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![Low rigidity-induced phosphorylation of ERK1/2 is associated with cell spreading. A: Immunofluorescence study to examine cell size of MDCK cells cultured on collagen gel-coated dish (Co) or collagen gel (G) for 1 h in the presence of inhibitors, including LY294002 (20 µM), U0126 (20 µM), SP600125 (50 µM), or SB203580 (20 µM). The actin filament was visualized through staining with phalloidin-TRITC. Bar = 10 µm. The areas of cells seeded on collagen gel-coated dish (Co) or collagen gel (G) for 1 h (B) or 4 h (C) were assessed by Image Tool software. About 150 cells in each condition were evaluated. Results are the mean ± SE of three independent experiments. *P < 0.05 versus cells cultured on collagen gel with treatment of DMSO. #P < 0.05 versus cells cultured on collagen gel-coated dish. D: Immunofluorescence image of cells transiently transfected with the plasmid of GFP and ERK2 kinase mutant (ERK2KD) were cultured on collagen gel-coated dish (Co) or collagen gel (G) for 4 h. Bar = 10 µm (D) Areas of transfected cells were measured by Image Tool software. About 100 cells in each condition were evaluated. The result shows the mean ± SE of three independent experiments. *P < 0.05 versus cells transfected with ERK2KD were cultured on collagen gel. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]](profile/Chau-Zen-Wang/publication/5819219/figure/fig3/AS:267738736230468@1440845297469/Low-rigidity-induced-phosphorylation-of-ERK1-2-is-associated-with-cell-spreading-A.png)
Low rigidity-induced phosphorylation of ERK1/2 is associated with cell spreading. A: Immunofluorescence study to examine cell size of MDCK cells cultured on collagen gel-coated dish (Co) or collagen gel (G) for 1 h in the presence of inhibitors, including LY294002 (20 µM), U0126 (20 µM), SP600125 (50 µM), or SB203580 (20 µM). The actin filament was visualized through staining with phalloidin-TRITC. Bar = 10 µm. The areas of cells seeded on collagen gel-coated dish (Co) or collagen gel (G) for 1 h (B) or 4 h (C) were assessed by Image Tool software. About 150 cells in each condition were evaluated. Results are the mean ± SE of three independent experiments. *P < 0.05 versus cells cultured on collagen gel with treatment of DMSO. #P < 0.05 versus cells cultured on collagen gel-coated dish. D: Immunofluorescence image of cells transiently transfected with the plasmid of GFP and ERK2 kinase mutant (ERK2KD) were cultured on collagen gel-coated dish (Co) or collagen gel (G) for 4 h. Bar = 10 µm (D) Areas of transfected cells were measured by Image Tool software. About 100 cells in each condition were evaluated. The result shows the mean ± SE of three independent experiments. *P < 0.05 versus cells transfected with ERK2KD were cultured on collagen gel. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Source publication
Previous study demonstrated that low substratum rigidity down-regulates focal adhesion proteins. In this study we found that cells cultured on collagen gel exhibited higher migration capacity than those cultured on collagen gel-coated dishes. Low rigidity of collagen gel induced delayed but persistent phosphorylation of ERK1/2. Inhibition of collag...
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... the MEK inhibitor, U0126, was employed. MDCK cells were pre-treated with U0126 for 1 h and then cultured on collagen gel-coated dish or collagen gel in the presence or absence of U0126. U0126 did not affect the ability of cell spreading on collagen gel-coated dish. However, it inhibited spreading of MDCK cells cultured on collagen gel (Fig. 3A). Different chemical inhibitors for phosphorylation of JNK, P38, and PI3K were also used to test whether these signal pathways affected the cell spreading on collagen gel. None of these inhibitors affected cell spreading on collagen gel or collagen gel-coated dish (Fig. 3A). Quantification of cell spreading was performed by measuring ...
