Yes-associated Protein Nuclear Localization Research Articles

High-viscosity driven modulation of biomechanical properties of human mesenchymal stem cells promotes osteogenic lineage

Biomechanical cues could effectively govern cell gene expression to direct the differentiation of specific stem cell lineage. Recently, the medium viscosity has emerged as a significant mechanical stimulator that regulates the cellular mechanical properties and various physiological functions. However, whether the medium viscosity can regulate the mechanical properties of human mesenchymal stem cells (hMSCs) to effectively trigger osteogenic differentiation remains uncertain. The mechanism by which cells sense and respond to changes in medium viscosity, and regulate cell mechanical properties to promote osteogenic lineage, remains elusive. In this study, we demonstrated that hMSCs, cultured in a high-viscosity medium, exhibited larger cell spreading area and higher intracellular tension, correlated with elevated formation of actin stress fibers and focal adhesion maturation. Furthermore, these changes observed in hMSCs were associated with activation of TRPV4 (transient receptor potential vanilloid sub-type 4) channels on the cell membrane. This feedback loop among TRPV4 activation, cell spreading and intracellular tension results in calcium influx, which subsequently promotes the nuclear localization of NFATc1 (nuclear factor of activated T cells 1). Concomitantly, the elevated intracellular tension induced nuclear deformation and promoted the nuclear localization of YAP (YES‐associated protein). The concurrent activation of NFATc1 and YAP significantly enhanced alkaline phosphatase (ALP) for pre-osteogenic activity. Taken together, these findings provide a more comprehensive view of how viscosity-induced alterations in biomechanical properties of MSCs impact the expression of osteogenesis-related genes, and ultimately promote osteogenic lineage.

FAK, vinculin, and talin control mechanosensitive YAP nuclear localization

Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity.

Nuclear Localization of Yes-Associated Protein is Associated With Tumor Progression in Cutaneous Melanoma

Yes-associated protein (YAP), an effector molecule of the Hippo signaling pathway, is expressed at high levels in cutaneous melanoma. However, the role of YAP in melanoma progression according to cellular localization is poorly understood. Tissues from 140 patients with invasive melanoma were evaluated by immunohistochemistry. Flow cytometry, western blotting, viability assays, wound healing assays, verteporfin treatment, and xenograft assays were conducted using melanoma cell lines B16F1 and B16F10 subjected to YapS127A transfection and siYap knockdown. Nuclear YAP localization was identified in 63 tumors (45.0%) and was more frequent than cytoplasmic YAP in acral lentiginous and nodular subtypes (P =.007). Compared with cytoplasmic YAP melanomas, melanomas with nuclear YAP had higher mitotic activity (P =.016), deeper invasion (P <.001), and more frequently metastasized to lymph nodes (P <.001) and distant organs (P <.001). Patients with nuclear YAP melanomas had poorer disease-free survival (P <.001) and overall survival (P <.001). Nuclear YAP was an independent risk factor for distant metastasis (hazard ratio: 3.206; 95% CI: 1.032-9.961; P =.044). Proliferative ability was decreased in siYapB16F1 (P <.001) and siYapB16F10 (P =.001) cells and increased in YapS127AB16F1 (P =.003) and YapS127AB16F10 (P =.002) cells. Cell cycle analysis demonstrated relative G1 retention in siYapB16F1 (P <.001) and siYapB16F10 (P <.001) cells and S retention in YapS127AB16F1 cells (P =.008). Wound healing assays showed that Yap knockdown inhibited cell invasion (siYapB16F1, P =.001; siYapB16F10, P <.001), whereas nuclear YAP promoted it (YapS127AB16F, P <.001; YapS127AB16F1, P =.017). Verteporfin, a direct YAP inhibitor, reduced cellular proliferation in B16F1 (P =.003) and B16F10 (P <.001) cells. Proliferative effects of nuclear YAP were confirmed in xenograft mice (P <.001). In conclusion, nuclear YAP in human melanomas showed subtype specificity and correlated with proliferative activity and proinvasiveness. It is expected that YAP becomes a useful prognostic marker, and its inhibition may be a potential therapy for melanoma patients.

