Ependymal Yes-Associated Protein Promotes the Neural Regeneration Through Enhancing the Ependymal Cell-Derived Perilesional Glial Borders in Mice After Spinal Cord Injury.

Yes-associated protein (YAP) transcriptional coactivator is negatively regulated by the Hippo pathway and functions in controlling the size of multiple organs, such as liver during development. However, it is not clear whether YAP signaling participates in the process of the formation of glia scars after spinal cord injury (SCI). In this study, we found that YAP was upregulated and activated in astrocytes of C57BL/6 male mice after SCI in a Hippo pathway-dependent manner. Conditional knockout (KO) of yap in astrocytes significantly inhibited astrocytic proliferation, impaired the formation of glial scars, inhibited the axonal regeneration, and impaired the behavioral recovery of C57BL/6 male mice after SCI. Mechanistically, the bFGF was upregulated after SCI and induced the activation of YAP through RhoA pathways, thereby promoting the formation of glial scars. Additionally, YAP promoted bFGF-induced proliferation by negatively controlling nuclear distribution of p27Kip1 mediated by CRM1. Finally, bFGF or XMU-MP-1 (an inhibitor of Hippo kinase MST1/2 to activate YAP) injection indeed activated YAP signaling and promoted the formation of glial scars and the functional recovery of mice after SCI. These findings suggest that YAP promotes the formation of glial scars and neural regeneration of mice after SCI, and that the bFGF-RhoA-YAP-p27Kip1 pathway positively regulates astrocytic proliferation after SCI.SIGNIFICANCE STATEMENT Glial scars play critical roles in neuronal regeneration of CNS injury diseases, such as spinal cord injury (SCI). Here, we provide evidence for the function of Yes-associated protein (YAP) in the formation of glial scars after SCI through regulation of astrocyte proliferation. As a downstream of bFGF (which is upregulated after SCI), YAP promotes the proliferation of astrocytes through negatively controlling nuclear distribution of p27Kip1 mediated by CRM1. Activation of YAP by bFGF or XMU-MP-1 injection promotes the formation of glial scar and the functional recovery of mice after SCI. These results suggest that the bFGF-RhoA-YAP-p27Kip1 axis for the formation of glial scars may be a potential therapeutic strategy for SCI patients.

Impaired angiogenesis and wound healing carry significant morbidity and mortality in diabetic patients. Metabolic stress from hyperglycemia and elevated free fatty acids have been shown to inhibit endothelial angiogenesis. However, the underlying mechanisms remain poorly understood. In this study, we show that dysregulation of the Hippo-Yes-associated protein (YAP) pathway, an important signaling mechanism in regulating tissue repair and regeneration, underlies palmitic acid (PA)-induced inhibition of endothelial angiogenesis. PA inhibited endothelial cell proliferation, migration, and tube formation, which were associated with increased expression of mammalian Ste20-like kinases 1 (MST1), YAP phosphorylation/inactivation, and nuclear exclusion. Overexpression of YAP or knockdown of MST1 prevented PA-induced inhibition of angiogenesis. When searching upstream signaling mechanisms, we found that PA dysregulated the Hippo-YAP pathway by inducing mitochondrial damage. PA treatment induced mitochondrial DNA (mtDNA) release to cytosol, and activated cytosolic DNA sensor cGAS-STING-IRF3 signaling. Activated IRF3 bound to the MST1 gene promoter and induced MST1 expression, leading to MST1 up-regulation, YAP inactivation, and angiogenesis inhibition. Thus, mitochondrial damage and cytosolic DNA sensor cGAS-STING-IRF3 signaling are critically involved in PA-induced Hippo-YAP dysregulation and angiogenesis suppression. This mechanism may have implication in impairment of angiogenesis and wound healing in diabetes.

