Integrin Activation Signaling Research Articles

Peripheral glia and neurons jointly regulate activity-induced synaptic remodeling at the Drosophila neuromuscular junction.

In the nervous system, reliable communication depends on the ability of neurons to adaptively remodel their synaptic structure and function in response to changes in neuronal activity. While neurons are the main drivers of synaptic plasticity, glial cells are increasingly recognized for their roles as active modulators. However, the underlying molecular mechanisms remain unclear. Here, using Drosophila neuromuscular junction as a model system for a tripartite synapse, we show that peripheral glial cells collaborate with neurons at the NMJ to regulate activity-induced synaptic remodeling, in part through a protein called shriveled (Shv). Shv is an activator of integrin signaling previously shown to be released by neurons during intense stimulation at the fly NMJ to regulate activity-induced synaptic remodeling. We demonstrate that Shv is also present in peripheral glia, and glial Shv is both necessary and sufficient for synaptic remodeling. However, unlike neuronal Shv, glial Shv does not activate integrin signaling at the NMJ. Instead, it regulates synaptic plasticity in two ways: 1) maintaining the extracellular balance of neuronal Shv proteins to regulate integrin signaling, and 2) controlling ambient extracellular glutamate concentration to regulate postsynaptic glutamate receptor abundance. Loss of glial cells showed the same phenotype as loss of Shv in glia. Together, these results reveal that neurons and glial cells homeostatically regulate extracellular Shv protein levels to control activity-induced synaptic remodeling. Additionally, peripheral glia maintains postsynaptic glutamate receptor abundance and contribute to activity-induced synaptic remodeling by regulating ambient glutamate concentration at the fly NMJ.

RNA-binding protein-regulated fibronectin is essential for EGFR-activated metastasis of head and neck squamous cell carcinoma.

There is a higher expression level of epidermal growth factor receptor (EGFR) in up to 90% of advanced head and neck squamous cell carcinoma (HNSCC) tissue than in normal surrounding tissues. However, the role of RNA-binding proteins (RBPs) in EGFR-associated metastasis of HNSCC remains unclear. In this study, we reveal that RBPs, specifically nucleolin (NCL) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), correlated with the mesenchymal phenotype of HNSCC. The depletion of RBPs significantly attenuated EGF-induced HNSCC metastasis. Intriguingly, the EGF-induced EMT markers, such as fibronectin, were regulated by RBPs through the ERK and NF-κB pathway, followed by the enhancement of mRNA stability of fibronectin through the 5' untranslated region (5'-UTR) of the gene. The upregulation of fibronectin triggered the integrin signaling activation to enhance tumor cells' attachment to endothelial cells and increase endothelial permeability. In addition, the concurrence of EGFR and RBPs or EGFR and fibronectin was associated with overall survival and disease-free survival of HNSCC. The in vivo study showed that depletion of NCL, hnRNPA2B1, and fibronectin significantly inhibited EGF-promoted extravasation of tumor cells into lung tissues. The depletion of fibronectin or treatment with integrin inhibitors dramatically attenuated EGF-induced HNSCC metastatic nodules in the lung. Our data suggest that the RBPs/fibronectin axis is essential for EGF-induced tumor-endothelial cell interactions to enhance HNSCC cell metastasis.

AFAP1L1 promotes gastric cancer progression by interacting with VAV2 to facilitate CDC42-mediated activation of ITGA5 signaling pathway