Exercise preconditioning inhibits doxorubicin-induced cardiotoxicity via YAP/STAT3 signaling

Doxorubicin (DOX) possesses strong anti-tumor effects but is limited by its irreversible cardiac toxicity. The relationship between exercise, a known enhancer of cardiovascular health, and DOX-induced cardiotoxicity has been a focus of recent research. Exercise has been suggested to mitigate DOX's cardiac harm by modulating the Yes-associated protein (YAP) and Signal transducer and activator of transcription 3 (STAT3) pathways, which are crucial in regulating cardiac cell functions and responses to damage. This study aimed to assess the protective role of exercise preconditioning against DOX-induced cardiac injury. We used Sprague–Dawley rats, divided into five groups (control, DOX, exercise preconditioning (EP), EP-DOX, and verteporfin + EP + DOX), to investigate the potential mechanisms. Our findings, including echocardiography, histological staining, Western blot, and q-PCR analysis, demonstrated that exercise preconditioning could alleviate DOX-induced cardiac dysfunction and structural damage. Notably, exercise preconditioning enhanced the nuclear localization and co-localization of YAP and STAT3. Our study suggests that exercise preconditioning may counteract DOX-induced cardiotoxicity by activating the YAP/STAT3 pathway, highlighting a potential therapeutic approach for reducing DOX's cardiac side effects.

3D-bioprinted hydrogels with instructive niches for dental pulp regeneration

Infections to dental pulp commonly result in pulpitis and pulp necrosis, and surgical removal of the infected tissues is the only therapeutic approach. Dental pulp injury remains a challenging medical issue due to the limited regenerative capability of dental pulp. In this work, a dental pulp guidance construct (DPGC) with the instructive niche was bioprinted to mimic native teeth for dentin and neovascular-like structure reconstruction. GelMA-Dextran aqueous emulsion was used as an ink for in situ printing of porous DPGC to induce predominant nuclear localization of Yes-associated protein (YAP) in the encapsulated dental pulp stem cells (DPSCs) and enhance their stemness properties. Furthermore, the DPSCs encapsulated in DPGC with microporous structures exhibited enhanced viability, migration, and spreading. Meanwhile, we found that DPGC could promote capillary tube formation and induce neurogenesis. In a mouse subcutaneous implant model, the DPGC consisted of porous structures, such as odontoblasts and newly formed vascular structures, that mimic dental pulp characteristics. This study demonstrated a new strategy to design DPGC with instructive niche for dental pulp regeneration, presenting a potential treatment alternative to root canal therapy.

Inhibition of transcriptional coactivator YAP Impairs the expression and function of transcription factor WT1 in diabetic podocyte injury

Podocyte injury and loss are hallmarks of diabetic nephropathy (DN). However, the molecular mechanisms underlying these phenomena remain poorly understood. YAP (Yes-associated protein) is an important transcriptional coactivator that binds with various other transcription factors, including the TEAD family members (nuclear effectors of the Hippo pathway), that regulate cell proliferation, differentiation, and apoptosis. The present study found an increase in YAP phosphorylation at S127 of YAP and a reduction of nuclear YAP localization in podocytes of diabetic mouse and human kidneys, suggesting dysregulation of YAP may play a role in diabetic podocyte injury. Tamoxifen-inducible podocyte-specific Yap gene knockout mice (YappodKO) exhibited accelerated and worsened diabetic kidney injury. YAP inactivation decreased transcription factor WT1 expression with subsequent reduction of Tead1 and other well-known targets of WT1 in diabetic podocytes. Thus, our study not only sheds light on the pathophysiological roles of the Hippo pathway in diabetic podocyte injury but may also lead to the development of new therapeutic strategies to prevent and/or treat DN by targeting the Hippo signaling pathway.

Coupling of Perinuclear Actin Cap and Nuclear Mechanics in Regulating Flow-Induced Yap Spatiotemporal Nucleocytoplasmic Transport.

