Mechanochemical Effects on Extracellular Signal-Regulated Kinase Dynamics in Stem Cell Differentiation

Abstract

Understanding how key signaling molecules are coregulated by biochemical agents and physical stimuli during stem cell differentiation is critical but often lacking. Due to the important role of extracellular signal-regulated kinase (ERK), this study has examined its temporal dynamics to determine the coregulation of mechanochemical cues on ERK phosphorylation for smooth muscle cell (SMC) differentiation. To assess ERK1/2 activity, a fluorescence resonance energy transfer-based biosensor was transfected into mesenchymal stem cells. The influences of nanopatterned substrates, growth factors, and drugs on ERK activities were related to their effects on SMC differentiation. Results revealed that nanopatterned substrates significantly increased ERK activity in cells, overriding ERK response from administered biochemical factors. The nanopatterned substrates reduced expression of SMC markers after a 48-h biochemical treatment, except for the combination with ERK inhibitor PD98059 treatment, which enhanced expression of mature SMC marker MYH11. Immunofluorescent staining for focal adhesion proteins, vinculin and zyxin, indicated no significant differences in vinculin cluster distribution or dimension, while the location of zyxin changed from adhesion sites of cell periphery on nonpatterned substrate to actin filaments on nanopatterned substrate. The zyxin-reinforced stress fibers likely enhanced the cytoskeletal tension to increase ERK dynamics. Collectively, results suggest that physical stimuli play a dominating role in initial ERK signaling and early-stage differentiation through focal adhesion changes, and the capability of monitoring signaling events in real time could be exploited to guide the engineering of cell microenvironment.

Keywords: ERK phosphorylation; FRET; mechanochemical stimuli; nanopattern.

FIG. 1.

Experimental strategy to study ERK response to mechanochemical stimuli. (A) Schematic illustration of FRET biosensor mechanism for studying cell mechanochemical transduction from membrane receptors to ERK signaling. (B) Fluorescent image of transfected MSCs. (C) SEM image of nanoimprint-patterned PDMS substrate. ERK, extracellular signal-regulated kinase; FRET, fluorescence resonance energy transfer; MSC, mesenchymal stem cells; PDMS, polydimethylsiloxane; SEM, scanning electron microscope.

FIG. 2.

Temporal profiles of ERK phosphorylation dynamics in response to mechanochemical stimuli. (A) Normalized FRET emission ratio of NES-EKAR biosensor in MSCs on a flat substrate with different biochemical treatments. (B) Changes in FRET ratio in the control, showing small signal fluctuations from mechanical stimulation of pipetting alone. Data are shown with mean ± SEM.

FIG. 3.

Nanopatterning upregulates ERK activity. (A–E) Normalized FRET emission ratio of ERK biosensor (NES-EKAR) in MSCs, showing the ERK phosphorylation dynamics in MSCs in response to different biochemical treatments (U46619, PD98059, control, EGF, and TGF-β1) on the PDMS substrata with (NIL+) or without (NIL−) nanopatterns. Results are shown with mean ± SEM for normalized FRET ratio, and “*” indicates significant difference (p < 0.05) between NIL+ and NIL− groups for greater than 50% of time course according to t-test. Fluctuation at the 24-h time point was due to the computer automatic resetting. NIL, nanoimprint lithography; EGF, epidermal growth factor.

FIG. 4.

The expression of SMC markers, including α-SMA, calponin-1, and MYH11, in MSCs on NIL+/− PDMS after 48 h. (A, C, E) Representative images of immunofluorescent staining of α-SMA (A), calponin-1 (C), and MYH11 (E). Scale bar = 150 μm. (B, D, F) Distribution of fluorescence of α-SMA (B), calponin-1 (D), and MYH11 (F) in MSCs. Individual cells are denoted by symbols and mean ± SEM indicated by lines. “*,” “**,” and “***” show p < 0.05, p < 0.01, and p < 0.001, respectively, for the significant differences in biochemical-treated conditions versus control within NIL+ or NIL− groups. “^#,” “^##”, and “^####” indicate p < 0.05, p < 0.01, and p < 0.0001, respectively, for the significant differences between NIL+ and NIL− conditions for each biochemical treatment. Two-way ANOVA was used here for statistics analyses. α-SMA, α-smooth muscle actin; SMC, smooth muscle cell.

FIG. 5.

PCR assessment of select vascular markers of MSCs cultured on NIL+ and NIL− substrates with or without PD98059 treatment: (A) α-SMA and (B) MYH11. “*” indicates significant difference in gene expression between groups (p < 0.05).

FIG. 6.

Vinculin expression and quantitation. (A, B) Immunofluorescent staining of vinculin in MSCs on NIL+ (A) or NIL− (B) PDMS substrates, scale bar = 25 μm. (C) Area and major axis length comparison of vinculin clusters. Mean ± SEM are normalized to NIL− length. (D) Frequency distribution of vinculin cluster length per cell. Mean ± SEM denoted at each bin.

FIG. 7.

Translocation of zyxin from focal adhesions to actin stress fibers in nanopatterned substrate. (A–F) F-actin filaments costained with zyxin and DAPI for NIL+/− PDMS substrates. White arrows indicate zyxin cluster position. Scale bar = 50 μm.