scholarly journals Critical role of PIP5KIγ87 in InsP3-mediated Ca2+ signaling

2004 ◽  
Vol 167 (6) ◽  
pp. 1005-1010 ◽  
Author(s):  
Ying Jie Wang ◽  
Wen Hong Li ◽  
Jing Wang ◽  
Ke Xu ◽  
Ping Dong ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Critical Role ◽  
Adhesion Formation ◽  
Type I ◽  
Focal Adhesion Formation ◽  
Inositol 1,4,5 Trisphosphate ◽  
Phosphatidylinositol 4 ◽  
G Protein Coupled ◽  
Short Splice Variant

Phosphatidylinositol 4,5-bisphosphate (PIP2) is the obligatory precursor of inositol 1,4,5-trisphosphate (InsP3 or IP3) and is therefore critical to intracellular Ca2+ signaling. Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase γ (PIP5KIγ87) as the major contributor of the PIP2 pool that supports G protein–coupled receptor (GPCR)-mediated IP3 generation. PIP5KIγ87 RNAi decreases the histamine-induced IP3 response and Ca2+ flux by 70%. Strikingly, RNAi of other PIP5KI isoforms has minimal effect, even though some of these isoforms account for a larger percent of total PIP2 mass and have previously been implicated in receptor mediated endocytosis or focal adhesion formation. Therefore, PIP5KIγ87's PIP2 pool that supports GPCR-mediated Ca2+ signaling is functionally compartmentalized from those generated by the other PIP5KIs.

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.

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.

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.

Syndecan-4 and integrins: combinatorial signaling in cell adhesion

1999 ◽  
Vol 112 (20) ◽  
pp. 3415-3420 ◽  
Author(s):  
J.R. Couchman ◽  
A. Woods
Keyword(s):  
Extracellular Matrix ◽  
Heparan Sulfate ◽  
Focal Adhesion ◽  
Core Protein ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Dominant Negative ◽  
Focal Adhesion Formation ◽  
Dominant Negative Inhibitor ◽  
Phosphatidylinositol 4

It is now becoming clear that additional transmembrane components can modify integrin-mediated adhesion. Syndecan-4 is a transmembrane heparan sulfate proteoglycan whose external glycosaminoglycan chains can bind extracellular matrix ligands and whose core protein cytoplasmic domain can signal during adhesion. Two papers in this issue of JCS demonstrate, through transfection studies, that syndecan-4 plays roles in the formation of focal adhesions and stress fibers. Overexpression of syndecan-4 increases focal adhesion formation, whereas a partially truncated core protein that lacks the binding site for protein kinase C(α) and phosphatidylinositol 4, 5-bisphosphate acts as a dominant negative inhibitor of focal adhesion formation. Focal adhesion induction does not require interaction between heparan sulfate glycosaminoglycan and ligand but can occur when non-glycanated core protein is overexpressed; this suggests that oligomerization of syndecan-4 plays a major role in signaling from the extracellular matrix in adhesion.

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
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