Mechanical forces, including flow shear stress, govern fundamental cellular processes by modulating nucleocytoplasmic transport of transcription factors like Yes-associated Protein (YAP). However, the underlying mechanical mechanism remains elusive. In this study, it is reported that unidirectional flow induces biphasic YAP transport with initial nuclear import, followed by nuclear export as actin cap formation and nuclear stiffening. Conversely, pathological oscillatory flow induces slight actin cap formation, nuclear softening, and sustained YAP nuclear localization. To elucidate the disparately YAP spatiotemporal distribution, a 3D mechanochemical model is developed, which integrates flow sensing, cytoskeleton organization, nucleus mechanotransduction, and YAP transport. The results unveiled that despite the significant localized nuclear stress imposed by the actin cap, its inherent stiffness counteracts the dispersed contractile stress exerted by conventional fibers on the nuclear membrane. Moreover, alterations in nuclear stiffness synergistically regulate nuclear deformation, thereby governing YAP transport. Furthermore, by expanding the single-cell model to a collective vertex framework, it is revealed that the irregularities in actin cap formation within individual cells have the potential to induce topological defects and spatially heterogeneous YAP distribution in the cellular monolayer. This work unveils a unified mechanism of flow-induced nucleocytoplasmic transport, providing a linkage between transcription factor localization and mechanical stimulation.

Hippo Signaling Mediates TGFβ-Dependent Transcriptional Inputs in Cardiac Cushion Mesenchymal Cells to Regulate Extracellular Matrix Remodeling.

The transforming growth factor beta (TGFβ) and Hippo signaling pathways are evolutionarily conserved pathways that play a critical role in cardiac fibroblasts during embryonic development, tissue repair, and fibrosis. TGFβ signaling and Hippo signaling are also important for cardiac cushion remodeling and septation during embryonic development. Loss of TGFβ2 in mice causes cardiac cushion remodeling defects resulting in congenital heart disease. In this study, we used in vitro molecular and pharmacologic approaches in the cushion mesenchymal cell line (tsA58-AVM) and investigated if the Hippo pathway acts as a mediator of TGFβ2 signaling. Immunofluorescence staining showed that TGFβ2 induced nuclear translocation of activated SMAD3 in the cushion mesenchymal cells. In addition, the results indicate increased nuclear localization of Yes-associated protein 1 (YAP1) following a similar treatment of TGFβ2. In collagen lattice formation assays, the TGFβ2 treatment of cushion cells resulted in an enhanced collagen contraction compared to the untreated cushion cells. Interestingly, verteporfin, a YAP1 inhibitor, significantly blocked the ability of cushion cells to contract collagen gel in the absence or presence of exogenously added TGFβ2. To confirm the molecular mechanisms of the verteporfin-induced inhibition of TGFβ2-dependent extracellular matrix (ECM) reorganization, we performed a gene expression analysis of key mesenchymal genes involved in ECM remodeling in heart development and disease. Our results confirm that verteporfin significantly decreased the expression of α-smooth muscle actin (Acta2), collagen 1a1 (Col1a1), Ccn1 (i.e., Cyr61), and Ccn2 (i.e., Ctgf). Western blot analysis indicated that verteporfin treatment significantly blocked the TGFβ2-induced activation of SMAD2/3 in cushion mesenchymal cells. Collectively, these results indicate that TGFβ2 regulation of cushion mesenchymal cell behavior and ECM remodeling is mediated by YAP1. Thus, the TGFβ2 and Hippo pathway integration represents an important step in understanding the etiology of congenital heart disease.

