Defined spatiotemporal features of RAS-ERK signals dictate cell fate in MCF-7 mammary epithelial cells

Abstract

Signals conveyed through the RAS-ERK pathway are essential for the determination of cell fate. It is well established that signal variability is achieved in the different microenvironments in which signals unfold. It is also known that signal duration is critical for decisions concerning cell commitment. However, it is unclear how RAS-ERK signals integrate time and space in order to elicit a given biological response. To investigate this, we used MCF-7 cells, in which EGF-induced transient ERK activation triggers proliferation, whereas sustained ERK activation in response to heregulin leads to adipocytic differentiation. We found that both proliferative and differentiating signals emanate exclusively from plasma membrane-disordered microdomains. Of interest, the EGF signal can be transformed into a differentiating stimulus by HRAS overexpression, which prolongs ERK activation, but only if HRAS localizes at disordered membrane. On the other hand, HRAS signals emanating from the Golgi complex induce apoptosis and can prevent heregulin-induced differentiation. Our results indicate that within the same cellular context, RAS can exert different, even antagonistic, effects, depending on its sublocalization. Thus cell destiny is defined by the ability of a stimulus to activate RAS at the appropriate sublocalization for an adequate period while avoiding switching on opposing RAS signals.

FIGURE 1:

ERK activation kinetics induced by HRG and EGF in MCF-7 cells. (A) HRG-induced differentiation is dependent on ERK activation. Cells were treated with HRG (30 ng/ml) in the presence or absence of U0126 (10 μm) for 7 d before fixing and staining with Oil Red O. (B) Time course of ERK phosphorylation induced by treatment with EGF (50 ng/ml) or HRG (30 ng/ml) for the indicated times. Graphs are quantifications of the results obtained in three independent experiments (mean ± SEM). (C) Comparison of the intensity of ERK phosphorylation in response to EGF and HRG. Bar chart shows mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 with 95% confidence interval.

FIGURE 2:

Activation kinetics for components of the RAS-ERK pathway induced by HRG and EGF. (A) Time course of MEK phosphorylation induced by treatment with EGF (50 ng/ml) or HRG (30 ng/ml) for the indicated times. (B) Time course of RAS GTP loading. Graphs are quantifications of the results obtained in three independent experiments (mean ± SEM). (C) Comparison of intensity of RAS GTP loading in response to EGF and HRG. Bar chart shows mean ± SEM of three independent experiments. *p < 0.05 with 95% confidence interval.

FIGURE 3:

Activation kinetics of ERK cytoplasmic and nuclear effectors induced by HRG and EGF. (A) Time course of RSK1 and ELK1 phosphorylation induced by treatment with EGF (50 ng/ml) or HRG (30 ng/ml) for the indicated times. (B) Time course of ERK phosphorylation in nuclear and cytoplasmic fractions of cells stimulated with EGF or HRG for the indicated times. The purity of the fractions was ascertained by immunoblotting with lamin A and RhoGDI as nuclear and cytoplasmic markers, respectively.

FIGURE 4:

Differential activation of RAS at different microdomains. (A) RAS distribution in plasma membrane and endomembranes of MCF-7 cells. Blots for transferrin receptor (Tfr) and calreticulin are used as respective markers. (B) Distribution of RAS isoforms in plasma membrane microdomains. Blots for Tfr (disordered membrane) and caveolin (lipid rafts) were used as microdomain markers. (C) RAS is not activated at ER. Cells transfected with HA-tagged PTP HRAS (0.5 μg), except (-), were left unstimulated (U) or treated with EGF (50 ng/ml) or HRG (30 ng/ml) for the indicated times. Cells transfected with RASGRF1 (1 μg) served as positive control. GTP loading was assayed by GST-RBD (RAF) pull down (PD). TL, total lysates. (D) RAS is not activated at GC. As in C, but cells were transfected with KDELr HRAS (0.5 μg). Cells transfected with RASGRP1 (1 μg) served as positive control. (E) RAS is activated in DM microdomains. As before, but in cells transfected with CD8 HRAS (0.5 μg).

