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Biomedicine & Pharmacotherapy =... Jul 2015Calreticulin (CRT) protein has multifaceted role in carcinogenesis, however its role in breast cancer remains unidentified. In this study, we attempted to evaluate the...
OBJECTIVE
Calreticulin (CRT) protein has multifaceted role in carcinogenesis, however its role in breast cancer remains unidentified. In this study, we attempted to evaluate the effect of overexpressed CRT on breast cancer cells viability and proliferation.
METHODS
Levels of mRNA and protein expression for CRT and CD47 in cells were determined by Quantitative RT-PCR analysis and Western blot, respectively. Cells apoptosis was evaluated using Annexin V-FITC assay with flow cytometry. Cell viability was assessed using MTT assay. Cell migration and autophagy were also evaluated.
RESULTS
In breast cancer cells of MCF-7 and MDA-MB-231, both CRT and CD47 expression were enhanced, compared with that in normal breast cells of MCF-10A. Overexpression of CRT by MCF-7 and MDA-MB-231 cells transfected with significantly suppressed cell migration, viability as well as promote cell apoptosis while exerted no effected on cell autophagy. Interestingly, combining of thrombospondin (TSP) and overexpression of CRT significantly induced cell autophagy and inhibited tumor growth in MCF-7 cells xenograft. In result of chip assay, we observed that TSP treatment promoted interaction of TSP with CRT and CD47.
CONCLUSION
TSP promoted anti-tumor of adenovirus-mediated CRT via forming complexes with CRT and CD47 in breast cancer.
Topics: Adenoviridae; Animals; Breast Neoplasms; CD47 Antigen; Calreticulin; Cell Survival; Female; Humans; MCF-7 Cells; Mice; Mice, Inbred C57BL; Thrombospondins; Xenograft Model Antitumor Assays
PubMed: 26211590
DOI: 10.1016/j.biopha.2015.05.017 -
Bone Feb 2010The matricellular protein thrombospondin-2 (TSP2) has context-dependent effects on osteoblast lineage proliferation and differentiation. Mice lacking TSP2 display...
The matricellular protein thrombospondin-2 (TSP2) has context-dependent effects on osteoblast lineage proliferation and differentiation. Mice lacking TSP2 display increased endocortical bone thickness, which is associated with increased marrow stromal cell (MSC) number and in vitro proliferation. TSP2-null MSC also exhibit delayed osteoblastogenesis and enhanced adipogenesis compared to cells harvested from wild type mice. The goal of the present work was to more precisely characterize the contribution that TSP2 makes to the maturation of osteoblast-derived extracellular matrix (ECM) using a highly characterized pre-osteoblast cell line. Specifically, we asked whether TSP2 influences mineralization indirectly through its known effects on proliferation, or whether TSP2 directly promotes osteoblast differentiation. To pursue these questions, we used RNA-interference (RNAi) to inhibit TSP2 gene expression in MC3T3-E1 pre-osteoblasts. Introduction of siRNA oligonucleotides resulted in reduced TSP2 mRNA expression as early as 24 h post-transfection, and TSP2 mRNA levels remained low for 10 days. Similarly, TSP2 protein levels in both conditioned medium and the cell-matrix layer were reduced at 24 h post-transfection and remained reduced for 7 days. At day 21, mineralization was significantly reduced in cells transfected with TSP2 siRNA when compared to cells treated with scrambled siRNA. This decrease in mineralization occurred without a concomitant change in cell number. Twenty-four hours after transfection, runx2 gene expression was transiently enhanced in TSP2 siRNA-treated cultures. Between 6 and 14 days post-transfection, runx2, osterix, alkaline phosphatase, type I collagen, osteocalcin and bone sialoprotein all displayed moderate increases in gene expression with TSP2 RNAi. As well, soluble osteocalcin levels were markedly higher in the conditioned medium of cells treated with TSP2 siRNA than in control siRNA-treated cells. Increased soluble osteocalcin occurred without a concomitant change in the levels of osteocalcin in the cell-ECM layer. TSP2 reduction also elicited a transient change in the distribution of collagen between the acid soluble cell-ECM protein fraction and the insoluble matrix. Together, our data suggest that TSP2 may promote mineralization, by facilitating proper organization of the osteoblast-derived ECM.
