Macrophage alternative activation is involved in kidney fibrosis. Previous researches have documented that the transcriptional regulators Yes-associated protein (Yap)/transcriptional coactivator with PDZ-binding motif (Taz) are linked to organ fibrosis. However, limited knowledge exists regarding the function and mechanisms of their downstream molecules in regulating macrophage activation and kidney fibrosis. In this paper, we observed that the Hippo pathway was suppressed in macrophages derived from fibrotic kidneys in mice. Knockout of Taz or Tead1 in macrophages inhibited the alternative activation of macrophages and reduced kidney fibrosis. Additionally, by using bone marrow-derived macrophages (BMDMs), we investigated that knockout of Taz or Tead1 in macrophages impeded both cell proliferation and migration. Moreover, deletion of Tead1 reduces p-Smad3 and Smad3 abundance in macrophages. And chromatin immunoprecipitation (ChIP) assays showed that Tead1 could directly bind to the promoter region of Smad3. Collectively, these results indicate that Tead1 knockout in macrophages could reduce TGFβ1-induced phosphorylation Smad3 via transcriptional downregulation of Smad3, thus suppressing macrophage alternative activation and IRI-induced kidney fibrosis.

The Hippo pathway is generally understood to inhibit tumor growth by phosphorylating the transcriptional cofactor YAP to sequester it to the cytoplasm and reduce the formation of YAP-TEAD transcriptional complexes. Aberrant activation of YAP occurs in various cancers. However, we found a tumor-suppressive function of YAP in clear cell renal cell carcinoma (ccRCC). Using cell cultures, xenografts, and patient-derived explant models, we found that the inhibition of upstream Hippo-pathway kinases MST1 and MST2 or expression of a constitutively active YAP mutant impeded ccRCC proliferation and decreased gene expression mediated by the transcription factor NF-κB. Mechanistically, the NF-κB subunit p65 bound to the transcriptional cofactor TEAD to facilitate NF-κB-target gene expression that promoted cell proliferation. However, by competing for TEAD, YAP disrupted its interaction with NF-κB and prompted the dissociation of p65 from target gene promoters, thereby inhibiting NF-κB transcriptional programs. This cross-talk between the Hippo and NF-κB pathways in ccRCC suggests that targeting the Hippo-YAP axis in an atypical manner-that is, by activating YAP-may be a strategy for slowing tumor growth in patients.

Glioblastoma (GBM) is a highly angiogenic malignancy of the central nervous system that resists standard antiangiogenic therapy, in part because of an alternative process to angiogenesis termed vasculogenic mimicry. Intricately linked to GBM, dysregulation of the Hippo signaling pathway leads to overexpression of YAP/TEAD and several downstream effectors involved in therapy resistance. Little is known about whether vasculogenic mimicry and the Hippo pathway intersect in the GBM chemoresistance phenotype. This study seeks to investigate the expression patterns of Hippo pathway regulators within clinically annotated GBM samples, examining their involvement in vitro regarding vasculogenic mimicry. In addition, it aims to assess the potential for pharmacological targeting of this pathway. In-silico analysis of the Hippo signaling members YAP1 , TEAD1 , AXL , NF2 , CTGF , and CYR61 transcript levels in low-grade GBM and GBM tumor tissues was done by Gene Expression Profiling Interactive Analysis. Gene expression was analyzed by real-time quantitative PCR from human U87, U118, U138, and U251 brain cancer cell lines and in clinically annotated brain tumor cDNA arrays. Transient gene silencing was performed with specific small interfering RNA. Vasculogenic mimicry was assessed using a Cultrex matrix, and three-dimensional capillary-like structures were analyzed with Wimasis. CYR61 and CTGF transcript levels were elevated in GBM tissues and were further induced when in-vitro vasculogenic mimicry was assessed. Silencing of CYR61 and CTGF , or treatment with a small-molecule TEAD inhibitor LM98 derived from flufenamic acid, inhibited vasculogenic mimicry. Silencing of SNAI1 and FOXC2 also altered vasculogenic mimicry and reduced CYR61 / CTGF levels. Pharmacological targeting of the Hippo pathway inhibits in-vitro vasculogenic mimicry. Unraveling the connections between the Hippo pathway and vasculogenic mimicry may pave the way for innovative therapeutic strategies.