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... However, it inhibited spreading of MDCK cells cultured on collagen gel (Fig. 3A). Different chemical inhibitors for phosphorylation of JNK, P38, and PI3K were also used to test whether these signal pathways affected the cell spreading on collagen gel. None of these inhibitors affected cell spreading on collagen gel or collagen gel-coated dish (Fig. 3A). Quantification of cell spreading was performed by measuring cell area on collagen gel or collagen gel-coated dish in the presence or absence of different inhibitors. We found that cells cultured on collagen gel displayed a smaller area than cells cultured on collagen gel-coated dish. However, U0126 markedly inhibited cell spreading ...
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... was performed by measuring cell area on collagen gel or collagen gel-coated dish in the presence or absence of different inhibitors. We found that cells cultured on collagen gel displayed a smaller area than cells cultured on collagen gel-coated dish. However, U0126 markedly inhibited cell spreading while cell were cultured on collagen gel (Fig. 3B). Similar results were obtained when U0126 treatment was extended to 4 h. U0126 decreased cell spreading only in those cells cultured on collagen gel (Fig. 3C). Furthermore, we applied ERK2 kinase mutant conjugated with GFP to confirm this observation. After transfection, control cells expressing GFP only spread out on collagen ...
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... cultured on collagen gel displayed a smaller area than cells cultured on collagen gel-coated dish. However, U0126 markedly inhibited cell spreading while cell were cultured on collagen gel (Fig. 3B). Similar results were obtained when U0126 treatment was extended to 4 h. U0126 decreased cell spreading only in those cells cultured on collagen gel (Fig. 3C). Furthermore, we applied ERK2 kinase mutant conjugated with GFP to confirm this observation. After transfection, control cells expressing GFP only spread out on collagen gel-coated dish or collagen gel as expected, but cells expressing the ERK2 kinase mutant displayed a roundish morphology (Fig. 3D). We further measured the cell area ...
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... only in those cells cultured on collagen gel (Fig. 3C). Furthermore, we applied ERK2 kinase mutant conjugated with GFP to confirm this observation. After transfection, control cells expressing GFP only spread out on collagen gel-coated dish or collagen gel as expected, but cells expressing the ERK2 kinase mutant displayed a roundish morphology (Fig. 3D). We further measured the cell area in MDCK cells expressing ERK2 kinase mutant cultured on collagen gel. The results showed that inhibition of ERK2 kinase markedly lowered low rigidity-triggered cell spreading (Fig. 3E), indicating that activation of ERK is required for cell spreading on collagen gel. ...
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... gel-coated dish or collagen gel as expected, but cells expressing the ERK2 kinase mutant displayed a roundish morphology (Fig. 3D). We further measured the cell area in MDCK cells expressing ERK2 kinase mutant cultured on collagen gel. The results showed that inhibition of ERK2 kinase markedly lowered low rigidity-triggered cell spreading (Fig. 3E), indicating that activation of ERK is required for cell spreading on collagen gel. ...
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Citations
... Likewise, immunoelectron microscopy studies have shown that ERK1/2 is concentrated in plasma membrane caveolae (17). In addition, research based on epithelial cells has revealed translocation of phosphorylated ERK1/2 to caveolin lipid rafts due to reduction in substrate rigidity (44). As cells in 3D culture are surrounded by naturally soft ECM, all these results suggest that accumulation of activated ERK1/2 into lipid rafts of different cell types may be a common phenomenon, taking place when the substrate stiffness is low. ...
Objectives:
Regulatory mechanisms of cell proliferation have been extensively studied as they represent major challenges when dealing with pathologies such as fibrosis, tumourigenesis or tissue regeneration. Numerous in vitro studies still exploit conventional, two-dimensional cell cultures where cells are forced to adhere to unnaturally stiff and flat surfaces of culture dishes. In the living organism, however, each cell is in contact with components of the extracellular matrix and/or neighbouring cells, thus creating a complex three-dimensional (3D) tissue structure. The current paper describes a native 3D culture of cells, based on the GD25β1 fibroblast cell line, and its use for investigating cell proliferation in in vivo-like conditions.
