Publications by authors named "Brian G Petrich"

39 Publications

Mechanically Triggered Hybridization Chain Reaction.

Angew Chem Int Ed Engl 2021 Jul 9. Epub 2021 Jul 9.

Department of Chemistry, Emory University, Atlanta, GA, 30322, USA.

Cells transmit piconewton forces to receptors to mediate processes such as migration and immune recognition. A major challenge in quantifying such forces is the sparsity of cell mechanical events. Accordingly, molecular tension is typically quantified with high resolution fluorescence microscopy, which hinders widespread adoption and application. Here, we report a mechanically triggered hybridization chain reaction (mechano-HCR) that allows chemical amplification of mechanical events. The amplification is triggered when a cell receptor mechanically denatures a duplex revealing a cryptic initiator to activate the HCR reaction in situ. Importantly, mechano-HCR enables direct readout of pN forces using a plate reader. We leverage this capability and measured mechano-IC for aspirin, Y-27632, and eptifibatide. Given that cell mechanical phenotypes are of clinical importance, mechano-HCR may offer a convenient route for drug discovery, personalized medicine, and disease diagnosis.
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http://dx.doi.org/10.1002/anie.202107660DOI Listing
July 2021

DNA-Based Microparticle Tension Sensors (μTS) for Measuring Cell Mechanics in Non-planar Geometries and for High-Throughput Quantification.

Angew Chem Int Ed Engl 2021 Aug 28;60(33):18044-18050. Epub 2021 Jun 28.

Department of Chemistry, Emory University, Atlanta, GA, 30322, USA.

Mechanotransduction, the interplay between physical and chemical signaling, plays vital roles in many biological processes. The state-of-the-art techniques to quantify cell forces employ deformable polymer films or molecular probes tethered to glass substrates. However, the applications of these assays in fundamental and clinical research are restricted by the planar geometry and low throughput of microscopy readout. Herein, we develop a DNA-based microparticle tension sensor, which features a spherical surface and thus allows for investigation of mechanotransduction at curved interfaces. The micron-scale of μTS enables flow cytometry readout, which is rapid and high throughput. We applied the method to map and measure T-cell receptor forces and platelet integrin forces at 12 and 56 pN thresholds. Furthermore, we quantified the inhibition efficiency of two anti-platelet drugs providing a proof-of-concept demonstration of μTS to screen drugs that modulate cellular mechanics.
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http://dx.doi.org/10.1002/anie.202102206DOI Listing
August 2021

Dual role of endothelial in tumor angiogenesis and tumor immunity.

Sci Transl Med 2021 03;13(583)

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110-1093, USA.

The cross-talk between angiogenesis and immunity within the tumor microenvironment (TME) is critical for tumor prognosis. While pro-angiogenic and immunosuppressive TME promote tumor growth, anti-angiogenic and immune stimulatory TME inhibit tumor progression. Therefore, there is a great interest in achieving vascular normalization to improve drug delivery and enhance antitumor immunity. However, anti-vascular endothelial growth factor (VEGF) mechanisms to normalize tumor vessels have offered limited therapeutic efficacies for patients with cancer. Here, we report that , a direct target of ETV2, was nearly exclusively expressed in endothelial cells. In preclinical mouse tumor models, deficiency reduced angiogenesis, enhanced high endothelial venule formation, and promoted antitumor immunity, leading to restricted tumor progression. Analysis of The Cancer Genome Atlas (TCGA) datasets revealed a significant ( < 0.05) correlation between expression, angiogenesis, and antitumor immunity in human cancers, as suggested by decreased expression and increased antitumor macrophages in patients with low expression. Mechanistically, MYCT1 interacted with tight junction protein Zona Occludens 1 and regulated Rho GTPase-mediated actin cytoskeleton dynamics, thereby promoting endothelial motility in the angiogenic environment. -deficient endothelial cells facilitated trans-endothelial migration of cytotoxic T lymphocytes and polarization of M1 macrophages. targeting combined with anti-PD1 treatment significantly ( < 0.05) increased complete tumor regression and long-term survival in anti-PD1-responsive and -refractory tumor models in mice. Our data collectively support a critical role for in controlling tumor angiogenesis and reprogramming tumor immunity. -targeted vascular control, in combination with immunotherapy, may become an exciting therapeutic strategy.
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http://dx.doi.org/10.1126/scitranslmed.abb6731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8252962PMC
March 2021

Integrin affinity modulation critically regulates atherogenic endothelial activation in vitro and in vivo.

Matrix Biol 2021 02 4;96:87-103. Epub 2020 Nov 4.

Departments of Molecular and Cellular Physiology, LSU Health Sciences Center - Shreveport, Shreveport, LA 71130, United States.; Cell Biology and Anatomy,LSU Health Sciences Center - Shreveport, Shreveport, LA 71130, United States.; Pathology and Translational Pathobiology,LSU Health Sciences Center - Shreveport, Shreveport, LA 71130, United States.; Department of Pathology and Translational Pathobiology, 1501 Kings Hwy, Biomedical Research Institute, Rm. 6-21, LSU Health Sciences Center - Shreveport, Shreveport, LA 71130, United States. Electronic address:

While vital to platelet and leukocyte adhesion, the role of integrin affinity modulation in adherent cells remains controversial. In endothelial cells, atheroprone hemodynamics and oxidized lipoproteins drive an increase in the high affinity conformation of α5β1 integrins in endothelial cells in vitro, and α5β1 integrin inhibitors reduce proinflammatory endothelial activation to these stimuli in vitro and in vivo. However, the importance of α5β1 integrin affinity modulation to endothelial phenotype remains unknown. We now show that endothelial cells (talin1 L325R) unable to induce high affinity integrins initially adhere and spread but show significant defects in nascent adhesion formation. In contrast, overall focal adhesion number, area, and composition in stably adherent cells are similar between talin1 wildtype and talin1 L325R endothelial cells. However, talin1 L325R endothelial cells fail to induce high affinity α5β1 integrins, fibronectin deposition, and proinflammatory responses to atheroprone hemodynamics and oxidized lipoproteins. Inducing the high affinity conformation of α5β1 integrins in talin1 L325R endothelial cells suggest that NF-κB activation and maximal fibronectin deposition require both integrin activation and other integrin-independent signaling. In endothelial-specific talin1 L325R mice, atheroprone hemodynamics fail to promote inflammation and macrophage recruitment, demonstrating a vital role for integrin activation in regulating endothelial phenotype.
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http://dx.doi.org/10.1016/j.matbio.2020.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902378PMC
February 2021

Talin-dependent integrin activation is required for endothelial proliferation and postnatal angiogenesis.

Angiogenesis 2021 02 28;24(1):177-190. Epub 2020 Oct 28.

Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Emory University School of Medicine, Atlanta, GA, USA.

Integrin activation contributes to key blood cell functions including adhesion, proliferation and migration. An essential step in the cell signaling pathway that activates integrin requires the binding of talin to the β-integrin cytoplasmic tail. Whereas this pathway is understood in platelets in detail, considerably less is known regarding how integrin-mediated adhesion in endothelium contributes to postnatal angiogenesis. We utilized an inducible EC-specific talin1 knock-out mouse (Tln1 EC-KO) and talin1 L325R knock-in mutant (Tln1 L325R) mouse, in which talin selectively lacks the capacity to activate integrins, to assess the role of integrin activation during angiogenesis. Deletion of talin1 during postnatal days 1-3 (P1-P3) caused lethality by P8 with extensive defects in retinal angiogenesis and widespread hemorrhaging. Tln1 EC-KO mice displayed reduced retinal vascular area, impaired EC sprouting and proliferation relative to Tln1 CTRLs. In contrast, induction of talin1 L325R in neonatal mice resulted in modest defects in retinal angiogenesis and mice survived to adulthood. Interestingly, deletion of talin1 or expression of talin1 L325R in ECs increased MAPK/ERK signaling. Strikingly, B16-F0 tumors grown in Tln1 L325R adult mice were 55% smaller and significantly less vascularized than tumors grown in littermate controls. EC talin1 is indispensable for postnatal development angiogenesis. The role of EC integrin activation appears context-dependent as its inhibition is compatible with postnatal development with mild defects in retinal angiogenesis but results in marked defects in tumor growth and angiogenesis. Inhibiting EC pan-integrin activation may be an effective approach to selectively target tumor blood vessel growth.
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http://dx.doi.org/10.1007/s10456-020-09756-4DOI Listing
February 2021

Live-cell super-resolved PAINT imaging of piconewton cellular traction forces.

Nat Methods 2020 10 14;17(10):1018-1024. Epub 2020 Sep 14.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.

Despite the vital role of mechanical forces in biology, it still remains a challenge to image cellular force with sub-100-nm resolution. Here, we present tension points accumulation for imaging in nanoscale topography (tPAINT), integrating molecular tension probes with the DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique to map piconewton mechanical events with ~25-nm resolution. To perform live-cell dynamic tension imaging, we engineered reversible probes with a cryptic docking site revealed only when the probe experiences forces exceeding a defined mechanical threshold (~7-21 pN). Additionally, we report a second type of irreversible tPAINT probe that exposes its cryptic docking site permanently and thus integrates force history over time, offering improved spatial resolution in exchange for temporal dynamics. We applied both types of tPAINT probes to map integrin receptor forces in live human platelets and mouse embryonic fibroblasts. Importantly, tPAINT revealed a link between platelet forces at the leading edge of cells and the dynamic actin-rich ring nucleated by the Arp2/3 complex.
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http://dx.doi.org/10.1038/s41592-020-0929-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574592PMC
October 2020

Topological Adaptation of Transmembrane Domains to the Force-Modulated Lipid Bilayer Is a Basis of Sensing Mechanical Force.

Curr Biol 2020 05 12;30(9):1614-1625.e5. Epub 2020 Mar 12.