BackgroundThe actin filament-associated protein (AFAP) family genes include AFAP1/AFAP-110, AFAP1L1 and AFAP1L2/XB130. Increasing evidence indicates these three AFAP family members participate in tumor progression, but their clinical significance and molecular mechanisms in gastric cancer (GC) remain unclear.MethodsWe first analyzed expression of AFAP family genes using public datasets and verified the results. The clinical significance of AFAP family genes in GC patients was also analyzed. In vitro and in vivo experiments were applied to explore the function of AFAP1L1. Enrichment analysis was used to explore potential molecular mechanisms. We then performed additional experiments, such as cell adhesion assay, co-immunoprecipitation and so on to confirm the downstream molecular mechanisms of AFAP1L1.ResultsPublic data analyses and our verification both showed AFAP1L1 was the only AFAP family members that was significantly upregulated in GC compared with normal gastric tissues. Besides, only AFAP1L1 could predict poor prognosis and act as an independent risk factor for GC patients. In addition, AFAP1L1 promotes GC cells proliferation, migration, invasion in vitro and tumor growth, metastasis in vivo by inducing epithelial-to-mesenchymal transition (EMT). In terms of mechanism, AFAP1L1 interacts with VAV guanine nucleotide exchange factor 2 (VAV2) to activate Rho family GTPases CDC42, which finally promotes expression of integrin subunit alpha 5 (ITGA5) and activation of integrin signaling pathway.ConclusionAFAP1L1 promotes GC progression by inducing EMT through VAV2-mediated activation of CDC42 and ITGA5 signaling pathway, indicating AFAP1L1 may be a promising prognostic biomarker and therapeutic target for GC patients.

Filamentous recombinant human Tau activates primary astrocytes via an integrin receptor complex

Microtubule-associated protein Tau can form protein aggregates transmissible within the brain, correlating with the progression of tauopathies in humans. The transmission of aggregates requires neuron-released Tau to interact with surface receptors on target cells. However, the underlying molecular mechanisms in astrocytes and downstream effects are unclear. Here, using a spatially resolved proteomic mapping strategy, we show that integrin αV/β1 receptor binds recombinant human Tau, mediating the entry of Tau fibrils in astrocytes. The binding of distinct Tau species to the astrocytic αV/β1 receptor differentially activate integrin signaling. Furthermore, Tau-mediated activation of integrin signaling results in NFκB activation, causing upregulation of pro-inflammatory cytokines and chemokines, induction of a sub-group of neurotoxic astrocytic markers, and release of neurotoxic factors. Our findings suggest that filamentous recombinant human Tau-mediated activation of integrin signaling induces astrocyte conversion towards a neurotoxic state, providing a mechanistic insight into tauopathies.

Integration of single-cell and bulk RNA sequencing data reveals key cell types and regulators in traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of disability and mortality worldwide, whose symptoms ranging from mild to severe, even life-threatening. However, specific cell types and key regulators involved in traumatic brain injury have not been well elucidated. In this study, utilizing single-cell RNA-seq (scRNA-seq) data from mice with TBI, we have successfully identified and characterized 13 cell populations including astrocytes, oligodendrocyte, newly formed oligodendrocytes, microglia, two types of endothelial cells, five types of excitatory and two types of inhibitory neurons. Differential expression analysis and gene set enrichment analysis (GSEA) revealed the upregulation of microglia and endothelial markers, along with the downregulation of markers of excitatory neurons in TBI. The cell-cell communication analysis revealed that microglia and endothelial cell might interact through the interaction of Icam1-Il2rg and C1qa-Cd93, and microglia might also communicate with each other via Icam1-Itagm. The autocrine ligand-receptor in microglia might result in activation of TYROBP causal network via Icam1-Itgam. The cell-cell contact between microglia and endothelial cell might activate integrin signaling pathways. Moreover, we also found that genes involved in microglia activation were highly downregulated in Tyrobp/Dap12-deficient microglia, indicating that the upregulation of Tyrobp and TYROBP causal network in microglia might be a candidate therapeutic target in TBI. In contrast, the excitatory neurons were involved in maintaining normal brain function, and their inactivation might cause dysfunction of nervous system in TBI patients. In conclusion, the present study has discerned major cell types such as microglia, endothelial cells and excitatory neurons, and revealed key regulator such as TYROBP, C1QA, and CD93 in TBI, which shall improve our understanding of the pathogenesis of TBI.