RNF214 is an understudied ubiquitin ligase with little knowledge of its biological functions or protein substrates. Here we show that the TEAD transcription factors in the Hippo pathway are substrates of RNF214. RNF214 induces non-proteolytic ubiquitylation at a conserved lysine residue of TEADs, enhances interactions between TEADs and YAP, and promotes transactivation of the downstream genes of the Hippo signaling. Moreover, YAP and TAZ could bind polyubiquitin chains, implying the underlying mechanisms by which RNF214 regulates the Hippo pathway. Furthermore, RNF214 is overexpressed in hepatocellular carcinoma (HCC) and inversely correlates with differentiation status and patient survival. Consistently, RNF214 promotes tumor cell proliferation, migration, and invasion, and HCC tumorigenesis in mice. Collectively, our data reveal RNF214 as a critical component in the Hippo pathway by forming a signaling axis of RNF214-TEAD-YAP and suggest that RNF214 is an oncogene of HCC and could be a potential drug target of HCC therapy.

Patients with colorectal cancer (CRC) and diabetes share many risk factors. Despite a strong association between diabetes and CRC being widely studied and confirmed, further genetic research is needed. This study found higher AL049796.1 and TEA domain transcription factor 1 (TEAD1) levels (both mRNA and protein) in CRC tissues of diabetic patients compared with nondiabetics, but no significant difference in miR-200b-3p levels. A positive correlation between AL049796.1 and TEAD1 protein existed regardless of diabetes status, whereas miR-200b-3p was only negatively correlated with TEAD1 protein in nondiabetic CRC tissues. In vitro experiments have shown that high glucose (HG) treatment increased AL049796.1 in CRC cells, and AL049796.1 silencing reduced HG-induced proliferation, migration and invasion, as well as connective tissue growth factor, cysteine-rich angiogenic inducer 61, and epidermal growth factor receptor protein expression. Mechanistic investigations indicated that AL049796.1 could mitigate suppression of miR-200b-3p on TEAD1 posttranscriptionally by acting as a competitive binder. In vivo, subcutaneous CRC tumors in streptozotocin (STZ)-induced mice grew significantly faster; AL049796.1 silencing did not affect the growth of subcutaneous CRC tumors but significantly reduced that of STZ-induced mice. Our study suggests that AL049796.1 independently contributes to the risk of CRC in diabetic patients, highlighting its potential as both a therapeutic target and a novel biomarker for CRC among individuals with diabetes.

Tissue injury causes activation of mesenchymal lineage cells into wound-repairing myofibroblasts (MFs), whose uncontrolled activity ultimately leads to fibrosis. Although this process is triggered by deep metabolic and transcriptional reprogramming, functional links between these two key events are not yet understood. Here, we report that the metabolic sensor post-translational modification O-linked β-D-N-acetylglucosaminylation (O-GlcNAcylation) is increased and required for myofibroblastic activation. Inhibition of protein O-GlcNAcylation impairs archetypal myofibloblast cellular activities including extracellular matrix gene expression and collagen secretion/deposition as defined in vitro and using ex vivo and in vivo murine liver injury models. Mechanistically, a multi-omics approach combining proteomic, epigenomic, and transcriptomic data mining revealed that O-GlcNAcylation controls the MF transcriptional program by targeting the transcription factors Basonuclin 2 (BNC2) and TEA domain transcription factor 4 (TEAD4) together with the Yes-associated protein 1 (YAP1) co-activator. Indeed, inhibition of protein O-GlcNAcylation impedes their stability leading to decreased functionality of the BNC2/TEAD4/YAP1 complex towards promoting activation of the MF transcriptional regulatory landscape. We found that this involves O-GlcNAcylation of BNC2 at Thr455 and Ser490 and of TEAD4 at Ser69 and Ser99. Altogether, this study unravels protein O-GlcNAcylation as a key determinant of myofibroblastic activation and identifies its inhibition as an avenue to intervene with fibrogenic processes.

