Activation Of Integrin Receptors Research Articles

Abstract 1261: In vitro medulloblastoma leptomeningeal metastasis models reveal adhesion signaling as a therapeutic vulnerability

Abstract Introduction: How tumor cells colonize an inherently distinct environment and survive the pressures of therapeutic treatment during metastasis is incompletely understood. In patients with medulloblastoma, the most common malignant pediatric brain tumor, mortality is almost exclusively dictated by metastasis to the leptomeninges, the inner covering of the brain and spinal cord. We aim to mechanistically understand the molecular drivers of leptomeningeal colonization, including how leptomeningeal adhesion mediates therapy resistance. Experimental approach: Progress in leptomeningeal metastasis research has been hindered by the scarcity of matched primary and metastatic tissue from patients given the lack of clinical benefit to obtaining metastatic tissue. Furthermore, end points from pre-clinical human xenograft mouse models are often driven by primary cerebellar tumors, not leptomeningeal dissemination. These barriers underscore the necessity for novel in vitro leptomeningeal metastasis models that allow for rigorous experimental analysis. These models must simultaneously recapitulate tumor cell-host and tumor cell-environment interactions driving colonization to produce translatable findings. Accordingly, our lab has developed two novel in vitro leptomeningeal metastasis models that allow for cell-based experimental analysis: a primary meningeal cell-medulloblastoma organotypic co-culture and an acellular leptomeningeal matrix mimetic monoculture. Results: Proteomic analysis identified that leptomeningeal cells promote medulloblastoma chemotactic honing to the leptomeninges through CXCL12-CXCR4 signaling. Additionally, loss of CXCL12/CXCR4 signaling impaired medulloblastoma adhesion to the leptomeninges. CXCL12/CXCR4 are known to mediate adhesion of stem cells in stromal niches via activation of integrin receptor ITGA4/ITGB1 (VLA4) binding to stromal VCAM1. Indeed, the VLA4-VCAM1 axis is required for medulloblastoma leptomeningeal adhesion and requires activation by CXCL12/CXCR4 signaling. From a translational perspective, we found that leptomeningeal adhesion promotes therapy resistance and that inhibition of adhesion signaling sensitized adherent medulloblastoma cells to therapy. Conclusions: These findings provide proof-of-concept that appropriate in vitro models of metastasis can be developed and utilized to define the biology driving adhesion and chemotherapy resistance in medulloblastoma metastasis. Furthermore, we show that medulloblastoma leptomeningeal adhesion is mediated by a CXCL12/CXCR4-VLA4/VCAM1 axis. Finally, we demonstrate the therapeutic potential of targeting this axis in sensitizing medulloblastoma metastases to current therapies. Citation Format: Leyre Jimenez Garcia, Amy C. Gross, Ryan D. Roberts, James B. Reinecke. In vitro medulloblastoma leptomeningeal metastasis models reveal adhesion signaling as a therapeutic vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1261.

A magnetically powered nanomachine with a DNA clutch.

Machines found in nature and human-made machines share common components, such as an engine, and an output element, such as a rotor, linked by a clutch. This clutch, as seen in biological structures such as dynein, myosin or bacterial flagellar motors, allows for temporary disengagement of the moving parts from the running engine. However, such sophistication is still challenging to achieve in artificial nanomachines. Here we present a spherical rotary nanomotor with a reversible clutch system based on precise molecular recognition of built-in DNA strands. The clutch couples and decouples the engine from the machine's rotor in response to encoded inputs such as DNA or RNA. The nanomotor comprises a porous nanocage as a spherical rotor to confine the magnetic engine particle within the nanospace (∼0.004 μm3) of the cage. Thus, the entropically driven irreversible disintegration of the magnetic engine and the spherical rotor during the disengagement process is eliminated, and an exchange of microenvironmental inputs is possible through the nanopores. Our motor is only 200 nm in size and the clutch-mediated force transmission powered by an embedded ferromagnetic nanocrystal is high enough (∼15.5 pN at 50 mT) for the in vitro mechanical activation of Notch and integrin receptors, demonstrating its potential as nano-bio machinery.

