Hyperosmolar Stress Induces Global mRNA Responses in Placental Trophoblast Stem Cells that Emulate Early Post-implantation Differentiation

Abstract 

Hyperosmolar stress acts in two ways on the implanting embryo and its major constituent, placental trophoblast stem cells (TSC). Stress causes homeostasis that slows development with lesser cell accumulation, increased cell cycle arrest, and apoptosis. Stress may also cause placental differentiation at implantation. To test for the homeostatic and differentiation-inducing consequences of stress, TSC were exposed to hyperosmolar stress for 24h and tested using whole mouse genome arrays and Real-time quantitative (Q)PCR. At 0.5h, all 31 highly changing mRNA (>1.5-fold compared with unstressed TSC) decreased, but by 24h 158/288 genes were upregulated. Many genes upregulated at 24h were near baseline levels in unstressed TSC, suggesting new transcription. Thus few genes change during the early stress response, but by 24h TSC have adapted to start new transcription with large gene sets. Types of genes upregulated at 24h included homeostatic genes regulating growth and DNA damage induced (GADD45β/γ), activator protein (AP)-1 (junB/junC/ATF3/4), heat shock proteins (HSP22/68), and cyclin-dependent kinase inhibitor [CDKI; p15, p21]. But, stress also induced transcription factors that mediate TSC differentiation to trophoblast giant cells (TGC) (Stra13, HES1, GATA-binding2), placental hormones [proliferin, placental lactogen (PL)1, prolactin-like protein (PLP)M], and extracellular matrix genes (CCN1/2). Transcription factors for later placental cell lineages, spongiotrophoblast (MASH2, TPBPα) and syncytiotrophoblast (GCM1, TEF5) and placental hormones (PLPA, PLII) were not induced by 24h stress. Thus stress induced the temporal and spatial placental differentiation normal after implantation. Although differentiation was induced, markers of TSC stemness such as inhibitor of differentiation (ID)2 remained at 100% of levels of unstressed TSC, suggesting that retained mRNA might mediate dedifferentiation were stress to subside.

Keywords: Microarray, Placental trophoblast stem cells, Hyperosmolar stress, Differentiation

Abbreviations: AMPK, AMP-activated protein kinase, AP1, activator protein, AP2γ, activating enhancer-binding protein, ATF, activating transcription factor, CDKI, cyclin-dependent kinase inhibitor, Cdx2, caudal type homeobox, CTGF/CCN, connective tissue growth factor, DDIT, DNA damage inducible, DES, diethyl stilbestrol, EED, extra-embryonic development, Errβ, estrogen receptor related, FGF4, fibroblast growth factor, GADD45, growth and DNA damage induced, GCM1, glial cells missing, HAND1, heart and mesoderm inducer, HES1, Hairy enhancer of split, HSP, heat shock protein, LIF, leukemia inhibitory factor, MASH2, mammalian achaete scute complex homologue, MSX2/Hox8, msh homeobox, p38MAPK, mitogen-activated protein kinase, PLII, placental lactogen 2, PLF, proliferin, PLPA, prolactin-like protein A, STRA13, stimulated by retinoic acid, TEF5, TPBPα, trophoblast specific protein, SAPK/JNK, stress-activated protein kinase/Jun Kinase