Materials and methods:
Four-day post-confluent culture of GD25β1 fibroblasts resulted in formation of a 3D system of cells embedded in naturally synthesized extracellular matrix. Morphological characterization of the culture was performed by histochemistry, immunohistochemistry and immunofluorescence. Viability/proliferation was assayed by MTT testing, FACS analysis and Western blotting for determination of expression levels and activation status of the relevant signalling molecules.
Results:
GD25b1 fibroblasts, grown as 3D culture, gave rise to tissue-like structures characterized by low level of apoptosis, low senescence and development of 3D matrix adhesions, typical of living tissues. Transition to three-dimensionality led to a switch from exponential to linear culture growth, accompanied by accumulation of activated ERK1/2 into caveolin-containing raft domains. Disruption of raft domains as well as reverse transition from 3D back to monolayer culture led to release of phosphorylated ERK1/2 from rafts, activation of cyclin D1 expression and increase in proliferation levels.
Conclusions:
These results imply that under in vivo-like conditions, cells might achieve reduction of their proliferation level by sequestering activated ERK1/2 to lipid rafts.
... This coupling is an initiation signal for focal adhesion proteins to cluster underneath the cell membrane to focal contacts and focal adhesions [13]. Thus, focal adhesion proteins such as vinculin and focal adhesion kinase are critical for the process of cellular motility into ECM [14][15][16][17]. In focal adhesions, cell surface transmembrane receptors of the integrin family promote the extracellular interaction with ECM ligands and couple the microenvironment with the cytoskeletal actin-microfilament system [18][19]. ...
... Integrins are known to be involved in cell adhesion, transmigration and invasion processes and in coupling of the actomyosin cell cytoskeleton to the microenvironment. The activation of integrin receptors could be through conformational changes after ligand binding and possibly through biomechanical stimulation [17,50]. The activation of integrins may be regulated through either increased affinity to ligands by enhancing the number of activated integrins and total integrins on the cell's surface or integrin translocation into lipid rafts, whereas this has only been reported for β1 integrin subunits [50]. ...
The process of cancer cell invasion through the extracellular matrix (ECM) of connective tissue plays a prominent role in tumor progression and is based fundamentally on biomechanics. Cancer cell invasion usually requires cell adhesion to the ECM through the cell-matrix adhesion receptors integrins. The expression of the αvβ3 integrin is increased in several tumor types and is consistently associated with increased metastasis formation in patients. The hypothesis was that the αvβ3 integrin expression increases the invasiveness of cancer cells through increased cellular stiffness, and increased cytoskeletal remodeling dynamics. Here, the invasion of cancer cells with different αvβ3 integrin expression levels into dense three-dimensional (3D) ECMs has been studied. Using a cell sorter, two subcell lines expressing either high or low amounts of αvβ3 integrins (αvβ3high or αvβ3low cells, respectively) have been isolated from parental MDA-MB-231 breast cancer cells. αvβ3high cells showed a threefold increased cell invasion compared to αvβ3low cells. Similar results were obtained for A375 melanoma, 786-O kidney and T24 bladder carcinoma cells, and cells in which the β3 integrin subunit was knocked down using specific siRNA. To investigate whether contractile forces are essential for αvβ3 integrin-mediated increased cellular stiffness and subsequently enhanced cancer cell invasion, invasion assays were performed in the presence of myosin light chain kinase inhibitor ML-7 and Rho kinase inhibitor Y27632. Indeed, cancer cell invasiveness was reduced after addition of ML-7 and Y27632 in αvβ3high cells but not in αvβ3low cells. Moreover, after addition of the contractility enhancer calyculin A, an increase in pre-stress in αvβ3low cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase, STAT3 or Rac1 strongly reduced the invasiveness of αvβ3high cells, whereas the invasiveness of β3 specific knock-down cells and αvβ3low cells was not altered. In summary, these results suggest that the αvβ3 integrin enhances cancer cell invasion through increased cellular stiffness and enhanced cytoskeletal remodeling dynamics, which enables the cells to generate and transmit contractile forces to overcome the steric hindrance of 3D ECMs.
