Sarcopenia-related changes in serum GLP-1 level affect myogenic differentiation.

BACKGROUND: Sarcopenia, a group of muscle-related disorders, leads to the gradual decline and weakening of skeletal muscle over time. Recognizing the pivotal role of gastrointestinal conditions in maintaining metabolic homeostasis within skeletal muscle, we hypothesize that the effectiveness of the myogenic programme is influenced by the levels of gastrointestinal hormones in the bloodstream, and this connection is associated with the onset of sarcopenia. METHODS: We first categorized 145 individuals from the Emergency Room of Taipei Veterans General Hospital into sarcopenia and non-sarcopenia groups, following the criteria established by the Asian Working Group for Sarcopenia. A thorough examination of specific gastrointestinal hormone levels in plasma was conducted to identify the one most closely associated with sarcopenia. Techniques, including immunofluorescence, western blotting, glucose uptake assays, seahorse real-time cell metabolic analysis, flow cytometry analysis, kinesin-1 activity assays and qPCR analysis, were applied to investigate its impacts and mechanisms on myogenic differentiation. RESULTS: Individuals in the sarcopenia group exhibited elevated plasma levels of glucagon-like peptide 1 (GLP-1) at 1021.5 ± 313.5 pg/mL, in contrast to non-sarcopenic individuals with levels at 351.1 ± 39.0 pg/mL (P < 0.05). Although it is typical for GLP-1 levels to rise post-meal and subsequently drop naturally, detecting higher GLP-1 levels in starving individuals with sarcopenia raised the possibility of GLP-1 influencing myogenic differentiation in skeletal muscle. Further investigation using a cell model revealed that GLP-1 (1, 10 and 100 ng/mL) dose-dependently suppressed the expression of the myogenic marker, impeding myocyte fusion and the formation of polarized myotubes during differentiation. GLP-1 significantly inhibited the activity of the microtubule motor kinesin-1, interfering with the translocation of glucose transporter 4 (GLUT4) to the cell membrane and the dispersion of mitochondria. These impairments subsequently led to a reduction in glucose uptake to 0.81 ± 0.04 fold (P < 0.01) and mitochondrial adenosine triphosphate (ATP) production from 25.24 ± 1.57 pmol/min to 18.83 ± 1.11 pmol/min (P < 0.05). Continuous exposure to GLP-1, even under insulin induction, attenuated the elevated glucose uptake. CONCLUSIONS: The elevated GLP-1 levels observed in individuals with sarcopenia are associated with a reduction in myogenic differentiation. The impact of GLP-1 on both the membrane translocation of GLUT4 and the dispersion of mitochondria significantly hinders glucose uptake and the production of mitochondrial ATP necessary for the myogenic programme. These findings point us towards strategies to establish the muscle-gut axis, particularly in the context of sarcopenia. Additionally, these results present the potential of identifying relevant diagnostic biomarkers.

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.

Matrix mechanics regulates muscle regeneration by modulating kinesin-1 activity.

Sarcopenia, a prevalent muscle disease characterized by muscle mass and strength reduction, is associated with impaired skeletal muscle regeneration. However, the influence of the biomechanical properties of sarcopenic skeletal muscle on the efficiency of the myogenic program remains unclear. Herein, we established a mouse model of sarcopenia and observed a reduction in stiffness within the sarcopenic skeletal muscle in vivo. To investigate whether the biomechanical properties of skeletal muscle directly impact the myogenic program, we established an in vitro system to explore the intrinsic mechanism involving matrix stiffness control of myogenic differentiation. Our findings identify the microtubule motor protein, kinesin-1, as a mechano-transduction hub that senses and responds to matrix stiffness, crucial for myogenic differentiation and muscle regeneration. Specifically, kinesin-1 activity is positively regulated by stiff matrices, facilitating its role in transporting mitochondria and enhancing translocation of the glucose transporter GLUT4 to the cell surface for glucose uptake. Conversely, the softer matrices significantly suppress kinesin-1 activity, leading to the accumulation of mitochondria around nuclei and hindering glucose uptake by inhibiting GLUT4 membrane translocation, consequently impairing myogenic differentiation. The insights gained from the in-vitro system highlight the mechano-transduction significance of kinesin-1 motor proteins in myogenic differentiation. Furthermore, our study confirms that enhancing kinesin-1 activity in the sarcopenic mouse model restores satellite cell expansion, myogenic differentiation, and muscle regeneration. Taken together, our findings provide a potential target for improving muscle regeneration in sarcopenia.

PAICS ubiquitination recruits UBAP2 to trigger phase separation for purinosome assembly.

Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.

Development of vessel mimicking microfluidic device for studying mechano-response of endothelial cells.

The objective of this study is to develop a device to mimic a microfluidic system of human arterial blood vessels. The device combines fluid shear stress (FSS) and cyclic stretch (CS), which are resulting from blood flow and blood pressure, respectively. The device can reveal real-time observation of dynamic morphological change of cells in different flow fields (continuous flow, reciprocating flow and pulsatile flow) and stretch. We observe the effects of FSS and CS on endothelial cells (ECs), including ECs align their cytoskeleton proteins with the fluid flow direction and paxillin redistribution to the cell periphery or the end of stress fibers. Thus, understanding the morphological and functional changes of endothelial cells on physical stimuli can help us to prevent and improve the treatment of cardiovascular diseases.

Early committed polarization of intracellular tension in response to cell shape determines the osteogenic differentiation of mesenchymal stromal cells.

Within the heterogeneous tissue architecture, a comprehensive understanding of how cell shapes regulate cytoskeletal mechanics by adjusting focal adhesions (FAs) signals to correlate with the lineage commitment of mesenchymal stromal cells (MSCs) remains obscure. Here, via engineered extracellular matrices, we observed that the development of mature FAs, coupled with a symmetrical pattern of radial fiber bundles, appeared at the right-angle vertices in cells with square shape. While circular cells aligned the transverse fibers parallel to the cell edge, and moved them centripetally in a counter-clockwise direction, symmetrical bundles of radial fibers at the vertices of square cells disrupted the counter-clockwise swirling and bridged the transverse fibers to move centripetally. In square cells, the contractile force, generated by the myosin IIA-enriched transverse fibers, were concentrated and transmitted outwards along the symmetrical bundles of radial fibers, to the extracellular matrix through FAs, and thereby driving FA organization and maturation. The symmetrical radial fiber bundles concentrated the transverse fibers contractility inward to the linkage between the actin cytoskeleton and the nuclear envelope. The tauter cytoskeletal network adjusted the nuclear-actomyosin force balance to cause nuclear deformability and to increase nuclear translocation of the transcription co-activator YAP, which in turn modulated the switch in MSC commitment. Thus, FAs dynamically respond to geometric cues and remodel actin cytoskeletal network to re-distribute intracelluar tension towards the cell nucleus, and thereby controlling YAP mechanotransduction signaling in regulating MSC fate decision. STATEMENT OF SIGNIFICANCE: We decipher how cellular mechanics is self-organized depending on extracellular geometric features to correlate with mesenchymal stromal cell lineage commitment. In response to geometry constrains on cell morphology, symmetrical radial fiber bundles are assembled and clustered depending on the maturation state of focal adhesions and bridge with the transverse fibers, and thereby establishing the dynamic cytoskeletal network. Contractile force, generated by the myosin-IIA-enriched transverse fibers, is transmitted and dynamically drives the retrograde movement of the actin cytoskeletal network, which appropriately adjusts the nuclear-actomyosin force balance and deforms the cell nucleus for YAP mechano-transduction signaling in regulating mesenchymal stromal cell fate decision.

Phase separation driven by interchangeable properties in the intrinsically disordered regions of protein paralogs.

Paralogs, arising from gene duplications, increase the functional diversity of proteins. Protein functions in paralog families have been extensively studied, but little is known about the roles that intrinsically disordered regions (IDRs) play in their paralogs. Without a folded structure to restrain them, IDRs mutate more diversely along with evolution. However, how the diversity of IDRs in a paralog family affects their functions is unexplored. Using the RNA-binding protein Musashi family as an example, we applied multiple structural techniques and phylogenetic analysis to show how members in a paralog family have evolved their IDRs to different physicochemical properties but converge to the same function. In this example, the lower prion-like tendency of Musashi-1's IDRs, rather than Musashi-2's, is compensated by its higher α-helical propensity to assist their assembly. Our work suggests that, no matter how diverse they become, IDRs could evolve different traits to a converged function, such as liquid-liquid phase separation.

Snail Augments Nuclear Deformability to Promote Lymph Node Metastasis of Head and Neck Squamous Cell Carcinoma.

Up to 50% of head and neck squamous cell carcinoma (HNSCC) patients have lymph node (LN) metastasis, resulting in poor survival rate. Numerous studies have supported the notion that the alterations of gene expression and mechanical properties of cancer cells play an important role in cancer metastasis. However, which genes and how they regulate the biomechanical properties of HNSCC cells to promote LN metastasis remains elusive. In this study, we used an LN-metastatic mouse model in vivo to generate an LN-metastatic head and neck squamous cell carcinoma cell line and compared the differences in the biomolecular and biomechanical properties of LN-metastatic and non-metastatic cells. Our results showed that LN-metastatic cells had a higher level of Snail expression compared to non-LN-metastatic cells. The higher Snail expression promoted the cellular invasion capability in confined environments, mainly by increasing the longitudinal strain of the cell nuclei, which could be attributed to the stronger cell traction force and softer nuclear stiffness. These two biomechanical changes were correlated, respectively, to a larger amount of focal adhesion and less amount of nuclear lamins. Taken together, our works revealed not only the biomechanical profiles of LN-metastatic cells but also the corresponding biomolecular expressions to pinpoint the key process in LN metastasis.

Dexamethasone accelerates muscle regeneration by modulating kinesin-1-mediated focal adhesion signals.

During differentiation, skeletal muscle develops mature multinucleated muscle fibers, which could contract to exert force on a substrate. Muscle dysfunction occurs progressively in patients with muscular dystrophy, leading to a loss of the ability to walk and eventually to death. The synthetic glucocorticoid dexamethasone (Dex) has been used therapeutically to treat muscular dystrophy by an inhibition of inflammation, followed by slowing muscle degeneration and stabilizing muscle strength. Here, in mice with muscle injury, we found that Dex significantly promotes muscle regeneration via promoting kinesin-1 motor activity. Nevertheless, how Dex promotes myogenesis through kinesin-1 motors remains unclear. We found that Dex directly increases kinesin-1 motor activity, which is required for the expression of a myogenic marker (muscle myosin heavy chain 1/2), and also for the process of myoblast fusion and the formation of polarized myotubes. Upon differentiation, kinesin-1 mediates the recruitment of integrin ß1 onto microtubules allowing delivery of the protein into focal adhesions. Integrin ß1-mediated focal adhesion signaling then guides myoblast fusion towards a polarized morphology. By imposing geometric constrains via micropatterns, we have proved that cell adhesion is able to rescue the defects caused by kinesin-1 inhibition during the process of myogenesis. These discoveries reveal a mechanism by which Dex is able to promote myogenesis, and lead us towards approaches that are more efficient in improving skeletal muscle regeneration.

Cell-Cell Adhesion-Driven Contact Guidance and Its Effect on Human Mesenchymal Stem Cell Differentiation.

Contact guidance has been extensively explored using patterned adhesion functionalities that predominantly mimic cell-matrix interactions. Whether contact guidance can also be driven by other types of interactions, such as cell-cell adhesion, still remains a question. Herein, this query is addressed by engineering a set of microstrip patterns of (i) cell-cell adhesion ligands and (ii) segregated cell-cell and cell-matrix ligands as a simple yet versatile set of platforms for the guidance of spreading, adhesion, and differentiation of mesenchymal stem cells. It was unprecedently found that micropatterns of cell-cell adhesion ligands can induce contact guidance. Surprisingly, it was found that patterns of alternating cell-matrix and cell-cell strips also induce contact guidance despite providing a spatial continuum for cell adhesion. This guidance is believed to be due to the difference between the potencies of the two adhesions. Furthermore, patterns that combine the two segregated adhesion functionalities were shown to induce more human mesenchymal stem cell osteogenic differentiation than monofunctional patterns. This work provides new insight into the functional crosstalk between cell-cell and cell-matrix adhesions and, overall, further highlights the ubiquitous impact of the biochemical anisotropy of the extracellular environment on cell function.

