The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors

The transforming growth factor-β (TGF-β) signaling pathway is often misregulated during cancer progression. In early stages of tumorigenesis, TGF-β acts as a tumor suppressor by inhibiting proliferation and inducing apoptosis. However, as the disease progresses, TGF-β switches to promote tumorigenic cell functions, such as epithelial-mesenchymal transition (EMT) and increased cell motility. Dramatic changes in the cellular microenvironment are also correlated with tumor progression, including an increase in tissue stiffness. However, it is unknown whether these changes in tissue stiffness can regulate the effects of TGF-β. To this end, we examined normal murine mammary gland cells and Madin-Darby canine kidney epithelial cells cultured on polyacrylamide gels with varying rigidity and treated with TGF-β1. Varying matrix rigidity switched the functional response to TGF-β1. Decreasing rigidity increased TGF-β1-induced apoptosis, whereas increasing rigidity resulted in EMT. Matrix rigidity did not change Smad signaling, but instead regulated the PI3K/Akt signaling pathway. Direct genetic and pharmacologic manipulations further demonstrated a role for PI3K/Akt signaling in the apoptotic and EMT responses. These findings demonstrate that matrix rigidity regulates a previously undescribed switch in TGF-β-induced cell functions and provide insight into how changes in tissue mechanics during disease might contribute to the cellular response to TGF-β.

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Matrix rigidity regulates a switch between TGF-β1-induced apoptosis and epithelial-mesenchymal transition.

Magnetorheological (MR) materials are classified as smart materials due to their responsiveness to external magnetic stimuli. Intensive research on MR materials has led to broad applications in several potential fields. A solid carrier matrix state called MR elastomer with its exceptional magnetic responsive feature is obtained by merging magnetizable particles within an elastomeric polymer. This integration results in outstanding characteristics on the rheological performances. Special prominence is given to the understanding of the base materials and fabrication as well as the functional behavior through various characterization methods. Broad applications of MREs are also explored to provide a profound market picture and to motivate researchers to develop novel technology. The functional behavior of MREs is briefly explained. The art of the materials provides the current position and mapping of the matrix and filler particles. Types of matrix and particles are mentioned together with the level of the research interest on MREs. Description of the fabrication is provided in simple diagrams as summarized from previous works to enhance the MREs performance. The possible tests to reveal the characteristics of the MREs are delivered with the global experimental setup. The review also explains the applications of MREs as well as discussion on the MREs future promising applications.

Recent Progress on Magnetorheological Solids: Materials, Fabrication, Testing, and Applications†

Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma.

Optic nerve degeneration caused by glaucoma is a leading cause of blindness worldwide. Patients affected by the normalpressure form of glaucoma are more likely to harbor risk alleles for glaucoma-related optic nerve disease. We have performed a meta-analysis of two independent genome-wide association studies for primary open angle glaucoma (POAG) followed by a normal-pressure glaucoma (NPG, defined by intraocular pressure (IOP) less than 22 mmHg) subgroup analysis. The single-nucleotide polymorphisms that showed the most significant associations were tested for association with a second form of glaucoma, exfoliation-syndrome glaucoma. The overall meta-analysis of the GLAUGEN and NEIGHBOR dataset results (3,146 cases and 3,487 controls) identified significant associations between two loci and POAG: the CDKN2BAS region on 9p21 (rs2157719 [G], OR=0.69 [95%CI 0.63–0.75], p=1.86610 218 ), and the SIX1/SIX6 region on chromosome 14q23 (rs10483727 [A], OR=1.32 [95%CI 1.21–1.43], p=3.87610 211 ). In sub-group analysis two loci were significantly associated with NPG: 9p21 containing the CDKN2BAS gene (rs2157719 [G], OR=0.58 [95% CI 0.50–0.67], p=1.17610 212 ) and a probable regulatory region on 8q22 (rs284489 [G], OR=0.62 [95% CI 0.53–0.72], p=8.88610 210 ). Both NPG loci were also nominally associated with a second type of glaucoma, exfoliation syndrome glaucoma (rs2157719 [G], OR=0.59 [95% CI 0.41–0.87], p=0.004 and rs284489 [G], OR=0.76 [95% CI 0.54–1.06], p=0.021), suggesting that these loci might contribute more generally to optic nerve degeneration in glaucoma. Because both loci influence transforming growth factor beta (TGF-beta) signaling, we performed a genomic pathway analysis that showed an association between the TGF-beta pathway and NPG (permuted p=0.009). These results suggest that neuro-protective therapies targeting TGFbeta signaling could be effective for multiple forms of glaucoma.

