Journal of Cell Science RESEARCH ARTICLE STAT3 and MCL-1 associate to cause a mesenchymal epithelial transition A. P. Renjini, Shiny Titus, Prashanth Narayan, Megha Murali, Rajesh Kumar Jha and Malini Laloraya* ABSTRACT Embryo implantation is effected by a myriad of signaling cascades acting on the embryo–endometrium axis. Here we show, by using MALDI TOF analysis, far-western analysis and colocalization and co-transfection studies, that STAT3 and MCL-1 are interacting partners during embryo implantation. We show in vitro that the interaction between the two endogenous proteins is strongly regulated by estrogen and progesterone. Implantation, pregnancy and embryogenesis are distinct from any other process in the body, with extensive, but controlled, proliferation, cell migration, apoptosis, cell invasion and differentiation. Cellular plasticity is vital during the early stages of development for morphogenesis and organ homeostasis, effecting the epithelial to mesenchymal transition (EMT) and, the reverse process, mesenchymal to epithelial transition (MET). STAT3 functionally associates with MCL-1 in the mammalian breast cancer cell line MCF7 that overexpresses STAT3 and MCL-1, which leads to an increased rate of apoptosis and decreased cellular invasion, disrupting the EMT. Association of MCL-1 with STAT3 modulates the normal, anti- apoptotic, activity of MCL-1, resulting in pro-apoptotic effects. Studying the impact of the association of STAT3 with MCL-1 on MET could lead to an enhanced understanding of pregnancy and infertility, and also metastatic tumors. KEY WORDS: STAT3–MCL-1 interaction, Apoptosis, Invasion, Epithelial mesenchymal transition, MET INTRODUCTION Successful implantation, and thereby pregnancy, is a hormone- driven process. Several essential cytokines and growth factors regulate the steroidal actions of hormones in order to prepare the endometrium for implantation. STAT3, a versatile member of the family known as ‘signal transducers and activators of transcription’ (STAT), mediates the axial responses of cytokines. STAT3 is involved in normal cellular responses, as well as oncogenesis (Takeda and Akira, 2000). In resting cells, STATs, including STAT3, are localized to the cytoplasm. Upon activation by cytokines, STATs are known to be phosphorylated by Janus kinases, which leads to formation of homo- or heterodimers through interactions between the Src homology 2 (SH2) domains and phosphorylated tyrosine residues. STAT3 then rapidly translocates to the nucleus, where it increases the expression of target genes. STAT3 plays an essential role in interleukin (IL)-9- induced expression of primary response genes, such as c-Myc and Cited2 (Zhu et al., 1997). Increased phosphorylation of STAT3 is known to be associated with elevated expression of potential downstream targets of STAT3 – these include the genes encoding apoptosis inhibitors [survivin, MCL-1, HSP27 (also known as HSPB1), adrenomedullin and Bcl-xL], cell cycle regulators (cyclin D1, cyclin-dependent kinase inhibitor 1, c-Fos, MAP2K5 and c- Myc) and inducers of tumor angiogenesis [vascular endothelial growth factor, COX-2 (also known as PTGS2) and the matrix metalloproteases (MMP)-2, MMP-10 and MMP-1] in invasive breast cancer tissues (Hsieh et al., 2005; Sinibaldi et al., 2001). The pleiotropic response of STAT3 has been attributed to its ability to act downstream of receptors for a number of IL-6-family cytokines, including IL-6, IL-11, ciliary neuorotrophic factor, oncostatin M, leukemia inhibitory factor (LIF) and interferon family members (IL-10, IFN-c and IFN-a) (Heinrich et al., 1998; Kisseleva et al., 2002). Furthermore, STAT3 has been shown to mediate transcriptional responses to granulocyte colony- stimulating factor, leptin (Akira, 1997), receptor tyrosine kinases (epidermal growth factor, colony stimulating factor 1 and platelet- derived growth factor) and IL-2 family members (IL-2, IL-7 and IL-15) (Zhong et al., 1994). Members of the IL-6 family of cytokines are expressed during implantation in the mouse (Bhatt et al., 1991) and human (Charnock-Jones et al., 1994; Nachtigall et al., 1996). In vivo STAT3 activation, induced by LIF alone, is restricted to day 4 of pregnancy, resulting in the localization of STAT3 specifically to the nuclei of cells in the luminal epithelium, which coincides with the onset of uterine receptivity (Cheng Jr et al., 2001). Targeted disruption of Stat3 revealed that this protein is essential for early embryonic development (Takeda et al., 1997). STAT3 signaling also plays a crucial role in the induction of antigen-specific T-cell tolerance (Cheng et al., 2003). Thus, in order to elucidate the role of STAT3 in the process of embryo implantation, we investigated which proteins this transcription factor interacts with at the time of embryo implantation. In this study, we show that STAT3 physically and functionally interacts with its downstream target myeloid cell leukemia-1 (MCL-1) during the ‘window of implantation’. We demonstrate the interaction using co-immunoprecipitation, far-western analysis, colocalization of the two proteins in uterine sections, and with co- transfection studies in MCF7 cells, and show that the interaction is strongly mediated by the ovarian hormones estrogen and progesterone. The epithelial mesenchymal transition (EMT) is characterized by changes in protein expression, increased invasion and resistance to apoptosis. In this study, we show that STAT3 interacts with MCL-1, which leads to increased apoptosis, decreased invasion and upregulation of certain proteins, such as E-cadherin (also known as cadherin-1) and cytokeratins, which are normally downregulated during the EMT. Utero-Embryo Repromics Lab, Division of Molecular Reproduction, Rajiv Gandhi Centre for Biotechnology, Thycaud PO, Poojappura, Thiruvananthapuram 695 014, Kerala, India. *Author for correspondence (laloraya@rgcb.res.in; laloraya@gmail.com) Received 10 July 2013; Accepted 1 January 2014 ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1738–1750 doi:10.1242/jcs.138214 1738