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Methods in Cell Biology 2018Thrombospondins are a family of five secreted proteins that have diverse roles in modulating cellular function. Thrombospondins-1 and 2 were identified as matricellular...
Thrombospondins are a family of five secreted proteins that have diverse roles in modulating cellular function. Thrombospondins-1 and 2 were identified as matricellular proteins based on their functional roles combined with their transient appearance or accumulation in extracellular matrix at specific times during development and in response to injury or stress in mature tissues. Thrombospondin-1 is a major component of platelet α-granules, which provides a convenient source for purification of the protein. Methods are described to prepare thrombospondin-1 from human platelets in a biologically active form with minimal degradation or contamination with other platelet proteins. A nondenaturing method is described for removing bound transforming growth factor-β1.
Topics: Blood Platelets; Chromatography, Affinity; Extracellular Matrix; Fibronectins; Heparin; Humans; Thrombospondin 1; Transforming Growth Factor beta1
PubMed: 29310787
DOI: 10.1016/bs.mcb.2017.08.021 -
FASEB Journal : Official Publication of... Jun 2010Thrombospondin 1 (TSP1), an oligomeric matrix protein, is known for its antiangiogenic activity. Recently, TSP1 has been shown to regulate synaptogenesis in the...
Thrombospondin 1 (TSP1), an oligomeric matrix protein, is known for its antiangiogenic activity. Recently, TSP1 has been shown to regulate synaptogenesis in the developing brain. In this study, we examine another role of TSP1 in the CNS, namely, in proliferation and differentiation of neural progenitor cells (NPCs). We found that adult mice deficient in TSP1 exhibit reduced proliferation of NPCs in vivo [13,330+/-826 vs. 4914+/-455 (mean+/-se wt vs. TSP1(-/-)); P<0.001, Student's t test] and impaired neuronal differentiation (1382+/-83 vs. 879+/-79; P<0.001). In vitro, NPC obtained from adult TSP1(-/-) mice display decreased proliferation in BrdU assay (48+/-8 vs. 24+/-3.5%; P<0.01) and decreased neuronal fate commitment (8+/-0.85 vs. 4.6+/-0.5%; P<0.05) in contrast to wild-type NPCs. Both proliferation and neuronal differentiation deficits are remediable in vitro by exogenous TSP1. Notably, conditioned medium from TSP1(-/-) astrocytes, unlike that from control astrocytes, fails to promote neurogenesis in wild-type NPCs, suggesting that TSP1 is one of the key molecules responsible for astrocyte-induced neurogenesis. Our data demonstrate that TSP1 is a critical participant in maintenance of the adult NPC pool and in neuronal differentiation.
Topics: Animals; Astrocytes; Blotting, Western; Bromodeoxyuridine; Cell Differentiation; Cell Proliferation; Cells, Cultured; Culture Media, Conditioned; Flow Cytometry; Fluorescent Antibody Technique; Immunoenzyme Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurogenesis; Neurons; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Thrombospondin 1; Thrombospondins
PubMed: 20124433
DOI: 10.1096/fj.09-150573 -
Journal of Neurochemistry Aug 2021Lysophosphatidic acid (LPA), a brain membrane-derived lipid mediator, plays important roles including neural development, function, and behavior. In the present study,...
Lysophosphatidic acid (LPA), a brain membrane-derived lipid mediator, plays important roles including neural development, function, and behavior. In the present study, the effects of LPA on astrocyte-derived synaptogenesis factor thrombospondins (TSPs) production were examined by real-time PCR and western blotting, and the mechanism underlying this event was examined by pharmacological approaches in primary cultured rat cortical astrocytes. Treatment of astrocytes with LPA increased TSP-1 mRNA, and TSP-2 mRNA, but not TSP-4 mRNA expression. TSP-1 protein expression and release were also increased by LPA. LPA-induced TSP-1 production were inhibited by AM966 a LPA receptor antagonist, and Ki16425, LPA receptors antagonist, but not by H2L5146303, LPA receptor antagonist. Pertussis toxin, Gi/o inhibitor, but not YM-254890, Gq inhibitor, and NF499, Gs inhibitor, inhibited LPA-induced TSP-1 production, indicating that LPA increases TSP-1 production through Gi/o-coupled LPA and LPA receptors. LPA treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). LPA-induced TSP-1 mRNA expression was inhibited by U0126, MAPK/ERK kinase (MEK) inhibitor, but not SB202190, p38 MAPK inhibitor, or SP600125, JNK inhibitor. However, LPA-induced TSP-1 protein expression was diminished with inhibition of all three MAPKs, indicating that these signaling molecules are involved in TSP-1 protein production. Treatment with antidepressants, which bind to astrocytic LPA receptors, increased TSP-1 mRNA and protein production. The current findings show that LPA/LPA receptors signaling increases TSP-1 production in astrocytes, which could be important in the pathogenesis of affective disorders and could potentially be a target for the treatment of affective disorders.
