To examine the effect of PKC- inhibitors on LIFR-STAT3 signaling, we cultivated mESCs in the presence of LIF with two kinds of PKC- inhibitors, rotttlerin and GF under hypoxic conditions. these results suggest that PKC- inhibitors block the early differentiation of mESCs via destabilization of HIF-1 under hypoxia. = 3). *, < 0.05; **, < 0.01; #, < 0.001. (B) The expression level of HIF-1 mRNA was examined using RT-PCR. N, normoxia; H, hypoxia. Tubulin and gapdh were used as internal controls. Results are representative of three independent experiments. PKC- inhibitors block the attenuation of LIFR-STAT3 pathway under hypoxia Our previous data clearly suggest that HIF-1 binds to reverse HREs (rHREs) of the LIFR promoter, which leads to a downregulation of LIFR-STAT3 signaling in mESCs under hypoxia (Jeong et al., 2007). To examine the effect of PKC- inhibitors on LIFR-STAT3 signaling, we cultivated mESCs in the presence of LIF with two kinds of PKC- inhibitors, rotttlerin and GF under hypoxic conditions. As indicated, expression of LIFR and phosphorylated-STAT3 was downregulated under hypoxia, whereas treatment with PKC- inhibitors effectively blocked the hypoxia-induced reduction of LIFR and phosphorylated-STAT3, but not of total STAT3 levels (Figure 2A). Importantly, rottlerin markedly upregulated phosphorylated-STAT3 under hypoxia to expression levels similar to those of the control (normoxia). We further confirmed the effect RS 127445 of PKC- inhibitors on the hypoxia-induced differentiation of mESCs using immunofluorescent staining of LIFR and phosphorylated-STAT3. Undifferentiated mESCs cultured under normoxia showed an abundant expression of LIFR in the cytosol and of phosphorylated-STAT3 in the nucleus, whereas the expression of these proteins was decreased under hypoxia (Figure 2B). Interestingly, treatment of mESCs with PKC- inhibitors Rabbit Polyclonal to TAS2R49 under hypoxia sustained the expression of LIFR and phosphorylated-STAT3; therefore, these results suggest that PKC- inhibitors may maintain LIFR-STAT3 signaling under hypoxia. Open in a separate window Figure 2 PKC inhibitors blocked the down-regulation of LIF-STAT3 pathway under hypoxia in mESCs. (A) CCE cells were treated with 5 M GF and 5 M rotttlerin (Rot), and were then exposed immediately to normoxia (N) or hypoxia (H) for 24 h. Western blot analysis of LIF receptor (LIFR), phosphorylated-STAT3 (p-STAT3, at RS 127445 tyrosine 705 residue), and endogenous STAT3 in CCE cells treated with PKC inhibitors. Tubulin was used as internal control. Graph represents mean values S.D. (= 3). RS 127445 *, RS 127445 < 0.05; #, < 0.001 (B) Immunofluorescent staining with LIFR (red) and phosphorylated-STAT3 (at tyrosine 705 residue, green) of cells treated for 24 h with 5 M GF and 5 M rotttlerin and grown under normoxic conditions (N), hypoxic conditions (H) in the presence of LIF. Nuclei are stained with DAPI (blue). Scale bar is 50 m. Maintenance of self-renewal activity in mESCs treated with PKC- inhibitors Based on the effect of PKC- inhibitors on LIFR-STAT3, RT-PCR was conducted to access the state of mESCs. Rex1 and fgf4 are represented markers for mESC stemness and self-renewal activity, whereas fgf5 and STAT5a are related to the early differentiation of mESCs (Jeong et al., 2007). Expression levels of rex1 and fgf4 were decreased under hypoxia, whereas treatment with PKC- inhibitors blocked this suppression of the rex1 and fgf4 (Figure 3). In contrast to self-renewal markers, expression levels of fgf5 and STAT5a were increased under hypoxia, whereas treatment with PKC- inhibitors blocked the increase in fgf5 and STAT5a expression levels. These results demonstrate that PKC- inhibitors maintain the self-renewal state of mESCs and block the early differentiation of mESCs under hypoxia. Open in a separate window Figure 3 PKC inhibitors maintained the self-renewal and blocked the early differentiation of mESCs under hypoxia. CCE cells were treated with 5 M GF and 5 M rottlerin (Rot), and were then exposed immediately to normoxia (N) or hypoxia (H) for 24.