The tyrosine phosphatase, SHP-1, acts on multiple tyrosine kinase receptors to negatively regulate human cytotrophoblast proliferation
Karen Forbes, Laura Skinner, John Aplin & Melissa Westwood
Pregnancy complications such as fetal growth restriction are associated with abnormal placental cell (cytotrophoblast) proliferation and apoptosis. Regulation of these events is unclear but recently we have used a placental explant model to demonstrate that IGFs influence cytotrophoblast kinetics and demonstrated that the protein tyrosine phosphatase (PTP) SHP-2 is required for IGF actions in the placenta. However, SHP-2 accounts for only 20% of total PTP activity, suggesting other PTPs may also be important. mRNA for a closely related PTP, SHP-1, has been reported in the placenta but its actions are unknown; in other systems it functions as a negative regulator of signalling events. We examined the hypothesis that SHP-1 negatively regulates IGF actions by measuring cytotrophoblast proliferation following siRNA-mediated knockdown of SHP-1.
SHP-1 siRNA or non-targeting siRNA (500 nM) was delivered to BeWo cells or first trimester villous tissue explants. Cells and tissue were maintained in culture for 72 h, then treated with IGF1 or IGF2 (10 nM) for a further 24 h before western blot, immunohistochemical (IHC) and QPCR analysis. Following exposure to SHP-1 siRNA, SHP-1 expression was reduced in both BeWo cells (~85% knockdown) and in first trimester explants (~73%). IHC analysis for cell proliferation (Ki67) demonstrated that SHP-1 siRNA had no effect on IGF-induced proliferation but significantly enhanced levels of basal (serum-free) proliferation in both BeWo cells (from 19.7.4±2.6 to 52.3±2.9%; P<0.05, n=4) and first trimester explants (from 22.3±3.7 to 63.8±2.7%; P<0.05, n=4). To elucidate the mechanism(s) by which SHP-1 regulates basal but not IGF-induced proliferation, the activation status of multiple receptor tyrosine kinases (RTKs) was examined using antibody arrays and IHC following depletion of SHP-1. Following SHP-1 knockdown there was enhanced activation of EGFR and TrkB, suggesting that under basal conditions SHP-1 may interact with these molecules to inhibit proliferation.
This study demonstrates a role for SHP-1 in human trophoblast and establishes SHP-1 as negative regulator of multiple RTKs that regulate placental growth.