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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

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.
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fig1: 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.

Mentions: To investigate the role of GPCRs in the maintenance of pluripotency in mESCs, we analyzed the expression profiles of various GPCRs in E14 cells cultured in self-renewal (mES medium with LIF or mES medium without LIF but supplemented with 2i) or differentiation (mES medium without LIF) conditions using a PCR array. As shown in Figure 1A, compared with E14 cells cultured in mES medium supplemented with LIF, cells cultured in LIF-deprived medium lost the typical compact colony morphology of mESCs on d 4. Two small-molecule inhibitors (2i, CHIR99021 and PD0325901) could replace LIF and maintain the typical morphology of the mESCs. Using PCR arrays, we found that some GPCRs that had been reported to be involved in stemness maintenance, such as the Wnt/Frizzled (FZDs)10, were down-regulated in mESCs cultured in LIF-deprived medium and accompanied by a significant decrease in the typical pluripotency biomarkers, including Oct4, Sox2, Rex1, and Nanog. Another GPCR, SSTR2, was also found to be significantly down-regulated after mESC differentiation (data not shown). Because SSTR2 has never been reported to be involved in ESC self-renewal, we decided to further explore its function. The PCR array result was confirmed by quantitative RT-PCR analysis, which also demonstrated a significantly reduced expression of SSTR2 in E14 cells cultured in LIF-deprived mES medium (Figure 1B). The RT-PCR results also confirmed the down-regulation of pluripotency genes, including Sox2, Nanog, and Rex1, after LIF deprivation (Figure 1B). We then analyzed the protein level of SSTR2 in E14 cells cultured in various conditions. As shown in Figure 1C, the protein level of SSTR2 was also reduced in cells cultured in the LIF-deprived medium, while 2i restored the SSTR2 protein level. These data suggest that SSTR2 may play a role in the maintenance of pluripotency.


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

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

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.
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Related In: Results  -  Collection

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fig1: 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.
Mentions: To investigate the role of GPCRs in the maintenance of pluripotency in mESCs, we analyzed the expression profiles of various GPCRs in E14 cells cultured in self-renewal (mES medium with LIF or mES medium without LIF but supplemented with 2i) or differentiation (mES medium without LIF) conditions using a PCR array. As shown in Figure 1A, compared with E14 cells cultured in mES medium supplemented with LIF, cells cultured in LIF-deprived medium lost the typical compact colony morphology of mESCs on d 4. Two small-molecule inhibitors (2i, CHIR99021 and PD0325901) could replace LIF and maintain the typical morphology of the mESCs. Using PCR arrays, we found that some GPCRs that had been reported to be involved in stemness maintenance, such as the Wnt/Frizzled (FZDs)10, were down-regulated in mESCs cultured in LIF-deprived medium and accompanied by a significant decrease in the typical pluripotency biomarkers, including Oct4, Sox2, Rex1, and Nanog. Another GPCR, SSTR2, was also found to be significantly down-regulated after mESC differentiation (data not shown). Because SSTR2 has never been reported to be involved in ESC self-renewal, we decided to further explore its function. The PCR array result was confirmed by quantitative RT-PCR analysis, which also demonstrated a significantly reduced expression of SSTR2 in E14 cells cultured in LIF-deprived mES medium (Figure 1B). The RT-PCR results also confirmed the down-regulation of pluripotency genes, including Sox2, Nanog, and Rex1, after LIF deprivation (Figure 1B). We then analyzed the protein level of SSTR2 in E14 cells cultured in various conditions. As shown in Figure 1C, the protein level of SSTR2 was also reduced in cells cultured in the LIF-deprived medium, while 2i restored the SSTR2 protein level. These data suggest that SSTR2 may play a role in the maintenance of pluripotency.

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