Stem cell factor-induced AKT cell signaling pathway: Effects on porcine trophectoderm and uterine luminal epithelial cells

Highlights

• SCF transcripts increase in endometrium during early pregnancy.

• SCF is a strong inducer of AKT activation in pTr and pLE cells.

• SCF-stimulated AKT activates P70RSK and RPS6 proteins in pTr cells.

• SCF induces transient and long-lasting activation of AKT cascade in pLE cells.

• SCF has stimulatory effect on cell migration through activation of PI3K/AKT cascade.

Abstract

Stem cell factor (SCF) is a multipotent growth factor that elicits diverse biological actions in various aspects of embryogenesis and animal development. The aim of the present study was to assess SCF-induced intracellular signaling and cellular activities in porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells which are well known as useful to elucidate developmental events. SCF induced abundances of p-AKT, p-P70RSK and RPS6 proteins in pTr cells reached to their maximum, and then returned to basal levels by 120 min. In pLE cells, SCF induced protracted effect to increase AKT phosphorylation which was well correlated with the time course for P70RSK and RPS6 phosphorylation. LY294002 (an inhibitor of AKT) decreased SCF-induced p-AKT, p-P70RSK and p-RPS6 proteins. Also, immunofluorescence analyses revealed that p-RPS6 was abundant within the cytoplasm of SCF-treated cells, but p-RPS6 was present only at basal levels in cells treated with LY294002. In the presence of LY294002, both SCF-stimulated transient and sustained AKT phosphorylation were inhibited in pLE cells. Furthermore, SCF increased migration of pTr and pLE cells, but LY294002 significantly reduced this effect of SCF. In conclusion, results of the present study suggest that SCF secreted by the endometrium induces autocrine/paracrine signaling responses that stimulate migration of pTr and pLE cells through activation of the AKT cell signaling pathway. Those results support the hypothesis that SCF is a critical regulatory factor for conceptus development and implantation during pregnancy in pigs.

Introduction

In mammals, the establishment of pregnancy requires bi-directional communication between the developing conceptus and the uterine endometrium. The close association of maternal reproductive tissues with the developing embryo/conceptus (embryo and its associated extra-embryonic membranes) allows for the maternal uterine environment to contribute to growth, development, and differentiation of the embryo/conceptus. One mechanism through which maternal-conceptus signaling can occur is by the exchange of diffusible factors synthesized by the maternal tissues and taken up by the conceptus. An increasing number of growth factors are being discovered that are produced by utero-placental tissues. In pigs, implantation of the conceptus begins on approximately Day 13 of pregnancy, and conceptus-maternal communication is especially pivotal for ensuring viability and successful implantation of the conceptus (Anderson, 1978, Dantzer, 1985, Geisert et al., 1982, Perry et al., 1976). It is during this period that the majority of embryonic loss occurs in the pig which suggests that a well-coordinated network between the conceptus and maternal tissues and regulated by uterine factors is essential for development of the conceptus (Ross et al., 2009; Spencer and Bazer, 2004a, Spencer and Bazer, 2004b).

Among the many growth factors, stem cell factor (SCF, also called steel factor, mast cell growth factor or kit ligand), a primary ligand for c-kit, is known for its powerful pleiotropic roles. SCF is produced as two alternative splice forms; a soluble- and a membrane-bound form (Anderson et al., 1991, Huang et al., 1990, Williams et al., 1990). Both forms of SCF expressed in a variety of tissues bind to SCF receptor (c-kit) and cause dimerization of the receptor which, in turn, activates tyrosine kinase activity and intracellular signal transduction molecules (Blume-Jensen et al., 1991). Activation of SCF/SCF receptor system plays a crucial role in a variety of cells to regulates differentiation, proliferation, resistance to apoptosis, and migration of cells through downstream signaling molecules (Liang et al., 2013). Failure of proper SCF/c-kit signaling results in angiogenesis, proliferation, invasion, and survival of c-kit-expressing tumor cells; therefore, the c-kit gene is also well known as a proto-oncogene (Yasuda et al., 2006). In the reproductive system, c-kit is expressed in a various cell types such as germ cells and stem cells, and SCF appears to regulate these c-kit-expressing cells by accelerating proliferation and increasing survival. Dolci et al. (Dolci et al., 1991) and Matsui et al. (Matsui et al., 1991) have shown that SCF is required for survival of primordial germ cells in vitro. Mice with loss-of-function mutations in c-kit and the SCF loci have altered function in their reproductive system (Blume-Jensen et al., 2000, Kissel et al., 2000, Loveland and Schlatt, 1997). For example, c-kit mutant male and female mice displayed sterility or reduced fertility (Blume-Jensen et al., 2000, Kissel et al., 2000).

SCF/c-kit is detected from the late two-cell stage embryo to hatched blastocysts in both embryonic disc and trophectoderm, as well as in endometrial cells during early pregnancy (Arceci et al., 1992, Mitsunari et al., 1999). SCF is produced by the uterine endometrium of non-pregnant and pregnant gilts (Zhang and Anthony, 1994), but the function of SCF/c-kit system in development of the pre- and peri-implantation porcine conceptus is not known. The aim of present study was to determine changes in cellular activities induced by SCF and to assess SCF-induced intracellular signaling in a porcine primary trophectoderm (pTr) cell line and in uterine endometrial epithelial (pLE) cells during the peri-implantation period of pregnancy. Results of this study indicate differential expression of SCF transcripts in maternal uterine tissues during early pregnancy and suggest that stimulatory effects of SCF on activation of the PI3K/AKT cascades promote migration of pTr and pLE cells.