Mechanotransduction in high aspect ratio nanostructured meta-biomaterials: The role of cell adhesion, contractility, and transcriptional factors.

Bone formation is linked with osteogenic differentiation of mesenchymal stem cells (MSCs) in the bone marrow. Microgravity in spaceflight is known to reduce bone formation. In this study, we used a real microgravity environment of the SJ-10 Recoverable Scientific Satellite to examine the effects of space microgravity on the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). hMSCs were induced toward osteogenic differentiation for 2 and 7 d in a cell culture device mounted on the SJ-10 satellite. The satellite returned to Earth after going through space experiments in orbit for 12 d, and cell samples were harvested and analyzed for differentiation potentials. The results showed that space microgravity inhibited osteogenic differentiation and resulted in adipogenic differentiation, even under osteogenic induction conditions. Under space microgravity, the expression of 10 genes specific for osteogenesis decreased, including collagen family members, alkaline phosphatase ( ALP), and runt-related transcription factor 2 ( RUNX2), whereas the expression of 4 genes specific for adipogenesis increased, including adipsin ( CFD), leptin ( LEP), CCAAT/enhancer binding protein β ( CEBPB), and peroxisome proliferator-activated receptor-γ ( PPARG). In the analysis of signaling pathways specific for osteogenesis, we found that the expression and activity of RUNX2 was inhibited, expression of bone morphogenetic protein-2 ( BMP2) and activity of SMAD1/5/9 were decreased, and activity of focal adhesion kinase (FAK) and ERK-1/2 declined significantly under space microgravity. These data indicate that space microgravity plays a dual role by decreasing RUNX2 expression and activity through the BMP2/SMAD and integrin/FAK/ERK pathways. In addition, we found that space microgravity increased p38 MAPK and protein kinase B (AKT) activities, which are important for the promotion of adipogenic differentiation of hMSCs. Space microgravity significantly decreased the expression of Tribbles homolog 3 ( TRIB3), a repressor of adipogenic differentiation. Y15, a specific inhibitor of FAK activity, was used to inhibit the activity of FAK under normal gravity; Y15 decreased protein expression of TRIB3. Therefore, it appears that space microgravity decreased FAK activity and thereby reduced TRIB3 expression and derepressed AKT activity. Under space microgravity, the increase in p38 MAPK activity and the derepression of AKT activity seem to synchronously lead to the activation of the signaling pathway specifically promoting adipogenesis.-Zhang, C., Li, L., Jiang, Y., Wang, C., Geng, B., Wang, Y., Chen, J., Liu, F., Qiu, P., Zhai, G., Chen, P., Quan, R., Wang, J. Space microgravity drives transdifferentiation of human bone marrow-derived mesenchymal stem cells from osteogenesis to adipogenesis.

RhoA a small G-protein that has an established role in cell growth and in regulation of the actin cytoskeleton. Far less is known about whether RhoA can modulate cell fate. We previously reported that sustained RhoA activation induces cardiomyocyte apoptosis (Del Re, D. P., Miyamoto, S., and Brown, J. H. (2007) J. Biol. Chem. 282, 8069-8078). Here we demonstrate that less chronic RhoA activation affords a survival advantage, protecting cardiomyocytes from apoptotic insult induced by either hydrogen peroxide treatment or glucose deprivation. Under conditions where RhoA is protective, we observe Rho kinase-dependent cytoskeletal rearrangement and activation of focal adhesion kinase (FAK). Activation of endogenous cardiomyocyte FAK leads to its increased association with the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and to concomitant activation of Akt. Treatment of isolated perfused hearts with sphingosine 1-phosphate recapitulates this response. The pathway by which RhoA mediates cardiomyocyte Akt activation is demonstrated to require Rho kinase, FAK and PI3K, but not Src, based on studies with pharmacological inhibitors (Y-27632, LY294002, PF271 and PP2) and inhibitory protein expression (FAK-related nonkinase). Inhibition of RhoA-mediated Akt activation at any of these steps, including inhibition of FAK, prevents RhoA from protecting cardiomyocytes against apoptotic insult. We further demonstrate that stretch of cardiomyocytes, which activates endogenous RhoA, induces the aforementioned signaling pathway, providing a physiologic context in which RhoA-mediated FAK phosphorylation can activate PI3K and Akt. We suggest that RhoA-mediated effects on the cardiomyocyte cytoskeleton provide a novel mechanism for protection from apoptosis.

