TY - JOUR
T1 - Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation
AU - Gupta, Manoj K.
AU - De Jesus, Dario F.
AU - Kahraman, Sevim
AU - Valdez, