An EGFR autocrine loop encodes a slow-reacting but dominant mode of mechanotransduction in a polarized epithelium

Abstract

The mechanical landscape in biological systems can be complex and dynamic, with contrasting sustained and fluctuating loads regularly superposed within the same tissue. How resident cells discriminate between these scenarios to respond accordingly remains largely unknown. Here, we show that a step increase in compressive stress of physiological magnitude shrinks the lateral intercellular space between bronchial epithelial cells, but does so with strikingly slow exponential kinetics (time constant approximately 110 s). We confirm that epidermal growth factor (EGF)-family ligands are constitutively shed into the intercellular space and demonstrate that a step increase in compressive stress enhances EGF receptor (EGFR) phosphorylation with magnitude and onset kinetics closely matching those predicted by constant-rate ligand shedding in a slowly shrinking intercellular geometry. Despite the modest degree and slow nature of EGFR activation evoked by compressive stress, we find that the majority of transcriptomic responses to sustained mechanical loading require ongoing activity of this autocrine loop, indicating a dominant role for mechanotransduction through autocrine EGFR signaling in this context. A slow deformation response to a step increase in loading, accompanied by synchronous increases in ligand concentration and EGFR activation, provides one means for cells to mount a selective and context-appropriate response to a sustained change in mechanical environment.

Figure 1.

Constitutive ligand shedding and autocrine EGFR activity. A, B) Time-dependent accumulation of EGF (A) and TGF-α (B)in basal medium of ALI-cultured NHBE cells in the absence (control) and presence of an EGFR neutralizing antibody (αEGFR; 20 μg/ml). Dashed lines indicate ELISA sensitivities. C) Dose-response effect of EGFR-neutralizing antibody on EGF and TGF-α accumulation over 48 h, demonstrating plateau at 20 μg/ml. D) Time-dependent loss of exogenous EGF added to basal medium of NHBE cells without EGFR antibody. E) Omission of EGF/BPE from full-growth medium does not significantly decrease baseline levels of phosphorylated (active) fraction of EGFR. However, addition of the MMP inhibitor GM6001 (10 μM) for 1 h significantly reduces phosphoEGFR (tyr 1068; P=0.005) relative to full growth medium condition. F) Addition of AG1478 (1 μM), an EGFR-kinase inhibitor, for 2 h selectively attenuates expression of genes encoding EGF-family ligands AREG (P=0.013), EREG (P=0.011), and HBEGF (P=0.006), but not TGF-α, relative to GAPDH expression. Data are means ± sd from 3 or 4 wells from one of ≥2 replicate experiments. *P ≤ 0.05.

Figure 2.

Visualizing intercellular deformations under sustained compressive stress. A) Comparison of raw and segmented images for matched optical sections at 0, 60, and 600 s after onset of continuous transcellular pressure (30 cmH2O). B) Composite image shows comparative LIS geometry at 0, 60, and 600 s after onset of continuous pressure gradient. Note that the NHBE cells form a 3-dimensional structure; hence, out-of-plane motions affect the degree to which the optical sections are superimposed.

Figure 3.

Mechanical characterization of LIS deformations. A) Time-dependent changes in LIS volume (normalized to time 0) for 3 cell donors over 600 s of 30 cmH₂O continuous compressive stress. Best-fit exponentials for each donor yielded time constants of 100, 116, and 113 s for donors 10, 11 and 12, respectively. B) Comparison of time-dependent LIS volume changes in NHBE 11 cells under 10, 30, and 50 cmH₂O continuous compressive stress. Best-fit exponentials yielded time constants of 100, 109, and 114 s for the 10, 30, and 50 cmH₂O pressure loads, respectively. C) LIS volume changes during and after continuous compressive stress for 600 s; NHBE 11 cells at 10 and 50 cmH₂O, as well as time-matched control. Vertical dashed line indicates time at which applied compressive stress was removed (600 s). Best-fit exponential time constants during the loading phase were 111 and 116 s, and during the recovery phase 154 and 312 s for 10 and 50 cmH₂O loads, respectively. D) Change in LIS volume at 600 s vs. applied pressure gradient for donor 11. Data are means ± sd from 3–4 independent replicate experiments.

Figure 4.

Comparing kinetics of ligand accumulation and EGFR activation. A) Time-dependent changes in phospho-EGFR (tyr 1068) during continuous compressive stress (30 cmH₂O). Phospho-EGFR results were normalized to time 0 controls within each experiment and expressed as means ± sd, n = 4 experiments. B) Schematic of the LIS and the governing ligand diffusion-convection equations used to compute spatial and temporal changes in LIS ligand concentration C(t) that occur with changes in LIS width w(t). C) Comparison of temporal (normalized to time 0) changes in LIS width (means±sd from 3 cell donors in Fig. 3A), calculated ligand concentration [C_{LIS, mean} is C(t) averaged over LIS height], and measured phospho-EGFR from A. D) Phospho-EGFR levels at 180 s after exposure to compressive stress (30 cmH₂O) for 3, 10, or 180 s, using an independent donor from experiments in A and C. Data are means ± sd from 3, 2, and 5 experiments, respectively for 3, 10, and 180 s conditions.

Figure 5.

EGFR-dependent transcriptomic response to compressive stress. A) Comparison of natural log fold changes in gene expression of pressure samples vs. time matched controls (P/C) measured by microarray anlaysis of pooled samples, and qPCR analysis of replicate samples (means of 3 independent experiments). Open circles indicate probes that reached statistical significance by qPCR. Cutoff for inclusion in mechanoresponsive gene list was 0.52 on the y axis. B) Rank-ordered mechanoresponsive probes (P/C) after 1 h continuous compressive stress, and their response to compressive stress in the presence of AG1478 (P+A/A). Criteria used to assess EGFR-independence are shown as a dashed line (P+A/A<0.5∗P/C). C) Comparison of qPCR results to microarray values that led to classification of 3 EGFR-dependent (FOS, SPRED2, and DUSP5) and 1 EGFR-independent mechanoresponsive genes (ATF3). *P < 0.05 for P vs. C or P+A vs. A. D) Changes in transcript levels for FOS and HBEGF, measured by qPCR, after exposure to compressive stress for 1 h in the presence of a neutralizing antibody against EGFR (20 μg/ml), or isotype control (IgG, 20 μg/ml). Cells from same donor as Fig. 4D were used. Data are means ± sd representative of 2 independent experiments; n = 2 wells/condition.