Liquid-liquid phase separation and extracellular multivalent interactions in the tale of galectin-3.

Liquid-liquid phase separation (LLPS) explains many intracellular activities, but its role in extracellular functions has not been studied to the same extent. Here we report how LLPS mediates the extracellular function of galectin-3, the only monomeric member of the galectin family. The mechanism through which galectin-3 agglutinates (acting as a "bridge" to aggregate glycosylated molecules) is largely unknown. Our data show that its N-terminal domain (NTD) undergoes LLPS driven by interactions between its aromatic residues (two tryptophans and 10 tyrosines). Our lipopolysaccharide (LPS) micelle model shows that the NTDs form multiple weak interactions to other galectin-3 and then aggregate LPS micelles. Aggregation is reversed when interactions between the LPS and the carbohydrate recognition domains are blocked by lactose. The proposed mechanism explains many of galectin-3's functions and suggests that the aromatic residues in the NTD are interesting drug design targets.

Fibroblast Promotes Head and Neck Squamous Cell Carcinoma Cell Invasion through Mechanical Barriers in 3D Collagen Microenvironments.

Cancer metastasis involves not only cancer cells but also fibroblasts and the surrounding collagen matrices. Previous studies have reported that in tumor tissues, cancer cells and fibroblasts surrounded by dense collagen are often associated with a high risk of cancer metastasis. However, the mechanism of the interaction between the cancer cells, fibroblasts, and the surrounding collagen matrices in vivo to promote cancer cell invasion in different collagen concentration environments remains unclear. To address this issue, we cocultured head and neck squamous cell carcinoma (OECM-1 cells) and human dermal fibroblasts (HDFs) to form 3D spheroids, embedded in collagen gel with different concentrations to delineate their roles and their interactions in cancer cell invasion. We showed that in single-species spheroids, the OECM-1 cells could not remodel the high-concentration (8 mg/mL) collagen matrices to invade into the surrounding collagen. In contrast, in the coculture spheroids, the HDF cells could remodel the collagen matrices, via MMP-meditated collagen degradation, to increase the invasion capability of OECM-1 cells. In the case of low-concentration (2 mg/mL) collagen matrices, both HDF and OECM-1 cells in the coculture spheroids could independently invade into the surrounding collagen via force remodeling of collagen. Our results revealed that the assistance of HDFs was critical for OECM-1 cell invasion into the surrounding extracellular matrix with high collagen concentration, high storage modulus, and small pore sizes. These insightful results shed light on the possible optimal invasion strategy of cancer tumors in vivo in response to different storage moduli of surrounding collagen matrices.

Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis.

X-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model.

Centrosome guides spatial activation of Rac to control cell polarization and directed cell migration.

Directed cell migration requires centrosome-mediated cell polarization and dynamical control of focal adhesions (FAs). To examine how FAs cooperate with centrosomes for directed cell migration, we used centrosome-deficient cells and found that loss of centrosomes enhanced the formation of acentrosomal microtubules, which failed to form polarized structures in wound-edge cells. In acentrosomal cells, we detected higher levels of Rac1-guanine nucleotide exchange factor TRIO (Triple Functional Domain Protein) on microtubules and FAs. Acentrosomal microtubules deliver TRIO to FAs for Rac1 regulation. Indeed, centrosome disruption induced excessive Rac1 activation around the cell periphery via TRIO, causing rapid FA turnover, a disorganized actin meshwork, randomly protruding lamellipodia, and loss of cell polarity. This study reveals the importance of centrosomes to balance the assembly of centrosomal and acentrosomal microtubules and to deliver microtubule-associated TRIO proteins to FAs at the cell front for proper spatial activation of Rac1, FA turnover, lamillipodial protrusion, and cell polarization, thereby allowing directed cell migration.

Early stage mechanical remodeling of collagen surrounding head and neck squamous cell carcinoma spheroids correlates strongly with their invasion capability.