Topics: Animals; Astrocytes; Cerebral Cortex; Female; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; MAP Kinase Signaling System; Mood Disorders; Pregnancy; Primary Cell Culture; Protein Kinase Inhibitors; Rats; Rats, Wistar; Receptors, Lysophosphatidic Acid; Thrombospondin 1; Thrombospondins
PubMed: 33118159
DOI: 10.1111/jnc.15227 -
American Journal of Physiology. Heart... Jun 2016
Topics: Aortic Aneurysm; Arteries; Cardiomegaly; Humans; Thrombospondins; Vasodilation
PubMed: 27106043
DOI: 10.1152/ajpheart.00273.2016 -
Circulation Research Nov 2010Thrombospondin (TSP)-4 is an extracellular protein that has been linked to several cardiovascular pathologies. However, a role for TSP-4 in vascular wall biology remains... (Comparative Study)
Comparative Study
RATIONALE
Thrombospondin (TSP)-4 is an extracellular protein that has been linked to several cardiovascular pathologies. However, a role for TSP-4 in vascular wall biology remains unknown.
OBJECTIVE
We have examined the effects of TSP-4 gene (Thbs4) knockout on the development of atherosclerotic lesions in ApoE(-/-) mice.
METHODS AND RESULTS
Deficiency in TSP-4 reduced atherosclerotic lesions: at 20 weeks of age, the size of the aortic root lesions in Thbs4(-/-)/ApoE(-/-) mice was decreased by 48% in females and by 39% in males on chow diets; in mice on Western diets, lesions in the descending aorta were reduced by 30% in females and 33% in males. In ApoE(-/-) mice, TSP-4 was abundant in vessel areas prone to lesion development and in the matrix of the lesions themselves. TSP-4 deficiency reduced the number of macrophages in lesions in all groups by ≥ 2-fold. In addition, TSP-4 deficiency reduced endothelial cell activation (expression of surface adhesion molecules) and other markers of inflammation in the vascular wall (decreased production of monocyte chemoattractant protein-1 and activation of p38). In vitro, both the adhesion and migration of wild-type macrophages increased in the presence of purified recombinant TSP-4 in a dose-dependent manner (up to 7- and 4.7-fold, respectively). These responses led to p38-MAPkinase activation and were dependent on β(2) and β(3) integrins, which recognize TSP-4 as a ligand.
CONCLUSIONS
TSP-4 is abundant in atherosclerotic lesions and in areas prone to development of lesions and may influence the recruitment of macrophages by activating endothelial cells and directly interacting with macrophages to increase their adhesion and migration. Our observations suggest an important role for this matricellular protein in the local regulation of inflammation associated with atherogenesis.
Topics: Animals; Atherosclerosis; Cells, Cultured; Endothelium, Vascular; Female; Inflammation Mediators; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Thrombospondins; Vascular Diseases
PubMed: 20884877
DOI: 10.1161/CIRCRESAHA.110.232371 -
Scientific Reports Apr 2016The α2δ proteins are auxiliary subunits of voltage-gated calcium channels, and influence their trafficking and biophysical properties. The α2δ ligand gabapentin...
Thrombospondin-4 reduces binding affinity of [(3)H]-gabapentin to calcium-channel α2δ-1-subunit but does not interact with α2δ-1 on the cell-surface when co-expressed.