Nuclear deformation regulates YAP dynamics in cancer associated fibroblasts

Cells cultured on stiff 2D substrates exert high intracellular force, resulting in mechanical deformation of their nuclei. This nuclear deformation (ND) plays a crucial role in the transport of Yes Associated Protein (YAP) from the cytoplasm to the nucleus. However, cells in vivo are in soft 3D environment with potentially much lower intracellular forces. Whether and how cells may deform their nuclei in 3D for YAP localization remains unclear. Here, by culturing human colon cancer associated fibroblasts (CAFs) on 2D, 2.5D, and 3D substrates, we differentiated the effects of stiffness, force, and ND on YAP localization. We found that nuclear translocation of YAP depends on the degree of ND irrespective of dimensionality, stiffness and total force. ND induced by the perinuclear force, not the total force, and nuclear membrane curvature correlate strongly with YAP activation. Immunostained slices of human tumors further supported the association between ND and YAP nuclear localization, suggesting ND as a potential biomarker for YAP activation in tumors. Additionally, we conducted quantitative analysis of the force dynamics of CAFs on 2D substrates to construct a stochastic model of YAP kinetics. This model revealed that the probability of YAP nuclear translocation, as well as the residence time in the nucleus follow a power law. This study provides valuable insights into the regulatory mechanisms governing YAP dynamics and highlights the significance of threshold activation in YAP localization. Statement of SignificanceYes Associated Protein (YAP), a transcription cofactor, has been identified as one of the drivers of cancer progression. High tumor stiffness is attributed to driving YAP to the nucleus, wherein it activates pro-metastatic genes. Here we show, using cancer associated fibroblasts, that YAP translocation to the nucleus depends on the degree of nuclear deformation, irrespective of stiffness. We also identified that perinuclear force induced membrane curvature correlates strongly with YAP nuclear transport. A novel stochastic model of YAP kinetics unveiled a power law relationship between the activation threshold and persistence time of YAP in the nucleus. Overall, this study provides novel insights into the regulatory mechanisms governing YAP dynamics and the probability of activation that is of immense clinical significance.

Regulation of the integrin αVβ3- actin filaments axis in early osteogenic differentiation of human mesenchymal stem cells under cyclic tensile stress

BackgroundIntegrins are closely related to mechanical conduction and play a crucial role in the osteogenesis of human mesenchymal stem cells. Here we wondered whether tensile stress could influence cell differentiation through integrin αVβ3.MethodsWe inhibited the function of integrin αVβ3 of human mesenchymal stem cells by treating with c(RGDyk). Using cytochalasin D and verteporfin to inhibit polymerization of microfilament and function of nuclear Yes-associated protein (YAP), respectively. For each application, mesenchymal stem cells were loaded by cyclic tensile stress of 10% at 0.5 Hz for 2 h daily. Mesenchymal stem cells were harvested on day 7 post-treatment. Western blotting and quantitative RT-PCR were used to detect the expression of alkaline phosphatase (ALP), RUNX2, β-actin, integrin αVβ3, talin-1, vinculin, FAK, and nuclear YAP. Immunofluorescence staining detected vinculin, actin filaments, and YAP nuclear localization.ResultsCyclic tensile stress could increase the expression of ALP and RUNX2. Inhibition of integrin αVβ3 activation led to rearrangement of actin filaments and downregulated the expression of ALP, RUNX2 and promoted YAP nuclear localization. When microfilament polymerization was inhibited, ALP, RUNX2, and nuclear YAP nuclear localization decreased. Inhibition of YAP nuclear localization could reduce the expression of ALP and RUNX2.ConclusionsCyclic tensile stress promotes early osteogenesis of human mesenchymal stem cells via the integrin αVβ3-actin filaments axis. YAP nuclear localization participates in this process of human mesenchymal stem cells.7pqm7m_g_KeHaQ8nD7WfNiVideo

Rapid responses of human pluripotent stem cells to cyclic mechanical strains applied to integrin by acoustic tweezing cytometry

Acoustic tweezing cytometry (ATC) is an ultrasound-based biophysical technique that has shown the capability to promote differentiation of human pluripotent stem cells (hPSCs). This study systematically examined how hPSCs respond to cyclic mechanical strains applied by ATC via displacement of integrin-bound microbubbles (averaged diameter of 4.3 µm) using ultrasound pulses (acoustic pressure 0.034 MPa, center frequency 1.24 MHz and pulse repetition frequency 1 Hz). Our data show downregulation of pluripotency marker Octamer-binding transcription factor 4 (OCT4) by at least 10% and increased nuclear localization of Yes-associated protein (YAP) by almost 100% in hPSCs immediately after ATC application for as short as 1 min and 5 min respectively. Analysis of the movements of integrin-anchored microbubbles under ATC stimulations reveals different stages of viscoelastic characteristic behavior and increasing deformation of the integrin-cytoskeleton (CSK) linkage. The peak displacement of integrin-bound microbubbles increased from 1.45 ± 0.16 to 4.74 ± 0.67 μm as the duty cycle of ultrasound pulses increased from 5% to 50% or the duration of each ultrasound pulse increased from 0.05 to 0.5 s. Real-time tracking of integrin-bound microbubbles during ATC application detects high correlation of microbubble displacements with OCT4 downregulation in hPSCs. Together, our data showing fast downregulation of OCT4 in hPSCs in respond to ATC stimulations highlight the unique mechanosensitivity of hPSCs to integrin-targeted cyclic force/strain dependent on the pulse duration or duty cycle of ultrasound pulses, providing insights into the mechanism of ATC-induced accelerated differentiation of hPSCs.

