Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation
Objectives: Insulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells; however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR s...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2018-12-01
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| Series: | Molecular Metabolism |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2212877818306999 |
| _version_ | 1831812592076587008 |
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| author | Manoj K. Gupta Dario F. De Jesus Sevim Kahraman Ivan A. Valdez Farnaz Shamsi Lian Yi Adam C. Swensen Yu-Hua Tseng Wei-Jun Qian Rohit N. Kulkarni |
| author_facet | Manoj K. Gupta Dario F. De Jesus Sevim Kahraman Ivan A. Valdez Farnaz Shamsi Lian Yi Adam C. Swensen Yu-Hua Tseng Wei-Jun Qian Rohit N. Kulkarni |
| author_sort | Manoj K. Gupta |
| collection | DOAJ |
| description | Objectives: Insulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells; however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR signaling in pluripotency and lineage development. Methods: To explore the role of IR signaling, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors: Oct4, Sox2, Klf4, cMyc. We performed pluripotency characterization and directed the differentiation of control and IRKO iPSCs into neural progenitors (ectoderm), adipocyte progenitors (mesoderm), and pancreatic beta-like cells (endoderm). We mechanistically confirmed these findings via phosphoproteomics analyses of control and IRKO iPSCs. Results: Interestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3, and cMyc were upregulated, while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold, respectively, in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, p-mTor and p-Erk and an increase in the total mTor and Erk proteins in IRKO iPSCs in the basal unstimulated state. Stimulation with leukemia inhibitory factor (LIF) showed a ∼33% decrease of phospho-ERK in IRKO iPSCs. On the contrary, Erk phosphorylation was increased during in vitro spontaneous differentiation of iPSCs lacking IRs. Lineage-specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Ascl1 and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Pparγ, Fabp4, C/ebpα, and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1, and Sox9). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network including several key proteins involved in diverse aspects of growth and embryonic development. Conclusion: We report, for the first time to our knowledge, the phosphoproteome of insulin, IGF1, and LIF stimulation in mouse iPSCs to reveal the importance of insulin receptor signaling for the maintenance of pluripotency and lineage determination. Keywords: Insulin receptor signaling, Pluripotency, Lineage differentiation, Adipocyte, Beta cells, Neurons, Stem cells, Phosphoproteomics, Reprogramming |
| first_indexed | 2024-12-22T21:39:05Z |
| format | Article |
| id | doaj.art-ed089cc8fe424187a8b0aa0f368d3df0 |
| institution | Directory Open Access Journal |
| issn | 2212-8778 |
| language | English |
| last_indexed | 2024-12-22T21:39:05Z |
| publishDate | 2018-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Metabolism |
| spelling | doaj.art-ed089cc8fe424187a8b0aa0f368d3df02022-12-21T18:11:39ZengElsevierMolecular Metabolism2212-87782018-12-0118153163Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiationManoj K. Gupta0Dario F. De Jesus1Sevim Kahraman2Ivan A. Valdez3Farnaz Shamsi4Lian Yi5Adam C. Swensen6Yu-Hua Tseng7Wei-Jun Qian8Rohit N. Kulkarni9Islet Cell and Regenerative Biology, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02215, USAIslet Cell and Regenerative Biology, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02215, USA; Graduate Program in Areas of Basic and Applied Biology (GABBA), Abel Salazar Biomedical Sciences Institute, University of Porto, 5000 Porto, PortugalIslet Cell and Regenerative Biology, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02215, USAIslet Cell and Regenerative Biology, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02215, USASection of Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USABiological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USABiological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USASection of Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USABiological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USAIslet Cell and Regenerative Biology, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02215, USA; Corresponding author. Islet Cell and Regenerative Biology, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, USA. Fax: +1 617 309 3476.Objectives: Insulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells; however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR signaling in pluripotency and lineage development. Methods: To explore the role of IR signaling, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors: Oct4, Sox2, Klf4, cMyc. We performed pluripotency characterization and directed the differentiation of control and IRKO iPSCs into neural progenitors (ectoderm), adipocyte progenitors (mesoderm), and pancreatic beta-like cells (endoderm). We mechanistically confirmed these findings via phosphoproteomics analyses of control and IRKO iPSCs. Results: Interestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3, and cMyc were upregulated, while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold, respectively, in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, p-mTor and p-Erk and an increase in the total mTor and Erk proteins in IRKO iPSCs in the basal unstimulated state. Stimulation with leukemia inhibitory factor (LIF) showed a ∼33% decrease of phospho-ERK in IRKO iPSCs. On the contrary, Erk phosphorylation was increased during in vitro spontaneous differentiation of iPSCs lacking IRs. Lineage-specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Ascl1 and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Pparγ, Fabp4, C/ebpα, and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1, and Sox9). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network including several key proteins involved in diverse aspects of growth and embryonic development. Conclusion: We report, for the first time to our knowledge, the phosphoproteome of insulin, IGF1, and LIF stimulation in mouse iPSCs to reveal the importance of insulin receptor signaling for the maintenance of pluripotency and lineage determination. Keywords: Insulin receptor signaling, Pluripotency, Lineage differentiation, Adipocyte, Beta cells, Neurons, Stem cells, Phosphoproteomics, Reprogramminghttp://www.sciencedirect.com/science/article/pii/S2212877818306999 |
| spellingShingle | Manoj K. Gupta Dario F. De Jesus Sevim Kahraman Ivan A. Valdez Farnaz Shamsi Lian Yi Adam C. Swensen Yu-Hua Tseng Wei-Jun Qian Rohit N. Kulkarni Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation Molecular Metabolism |
| title | Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation |
| title_full | Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation |
| title_fullStr | Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation |
| title_full_unstemmed | Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation |
| title_short | Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation |
| title_sort | insulin receptor mediated signaling regulates pluripotency markers and lineage differentiation |
| url | http://www.sciencedirect.com/science/article/pii/S2212877818306999 |
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