The effect of purmorphamine and sirolimus on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

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

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

FAK, vinculin, and talin control mechanosensitive YAP nuclear localization

Yes-associated protein (YAP) is a mechanosensory protein that serves as a critical transcriptional coactivator involved in various cellular processes. Thus, the precise regulation of YAP is vital for effectively manipulating cellular functions. Despite small molecular inhibitors and siRNA being available, these approaches often fail to achieve precise and reversible regulation of YAP dynamics within complex biological systems. This often leads to suboptimal therapeutic results or collateral damage to healthy tissues. Here, we introduce a method for quantitative and reversible regulation of YAP through nanoparticle-mediated magnetothermal stimulation of F-actin, which is crucial for transmitting mechanical signals to YAP. We utilized actin-binding peptide-modified ultrasmall zinc-doped ferrite nanoparticles to specifically and effectively disrupt intracellular F-actin structures by generating localized heat upon exposure to an alternating magnetic field (AMF). In our in vitro studies, increasing the AMF intensity from 0 to 313 Oe led to a progressive deterioration of F-actin integrity, which was defined as Fint. Fint gradually decreased from 0.91 to 0.38 as the AMF intensity increased from 0 to 313 Oe. Concurrently, YAP nuclear localization also decreased, with the most rapid decline occurring within the range of 243-313 Oe. Importantly, cells were able to completely F-actin self-repair within 24 h, allowing YAP to re-enter the nucleus. This ability facilitates the quantitative and reversible control over YAP nuclear translocation. Further in vivo studies using MCF-7 tumor-bearing mouse models demonstrated the efficacy of this method in inhibiting glycolysis, reducing the 'Warburg effect', and reprogramming the tumor metabolic microenvironment. These results not only provide a promising strategy for precise manipulation of YAP nuclear localization but also establish a therapeutic framework for treating intractable diseases driven by YAP dysregulation.

Nuclear deformation regulates YAP dynamics in cancer associated fibroblasts

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.

This study aims to investigate the effect and mechanism of nystose on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). hBMSCs were cultured and divided into 5 groups: a control group that received no treatment, and 4 groups treated with varying concentrations of nystose solution (0.5, 1, 5, and 10 μg/mL). The proliferation ability of hBMSCs was tested at different culture times and different concentrations of nystose solution using cell counting kit-8 assay. Alizarin red staining was employed to examine ALP expression and calcium nodule formation in hBMSCs. Western blotting and RT-PCR were conducted to analyze the expressions of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2), as well as the levels of c-Jun N-terminal Kinase (JNK)/extracellular regulated protein kinases (ERK) pathway genes. Our findings demonstrated that increasing nystose concentration enhanced cell proliferation. Notably, compared to the control group, nystose intervention significantly elevated ALP activity and expression of osteoblast-related genes. Additionally, it was observed that nystose intervention increased phosphorylation levels of JNK1 and ERK1/2. Conversely, inhibition of JNK/ERK pathway activity reduced ALP and RUNX2 expression and calcium nodule formation, thereby inhibiting osteogenic differentiation of hBMSCs. Nystose intervention effectively regulated the osteogenic differentiation ability of hBMSCs through modulation of the JNK/ERK pathway. These results provide evidence supporting the promotion of osteogenic differentiation of hBMSCs by nystose, thus offering a promising therapeutic approach for bone-related diseases such as osteoporosis.

Antibody (Ab) crosslinking of HLA class II (HLA II) molecules on the surface of endothelial cells (ECs) triggers proliferative and prosurvival intracellular signaling, which are implicated in promoting chronic Ab-mediated rejection (cAMR). Despite the importance of cAMR in transplant medicine, the mechanisms involved remain incompletely understood. Here, we examined the regulation of yes-associated protein (YAP) nuclear cytoplasmic localization and phosphorylation in human ECs challenged with Abs that bind HLA II, which are strongly associated with cAMR. To examine changes in YAP localization in response to Ab-mediated engagement of HLA II, we used an adenoviral vector to express the class II transactivator or treatment with interferon γ. In unstimulated ECs expressing HLA II, YAP localized mainly in the cytoplasm. Stimulation with HLA II Ab (0.1–1 µg/mL) induced marked translocation of YAP to the nucleus. HLA II signaling triggered by high concentrations of HLA II Ab (1 µg/mL) also induced prominent YAP localization in cytoplasmic punctate structures that were disassembled by exposure to 1,6-hexanediol, suggesting that these structures are biomolecular condensates. Using multiple treatments, including stimulation with serum, thrombin or HLA I Ab and conditions (eg ECs plated at different densities) indicate that formation of YAP cytoplasmic puncta can be dissociated from YAP nuclear localization and phosphorylation at Ser127, a site in YAP targeted by the Hippo kinases LATS1/2. The results revealed that HLA II signaling regulates YAP subcellular distributions in ECs and demonstrate, for the first time, that HLA II Ab selectively stimulates YAP concentration in punctate structures.