Topics: 3T3 Cells; Animals; Biomarkers; Calcification, Physiologic; Cell Count; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; Extracellular Matrix; Gene Expression Regulation; Mice; Organ Specificity; Osteoblasts; RNA Interference; RNA, Messenger; Sp7 Transcription Factor; Thrombospondins; Time Factors; Transcription Factors
PubMed: 19744582
DOI: 10.1016/j.bone.2009.08.058 -
Journal of Vascular Surgery May 2010Diabetes is associated with a more aggressive form of atherosclerosis. Thrombospondin-1 (TSP-1), an extracellular matrix protein, is an acute-phase reactant that induces... (Comparative Study)
Comparative Study
OBJECTIVE
Diabetes is associated with a more aggressive form of atherosclerosis. Thrombospondin-1 (TSP-1), an extracellular matrix protein, is an acute-phase reactant that induces vascular smooth muscle (VSMC) migration and proliferation in areas of vascular injury and is also up-regulated in VSMCs exposed to hyperglycemia. This study tested the hypothesis that hyperglycemia amplifies the expression of genes induced by TSP-1 in VSMCs.
METHODS
Human aortic VSMCs were cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were used between passages three and five. VSMCs were preincubated in DMEM containing 0.2% FBS with 5 mM glucose (normoglycemia), 25 mM glucose (hyperglycemia), 25 mM mannose (osmotic control), TSP-1 (20 microg/mL), 25 mM glucose + TSP-1 (20 microg/mL), or 25 mM mannose + TSP-1 (20 microg/mL). Total RNA was extracted. Microarray analysis was performed and analyzed by analysis of variance. P < .05 was considered significant. Quantitative real-time polymerase chain reaction (rtPCR) was used to confirm selected up-regulated genes.
RESULTS
Microarray analysis revealed: (1) hyperglycemia altered 30 genes; (2) TSP-1 altered 212 genes, of which 8 were altered similarly to VSMCs exposed to 25 mM glucose; (3) TSP-1 up-regulated 10 genes associated with atherosclerosis and 4 others with diabetic vascular disease; (4) hyperglycemia combined with TSP-1 altered expression of 2822 genes. The three genes most up-regulated by TSP-1 in a normoglycemic environment were uridine 5'-diphosphoglucose (UDP-glucose) dehydrogenase (UGDH, 127%), transforming growth factor beta-2 (TGFbeta2, 116%), and hyaluronan synthase 2 (HAS2, 113%). Further, TSP-1 altered the expression of genes in 13 canonical pathways; however, when combined with hyperglycemia, 53 canonical pathways were affected.
CONCLUSION
Quantitative rtPCR confirmed that genes in several of these pathways for TSP-1 and hyperglycemia combined with TSP-1 were up-regulated. These findings suggest that TSP-1 may be germane to the progression of atherosclerosis and may have a large effect with concurrent hyperglycemia.
Topics: Analysis of Variance; Animals; Atherosclerosis; Cattle; Cell Movement; Cells, Cultured; Gene Expression Regulation; Glucose; Humans; Hyperglycemia; Muscle, Smooth, Vascular; Oligonucleotide Array Sequence Analysis; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thrombospondins; Up-Regulation
PubMed: 20299182
DOI: 10.1016/j.jvs.2009.11.073 -
Journal of Neurochemistry Feb 2017Read the highlighted article 'Defective synthesis and release of astrocytic thrombospondin-1 mediates the neuronal TDP-43 proteinopathy, resulting in defects in neuronal...
An in vitro model yields 'importin' new insights into chronic traumatic encephalopathy: damaged astrocytes stop 'thrombospondin' to the injury: An Editorial Highlight for 'Defective synthesis and release of astrocytic thrombospondin-1 mediates the neuronal TDP-43 proteinopathy, resulting in...