Yes-associated protein (YAP), a transcription coactivator, is the major downstream effector of the Hippo pathway, which plays a critical role in organ size control and cancer development. However, how YAP is regulated by extracellular stimuli in tumorigenesis remains incompletely understood. Netrin-1, a laminin-related secreted protein, displays proto-oncogenic activity in cancers. Nonetheless, the downstream signaling mediating its oncogenic effects is not well defined. Here we show that netrin-1 via its transmembrane receptors, deleted in colorectal cancer and uncoordinated-5 homolog, up-regulates YAP expression, escalating YAP levels in the nucleus and promoting cancer cell proliferation and migration. Inactivating netrin-1, deleted in colorectal cancer, or uncoordinated-5 homolog B (UNC5B) decreases YAP protein levels, abrogating cancer cell progression by netrin-1, whereas knockdown of mammalian STE20-like protein kinase 1/2 (MST1/2) or large tumor suppressor kinase 1/2 (Lats1/2), two sets of upstream core kinases of the Hippo pathway, has no effect in blocking netrin-1-induced up-regulation of YAP. Netrin-1 stimulates phosphatase 1A to dephosphorylate YAP, which leads to decreased ubiquitination and degradation, enhancing YAP accumulation and signaling. Hence, our findings support that netrin-1 exerts oncogenic activity through YAP signaling, providing a mechanism coupling extracellular signals to the nuclear YAP oncogene.

Trophoblast Stem-Cell-Derived Exosomes Improve Doxorubicin-Induced Dilated Cardiomyopathy by Modulating the let-7i/YAP Pathway

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Titanium is widely used in implant materials, while excessive fluoride may have negative effects on the osseointegration between the titanium and osteoblasts. Although the underlying mechanisms are still not clear, the mitogen-activated protein kinase (MAPK) or Yes-associated protein (YAP) signaling pathways are thought to be involved. This study evaluated the role of Hippo/YAP and MAPK signaling pathway in osteoblast behaviors under excessive fluoride exposure in vitro and in vivo. Commercially pure Ti (cp-Ti) samples were exposed to fluoride (0, 0.1, and 1.0mM NaF) for 7days. Cell adhesion was observed using a laser scanning confocal microscope. Cell viability and apoptosis were evaluated by CCK-8 assay and flow cytometry, respectively. The expressions of osteoblast markers and key molecules in MAPK and YAP pathway were detected by Western blot. In vivo studies were evaluated by histology methods in C57/BL6 mice model. Our results showed that 1.0mM NaF destroyed the passivation film on cp-Ti surface, which further inhibited the osteoblast adhesion and spreading. Meanwhile, compared to other groups, 1.0mM NaF led to a remarkable reduction in cell viability (P < 0.05), as well as increased apoptosis (P < 0.05) and downregulation of osteogenesis protein expression (P < 0.05). MAPK and YAP signaling pathways were also activated under 1.0mM NaF exposure, and JNK seemed to regulate YAP phosphorylation in response to NaF impacts on osteoblasts. In vivo fluorosis mouse model further indicated that 100ppm NaF group (high fluoride group) increased bone resorption and inhibited the nuclear translocation of YAP. The osteoblast behaviors were negatively altered under excessive fluoride, and MAPK/JNK axis contributed to YAP signaling activation in regulating NaF-induced osteoblast behaviors. KEY MESSAGES: • Excessive fluoride inhibited osteoblast behaviors and bone formation. • YAP and MAPK signaling pathways were activated in osteoblasts under fluoride exposure. • Fluoride regulated osteoblast behaviors via the cross-talk between YAP and MAPK.

The epidermal growth factor receptor (EGFR) pathway and Hippo signaling play an important role in the carcinogenesis of hepatocellular carcinoma (HCC). However, the crosstalk between these two pathways and its implications in targeted therapy remains unclear. We found that the activated EGFR signaling could bypass RhoA to promote the expression of YAP(Yes-associated protein), the core effector of the Hippo signaling, and its downstream target Cyr61. Further studies indicated that EGFR signaling mainly acted through the PI3K-PDK1 (Phosphoinositide 3-kinase-Phosphoinositide-dependent kinase-1) pathway to activate YAP, but not the AKT and MAPK pathways. While YAP knockdown hardly affected the EGFR signaling. In addition, EGF could promote the proliferation of HCC cells in a YAP-independent manner. Combined targeting of YAP and EGFR signaling by simvastatin and the EGFR signaling inhibitors, including the EGFR tyrosine kinase inhibitor (TKI) gefitinib, the RAF inhibitor sorafenib and the MEK inhibitor trametinib, presented strong synergistic cytotoxicities in HCC cells. Therefore, the EGFR-PI3K-PDK1 pathway could activate the YAP signaling, and the activated EGFR signaling could promote the HCC cell growth in a YAP-independent manner. Combined use of FDA-approved inhibitors to simultaneously target YAP and EGFR signaling presented several promising therapeutic approaches for HCC treatment.