The α2δ proteins are auxiliary subunits of voltage-gated calcium channels, and influence their trafficking and biophysical properties. The α2δ ligand gabapentin interacts with α2δ-1, and inhibits calcium channel trafficking. However, α2-1 has also been proposed to play a synaptogenic role, independent of calcium channel function. In this regard, α2δ-1 was identified as a ligand of thrombospondins, with the interaction involving the thrombospondin synaptogenic domain and the α2δ-1 von-Willebrand-factor domain. Co-immunoprecipitation between α2δ-1 and the synaptogenic domain of thrombospondin-2 was prevented by gabapentin. We therefore examined whether interaction of thrombospondin with α2δ-1 might reciprocally influence (3)H-gabapentin binding. We concentrated on thrombospondin-4, because, like α2δ-1, it is upregulated in neuropathic pain models. We found that in membranes from cells co-transfected with α2δ-1 and thrombospondin-4, there was a Mg(2+) -dependent reduction in affinity of (3)H-gabapentin binding to α2δ-1. This effect was lost for α2δ-1 with mutations in the von-Willebrand-factor-A domain. However, the effect on (3)H-gabapentin binding was not reproduced by the synaptogenic EGF-domain of thrombospondin-4. Partial co-immunoprecipitation could be demonstrated between thrombospondin-4 and α2δ-1 when co-transfected, but there was no co-immunoprecipitation with thrombospondin-4-EGF domain. Furthermore, we could not detect any association between these two proteins on the cell-surface, indicating the demonstrated interaction occurs intracellularly.
Topics: Amines; Analgesics; Calcium Channels; Cyclohexanecarboxylic Acids; Gabapentin; Immunoprecipitation; Protein Binding; Thrombospondins; gamma-Aminobutyric Acid
PubMed: 27076051
DOI: 10.1038/srep24531 -
IUBMB Life Oct 2022Thrombospondins are a family of matricellular proteins with a multimeric structure that is known to be involved in several biological and pathological processes. Their... (Review)
Review
Thrombospondins are a family of matricellular proteins with a multimeric structure that is known to be involved in several biological and pathological processes. Their relationship with vascular disorders has raised special interest recently. Aortic aneurysms are related to the impairment of vascular remodeling, in which extracellular matrix proteins seem to play an important role. Thus, research in thrombospondins, and their potential role in aneurysm development is progressively gaining importance. Nevertheless, studies showing thrombospondin dysregulation in human samples are still scarce. Although studies performed in vitro and in vivo models are essential to understand the molecular mechanisms and pathways underlying the disorder, descriptive studies in human samples are also necessary to ascertain their real value as biomarkers and/or novel therapeutic targets. The present article reviews the latest findings regarding the role of thrombospondins in aortic aneurysm development, paying particular attention to the studies performed in human samples.
Topics: Aortic Aneurysm; Biomarkers; Extracellular Matrix Proteins; Humans; Thrombospondins
PubMed: 35293116
DOI: 10.1002/iub.2610 -
Peptides Apr 2010C21, the C-terminal residue of thrombospondin-4 (TSP-4), was identified as a peptide growth factor during an investigation concerning erythropoietin-dependent, erythroid... (Review)
Review
C21, the C-terminal residue of thrombospondin-4 (TSP-4), was identified as a peptide growth factor during an investigation concerning erythropoietin-dependent, erythroid stimulating factors of endothelial origin. It is active in cultures of several human hematopoietic stem cells, skin fibroblasts and kidney epithelial cells and stimulates red cell formation in anemic mice. A method of affinity chromatography in the presence of high concentrations of Triton X-100, previously developed for identifying proteins associated with the TSP-1 receptor CD47, was utilized for the detection of C21 binding molecules and their detergent-resistant, associated partners. These experiments helped to delineate two different mechanisms of C21 action, which are compatible with its cell proliferating activity. As a cell matrix peptide, C21 binds to the osteopontin receptor CD44 and could act as an osteopontin antagonist, preventing the inhibition of primitive hematopoietic stem cell proliferation. TSP-1, another matrix protein, binds to C21 and could indirectly act as an antagonist, by shunting C21-CD44 interactions. The second mechanism is a direct effect of C21 on cell proliferation. The extremely rapid internalization and nuclear localization of the peptide could be explained by CD44-mediated internalization, followed by a microtubule-mediated transport towards the nucleus, or, eventually, direct membrane insertion. These alternative hypotheses are supported by previously observed membrane insertion of similar synthetic and viral acidic amphipathic peptides, the presence of microtubule-associated protein 1B (MAP1B) and dynactin in the triton-soluble complexes associated with C21 and the presence in such complexes of dual compartment proteins for nuclei and plasma membranes, such as MAP1B, AHNAK and CD44.