Department of Life Sciences, Korea University, Seoul 02841, Republic of Korea. Electronic address:

Cells can sense and respond to various mechanical stimuli from their surrounding environment. One of the explanations for mechanosensitivity, a lipid-bilayer model, suggests that a stretch of the membrane induced by mechanical force alters the physical state of the lipid bilayer, driving mechanosensors to assume conformations better matched to the altered membrane. However, mechanosensors of this class are restricted to ion channels. Here, we reveal that integrin αIIbβ3, a prototypic adhesion receptor, can be activated by various mechanical stimuli including stretch, shear stress, and osmotic pressure. The force-induced integrin activation was not dependent on its known intracellular activation signaling events and was even observed in reconstituted cell-free liposomes. Instead, these mechanical stimuli were found to alter the lipid embedding of the integrin β3 transmembrane domain (TMD) and subsequently weaken the αIIb-β3 TMD interaction, which results in activation of the receptor. Moreover, artificial modulation of the membrane curvature near integrin αIIbβ3 can induce its activation in cells as well as in lipid nanodiscs, suggesting that physical deformation of the lipid bilayer, either by mechanical force or curvature, can induce integrin activation. Thus, our results establish the adhesion receptor as a bona fide mechanosensor that directly senses and responds to the force-modulated lipid environment. Furthermore, this study expands the lipid-bilayer model by suggesting that the force-induced topological change of TMDs and subsequent alteration in the TMD interactome is a molecular basis of sensing mechanical force transmitted via the lipid bilayer.
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http://dx.doi.org/10.1016/j.cub.2020.02.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202955PMC
May 2020

Integrin-dependent regulation of the endothelial barrier.

Tissue Barriers 2019 5;7(4):1685844. Epub 2019 Nov 5.

Department of Pediatrics, Aflac Cancer and Blood Disorders Center (FEP, BGP) and Cancer Biology Graduate Program (FEP), Emory University School of Medicine, Atlanta, GA, USA.

The endothelium physically separates blood from surrounding tissue and yet allows for the regulated passage of nutrients, waste, and leukocytes into and out of the circulation. Trans-endothelium flux occurs across endothelial cells (transcellular) and between endothelial cells (paracellular). Paracellular endothelial barrier function depends on the regulation of cell-cell junctions. Interestingly, a functional relationship between cell-cell junctions and cell-matrix adhesions has long been appreciated but the molecular mechanisms underpinning this relationship are not fully understood. Here we review the evidence that supports the notion that cell-matrix interactions contribute to the regulation of cell-cell junctions, focusing primarily on the important adherens junction protein VE-cadherin. In particular, we will discuss recent insights gained into how integrin signaling impacts VE-cadherin stability in adherens junctions and endothelial barrier function.
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http://dx.doi.org/10.1080/21688370.2019.1685844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6866814PMC
July 2020

Talin-Dependent Integrin Activation Regulates VE-Cadherin Localization and Endothelial Cell Barrier Function.

Circ Res 2019 03;124(6):891-903

From the Department of Pediatrics, Aflac Cancer and Blood Disorders Center (F.E.P., S.K., B.G.P.), Emory University School of Medicine, Atlanta, GA.

Rationale: Endothelial barrier function depends on the proper localization and function of the adherens junction protein VE (vascular endothelial)-cadherin. Previous studies have suggested a functional relationship between integrin-mediated adhesion complexes and VE-cadherin yet the underlying molecular links are unclear. Binding of the cytoskeletal adaptor protein talin to the β-integrin cytoplasmic domain is a key final step in regulating the affinity of integrins for extracellular ligands (activation) but the role of integrin activation in VE-cadherin mediated endothelial barrier function is unknown.

Objective: To test the requirement of talin-dependent activation of β1 integrin in VE-cadherin organization and endothelial cell (EC) barrier function.

Methods And Results: EC-specific deletion of talin in adult mice resulted in impaired stability of intestinal microvascular blood vessels, hemorrhage, and death. Talin-deficient endothelium showed altered VE-cadherin organization at EC junctions in vivo. shRNA (short hairpin RNA)-mediated knockdown of talin1 expression in cultured ECs led to increased radial actin stress fibers, increased adherens junction width and increased endothelial monolayer permeability measured by electrical cell-substrate impedance sensing. Restoring β1-integrin activation in talin-deficient cells with a β1-integrin activating antibody normalized both VE-cadherin organization and EC barrier function. In addition, VE-cadherin organization was normalized by reexpression of talin or integrin activating talin head domain but not a talin head domain mutant that is selectively deficient in activating integrins.

Conclusions: Talin-dependent activation of EC β1-integrin stabilizes VE-cadherin at endothelial junctions and promotes endothelial barrier function.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.314560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521868PMC
March 2019

Integrin Activation Controls Regulatory T Cell-Mediated Peripheral Tolerance.

J Immunol 2018 06 27;200(12):4012-4023. Epub 2018 Apr 27.