Read the highlighted article 'Defective synthesis and release of astrocytic thrombospondin-1 mediates the neuronal TDP-43 proteinopathy, resulting in defects in neuronal integrity associated with chronic traumatic encephalopathy: in vitro studies' on page 645.
Topics: Astrocytes; Chronic Traumatic Encephalopathy; Karyopherins; TDP-43 Proteinopathies; Thrombospondin 1; Thrombospondins
PubMed: 28074610
DOI: 10.1111/jnc.13906 -
Clinica Chimica Acta; International... Jul 2021Distribution of serum thrombospondin-2 in general population and cancer patients in China have not been reported.
PURPOSE
Distribution of serum thrombospondin-2 in general population and cancer patients in China have not been reported.
METHODS
This study evaluated the expression level of serum thrombospondin-2 in general population and various cancer patients, the 95% confidence interval was used for the derivation of reference range. The comparison of the expression levels in controls for age and gender was performed. The associations between candidate biomarkers (thrombospondin-2 [THBS2]) expression and tumor metastasis status were also explored.
RESULTS
125 healthy controls and 193 various cancer patients were enrolled. The mean ± SD in serum THBS2 levels in general population was 42.37 ± 12.24 ng/ml, there was no significant sex and age difference, the reference range is 18.37-66.36 ng/ml. Most cancer patients present a decreased serum THBS2 level except hepatoma and lymphoma which most patients showed a relatively high level of THBS2. There was no statistical difference of serum THBS2 level between metastasis and non-metastasis group in breast, lung, cervical, colorectal cancer, nasopharyngeal carcinoma and hepatoma (P > 0.05) while a significant negative correlation was observed in ovarian cancer (P = 0.0209).
CONCLUSIONS
The distribution of serum THBS2 displayed an obvious heterogeneity among various cancers comparing to health controls, ovarian cancer patients detected with low THBS2 expression may be more prone to develop metastasis in China.
Topics: Biomarkers, Tumor; Carcinoma, Hepatocellular; China; Female; Humans; Liver Neoplasms; Thrombospondins
PubMed: 33794141
DOI: 10.1016/j.cca.2021.03.022 -
FASEB Journal : Official Publication of... Jun 2012Thrombospondin-4 (TSP-4) expression increases dramatically in hypertrophic and failing hearts in rodent models and in humans. The aim of this study was to address the...
Thrombospondin-4 (TSP-4) expression increases dramatically in hypertrophic and failing hearts in rodent models and in humans. The aim of this study was to address the function of TSP-4 in the heart. TSP-4-knockout (Thbs4(-/-)) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) to increase left ventricle load. After 2 wk, Thbs4(-/-) mice had a significantly higher heart weight/body weight ratio than WT mice. The additional increase in the heart weight in TAC Thbs4(-/-) mice was due to increased deposition of extracellular matrix (ECM). The levels of interstitial collagens were higher in the knockout mice, but the size of cardiomyocytes and apoptosis in the myocardium was unaffected by TSP-4 deficiency, suggesting that increased reactive fibrosis was the primary cause of the higher heart weight. The increased ECM deposition in Thbs4(-/-) mice was accompanied by changes in functional parameters of the heart and decreased vessel density. The expression of inflammatory and fibrotic genes known to be influential in myocardial remodeling changed as a result of TSP-4 deficiency in vivo and as a result of incubation of cells with recombinant TSP-4 in vitro. Thus, TSP-4 is involved in regulating the adaptive responses of the heart to pressure overload, suggesting its important role in myocardial remodeling. Our study showed a direct influence of TSP-4 on heart function and to identify the mechanism of its effects on heart remodeling.