Topics: Amino Acid Sequence; Animals; Cell Adhesion Molecules; Cell Membrane; Cell Nucleus; Chromatography, Affinity; Humans; Hyaluronan Receptors; Models, Molecular; Molecular Sequence Data; Multiprotein Complexes; Peptides; Protein Structure, Tertiary; Signal Transduction; Surface-Active Agents; Thrombospondins
PubMed: 20006665
DOI: 10.1016/j.peptides.2009.12.011 -
Cold Spring Harbor Perspectives in... Oct 2011Thrombospondins are evolutionarily conserved, calcium-binding glycoproteins that undergo transient or longer-term interactions with other extracellular matrix... (Review)
Review
Thrombospondins are evolutionarily conserved, calcium-binding glycoproteins that undergo transient or longer-term interactions with other extracellular matrix components. They share properties with other matrix molecules, cytokines, adaptor proteins, and chaperones, modulate the organization of collagen fibrils, and bind and localize an array of growth factors or proteases. At cell surfaces, interactions with an array of receptors activate cell-dependent signaling and phenotypic outcomes. Through these dynamic, pleiotropic, and context-dependent pathways, mammalian thrombospondins contribute to wound healing and angiogenesis, vessel wall biology, connective tissue organization, and synaptogenesis. We overview the domain organization and structure of thrombospondins, key features of their evolution, and their cell biology. We discuss their roles in vivo, associations with human disease, and ongoing translational applications. In many respects, we are only beginning to appreciate the important roles of these proteins in physiology and pathology.
Topics: Amino Acid Sequence; Animals; Cell Movement; Cell Proliferation; Drosophila; Evolution, Molecular; Genetic Predisposition to Disease; Humans; Mice; Mice, Knockout; Molecular Sequence Data; Protein Structure, Tertiary; Sequence Alignment; Signal Transduction; Thrombospondins
PubMed: 21875984
DOI: 10.1101/cshperspect.a009712 -
Archives of Biochemistry and Biophysics Jul 2023Intercellular communication is pivotal in various stages of cancer progression. For smart and effective communication, cancer cells employ diverse modes of messaging...
Intercellular communication is pivotal in various stages of cancer progression. For smart and effective communication, cancer cells employ diverse modes of messaging that may be further fine-tuned by the microenvironmental changes. Extracellular matrix (ECM) stiffening due to excess deposition and crosslinking of collagen is one of the crucial tumor-microenvironmental changes that influence a plethora of cellular processes, including cell-cell communication. We herein studied the crosstalk between exosomes and tunneling nanotubes (TNT), the two distinct means of cell-cell communication under varying ECM-stiffness conditions. We show that exosomes promote the formation of tunneling nanotubes in breast cancer cells, which results in cellular internet. Interestingly, exosomes drastically increased the fraction of cells connected by TNT; however, they elicited no effect on the number of TNTs per pair of connected cells or the length of TNT. The observed pro-TNT effects of exosomes were found to be ECM-stiffness dependent. ECM-stiffness tuned exosomes were found to promote TNT formation predominantly via the 'cell dislodgment model'. At the molecular level, exosomal thrombospondin-1 was identified as a critical pro-TNT factor. These findings underline the influence of ECM stiffening on two diverse modes of cell communication and their interdependence, which may have significant implications in cancer biomedical research.
Topics: Humans; Female; Breast Neoplasms; Nanotubes; Cell Communication; Extracellular Matrix; Thrombospondins
PubMed: 37146866
DOI: 10.1016/j.abb.2023.109624