Neuropathic pain has been reported as a type of chronic pain due to the primary dysfunction of the somatosensory nervous system. It is the most serious types of chronic pain, which can lead to a significant public health burden. But, the understanding of the cellular and molecular pathogenesis of neuropathic pain is barely complete. Long noncoding RNAs (lncRNAs) have recently been regarded as modulators of neuronal functions. Growing studies have indicated lncRNAs can exert crucial roles in the development of neuropathic pain. Therefore, our present study focused on the potential role of the lncRNA Colorectal Neoplasia Differentially Expressed (CRNDE) in neuropathic pain progression. Firstly, a chronic constrictive injury (CCI) rat model was built. CRNDE was obviously increased in CCI rats. Interestingly, overexpression of CRNDE enhanced neuropathic pain behaviors. Neuroinflammation was induced by CRNDE and as demonstrated, interleukin-10 (IL-10), IL-1, IL-6, and tumor necrosis factor-α (TNF-α) protein levels in CCI rats were activated by LV-CRNDE. For another, miR-136 was obviously reduced in CCI rats. Previously, it is indicated that miR-136 participates in the spinal cord injury via an inflammation in a rat model. Here, firstly, we verified miR-136 could serve as CRNDE target. Loss of miR-136 triggered neuropathic pain remarkably via the neuroinflammation activation. Additionally, IL6R was indicated as a target of miR-136 and miR-136 regulated its expression. Subsequently, we confirmed that CRNDE could induce interleukin 6 receptor (IL6R) expression positively. Overall, it was implied that CRNDE promoted neuropathic pain progression via modulating miR-136/IL6R axis in CCI rat models.

Yes-associated protein (YAP) signaling regulates lipopolysaccharide-induced tissue factor expression in human endothelial cells