Mechanical remodeling of stromal collagen, such as reorientation and deformation of collagen matrix, generated by invading cancer cells, plays an important role in the progression of cancer invasion and metastasis. In this study, we applied time-lapse microscopy in conjunction with particle displacement mapping to analyze time-dependent contraction and expansion deformations of collagen surrounding individual spheroids of head and neck squamous cell carcinoma cells (HNSCC), OECM-1 & SAS, as the cancer cells detached from the spheroid and invaded into the surrounding 3D collagen matrix. Our results revealed that highly-invasive HNSCC spheroids, stimulated by epidermal growth factor (EGF), generated a strong contraction deformation of the surrounding collagen in the very early stage, and aligned the collagen fibers radially with respect to the center of the spheroid. This initial collagen contraction deformation generated by the HNSCC spheroid bears a strong positive correlation with the overall extent of subsequent cancer cells invasion; hence, it may serve as an early indicator of the invasion capability of the HNSCC spheroids. STATEMENT OF SIGNIFICANCE: Mechanical remodeling of extracellular matrix (ECM) generated by cancer cells plays an important role in the progression of cancer invasion and metastasis. We observed that the extent of initial contraction deformation of collagen surrounding a head and neck squamous cell carcinoma cell (HNSCC) spheroid played an indispensable role in early stage to promote cancer cells invasion into the surrounding ECM. Our results revealed that more invasive HNSCC spheroids generated a larger extent of initial collagen contraction to align the surrounding collagen and to promote cancer cells invasion. This initial collagen contraction deformation generated by the HNSCC spheroids bears a strong positive correlation with the overall extent of cancer cells invasion; hence, it may serve as an early indicator of the invasion capability of the HNSCC spheroids.

ARAP2 inhibits Akt independently of its effects on focal adhesions.

BACKGROUND INFORMATION: ARAP2, an Arf GTPase-activating protein (Arf GAP) that binds to adaptor protein with PH domain, PTB domain and leucine zipper motifs 1 (APPL1), regulates focal adhesions (FAs). APPL1 affects FA dynamics by regulating Akt. Here, we tested the hypothesis that ARAP2 affects FAs in part by regulating Akt through APPL1. RESULTS: We found that ARAP2 controlled FA dynamics dependent on its enzymatic Arf GAP activity. In some cells, ARAP2 also regulated phosphoAkt (pAkt) levels. However, ARAP2 control of FAs did not require Akt and conversely, the effects on pAkt were independent of FAs. Reducing ARAP2 expression reduced the size and number of FAs in U118, HeLa and MDA-MB-231 cells. Decreasing ARAP2 expression increased pAkt in U118 cells and HeLa cells and overexpressing ARAP2 decreased pAkt in U118 cells; in contrast, ARAP2 had no effect on pAkt in MDA-MB-231 cells. An Akt inhibitor did not block the effect of reduced ARAP2 on FAs in U118. Furthermore, the effect of ARAP2 on Akt did not require Arf GAP activity, which is necessary for effects on FAs and integrin traffic. Altering FAs by other means did not induce the same changes in pAkt as those seen by reducing ARAP2 in U118 cells. In addition, we discovered that ARAP2 and APPL1 had co-ordinated effects on pAkt in U118 cells. Reduced APPL1 expression, as for ARAP2, increased pAkt in U118 and the effect of reduced APPL1 expression was reversed by overexpressing ARAP2. Conversely, the effect of reduced ARAP2 expression was reversed by overexpressing APPL1. ARAP2 is an Arf GAP that has previously been reported to affect FAs by regulating Arf6 and integrin trafficking and to bind to the adaptor proteins APPL1. Here, we report that ARAP2 suppresses pAkt levels in cells co-ordinately with APPL1 and independently of GAP activity and its effect on the dynamic behaviour of FAs. CONCLUSIONS: We conclude that ARAP2 affects Akt signalling in some cells by a mechanism independent of FAs or membrane traffic. SIGNIFICANCE: Our results highlight an Arf GAP-independent function of ARAP2 in regulating Akt activity and distinguish the effect of ARAP2 on Akt from that on FAs and integrin trafficking, which requires regulation of Arf6.

Fibronectin in cell adhesion and migration via N-glycosylation.

