Two phases of mitogenic signaling unveil roles for p53 and EGR1 in elimination of inconsistent growth signals

Abstract

Normal cells require continuous exposure to growth factors in order to cross a restriction point and commit to cell-cycle progression. This can be replaced by two short, appropriately spaced pulses of growth factors, where the first pulse primes a process, which is completed by the second pulse, and enables restriction point crossing. Through integration of comprehensive proteomic and transcriptomic analyses of each pulse, we identified three processes that regulate restriction point crossing: (1) The first pulse induces essential metabolic enzymes and activates p53-dependent restraining processes. (2) The second pulse eliminates, via the PI3K/AKT pathway, the suppressive action of p53, as well as (3) sets an ERK-EGR1 threshold mechanism, which digitizes graded external signals into an all-or-none decision obligatory for S phase entry. Together, our findings uncover two gating mechanisms, which ensure that cells ignore fortuitous growth factors and undergo proliferation only in response to consistent mitogenic signals.

Figure 1

Human mammary epithelial cells commit to proliferation upon two timed pulses of EGF. (A) 184A1 human mammary cells were GF-starved for 16 hours. They were then either pulsed with EGF (“1E”, red) for 1 hour, or mock pulsed (“1S”, green). Thereafter, cells were washed and incubated in starvation medium for 7 hours, as indicated, either followed by a second, one hour pulse of EGF, or not. Cells were then washed and incubated for 3 hours with BrdU in starvation medium. Thereafter, the cells were fixed, stained, and counted under a fluorescent microscope. BrdU incorporation into DNA was measured by determining the ratio of BrdU- to DAPI-stained nuclei, and normalized according to the starvation control (1S-7S-1S). Bars represent standard errors calculated from at least 15 non-overlapping photomicrograph fields (>500 nuclei). Significant p-values of two-tailed student’s T-test are indicated. The experiment was repeated thrice. (B) 184A1 cells were GF-starved as in A and then treated with two pulses of EGF, or continuously stimulated with EGF for 9 hours. Cells were then washed and incubated for 3 hours with BrdU, fixed and BrdU incorporation analysed as in A. Bars represent standard errors calculated from at least 15 non-overlapping photomicrograph fields (>500 nuclei). p-values of two-tailed student’s T-test are indicated. The experiment was repeated twice. (C) 184A1 cells were GF-starved and treated as in A. Following the second pulse, cells were left in starvation medium for 17 hours, fixed and stained with methyl violet. Cell-occupied area was then measured from four light photomicrographs of non-overlapping fields. Bars represent the standard errors calculated from triplicates. Significant p-values of two-tailed student’s T-test are indicated. Representative light photomicrographs are presented. The experiment was repeated twice. (D) Cells were GF-starved and treated as in A. At the indicated time points, cells were harvested, lysed, and cleared extracts electroblotted. Phosphorylation of Rb and abundance of c-MYC were determined (left panel), and quantified by densitometry. Signals were normalized to actin and fold phosphorylation or expression calculated (presented under each lane). The right panel presents the corresponding signals determined at the time of BrdU measurement (marked by Roman numbers). The experiment was repeated twice.

Figure 2

Comparative analyses of phosphorylation cascades stimulated by each EGF pulse. (A) A scheme presenting phosphorylation cascades activated by EGFR. Light blue labelled proteins were analysed using RPPA. (B) 184A1 cells were GF-starved for 16 hours. Thereafter they were pulsed for 1 hour with EGF, washed, and incubated in starvation medium for 7 hours, followed by a second pulse with EGF (red) or no treatment (green). At the indicated time points, cells were harvested and equal amounts of protein were used for RPPA analysis using the indicated antibodies. The mean of phosphorylation signals normalized to the respective total expression level (in triplicates) was calculated. The heatmap presents the means in log₂ scale, centered to the corresponding mean across all samples. The fold-change in phosphorylation between the second pulse peak and the first pulse peak is indicated (right column), if the difference between the peaks was significant. (C) 184A1 cells were GF-starved and treated as in B. Cells were harvested at the indicated time points, lysed, and analyzed by immunoblotting. Quantified and normalized signals are presented under each lane. The experiment was repeated thrice. (D) 184A1 cells were treated and BrdU incorporation measured as in Figure 1A. Cells were treated with U0126 at the indicated concentrations, 30 minutes prior to and throughout the second pulse (shaded area). Bars represent standard error values (>500 nuclei). ERK activation was calculated according to Figure S2D. The experiment was repeated thrice. (E) 184A1 cells were GF-starved, treated, and BrdU incorporation measured as in D. Cells were treated with LY294002 as indicated, 30 minutes prior to and throughout the second pulse (shaded). The experiment was repeated thrice.

Figure 3

A short pulse of EGF is sufficient for the induction of metabolic enzymes essential for cell proliferation. (A) A scheme depicting the setup of the microarray experiment. mRNA samples were isolated at the indicated time points (blue triangles). Red and green segments indicate EGF pulses and intervals, respectively. (B) 184A1 cells were treated as in Figure 1A. mRNA abundance was measured at the indicated time points using Affymetrix GeneSet microarrays. Shown are centered and normalized expression patterns of genes associated with cholesterol biosynthetic processes included in the “persistently induced” profile (see Figure S3B). (C) 184A1 cells were grown and processed as in Figure 1A except that cells were treated with Metformin (0.1 mM), Mevastatin (1µM), or AICAR (0.5mM) during the interval (shaded). Bars represent standard errors calculated from at least 15 non-overlapping photomicrograph fields (>500 nuclei). A significant p-value of two-tailed student’s T-test is indicated. The experiment was repeated thrice.

Figure 4

Differential induction of immediate-early transcription factors by the two pulses. (A) 184A1 cells were treated with two pulses of EGF, as in Figure 3B. Shown are centered and normalized expression levels of the indicated immediate-early induced TFs from the profile denoted 1^st<2^nd (Figure S3D). (B) 184A1 cells were treated with two pulses of EGF. At the end of each pulse, mRNA was isolated, followed by cDNA synthesis and RT-qPCR with primers for either c-FOS or EGR1. Presented are the average ratios of expression, calculated from four biological repeats. (C) GF-starved 184A1 cells were pulsed for 1 hour with EGF, washed, and incubated for 7 hours in starvation medium, followed by a second EGF pulse. Cells were treated with U0126 at the indicated concentrations, 30 minutes prior to and throughout the second pulse. Immunoblotting was used to quantify EGR1 levels of induction relative to the level at 1E-7S-1E, normalized to actin. (D) GF-starved 184A1 cells were pulsed for 1 hour with EGF (“1E”, red), IGF-1 (“1IGF”, purple), or left untreated (“1S”, green). Following GF removal, cells were incubated in starvation medium for 7 hours, followed by a second pulse of EGF or IGF-1, as indicated. For BrdU incorporation analysis see Figure S4D. To determine EGR1 fold induction, mRNA was isolated at the end of each pulse (blue triangles), and cDNA analysed by RT-qPCR. The right column schematically presents time profiles of EGR1’s patterns of expression. (E) GF-starved 184A1 cells were treated and analyzed as in Figure 1A. The shaded rectangle indicates transfection with control or EGR1-specific siRNA oligonucleotides.

Figure 5

The second pulse down-regulates anti-proliferative genes induced by the first pulse. (A) 184A1 cells were treated with two pulses of EGF. Shown are centered and normalized levels of anti-proliferation genes (marked in black are known p53 targets) from the profile “down-regulated by a second pulse” (see Figure S3F). (B) 184A1 cells were treated as in Figure 4D. Where indicated, cells were treated for 30 minutes with LY294002. BrdU incorporation results are presented in Figure 2E and Figure S4D. To determine fold change, mRNA was isolated before the second pulse and 60 minutes after completion of the pulse (blue triangles), and cDNA analysed by RT-qPCR. Listed are the ratios of expression levels after and before the second pulse. (C) GF-starved cells were pulsed for 60 minutes with EGF, washed, and incubated in starvation medium for 7 hours, followed by a second pulse. At the indicated time points, cells were harvested for a chromatin association assay and DNA-bound proteins isolated and analysed by immunoblotting. (D) Cells were transfected with control or p53-specific siRNAs. Twenty-four hours later, the cells were re-plated on cover-slips, and 24 hours later, they were GF-starved for 16 hours, treated with EGF, and BrdU incorporation measured. Bars represent standard errors calculated from 15 non-overlapping photomicrograph fields. P-values of two-tailed student’s T-test are indicated. (E) NIH-3T3 cells were transfected with control or p53-specific siRNAs. Twenty-four hours later, the cells were re-plated, and 24 hours later they were GF-starved for 24 hours, treated for 1 hour without (“1S”, green) or with PDGF (“1P”, blue), washed, and incubated in starvation medium for 7 hours, followed by a second PDGF pulse. Cells were left in starvation medium or PDGF-containing medium for additional 18 hours, and then fixed and stained with methyl violet. Representative photomicrographs are shown (right part). Cell-occupied area was measured from five photomicrographs (left part). Bars represent standard errors of triplicates. The experiment was repeated twice.

Figure 6

Schematic representations of the proposed biochemical events elicited by a single and a dual EGF pulse (shown in red). The first pulse of EGF induces expression of lipid biosynthesis-associated genes, along with activation of p53. The latter propels expression of anti-proliferation genes, such as BTG2 and SESN1. When cells are treated with a second pulse of EGF, enhanced activation of ERK and subsequent induction of EGR1 exceed a critical threshold. In parallel, signaling through PI3K, and the resulting suppressed expression of anti-proliferative genes, permit cells to cross the restriction point (R) and enter the S-phase.