The endothelial-to-mesenchymal transition changes the focal adhesion site proteins levels and the SLRP-lumican level in HMEC-1 cell line

Scleroderma, the chronic autoimmune disease is a consequence of inflammation in the connective tissue. Prolonged duration affects formation of compact connective tissue strands (scarring) within the target organ. Endothelial cells undergoing endothelial-to-mesenchymal transition (EndMT) are the source of fibroblast phenotype-resembling cells. EndMT contributes to reorganization of the focal adhesion proteins (FA), including integrins, and intensive extracellular matrix (ECM) remodelling. However, in endothelial cells, the relationship between EndMT and the interaction of integrin receptors with lumican - a component of ECM, is still unclear. Our findings indicate that at the early stages of EndMT caused by Snail-1 transcription factor overexpression, the level of the β1 integrin subunit and its phosphorylation are elevated. Simultaneously, the changes in the level of proteins that build FAs and promote activation of integrin receptors as well as a decrease in lumican quantity were observed. These modulations contributed to increased migration of human microvascular endothelial cells, HMEC-1. Our findings were achieved by WB, ELISA and wound healing assay. Taken altogether, transfection of HMEC-1 cells with Snail-1 plasmids inducing the early stages of EndMT results in the increase of total FAK and integrin β1 phosphorylation as well as cell migration: phenomena which are modulated by interaction with lumican.

Extracellular matrix remodelling in obesity and metabolic disorders.

Obesity causes extracellular matrix (ECM) remodelling which can develop into serious pathology and fibrosis, having metabolic effects in insulin-sensitive tissues. The ECM components may be increased in response to overnutrition. This review will focus on specific obesity-associated molecular and pathophysiological mechanisms of ECM remodelling and the impact of specific interactions on tissue metabolism. In obesity, complex network of signalling molecules such as cytokines and growth factors have been implicated in fibrosis. Increased ECM deposition contributes to the pathogenesis of insulin resistance at least in part through activation of cell surface integrin receptors and CD44 signalling cascades. These cell surface receptors transmit signals to the cell adhesome which orchestrates an intracellular response that adapts to the extracellular environment. Matrix proteins, glycoproteins, and polysaccharides interact through ligand-specific cell surface receptors that interact with the cytosolic adhesion proteins to elicit specific actions. Cell adhesion proteins may have catalytic activity or serve as scaffolds. The vast number of cell surface receptors and the complexity of the cell adhesome have made study of their roles challenging in health and disease. Further complicating the role of ECM-cell receptor interactions is the variation between cell types. This review will focus on recent insights gained from studies of two highly conserved, ubiquitously axes and how they contribute to insulin resistance and metabolic dysfunction in obesity. These are the collagen-integrin receptor-IPP (ILK-PINCH-Parvin) axis and the hyaluronan-CD44 interaction. We speculate that targeting ECM components or their receptor-mediated cell signalling may provide novel insights into the treatment of obesity-associated cardiometabolic complications.

Thyroid hormone-dependent oligodendroglial cell lineage genomic and non-genomic signaling through integrin receptors.

Multiple sclerosis (MS) is a heterogeneous autoimmune disease whereby the pathological sequelae evolve from oligodendrocytes (OLs) within the central nervous system and are targeted by the immune system, which causes widespread white matter pathology and results in neuronal dysfunction and neurological impairment. The progression of this disease is facilitated by a failure in remyelination following chronic demyelination. One mediator of remyelination is thyroid hormone (TH), whose reliance on monocarboxylate transporter 8 (MCT8) was recently defined. MCT8 facilitates the entry of THs into oligodendrocyte progenitor cell (OPC) and pre-myelinating oligodendrocytes (pre-OLs). Patients with MS may exhibit downregulated MCT8 near inflammatory lesions, which emphasizes an inhibition of TH signaling and subsequent downstream targeted pathways such as phosphoinositide 3-kinase (PI3K)-Akt. However, the role of the closely related mammalian target of rapamycin (mTOR) in pre-OLs during neuroinflammation may also be central to the remyelination process and is governed by various growth promoting signals. Recent research indicates that this may be reliant on TH-dependent signaling through β1-integrins. This review identifies genomic and non-genomic signaling that is regulated through mTOR in TH-responsive pre-OLs and mature OLs in mouse models of MS. This review critiques data that implicates non-genomic Akt and mTOR signaling in response to TH-dependent integrin receptor activation in pre-OLs. We have also examined whether this can drive remyelination in the context of neuroinflammation and associated sequelae. Importantly, we outline how novel therapeutic small molecules are being designed to target integrin receptors on oligodendroglial lineage cells and whether these are viable therapeutic options for future use in clinical trials for MS.

