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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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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).
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fig3: 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).

Mentions: To rule out the possibility of off-target effects by octreotide and S4, we examined the effect of SSTR2 knock-down in mESCs. An shRNA-based technique was employed to specifically knock-down the SSTR2 gene in mESCs cultured in medium containing LIF. Quantitative RT-PCR results revealed that, relative to the scrambled shRNA, the transfection of shRNA targeting SSTR2 led to a dramatic reduction of the SSTR2 mRNA in mESCs 24 h after transfection (Figure 3A). Knock-down of SSTR2 significantly decreased the ratio of undifferentiated E14 cells, even in the presence of LIF (Figure 3B). Cells expressing SSTR2 shRNA displayed a lower expression level of pluripotency genes (Oct4, Sox2, Nanog, and Rex1) (Figure 3C) and showed weak or negative staining for AP and other pluripotent markers, including Oct4, Nanog, and SSEA1 (Figure 3D). Adding LIF or the combination of LIF and octreotide did not rescue the spontaneous differentiation of these SSTR2 knock-down cells (Figure 3E), although the differentiation could be rescued by 2i (Figure 3B). These data confirmed that SSTR2 contributes to mES cell self-renewal and that knock-down of SSTR2 leads to spontaneous differentiation, even in the presence of LIF.


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

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

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).
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fig3: 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).
Mentions: To rule out the possibility of off-target effects by octreotide and S4, we examined the effect of SSTR2 knock-down in mESCs. An shRNA-based technique was employed to specifically knock-down the SSTR2 gene in mESCs cultured in medium containing LIF. Quantitative RT-PCR results revealed that, relative to the scrambled shRNA, the transfection of shRNA targeting SSTR2 led to a dramatic reduction of the SSTR2 mRNA in mESCs 24 h after transfection (Figure 3A). Knock-down of SSTR2 significantly decreased the ratio of undifferentiated E14 cells, even in the presence of LIF (Figure 3B). Cells expressing SSTR2 shRNA displayed a lower expression level of pluripotency genes (Oct4, Sox2, Nanog, and Rex1) (Figure 3C) and showed weak or negative staining for AP and other pluripotent markers, including Oct4, Nanog, and SSEA1 (Figure 3D). Adding LIF or the combination of LIF and octreotide did not rescue the spontaneous differentiation of these SSTR2 knock-down cells (Figure 3E), although the differentiation could be rescued by 2i (Figure 3B). These data confirmed that SSTR2 contributes to mES cell self-renewal and that knock-down of SSTR2 leads to spontaneous differentiation, even in the presence of LIF.

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