Directed cell migration is an important step in effective wound healing and requires the dynamic control of the formation of cell-extracellular matrix interactions. Plasma fibronectin is an extracellular matrix glycoprotein present in blood plasma that plays crucial roles in modulating cellular adhesion and migration and thereby helping to mediate all steps of wound healing. In order to seek safe sources of plasma fibronectin for its practical use in wound dressing, we isolated fibronectin from human (homo) and porcine plasma and demonstrated that both have a similar ability as a suitable substrate for the stimulation of cell adhesion and for directing cell migration. In addition, we also defined the N-glycosylation sites and N-glycans present on homo and porcine plasma fibronectin. These N-glycosylation modifications of the plasma fibronectin synergistically support the integrin-mediated signals to bring about mediating cellular adhesion and directed cell migration. This study not only determines the important function of N-glycans in both homo and porcine plasma fibronectin-mediated cell adhesion and directed cell migration, but also reveals the potential applications of porcine plasma fibronectin if it was applied as a material for clinical wound healing and tissue repair.

Suppression of LPS-induced inflammatory responses by the hydroxyl groups of dexamethasone.

The innate immune response is a central process that is activated during pathogenic infection in order to maintain physiological homeostasis. It is well known that dexamethasone (Dex), a synthetic glucocorticoid, is a potent immunosuppressant that inhibits the cytokine production induced by bacterial lipopolysaccharides (LPS). Nevertheless, the extent to which the functional groups of Dex control the excessive activation of inflammatory reactions remains unknown. Furthermore, importantly, the role of Dex in the innate immune response remains unclear. Here we explore the mechanism of LPS-induced TNF-α secretion and reveal p38 MAPK signaling as a target of Dex that is involved in control of tumor necrosis factor-α (TNF-α)-converting enzyme (TACE) activity; that later mediates the shedding of TNF-α that allows its secretion. We further demonstrate that the 11-hydroxyl and 21-hydroxyl groups of Dex are the main groups that are involved in reducing LPS-induced TNF-α secretion by activated macrophages. Blockage of the hydroxyl groups of Dex inhibits immunosuppressant effect of Dex during LPS-induced TNF-α secretion and mouse mortality. Our findings demonstrate Dex signaling is involved in the control of innate immunity.

Epigenetic repression of ribosomal RNA transcription by ROCK-dependent aberrant cytoskeletal organization.

It is known that ribosomal RNA (rRNA) synthesis is regulated by cellular energy and proliferation status. In this study, we investigated rRNA gene transcription in response to cytoskeletal stress. Our data revealed that the cell shape constrained by isotropic but not elongated micropatterns in HeLa cells led to a significant reduction in rRNA transcription dependent on ROCK. Expression of a dominant-active form of ROCK also repressed rRNA transcription. Isotropic constraint and ROCK over-activation led to different types of aberrant F-actin organization, but their suppression effects on rRNA transcription were similarly reversed by inhibition of histone deacetylase (HDAC) or overexpression of a dominant negative form of Nesprin, which shields the signal transmitted from actin filament to the nuclear interior. We further showed that the binding of HDAC1 to the active fraction of rDNA genes is increased by ROCK over-activation, thus reducing H3K9/14 acetylation and suppressing transcription. Our results demonstrate an epigenetic control of active rDNA genes that represses rRNA transcription in response to the cytoskeletal stress.

Focal adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration.

Directed cell migration requires dynamical control of the protein complex within focal adhesions (FAs) and this control is regulated by signaling events involving tyrosine phosphorylation. We screened the SH2 domains present in tyrosine-specific kinases and phosphatases found within FAs, including SRC, SHP1 and SHP2, and examined whether these enzymes transiently target FAs via their SH2 domains. We found that the SRC_SH2 domain and the SHP2_N-SH2 domain are associated with FAs, but only the SRC_SH2 domain is able to be regulated by focal adhesion kinase (FAK). The FAK-dependent association of the SRC_SH2 domain is necessary and sufficient for SRC FA targeting. When the targeting of SRC into FAs is inhibited, there is significant suppression of SRC-mediated phosphorylation of paxillin and FAK; this results in an inhibition of FA formation and maturation and a reduction in cell migration. This study reveals an association between FAs and the SRC_SH2 domain as well as between FAs and the SHP2_N-SH2 domains. This supports the hypothesis that the FAK-regulated SRC_SH2 domain plays an important role in directing SRC into FAs and that this SRC-mediated FA signaling drives cell migration.