Gastric cancer (GC) exhibits significant heterogeneity and its prognosis remains dismal. Therefore, it is essential to investigate new approaches for diagnosing and treating GC. Desmosome proteins are crucial for the advancement and growth of cancer. Plakophilin-2 (PKP2), a member of the desmosome protein family, frequently exhibits aberrant expression and is strongly associated with many tumor types\' progression. In this study, we found upregulation of PKP2 in GC. Further correlation analysis showed a notable association between increased PKP2 expression and both tumor stage and poor prognosis in individuals diagnosed with gastric adenocarcinoma. In addition, our research revealed that the Yes-associated protein1 (YAP1)/TEAD4 complex could stimulate the transcriptional expression of PKP2 in GC. Elevated PKP2 levels facilitate activation of the AKT/mammalian target of rapamycin signaling pathway, thereby promoting the malignant progression of GC. By constructing a mouse model, we ultimately validated the molecular mechanism and function of PKP2 in GC. Taken together, these discoveries suggest that PKP2, as a direct gene target of YAP/TEAD4 regulation, has the potential to be used as an indication of GC progression and prognosis. PKP2 is expected to be a promising therapeutic target for GC.

The Hippo pathway controls organ size and homeostasis and is linked to numerous diseases, including cancer. The transcriptional enhanced associate domain (TEAD) family of transcription factors acts as a receptor for downstream effectors, namely yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), which binds to various transcription factors and is essential for stimulated gene transcription. YAP/TAZ-TEAD facilitates the upregulation of multiple genes involved in evolutionary cell proliferation and survival. TEAD1-4 overexpression has been observed in different cancers in various tissues, making TEAD an attractive target for drug development. The central drug-accessible pocket of TEAD is crucial because it undergoes a post-translational modification called auto-palmitoylation. Crystal structures of the C-terminal TEAD complex with small molecules are available in the Protein Data Bank, aiding structure-based drug design. In this study, we utilized the fragment molecular orbital (FMO) method, molecular dynamics (MD) simulations, shape-based screening, and molecular mechanics-generalized Born surface area (MM-GBSA) calculations for virtual screening, and we identified a novel non-covalent inhibitor-BC-001-with IC50 = 3.7 μM in a reporter assay. Subsequently, we optimized several analogs of BC-001 and found that the optimized compound BC-011 exhibited an IC50 of 72.43 nM. These findings can be used to design effective TEAD modulators with anticancer therapeutic implications.

The mouse and human embryo gradually loses totipotency before diversifying into the inner cell mass (ICM, future organism) and trophectoderm (TE, future placenta). The transcription factors TFAP2C and TEAD4 with activated RHOA accelerate embryo polarization. Here we show that these factors also accelerate the loss of totipotency. TFAP2C and TEAD4 paradoxically promote and inhibit Hippo signaling before lineage diversification: they drive expression of multiple Hippo regulators while also promoting apical domain formation, which inactivates Hippo. Each factor activates TE specifiers in bipotent cells, while TFAP2C also activates specifiers of the ICM fate. Asymmetric segregation of the apical domain reconciles the opposing regulation of Hippo signaling into Hippo OFF and the TE fate, or Hippo ON and the ICM fate. We propose that the bistable switch established by TFAP2C and TEAD4 is exploited to trigger robust lineage diversification in the developing embryo.

Microcystins (MCs) are toxic to the central nervous system of mammals. However, the direct toxicity of MCs on mammalian brain cells and the involved molecular mechanisms are not fully elucidated. Here, we incubated primary astrocytes, the major glial cell-type in the brain, with 0-12.5 μM concentrations of MC-LR for 48 h, and the impairment was evaluated. We found that MC-LR caused significant increases in the cell viability at the range of 0.05-1 μM concentrations with the highest density at 0.1 μM concentration. Treatment with 0.1 μM MC-LR induced YAP nuclear translocation and decreased the ratio of p-YAP to YAP. It also decreased mRNA levels of the upstream regulator (AMOT), and enhanced expressions of YAP interacted genes (Egfr, Tead1, and Ctgf) in primary astrocytes. Overexpression of AMOT significantly attenuated the increase of MC-LR-induced astrocyte proliferation and the expression of YAP downstream genes. These results indicate that Hippo signaling contributed to MC-LR-caused astrocyte proliferation. Further, reactive astrogliosis was observed in the mice brain after MC-LR exposure to environmentally relevant concentrations (20 or 100 μg/L) through drinking water for 16 weeks. Pathological observations revealed that 100 μg/L MC-LR exposure caused neuronal damages with characteristics of shrunken or vacuolation in the region of the cerebral cortex, striatum and cerebellum. These results were accompanied with increased oxidative stress and inflammatory response. Our data reveal the potential astrocytic mechanisms in MC-induced neurotoxicity and raise an alarm for neurodegenerative disease risk following daily exposure to MC-LR.