A Sampling of Highlights from the Literature

Mimicking STING–cGAMP binding can activate signaling (by Kissiovd via Wikimedia Commons)How to effectively induce STING activation in the tumor microenvironment has not yet been determined. Lu et al. developed microparticles that can release an encapsulated STING agonist in waves, allowing for timed release into the tumor microenvironment (TME) without having to perform multiple intratumoral injections. Treatment of tumor-bearing mice reduces tumor growth and metastases. Chin et al. identified SR-717, a non-nucleotide small-molecule STING agonist, that mimics cGAMP and can confer the active conformation of STING to promote antitumor responses by CD8+ T cells and NK cells. Pan et al. identified MSA-2, which binds and activates human and mouse STING preferentially in the acidified TME, can be given safely as a subcutaneous and oral treatment, induces clearance of tumors, and generates long-lasting antitumor immunity. These studies highlight the potential utility of new classes of STING agonists.Lu X, …, Jaklenec A. Sci Transl Med 2020 Aug 12;12:eaaz6606.Chin EN, …, Lairson LL. Science 2020 Aug 21;369:993–9.Pan B-S, …, Addona GH. Science 2020 Aug 21;369:eaba6098.scRNA-seeking for TREM2 effects (by Mohamed Hassan via PxHere)Immune-suppressive cells support tumor growth, thus identification and targeting of these cells are crucial to induce antitumor immunity. Katzenelenbogen et al. use a novel technology that integrates scRNA sequencing with intracellular protein activity (INs-seq) to establish that TREM2 expression positively correlates with arginase-1+ myeloid cells in tumors. Knockout of TREM2 decreases myeloid cells and improves T-cell function in tumors, delaying tumor growth. Molgora et al. demonstrate that TREM2 knockout or blockade delays tumor growth in a T cell–mediated manner and improves checkpoint blockade efficacy. Thus, TREM2+ tumor-infiltrating myeloid cells inhibit antitumor immunity and are targetable.Katzenelenbogen Y, …, Amit I. Cell 2020 Aug 20;182;872–85.E19.Molgora M, …, Colonna M. Cell 2020 Aug 20;182:886–900.E17.BTN3A1 can be overexpressed in cancer (from Fig. 2 of Gu et al., Front Immunol 2015)Butyrophilins (BTNs) have a structure comparable to some B7 family inhibitory members but regulate T-cell responses through unclear mechanisms. Payne et al. show that BTN3A1 is overexpressed in high-grade ovarian cancer and disrupts αβ T-cell activation by preventing segregation of the phosphatase, CD45, from the immunologic synapse. Stabilizing BTN3A1's extracellular domain with CD277 antibodies or BTN3A1 blockade reversed this, unleashing both αβ and γδ T-cell responses. Thus, targeting inhibitory BTNs could be a potential strategy to boost antitumor responses and increase efficacy of immunotherapy.Payne KK, …, Conejo-Garcia JR. Science 2020 Aug 21;369:942–9.Altering CARs to inhibit ubiquitination (image from PickPik)Despite the efficacy of chimeric antigen receptor (CAR) T-cell therapies, many cancer patients do not respond or eventually fail with poor CAR T-cell persistence. Upon antigen binding, CARs are ubiquitinated and downregulated from the surface of T cells, and subsequently degraded intracellularly. Ubiquitination-blocking mutations in CARs reverse degradation, and lead to enhanced signaling and recycling with increased CAR surface expression. These effects improve tumor control, increase the persistence of CAR T cells in mice, and promote differentiation to central memory cells. Thus, inhibiting the ubiquitination of CARs improves the efficacy and longevity of CAR T-cell therapy.Li W, …, Wang H. Immunity 2020 Aug 18;53:456–70.E6.Activating NK cells can reduce tumor spread (by NIAID via Wikimedia Commons)How tumors evade NK-cell surveillance is not yet fully elucidated. With exposure to tumor cells, NK cells acquire a resting, rather than active, phenotype and not only lose cytotoxic ability but promote metastasis. Sequencing and receptor–ligand analysis show tumor-exposed NK cells upregulate inhibitory receptors, as well as DNA methyltransferases (DNMTs), and bind tumor cells via KLRG1 or TIGIT. Blocking tumor interaction through TIGIT or KLRG1, or inhibiting DNMTs, reduces tumor cell spread.Chan IS, …, Ewald AJ. J Cell Biol 2020 Jul 9;219:e202001134.Don't eat me! (adapted from “Children Eating and Enjoying Watermelon Fruit” via Wannapik Studio)CD47 is a “don't eat me” signal used by tumor cells to avoid being phagocytosed by macrophages via CD47 ligation of SIRPA. The biochemical basis of this “don't eat me” signal remains unclear. CD47 ligation re-localizes SIRPA to the phagocytic synapse, where it interferes with macrophage cell spreading by inhibiting inside-out activation of integrin signaling. Reactivating integrins via manganese re-induces engulfment and phagocytosis of targets with or without CD47 conjugation. Thus, activating integrins may hold utility in reversing the protective impact of tumor expression of CD47.Morrissey MA, …, Vale RD. Immunity 2020 Aug 18;53:290–302.E6.