Department of Medicine, University of California, San Diego, La Jolla, CA 92093;

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and β subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact-mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell-specific loss of talin, a β integrin-binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin αβ led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.
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http://dx.doi.org/10.4049/jimmunol.1800112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988969PMC
June 2018

Talin Plays a Critical Role in the Maintenance of the Regulatory T Cell Pool.

J Immunol 2017 06 17;198(12):4639-4651. Epub 2017 May 17.

Department of Medicine, University of California San Diego, La Jolla, CA 92093;

Talin, a cytoskeletal protein essential in mediating integrin activation, has been previously shown to be involved in the regulation of T cell proliferation and function. In this study, we describe a role for talin in maintaining the homeostasis and survival of the regulatory T (Treg) cell pool. T cell-specific deletion of talin in mice resulted in spontaneous lymphocyte activation, primarily due to numerical and functional deficiencies of Treg cells in the periphery. Peripheral talin-deficient Treg cells were unable to maintain high expression of IL-2Rα, resulting in impaired IL-2 signaling and ultimately leading to increased apoptosis through downregulation of prosurvival proteins Bcl-2 and Mcl-1. The requirement for talin in maintaining high IL-2Rα expression by Treg cells was due, in part, to integrin LFA-1-mediated interactions between Treg cells and dendritic cells. Collectively, our data suggest a critical role for talin in Treg cell-mediated maintenance of immune homeostasis.
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http://dx.doi.org/10.4049/jimmunol.1601165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507362PMC
June 2017

Blocking neutrophil integrin activation prevents ischemia-reperfusion injury.

J Exp Med 2015 Jul 13;212(8):1267-81. Epub 2015 Jul 13.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104

Neutrophil recruitment, mediated by β2 integrins, combats pyogenic infections but also plays a key role in ischemia-reperfusion injury and other inflammatory disorders. Talin induces allosteric rearrangements in integrins that increase affinity for ligands (activation). Talin also links integrins to actin and other proteins that enable formation of adhesions. Structural studies have identified a talin1 mutant (L325R) that perturbs activation without impairing talin's capacity to link integrins to actin and other proteins. Here, we found that mice engineered to express only talin1(L325R) in myeloid cells were protected from renal ischemia-reperfusion injury. Dissection of neutrophil function in vitro and in vivo revealed that talin1(L325R) neutrophils had markedly impaired chemokine-induced, β2 integrin-mediated arrest, spreading, and migration. Surprisingly, talin1(L325R) neutrophils exhibited normal selectin-induced, β2 integrin-mediated slow rolling, in sharp contrast to the defective slow rolling of neutrophils lacking talin1 or expressing a talin1 mutant (W359A) that blocks talin interaction with integrins. These studies reveal the importance of talin-mediated activation of integrins for renal ischemia-reperfusion injury. They further show that neutrophil arrest requires talin recruitment to and activation of integrins. However, although neutrophil slow rolling requires talin recruitment to integrins, talin-mediated integrin activation is dispensable.
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http://dx.doi.org/10.1084/jem.20142358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516797PMC
July 2015

Caspase-1-mediated pathway promotes generation of thromboinflammatory microparticles.

J Clin Invest 2015 Apr 23;125(4):1471-84. Epub 2015 Feb 23.

Extracellular ATP is a signal of tissue damage and induces macrophage responses that amplify inflammation and coagulation. Here we demonstrate that ATP signaling through macrophage P2X7 receptors uncouples the thioredoxin (TRX)/TRX reductase (TRXR) system and activates the inflammasome through endosome-generated ROS. TRXR and inflammasome activity promoted filopodia formation, cellular release of reduced TRX, and generation of extracellular thiol pathway-dependent, procoagulant microparticles (MPs). Additionally, inflammasome-induced activation of an intracellular caspase-1/calpain cysteine protease cascade degraded filamin, thereby severing bonds between the cytoskeleton and tissue factor (TF), the cell surface receptor responsible for coagulation activation. This cascade enabled TF trafficking from rafts to filopodia and ultimately onto phosphatidylserine-positive, highly procoagulant MPs. Furthermore, caspase-1 specifically facilitated cell surface actin exposure, which was required for the final release of highly procoagulant MPs from filopodia. Together, the results of this study delineate a thromboinflammatory pathway and suggest that components of this pathway have potential as pharmacological targets to simultaneously attenuate inflammation and innate immune cell-induced thrombosis.
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http://dx.doi.org/10.1172/JCI79329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396490PMC
April 2015

A talin mutant that impairs talin-integrin binding in platelets decelerates αIIbβ3 activation without pathological bleeding.

Blood 2014 Apr 28;123(17):2722-31. Epub 2014 Feb 28.

Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC;

Tight regulation of integrin affinity is critical for hemostasis. A final step of integrin activation is talin binding to 2 sites within the integrin β cytoplasmic domain. Binding of talin to a membrane-distal NPxY sequence facilitates a second, weaker interaction of talin with an integrin membrane-proximal region (MPR) that is critical for integrin activation. To test the functional significance of these distinct interactions on platelet function in vivo, we generated knock-in mice expressing talin1 mutants with impaired capacity to interact with the β3 integrin MPR (L325R) or NPLY sequence (W359A). Both talin1(L325R) and talin1(W359A) mice were protected from experimental thrombosis. Talin1(L325R) mice, but not talin(W359A) mice, exhibited a severe bleeding phenotype. Activation of αIIbβ3 was completely blocked in talin1(L325R) platelets, whereas activation was reduced by approximately 50% in talin1(W359A) platelets. Quantitative biochemical measurements detected talin1(W359A) binding to β3 integrin, albeit with a 2.9-fold lower affinity than wild-type talin1. The rate of αIIbβ3 activation was slower in talin1(W359A) platelets, which consequently delayed aggregation under static conditions and reduced thrombus formation under physiological flow conditions. Together our data indicate that reduction of talin-β3 integrin binding affinity results in decelerated αIIbβ3 integrin activation and protection from arterial thrombosis without pathological bleeding.
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http://dx.doi.org/10.1182/blood-2013-12-543363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3999757PMC
April 2014

Nov/CCN3 regulates long-term repopulating activity of murine hematopoietic stem cells via integrin αvβ3.

Int J Hematol 2014 Apr 22;99(4):393-406. Epub 2014 Feb 22.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan.

Throughout life, hematopoietic stem cells (HSCs) sustain the blood cell supply through their capacities for self-renewal and multilineage differentiation. These processes are regulated within a specialized microenvironment termed the 'niche'. Here, we show a novel mechanism for regulating HSC function that is mediated by nephroblastoma overexpressed (Nov/CCN3), a matricellular protein member of the CCN family. We found that Nov contributes to the maintenance of long-term repopulating (LTR) activity through association with integrin αvβ3 on HSCs. The resultant β3 integrin outside-in signaling is dependent on thrombopoietin (TPO), a crucial cytokine involved in HSC maintenance. TPO was required for Nov binding to integrin αvβ3, and stimulated Nov expression in HSCs. However, in the presence of IFNγ, a cytokine known to impair HSC function, not only was TPO-induced expression of Nov suppressed, but the LTR activity was conversely impaired by TPO-mediated ligation of integrin αvβ3 with exogenous ligands, including Nov, as well. Thus, Nov/integrin αvβ3-mediated maintenance of HSCs appears to be modulated by simultaneous stimulation by other cytokines. Our finding suggests that this system contributes to the regulation of HSCs within the bone marrow niche.
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http://dx.doi.org/10.1007/s12185-014-1534-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4412171PMC
April 2014

The mechanism of kindlin-mediated activation of integrin αIIbβ3.

Curr Biol 2013 Nov 7;23(22):2288-2295. Epub 2013 Nov 7.

Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Increased ligand binding to cellular integrins ("activation") plays important roles in processes such as development, cell migration, extracellular matrix assembly, tumor metastasis, hemostasis, and thrombosis. Integrin activation encompasses both increased integrin monomer affinity and increased receptor clustering and depends on integrin-talin interactions. Loss of kindlins results in reduced activation of integrins. Kindlins might promote talin binding to integrins through a cooperative mechanism; however, kindlins do not increase talin association with integrins. Here, we report that, unlike talin head domain (THD), kindlin-3 has little effect on the affinity of purified monomeric αIIbβ3, and it does not enhance activation by THD. Furthermore, studies with ligands of varying valency show that kindlins primarily increase cellular αIIbβ3 avidity rather than monomer affinity. In platelets or nucleated cells, loss of kindlins markedly reduces αIIbβ3 binding to multivalent but not monovalent ligands. Finally, silencing of kindlins reduces the clustering of ligand-occupied αIIbβ3 as revealed by total internal reflection fluorescence and electron microscopy. Thus, in contrast to talins, kindlins have little primary effect on integrin αIIbβ3 affinity for monovalent ligands and increase multivalent ligand binding by promoting the clustering of talin-activated integrins.
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http://dx.doi.org/10.1016/j.cub.2013.09.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3912999PMC
November 2013

Requirement for integrin-linked kinase in neural crest migration and differentiation and outflow tract morphogenesis.

BMC Biol 2013 Oct 16;11:107. Epub 2013 Oct 16.

Key Laboratory of Arrhythmia, Ministry of Education, East Hospital, Tongji University School of Medicine, 150 Jimo Road, Shanghai 200120, China.

Background: Neural crest defects lead to congenital heart disease involving outflow tract malformation. Integrin-linked-kinase (ILK) plays important roles in multiple cellular processes and embryogenesis. ILK is expressed in the neural crest, but its role in neural crest and outflow tract morphogenesis remains unknown.