... Cell adhesion based mechanosensors play a prominent role in development [53, 73]. Epithelial cells require 3-D matrix attachments to complete differentiation and can sense the rigidity of their environment and respond in context dependent ways747576777879. ADPKD cells often exhibit a partially " de-differentiated " phenotype, suggesting that disease causing mutations in polycystins allow cells to revert to embryonic patterns of gene expression [3]. ...
Polycystic kidney disease is the most common heritable disease in humans. In addition to epithelial cysts in the kidney, liver and pancreas, patients with autosomal dominant polycystic kidney disease (ADPKD) also suffer from abdominal hernia, intracranial aneurysm, gastrointestinal cysts, and cardiac valvular defects, conditions often associated with altered extracellular matrix production or integrity. Despite more than a decade of work on the principal ADPKD genes, PKD1 and PKD2, questions remain about the basis of cystic disease and the role of extracellular matrix in ADPKD pathology. This review explores the links between polycystins, focal adhesions, and extracellular matrix gene expression. These relationships suggest roles for polycystins in cell-matrix mechanosensory signaling that control matrix production and morphogenesis. This article is part of a Special Issue entitled: Polycystic Kidney Disease.
... Interestingly, adenoviral transduction of a DNA segment encoding the FAT domain resulted in cellular rounding and loss of adhesion of breast cancer cells [28]. By contrast, overexpression of FAK C-terminal fragment in MDCK cells triggers Erk phosphorylation, which in turn facilitates cells spreading and lipid raft-dependent migration [29]. These results and our contribution suggest that FAK Cterminal domains are important in cell adhesion and consecutive spreading. ...
Integrin-dependent interaction of epithelial tumor cells with extracellular matrix (ECM) is critical for their migration, but also for hematogenous dissemination. Elevated expression and activity of Src family kinases (SFKs) in colon cancer cells is often required in the disease progression. In this work, we highlighted how focal adhesion kinase (FAK) and SFKs interacted and we analyzed how PI3K/Akt and MAPK/Erk1/2 signaling pathways were activated in early stages of colon cancer cell adhesion. During the first hour, integrin engagement triggered FAK-Y397 phosphorylation and a fraction of FAK was located in lipid rafts/caveolae domains where it interacted with Fyn. The FAK-Y861 and/or -Y925 phosphorylations led to a subsequently FAK translocation out of lipid domains. In parallel, a PI3K/Akt pathway dependent of lipid microdomain integrity was activated. In contrast, the MAPK/Erk1/2 signaling triggered by adhesion increased during at least 4 h and was independent of cholesterol disturbing. Thus, FAK/Fyn interaction in lipid microdomains and a Akt-1 activation occurred at the same time during early contact with ECM suggesting a specific signaling dependent of lipid rafts/caveolae domains.
The focal adhesion kinase (FAK) regulates the dynamics of integrin-based cell adhesions important for motility. FAK’s activity regulation is involved in stress-sensing and focal-adhesion turnover. The effect of FAK on 3D migration and cellular mechanics is unclear. We analyzed FAK knock-out mouse embryonic fibroblasts and cells expressing a kinase-dead FAK mutant, R454-FAK, in comparison to FAK wild-type cells. FAK knock-out and FAKR454/R454 cells invade dense 3D matrices less efficiently. These results are supported by FAK knock-down in wild-type fibroblasts and MDA-MB-231 human breast cancer cells showing reduced invasiveness. Pharmacological interventions indicate that in 3D matrices, cells deficient in FAK or kinase-activity behave similarly to wild-type cells treated with inhibitors of Src-activity or actomyosin-contractility. Using magnetic tweezers experiments, FAKR454/R454 cells are shown to be softer and exhibit impaired adhesion to fibronectin and collagen, which is consistent with their reduced 3D invasiveness. In line with this, FAKR454/R454 cells cannot contract the matrix in contrast to FAK wild-type cells. Finally, our findings demonstrate that active FAK facilitates 3D matrix invasion through increased cellular stiffness and transmission of actomyosin-dependent contractile force in dense 3D extracellular matrices.