Neutrophil elastase promotes macrophage cell adhesion and cytokine production through the integrin-Src kinases pathway

There are a number of respiratory diseases characterized by the presence of excess neutrophil elastase (NE) activity in tissues, including cystic fibrosis and chronic obstructive pulmonary disease (COPD). NE is considered a primary contributor to disease development, but the precise mechanism has yet to be fully determined. We hypothesized that NE alters the function of macrophages (Mɸ) which play a critical role in many physiological processes in healthy lungs. We demonstrate that monocyte-derived Mɸ exposed to NE releases active matrix metalloproteinases (MMPs), increase expression of pro-inflammatory cytokines TNFα, IL-1β, and IL-8, and reduce capacity to phagocytose bacteria. Changes in Mɸ function following NE treatment were accompanied by increased adhesion and cytoskeleton re-arrangement, indicating the possibility of integrin involvement. To support this observation, we demonstrate that NE induces phosphorylation of kinases from the Src kinase family, a hallmark of integrin signaling activation. Moreover, pretreatment of Mɸ with a specific Src kinase inhibitor, PP2 completely prevents NE-induced pro-inflammatory cytokine production. Taken together these findings indicate that NE participates in lung destruction not only through direct proteolytic degradation of matrix proteins, but also through activation of Mɸ inflammatory and proteolytic functions.

CD47 Ligation Repositions the Inhibitory Receptor SIRPA to Suppress Integrin Activation and Phagocytosis

CD47 acts as a "don't eat me" signal that protects cells from phagocytosis by binding and activating its receptor SIPRA on macrophages. CD47 suppresses multiple different pro-engulfment "eat me" signals, including immunoglobulin G (IgG), complement, and calreticulin, on distinct target cells. This complexity has limited understanding of how the "don't eat me" signal is transduced biochemically. Here, we utilized a reconstituted system with a defined set of signals to interrogate the mechanism of SIRPA activation and its downstream targets. CD47 ligation altered SIRPA localization, positioning SIRPA for activation at the phagocytic synapse. At the phagocytic synapse, SIRPA inhibited integrin activation to limit macrophage spreading across the surface of the engulfment target. Chemical reactivation of integrin bypassed CD47-mediated inhibition and rescued engulfment, similar to the effect of a CD47 function-blocking antibody. Thus, the CD47-SIRPA axis suppresses phagocytosis by inhibiting inside-out activation of integrin signaling in the macrophage, with implications to cancer immunotherapy applications.

Phosphoproteomic Profiling Identifies Aberrant Activation of Integrin Signaling in Aggressive Non-Type Bladder Carcinoma.

Bladder carcinoma is highly heterogeneous and its complex molecular landscape; thus, poses a significant challenge for resolving an effective treatment in metastatic tumors. We computed the epithelial-mesenchymal transition (EMT) scores of three bladder carcinoma subtypes—luminal, basal, and non-type. The EMT score of the non-type indicated a “mesenchymal-like” phenotype, which correlates with a relatively more aggressive form of carcinoma, typified by an increased migration and invasion. To identify the altered signaling pathways potentially regulating this EMT phenotype in bladder cancer cell lines, we utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based phosphoproteomic approach. Bioinformatics analyses were carried out to determine the activated pathways, networks, and functions in bladder carcinoma cell lines. A total of 3125 proteins were identified, with 289 signature proteins noted to be differentially phosphorylated (p ≤ 0.05) in the non-type cell lines. The integrin pathway was significantly enriched and five major proteins (TLN1, CTTN, CRKL, ZYX and BCAR3) regulating cell motility and invasion were hyperphosphorylated. Our study reveals GSK3A/B and CDK1 as promising druggable targets for the non-type molecular subtype, which could improve the treatment outcomes for aggressive bladder carcinoma.

