Abstract 395: The Role of Transglutaminase 2 (tg2) in Regulation of Macrophage Phenotype

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Arti V Shinde ◽  
Ya Su ◽  
Nikolaos G Frangogiannis
Keyword(s):  
Focal Adhesion ◽  
Adhesion Formation ◽  
Myeloid Cell ◽  
Transglutaminase 2 ◽  
M2 Macrophages ◽  
Macrophage Phenotype ◽  
Caveolin 1 ◽  
Infarcted Heart ◽  
Focal Adhesion Formation

Transglutaminase 2 (TG2) expression is increased in infarcted and remodeling hearts and modulates cellular responses through enzymatic effects and non-enzymatic actions. In monocytic cells, TG2 is predominantly expressed by M2 macrophages in both mice and humans. Although TG2 is considered a preserved and reliable marker of M2 macrophages, its role in regulation of macrophage phenotype remains unknown. In order to study the role of TG2 in macrophage function in homeostasis and disease, we generated mice with myeloid cell-specific loss of TG2 (MyTG2KO), and examined their response to experimental myocardial infarction (MI). Using myeloid cell reporter CSF1R EGFP mice, we found that TG2 is highly expressed in the majority of spleen, liver and lung macrophages, and is markedly upregulated in M2-like Arg1+ macrophages infiltrating the infarcted heart in contrast to infarct neutrophils. MyTG2KO mice had no baseline defects. Following MI, MyTG2KO mice and TG2 fl/fl controls had comparable chamber dilation, systolic dysfunction and infarct size, but exhibited attenuated diastolic dysfunction and reduced atrial size, reflecting lower filling pressures. Macrophages harvested from infarcted MyTG2KO mice had comparable pro-inflammatory and anti-inflammatory cytokine synthesis, but markedly increased expression of caveolin-1, the integrins a1, b1 and b3, and of the anti-fibrotic proteoglycan decorin. In vitro, we examined the effects of TG2 loss on the transcriptomic profile of bone marrow macrophages using RNAseq. Reactome and KEGG analysis showed that TG2 loss did not affect the baseline macrophage transcriptome differences. In contrast, following TGF-b stimulation, TG2 KO and WT cells had differential expression of genes regulating focal adhesion formation and the actin cytoskeleton. Consistent with the in vivo findings, TGF-b stimulation resulted in higher caveolin-1 and decorin in TG2 KO macrophages. In summary, TG2 expression is induced in M2-like macrophages infiltrating the infarcted heart and mediates diastolic dysfunction. The effects of TG2 in macrophages are not due to actions on their inflammatory profile, but may involve modulation of focal adhesion formation and cell migration, and subsequent expression of anti-fibrotic genes.

Abstract 110: Thrombospondin Type-1 Domain-Containing Protein 1 (THSD1), an Intracranial Aneurysm Susceptibility Gene, Mediates Cell Adhesion in Human Umbilical Vein Endothelial Cells

Stroke ◽  
2015 ◽  
Vol 46 (suppl_1) ◽  
Author(s):  
Xiaoqian Fang ◽  
Dong H Kim ◽  
Teresa Santiago-Sim
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Focal Adhesion ◽  
Umbilical Vein ◽  
Adhesion Formation ◽  
Wild Type ◽  
Focal Adhesion Formation ◽  
Umbilical Vein Endothelial Cells

Introduction: An intracranial aneurysm (IA) is a weak spot in cerebral blood vessel wall that can lead to its abnormal bulging. Previously, we reported that mutations in THSD1 , encoding thrombospondin type-1 domain-containing protein 1, are associated with IA in a subset of patients. THSD1 is a transmembrane molecule with a thrombospondin type-1 repeat (TSR). Proteins with TSR domain have been implicated in a variety of processes including regulation of matrix organization, cell adhesion and migration. We have shown that in mouse brain Thsd1 is expressed in endothelial cells. Hypothesis: THSD1 plays an important role in maintaining the integrity of the endothelium by promoting adhesion of endothelial cells to the underlying basement membrane. Methods: Human umbilical vein endothelial cells are used to investigate the role of THSD1 in vitro . THSD1 expression was knocked-down by RNA interference. Cell adhesion assay was done on collagen I-coated plates and focal adhesion formation was visualized using immunofluorescence by paxillin and phosphorylated focal adhesion kinase (pFAK) staining. THSD1 re-expression is accomplished by transfection with a pCR3.1-THSD1-encoding plasmid. Results: Knockdown of THSD1 caused striking change in cell morphology and size. Compared to control siRNA-treated cells that exhibited typical cobblestone morphology, THSD1 knockdown cells were narrow and elongated, and were significantly smaller ( p <0.01). Cell adherence to collagen I-coated plates was also attenuated in THSD1 knockdown cells ( p <0.01). Consistent with this finding is the observation that the number and size of focal adhesions, based on paxillin and pFAK staining, were significantly reduced after THSD1 knockdown ( p <0.01). These defects in cell adhesion and focal adhesion formation were rescued by re-expression of wild type THSD1 ( p <0.05). In contrast, initial studies indicate that expression of mutated versions of THSD1 as seen in human patients (L5F, R450*, E466G, P639L) could not restore cell adhesion and focal adhesion formation to wild type levels. Conclusions: Our studies provide evidence for a role of THSD1 and THSD1 mutations in endothelial cell adhesion and suggest a possible mechanism underlying THSD1 -mediated aneurysm disease.