FIGURE 5:

Live-cell imaging of RAS activation in response to EGF and HRG. MCF-7 cells were transfected with constructs expressing Cherry-HRAS and the RAS-GTP biosensor E3-R3(A/D) (1 μg each) and stimulated with (top) EGF (50 ng/ml) or (bottom) HRG (30 ng/ml) for the indicated times. Scale bars, 10 μm. Arrowheads show areas of prominent RAS-GTP accumulation.

FIGURE 6:

Effects of site-specific RAS blockade on differentiation and proliferation. (A) MCF-7 cells transfected with constructs expressing the indicated site-specific HRAS N17 mutants (1 μg) were stimulated with HRG (30 ng/ml) for 7 d before fixing and staining with Oil Red O. Bar chart shows the degree of differentiation quantified by extraction of the Oil Red O stain with isopropanol. Results show mean ± SEM of three independent experiments. **p < 0.01 and ***p < 0.001 with 95% confidence interval. Bottom, expression levels of the indicated HRAS N17 mutants. (B) The proliferation rate of cells expressing the indicated constructs was monitored over the indicated period of time. Results show mean ± SEM of three independent experiments. *p < 0.05; ns, not significant; 95% confidence interval.

FIGURE 7:

Overexpression of upstream components of the RAS-ERK pathway prolongs ERK phosphorylation. (A) MCF-7 cells transfected with constructs expressing the indicated proteins (5 μg) were stimulated with EGF (50 ng/ml) for the indicated times, and ERK phosphorylation was assayed by immunoblotting. Graph shows results mean from three independent experiments. (B) For comparative purposes, ERK phosphorylation levels after 2 and 15 min of EGF stimulation from cells expressing the indicated proteins were evaluated in the same gel. Graph quantitates mean ± SEM of three independent experiments relative to the levels in unstimulated cells (0).

FIGURE 8:

Overexpression of upstream components of the RAS-ERK pathway facilitates EGF-induced differentiation. MCF-7 cells transfected with constructs expressing the indicated proteins (5 μg) were stimulated with EGF (50 ng/ml) or HRG (30 ng/ml) for 7 d before fixing and staining with Oil Red O. Bar chart shows the degree of differentiation quantified by extraction of the Oil Red O stain with isopropanol. Results show mean ± SEM of three independent experiments. **p < 0.01 and ***p < 0.001 with 95% confidence interval.

FIGURE 9:

Effects of sustaining ERK phosphorylation at the cytoplasm and nucleus on differentiation. (A) Time course of ERK phosphorylation in nuclear and cytoplasmic fractions of cells transfected with MXI2, PEA15 (1 μg), or shRNAs against DUSP6 and treated with EGF (50 ng/ml) for the indicated times. The purity of the fractions was ascertained by immunoblotting with lamin A and RhoGDI as nuclear and cytoplasmic markers, respectively. Left, evaluation of DUSP-6 down-regulation by shRNAs (sh). (B) Effects of the indicated constructs plus EGF treatment on differentiation. Bar chart shows the degree of differentiation quantified by extraction of the Oil Red O stain with isopropanol. Results show mean ± SEM of three independent experiments. ***p < 0.001 with 95% confidence interval.

FIGURE 10:

Oncogenic HRAS induces apoptosis in MCF-7 cells. (A) Induction of apoptosis in cells transfected with RAS V12 oncogenic isoforms (1 μg), as evaluated by assaying for cleaved caspase 3. (B) Effects of the indicated site-specific HRAS V12 constructs (1 μg) on cell viability, scored in soft agar colonies. (C) Activation of endogenous RAS at the GC prevents HRG-induced differentiation. Cells transfected with RASGRP1 or KDEL-cdc25 (1 μg) were treated with HRG (30 ng/ml) for 7 f before fixing and staining with Oil Red. **p < 0.01, ***p < 0.001 with 95% confidence interval.