THE ROLE OF MATRIX METALLOPROTEINASE (MMP)-2 TO TYPE I COLLAGEN TURNOVER CONTRIBUTING TO HYPERTROPHIC ARTERIAL REMODELING IN RENOVASCULAR HYPERTENSION

Objective: Type I collagen proteolytic products contribute to VSMC proliferation via integrin receptor and focal adhesion kinase (FAK) activation. We then evaluated whether the early activation of MMP-2 in hypertension induces VSMC proliferation, due to type I collagen proteolysis, which may activate integrin and FAK to develop hypertrophic arterial remodeling. Design and method: Sprague Dawley rats were submitted to 2K1C surgery to induce hypertension and they were orally treated with doxycycline or vehicle (five days at first week (acute) and eight weeks at ten weeks of hypertension (chronic)); (CEUA-USP 165/2019). In the aorta, we evaluated the expression of MMP-2 and type I collagen by Western blotting; MMP-2 activity by gelatin and in situ zymography, arterial morphology by HE, and collagen deposition by picrosirius red. All statistical analyzes used two-way ANOVA followed by Tukey post hoc test with p < 0.05. Results: Our results showed that 2K1C rats developed high systolic blood pressure at the first week (156 ± 13 mmHg vs. Sham (p < 0,001)), which was progressively higher at ten weeks (219 ± 6 mmHg, p < 0,0001). Moreover, there was a significant increase in the MMP-2 expression and activity in the 2K1C rats at the first and tenth weeks of hypertension (p < 0.05). Treatment with doxycycline reduced MMP-2 activity only in the acute situation (p < 0.05). The 2K1C showed increased levels of type I collagen as well as its potential degradation products (p < 0.05) in the aorta during the first week of hypertension. However, the deposition of collagen was notably resynthesized in the aortas of chronic 2K1C rats (p < 0.05). Conclusions: Treatment with doxycycline reduced such effects in hypertensive rats. At this moment, we concluded that increased activity of MMP-2 may contribute to type I collagen proteolysis in aortas at early stages of hypertension, with its potential re-synthesis at late stages. This proteolytic effect of MMP-2 on collagen may contribute to stimulating VSMCs proliferation through integrins and FAK, mechanisms that we are currently investigating.

Yersinia pseudotuberculosisYopH targets SKAP2-dependent and independent signaling pathways to block neutrophil antimicrobial mechanisms during infection.

Yersinia suppress neutrophil responses by using a type 3 secretion system (T3SS) to inject 6–7 Yersinia effector proteins (Yops) effectors into their cytoplasm. YopH is a tyrosine phosphatase that causes dephosphorylation of the adaptor protein SKAP2, among other targets in neutrophils. SKAP2 functions in reactive oxygen species (ROS) production, phagocytosis, and integrin-mediated migration by neutrophils. Here we identify essential neutrophil functions targeted by YopH, and investigate how the interaction between YopH and SKAP2 influence Yersinia pseudotuberculosis (Yptb) survival in tissues. The growth defect of a ΔyopH mutant was restored in mice defective in the NADPH oxidase complex, demonstrating that YopH is critical for protecting Yptb from ROS during infection. The growth of a ΔyopH mutant was partially restored in Skap2-deficient (Skap2KO) mice compared to wild-type (WT) mice, while induction of neutropenia further enhanced the growth of the ΔyopH mutant in both WT and Skap2KO mice. YopH inhibited both ROS production and degranulation triggered via integrin receptor, G-protein coupled receptor (GPCR), and Fcγ receptor (FcγR) stimulation. SKAP2 was required for integrin receptor and GPCR-mediated ROS production, but dispensable for degranulation under all conditions tested. YopH blocked SKAP2-independent FcγR-stimulated phosphorylation of the proximal signaling proteins Syk, SLP-76, and PLCγ2, and the more distal signaling protein ERK1/2, while only ERK1/2 phosphorylation was dependent on SKAP2 following integrin receptor activation. These findings reveal that YopH prevents activation of both SKAP2-dependent and -independent neutrophilic defenses, uncouple integrin- and GPCR-dependent ROS production from FcγR responses based on their SKAP2 dependency, and show that SKAP2 is not required for degranulation.