Extracellular signal-regulated kinase1/2 activated by fluid shear stress promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells through novel signaling pathways

Idiopathic pulmonary fibrosis is a progressive lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. We previously identified HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (statins) as YAP inhibitors based on a high-throughput small-molecule screen in primary human lung fibroblasts. Here we report that several Aurora kinase inhibitors were also identified from the top hits of this screen. MK-5108, a highly selective inhibitor for AURKA (Aurora kinase A), induced YAP phosphorylation and cytoplasmic retention and significantly reduced profibrotic gene expression in human lung fibroblasts. The inhibitory effect on YAP nuclear translocation and profibrotic gene expression is specific to inhibition of AURKA, but not Aurora kinase B or C, and is independent of the Hippo pathway kinases LATS1 and LATS2 (Large Tumor Suppressor 1 and 2). Further characterization of the effects of MK-5108 demonstrate that it inhibits YAP nuclear localization indirectly via effects on actin polymerization and TGFβ (Transforming Growth Factor β) signaling. In addition, MK-5108 treatment reduced lung collagen deposition in the bleomycin mouse model of pulmonary fibrosis. Our results reveal a novel role for AURKA in YAP-mediated profibrotic activity in fibroblasts and highlight the potential of small-molecule screens for YAP inhibitors for identification of novel agents with antifibrotic activity.

We demonstrate that Ha-RasV12 overexpression induces the nuclear translocation of Hippo effector Yes-associated protein (YAP) in MDCK cells via the hippo-independent pathway at the confluent stage. Ha-RasV12 overexpression leads to the downregulation of Caveolin-1 (Cav1) and the disruption of junction integrity. It has been shown that the disruption of actin belt integrity causes YAP nuclear translocation in epithelial cells at high density. Therefore, we hypothesized that Ha-RasV12-decreased Cav1 leads to the disruption of cell junction integrity, which subsequently facilitates YAP nuclear retention. We revealed that Ha-RasV12 downregulated Cav1 through the ERK pathway. Furthermore, the distribution and expression of Cav1 mediated the cell junction integrity and YAP nuclear localization. This suggests that the downregulation of Cav1 induced by Ha-RasV12 disrupted the cell junction integrity and promoted YAP nuclear translocation. We further indicated the consequence of Ha-RasV12-induced YAP activation. Surprisingly, the activation of YAP is not required for Ha-RasV12-induced multilayer cellular aggregates. Instead, Ha-RasV12 triggered the ERK-Rac pathway to promote cellular aggregate formation. Moreover, the overexpression of constitutively active Rac is sufficient to trigger cellular aggregation in MDCK cells at the confluent stage. This highlights that Rac activity is essential for cellular aggregates.

MicroRNAs play an important role in the development and function of neuron cells. Among these, the miRNA known as MIR96 is abundantly expressed in mammalian retina and significantly affects differentiation, maturation, and survival of human photoreceptor cells. In this study, a mimic to miRNA-96 was transfected into human bone marrowderived mesenchymal stem cells to explore the biological functions of MIR96 at differentiation processing. A mimic to miRNA-96 and a competitive control were transfected into human bone marrow-derived mesenchymal stem cells using Lipofectamine. After 24 and 48 hours, we evaluated changes in expression levels of genes associated with neural progenitor and photoreceptor differentiation (OTX2, NRL, protein kinase C, SLC1A1, and recoverin) by real-time polymerase chain reaction. In addition, we measured expression of mRNA and protein of the CRX gene (neuroretinal progenitor cell marker) and the RHO gene (terminal differentiation marker) using real-time polymerase chain reaction and immunocytochemistry, respectively. Real-time polymerase chain reaction results showed increased levels of RHO and recoverin mRNA after 24 hours in transfected cells. In addition, mRNA levels of OTX2, CRX, NRL, RHO, recoverin, and protein kinase C increased after 48 hours in transfected cells. Immunocytochemistry results confirmed these findings by demonstrating RHO and CRX at both 24 and 48 hours in transfected cells. Control of the expression of MIR96 can be a good strategy to promote cell differentiation and can be used in cell therapy for retinal degeneration. Our results showed that human bone marrow-derived mesenchymal stem cells can differentiate into photoreceptor cells after transfection with MIR96. These results support therapeutic use of MIR96 in retinal degeneration and suggest human bone marrowderived mesenchymal stem cells as a promising tool for interventions.