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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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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.
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fig4: 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.

Mentions: Activation of the JAK/STAT3 pathway is one of the major mechanisms by which LIF promotes the self-renewal of mESCs4. Our study demonstrated that octreotide and seglitide, two SSTR2 agonists, prevented the mESC differentiation induced by LIF deprivation. We wondered whether the activation of SSTR2 leads to phosphorylation and activation of STAT3. Western blot analysis revealed that LIF (1000 U/mL) induced a strong but transient phosphorylation of STAT3 at residue Y705, and p-STAT peaked at 10 min after stimulation. In contrast, octreotide (1 μmol/L) induced a mild but relatively long-lasting phosphorylation of STAT3, and p-STAT peaked at 60 min after stimulation (Figure 4A, 4B). As expected, the SSTR2 antagonist S4 blocked octreotide-induced STAT3 phosphorylation but did not affect LIF-mediated STAT3 phosphorylation (Figure 4A, 4B). Nuclear translocation of STAT3 is another phenomenon indicating activation of the STAT3 pathway14. Indeed, immunofluorescent staining indicated that both LIF and octreotide induced the nuclear translocation of STAT3 in mESCs (Figure 4C, 4D). Next, we measured the total STAT3 and phosphorylated STAT3 levels in the nuclei and cytoplasm of the mESCs. As shown in Figure 4E, stimulation with LIF or octreotide induced phosphorylation of STAT3 and promoted its nuclear translocation. Oct4 and GAPDH were used as markers of nuclear and cytoplasmic proteins, respectively. Taken together, these data suggest that activation of SSTR2 may contribute to the pluripotency and self-renewal of mESCs via activation of the STAT3 pathway.


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 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.
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fig4: 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.
Mentions: Activation of the JAK/STAT3 pathway is one of the major mechanisms by which LIF promotes the self-renewal of mESCs4. Our study demonstrated that octreotide and seglitide, two SSTR2 agonists, prevented the mESC differentiation induced by LIF deprivation. We wondered whether the activation of SSTR2 leads to phosphorylation and activation of STAT3. Western blot analysis revealed that LIF (1000 U/mL) induced a strong but transient phosphorylation of STAT3 at residue Y705, and p-STAT peaked at 10 min after stimulation. In contrast, octreotide (1 μmol/L) induced a mild but relatively long-lasting phosphorylation of STAT3, and p-STAT peaked at 60 min after stimulation (Figure 4A, 4B). As expected, the SSTR2 antagonist S4 blocked octreotide-induced STAT3 phosphorylation but did not affect LIF-mediated STAT3 phosphorylation (Figure 4A, 4B). Nuclear translocation of STAT3 is another phenomenon indicating activation of the STAT3 pathway14. Indeed, immunofluorescent staining indicated that both LIF and octreotide induced the nuclear translocation of STAT3 in mESCs (Figure 4C, 4D). Next, we measured the total STAT3 and phosphorylated STAT3 levels in the nuclei and cytoplasm of the mESCs. As shown in Figure 4E, stimulation with LIF or octreotide induced phosphorylation of STAT3 and promoted its nuclear translocation. Oct4 and GAPDH were used as markers of nuclear and cytoplasmic proteins, respectively. Taken together, these data suggest that activation of SSTR2 may contribute to the pluripotency and self-renewal of mESCs via activation of the STAT3 pathway.

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