A scaffold for signaling of Tim-4-mediated efferocytosis is formed by fibronectin.

An essential step during clearance of apoptotic cells is the recognition of phosphatidylserine (PS) exposed on apoptotic cells by its receptors on phagocytes. Tim-4 directly binding to PS and functioning as a tethering receptor for phagocytosis of apoptotic cells has been extensively studied over the past decade. However, the molecular mechanisms by which Tim-4 collaborates with other engulfment receptors during efferocytosis remain elusive. By comparing efferocytosis induced by Tim-4 with that by Anxa5-GPI, an artificial tethering receptor, we found that Tim-4 possesses auxiliary machinery to induce a higher level of efferocytosis than Anxa5-GPI. To search for that, we performed a yeast two-hybrid screen and identified Fibronectin (Fn1) as a novel Tim-4-associating protein. Tim-4 directly associated with Fn1 and formed a complex with integrins via the association of Fn1. Through Tim-4-/- mice and cell-based assays, we found that modulation of the Fn1 level affected efferocytosis induced by Tim-4 and disruption of the interaction between Tim-4 and Fn1 abrogated Tim-4-mediated efferocytosis. In addition, Tim-4 depletion attenuated integrin signaling activation and perturbation of integrin signaling suppressed Tim-4-promoted efferocytosis. Taken together, the data suggest that Fn1 locates Tim-4 and integrins in close proximity by acting as a scaffold, resulting in synergistic cooperation of Tim-4 with integrins for efficient efferocytosis.

Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis.

Signal transducer and activator of transcription 3 (Stat3) has been shown to play a role in intestinal regeneration and colitis-associated colon carcinogenesis. However, the role of Stat3 in the Wnt-driven sporadic intestinal tumorigenesis remains poorly understood. We examined the roles of Stat3 in intestinal regeneration and tumorigenesis by organoid culture experiments using Stat3∆IEC mouse–derived intestinal epithelial cells in which Stat3 was disrupted. The regeneration of intestinal mucosa and organoid formation were significantly suppressed by Stat3 disruption, which was compensated by Wnt activation. Furthermore, once organoids were recovered, Stat3 was no longer required for organoid growth. These results indicate that Stat3 and Wnt signaling cooperatively protect epithelial cells at the early phase of intestinal regeneration. In contrast, intestinal tumorigenesis was not suppressed by Stat3 disruption in adenomatous polyposis coli (Apc)Δ716 and Apc∆716 Tgfbr2∆IEC mice, thus indicating that Stat3 is not required for Wnt activation–driven intestinal tumorigenesis. Mechanistically, Itga5 and Itga6 were down-regulated by Stat3 disruption, and focal adhesion kinase (FAK) activation was also suppressed. Notably, FAK inhibitor suppressed the organoid formation of wild-type epithelial cells. These results indicate that Stat3 is indispensable for the survival of epithelial cells through the activation of integrin signaling and the downstream FAK pathway; however, it is not required for the Wnt signaling-activated normal or tumor epithelial cells.—Oshima, H., Kok, S.-Y., Nakayama, M., Murakami, K., Voon, D. C.-C., Kimura, T., Oshima, M. Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis.

Glycine and succinic acid are effective indicators of the suppression of epithelial-mesenchymal transition by fucoxanthinol in colorectal cancer stem-like cells.

Fucoxanthinol (FxOH) is a strong anticancer metabolite of fucoxanthin that accumulates in abundance in edible brown algae and promises human health benefits. FxOH has been shown to suppress tumorigenicity and sphere formation in human colorectal cancer stem cell (CCSC)-like spheroids (colonospheres, Csps). In the present study, we aimed to clarify the inhibitory activity of FxOH on epithelial-mesenchymal transition (EMT), which is essential for cancer recurrence and distant metastasis, and to identify intracellular low-molecular-weight metabolites that may be useful for evaluating the cellular effects of FxOH on CCSCs. FxOH significantly suppressed sphere-forming activity, migration and invasion in a dose-dependent manner. In addition, treatment with 50µmol/l FxOH suppressed N-cadherin and vimentin expression and the activation of integrin signaling linked to EMT suppression by western blot analysis. MAPK signaling and STAT signaling related to cell growth and apoptosis in Csps derived from human CRC HT-29 and HCT116 cells were also altered. According to our metabolite profiling by GC-MS analysis, reduced glycine and succinic acid levels were correlated with EMT suppression and apoptosis induction in Csps. Our data indicate that simple amino acids such as glycine and succinic acid may be good prognostic indicators of physiological changes to CCSCs induced by FxOH treatment.