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Common Variants at 9p21 and 8q22 Are Associated with Increased Susceptibility to Optic Nerve Degeneration in Glaucoma

Purpose Extracellular matrix (ECM) composition, tension, and rigidity modulate cell-ECM interactions and have substantial impact on cell functions. The authors studied the effects of ECM rigidity on human trabecular meshwork (HTM) cells to assess ECM rigidity as a possible pathophysiologic factor in glaucoma. Methods Trabecular meshwork cells derived from donor cornea rings and passaged three to seven times were plated on collagen-coated tissue culture plastic or polyacrylamide gels of different rigidity. Cell spreading and focal adhesions were assessed by immunofluorescence microscopy. Expression of focal adhesion kinase (FAK), alpha-smooth muscle actin (alpha-SMA), tubulin, alpha-B-crystallin and GAPDH, as well as phosphorylation of FAK and serum-induced activation of ERK, were studied by Western blot. The subcellular distributions of alpha-SMA and fibronectin were examined by confocal immunofluorescence microscopy. Results ECM rigidity modulated cell spreading and focal adhesion size. FAK activation and serum-induced ERK phosphorylation increased with rising substrate rigidity. Expression of alpha-SMA and recruitment of alpha-SMA to stress fibers were enhanced on rigid substrates, whereas myocilin and alpha-B-crystallin expression increased on soft substrates. The structure of fibronectin deposits differed on stiff and soft matrices. Conclusions Extracellular matrix rigidity modulates cytoskeletal structures, protein expression patterns, signal transduction, and fibronectin deposition in HTM cells. ECM changes altering trabecular meshwork resiliency may therefore have significant effects on ocular outflow tract functions with implications in glaucoma.

Substrate rigidity modulates cell matrix interactions and protein expression in human trabecular meshwork cells.

PURPOSE. The transdifferentiation of Tenon fibroblasts to myofibroblasts is a pivotal step in filtering bleb scarring. It is mediated by the cytokine TGF-, Rho-dependent contractility, and cell‐matrix interactions in an interdependent fashion. HMG-CoA-reductase inhibitors (statins) have been shown to inhibit Rho-GTPase signaling; therefore, the authors studied the influence of lovastatin on TGF-‐mediated myofibroblast transdifferentiation to assess the potential use of statins in wound healing modulation. METHODS. Human Tenon fibroblasts were grown in culture, pretreated with lovastatin, lovastatin and mevalonate, or specific inhibitors of farnesyl transferase or geranylgeranyl transferase and were stimulated with TGF-1. -Smooth muscle actin (-SMA) and connective tissue growth factor (CTGF) transcription were assessed by real-time PCR. -SMA protein expression and localization were studied by Western blot and confocal immunofluorescence microscopy. Cell contractility was determined in collagen gel contraction assays. Phosphorylation of the signaling proteins Smad-2/3 and p38 were detected by Western blot, and Smad-2/3 localization was determined by confocal immunofluorescence microscopy. RESULTS. Lovastatin inhibited TGF-‐induced CTGF transcription, -SMA expression and incorporation into actin stress fibers, and subsequent collagen gel contraction. These effects were reversed by mevalonate. The inhibition of geranylgeranyl transferase but not farnesyl transferase blocked TGF-‐induced -SMA expression. Lovastatin decreased TGF-‐induced p38 activation, whereas Smad-2/3 phosphorylation and nuclear translocation were preserved. CONCLUSIONS. Lovastatin inhibits TGF-‐induced myofibroblast transdifferentiation in human Tenon fibroblasts, most likely by interfering with Rho-signaling. Statins may, therefore, serve to inhibit scarring after filtering glaucoma surgery. (Invest Ophthalmol Vis Sci. 2008;49:3955‐3960) DOI:10.1167/iovs.07-1610

Lovastatin Inhibits TGF-β-Induced Myofibroblast Transdifferentiation in Human Tenon Fibroblasts