Tumor Necrosis Factor‐α Activates Integrin Signaling in Vascular Smooth Muscle Cells via α5 Receptor

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2•−) to be produced extracellularly by NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). Extracellular superoxide dismutase (ecSOD) converts O2•− to hydrogen peroxide (H2O2). It binds to the cell surface by association with either heparin‐sulfate or fibulin‐5, an extracellular matrix protein that interacts with integrin receptors. We previously found that ecSOD knockdown (siRNA) impaired TNFα signal transduction as reflected by MAPK and NF‐κB activation. Targeting of ecSOD also inhibited TNFα‐dependent activation of focal adhesion kinase (FAK) which reflects integrin receptor activation. Here we investigated the mechanism by which TNFα activates integrin signaling in VSMC. We analyzed NF‐κB activation in the presence of selective blocking antibodies (Ab) for integrin receptor subtypes previously shown to be expressed in VSMCs including α4, α5, αv, β1, and β3. TNFα‐induced NF‐κB activation was not affected by blockade of integrin receptors α4, αv, β1, and β3. However blocking of α5 reduced TNFα‐induced NF‐κB activation (control IgG + TNFα 7.36 ± 0.7, anti‐α5 Ab + TNFα 4.78 ± 0.3 fold change, p < 0.05, n = 5). We next sought to determine if α5 integrin is associated with upstream steps of integrin signaling in VSMCs. FAK and integrin‐linked kinase (ILK) are downstream effectors of integrin receptor activation. After 10 min, TNFα induced phosphorylation of both FAK and Protein Kinase B (Akt) which was used as a surrogate for ILK activation. Blockade of α5 receptors decreased TNFα‐induced phosphorylation of both FAK and Akt. Thus, short term exposure of VSMCs to TNFα transactivates α5 integrins and subsequently FAK and ILK‐Akt. We speculate that ecSOD localizes TNFα‐induced H2O2 production to the vicinity of α5 integrins via physical association with fibulin‐5 and this redox environment affects activation of the receptor.

Thyroid hormones stimulate L-arginine transport in human endothelial cells

Thyroid hormone activity is associated with L-arginine metabolism and nitric oxide (NO) production, which participate in the cardiovascular manifestations of thyroid disorders. L-arginine transporters play an important role in activating L-arginine uptake and NO production. However, the effects of thyroid hormones on L-arginine transporters in endothelial cells have not yet been evaluated. The following methods were used. We measured L-arginine uptake, mRNA expression of L-arginine transporters, endothelial nitric oxide synthase (eNOS) mRNA and NO generation after the administration of T3, T4 and the T3 analog, 3,3′,5-triiodothyroacetic acid TRIAC in human umbilical vein endothelial cells (HUVECs). We also analyzed the role of αvβ3 integrin and of phosphatidyl-inositol-3 kinase (PI3K), mitogen-activated protein kinases (MAPKs: ERK1/2, p38 and SAPK-JNK) and intracellular calcium signaling pathways as underlying mechanisms. To this end, αvβ3 integrin was pharmacologically inhibited by tetraiodothyroacetic acid (TETRAC) or genetically blocked by silencing αv mRNA and PI3K, MAPKs and intracellular calcium by selective inhibitors. The following results were obtained. Thyroid hormones and the T3 analog TRIAC increased L-arginine uptake in HUVECs, the sodium-independent y+/CAT isoforms, except CAT2b, sodium-dependent y+L system and sodium-independent system b0,+L-arginine transporters, eNOS mRNA and NO production. These effects were suppressed by αvβ3 integrin inhibition with TETRAC or αv integrin downregulation or by PI3K, MAPK or intracellular Ca2+ signaling inhibitors. In conclusion, we report for the first time that activation of L-arginine uptake by thyroid hormones is related to an upregulation of L-arginine transporters. This effect seems to be mediated by activation of αvβ3 integrin receptor and subsequent PI3K, MAPK and intracellular Ca2+ signaling pathways.

Kindlin-1 Regulates Epidermal Growth Factor Receptor Signaling

Kindler syndrome is an autosomal recessive genodermatosis that results from mutations in the FERMT1 gene encoding t kindlin-1. Kindlin-1 localizes to focal adhesion and is known to contribute to the activation of integrin receptors. Most cases of Kindler syndrome show a reduction or complete absence of kindlin-1 in keratinocytes, resulting in defective integrin activation, cell adhesion, and migration. However, roles for kindlin-1 beyond integrin activation remain poorly defined. In this study we show that skin and keratinocytes from Kindler syndrome patients have significantly reduced expression levels of the EGFR, resulting in defective EGF-dependent signaling and cell migration. Mechanistically, we show that kindlin-1 can associate directly with EGFR in vitro and in keratinocytes in an EGF-dependent, integrin-independent manner and that formation of this complex is required for EGF-dependent migration. We further show that kindlin-1 acts to protect EGFR from lysosomal-mediated degradation. This shows a new role for kindlin-1 that has implications for understanding Kindler syndrome disease pathology.