scholarly journals Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts

2017 ◽  
Author(s):  
Kazuo Katoh
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Rho Kinase ◽  
Focal Adhesions ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Focal Adhesion Formation ◽  
Specific Regulation

Specific regulation and activation of focal adhesion kinase (FAK) are thought to be important for focal adhesion formation, and activation of Rho-kinase has been suggested to play a role in determining the effects of FAK on the formation of stress fibers and focal adhesions. To clarify the role of FAK in stress fiber formation and focal adhesion organization, we examined the formation of new stress fibers and focal adhesions by activation of Rho-kinase in FAK knockout (FAK–/–) fibroblasts. FAK–/– cells were elliptical in shape, and showed reduced numbers of stress fibers and focal adhesions in the central part of the cells along with large focal adhesions in the peripheral regions. Activation of Rho-kinase in FAK–/– cells transiently increased the actin filaments in the cell center, but these did not form typical thick stress fibers. Moreover, only plaque-like structures as the origins of newly formed focal adhesions were observed in the center of the cell. Furthermore, introduction of an exogenous GFP-labeled FAK gene into FAK–/– cells resulted in increased numbers of stress fibers and focal adhesions in the center of the cells, which showed typical fibroblast morphology. These results indicated that FAK plays an important role in the formation of stress fibers and focal adhesions as well as in regulation of cell shape and morphology with the activation of Rho-kinase.

scholarly journals Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts

2017 ◽  
Author(s):  
Kazuo Katoh
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Rho Kinase ◽  
Focal Adhesions ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Focal Adhesion Formation ◽  
Specific Regulation

Specific regulation and activation of focal adhesion kinase (FAK) are thought to be important for focal adhesion formation, and activation of Rho-kinase has been suggested to play a role in determining the effects of FAK on the formation of stress fibers and focal adhesions. To clarify the role of FAK in stress fiber formation and focal adhesion organization, we examined the formation of new stress fibers and focal adhesions by activation of Rho-kinase in FAK knockout (FAK–/–) fibroblasts. FAK–/– cells were elliptical in shape, and showed reduced numbers of stress fibers and focal adhesions in the central part of the cells along with large focal adhesions in the peripheral regions. Activation of Rho-kinase in FAK–/– cells transiently increased the actin filaments in the cell center, but these did not form typical thick stress fibers. Moreover, only plaque-like structures as the origins of newly formed focal adhesions were observed in the center of the cell. Furthermore, introduction of an exogenous GFP-labeled FAK gene into FAK–/– cells resulted in increased numbers of stress fibers and focal adhesions in the center of the cells, which showed typical fibroblast morphology. These results indicated that FAK plays an important role in the formation of stress fibers and focal adhesions as well as in regulation of cell shape and morphology with the activation of Rho-kinase.

Role of focal adhesion formation in migration and morphogenesis of endothelial cells

2004 ◽  
Vol 16 (11) ◽  
pp. 1273-1281 ◽  
Author(s):  
Shigeru Kanda ◽  
Yasuyoshi Miyata ◽  
Hiroshi Kanetake
Keyword(s):  
Endothelial Cells ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesion Formation

Abstract MP152: Smad1 Activation in Infarct Macrophages Restrains Angiogenesis and Accentuates Adverse Remodeling of the Infarcted Myocardium

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Bijun Chen ◽  
Ya Su ◽  
Nikolaos G Frangogiannis
Keyword(s):  
Systolic Dysfunction ◽  
Myeloid Cell ◽  
Left Ventricular ◽  
Macrophage Phenotype ◽  
Infarcted Myocardium ◽  
Infarcted Heart ◽  
Adverse Remodeling

