Findings suggest that FAK plays a role in the mechanical crosstalk between integrin- mediated rigidity sensing and the regulation of cadherin adhesions (220). Increased substrate rigidity increased basal FAK activation (FAK phosphorylation) in mammary gland tissue and facilitated tumor invasion (225). In order to investigate signaling mechanism(s) that might link rigidity sensing with the dynamic regulation of endothelial adherens junctions in response to thrombin, I grew BAEC monolayers on 0.25 kPa or 24 kPa fibronoectin-coated hydrogels and immunoblotted for FAK phosphorylation at Tyr 397 at various time points over thrombin stimulation.
As predicted, basal FAK activation was increased in cells on stiffer hydrogels (24 kPa) compared to 0.25 kPa hydrogels (Fig. 5.6). After 15 min of thrombin stimulation, pTyr397-FAK increased in cells on both hydrogels, however thrombin induced more robust phosphorylation of
Tyr397-FAK on softer hydrogels (p < 0.05). FAK activation decreased within 120 min of thrombin stimulation, but levels for the softer hydrogels remained significantly elevated than basal levels (p < 0.05). FAK activation on stiffer hydrogels returned to basal levels. The biphasic response to thrombin, characterized by an initial increase in pY397 after 15 min followed by a decrease after 120 min, corresponds with the time dependence of thrombin-induced adherens junction disruption and resealing, respectively. The decrease in FAK activation to basal levels on stiffer hydrogels occurred in conjunction with the re-annealing of adherens junctions (Fig. 5.5B). Softer substrates failed to fully re-anneal adherens junctions after thrombin stimulation compared to stiffer substrates because inter-endothelial gaps remained in BAEC monolayers cultured on hydrogels after 120 min of thrombin stimulation (Fig. 5.5B).
5.4 Discussion
Evidence suggests that compliant polyacrylamide hydrogel substrates control the global contractility of cells (148, 238, 247). Imaging BAEC monolayers cultured on hydrogels identified the impact of global cell tension on the architecture of adherens junctions and associated cytoskeletal elements. BAECs on rigid hydrogels exhibited disrupted adherens junctions and inter-endothelial gaps associated with increased permeability. BAEC monolayers displayed basal differences in cell morphology, adherens junction structure, and cytoskeletal organization in response to altered contractile stress associated with rigidity sensing. Further studies identified that endothelial rigidity sensing functions in a Rho-dependent mechanism, and substrate rigidity modulates the activation of FAK in thrombin-induced remodeling of adherens junctions.
of force-actuated junction remodeling depended on substrate rigidity. Cadherin-based traction forces exerted by MDCK cells were greater on rigid (34 kPa) hydrogels compared to soft (0.6 kPa) hydrogels coated with cadherins (248). The results of this study suggest that endogenous tension, as modulated by substrate rigidity, alters the tension-sensing function of cadherin junctions. This dependence is supported by reported increases in intercellular junction size and traction forces generated by cells on deformable substrates following thrombin-stimulated cell contractility (87, 155). Together, these results highlight the importance of endogenous mechanical tension in junction remodeling, however questions remain regarding conflicting cellular responses to force. One possible mechanism is that contractile forces biphasically modulate cadherin junctions such that low forces positively regulate barrier function while high forces destabilize junctions. Force-dependent FAK activation may promote Rac1 activation and barrier re-annealing on soft substrates; or may lead to RhoA activation and barrier disruption, on very stiff substrates.
5.5 Figures
!
Figure 5.1. Postulated pathways linking matrix rigidity, force sensing, cell mechanics, and endothelial barrier regulation. Integrin ligation activates focal adhesion kinase (FAK), which regulates Rho GTPases (Rac1, RhoA) and downstream actin remodeling. Thrombin stimulation also activates Rho signaling and triggers disruption of cell-cell junctions.
Figure 5.2. Elastic moduli of polyacrylamide hydrogels. Gels were polymerized from solutions of differing concentrations of acrylamide (AA) and bis-acrylamide (bis), and elastic moduli were measured by rheology. Data represent the mean of 30 measurements recorded per sample, and error bars represent s.d. Three independent experiments were performed for each condition.
Figure 5.3. BSA-FITC coverage on hydrogels. (A) Hydrogels covalently bound with BSA- FITC using sulfo-SANPAH and UV irradiation. The hydrogel surfaces were imaged by fluorescence microscopy. Ten different locations were imaged across gels using a 10x objective. Images shown are of two distinct locations on opposite sides of the gel and are representative of three independent experiments. (B) Hydrogels incubated with BSA-FITC but without covalent
hydrogels that were not incubated with protein. Scale bars represent 50 µm. (D) Mean fluorescence intensities of BSA-FITC bound to hydrogels. Error bars represent s.d. Data represent three independent experiments for each condition.
Figure 5.4. Confocal microscopy of endothelial junctions on hydrogel substrates. Cells were immunostained with anti-β-catenin antibody (green) to visualize adherens junctions, and phalloidin (red) to visualize F-actin organization. Scale bars represent 10 µm.
Figure 5.5. Impact of substrate rigidity on dynamic junction remodeling. (A) BAEC monolayers grown on glass substrates were stimulated with thrombin for the given time points. Nuclei were stained with DAPI (blue). (B) BAEC monolayers grown on 24 kPa gels and stimulated with thrombin. Rho activity was inhibited by treating cells with 10 µM Y-27632 for 1 h. Cells were stained for VE-cadherin (green) and F-actin (red). Images represent at least three independent experiments. Scale bars represent 20 µm.
Figure 5.6. Matrix rigidity modulates FAK activity. BAEC monolayers were grown on 0.25 kPa or 24 kPa hydrogels and stimulated with thrombin for given time points. Phosphorylation of Tyr 397 FAK was determined by immunoblotting, and levels of pY397-FAK as measured by densitometric analysis were normalized to total FAK levels. Histogram represents the mean +/- s.e.m. of two independent experiments. * indicates significant increase in phosphorylation above time zero (p < 0.05), and # indicates significant difference between 0.25 kPa and 24 kPa gels at 120 min (p < 0.05).