Role of Integrin Signaling Activation on the Development of Human T Cell Leukemia Virus-1 (HTLV-1)-Associated Myelopathy/Tropical Spastic Paraparesis: Its Relationship to HTLV-1-Infected CD4+ T Cell Transmigrating Activity into the Tissues.

The main clinical feature of human T cell leukemia virus-1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is slowly progressive spastic paraparesis with bladder dysfunction. HAM/TSP is induced by chronic inflammation in the spinal cord, mainly the lower thoracic cord. A long-standing bystander mechanism, such as the destruction of surrounding tissues by the interaction between infiltrated Th1-like, HTLV-1-infected CD4+ T cells and HTLV-1-specific CD8+ cytotoxic T cells (CTL), is probably critical for the induction of chronic inflammation. Although the HTLV-1-infected CD4+ T cells in HAM/TSP appear to play a crucial role in the initial pathogenesis of HAM/TSP, the exact mechanisms of how these cells acquire their function as the first responders in the pathogenesis of HAM/TSP still remain unresolved. Herein, we propose the importance of the activation of both outside-in signals from integrin signaling and inside-out signals for integrin signaling in the HTLV-1-infected CD4+ T cells of HAM/TSP patients.

Static Compression Induces ECM Remodeling and Integrin α2β1 Expression and Signaling in a Rat Tail Caudal Intervertebral Disc Degeneration Model

A three-level rat tail caudal intervertebral disc (IVD) degeneration (IVDD) model was established to study effects of static compression on extracellular matrix (ECM) remodeling and integrin signaling in IVDs during IVDD. The aim of this study was to investigate the effect of compression force on ECM remodeling and integrin signaling in IVDs during IVDD. Integrins sense mechanical environment alteration via binding to ECM ligands and trigger intracellular signaling for pathological ECM remodeling during IVDD. However, the role of compression force in ECM remodeling and integrin signaling during IVDD remains elusive. Compared with the classical one-level rat tail IVDD model that exerts axial stress on the 8th to 9th caudal vertebral bodies, a three-level model was established by using an Ilizarov-type apparatus to exert stress on the 7th to 10th caudal vertebral bodies in rat tails for four weeks. To exclude side effects from surgical stab injury on manipulated discs, intact coccygeal (Co) disc Co8-9 was analyzed. In three-level IVDD model, significant degeneration of the Co8-9 disc was observed. Quantitative real-time polymerase chain reaction (qRT-PCR) showed elevated mRNA expression of collagen types I, III, and V; matrix metalloproteinases (MMPs) 2, 3, 9, 13, 14; and decreased mRNA expression of collagen type II in Co8-9 disc. Compression loading altered the expression of integrin α2β1 (upregulated) and α10β1 (downregulated) in NP cells, and activated integrin downstream signaling. By contrast, one-level model showed more severe disc degeneration and ECM remodeling. Integrin α1, α2, α11, and β1 were upregulated, whereas α10 was downregulated. Similar activation of integrin signaling was observed. Static compression altered collagen and MMP expression, and promoted β1 integrin expression and signaling in IVD. Compared with one-level rat tail IVDD model, three-level model showed milder effects on disc degeneration, ECM remodeling, and integrin expression, suggesting one-level model might involve other causes that induce IVDD via mechanisms independent of compression force. N/A.

Activity-Induced Synaptic Structural Modifications by an Activator of Integrin Signaling at the Drosophila Neuromuscular Junction.

