Cell-cell adhesion impacts epithelia response to substrate stiffness: Morphology and gene expression

Abstract

Monolayer epithelial cells interact constantly with the substrate they reside on and their surrounding neighbors. As such, the properties of epithelial cells are profoundly governed by the mechanical and molecular cues that arise from both the substrate and contiguous cell neighbors. Although both cell-substrate and cell-cell interactions have been studied individually, these results are difficult to apply to native confluent epithelia, in which both jointly regulate the cell phenotype. Specifically, it remains poorly understood about the intertwined contributions from intercellular adhesion and substrate stiffness on cell morphology and gene expression, two essential microenvironment properties. Here, by adjusting the substrate modulus and altering the intercellular adhesion within confluent kidney epithelia, we found that cell-substrate and cell-cell interactions can mask each other's influence. For example, we found that epithelial cells exhibit an elongated morphological phenotype only when the substrate modulus and intercellular adhesions are both reduced, whereas their motility can be upregulated by either reduction. These results illustrate that combinatorial changes of the physical microenvironment are required to alter cell morphology and gene expression.

Figure 1

Substrates were produced by mixing various ratios of Sylgard 184 (S184) and Sylgard 527 (S527), and their stiffnesses were characterized. (A) These stiffnesses show a logarithmic trend as the percentage of S184 was increased. Pure S527 was shown to have a similar stiffness to in vivo kidney tissue (∼6 kPa). Tissue culture plastic (TCP) was also plotted for comparison. From top to bottom, the dotted lines denote substrate moduli for substrates of TCP (112.8 MPa), S184 (1123.2 kPa), 1:1 blend of S184 and S527 (450.7 kPa), and S527 (3.2 kPa). (B) Phase contrast images of confluent MDCK layers, five days postseeding, grown on TCP and S527 show no significant morphological change despite the five orders magnitude difference in the stiffness. Scale bar represents 30 μm. Volcano plots of log2 ratio versus p value compare genes expressed by cells grown on TCP and S527 for confluent (C) and nonconfluent, 24 h postseeding (D) conditions. For confluent culture, the substrate stiffness induced virtually no differences in gene expression. In contrast, nonconfluent cells grown on the soft substrate show upregulation of genes related to EMT, epithelial phenotype, cell adhesion, and solute transporters. The vast differences between confluence conditions suggest that intercellular contact may be a factor in determining cell phenotype. For (C)–(D), n = 6.

Figure 2

(A–D) Fluorescence images of Ca+/- treated samples grown on TCP and S527, stained for nuclei and actin, reveal significant morphological change only for MDCK cells grown on the softer substrate and subject to interference of intercellular adhesions. Scale bar represents 30 μm. Aspect ratio (E), perimeter (F), circularity (G), and cell area (H) were calculated by outlining and measuring individual cells from the obtained fluorescence images for each of the four conditions. This morphology quantification shows that Ca-/S527 cells had significant elongation, confirmed by a decrease in circularity and increases in aspect ratio and perimeter. For (E)–(H), n = 100. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001.

Figure 3

E-cadherin antibody, which specifically blocks cellular junctions, and DTT, which inactivates extracellular adhesions through disulfide exchange, were used to perturb cell-cell adhesions while maintaining extracellular calcium to validate our Ca- results. Phase contrast images show morphology for cells treated with control media (A and B), calcium-free media (C and D), antibody (E and F), and DTT (G and H). Scale bar represents 60μm. Morphology quantification (I) reveals that morphological changes induced by both antibody and DTT are consistent with our Ca- results. Cross-category statistics for cells grown on TCP were not included as they were all found to be nonsignificant. For (G), n = 50. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 4

(A) Volcano plot of log2 ratio versus p value compares genes expressed by cells grown on TCP and S527 under the Ca- treatment condition. Although ∼96% of tested genes show similar expression, SERPINE1 and TIMP3 were found to be significantly downregulated by the softer substrate. These genes are both related to wound healing and cell-extracellular matrix adhesion. Bar charts for SERPINE1 (B) and TIMP3 (C), using data from Figs. 1C and 3A, provide a visual comparison of relative expression across conditions. These illustrate that Ca- treatment exacerbates the difference in gene expression derived from the substrate stiffness effect. For (A), n = 6. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01.

Figure 5

Average unsupervised hierarchical clustering analysis was performed using the same data sets used for Figs. 1C and 4A to visualize overall trends in gene expression due to substrate stiffness and cell-cell adhesion. Ca- treatment was found to downregulate FN1 and VIM and upregulate CDH1, suggesting that interference of intercellular adhesions may not always induce EMT. Extreme outliers were removed.

Figure 6

(A) Cell mean velocity was measured by PIV analysis performed on time-lapse images of cell monolayers treated with Ca + or Ca- media. Regions of interest, with a field of view of 672 μm × 672 μm, were registered and imaged for 48 h post-Ca+/- treatment at an interval of ∼4 min. Both softening of the substrate modulus and disruption of cell-cell contacts were shown to increase cell motility to different degrees. (B–E) Representative heatmaps for each condition illustrate this trend. Additionally, samples with lower mean migration showed greater homogeneity in local velocity magnitudes. Scale bar represents 100 μm. For (A), n = 32. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001.

Figure 7

The present study identifies how substrate stiffness and cell-cell adhesion affect epithelial morphology, gene expression, and motility. Specifically, for morphology, we found that both factors are able to mask the influence of each other, requiring reduction of both substrate moduli and intercellular interactions for significant morphological change (AND). For motility, however, modulation of both factors was able to induce increases in cell mean velocity (OR). Lastly, SERPINE1 specifically was found to be consistently downregulated on soft substrates, suggesting that the intercellular adhesions are not as influential on SERPINE1 expression as the substrate.