Results: We ablated ILK specifically in the neural crest using the Wnt1-Cre transgene. ILK ablation resulted in abnormal migration and overpopulation of neural crest cells in the pharyngeal arches and outflow tract and a significant reduction in the expression of neural cell adhesion molecule (NCAM) and extracellular matrix components. ILK mutant embryos exhibited an enlarged common arterial trunk and ventricular septal defect. Reduced smooth muscle differentiation, but increased ossification and neurogenesis/innervation were observed in ILK mutant outflow tract that may partly be due to reduced transforming growth factor β2 (TGFβ2) but increased bone morphogenetic protein (BMP) signaling. Consistent with these observations, microarray analysis of fluorescence-activated cell sorting (FACS)-sorted neural crest cells revealed reduced expression of genes associated with muscle differentiation, but increased expression of genes of neurogenesis and osteogenesis.

Conclusions: Our results demonstrate that ILK plays essential roles in neural crest and outflow tract development by mediating complex crosstalk between cell matrix and multiple signaling pathways. Changes in these pathways may collectively result in the unique neural crest and outflow tract phenotypes observed in ILK mutants.
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http://dx.doi.org/10.1186/1741-7007-11-107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906977PMC
October 2013

Heparin rescues factor V Leiden-associated placental failure independent of anticoagulation in a murine high-risk pregnancy model.

Blood 2013 Mar 16;121(11):2127-34. Epub 2013 Jan 16.

Division of Pediatric Pathology, Department of Pathology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

Low molecular weight heparin (LMWH) is being tested as an experimental drug for improving pregnancy outcome in women with inherited thrombophilia and placenta-mediated pregnancy complications, such as recurrent pregnancy loss. The role of thrombotic processes in these disorders remains unproven, and the issue of antithrombotic prophylaxis is intensely debated. Using a murine model of factor V Leiden-associated placental failure, we show that treatment of the mother with LMWH allows placental development to proceed and affords significant protection from fetal loss. Nonetheless, the therapeutic effect of LMWH is not replicated by anticoagulation; fondaparinux and a direct Xa inhibitor, C921-78, achieve anticoagulation similar to LMWH but produce little or no improvement in pregnancy outcome. Genetic attenuation of maternal platelet aggregation is similarly ineffective. In contrast, even a partial loss of thrombin sensitivity of maternal platelets protects pregnancies. Neonates born from these pregnancies are growth retarded, suggesting that placental function is only partially restored. The placentae are smaller but do not reveal any evidence of thrombosis. Our data demonstrate an anticoagulation-independent role of LMWH in protecting pregnancies and provide evidence against the involvement of thrombotic processes in thrombophilia-associated placental failure. Importantly, thrombin-mediated maternal platelet activation remains central in the mechanism of placental failure.
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http://dx.doi.org/10.1182/blood-2012-08-448209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3952382PMC
March 2013

Talin1 and Rap1 are critical for osteoclast function.

Mol Cell Biol 2013 Feb 10;33(4):830-44. Epub 2012 Dec 10.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

To determine talin1's role in osteoclasts, we mated TLN1(fl/fl) mice with those expressing cathepsin K-Cre (CtsK-TLN1) to delete the gene in mature osteoclasts or with lysozyme M-Cre (LysM-TLN1) mice to delete TLN1 in all osteoclast lineage cells. Absence of TLN1 impairs macrophage colony-stimulating factor (M-CSF)-stimulated inside-out integrin activation and cytoskeleton organization in mature osteoclasts. Talin1-deficient precursors normally express osteoclast differentiation markers when exposed to M-CSF and receptor activator of nuclear factor κB (RANK) ligand but attach to substrate and migrate poorly, arresting their development into mature resorptive cells. In keeping with inhibited resorption, CtsK-TLN1 mice exhibit an ∼5-fold increase in bone mass. Osteoclast-specific deletion of Rap1 (CtsK-Rap1), which promotes talin/β integrin recognition, yields similar osteopetrotic mice. The fact that the osteopetrosis of CtsK-TLN1 and CtsK-Rap1 mice is substantially more severe than that of those lacking αvβ3 is likely due to added failed activation of β1 integrins. In keeping with osteoclast dysfunction, mice in whom talin is deleted late in the course of osteoclastogenesis are substantially protected from ovariectomy-induced osteoporosis and the periarticular osteolysis attending inflammatory arthritis. Thus, talin1 and Rap1 are critical for resorptive function, and their selective inhibition in mature osteoclasts retards pathological bone loss.
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http://dx.doi.org/10.1128/MCB.00790-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571341PMC
February 2013

Distinct roles for talin-1 and kindlin-3 in LFA-1 extension and affinity regulation.

Blood 2012 May 19;119(18):4275-82. Epub 2012 Mar 19.

Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA.

