Blocking Epidermal Growth Factor Receptor Signaling in HTR-8/SVneo First Trimester Trophoblast Cells Results in Dephosphorylation of PKBα/AKT and Induces Apoptosis

Abstract

We identified a major peptide signaling target of EGF/EGFR pathway and explored the consequences of blocking or activating this pathway in the first trimester extravillous trophoblast cells, HTR-8/SVneo. A global analysis of protein phosphorylation was undertaken using novel technology (Kinexus Kinetworks) that utilizes SDS-polyacrylamide minigel electrophoresis and multi-lane immunoblotting to permit specific and semiquantitative detection of multiple phosphoproteins. Forty-seven protein phosphorylation sites were queried, and the results reported based on relative phosphorylation at each site. EGF- and Iressa-(gefitinib, ZD1839, an inhibitor of EGFR) treated HTR-8/SVneo cells were subjected to immunoblotting and flow cytometry to confirm the phosphoprotein screen and to assess the effects of EGF versus Iressa on cell cycle and apoptosis. EGFR mediates the phosphorylation of important signaling proteins, including PKBα/AKT. This pathway is likely to be central to EGFR-mediated trophoblast survival. Furthermore, EGF treatment induces proliferation and inhibits apoptosis, while Iressa induces apoptosis.

STAT5 is Activated by Epidermal Growth Factor and Induces Proliferation and Invasion in Trophoblastic Cells

“…41 Previous studies in HTR-8/SVneo cells demonstrated that EGF triggers STAT5 phosphorylation but does not induce phosphorylation of either STAT1 or STAT3. 7 This study goes in line with our results demonstrating activation of STAT5 but not STAT3 after EGF treatment in HTR8/SVneo and JAR cells. These observations suggest that in trophoblast cells, EGF effects are mediated via STAT5 rather than via STAT3.…”

“…Epidermal growth factor can activate STAT1, STAT3, or STAT5 either simultaneously or individually in a cell typedependent manner. 7,37,38 Treatment of EGF results in increase in only p-STAT3 in kidney tubular epithelial cells 39 and only p-STAT5 in U87 glioma cells. 40 In human breast cancer, STAT3 and STAT5 are activated simultaneously by EGF, 38 while in squamous carcinoma cells, EGF activates STAT1 and STAT3.…”

“…5 One of the most relevant is the epidermal growth factor (EGF), which is important for embryo implantation, trophoblast differentiation, and endocrine functions of the placenta. 6,7 Several studies on different cell models have demonstrated that EGF influences positively or negatively a variety of fundamental cell properties such as proliferation, [8][9][10] differentiation, 8 inhibition of apoptosis, [11][12][13][14] motility, [15][16][17] secretion, 15,18 angiogenesis, 7 and invasion/ migration. 15,[19][20][21] However, the role of EGF in the regulation of trophoblast behavior and the intracellular mechanisms responsible for these effects remain poorly understood.…”

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

“…As expected, known pathways (e.g., ErbB and insulin signaling pathways; Table S2) were enriched. We identified 7 key upregulated phosphorylation sites in ErbB signaling pathways, such as T693 in EGFR 28 , Y427 in SHC1 29 , S10 in CDKN1B 30 , Y216 in GSK3B 31 , S241 in PDPK1 32 , and S226 33 and T394 in MAP2K2 34, 35 (Figure 6e). In addition, mRNA splicing was found to be activated after EGF treatment (Table S2).…”

Bisphenol S Impairs Invasion and Proliferation of Extravillous Trophoblasts Cells by Interfering with Epidermal Growth Factor Receptor Signaling

“…To test BPS’ EGFR antagonism, HTR-8/SVneo cells were used. HTR-8/SVneo is a first-trimester human extravillous trophoblast cell line with EGFR expression [ 63 ] that proliferates and invade in response to EGF [ 49 ]. HTR-8/SVneo were maintained in basic medium, Dulbecco’s modified Eagle’s medium/F12 medium (Cat#: 124000-024, MilliporeSigma, Saint Louis, MO, USA) supplemented with 10% of fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, 100 IU/mL penicillin, and 100 μg/mL streptomycin.…”