Topics: Animals; Aorta; Cardiomegaly; Collagen; Constriction, Pathologic; Extracellular Matrix; Heart Failure; Heart Ventricles; Mice; Mice, Knockout; Myocardium; Myocytes, Cardiac; Thrombospondins; Ventricular Remodeling
PubMed: 22362893
DOI: 10.1096/fj.11-190728 -
Pain Sep 2016Loss of high-voltage-activated (HVA) calcium current (ICa) and gain of low-voltage-activated (LVA) ICa after painful peripheral nerve injury cause elevated excitability...
Loss of high-voltage-activated (HVA) calcium current (ICa) and gain of low-voltage-activated (LVA) ICa after painful peripheral nerve injury cause elevated excitability in sensory neurons. Nerve injury is also accompanied by increased expression of the extracellular matrix glycoprotein thrombospondin-4 (TSP4), and interruption of TSP4 function can reverse or prevent behavioral hypersensitivity after injury. We therefore investigated TSP4 regulation of ICa in dorsal root ganglion (DRG) neurons. During depolarization adequate to activate HVA ICa, TSP4 decreases both N- and L-type ICa and the associated intracellular calcium transient. In contrast, TSP4 increases ICa and the intracellular calcium signal after low-voltage depolarization, which we confirmed is due to ICa through T-type channels. These effects are blocked by gabapentin, which ameliorates neuropathic pain by targeting the α2δ1 calcium subunit. Injury-induced changes of HVA and LVA ICa are attenuated in TSP4 knockout mice. In the neuropathic pain model of spinal nerve ligation, TSP4 application did not further regulate ICa of injured DRG neurons. Taken together, these findings suggest that elevated TSP4 after peripheral nerve injury may contribute to hypersensitivity of peripheral sensory systems by decreasing HVA and increasing LVA in DRG neurons by targeting the α2δ1 calcium subunit. Controlling TSP4 overexpression in peripheral sensory neurons may be a target for analgesic drug development for neuropathic pain.
Topics: Analysis of Variance; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cholera Toxin; Disease Models, Animal; Evoked Potentials; Ganglia, Spinal; Gene Expression Regulation; Mice; Mice, Knockout; Peripheral Nerve Injuries; Sensory Receptor Cells; Thrombospondins
PubMed: 27168360
DOI: 10.1097/j.pain.0000000000000612 -
Journal of Bone and Mineral Research :... Jun 2009Thrombospondin-2 (TSP2) is a matricellular protein with increased expression during growth and regeneration. TSP2-null mice show accelerated dermal wound healing and...
Thrombospondin-2 (TSP2) is a matricellular protein with increased expression during growth and regeneration. TSP2-null mice show accelerated dermal wound healing and enhanced bone formation. We hypothesized that bone regeneration would be enhanced in the absence of TSP2. Closed, semistabilized transverse fractures were created in the tibias of wildtype (WT) and TSP2-null mice. The fractures were examined 5, 10, and 20 days after fracture using microCT, histology, immunohistochemistry, quantitative RT-PCR, and torsional mechanical testing. Ten days after fracture, TSP2-null mice showed 30% more bone by microCT and 40% less cartilage by histology. Twenty days after fracture, TSP2-null mice showed reduced bone volume fraction and BMD. Mice were examined 5 days after fracture during the stage of neovascularization and mesenchymal cell influx to determine a cellular explanation for the phenotype. TSP2-null mice showed increased cell proliferation with no difference in apoptosis in the highly cellular fracture callus. Although mature bone and cartilage is minimal 5 days after fracture, TSP2-null mice had reduced expression of collagen IIa and Sox9 (chondrocyte differentiation markers) but increased expression of osteocalcin and osterix (osteoblast differentiation markers). Importantly, TSP2-null mice had a 2-fold increase in vessel density that corresponded with a reduction in vascular endothelial growth factor (VEGF) and Glut-1 (markers of hypoxia inducible factor [HIF]-regulated transcription). Finally, by expressing TSP2 using adenovirus starting 3 days after fracture, chondrogenesis was restored in TSP2-null mice. We hypothesize that TSP2 expressed by cells in the fracture mesenchyme regulates callus vascularization. The increase in vascularity increases tissue oxemia and decreases HIF; thus, undifferentiated cells in the callus develop into osteoblasts rather than chondrocytes. This leads to an alternative strategy for achieving fracture healing with reduced endochondral ossification and enhanced appositional bone formation. Controlling the ratio of cartilage to bone during fracture healing has important implications for expediting healing or promoting regeneration in nonunions.
