Somatostatin receptor type 2 contributes to the self-renewal of murine embryonic stem cells

Abstract

Aim: The roles of G-protein coupled receptors (GPCRs) in stem cell biology remain unclear. In this study, we aimed to identify GPCRs that might contribute to the self-renewal of mouse embryonic stem cells (mESCs).

Methods: The expression levels of pluripotent genes and GPCR gene were detected in E14 mESCs using PCR array and RT-PCR. Immunofluorescent staining was used to examine the expression of pluripotent markers and the receptor translocation. Western blot analysis was used to detect phosphorylation of signal proteins. Knock-down of receptor was conducted to confirm its role in pluripotency maintenance.

Results: In leukemia inhibitory factor (LIF)-free medium, mESCs lost the typical morphology of pluripotency, accompanied by markedly decreases in expression of somatostatin receptor type 2 (SSTR2), as well as the pluripotency biomarkers Oct4, Sox2, Rex1 and Nanog. Addition of the SSTR2 agonist octreotide or seglitide (0.1-30 μmol/L) in LIF-free medium dose-dependently promoted the self-renewal of mESCs, whereas the SSTR2 antagonist S4 (0.03-3 μmol/L) dose-dependently blocked octreotide-induced self-renewal. Knock-down of SSTR2 significantly decreased the self-renewal of mESCs even in the presence of LIF. Addition of LIF (1000 U/mL) or octreotide (1 μmol/L) in LIF-free medium significantly increased both phosphorylation and nuclear ocalization of STAT3.

Conclusion: The activation of SSTR2 contributes to the self-renewal of mESCs via activation of the STAT3 pathway.

Figure 1

Reduced expression of SSTR2 in mESCs cultured in LIF-deprived medium. (A) Morphology of E14 mouse ES cells cultured in basal mES medium (no LIF), or media supplemented with LIF (1000 U/mL) or 2i (1 μmol/L PD0325901 and 3 μmol/L CHIR99021) for 4 d (Scale bar: 50 μm). (B) Quantitative RT-PCR analysis of pluripotency genes and SSTR2 in the E14 cells described in (A). (C) Western blot analysis of SSTR2 in mESCs cultured in basal mES medium (no LIF) or media supplemented with LIF or 2i. The data are the mean±SEM (n=3). ^bP<0.05, ^cP<0.01 vs the LIF(−) group.

Figure 2

Activation of SSTR2 prevents mESC differentiation caused by LIF deprivation. (A) Morphology and alkaline phosphatase (AP) staining of E14 cells cultured in mES media containing LIF (1000 U/mL) or mES media without LIF but supplemented with 2i (1 μmol/L PD0325901 and 3 μmol/L CHIR99021) or the SSTR2 agonist octreotide (1 μmol/L) (Scale bar: 50 μm). (B) The percent of undifferentiated colonies (according to AP staining and morphology) of E14 cells cultured in mES media containing LIF, 2i, octreotide or seglitide (SSTR2 agonist) at various concentrations for 3 d. The data are the mean±SEM (n=3). ^cP<0.01 vs LIF(−) group. (C) Quantitative RT-PCR analysis of pluripotency genes and SSTR2 in cells corresponding to (A). Data are the mean±SEM (n=3). ^bP<0.05, ^cP<0.01 vs LIF(+) condition. ^eP<0.05, ^fP<0.01 vs LIF(−) condition. (D) Immunofluorescence staining of pluripotency markers (Oct4, Nanog, and SSEA1) in mESCs cultured in octreotide (1 μmol/L)-containing, LIF-free media. (Scale bar: 10 μm). (E) AP staining of E14 cells pre-incubated with various concentrations of the SSTR2 antagonist S4 for 24 h and then cultured in LIF-free media containing octreotide (1 μmol/L) (Scale bar: 50 μm). (F) The percent of undifferentiated colonies (according to AP staining and morphology) of E14 cells cultured in the indicated media. ^cP<0.01 vs LIF(−) condition. ^fP<0.01 vs octreotide alone.

Figure 3

Knock-down of SSTR2 induces mESC differentiation, even in the presence of LIF. (A) Validation of the knock-down efficiency by shRNA targeting SSTR2 using quantitative RT-PCR. (B) E14 cells (initial density of 10 000 cells/well in a 24-well plate) transfected with SSTR2 shRNA or scramble shRNA were cultured in mES media with or without LIF (1000 U/mL) or LIF-free mES medium supplemented with 2i for 3 d. The percent of undifferentiated colonies was calculated at passages 1 and 2. The data are the mean±SEM (n=3). ^bP<0.05, ^cP<0.01 vs scramble shRNA. (C) Quantitative RT-PCR analysis of pluripotency genes in mouse ES cells expressing SSTR3 shRNA or scramble shRNA cultured in mES media with LIF (passage 2). The data are the mean±SEM (n=3). ^bP<0.05, ^cP<0.01 vs scramble shRNA. (D) AP and immunofluorescence staining of pluripotency markers (Oct4, Nanog, and SSEA1) of the cells described in (C). (E) Morphology of E14 cells expressing the indicated shRNAs cultured in mES media supplemented with LIF and/or octreotide (1 μmol/L) (Scale bar: 50 μm).

Figure 4

Activation of SSTR2 induces phosphorylation of STAT3. (A, B) Representative Western blot and statistical analyses of STAT3 phosphorylation in E14 cells stimulated with LIF (1000 U/mL) (A) or octreotide (1 μmol/L) (B) for various durations. The effect of S4 (1 μmol/L) on LIF- or octreotide-induced phosphorylation was also tested. The data are the mean±SEM (n=3). ^bP<0.05, ^cP<0.01 vs control. ^fP<0.01 vs cells treated with LIF or octreotide. (C) Representative confocal images of immunofluorescent staining of STAT3 in E14 cells stimulated with LIF (1000 U/mL, 10 min) or octreotide (1 μmol/L, 1 h) (Scale bar: 10 μm). (D) Statistical analysis of the percent nuclear localization of STAT3 presented in (C). The data are the mean±SEM (n=10 cells). ^cP<0.01 vs LIF(−) control. (E) Western blot analysis of STAT3 phosphorylation in the nuclei and cytoplasm of mESCs stimulated with LIF (1000 U/mL, 10 min) or octreotide (1 μmol/L, 1 h). Oct4 was used as a nuclear marker, and GAPDH was used as a cytoplasmic marker.