Local Integrin Activation in Pancreatic β Cells Targets Insulin Secretion to the Vasculature

The extracellular matrix (ECM) critically affects β cell functions via integrin activation. But whether these ECM actions drive the spatial organization of β cells, as they do in epithelial cells, is unknown. Here, we show that within islets of Langerhans, focal adhesion activation in β cells occurs exclusively where they contact the capillary ECM (vascular face). In cultured β cells, 3D mapping shows enriched insulin granule fusion where the cells contact ECM-coated coverslips, which depends on β1 integrin receptor activation. Culture on micro-contact printed stripes of E-cadherin and fibronectin shows that β cell contact at the fibronectin stripe selectively activates focal adhesions and enriches exocytic machinery and insulin granule fusion. Culture of cells in high glucose, as a model of glucotoxicity, abolishes granule targeting. We conclude that local integrin activation targets insulin secretion to the islet capillaries. This mechanism might be important for islet function and may change in disease.

Sustained Diabetes Remission Induced by the Central Action of Fibroblast Growth Factor-1 (FGF-1) Requires Activation of Integrin Receptor aνß3

The brain is implicated in the antidiabetic effects of members of the fibroblast growth factor (FGF) peptide family. Previously, we reported that a single intracerebroventricular (icv) injection of FGF-1 induces remission of hyperglycemia that is sustained for weeks in rodent models of type 2 diabetes (T2D). The integrin receptor ανβ3 plays a key role in FGF-1 signaling, and crosstalk between ανβ3 and the FGF-1-FGF receptor (FGF-R) complex is necessary for increased DNA synthesis and proliferation in cultured cells. These cellular responses downstream of FGF-1-FGFR-ανβ3 result from sustained (e.g., lasting for 5 hours) activation of the extracellular signal-regulated kinase (ERK) signal transduction pathway. In cultured fibroblasts, prolonged ERK activation induced by FGF-1 is not replicated with a mutant FGF-1 peptide (R50E) that activates FGFR normally, but does not activate ανβ3 signaling. Similarly, we report that whereas icv FGF-1 (3 µg) induces phosphorylation of ERK1/2 at both early (20 minute) and late (5 hr) time points in hypothalamic punches obtained from wild type mice, icv injection of the same dose of R50E elicited this effect only at the early time point. Thus, R50E fails to activate hypothalamic FGF-1-FGFR-ανβ3 signaling in vivo. To investigate whether activation of ανβ3 is required for the prolonged antidiabetic effect of icv FGF-1, diabetic ob/ob mice received a single injection of either vehicle (Veh), recombinant FGF-1 (3 µg), or R50E (3 µg). Whereas R50E mimicked the effect of native FGF-1 to reduce food intake, body weight and blood glucose for the first week after icv injection (P<0.001 vs. Veh), the mutant peptide failed to elicit the sustained diabetes remission observed in animals receiving icv FGF-1. We conclude that signaling by the full FGF-1-FGFR-ανβ3 complex is necessary for the effect of FGF-1 signaling in the brain to elicit sustained remission of diabetic hyperglycemia in rodent models of T2D. Disclosure J. Scarlett: None. J.M. Brown: None. B.N. Phan: None. E.L. Khav: None. M.E. Matsen: None. N.K. Acharya: None. H.T. Nguyen: None. A. Secher: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. Employee; Spouse/Partner; Gubra. R. Jorgensen: Employee; Self; Novo Nordisk Inc.. G.J. Morton: None. M.W. Schwartz: Consultant; Self; Novo Nordisk A/S. Research Support; Self; Novo Nordisk A/S.