In inflammation, neutrophils and other leukocytes roll along the microvascular endothelium before arresting and transmigrating into inflamed tissues. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen-1 (LFA-1). Mutations of the FERMT3 gene encoding kindlin-3 underlie the human immune deficiency known as leukocyte adhesion deficiency-III. Both kindlin-3 and talin-1, another FERM domain-containing cytoskeletal protein, are required for integrin activation, but their individual roles in the induction of specific integrin conformers are unclear. Here, we induce differential LFA-1 activation in neutrophils through engagement of the selectin ligand P-selectin glycoprotein ligand-1 or the chemokine receptor CXCR2. We find that talin-1 is required for inducing LFA-1 extension, which corresponds to intermediate affinity and induces neutrophil slow rolling, whereas both talin-1 and kindlin-3 are required for induction of the high-affinity conformation of LFA-1 with an open headpiece, which results in neutrophil arrest. In vivo, both slow rolling and arrest are defective in talin-1-deficient neutrophils, whereas only arrest is defective in kindlin-3-deficient neutrophils. We conclude that talin-1 and kindlin-3 serve distinct functions in LFA-1 activation.
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http://dx.doi.org/10.1182/blood-2011-08-373118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3359742PMC
May 2012

Integrin-αvβ3 regulates thrombopoietin-mediated maintenance of hematopoietic stem cells.

Blood 2012 Jan 16;119(1):83-94. Epub 2011 Nov 16.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan.

Throughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-β3 signaling contributes to HSC maintenance. Specific ligation of β3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvβ3-integrin "inside-out" signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent "outside-in" signaling via phosphorylation of Tyr747 in the β3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between β3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.
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http://dx.doi.org/10.1182/blood-2011-02-335430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3251239PMC
January 2012

In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling.

Blood 2012 Feb 16;119(8):e45-56. Epub 2011 Nov 16.

Department of Cardiovascular Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan.

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.
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http://dx.doi.org/10.1182/blood-2011-09-381400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3351094PMC
February 2012

Kindlin: helper, co-activator, or booster of talin in integrin activation?

Curr Opin Hematol 2011 Sep;18(5):356-60

Department of Medicine, University of California San Diego, La Jolla, California, USA.

Purpose Of Review: The modulation of integrin affinity is central to platelet and leukocyte function. Two proteins, talin and kindlin, that interact with distinct regions of integrin cytoplasmic domains, have been shown to play essential roles in inducing the high affinity integrin conformation required for platelet and leukocyte adhesive interactions.Here we highlight some of the key studies that have described roles for talin and kindlin in integrin function and discuss several models that explain how talin and kindlins might work together to regulate integrin activation.

Recent Findings: Genetic deletion of kindlin-3 in mice results in platelet and leukocyte adhesive dysfunction associated with profoundly impaired activation of multiple classes of integrins, a phenotype similar to that observed in talin-deficient platelets and leukocytes. Since this initial report three years ago, numerous studies have provided important clues to how kindlins activate integrins and, in some cases, the relationship between kindlins and talin in integrin activation.

Summary: Clearly, talin and kindlins are key regulators of integrin affinity. Future experiments that define precisely how these molecules work in concert should provide important insights into the terminal signaling events that activate integrins.
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http://dx.doi.org/10.1097/MOH.0b013e3283497f09DOI Listing
September 2011

Live cell imaging of paxillin in rolling neutrophils by dual-color quantitative dynamic footprinting.

Microcirculation 2011 Jul;18(5):361-72

Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

Objective: Neutrophil recruitment to sites of inflammation involves P-selectin-dependent rolling. qDF is a useful tool to visualize the topography of the neutrophil footprint as it interacts with the substrate. However, elucidating the role of specific proteins in addition to topography requires simultaneous visualization of two fluorochromes.

Methods: To validate DqDF, mouse neutrophils were labeled with the membrane dyes DiO and DiI and perfused into microchannels coated with P-selectin-Fc. Footprints of rolling neutrophils were recorded as two separate images, one for each fluorochrome. To assess the localization of the cytoskeletal protein paxillin, we applied DqDF to DiO-stained neutrophils of mice expressing an mCherry-paxillin fusion protein.

Results: The footprint topographies obtained from DiO and DiI in the plasma membrane were identical. The z-coordinates of the microvilli tips obtained with the two fluorochromes in the footprint were also identical. Paxillin was found to be localized to some, but not all ridges in the neutrophil footprint.

Conclusions: Our data suggest that the spectral properties of the fluorochrome do not affect the results. DqDF will be useful for simultaneous visualization of two fluorochromes in the footprint of rolling cells.
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http://dx.doi.org/10.1111/j.1549-8719.2011.00090.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123727PMC
July 2011

Talin-dependent integrin activation is required for fibrin clot retraction by platelets.

Blood 2011 Feb 22;117(5):1719-22. Epub 2010 Oct 22.

Department of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton.
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http://dx.doi.org/10.1182/blood-2010-09-305433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056596PMC
February 2011

A small molecule that inhibits the interaction of paxillin and alpha 4 integrin inhibits accumulation of mononuclear leukocytes at a site of inflammation.

J Biol Chem 2010 Mar 22;285(13):9462-9469. Epub 2010 Jan 22.