Macrophages play multiple roles in repair and remodeling of the infarcted heart, contributing to phagocytosis of dead cells, regulation of inflammation, fibrosis and angiogenesis. TGF-β superfamily members are critically involved in regulation of macrophage phenotype through the Smad cascades. In most cell types, TGF-βs signal predominantly by activating Smad2/3 signaling, whereas BMPs act through Smad1/5-mediated pathways. We previously showed that TGF-β/Smad3 signaling in macrophages protects the infarcted heart from adverse remodeling by mediating phagocytotic activity and anti-inflammatory transition. However, the in vivo role of Smad1/5 signaling in macrophages remains unknown. We examined the role of macrophage-specific Smad1 signaling in a mouse model of myocardial infarction (MI). Smad1 is activated in a subset of myeloid cells infiltrating the infarcted myocardium. In vitro, TGF-β1, TGF-β3, BMP4, BMP7, but not TGF-β2, BMP2, BMP6 markedly activated Smad1/5 signaling in macrophages. In order to examine the role of Smad1 in regulation of macrophage phenotype we generated myeloid cell-specific Smad1 KO mice (MyS1KO). MyS1KO mice had better survival compared to S1fl/fl controls 28 days post MI ( p =0.0009, n=26-27). Echocardiographic data showed that MyS1KO mice had reduced left ventricular dilation and attenuated systolic dysfunction 28 days after MI. Although scar size was comparable between groups at the 7 day timepoint, MyS1KO animals had significantly smaller scars 28 days after MI, suggesting improved scar remodeling. MyS1KO mice exhibited increased microvessel density in the infarct, suggesting that Smad1 activation in macrophages may restrain angiogenesis. In order to explore the mechanisms, we isolated infarct macrophages from MyS1KO and S1fl/fl hearts and compared angiogenesis-related protein expression by performing a proteomic array. Smad1 loss in macrophages did not affect VEGF-A expression, but significantly increased levels of the angiogenic chemokine CXCL12. Our findings suggest that Smad1 is directly activated by TGF-βs in macrophages, and that Smad1 activation in infarct macrophages may promote adverse remodeling by restraining angiogenic effects through suppression of angiogenic chemokines.

Elmo1 Helps Dock180 to Regulate Rac1 Activity and Cell Migration of Ovarian Cancer

2014 ◽  
Vol 24 (5) ◽  
pp. 844-850 ◽  
Author(s):  
Jin Wang ◽  
Jie-min Dai ◽  
Ya-ling Che ◽  
Yi-meng Gao ◽  
Hui-juan Peng ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Motility ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Negative Control ◽  
Small G Protein ◽  
Rac1 Activity ◽  
Focal Adhesion Formation ◽  
Synergetic Action

ObjectiveEngulfment and cell motility 1 (Elmo1) has been reported to cooperate with dedicator of cytokinesis 1 (Dock180) and to be linked to the invasive phenotype of cancer cells through activating small G-protein Rac. We aimed to study the role of Elmo1 in the malignant migration of ovarian cancer.MethodsEngulfment and cell motility 1 expression was evaluated in specimens from 93 patients with serous ovarian cancer (SOC) by immunohistochemical staining. Next, Elmo1-RNAi cells were established by validated small interference RNAs. Cell proliferation and cell motility were observed and compared with Dock180-RNAi cells. To confirm their synergetic contribution to forming focal adhesion and activating Rac1, Rac1-GTP level was measured by GST pull-down assay and immunofluorescence was used to observe focal adhesion formation both in Elmo1-RNAi and Dock180-RNAi cells.ResultsEngulfment and cell motility 1 was mainly overexpressed in high-grade SOC tissues. Western blot analysis demonstrated that both Elmo1 and Dock180 expressions were hampered in Elmo1-RNAi cells. Compared with the negative control, decreased colony formation and cell invasion were observed in Elmo1-RNAi cells and Dock180-RNAi cells. Consistently, both exhibited reduced Rac1-GTP level and inhibited focal adhesion formation.ConclusionsEngulfment and cell motility 1 presents with synergetic action in helping Dock180 to activate Rac1 and promote cell motility, and thus promote untoward expansion and aggressiveness of SOC.

Control of morphology, cytoskeleton and migration by syndecan-4

1999 ◽  
Vol 112 (20) ◽  
pp. 3421-3431 ◽  
Author(s):  
R.L. Longley ◽  
A. Woods ◽  
A. Fleetwood ◽  
G.J. Cowling ◽  
J.T. Gallagher ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Core Protein ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Fiber Formation ◽  
Microfilament Bundle ◽  
Focal Adhesion Formation ◽  
And Migration

Syndecan-4 is a widely expressed transmembrane heparan sulfate proteoglycan which localizes to focal adhesions. Previous studies showed that the syndecan-4 cytoplasmic domain can associate with and potentiate the activity of protein kinase C, which is required for focal adhesion formation. To examine further the role of syndecan-4 in cell adhesion, we expressed syndecan-4 cDNA constructs in CHO-K1 cells. Syndecan-2 transfection was used to confirm effects seen were specific for syndecan-4. Cells overexpressing full length syndecan-4 core protein exhibited a more flattened, fibroblastic morphology, with increased focal adhesion formation and decreased cell motility. Expression of a syndecan-4 core protein with either a partial or complete deletion of the cytoplasmic domain or of an antisense construct led to markedly decreased spreading and focal adhesion formation, a more epithelioid morphology, and decreased motility. Overexpression of syndecan-2 changed the adhesive phenotype, but did not markedly alter focal adhesion and microfilament bundle formation. The data suggest that syndecan-4 is a regulator of focal adhesion and stress fiber formation, and influences both morphology and migration.

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