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

Xu XX, Zhang LH, Xie X - Acta Pharmacol. Sin. (2014)

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

View Article: PubMed Central - PubMed

Affiliation: 1] CAS Key Laboratory of Receptor Research, the National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China [2] Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptor-based Bio-medicine, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

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.

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Related in: MedlinePlus

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.
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fig2: 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.

Mentions: SSTR2 is one of the five somatostatin receptors (SSTR1-5)11. Various somatostatin analogues, such as octreotide and seglitide12, have been developed for clinical applications through specific activation of SSTR2. To investigate the role of SSTR2 in mESC self-renewal, we supplemented the LIF-free medium with various concentrations of octreotide or seglitide. As shown in Figure 2A and 2B, both agonists displayed dose-dependent promotion of mES cell self-renewal in the absence of LIF, with the most effective concentration being 1 μmol/L. In mES cell culture supplemented with 1 μmol/L octreotide, more than 80% of the colonies maintained the typical compact morphology and strong AP staining, even when LIF was removed (Figure 2A and 2B). RT-PCR revealed that octreotide nearly completely reversed the loss of pluripotency genes, including Oct4, Sox2, Nanog, and Rex1, that was induced by LIF withdrawal. Octreotide also prevented the down-regulation of SSTR2 that was induced by LIF deprivation (Figure 2C). Immunofluorescent staining confirmed that mESCs cultured in octreotide-containing, LIF-free medium expressed high levels of pluripotency markers, including Oct4, Nanog, and SSEA1 (Figure 2D).


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

Xu XX, Zhang LH, Xie X - Acta Pharmacol. Sin. (2014)

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.
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fig2: 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.
Mentions: SSTR2 is one of the five somatostatin receptors (SSTR1-5)11. Various somatostatin analogues, such as octreotide and seglitide12, have been developed for clinical applications through specific activation of SSTR2. To investigate the role of SSTR2 in mESC self-renewal, we supplemented the LIF-free medium with various concentrations of octreotide or seglitide. As shown in Figure 2A and 2B, both agonists displayed dose-dependent promotion of mES cell self-renewal in the absence of LIF, with the most effective concentration being 1 μmol/L. In mES cell culture supplemented with 1 μmol/L octreotide, more than 80% of the colonies maintained the typical compact morphology and strong AP staining, even when LIF was removed (Figure 2A and 2B). RT-PCR revealed that octreotide nearly completely reversed the loss of pluripotency genes, including Oct4, Sox2, Nanog, and Rex1, that was induced by LIF withdrawal. Octreotide also prevented the down-regulation of SSTR2 that was induced by LIF deprivation (Figure 2C). Immunofluorescent staining confirmed that mESCs cultured in octreotide-containing, LIF-free medium expressed high levels of pluripotency markers, including Oct4, Nanog, and SSEA1 (Figure 2D).

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

View Article: PubMed Central - PubMed

Affiliation: 1] CAS Key Laboratory of Receptor Research, the National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China [2] Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptor-based Bio-medicine, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

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.

Show MeSH
Related in: MedlinePlus