Topics: Animals; Apoptosis; Base Sequence; Bone Density; Cartilage; DNA Primers; Fracture Healing; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Mice, Knockout; Polymerase Chain Reaction; Thrombospondins; Tomography, X-Ray Computed
PubMed: 19123916
DOI: 10.1359/jbmr.090101 -
Technology and Health Care : Official... 2022Alveolar echinococcosis is an epidemic disease caused by the parasitism of Echinococcus multilocularis (Em) larvae in the intermediate or final host.
BACKGROUND
Alveolar echinococcosis is an epidemic disease caused by the parasitism of Echinococcus multilocularis (Em) larvae in the intermediate or final host.
OBJECTIVE
To identify and analyze B-cell and T-cell (Th1, Th2, and Th17) epitopes of the Em antigen protein thrombospondin 3 (TSP3).
METHODS
The amino acid sequence of TSP3 was obtained, and the secondary structural characteristics of TSP3 were predicted using bioinformatics software to further predict its potential T-cell and B-cell epitopes. The spleen lymphocytes of BALB/c mice, which were immunized with the TSP3 protein, were collected for co-culture with B-cell and T-cell antigen small peptides. The B-cell epitopes and T-cell epitope subtypes Th1, Th2, and Th17 were identified as having good immunogenicity.
RESULTS
After identification, it was found that the predominant epitopes of B cells existing in TSP3 were T18-33, T45-55, and T110-122. Furthermore, the predominant epitopes of T cells existing in TSP3 were T33-42, T45-55, T80-90, and T110-122 in the T1 subtype, T45-55, T68-77, and T92-104 in the Th2 subtype, and T53-63 and T80-90 in the Th17 subtype.
CONCLUSIONS
Six T-cell and eight B-cell dominant epitopes of the TSP3 antigen were revealed; these results may be applied in the development of a dominant epitope vaccine.
Topics: Animals; Echinococcosis; Echinococcus multilocularis; Epitopes, B-Lymphocyte; Mice; Thrombospondins
PubMed: 35068426
DOI: 10.3233/THC-212983 -
International Journal of Molecular... Jan 2019Osteoarthritis (OA) is a progressive joint disease characterized by a continuous degradation of the cartilage extracellular matrix (ECM). The expression of the...
Osteoarthritis (OA) is a progressive joint disease characterized by a continuous degradation of the cartilage extracellular matrix (ECM). The expression of the extracellular glycoprotein thrombospondin-4 (TSP-4) is known to be increased in injured tissues and involved in matrix remodeling, but its role in articular cartilage and, in particular, in OA remains elusive. In the present study, we analyzed the expression and localization of TSP-4 in healthy and OA knee cartilage by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot. We found that TSP-4 protein expression is increased in OA and that expression levels correlate with OA severity. TSP-4 was not regulated at the transcriptional level but we detected changes in the anchorage of TSP-4 in the altered ECM using sequential protein extraction. We were also able to detect pentameric and fragmented TSP-4 in the serum of both healthy controls and OA patients. Here, the total protein amount was not significantly different but we identified specific degradation products that were more abundant in sera of OA patients. Future studies will reveal if these fragments have the potential to serve as OA-specific biomarkers.
Topics: Aged; Aged, 80 and over; Biomarkers; Cartilage, Articular; Extracellular Matrix; Female; Gene Expression; Humans; Male; Middle Aged; Osteoarthritis, Knee; Protein Transport; Severity of Illness Index; Thrombospondins
PubMed: 30669608
DOI: 10.3390/ijms20020447