Absence of Suppressor of Cytokine Signalling 3 Reduces Self-Renewal and Promotes Differentiation in Murine Embryonic Stem Cells

Abstract

Abstract Leukemia inhibitory factor (LIF) is required to maintain pluripotency and permit self-renewal of murine embryonic stem (ES) cells. LIF binds to a receptor complex of LIFR-β and gp130 and signals via the Janus kinase–signal transducer and activator of transcription (JAK–STAT) pathway, with signalling attenuated by suppressor of cytokine signalling (SOCS) proteins. Recent in vivo studies have highlighted the role of SOCS-3 in the negative regulation of signalling via gp130. To determine the role of SOCS-3 in ES cell biology, SOCS-3–null ES cell lines were generated. When cultured in LIF levels that sustain self-renewal of wild-type cells, SOCS-3–null ES cell lines exhibited less self-renewal and greater differentiation into primitive endoderm. The absence of SOCS-3 enhanced JAK–STAT and extracellular signal–related kinase 1/2 (ERK-1/2)–mitogen-activated protein kinase (MAPK) signal transduction via gp130, with higher levels of phosphorylated STAT-1, STAT-3, SH-2 domain–containing cytoplasmic protein tyrosine phosphatase 2 (SHP-2), and ERK-1/2 in steady state and in response to LIF stimulation. Attenuation of ERK signalling by the addition of MAPK/ERK kinase (MEK) inhibitors to SOCS-3–null ES cell cultures rescued the differentiation phenotype, but did not restore proliferation to wild-type levels. In summary, SOCS-3 plays a crucial role in the regulation of the LIF signalling pathway in murine ES cells. Its absence perturbs the balance between activation of the JAK–STAT and SHP-2–ERK-1/2–MAPK pathways, resulting in less self-renewal and a greater potential for differentiation into the primitive endoderm lineage.