To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in primary culture of mice proximal tubular epithelial cells (mPTECs), we employed soft matrix made from monomeric type I collagen-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain the tubular-like morphology with differentiation and growth arrest and to evade TGF-β1-induced EMT. However, the potent effect of MG on mPTECs differentiation was suppressed by glutaraldehyde-induced crosslinking and subsequently stiffening MG or by the increasing ratio of collagen in soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain the tubular-like morphology and differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U0126, a MEK inhibitor, abolished the stiff matrix-induced de-differentiation and proliferation. These data suggest ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from matrix are required for the regulation of epithelial differentiation and proliferation. This study will provide the basic understandings of how physical and chemical cues derived from extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis.
Excessive collagen deposition plays a critical rolein tumor progression and metastasis. To understand how type IV collagen affects mechanical stiffness andmigration,low-collagen-IV-expressing transfectantsof B16F10, U118MG, and Huh7(denoted shCol cells) were established by the lentiviral-mediated delivery of small interfering RNA againsttype IV-α1 collagen (Col4A1). Although having similar growth rates, shCol cells showed a flatter morphology compared to that of thecorresponding controls. Notably, knocking down theCol4A1geneconferred the cells withhigher levels of elasticityand lower motility.Exposure to blocking antibodies against human β1integrinor α2β1integrinor the pharmacologicalinhibition ofSrc and ERK activity by PP1 and U0126, respectively, effectively reducedcell motility and raisedcell stiffness. Reduced Src and ERK activitiesin shCol cells indicate theinvolvement of a collagen IV/integrin signaling pathway. The forced expression ofβ1integrinsignificantly stimulated Src and ERK phosphorylation, reduced cell stiffness, and acceleratedcell motility. In an experimental metastasis assay using C57BL/6 mice, B16F10 shColcells formed significantly fewer and smaller lung nodules,confirming the contribution of collagen to metastasis.In summary, theintegrin signaling pathway activated in a tumor environment with collagendeposition is responsible for low cell elasticity and high metastatic ability.
Aim:
We investigate how the α7 nicotinic acetylcholine receptor (α7 nAChR), an essential regulator of inflammation, contributes to the α7 agonist nicotine-enhanced Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMECs) through lipid rafts/caveolae-mediated signaling.
Materials & methods:
α7 nAChR-mediated signaling and bacterial invasion were defined by lipid raft fractionation, immunofluorescence microscopy and siRNA knockdown.
Results:
Nicotine-enhanced bacterial invasion was dose-dependently inhibited by two raft-disrupting agents, nystatin and filipin. Significant accumulation of the lipid raft marker GM3 was observed in HBMEC induced by E. coli K1 and nicotine. The recruitment of α7 nAChR and related signaling molecules, including vimentin, and Erk1/2, to caveolin-1 enriched lipid rafts was increased upon treatment with E44 or E44 plus nicotine. Erk1/2 activation (phosphorylation), which is required for α7 nAChR-mediated signaling and E44 invasion, was associated with lipid rafts and nicotine-enhanced bacterial infection. Furthermore, E44 invasion, E44/nicotine-induced activation of Erk1/2 and clustering of α7 nAChR and caveolin-1 was specifically blocked by both siRNAs.
Conclusion:
α7 nAChR-mediated signaling through lipid rafts/caveolae is required for nicotine-enhanced E. coli K1 invasion of HBMEC.