Spinal cord injury (SCI) is a debilitating and devastating condition, and there are approximately 12,000 new cases in the USA each year and an estimated number of sufferers reaching 250,000–300,000 in the USA alone. Although important advances have been made in the medical treatment of SCI recently, it is not yet possible to completely restore neuronal function after SCI. In rodents and primates, SCI causes irreversible loss of function distal to the lesion as a result of axonal damage, demyelination, and death of oligodendrocytes, astrocytes and neurons (including both spinal cord interneurons and motor neurons) (Grossman et al., 2001). Replacing the lost cell types, and integrating newly transplanted or generated cells into the spinal cord circuitry, are key aims in designing potential therapies for patients that suffer from SCI. Recent advances in our understanding of stem cell biology are revealing important potential applications for these cells in treating patients with SCI.The majority of research into developing treatments for SCI patients currently focuses on the use of stem cells to regenerate damaged tissue. The plasticity and ability to self-renew of three different types of stem cells suggests that they hold potential for regeneration following SCI: human embryonic stem cells (hESCs), neural stem cells (NSCs) and induced pluripotent stem cells (iPSCs). hESCs are pluripotent cells that can differentiate into nearly all cell types, including oligodendrocytes and motor neurons. NSCs are multi-potent cells that can give rise to neurons, oligodendrocytes and astrocytes. Patient-derived iPSCs, which can be created by reprogramming adult somatic cells using a variety of methods, have recently been proposed for use in SCI therapy. iPSCs can differentiate into many cell types, including glia and motor neurons (for a review, see Ronaghi et al., 2010). In addition, unlike transplantation of foreign cells, patient-derived iPSCs should not be rejected by the immune system. However, it should be noted that all forms of stem cell-based therapy that involve transplantation require labor-intensive in vitro propagation and manipulation, followed by transplantation and the establishment of the cells into appropriate sites in injured patients. In addition, iPSCs bring other disadvantages, including the potential for teratoma formation, aberrant reprogramming and the presence of transgenes in iPSC populations (Yamanaka, 2009; Ronaghi et al., 2010).Ependymal stem cells (EpSCs) are multipotent stem cells found in the adult tissue surrounding the ependymal canal of the spinal cord (reviewed by Ronaghi et al., 2010). Direct reprogramming of this resident spinal cord stem cell population might be a promising way to avoid the need for stem cell transplantation to treat SCI. Work from Meletis et al. suggests that such an approach is possible: they reported that the EpSCs of the spinal cord proliferate in response to SCI in adult rodents (Meletis et al., 2008). After SCI, the EpSC progeny is recruited to the injury site (even when the injury does not affect the EpSCs or their processes) to give rise to scar-forming glial cells and, to a lesser degree, oligodendrocytes. Thus, the cells of an injured host can give rise to some of the cell types that are necessary to regenerate healthy spinal cord tissue.Meletis et al. generated two transgenic mouse lines expressing tamoxifen-dependent Cre recombinase under the control of FoxJ1 or nestin regulatory sequences, which allowed genetic labeling of cells of the ependymal layer in the adult spinal cord (Meletis et al., 2008). FoxJ1 is a specific marker of cells with motile cilia or flagella, whereas NSCs and neural progenitor cells express nestin. The authors used cell culture, immunohistochemistry and electron microscopy to characterize these populations and their progeny before and after SCI in mice. The EpSCs respond to SCI by proliferating and differentiating, and their progeny migrate towards the injury site; increasing numbers of cells accumulate in the forming glial scar over several weeks (Meletis et al., 2008). By using molecular markers, the authors showed that most of the EpSC progeny have an astrocyte-like morphology and were negative for GFAP (the major intermediate filament protein of mature astrocytes), and that a smaller sub-population expressed GFAP and nestin. None of the recombined cells in the scar tissue had neuronal morphology or was immunoreactive to NeuN (neuron-specific phenotype), whereas a population of cells expressed Olig2 (an oligodendrocyte lineage transcription factor). The first month after injury, the Olig2-expressing cells displayed an ultrastructural morphology that corresponded to immature oligodendrocytes. At 10 months after SCI, most of the ependymal-derived progeny were located in the scar tissue, but a substantial number of these cells were dispersed in the intact gray and white matter bordering the lesion. Most of these cells expressed Olig2 and displayed a mature oligodendrocyte morphology with processes that enwrapped myelin-basic-protein-immunoreactive myelin-ensheathing axons. Thus, EpSCs generate both astrocytes and myelinating oligodendrocytes (Meletis et al., 2008). The intrinsic potential of EpSCs to replace some of the cells in the spinal cord following injury opens up the opportunity for developing non-invasive therapies for patients with SCI, through activating the differentiation of EpSCs into various cell types. The transgenic animals generated by Meletis et al. could be used in further experiments to determine whether EpSCs can also function as NSCs.At this point, additional work is needed to increase our understanding of stem cell differentiation pathways to move forward in developing treatments for SCI. The use of animal models in which functional regeneration occurs after SCI will be of great help. The spontaneous regeneration of neuronal cells from stem cell progenitors after SCI has been reported in adult fish [e.g. serotonergic interneurons in goldfish (Takeda et al., 2008) and motor neurons in zebrafish (Reimer et al., 2008)], which is in marked contrast to the absence of neuronal replacement observed in mammals (Meletis et al., 2008). Reimer et al. showed that regeneration of motor neurons occurs following SCI in adult zebrafish, and that the new motor neurons are integrated into the spinal cord circuits. The plasticity of Olig2-expressing progenitor cells seems to allow them to generate motor neurons through activation of the transcription factors HB9, islet-1 (ISL1) and ISL2, which are also found in developing motor neurons of mammals (Tsuchida et al., 1994; William et al., 2003) and zebrafish (Cheesman et al., 2004; Park et al., 2007). An in vitro study in mice has shown that, with a specific differentiation protocol, 90% of differentiated cultures of EpSCs obtained after SCI stain positive for the motor-neuron-specific marker HB9, with 32% of these motor neurons displaying electrophysiological properties that resemble those of functional spinal motor neurons (Moreno-Manzano et al., 2009), which shows that the manipulation of EpSCs after SCI might be a viable strategy for restoring neuronal dysfunction in humans.The next step in studies of rodent or non-human primate models should focus on the in vivo manipulation of the EpSC population. Clues for moving forward are provided by zebrafish studies: in zebrafish, the Olig2-expressing progenitors respond to a Sonic hedgehog signal to regenerate motor neurons (Reimer et al., 2009). Intraperitoneal injection of cyclopamine inhibits Sonic hedgehog signaling and reduces ventricular proliferation and motor neuron regeneration in zebrafish (Reimer et al., 2009). This finding indicates the possibility that Olig2-expressing progenitors in mammals could be manipulated in vivo to stimulate generation of specific neuronal cells. Indeed, it has recently been shown in rats that application of an intravenous hedgehog agonist increases the size of the population of neural precursor cells after SCI (Bambakidis et al., 2009). Further research should not only focus on the pharmacological manipulation of the EpSC population, but also on other aspects of EpSCs: for example, enhanced physical activity in adult rats induces an endogenous response that leads to increased proliferation and differentiation of EpSCs, mainly into macroglia or cells that express nestin (Cizkova et al., 2009). Recent research has shown that physical exercise maintains nestin expression in the EpSCs and improves functional recovery in rats following SCI (Foret et al., 2010).Therefore, the study by Meletis et al. opens up the possibility that combined pharmacological and physiotherapeutic treatments could be used to manipulate the resident EpSC population in patients with SCI to improve functional recovery. This approach could bypass the significant risks associated with therapies involving transplantation of non-patient-derived donor cells. Since the report by Meletis et al. was published (Meletis et al., 2008), other studies have shown that it is indeed possible to manipulate activated EpSCs to improve neurological recovery after SCI (e.g. Moreno-Manzano et al., 2009; Reimer et al., 2009; Fortet et al., 2010). Potential use of EpSCs in spinal cord regenerative medicine will depend on the development of strategies for directed in vivo differentiation into different functional cell types (see Zhang et al., 2010). To acheive this aim, it will be important to advance our understanding of stem cell differentiation pathways in the mature nervous system. The use of animal models such as zebrafish, in which functional regeneration of neurons from EpSCs occurs after the SCI event, should facilitate progress towards this goal.I apologize to colleagues for omitting papers that could not be cited owing to space constraints. I thank Maria C. Rodicio and members of the laboratory for their support when preparing this manuscript. The work of the author is supported by the Xunta de Galicia Consellería de Economía e Industria (Grant numbers: INCITE08PXIB200063PR and INCITE09ENA200036ES), and the Spanish Ministry of Science and Innovation (Grant number: BFU2010-17174/BFI). A.B.-I. was also supported by a Shriners Hospital Postdoctoral Research Fellowship (2010–2011).

Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with β-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase β-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.

&lt;div&gt;Abstract&lt;p&gt;Given that Yes-associated protein (YAP) signaling acts as a critical survival input for hypoxic cancer cells in hepatocellular carcinoma (HCC), disruption of YAP function and the maintenance of hypoxia is an attractive way to treat HCC. Utilizing a cell-based YAP-TEAD luciferase reporter assay and functional analyses, we identified CT-707, a China-FDA approved multi-kinase inhibitor under clinical trial with remarkable inhibitory activity against YAP function. CT-707 exhibited prominent cytotoxicity under hypoxia on HCC cells, which was attributable to the inhibition of YAP signaling. CT-707 arrested tumor growth in HepG2, Bel-7402, and HCC patient-derived xenografts. Mechanistically, the inhibitory activity of CT-707 on YAP signaling was due to the interruption of hypoxia-activated IGF1R. Overall, these findings not only identify CT-707 as a promising hypoxia-targeting agent against HCC, but they also unveil IGF1R as a new modulator specifically regulating hypoxia-activated YAP signaling.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; CT-707 may represent a novel clinical approach for patients with HCC suffering poor drug response due to intratumor hypoxia. &lt;i&gt;Cancer Res; 78(14); 3995–4006. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Given that Yes-associated protein (YAP) signaling acts as a critical survival input for hypoxic cancer cells in hepatocellular carcinoma (HCC), disruption of YAP function and the maintenance of hypoxia is an attractive way to treat HCC. Utilizing a cell-based YAP-TEAD luciferase reporter assay and functional analyses, we identified CT-707, a China-FDA approved multi-kinase inhibitor under clinical trial with remarkable inhibitory activity against YAP function. CT-707 exhibited prominent cytotoxicity under hypoxia on HCC cells, which was attributable to the inhibition of YAP signaling. CT-707 arrested tumor growth in HepG2, Bel-7402, and HCC patient-derived xenografts. Mechanistically, the inhibitory activity of CT-707 on YAP signaling was due to the interruption of hypoxia-activated IGF1R. Overall, these findings not only identify CT-707 as a promising hypoxia-targeting agent against HCC, but they also unveil IGF1R as a new modulator specifically regulating hypoxia-activated YAP signaling.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; CT-707 may represent a novel clinical approach for patients with HCC suffering poor drug response due to intratumor hypoxia. &lt;i&gt;Cancer Res; 78(14); 3995–4006. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