Dynamic Stiffening Hydrogel with Instructive Stiffening Timing Modulates Stem Cell Fate In Vitro and Enhances Bone Remodeling In Vivo.

Biomechanical stimuli derived from the extracellular matrix (ECM) extremely tune stem cell fate through 3D and spatiotemporal changes in vivo. The matrix stiffness is a crucial factor during bone tissue development. However, most in vitro models to study the osteogenesis of mesenchymal stem cells (MSCs) are static or stiffening in a 2D environment. Here, a dynamic and controllable stiffening 3D biomimetic model is created to regulate the osteogenic differentiation of MSCs with a dual-functional gelatin macromer that can generate a double-network hydrogel by sequential enzymatic and light-triggered crosslinking reactions. The findings showthat these dynamic hydrogels allowed cells to spread and expand prior to the secondary crosslinking and to sense high stiffness after stiffening. The MSCs in the dynamic hydrogels, especially the hydrogel stiffened at the late period, presentsignificantly elevated osteogenic ECM secretion, gene expression, and nuclear localization of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). In vivo evaluation of animal experiments further indicatesthat the enhancement of dynamic stiffening on osteogenesis of MSCs substantially promotes bone remodeling. Consequently, this work reveals that the 3D dynamic stiffening microenvironment as a critical biophysical cue not only mediates the stem cell fate in vitro, but also augments bone restoration in vivo.

Nuclear compression regulates YAP spatiotemporal fluctuations in living cells

Yes-associated protein (YAP) is a key mechanotransduction protein in diverse physiological and pathological processes; however, a ubiquitous YAP activity regulatory mechanism in living cells has remained elusive. Here, we show that YAP nuclear translocation is highly dynamic during cell movement and is driven by nuclear compression arising from cell contractile work. We resolve the mechanistic role of cytoskeletal contractility in nuclear compression by manipulation of nuclear mechanics. Disrupting the linker of nucleoskeleton and cytoskeleton complex reduces nuclear compression for a given contractility and correspondingly decreases YAP localization. Conversely, decreasing nuclear stiffness via silencing of lamin A/C increases nuclear compression and YAP nuclear localization. Finally, using osmotic pressure, we demonstrated that nuclear compression even without active myosin or filamentous actin regulates YAP localization. The relationship between nuclear compression and YAP localization captures a universal mechanism for YAP regulation with broad implications in health and biology.

YAP/WNT5A/FZD4 axis regulates osteogenic differentiation of human periodontal ligament cells under cyclic stretch.

