The tyrosine phosphatase SHP-1 negatively regulates cytotrophoblast proliferation in first-trimester human placenta by modulating EGFR activation

Abstract

Insulin-like growth factors (IGFs) influence placental cell (cytotrophoblast) kinetics. We recently reported that the protein tyrosine phosphatase (PTP) SHP-2 positively regulates IGF actions in the placenta. In other systems, the closely related PTP, SHP-1, functions as a negative regulator of signaling events but its role in the placenta is still unknown. We examined the hypothesis that SHP-1 negatively regulates IGF actions in the human placenta. Immunohistochemical (IHC) analysis demonstrated that SHP-1 is abundant in cytotrophoblast. SHP-1 expression was decreased in first-trimester placental explants using siRNA; knockdown did not alter IGF-induced proliferation but it significantly enhanced proliferation in serum-free conditions, revealing that placental growth is endogenously regulated. Candidate regulators were determined by using antibody arrays, Western blotting, and IHC to examine the activation status of multiple receptor tyrosine kinases (RTKs) in SHP-1-depleted explants; amongst the alterations observed was enhanced activation of EGFR, suggesting that SHP-1 may interact with EGFR to inhibit proliferation. The EGFR tyrosine kinase inhibitor PD153035 reversed the elevated proliferation seen in the absence of SHP-1. This study demonstrates a role for SHP-1 in human trophoblast turnover and establishes SHP-1 as a negative regulator of EGFR activation. Targeting placental SHP-1 expression may provide therapeutic benefits in common pregnancy conditions with abnormal trophoblast proliferation.

Fig. 1

Knockdown of SHP-1 increases endogenous, but not-IGF-induced, proliferation in placental explants. First-trimester placental tissue fragments were transfected with two different SHP-1 siRNA sequences (SHP-1A and SHP-1B; 100–500 nM). Non-transfected (control), mock transfected (mock), or non-targeting siRNA (500 nM) were used as controls. SHP-1 mRNA expression (a) was analyzed by QPCR 72 h post-transfection and data are displayed as percentage of non-targeting siRNA (500 nM); bar represents median. SHP-1 protein expression was analyzed 72 h post-transfection by Western blotting (b), and immunostaining (c), using an anti-SHP-1 antibody; blots were re-probed using an anti-β actin antibody to confirm equal protein loading. Data were considered significant if p < 0.05 using Wilcoxon signed-rank test. Each panel is representative of at least five independent experiments. Scale bars represent 50 μm. ST syncytiotrophoblast, CT cytotrophoblast. Western blotting was used to assess first-trimester placental explants expression of cyclin-D1 72 h post-transfection (b); C non-transfected, M mock-transfected, NT transfection with non-targeting siRNA. Serum-starved first-trimester placental explants (d, e) were exposed to IGF-I (10 nM) IGF-II (10 nM) or an equal volume (10 μl) of phosphate buffered saline for 24 h. The number of Ki67-positive cytotrophoblast was expressed as a percentage of the total cell number. Bars represent the median (+ interquartile range; IQR). Differences between groups were determined using a Kruskal–Wallis test of variance followed by Dunn’s multiple comparison post hoc test. Data were considered significant when p < 0.05. a Significantly different from non-stimulated, non-transfected; b significantly different from non-stimulated, mock transfected; c significantly different from non-stimulated, non-targeting siRNA. Each image is representative of at least five independent experiments. ST syncytiotrophoblast, CT cytotrophoblast. Scale bars represent 50 μm

Fig. 2

Knockdown of SHP-1 increases endogenous, but not-IGF-induced, proliferation in BeWo cells. BeWo cells were transfected with two different SHP-1 siRNA sequences (SHP-1A and SHP-1B; 100–500nM. Non-transfected (control), mock transfected (mock), or non-targeting siRNA (500 nM) were used as controls; 48 h after transfection, cells were switched to serum-free medium for 24 h and then exposed to IGF-I (10 nM) IGF-II (10 nM) or an equal volume (10 μl) of phosphate buffered saline for a further 24 h. The number of Ki67-positive BeWo cells (a) was expressed as a percentage of the total cell number (b). Bars represent the median (+ interquartile range; IQR). Differences between groups were determined using a Kruskal–Wallis test of variance followed by Dunn’s multiple comparison post hoc test. Data were considered significant when P < 0.05. a significantly different from non-stimulated, non-transfected; b significantly different from non-stimulated, mock transfected; c significantly different from non-stimulated, non-targeting siRNA. Each image is representative of at least five independent experiments

Fig. 3

SHP-1 regulates endogenous receptor tyrosine kinase activation in BeWo cells and in placental explants. a BeWo cells were treated with non-targeting siRNA (500 nM) or SHP-1 siRNA (500 nM) for 72 h, lysed and then samples from four independent experiments were pooled and applied to human phosphorylated RTK array membranes (400 μg protein/array). Proteins on the array membranes were detected by chemiluminescence and the average pixel density for each protein on the membranes was measured using ImageJ software (National Institutes of Health) and normalized using non-RTK controls. Dotted line represents threshold of assay sensitivity, thus only proteins with a mean pixel density higher than 10 were considered. b Localization of IGF1R, EGFR, and TrkB was determined by performing immunohistochemistry on freshly isolated first-trimester placenta. The effect of SHP-1 knockdown on the activation of IGF1R, EGFR, and TrkB in first-trimester placental explants was assessed by Western blotting (c) and immunohistochemistry (d) 72 h after transfection. C untransfected, M mock-transfected, NT siRNA transfected with non-targeting siRNA (500 nM), SHP-1 siRNA transfected with SHP-1 siRNA (500 nM). Representative images from three independent experiments are shown. CT cytotrophoblast, ST syncytium, MVM microvillus membrane. Scale bars on images represent 50 μm

Fig. 4

TrkB does not influence endogenous trophoblast proliferation. First-trimester explants were cultured for 24 h in serum-free conditions and then incubated with the TrkB inhibitory peptide cyclotraxin-B (500 nM to 2 μM) for a further 24 h. Sections were stained using an anti-phospho-TrkB antibody to confirm the inhibitory effect of the peptide (a) or with an anti-Ki67 antibody to assess proliferation. Three random areas from each placenta were counted, and the number of Ki67-positive cells was expressed as a percentage of the total number of cytotrophoblasts (median and IQR) of at least five independent experiments (b). Kruskal–Wallis test of variance followed by Dunn’s multiple comparison post hoc test was used to assess significant (p < 0.05) differences between the groups. ST syncytiotrophoblast, CT cytotrophoblast. Scale bars 50 μm

Fig. 5

Enhanced proliferation following SHP-1 knockdown can be attributed to enhanced EGFR activation. First-trimester placental explants were transfected with non-targeting siRNA (500 nM) or SHP-1 siRNA (500 nM) for 48 h in serum-free conditions. Explants were exposed to the EGFR-specific inhibitor PD153035 (250 nM to 1 μM) for a further 24 h then EGFR activation (a) and cytotrophoblast proliferation (b) was assessed by immunohistochemistry. Three random areas from each placenta were counted, and the number of Ki67-positive cells is expressed as a percentage of the total number of cytotrophoblast (median and IQR) of at least five independent experiments (b). Kruskal–Wallis test of variance followed by Dunn’s multiple comparison post hoc test was used to assess significant (p < 0.05) differences between the groups. a Significantly different from non-treated, non-transfected; b, significantly different from non-treated, SHP-1A siRNA transfected; c significantly different from non-treated, siRNA B siRNA transfected. ST syncytiotrophoblast, CT cytotrophoblast. Scale bars 50 μm