AimsTANK‐binding kinase 1 (TBK1) is involved in regulating the pathological process of a variety of inflammatory diseases in the central nervous system. However, its role and underlying molecular mechanisms in spinal cord injury (SCI) remain largely unknown.MethodsWe employed the TBK1 inhibitor amlexanox (ALX) to address this question. An in vivo clip‐compressive SCI model and in vitro lipopolysaccharide (LPS)‐induced astrocyte inflammation model were established to examine the effects of TBK1 inhibition on the expression of proinflammatory cytokines.ResultsIn this study, we found that TBK1 and TBK1‐medicated innate immune pathways, such as TBK1/IRF3 and noncanonical NF‐κB signaling, were activated in astrocytes and neurons after SCI. Furthermore, inhibition of TBK1 by ALX alleviated neuroinflammation response, reduced the loss of motor neurons, and improved the functional recovery after SCI. Mechanistically, inhibition of TBK1 activity promoted the activation of the noncanonical NF‐κB signaling pathway and inhibited p‐IRF3 activity in LPS‐induced astrocytes, and the TBK1 activity was required for astrocytic activation through yes‐associated protein (YAP) signaling after SCI and in LPS‐induced astrocytes inflammation model.ConclusionTBK1‐medicated innate immune pathway in astrocytes through YAP signaling plays an important role in the pathogenesis of SCI and inhibition of TBK1 may be a potential therapeutic drug for SCI.

Mechanical forces regulate muscle development, hypertrophy, and homeostasis. Force-transmitting structures allow mechanotransduction at the sarcolemma, cytoskeleton, and nuclear envelope. There is growing evidence that Yes-associated protein (YAP) serves as a nuclear relay of mechanical signals and can induce a range of downstream signaling cascades. Dystrophin is a sarcolemma-associated protein, and its absence underlies the pathology in Duchenne muscular dystrophy. We tested the hypothesis that the absence of dystrophin in muscle would result in reduced YAP signaling in response to loading. Following in vivo contractile loading in muscles of healthy (wild-type; WT) mice and mice lacking dystrophin (mdx), we performed Western blots of whole and fractionated muscle homogenates to examine the ratio of phospho (cytoplasmic) YAP to total YAP and nuclear YAP, respectively. We show that in vivo contractile loading induced a robust increase in YAP expression and its nuclear localization in WT muscles. Surprisingly, in mdx muscles, active YAP expression was constitutively elevated and unresponsive to load. Results from qRT-PCR analysis support the hyperactivation of YAP in vivo in mdx muscles, as evidenced by increased gene expression of YAP downstream targets. In vitro assays of isolated myofibers plated on substrates with high stiffness showed YAP nuclear labeling for both genotypes, indicating functional YAP signaling in mdx muscles. We conclude that while YAP signaling can occur in the absence of dystrophin, dystrophic muscles have altered mechanotransduction, whereby constitutively active YAP results in a failure to respond to load, which could be attributed to the increased state of "pre-stress" with increased cytoskeletal and extracellular matrix stiffness.