To verify the role of YAP/WNT5A/FZD4 axis in stretch-induced osteogenic differentiation of hPDLCs. During orthodontic tooth movement, differentiation of human periodontal ligament cells (hPDLCs) at the tension side of the periodontal ligament mediates new bone formation. WNT5A promotes osteogenesis and its regulator Yes-associated protein (YAP) is responsive to mechanical stimulation in hPDLCs. However, the mechanisms of YAP and WNT5A in alveolar bone remodeling remain unclear. Cyclic stretch was applied to hPDLCs to mimic the orthodontic stretching force. Osteogenic differentiation was determined by alkaline phosphatase (ALP) activity, Alizarin Red staining, qRT-PCR and western blotting. To detect activation of YAP and expression of WNT5A and its receptor Frizzled-4 (FZD4), western blotting, immunofluorescence, qRT-PCR and ELISA were performed. Verteporfin, Lats-IN-1, small interfering RNAs and recombinant protein were used to explore the relationship of YAP, WNT5A and FZD4, and the effect of their relationship on stretch-induced osteogenesis of hPDLCs. WNT5A, FZD4 and nuclear localization of YAP were upregulated by cyclic stretch. YAP positively regulated WNT5A and FZD4 expression and osteogenic differentiation of hPDLCs under cyclic stretch by YAP inhibition or activation assay. Knockdown of WNT5A and FZD4 attenuated YAP-induced and stretch-induced osteogenic differentiation. Recombinant WNT5A rescued the suppressed osteogenic differentiation by YAP inhibitor in hPDLCs, whereas knockdown of FZD4 weakened the effect of WNT5A and amplified the suppression. WNT5A/FZD4 could be positively regulated by YAP and the YAP/WNT5A/FZD4 axis mediated osteogenic differentiation of hPDLCs under cyclic stretch. This study provided further insight into the biological mechanism of orthodontic tooth movement.

YAP Inhibition Alleviates Simulated Microgravity-Induced Mesenchymal Stem Cell Senescence via Targeting Mitochondrial Dysfunction.

Weightlessness in space leads to bone loss, muscle atrophy, and impaired immune defense in astronauts. Mesenchymal stem cells (MSCs) play crucial roles in maintaining the homeostasis and function of the tissue. However, how microgravity affects the characteristics MSCs and the related roles in the pathophysiological changes in astronauts remain barely known. Here we used a 2D-clinostat device to simulate microgravity. Senescence-associated-β-galactosidase (SA-β-gal) staining and the expression of senescent markers p16, p21, and p53 were used to evaluate the senescence of MSCs. Mitochondrial membrane potential (mΔΨm), reactive oxygen species (ROS) production, and ATP production were used to evaluate mitochondrial function. Western blot and immunofluorescence staining were used to investigate the expression and localization of Yes-associated protein (YAP). We found that simulated microgravity (SMG) induced MSC senescence and mitochondrial dysfunction. Mito-TEMPO (MT), a mitochondrial antioxidant, restored mitochondrial function and reversed MSC senescence induced by SMG, suggesting that mitochondrial dysfunction mediates SMG-induced MSC senescence. Further, it was found that SMG promoted YAP expression and its nuclear translocation in MSCs. Verteporfin (VP), an inhibitor of YAP, restored SMG-induced mitochondrial dysfunction and senescence in MSCs by inhibiting YAP expression and nuclear localization. These findings suggest that YAP inhibition alleviates SMG-induced MSC senescence via targeting mitochondrial dysfunction, and YAP may be a potential therapeutic target for the treatment of weightlessness-related cell senescence and aging.

The biophysical property of the limbal niche maintains stemness through YAP

The cell fate decisions of stem cells (SCs) largely depend on signals from their microenvironment (niche). However, very little is known about how biochemical niche cues control cell behavior in vivo. To address this question, we focused on the corneal epithelial SC model in which the SC niche, known as the limbus, is spatially segregated from the differentiation compartment. We report that the unique biomechanical property of the limbus supports the nuclear localization and function of Yes-associated protein (YAP), a putative mediator of the mechanotransduction pathway. Perturbation of tissue stiffness or YAP activity affects SC function as well as tissue integrity under homeostasis and significantly inhibited the regeneration of the SC population following SC depletion. In vitro experiments revealed that substrates with the rigidity of the corneal differentiation compartment inhibit nuclear YAP localization and induce differentiation, a mechanism that is mediated by the TGFβ−SMAD2/3 pathway. Taken together, these results indicate that SC sense biomechanical niche signals and that manipulation of mechano-sensory machinery or its downstream biochemical output may bear fruits in SC expansion for regenerative therapy.

WNT7A suppresses adipogenesis of skeletal muscle mesenchymal stem cells and fatty infiltration through the alternative Wnt-Rho-YAP/TAZ signaling axis.

