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Conclusions
Author contributions
Conflicts of interest
Acknowledgments
Introduction Self-renewal is an integrated proliferation control that maintains undifferentiated states of stem cells. The integrated control is achieved by multilayered regulatory networks involving complex interplays among an array of cell-intrinsic factors including transcription factors, miRNAs, and epigenetic factors (He et al., 2009). Several transcription factors (TFs), such as OCT4, SOX2, and NANOG, play critical roles to maintain the proliferative undifferentiated state, and their ectopic expression generates pluripotent EZ Cap Reagent GG (3\' OMe) from mouse and human stem cells (Boyer et al., 2005; Loh et al., 2006). Also, cell-extrinsic environmental factors, such as growth factors and cytokines, affect the self-renewal program through their interactions with the components in pluripotency signaling and/or transcriptional networks (Boiani and Scholer, 2005). Several intracellular signaling pathways, such as MAPK and JAK/STAT pathways, are critical to maintain the self-renewal program (Dreesen and Brivanlou, 2007; Hou et al., 2002; Raz et al., 1999). Heat shock is one of common environmental factors, which activates heat shock factors (HSFs) inducing heat shock proteins (HSPs) involved in stress responses. Human and mouse embryonic stem cells exhibit higher stress tolerance than differentiated cells (Prinsloo et al., 2009). Heat shock proteins are constitutively expressed and appear to be required for cellular development. For example, HSP90B knockout mouse embryos failed to develop past E9/9.5 (Voss et al., 2000). Heat shock proteins mediate folding events and also intracellular signaling by controlling nuclear targeting and delivery of signaling molecules, which can affect self-renewal and pluripotency of stem cells (Dasgupta and Momand, 1997; Davies et al., 2002; Galigniana et al., 2004). For example, in mouse embryonic stem cells (mESCs), heat shock proteins (HSP70 and HSP90) mediate dimerization and nuclear import of STAT3 activated by LIF receptor, leading to up-regulation of NANOG (Kretzschmar et al., 2004; Liu et al., 2005; Zhong et al., 1994). However, how heat shock is linked to molecular networks defining self-renewal of stem cells remains still unknown. For instance, whether heat shock affects self-renewal indirectly through HSP-dependent modulation of signaling molecules, as described above, or could also directly control core self-renewal regulators (e.g. OCT4, SOX2, or NANOG) via HSFs is unclear. Here, we investigated the effect of heat shock on self-renewal in H9 hESCs. We first showed that the increase of the culture temperature led to the exit of H9 hESCs from their undifferentiated state. To elucidate mRNA expression changes induced by heat shock, we performed gene expression analysis of hESCs and heat shocked hESCs. The analysis of gene expression data revealed that heat shock increased the expression of many genes involved in cellular processes related to differentiation of stem cells. Self-renewal networks in hESCs are governed by pluripotency inducing TFs, OCT4, SOX2, and NANOG. To investigate the link of heat shock to the self-renewal networks, we thus examined transcriptional control of the expression of the key TFs by heat shock-induced HSFs. The data showed that heat shocked hESCs induced mainly HSF1, which repressed the expression of OCT4. The comparison of HSF1-dependent and OCT4-dependent gene expression changes in hESCs revealed that the heat shock-induced exit of hESCs from their undifferentiated state could be caused by the negative regulation of OCT4 by HSF1. Heat shock activates MAP kinases, the upstream kinases of HSF1. Thus, we then examined the effects of the MAP kinases on the repression of OCT4 expression by HSF1. Among ERK1/2, SAPK/JNK, and p38 kinases activated by heat shock, SAPK/JNK pathway controlled mainly the repression of the OCT4 expression by HSF1. Taken together, the results demonstrate a direct link of heat shock to the self-renewal network by activating SAPK/JNK and HSF1 pathway, which negatively regulates a core stem cell core regulator, OCT4.