The role of Protein Disulfide Isomerase and thiol bonds modifications in activation of integrin subunit alpha11

Integrins belong to a family of transmembrane receptors that mediate cell migration and adhesion to ECM. Extracellular domains of integrin heterodimers contain cysteine-rich regions, which are potential sites of thiol-disulfide exchanges. Rearrangements of extracellular disulfide bonds regulate activation of integrin receptors by promoting transition from an inactive state into a ligand-binding competent state. Modifications of integrin disulfide bonds dependent on oxidation-reduction can be mediated by Protein Disulfide Isomerse (PDI). This paper provides evidences that binding to integrin ligands initiate changes in free thiol pattern on cell surface and that thiol-disulfide exchange mediated by PDI leads to activation of integrin subunit α11. By employing co-immunoprecipitation and confocal microscopy analysis we showed that α11β1 and PDI create complexes bounded by disulfide bonds. Using surface plasmon resonance we provide biochemical evidence that PDI can interact directly with integrin subunit α11.

Regulation of cardiac excitation-contraction coupling by fibroblasts in health and disease

Introduction: Cardiomyocyte structure and function are influenced directly via cell-cell communication, the ubiquitous extra-cellular matrix (ECM), and paracrine interactions. These interactions modulate cardiomyocytes in health and the changes that occur in disease. Clear mechanisms in these interactions are still lacking, particularly how fibroblasts may affect myocyte excitation-contraction coupling in a human model. Methods: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were co-cultured with human cardiac fibroblasts, isolated from heart failure patients, at a ratio of 2:1 fibroblasts:hiPSC-CMs for 24 hours in three different conditions: (1) hiPSC-CMs in fibroblast-conditioned medium, (2) co-culture with fibroblasts in tissue culture inserts, and (3) fibroblasts in direct contact with hiPSC-CMs. These conditions differentiate the various aspects of cardiomyocyte-fibroblast interaction. Optical mapping experiments were conducted to visualise changes in excitation-contraction coupling. Results: Human fibroblasts-conditioned medium altered excitation-contraction coupling by decreasing calcium transient amplitude and prolonging time to 80% decay vs baseline (p<0.001, -30±2.6% and +6±2.6%). Direct contact with fibroblasts further decreased calcium transient amplitude (p<0.001 vs. baseline, -46±4.3%) and further prolonged time to 80% decay (p<0.001 vs. baseline, +47±8.7%). Soluble Arginine-Glycine-Aspartic acid (RGD) motif-containing peptides present in the attachment site of many ECM proteins reduce the duration of transients and cause hiPSC-CM clustering and detachment from the glass surface substrate. These effects are not attenuated in the presence of anti-β1 or anti-β3-integrin receptor antibodies, but are also likely to prevent integrin receptor activation. Discussion:Human fibroblasts isolated from the ventricles of cardiomyopathy patients modulate calcium transients in hiPSC-CMs. The effects are dependent on whether the interaction is paracrine or direct, and unidirectional or bidirectional. Although it is unclear which interactions play the greatest role in modulating cardiomyocytes, it is accepted that multiple pathways and factors are involved. Further work is needed to identify mechanisms and understand their subsequent consequences in health and in disease.

Knockdown of HMGN2 increases the internalization of Klebsiellapneumoniae by respiratory epithelial cells through the regulation of α5β1 integrin expression.

Integrin receptors, a large family of adhesion receptors, are involved in the attachment of Klebsiella pneumoniae to respiratory epithelial cells, and subsequently cause the internalization of K. pneumoniae by host cells. Although a number of molecules have been reported to regulate the expression and activity of integrin receptors in respiratory epithelial cells, the specific underlying molecular mechanisms remain largely unknown. High mobility group nucleosomal binding domain 2 (HMGN2), a non-histone nuclear protein, is present in eukaryotic cells as a ubiquitous nuclear protein. Our previous studies have demonstrated that HMGN2 affects chromatin function and modulates the expression of antibacterial peptide in A549 cells exposed to lipopolysaccharide, which indicates the critical role of HMGN2 in innate immune responses. In addition, our cDNA microarray analysis suggested that HMGN2 knockdown induced the enhanced expression of α5β1 integrin in A549 cells. Therefore, we hypothesized that intercellular HMGN2 may mediate the internalization of K. pneumoniae by altering the expression of α5β1 integrin. Using the A549 cell line, we demonstrated that HMGN2 knockdown induced the increased expression of α5β1 integrin on cell membranes, which resulted in a significant increase in K. pneumoniae internalization. Further results revealed that HMGN2 silencing induced the expression of talin and the activation of α5β1 integrin, which led to actin polymerization following the phosphorylation of FAK and Src. This study suggests a possible therapeutic application for bacterial internalization by targeting HMGN2 in order to treat K. pneumoniae infection.

