N-cadherin adhesive interactions modulate matrix mechanosensing and fate commitment of mesenchymal stem cells

Abstract

During mesenchymal development, the microenvironment gradually transitions from one that is rich in cell-cell interactions to one that is dominated by cell-ECM (extracellular matrix) interactions. Because these cues cannot readily be decoupled in vitro or in vivo, how they converge to regulate mesenchymal stem cell (MSC) mechanosensing is not fully understood. Here, we show that a hyaluronic acid hydrogel system enables, across a physiological range of ECM stiffness, the independent co-presentation of the HAVDI adhesive motif from the EC1 domain of N-cadherin and the RGD adhesive motif from fibronectin. Decoupled presentation of these cues revealed that HAVDI ligation (at constant RGD ligation) reduced the contractile state and thereby nuclear YAP/TAZ localization in MSCs, resulting in altered interpretation of ECM stiffness and subsequent changes in downstream cell proliferation and differentiation. Our findings reveal that, in an evolving developmental context, HAVDI/N-cadherin interactions can alter stem cell perception of the stiffening extracellular microenvironment.

Figure 1. Decoupled presentation of N-Cadherin and Fibronectin adhesive domains to study ECM mechanosensing

(a) Schematic representation of the evolution of the mechanical microenvironment during mesenchymal development. (b) Schematic of methacrylated hyaluronic acid (MeHA) modified with N-Cadherin and Fibronectin adhesive domains and formation into hydrogels via UV-light initiated crosslinking. ‘scram/RGD’ substrates allow for only integrin-material interactions, while ‘HAVDI/RGD’ substrates allow for material integrin and cadherin interactions. (c) Control of substrate mechanical properties was achieved by altering UV crosslinking time, as verified by AFM measurements of Young’s Modulus (mean ± SD), and normalized comparisons of scram/RGD and HAVDI/RGD moduli across three UV times (inset). (d) MSC spread area on increasingly stiff substrates (n>102 cells/group, p>0.6145, mean ± SEM). Scale bars = 10μm.

Figure 2. HAVDI ligation reduces the mechanical threshold for YAP/TAZ signaling, altering MSC interpretation of substrate stiffness

(a) Representative quantifications of high and low YAP/TAZ ratios, which were obtained by taking the average intensity in the nucleus and divided by the average intensity of the cytoplasm. (b) YAP/TAZ nuclear to cytoplasmic ratios across a range of substrate stiffness (n>31 cells/group, *=p<0.05, ***=p<0.001 by 2-way ANOVA, mean ± SEM) with corresponding sigmoidal curve-fits. (c) YAP/TAZ ratios after blocking cellular N-Cadherin with a neutralizing antibody prior to seeding on 10 kPa substrates (n>23 cells/group, ***=p<0.001 by 1-Way ANOVA with Bonferroni post-hoc, mean ± SEM). (d) Schematic for assaying dose-dependence of HAVDI presentation on 10 kPa substrates and (e) representative YAP/TAZ images for each group. (f) Quantification of YAP/TAZ ratios with increased presentation of functional HAVDI (n>100 cells/group, **=p<0.01 compared to 0%, by 1-Way ANOVA with Bonferroni post-hoc, mean ± SEM). (g) YAP/TAZ nuclear-to-cytoplasmic ratios following competition with either no peptide (CTL) or 1 mM of soluble peptide in growth media (n>30 cells/group. ***=p<0.001, ** = p<0.01 by 1-Way ANOVA with Bonferroni post-hoc testing, mean ± SEM). (h) (left) Schematic for the early osteoinduction experiments where GM (growth media) or OM (osteoinductive media) were added over three days and representative images of RUNX2 localization. (right) Quantification of RUNX2 nuclear-to- cytoplasmic ratios. (n>43 cells/group, *** = p<0.001 by 1-Way ANOVA with Bonferroni’s post-hoc testing, mean ± SEM). (i) MSC proliferation rate after 48 hrs of culture on 10 kPa substrates, as assayed by the fraction of EdU+ cells (n=3 replicates, measured in 200+ cells per replicate, *=p<0.05, mean ± SEM). Scale bars = 10 μm.

Figure 3. HAVDI/RGD co-presentation attenuates the generation of contractile force

(a) Representative images of F-actin immunofluorescence on the apical and basal planes of MSCs cultured on 10 kPa substrates. Actin organization and polarization was quantified via anisotropy ratios, where more polarized actin yields higher anisotropy ratios (n=14 cells/group, ** = p<0.01, * = p<0.05 when comparing within planes, mean ± SEM). (b) Paxillin immunostaining for focal adhesions in MSCs on 10 kPa substrates (n=18 cells/group for adhesion number, n>1275 adhesions/group for adhesion area/aspect ratio/length, box plots show 25/50/75^th percentile, whiskers show min/max). (c) Representative traction stress vector maps for MSCs plated on 10 kPa substrates. Quantification shows average traction stress generation per cell (n>26 cells/group, mean ± SEM). Scale bars = 10μm.

Figure 4. HAVDI ligation alters ECM mechanosensing at intermediate substrate stiffness through Rac1 and Myosin-IIA control of focal adhesion maturation

(a) Representative YAP/TAZ staining and quantification of nuclear to cytoplasmic ratios with pharmacologic inhibition of Rac1 (50 μM NSC-23766) or ROCK (10 μM Y-27632) in MSCs cultured on 10 kPa substrates (n>51 cells/group, ***=p<0.001 by 1-Way ANOVA with Bonferroni post-doc, mean ± SEM). (b) YAP/TAZ ratios after adenoviral transduction of LacZ controls or constitutively active Rac1 on 10 kPa substrates (n>64 cells/group, **=p<0.01, ***=p<0.001 by 1-Way ANOVA with Bonferroni post-hoc, mean ± SEM). (c) Representative confocal images of paxillin and myosin-IIA in MSCs cultured on 10 kPa substrates. Yellow lines in second column indicate pixel regions used to generate intensity profiles. (d) Quantification of focal adhesion area positive for myosin-IIA (n=22 cells/group, **=p<0.01, mean ± SEM). (e) YAP/TAZ ratios following pharmacologic inhibition of PKCβII with 50/200 nM of LY-333531 for 1 hour (n>55 cells/group, ***=p<0.001 by 1-Way ANOVA with Bonferroni post-hoc, mean ± SEM). Scale bars = 10 μm.

Figure 5. A summary of how HAVDI (from N-Cadherin) ligation can alter MSC mechanosensing of ECM stiffness cues

In normal cell-ECM mechanosensing (in this case, with fibronectin & RGD), as the stiffness of the underlying matrix increases, so does the nuclear-to-cytoplasmic localization of YAP/TAZ. This nuclear YAP/TAZ is a transcriptional regulator of downstream functional outcomes important in progenitor cells. Additional cell-cell contact via N-Cadherin in mesenchymal progenitors acts to attenuate ECM mechanosensing of MSCs by regulating the contractile sate of the cell. In this scenario, N-Cadherin inhibits Rac1-GTP levels, which then results in decreased myosin-IIA incorporation into focal adhesions and reduced contractile force generation. This reduced contractility leads to reduced YAP/TAZ nuclear localization, which thereby influences cell behavior. This attenuation of cell-ECM mechanosensing can lead to progenitor cells behaving as if they were in a different biophysical niche, with cells on intermediate stiffness substrates (~15 kPa) with N-Cadherin and ECM ligation behaving as if they were on a much softer substrate (~6 kPa) with only ECM ligands.