Activity-induced synaptic structural modification is crucial for neural development and synaptic plasticity, but the molecular players involved in this process are not well defined. Here, we report that a protein named Shriveled (Shv) regulates synaptic growth and activity-dependent synaptic remodeling at the Drosophila neuromuscular junction. Depletion of Shv causes synaptic overgrowth and an accumulation of immature boutons. We find that Shv physically and genetically interacts with βPS integrin. Furthermore, Shv is secreted during intense, but not mild, neuronal activity to acutely activate integrin signaling, induce synaptic bouton enlargement, and increase postsynaptic glutamate receptor abundance. Consequently, loss of Shv prevents activity-induced synapse maturation and abolishes post-tetanic potentiation, a form of synaptic plasticity. Our data identify Shv as a novel trans-synaptic signal secreted upon intense neuronal activity to promote synapse remodeling through integrin receptor signaling.SIGNIFICANCE STATEMENT The ability of neurons to rapidly modify synaptic structure in response to neuronal activity, a process called activity-induced structural remodeling, is crucial for neuronal development and complex brain functions. The molecular players that are important for this fundamental biological process are not well understood. Here we show that the Shriveled (Shv) protein is required during development to maintain normal synaptic growth. We further demonstrate that Shv is selectively released during intense neuronal activity, but not mild neuronal activity, to acutely activate integrin signaling and trigger structural modifications at the Drosophila neuromuscular junction. This work identifies Shv as a key modulator of activity-induced structural remodeling and suggests that neurons use distinct molecular cues to differentially modulate synaptic growth and remodeling to meet synaptic demand.

Abstract 4395: Tissue stiffness and hypoxia regulate breast cancer stem cells through ILK

Abstract The mechanical and chemical properties of the cellular microenvironment can influence the phenotype of cancer cells. The hypoxic conditions within solid breast tumors can contribute to invasiveness and the formation of breast cancer stem cells (CSCs). Integrin-linked kinase (ILK) is a critical mediator for mechanotransduction, a process that converts exogenous mechanical stimuli into biochemical signals. Yet, it is unclear how the physical properties of the tumor microenvironment, including matrix stiffness and hypoxia, integrate to direct the formation of the breast CSC niche. Using an innovative engineered culture model, we found that stiff and hypoxic microenvironments activate integrin signaling and a stem-like gene signature in breast cancer cells, suggesting that integrin signaling, matrix mechanics and oxygen tension cooperatively promote the formation of breast CSCs. Stiff and hypoxic microenvironments promote integrin signaling and the expression of CSC markers including CD44 and Nanog. Knocking down ILK reverts the CSC phenotype of invasive breast cancer cells on stiff matrix, and abrogates their ability to form mammospheres and colonies in soft agar. In contrast, ectopic expression of ILK enhances CSC properties. ILK promotes integrin signaling and the CSC phenotype through the PI3K/Akt pathway. Stiff microenvironments promote tumor formation and metastasis in ovo. ILK depletion significantly abrogates the tumorigenenic and metastatic potential of invasive breast cancer cells in ovo. Importantly, we found that breast cancer cells expressing ILK and the CSC marker CD44 were only present in the regions of tumors predicted to be stiff in breast cancer patient samples. Our data suggest that ILK act as an essential mechanosensor that regulates breast CSC phenotype in response to matrix mechanics and oxygen tension to regulate the formation of breast CSCs. Citation Format: Mei-Fong Pang, Melody Stallings-Mann, Michael J. Siedlik, Victor D. Varner, Siyang Han, Derek C. Radisky, Celeste M. Nelson. Tissue stiffness and hypoxia regulate breast cancer stem cells through ILK. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4395.

