Protein Phosphatase Magnesium Dependent 1A (PPM1A) Plays a Role in the Differentiation and Survival Processes of Nerve Cells

The serine/threonine phosphatase type 2C (PPM1A) has a broad range of substrates, and its role in regulating stress response is well established. We have investigated the involvement of PPM1A in the survival and differentiation processes of PC6-3 cells, a subclone of the PC12 cell line. This cell line can differentiate into neuron like cells upon exposure to nerve growth factor (NGF). Overexpression of PPM1A in naive PC6-3 cells caused cell cycle arrest at the G2/M phase followed by apoptosis. Interestingly, PPM1A overexpression did not affect fully differentiated cells. Using PPM1A overexpressing cells and PPM1A knockdown cells, we show that this phosphatase affects NGF signaling in PC6-3 cells and is engaged in neurite outgrowth. In addition, the ablation of PPM1A interferes with NGF-induced growth arrest during differentiation of PC6-3 cells

PPM1A overexpression and PC6-3 cells differentiation.

<p><i>(</i><b><i>A</i></b><i>)</i> Cell proliferation assay. PC6-3 PPM1A wt cells were incubated with or without NGF and with or without Tet. The cells were then assayed for viability at the indicated times using XTT assay. The average of 6 different wells ± S.D. from a representative experiment was plotted. <i>(</i><b><i>B</i></b><i>)</i> FACS analysis of PC6-3 cells preincubated with Tet for 24 hours followed by addition of NGF for the indicated times. The table represents % of total counted cells of one experiments out of three performed. <i>(</i><b><i>C</i></b><i>)</i> Western blot analysis of extracts from cells treated for 144 hours with NGF with or without Tet, and assayed for PPM1A expression level with anti-PPM1A antibodies.</p

PPM1A overexpression induces cell cycle arrest.

<p>FACS analysis <i>(</i><b><i>A</i></b><i>)</i>, and BrdU labeling <i>(</i><b><i>B</i></b><i>)</i> of PC6-3 cells overexpressing PPM1A wt. Cells were treated as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032438#s2" target="_blank">Materials and Methods</a>” and analyzed for DNA content by propidium iodide (PI) staining <i>(</i><b><i>A</i></b><i>)</i> and for BrdU labeling by anti-BrdU antibodies as represented by the dot plot of DNA content (PI) against BrdU incorporation <i>(</i><b><i>B</i></b><i>)</i>. <i>(</i><b><i>C</i></b><i>)</i> FACS analysis of PC6-3 cells overexpressing PPM1A mutant. Cells were incubated with or without Tet, and then analyzed for DNA content by propidium iodide (PI) staining. The numbers represented in the tables indicate % of total cells in <i>(</i><b><i>A</i></b><i>)</i> and <i>(</i><b><i>C</i></b><i>)</i>, and % of total BrdU labeled cells in <i>(</i><b><i>B</i></b><i>)</i>. The experiments in <i>(</i><b><i>A</i></b><i>)</i>, <i>(</i><b><i>B</i></b><i>)</i> and <i>(</i><b><i>C</i></b><i>)</i> were performed at least three times.</p

PPM1A expression affects neurite outgrowth.

<p><i>(</i><b><i>A</i></b><i>)</i> PC6-3 PPM1A wt cells were seeded on collagen coated plates and incubated with or without NGF and Tet. <i>(</i><b><i>B</i></b><i>)</i> PC6-3 siLacZ and siα cells were seeded in the same manner and treated with or without NGF. Phase contrast images were taken at the indicated time periods. The histogram represents quantification of neurite formation from one out of three independent experiments. Cells with processes twice longer than cell body were counted as positive. At least 100 cells were counted from 5 different areas of each plate and the average numbers are represented with standard deviation. P-Values: * P-Values<0.01; ** P-Values<0.0005; *** P-Values<0.03; **** P-Values<0.03; ***** P-Values<0.05.</p

PPM1A over and underexpression and cell signaling during PC6-3 cells differentiation.

<p><i>(</i><b><i>A</i></b><i>)</i> PPM1A wt cells were incubated with or without Tet for 24 hours and then NGF was added for the indicated times. <i>(</i><b><i>B</i></b><i>)</i> PC6-3 PPM1A knock-down cells and control cells were incubated with NGF for the indicated times. The cells were harvested and lysed. Cell extracts were analyzed by western blot using the indicated antibodies. Please note that the AKT and PPM1A panels in <i>(</i><b><i>B</i></b><i>)</i> are from different gels of the same experiments. The graphs represent quantification of the blots shown in <i>(</i><b><i>A</i></b><i>)</i> and <i>(</i><b><i>B</i></b><i>)</i>. The results shown are from representative experiments out of 3 independent that were performed.</p

PPM1A overexpression affects cell cycle and survival markers.

<p><i>(</i><b><i>A</i></b><i>)</i> PC6-3 cells overexpressing wt or mutant PPM1A were incubated with or without Tet for the indicated times. The cells were then harvested and lysed. Cell extracts were analyzed by western blot for levels of phosphorylated AKT, total AKT and PPM1A. Note that PPM1A mutant migrates faster than the wt on the gel (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032438#pone.0032438-Ofek1" target="_blank">[10]</a>) and that even in the absence of Tet there was a slight expression due to leakiness of PPM1A mutant. PPM1A wt cells were analyzed in the same manner for levels of <i>(</i><b><i>B</i></b><i>)</i> cleaved caspase 3 and p38. <i>(</i><b><i>C</i></b><i>)</i> PC6-3 cells overexpressing PPM1A were incubated with or without Tet for 24 hours. The cells were then harvested and lysed and cell extracts were analyzed for levels of p53, Mdm2, PPM1A and p38 by western blot. The numbers represent the ratio of p53/p38 (0.1, 0.07) and Mdm2/p38 levels (0.5, 0.5). The blots presented in <i>(</i><b><i>A</i></b><i>) (</i><b><i>B</i></b><i>)</i> and <i>(</i><b><i>C</i></b><i>)</i> are from representative experiments out of three performed.</p

Phosphorylation of p38 during NGF treatment is increased in PPM1A underexpressed PC6-3 cells.

<p>PC6-3 PPM1A knock-down cells and control cells were incubated with NGF for the indicated times. The cells were harvested and lysed. Cell extracts were analyzed by western blot using the indicated antibodies. The graph represents quantification of the phospho-p38 levels. The results shown are from representative experiments out of 3 independent that were performed.</p

Proliferation assay of PPM1A knock-down cells.

<p><i>(</i><b><i>A</i></b><i>)</i> PC6-3 cells were transduced with pSuperRetro-PPM1A or pSuperRetro-LacZ. Forty eight hours later cells were selected by the addition of puromycin. The protein levels of PPM1A were analyzed by western blot. The graph represents the quantification of PPM1A levels. <i>(</i><b><i>B</i></b><i>)</i> and <i>(</i><b><i>C</i></b><i>)</i> Proliferation assay of PPM1A knock-down cells compared to normal cells in the absence <i>(</i><b><i>B</i></b><i>)</i> or presence <i>(</i><b><i>C</i></b><i>)</i> of NGF. The average of 6 different wells ± standard deviation from a representative experiment out of 3 independent experiments that were performed was plotted. * P-value of 120 hours <0.002.</p