Conformational equilibrium of talin is regulated by anionic lipids

A critical step in the activation of integrin receptors is the binding of talin to the cytoplasmic domain of the β subunits. This interaction leads to separation of the integrin α and β transmembrane domains and significant conformational changes in the extracellular domains, resulting in a dramatic increase in integrin's affinity for ligands. It has long been shown that the membrane bilayer also plays a critical role in the talin–integrin interaction. Anionic lipids are required for proper interaction, yet the specificity for specific anionic headgroups is not clear. In this report, we document talin–membrane interactions with bilayers of controlled composition using Nanodiscs and a FRET based binding and structural assay. We confirm that recruitment of the talin head domain to the membrane surface is governed by charge in the absence of other adapter proteins. In addition, measurement of the donor–acceptor distance is consistent with the hypothesis that anionic lipids promote a conformational change in the talin head domain allowing interaction of the F3 domain with the phospholipid bilayer. The magnitude of the F3 domain movement is altered by the identity of the phospholipid headgroup with phosphatidylinositides promoting the largest change. Our results suggest that phoshpatidylinositol-4,5-bisphosphate plays key a role in converting talin head domain to a conformation optimized for interactions with the bilayer and subsequently integrin cytoplasmic tails.

Clinical applications of low-intensity pulsed ultrasound and its potential role in urology.

Low-intensity pulsed ultrasound (LIPUS) is a form of ultrasound that delivered at a much lower intensity (<3 W/cm2) than traditional ultrasound energy and output in the mode of pulse wave, and it is typically used for therapeutic purpose in rehabilitation medicine. LIPUS has minimal thermal effects due to its low intensity and pulsed output mode, and its non-thermal effects which is normally claimed to induce therapeutic changes in tissues attract most researchers’ attentions. LIPUS have been demonstrated to have a rage of biological effects on tissues, including promoting bone-fracture healing, accelerating soft-tissue regeneration, inhibiting inflammatory responses and so on. Recent studies showed that biological effects of LIPUS in healing morbid body tissues may be mainly associated with the upregulation of cell proliferation through activation of integrin receptors and Rho/ROCK/Src/ERK signaling pathway, and with promoting multilineage differentiation of mesenchyme stem/progenitor cell lines through ROCK-Cot/Tpl2-MEK-ERK signaling pathway. Hopefully, LIPUS may become an effective clinical procedure for the treatment of urological diseases, such as chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), erectile dysfunction (ED), and stress urinary incontinence (SUI) in the field of urology. It still needs an intense effort for basic-science and clinical investigators to explore the biomedical applications of ultrasound.

The Membrane Lipid Composition Regulates The Activity of Talin

Integrins plays a central role in the dynamics of cell‐cell and cell to extra‐cellular matrix interactions and signaling. A key step in the activation of integrin receptors is the binding of the cytoplasmic domain of integrin subunits by talin. This interaction leads to separation of the integrin transmembrane domains and significant conformational changes in the extracellular domains, resulting in a dramatic increase in integrin's affinity for ligands. Talin is a 470 kDa adapter protein that consists of a N‐terminal head domain (THD) and a C‐terminal rod domain. THD is homologous to to the domain found in band 4.1/ezrin/radixin/moesin family of proteins (FERM domain) with uncanonical linear arrangement of F0–F3 domains. A second mechanism in the regulation of integrin activation is directly related to the fact that full‐length talin adopts an auto‐inhibited conformation. In the cytosol, the rod domain binds THD, preventing talin interactions with the membrane surface and the integrin cytoplasmic domain. The membrane bilayer also plays a critical role in the talin ‐integrin interaction, where anionic lipids are required for proper interaction, yet a detailed picture of the interplay between talin and the membrane has remained elusive. We describe a new fluorescence based assay for probing talin – membrane interactions with Nanodisc bilayers of controlled composition. We show that recruitment of THD to the membrane surface is governed by the absolute charge. Measurement of the donor‐acceptor distances reveals that anionic lipids promote a conformational change in the THD favoring a direct interaction of the F3 domain with the phospholipid bilayer. The magnitude of this conformational change is regulated by the identity of the phospholipid headgroup with phosphatidylinositides (e.g. PI4P and PIP2) promoting the largest conformational motions. In addition, the binding of THD to bilayers containing PIP2 is insensitive to the presence of the inhibitory segments of the talin rod domain, while the association of THD to other tested anionic lipid bilayers are strongly inhibited by purified talin rod domains. Furthermore, we have mapped the activation to the interaction of PIP2 with the F2–F3 domain of the THD. Thus, we show that PIP2 plays a central role in the converting talin to a conformation optimized for interactions with integrin cytoplasmic tails and regulation of the auto‐inhibited form of talin, which are essential for integrin inside‐out activation.Support or Funding InformationSupported by NIH grant GM101048.

