PKC-δ inhibitors sustain self-renewal of mouse embryonic stem cells under hypoxiain vitro

Hyo-Jong Lee,Kyu-Won Kim,Jong-Ho Cha,Chul-Ho Jeong

doi:10.3858/emm.2010.42.4.028

Abstract

Under hypoxia, mouse embryonic stem cells (mESCs) lose their self-renewal activity and display an early differentiated morphology mediated by the hypoxia-inducible factor-1 alpha (HIF-1 alpha). Previous studies have demonstrated that PKC-delta is activated by hypoxia and increases the protein stability and transcriptional activity of HIF-1 alpha in human cancer cells. Furthermore, activation of PKC-delta mediates cardiac differentiation of ESCs and hematopoietic stem cells. However, the role of PKC-delta in hypoxia-induced early differentiation of mESCs remains largely unknown. Here, we show the inhibition of PKC-delta activity prevents the early differentiation of mESCs under hypoxia using PKC-delta inhibitors, GF 109203X and rottlerin. Reduction of PKC-delta activity under hypoxia effectively decreased HIF-1 alpha protein levels and substantially recovered the expression of LIF-specific receptor (LIFR) and phosphorylated-STAT3 in mESCs. Furthermore, PKC-delta inhibitors aid to sustain the expression of self-renewal markers and suppress the expression of early differentiation markers in mESCs under hypoxia. Taken together, these results suggest that PKC-delta inhibitors block the early differentiation of mESCs via destabilization of HIF-1 alpha under hypoxia.

Highlights

The mouse embryonic stem cells (mESCs) derived from the inner cell mass of blastocysts have been used for the research fields including molecular mechanism and cell-fate in early mammalian development, because of their potentials to generate any type of cell (Evans and Kaufman, 1981)
The effect of PKC-δ and their specific inhibitors varies depending on the dose, the duration of the treatment, and the types of the treated cells, we examined the role of PKC inhibitors on the hypoxia-induced differentiation of mESCs
These results suggest that PKC-δ inhibitors destabilize hypoxia-inducible factor-1α (HIF-1α) protein levels under hypoxia in mESCs

Summary

Introduction

The mESCs derived from the inner cell mass of blastocysts have been used for the research fields including molecular mechanism and cell-fate in early mammalian development, because of their potentials to generate any type of cell (Evans and Kaufman, 1981). In a related signaling pathway, LIF binds to the LIFR with low-affinity, followed by heterodimerization with glycoprotein 130 This high-affinity complex activates the Janus kinase (JAK) and the signal transducer and activator of transcription 3 (STAT3) signaling pathways (Ernst et al, 1999; Tighe and Gudas, 2004). Embryos and their tissues are generally exposed to pO2 values (∼5% O2, or hypoxia) that are lower than atmospheric pO2 values (20% O2, or normoxia) (Land, 2004; Powers et al, 2008). Hypoxic regions in normal embryo development (Lee et al, 2001) and even after a well-developed vascularization (Powers et al, 2008) include reproductive tract (Masuda et al, 2000), the microvasculature (Intaglietta et al, 1996), and the ocular vitreous (Shui et al, 2006)

Methods

Results

Conclusion