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Actin and Actin-Associated Proteins in Extracellular Vesicles Shed by Osteoclasts

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Actin and Actin-Associated Proteins in Extracellular Vesicles Shed by Osteoclasts International Journal of Molecular Sciences Review Actin and Actin-Associated Proteins in Extracellular Vesicles Shed by Osteoclasts L. Shannon Holliday 1,* , Lorraine Perciliano de Faria 2 and Wellington J. Rody Jr. 3 1 Department of Orthodontics, College of Dentistry, University of Florida, Gainesville, FL 32610, USA 2 Department of Biomaterials and Oral Biology, School of Dentistry, University of São Paulo, São Paulo 01000, Brazil; [email protected]fl.edu 3 Department of Orthodontics and Pediatric Dentistry, Stony Brook University School of Dental Medicine, Stony Brook, NY 11794, USA; [email protected] * Correspondence: [email protected]fl.edu Received: 21 November 2019; Accepted: 16 December 2019; Published: 25 December 2019 Abstract: Extracellular vesicles (EVs) are shed by all eukaryotic cells and have emerged as important intercellular regulators. EVs released by osteoclasts were recently identified as important coupling factors in bone remodeling. They are shed as osteoclasts resorb bone and stimulate osteoblasts to form bone to replace the bone resorbed. We reported the proteomic content of osteoclast EVs with data from two-dimensional, high resolution liquid chromatography/mass spectrometry. In this article, we examine in detail the actin and actin-associated proteins found in osteoclast EVs. Like EVs from other cell types, actin and various actin-associated proteins were abundant. These include components of the polymerization machinery, myosin mechanoenzymes, proteins that stabilize or depolymerize microfilaments, and actin-associated proteins that are involved in regulating integrins. The selective incorporation of actin-associated proteins into osteoclast EVs suggests that they have roles in the formation of EVs and/or the regulatory signaling functions of the EVs. Regulating integrins so that they bind extracellular matrix tightly, in order to attach EVs to the extracellular matrix at specific locations in organs and tissues, is one potential active role for actin-associated proteins in EVs. Keywords: exosome; microvesicle; microfilament; integrins; bone remodeling; myosins; actin-related protein; proteomics; extracellular vesicles 1. Introduction Extracellular vesicles (EVs) are 30–150 nm in diameter vesicles that are released by eukaryotic cells and function in intercellular signaling [1,2]. The term EVs encompasses exosomes and microvesicles (Figure1)[ 3]. Exosomes develop as inward buds into endocytic compartments, which pinch off into the lumen of the compartment, resulting in the formation of multivesicular bodies. Multivesicular bodies can then fuse with the plasma membrane to shed the exosomes from the cell. Microvesicles bud off directly from the plasma membrane. The two types of EVs have similar size, composition, and regulatory functions and are difficult to distinguish in extracellular vesicle populations, although some articles suggest that microvesicles may be on average larger and may have some differing components [4]. In addition, some non-vesicular particles are probably often isolated in EV preps, including exomeres and lipoproteins [5]. Unless the type of vesicle being studied is known, which is usually not the case at the present time, the term EVs is preferred [3]. Int. J. Mol. Sci. 2020, 21, 158; doi:10.3390/ijms21010158 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 158 2 of 19 Int. J. Mol. Sci. 2020, 20, x FOR PEER REVIEW 2 of 19 Figure 1. Extracellular vesicles include exosomes which are derived from multivesicular bodies Figure 1. Extracellular vesicles include exosomes which are derived from multivesicular bodies (MVB) (MVB) and microvesicles (ectosomes) which bud directly from the plasma membrane. Both may bind and microvesicles (ectosomes) which bud directly from the plasma membrane. Both may bind surface surface receptors of target cells to stimulate signaling pathways, or to fuse with the plasma receptors of target cells to stimulate signaling pathways, or to fuse with the plasma membrane or membranemembranes or of membranes endocytic compartments. of endocytic compartments. Fusion releases Fusion their luminalreleases contentstheir luminal into thecontents cytosol into of the cytosoltarget cell, of andthe target membrane cell, proteinsand membrane into either proteins the plasma into either membrane the plasma or endocytic membrane membrane. or endocytic membrane. Exosomes were first identified and characterized due to their role in the removal of the transferrin receptorExosomes from reticulocytes were first asidentified they diff erentiatedand characteri [6,7].zed For manydue to years, their exosomes role in werethe removal mostly thought of