Intramuscular fatty infiltration in muscle injuries and diseases, caused by aberrant adipogenesis of fibro-adipogenic progenitors, negatively impacts function. Intramuscular delivery of wingless-type MMTV integration site family 7a (WNT7A) offers a promising strategy to stimulate muscle regeneration, but its effects on adipogenic conversion of fibro-adipogenic progenitors remain unknown. Here, we show that WNT7A decreases adipogenesis of fibro-adipogenic progenitors (FAPs) by inducing nuclear localization of Yes-associated protein (YAP) through Rho in a β-CATENIN-independent manner and by promoting nuclear retention of YAP and transcriptional co-activator with PDZ-binding motif (TAZ) in differentiating FAPs. Furthermore, intramuscular injection of WNT7A invivo effectively suppresses fatty infiltration in mice following glycerol-induced injury. Our results collectively suggest WNT7A as a potential protein-based therapeutic for diminishing adipogenesis of FAPs and intramuscular fatty infiltration in pathological muscle injuries or diseases.

Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

Stiffness-Modulation of Collagen Gels by Genipin-Crosslinking for Cell Culture.

The stiffness of extracellular matrices (ECMs) is critical for cellular functions. Therefore, modulating the stiffness of ECMs in vitro is necessary to investigate the role of stiffness in cellular phenomena. Collagen gels are widely used for cell culture matrices in vitro. However, modulation of the stiffness in collagen gels for cell culture is challenging owing to the limited knowledge of the method to increase the stiffness while maintaining low cytotoxicity. Here, we established a novel method to modulate collagen gel stiffness from 0.0292 to 12.5 kPa with low cytotoxicity. We prepared collagens with genipin, a low-cytotoxic crosslinker of amines, at different concentrations and successfully modulated the stiffness of the gels. In addition, on 10 mM genipin-mixed collagen gels (approximately 12.5 kPa), H1299 human lung cancer cells showed spreading morphology and nuclear localization of yes-associated protein (YAP), typical phenomena of cells on stiff ECMs. Mouse mesenchymal stromal cells on 10 mM genipin-mixed collagen gels differentiated to vascular smooth muscle cells. On the other hand, the cells on 0 mM genipin-mixed collagen gels (approximately 0.0292 kPa) differentiated to visceral smooth muscle cells. Our new method provides a novel way to prepare stiffness-modulated collagen gels with low cytotoxicity in cell culture.

Stretch-activated nuclear YAP localization in cardiomyocytes.

Yes Associated Protein (YAP) is a mechanosensitive transcriptional activator regulating a variety of cell fates, including proliferation and cell growth. YAP is activated (localized to the nucleus) in both physiological development (e.g., organ growth mediated by cell proliferation) and pathological heart diseases (e.g., organ growth mediated by cell enlargement). Cardiomyocytes (CMs) generate the mechanical work that powers each heartbeat, and each contraction shortens the CM and strains the nuclei. Mechanical forces and nuclear deformation are considered putative drivers of YAP activation. However, the mechanoregulation of YAP's dual roles in organ growth - through proliferation during development and via hypertrophic enlargement in adulthood - remain unknown. Both contractile output and strain increase as the heart works harder under increased load (e.g., with exercise, disease, aging) and over time can lead to remodeling characteristic of hypertrophic cardiomyopathy. To mimic this additional loading, we first patterned extracellular matrix (Matrigel) single-cell rectangles of 7:1 aspect ratio on silicon substrate of a previously published, microfabricated, silicone pneumatic cell-stretching device (Hart, CAMB, 2021). Then, we then seeded human induced pluripotent stem cell-derived CMs (hiPSC-CM) on these patterns to elicit an elongated, anisotropic subcellular structure. We applied 15% stretch strain in static and cyclic, at 1 Hz frequency, for 10 mins to hiPSC-CM aligned in perpendicular or in parallel to the direction of stretch. Finally, we fixed and immunostained with YAP antibodies to quantify YAP localization. We quantified the ratio of nuclear/non-nuclear YAP in hiPSC-CM and found an upregulation of nuclear YAP in parallel, 97±10% in static and 42±5% in cyclic, and no significant change in regulation in perpendicular. Ultimately, these experiments will help us understand how mechanical cues and directionality of forces drive nuclear YAP, and the role of YAP signaling in driving hypertrophic hiPSC-CM remodeling.