Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations

Abstract

Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

Figure 1. Image analysis protocol to study Ca²⁺ signaling.

A/Image of fluo5F calcium-dependent fluorescence after addition of 2 nM EGF. ROIs were drawn over two COS-7 cells (red and blue circles) and 3 areas outside cells (grey circles) in the same visual field. Scale bar: 100 µm. B/Raw fluorescence intensity (F) as a function of time for the two cell ROIs (red and blue lines), and the three background ROIs (grey lines) shown in A. 2 nM EGF application (represented by a white bar) was performed 25 s after the start of the video time-lapse. C/Grayscale-coded raster plot of fluorescence intensity over time of 41 control cells in response to buffer application (i.e. in the absence of EGF). Buffer was added 25 s after the start of the video time-lapse (as indicated by a white bar). D/Data (upper graph) obtained in cells subjected to control application of buffer in the absence of EGF (same data as in C). Background fluorescence (F_bkg), evaluated by averaging the fluorescence of three areas outside cells, was subtracted from the signal (F_cell), measured in 32 cells showing no fluorescence peak throughout the entire video time-lapse. The average fluorescence did not exhibit a flat baseline due to photobleaching. An F_bleach term was determined from a single exponential fit (lower graph) to the average of 32 traces calculated from F_cell -F_bkg/F(0) where F(0) is the average of the 25 images preceding buffer application (white bar, 25 s after the start of the video time-lapse). E/Normalized fluorescence intensity (ΔF/F) as a function of time for the two cell ROIs (red and blue lines) shown in A. EGF (white bar) was added to cells 25 s after the start of the video time-lapse. F/Histogram of fluorescence intensity values from the 32 control cells where no peak was detected when buffer was added. A centered value (t0) and a standard deviation (SD) were extracted from the Gaussian fit (red line) of the distribution and a threshold value (th) was set as t0+3 SD = 0.23, and was used for the detection of significant responses in further experiments.

Figure 2. Ca²⁺ single-cell microscopy measurements induced by 20 pM EGF in COS-7 cells.

A/Raster plot of normalized fluorescence intensity against time, grayscale coded according to fluorescence intensity. 20 pM EGF was applied 25 s after the start of the video time-lapse (white bar). B/Representative traces of fluorescence variation over time for four individual cells corresponding to the three classes of responses observed following 20 pM EGF application: from top to bottom panels, unresponsive cell (0 peak); cells displaying transient or sustained single response (1 peak); cell displaying oscillatory signals (>2 peaks). For each cell, the response is represented both as a grayscale coded raster plot (top, same representation as in A) and as line plot (bottom). C/Proportion of unresponsive (0 peak), single-peak responsive (1 peak) and oscillatory responsive (>2 peaks) cells following the addition of 20 pM EGF. D/Comparison of the average fluorescence signals in response to the addition of EGF-free buffer (n = 8 responsive cells over 41 tested, red trace) or of 20 pM EGF (n = 137 over 281 tested, black trace). Fluorescence signals were synchronized at the time the first fluorescence slope (time = 0 s), found by estimating the first derivative of the signal, and averaged over 150 s. E/Ca²⁺ signals are specifically triggered by EGFR activation. Population traces averaged over cells to which irrelevant (n = 32, black line) or antagonistic anti-EGFR (n = 19 cells, red line) antibodies were added (black bar) 200 s after the start of real-time fluorescence imaging. Empty and filled circles represent the median intensity during 176 s before and after the addition of antibodies respectively. EGF was applied 25 s after the start of the video time-lapse (white bar).

Figure 3. Comparative analysis of the Ca²⁺ responses following application of EGF at two different concentrations.

A/Proportion of cells not responding (white bar) or responding to 2 nM (grey bar, n = 40/43) or 20 pM (red bar, n = 137/281) EGF. B/Average of responsive cell Ca²⁺ signals time-locked on the first fluorescence peak and recorded over 150 sec in response to 2 nM (grey line, n = 40) or 20 pM (red line, n = 137) EGF application. C/Schematic representation of the rules used to define the properties of the fluorescence peaks during an oscillatory response. Peaks were defined as signals rising and falling through an intensity threshold (th) of 0.23, and delay, duration and inter-spike interval (ISI, difference between the starting time of 2 consecutive peaks) values were defined relative to the threshold crossing. The area under the first peak is shown in black. EGF was added 25 s after the start of the video time-lapse (white bar). D/Bar plot showing the distribution of first peak delays as defined in C elicited by 2 nM (grey box, n = 40/43) or 20 pM (red box, n = 137/281) EGF. E/Bar plot showing the distribution of first peak durations as defined in C elicited by 2 nM (grey box, n = 40/43) or 20 pM (red box, n = 137/281) EGF. F/Bar plot showing the distribution of first peak areas as defined in C elicited by 2 nM (grey box, n = 40/43) or 20 pM (red box, n = 137/281) EGF. G/Bar plots showing the distribution of average interspike intervals (Average ISI) for oscillatory cells responding to 2 nM (grey box, n = 22/43 cells) or 20 pM (red box, n =  98/281 cells) EGF.

Figure 4. External Ca²⁺ dependence and sensitivity to K⁺ channel blocker charybdotoxin of EGF Ca²⁺ transients.

A/Proportion of cells responding (grey bar) or not responding (white bar) to 2 nM EGF in 3 mM extracellular Ca²⁺ (n = 24) or in 0 mM Ca^2+/1 mM EGTA (n = 28) in the extracellular medium. B/Fluorescence intensity signaling of individual cells (each represented by a different color) during the application of 2 nM EGF (white bar) when 3 mM Ca²⁺ was present (n = 24). The averaged population signal is shown as a thick black trace. C/Fluorescence intensity of individual cells (each represented by a different color) during the application of 2 nM EGF (white bar) when Ca²⁺ was removed and 1 mM EGTA was added to the extracellular medium (n = 28). The averaged population signal is shown as a thick black trace. D/Average of all cell signals during 2 nM EGF application, synchronized at the time of the first fluorescence peak and averaged for 150 sec, when 3 mM Ca²⁺ was present (black line, n = 24) or when Ca²⁺ was removed from and 1 mM EGTA was added to the extracellular medium (red line, n = 28). E/Proportion of cells responding (grey bar) or not responding (white bar) to 20 pM EGF in 3 mM extracellular Ca²⁺ (n = 13) or in 0 mM Ca^2+/1 mM EGTA (n = 11) in the extracellular medium. F/Fluorescence intensity of individual cells (each represented by a different color) during the application of 20 pM EGF (white bar) when 3 mM Ca²⁺ was present (n = 13). The averaged population signal is shown as a thick black trace. G/Fluorescence intensity of individual cells (each represented by a different color) during the application of 20 pM EGF (white bar) when Ca²⁺ was removed from and 1 mM EGTA was added to the extracellular medium (n = 11). The averaged population signal is shown as a thick black trace. H/Average of all cell signals during 20 pM EGF application, synchronized at the time the first fluorescence peak and for 150 sec, when 3 mM Ca²⁺ was present (black line, n = 13) or when Ca²⁺ was removed from and 1 mM EGTA was added to the extracellular medium (red line, n = 11). I/Proportion of cells responding (grey bar) or not responding (white bar) to 2 nM EGF in the absence (0, n = 24/27) or in the presence (100, n = 16/19) of 100 nM charybdotoxin (chx) in the extracellular medium. J/Proportion of cells responding (grey bar) or not responding (white bar) to 20 pM EGF in the absence (0, n = 16/22) or in the presence (100, n = 6/22) of 100 nM charybdotoxin (chx) in the extracellular medium.