the transferrinof as “garbage receptor bags”, from although reticulocytes evidence as that they EVs diff coulderentiated present [6,7]. antigen For appearedmany years, during exosomes the 1990s were [8]. mostlyIn 2007, thought landmark of as articles “garbage showed bags”, that although exosomes evidence carried that mRNAs EVs andcould microRNAs, present antigen and could appeared fuse withduring target the cells1990s to introduce[8]. In 2007, the functionallandmark RNAsarticles into showed the cytosol that [exosomes9,10]. The carried concept mRNAs of EVs being and microRNAs,able to regulate and target could cells fuse acting with target at diff erentcells to regulatory introduce levels the functional stimulated RNAs the EV into field. the cytosol Subsequently, [9,10]. Themuch concept evidence of EVs has being accumulated able to regulate that by transferringtarget cells acting microRNAs, at different EVs modulateregulatorytarget levels cellstimulated protein theexpression. EV field. For Subsequently, example, two much groups evidence reported has that ac microRNAcumulated 214-3pthat by is transf founderring in EVs microRNAs, from osteoclasts, EVs modulateand is transferred target cell to protein osteoblasts, expression. where itFor inhibits exampl osteoblaste, two groups formation reported by reducing that microRNA the expression 214-3p ofis foundregulatory in EVs proteins from [11 osteoclasts,,12]. Despite and the plethorais transferre of articlesd to supportingosteoblasts, the where hypothesis it inhibits that microRNAs osteoblast formationin EVs are by crucial reducing to their the regulatoryexpression function,of regulatory some proteins studies [11,12]. have cast Despite doubt th one plethora whether of su articlesfficient supportingnumbers of microRNAsthe hypothesis are that present microRNAs in EVs to suppressin EVs are mRNA crucial translation to their regulatory [13]. function, some studiesFor have EVs cast to bind doubt and on stimulatewhether sufficient a target cell, numbers either of from microRNAs the outside are present through in traditional EVs to suppress signal mRNAtransduction translation pathways, [13]. or after fusing, the EVs must interact with the cell. Osteoclast EVs serve as a modelFor for EVs the to sorts bind of and interactions stimulate and a target regulation cell, thateither have from been the found outside in EVsthrough in general. traditional signal transductionIn osteoclasts, pathways, three or potential after fusing, modes the ofEVs interaction must interact have with been the identified, cell. Osteoclast namely EVs semaphorin serve as a model4D in EVs for the binding sorts plexin-B1of interactions on osteoblasts and regulati [11on], ephrin-B2that have been in EVs found binding in EVs ephB4 in general. [12], and receptor activatorIn osteoclasts, of nuclear three factor potential kappa B modes (RANK) ofin interaction EVs binding have RANK-ligand been identified, (RANKL) namely [14 semaphorin]. Semaphorin 4D in4D EVs and binding ephrinB2 plexin-B1 were reported on osteoblasts to be osteoclast-derived [11], ephrin-B2 signalingin EVs binding factors, ephB4 before [12], their and detection receptor in activatorEVs [15,16 ].of Bothnuclear were foundfactor tokappa tether B EVs (RANK) to the surface in EVs of osteoblastsbinding RANK-ligand prior to the fusion (RANKL) that delivers [14]. SemaphorinmicroRNA-214-3p 4D and from ephrinB2 the EVs were lumen reported to the cytosolto be osteoclast-derived of the osteoblast. signaling factors, before their detectionRANK in isEVs part [15,16]. of the Both central were signaling found to pathway tether EVs in bone to the remodeling surface of [17osteoblasts,18]. RANKL, prior whichto the fusionis found that on delivers osteoblasts microRNA-214-3p and osteocytes, from binds the EVs RANK lumen on theto the surface cytosol of of osteoclasts the osteoblast. to stimulate a signalingRANK pathway, is part resultingof the central in the signaling activation pathway of nuclear in factorbone kapparemodeling B and [17,18]. nuclear RANKL, factor of activatedwhich is Tfound cells on 1 signaling. osteoblasts These and pathwaysosteocytes, are binds required RANK for on osteoclasts the surface to diofff erentiateosteoclasts from to multipotentstimulate a signaling pathway, resulting in the activation of nuclear factor kappa B and nuclear factor of activated T cells 1 signaling. These pathways are required for osteoclasts to differentiate from multipotent hematopoietic precursors to osteoclasts, and for the bone resorbing activity by mature Int. J. Mol. Sci. 2020, 21, 158 3 of 19 hematopoietic precursors to osteoclasts, and for the bone resorbing activity by mature osteoclasts. A third protein, osteoprotegerin, is a soluble protein secreted by osteoblasts that binds RANKL and serves as a competitive inhibitor of binding between RANKL and RANK. The detection of
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