Department of Medicine, University of California at San Diego, La Jolla, California 92093. Electronic address:

Extracellular antagonists of alpha 4 integrin are an effective therapy for several autoimmune and inflammatory diseases; however, these agents that directly block ligand binding may exhibit mechanism-based toxicities. Inhibition of alpha 4 integrin signaling by mutations of alpha 4 that block paxillin binding inhibits inflammation while limiting mechanism-based toxicities. Here, we test a pharmacological approach by identifying small molecules that inhibit the alpha 4 integrin-paxillin interaction. By screening a large (approximately 40,000-compound) chemical library, we identified a noncytotoxic inhibitor of this interaction that impaired integrin alpha 4-mediated but not alpha L beta 2-mediated Jurkat T cell migration. The identified compound had no effect on alpha 4-mediated migration in cells bearing the alpha 4(Y991A) mutation that disrupts the alpha 4-paxillin interaction, establishing the specificity of its action. Administration of this compound to mice led to impaired recruitment of mononuclear leukocytes to a site of inflammation in vivo, whereas an isomer that does not inhibit the alpha 4-paxillin interaction had no effect on alpha 4-mediated cell migration, cell spreading, or recruitment of leukocytes to an inflammatory site. Thus, a small molecule inhibitor that interferes with alpha 4 integrin signaling reduces alpha 4-mediated T cell migration in vivo, thus providing proof of principle for inhibition of alpha 4 integrin signaling as a target for the pharmacological reduction of inflammation.
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http://dx.doi.org/10.1074/jbc.M109.066993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2843196PMC
March 2010

Talin-dependent integrin signalling in vivo.

Authors:
Brian G Petrich

Thromb Haemost 2009 Jun;101(6):1020-4

Division of Cardiology, Department of Medicine, University of California, San Diego, La Jolla, CA 92122, USA.

Integrins are heterodimeric adhesion receptors essential for metazoan life. In addition to mediating cell-extracellular matrix and cell-cell interactions, integrins are bona fide signalling receptors in that they transmit information in both directions across the plasma membrane. The affinity of integrins for extracellular ligands is regulated through a process termed integrin activation or "inside-out signalling". On the other hand, ligand binding to integrins can induce the recruitment and activation of a number of enzymes and adaptors such as pp125(FAK) and Src family kinases, to initiate "outside-in signalling". Intensive investigation into the mechanisms of integrin signalling has revealed many of the key players; amongst these, one of the most important is talin. Our understanding of how many of these molecules interact is now understood at the atomic level thanks to detailed structural studies. Indeed structural information and model cell systems have provided unique opportunities to dissect the molecular mechanisms of many aspects of integrin signalling. Recent studies have begun testing the biological significance of these mechanisms using in-vivo models, particular genetically modified mice. The generation and characterisation of in-vivo models to study integrin signalling has provided valuable information into the functional significance of integrin signalling in fundamental physiological processes as well as within the context of human disease. Here, I will review recent insights that have been gained into integrin signalling through the use of genetically modified mice focusing on integrin alphaIIbbeta3 (GPIIb-IIIa) and the regulation of its function in haemostasis and thrombosis.
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June 2009

Antithrombotic effects of targeting alphaIIbbeta3 signaling in platelets.

Blood 2009 Apr 12;113(15):3585-92. Epub 2008 Nov 12.

Department of Medicine, University of California San Diego, La Jolla, CA 92093-0726, USA.

alphaIIbbeta3 interaction with fibrinogen promotes Src-dependent platelet spreading in vitro. To determine the consequences of this outside-in signaling pathway in vivo, a "beta3(Delta760-762)" knockin mouse was generated that lacked the 3 C-terminal beta3 residues (arginine-glycine-threonine [RGT]) necessary for alphaIIbbeta3 interaction with c-Src, but retained beta3 residues necessary for talin-dependent fibrinogen binding. beta3(Delta760-762) mice were compared with wild-type beta3(+/+) littermates, beta3(+/-) heterozygotes, and knockin mice where beta3 RGT was replaced by beta1 C-terminal cysteine-glycine-lysine (EGK) to potentially enable signaling by Src kinases other than c-Src. Whereas beta3(+/+), beta3(+/-) and beta3/beta1(EGK) platelets spread and underwent tyrosine phosphorylation normally on fibrinogen, beta3(Delta760-762) platelets spread poorly and exhibited reduced tyrosine phosphorylation of c-Src substrates, including beta3 (Tyr(747)). Unlike control mice, beta3(Delta760-762) mice were protected from carotid artery thrombosis after vessel injury with FeCl(3). Some beta3(Delta760-762) mice exhibited prolonged tail bleeding times; however, none demonstrated spontaneous bleeding, excess bleeding after surgery, fecal blood loss, or anemia. Fibrinogen binding to beta3(Delta760-762) platelets was normal in response to saturating concentrations of protease-activated receptor 4 or glycoprotein VI agonists, but responses to adenosine diphosphate were impaired. Thus, deletion of beta3 RGT disrupts c-Src-mediated alphaIIbbeta3 signaling and confers protection from arterial thrombosis. Consequently, targeting alphaIIbbeta3 signaling may represent a feasible antithrombotic strategy.
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http://dx.doi.org/10.1182/blood-2008-09-180687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2668853PMC
April 2009
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