasizes the potential use of targeting the JNK/JUN pathway to suppress YAP expression in HCC patients.

Signalling of the neuropeptide calcitonin gene-related peptide (CGRP) through RAMP1 promotes liver fibrosis via TGFβ1/Smad2 and YAP pathways

The liver is innervated by primary sensory nerve fibres releasing the neuropeptide calcitonin gene-related peptide (CGRP). Elevated plasma levels of CGRP have been found in patients with liver fibrosis or cirrhosis. We hypothesised that signalling of CGRP and its receptors might regulate liver fibrosis and propose a novel potential target for the treatment. In this study, hepatic expression of CGRP and its receptor component, the receptor activity-modifying protein 1 (RAMP1), was dramatically increased in diseased livers of patients. In a murine liver fibrosis model, deficiency of RAMP1 resulted in attenuated fibrogenesis characterized by less collagen deposition and decreased activity of hepatic stellate cells (HSC). Mechanistically, activity of the TGFβ1 signalling core component Smad2 was severely impaired in the absence of RAMP1, and Yes-associated protein (YAP) activity was found to be diminished in RAMP1-deficient liver parenchyma. In vitro, stimulation of the HSC line LX-2 cells with CGRP induces TGFβ1 production and downstream signalling as well as HSC activation documented by increased α-SMA expression and collagen synthesis. We further demonstrate in LX-2 cells that CGRP promotes YAP activation and its nuclear translocation subsequent to TGFβ1/Smad2 signals. These data support a promotive effect of CGRP signalling in liver fibrosis via stimulation of TGFβ1/Smad2 and YAP activity.