Purpose To gain further insight into a possible role of biomechanical cues in glaucoma, the authors assessed the influence of extracellular matrix (ECM) elasticity on TGF-β2-induced changes in trabecular meshwork (TM) cells. Methods Human TM cells derived from donor cornea rings were plated on rigid collagen-coated tissue culture plastic or polyacrylamide gels of different elasticity and treated with vehicle or TGF-β2. Activation of Smad-2/3, ERK, and AKT signaling and expression of α-SMA and PAI-1 proteins were assessed by Western blot analysis. Subcellular localization of α-SMA was determined by confocal immunofluorescence microscopy. Transcription of collagen-I, -IV, and -VI, α-SMA, PAI-1, fibronectin, fibrillin-1, cochlin, and MGP-1 was studied by RT-qPCR. The contribution of non-Smad signaling pathways to TGF-β-induced α-SMA and PAI-1 expression was assessed using the small molecule inhibitors U0126 and LY294002. Results TGF-β2-induced activation of Smad-2/3, ERK, and AKT signaling as well as expression of collagen-1, α-SMA, fibulin, and MGP-1 were attenuated with increasing elasticity. In contrast, TGF-β2-induced collagen-6, fibronectin, PAI-1, and cochlin expression were enhanced on elastic substrates. The MEK-inhibitor U0126 blocked TGF-β-induced PAI-1 expression, whereas α-SMA expression was enhanced. PI3K inhibition with LY294002 reduced α-SMA expression. Conclusions ECM elasticity modulates TGF-β-induced signaling and protein expression in human TM cells. Non-Smad signaling contributes to TGF-β-induced alterations. Increasing ECM elasticity in vitro promotes protein expression patterns reminiscent of patterns reported in primary open-angle glaucoma. Therefore, changes in TM elasticity and mechanical load may have a significant role in primary open-angle glaucoma.

Elasticity-dependent modulation of TGF-β responses in human trabecular meshwork cells.

Mechanical cues such as extracellular matrix stiffness and movement have a major impact on cell differentiation and function. To replicate these biological features in vitro, soft substrata with tunable elasticity and the possibility for controlled surface translocation are desirable. Here we report on the use of ultra-soft (Young’s modulus <100 kPa) PDMS-based magnetoactive elastomers (MAE) as suitable cell culture substrata. Soft non-viscous PDMS (<18 kPa) is produced using a modified extended crosslinker. MAEs are generated by embedding magnetic microparticles into a soft PDMS matrix. Both substrata yield an elasticity-dependent (14 vs. 100 kPa) modulation of α-smooth muscle actin expression in primary human fibroblasts. To allow for static or dynamic control of MAE material properties, we devise low magnetic field (≈40 mT) stimulation systems compatible with cell-culture environments. Magnetic field-instigated stiffening (14 to 200 kPa) of soft MAE enhances the spreading of primary human fibroblasts and decreases PAX-7 transcription in human mesenchymal stem cells. Pulsatile MAE movements are generated using oscillating magnetic fields and are well tolerated by adherent human fibroblasts. This MAE system provides spatial and temporal control of substratum material characteristics and permits novel designs when used as dynamic cell culture substrata or cell culture-coated actuator in tissue engineering applications or biomedical devices.

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Ultra-soft PDMS-based magnetoactive elastomers as dynamic cell culture substrata.

PURPOSE Extracellular matrix and the cytokine TGF-β influence scar formation in an interdependent fashion. In this study, the impact of extracellular matrix elasticity on TGF-β-induced signal transduction and myofibroblast transdifferentiation was examined. METHODS Primary human tenon fibroblasts were seeded on collagen-coated glass coverslips (rigid environment) or collagen or polyacrylamide gels (elastic environment) of different compliance and stimulated with TGF-β. Myofibroblast transdifferentiation was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis for the marker gene α-smooth muscle actin (SMA), and SMA incorporation into stress fibers was determined by confocal immunofluorescence microscopy. CTGF transcription was assessed by RT-qPCR. Signaling pathways were examined by Western blot using phosphospecific antibodies and by immunofluorescence microscopy. RESULTS TGF-β-dependent myofibroblast transdifferentiation was enhanced in a stiff environment. Increasing matrix elasticity attenuated TGF-β-induced myofibroblast transdifferentiation and the associated CTGF expression. TGF-β-induced p38 activation was reduced on elastic substrates. CONCLUSIONS The results suggest that matrix elasticity influences TGF-β-dependent activation of p38 signaling and subsequent myofibroblast transdifferentiation. Biomechanical cues represent an important determinant of scarring processes. Therefore, cellular signals elicited by mechanotransduction deserve consideration in the design of novel antifibrotic strategies.

Hong Han

Papers

Substrate rigidity modulates cell matrix interactions and protein expression in human trabecular meshwork cells.

Lovastatin Inhibits TGF-β-Induced Myofibroblast Transdifferentiation in Human Tenon Fibroblasts

Elasticity-dependent modulation of TGF-β responses in human trabecular meshwork cells.

Ultra-soft PDMS-based magnetoactive elastomers as dynamic cell culture substrata.

Extracellular matrix elasticity modulates TGF-β-induced p38 activation and myofibroblast transdifferentiation in human tenon fibroblasts.