Integrin‐Linked Kinase Enhances Skeletal Muscle Fatty Acid Metabolism In Vivo

Accumulation of extracellular matrix (ECM) components and activation of specific integrin receptors contribute to skeletal muscle (SkM) insulin resistance in the high fat (HF)‐fed mouse. Integrin‐linked kinase (ILK) is an adaptor protein that serves as a hub for the integrin signaling but its role in metabolism is poorly defined. A shift from glucose to fatty acid (FA) oxidation is a hallmark of insulin resistance and diabetes, but the mechanisms responsible for this shift in nutrient utilization are unclear. Integrins have been implicated in the transcriptional regulation of mitochondrial biogenesis and are known to interact with FA transporters at the plasma membrane. The objective of this study was to test the novel hypothesis that integrins, through ILK signaling, enhance SkM FA metabolism under insulin resistant conditions.SkM‐specific ILK knockout mice (mILK‐KO) were generated using mice expressing Cre recombinase on the human SkM actin (HSA) promoter. Body weight was measured weekly and body composition was measured immediately prior to and following energy balance studies. mILK‐KO and their wild type (WT) littermates were fed a chow or HF diet (60% calories from fat) for 27 weeks then placed in metabolic cages for five days (n=6–9/group). Tissues were collected after a 5‐h fast. Pyruvate and FA‐supported mitochondrial respiratory capacity was measured in permeabilized SkM myofibers (PmFBs) from the soleus and EDL. Muscle free fatty acids (FFAs) and triglycerides (TGs) were measured in gastrocnemius with gas chromatography. Comparisons were made using Student's t‐test or 2‐way ANOVA as appropriate.HF‐fed mILK‐KO mice had a decreased reliance on FA oxidation compared to their WT littermates, as suggested by the increase in RQ. This RQ difference was independent of changes in whole body energy expenditure, body composition, food consumption or spontaneous activity. RQ was unchanged in mILK‐KO mice fed a chow diet. FA‐supported PmFB respiration was decreased in EDL, but not soleus, of HF‐fed mILK‐KO mice. Respiration in chow‐fed mice was not affected by muscle ILK deletion. Total muscle FFAs and TGs were increased by HF feeding but lower in mILK‐KO mice fed a HF diet. Muscle lipids were unaffected by ILK deletion in mice on a chow diet.These data are the first to demonstrate a mechanistic role for integrin signaling in the regulation of SkM FA metabolism in vivo and suggest that integrin signaling, through ILK, responds to nutrient overload by enhancing the uptake, storage and oxidation of FAs in SkM. These findings provide strong support for a model where extracellular cues (e.g. ECM expansion) cause a coordinated shift in cellular energetics, a paradigm that may permit for therapeutic targeting of oxidative metabolism through integrin receptors.Support or Funding InformationFunding for this project was provided by the following NIH grants: R01 DK054902, U24 DK059637, P30 DK020593, and T32 DK007061.

Tumor Cell-Derived Periostin Regulates Cytokines That Maintain Breast Cancer Stem Cells.

Basal-like breast cancer (BLBC) is an aggressive subtype of breast cancer which is often enriched with cancer stem cells (CSC), but the underlying molecular basis for this connection remains elusive. We hypothesized that BLBC cells are able to establish a niche permissive to the maintenance of CSCs and found that tumor cell-derived periostin (POSTN), a component of the extracellular matrix, as well as a corresponding cognate receptor, integrin α(v)β(3), are highly expressed in a subset of BLBC cell lines as well as in CSC-enriched populations. Furthermore, we demonstrated that an intact periostin-integrin β3 signaling axis is required for the maintenance of breast CSCs. POSTN activates the ERK signaling pathway and regulates NF-κB-mediated transcription of key cytokines, namely IL6 and IL8, which in turn control downstream activation of STAT3. In summary, these findings suggest that BLBC cells have an innate ability to establish a microenvironmental niche supportive of CSCs. The findings reported here indicate that POSTN produced by CSCs acts to reinforce the stem cell state through the activation of integrin receptors and the production of key cytokines.