Novel phosphatidylethanolamine derivatives accumulate in circulation in hyperlipidemic ApoE−/− mice and activate platelets via TLR2

AbstractA prothrombotic state and increased platelet reactivity are common in dyslipidemia and oxidative stress. Lipid peroxidation, a major consequence of oxidative stress, generates highly reactive products, including hydroxy-ω-oxoalkenoic acids that modify autologous proteins generating biologically active derivatives. Phosphatidylethanolamine, the second most abundant eukaryotic phospholipid, can also be modified by hydroxy-ω-oxoalkenoic acids. However, the conditions leading to accumulation of such derivatives in circulation and their biological activities remain poorly understood. We now show that carboxyalkylpyrrole-phosphatidylethanolamine derivatives (CAP-PEs) are present in the plasma of hyperlipidemic ApoE−/− mice. CAP-PEs directly bind to TLR2 and induces platelet integrin αIIbβ3 activation and P-selectin expression in a Toll-like receptor 2 (TLR2)-dependent manner. Platelet activation by CAP-PEs includes assembly of TLR2/TLR1 receptor complex, induction of downstream signaling via MyD88/TIRAP, phosphorylation of IRAK4, and subsequent activation of tumor necrosis factor receptor–associated factor 6. This in turn activates the Src family kinases, spleen tyrosine kinase and PLCγ2, and platelet integrins. Murine intravital thrombosis studies demonstrated that CAP-PEs accelerate thrombosis in TLR2-dependent manner and that TLR2 contributes to accelerate thrombosis in mice in the settings of hyperlipidemia. Our study identified the novel end-products of lipid peroxidation, accumulating in circulation in hyperlipidemia and inducing platelet activation by promoting cross-talk between innate immunity and integrin activation signaling pathways.

Maintenance of Stem Cell Niche Integrity by a Novel Activator of Integrin Signaling.

Stem cells depend critically on the surrounding microenvironment, or niche, for their maintenance and self-renewal. While much is known about how the niche regulates stem cell self-renewal and differentiation, mechanisms for how the niche is maintained over time are not well understood. At the apical tip of the Drosophila testes, germline stem cells (GSCs) and somatic stem cells share a common niche formed by hub cells. Here we demonstrate that a novel protein named Shriveled (Shv) is necessary for the maintenance of hub/niche integrity. Depletion of Shv protein results in age-dependent deterioration of the hub structure and loss of GSCs, whereas upregulation of Shv preserves the niche during aging. We find Shv is a secreted protein that modulates DE-cadherin levels through extracellular activation of integrin signaling. Our work identifies Shv as a novel activator of integrin signaling and suggests a new integration model in which crosstalk between integrin and DE-cadherin in niche cells promote their own preservation by maintaining the niche architecture.

Abstract 40: Prothrombotic Role of Platelet Tlr2 in Hyperlipidemia

A prothrombotic state and increased platelet reactivity are common in hyperlipidemia and oxidative stress. Lipid peroxidation, a major consequence of oxidative stress, generates highly reactive products including hydroxy-w-oxoalkenoic acids that modify autologous proteins generating biologically active derivatives. Phosphatidylethanolamine, the second most abundant eukaryotic phospholipid can also be modified by hydroxy-w-oxoalkenoic acids. However, the conditions leading to accumulation of such derivatives in circulation and their biological activities remain poorly understood. We now show that carboxyalkylpyrrole-phosphatidylethanolamine derivatives (CAP-PE) accumulate in plasma of hyperlipidemic ApoE -/- mice. CAP-PE directly bind to TLR2 and induce platelet integrin alpha 2b beta 3 activation and P-selectin expression in TLR2 dependent manner. Platelet activation by CAP-PE includes assembly of TLR2/TLR1 receptor complex, induction of downstream signaling via MyD88/TIRAP, phosphorylation of IRAK4, and subsequent activation of TRAF6. This in turn activates the Src family kinases, Syk and PLC gamma 2 and platelet integrins. By intravital thrombosis studies we have demonstrated that CAP-PE accelerate thrombosis in TLR2 dependent manner. Furthermore, we demonstrate that TLR2 deficient mice are protected from accelerated thrombosis induced by hyperlipidemia. Taken together, our studies demonstrate a cross-talk between innate immunity and integrin activation signaling pathways in platelets and reveal that TLR2 plays a key role in platelet hyperreactivity and prothrombotic state in hyperlipidemia.

Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electric impulse propagation velocity and immature action potential profiles. Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; n=9) and have mature cardiomyocyte action potential profiles, including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s; n=5 monolayers). In addition, the optimal extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1, and connexin43) and myofilament markers (cardiac troponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase activation prevented structural maturation. Maturation of human stem cell-derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers.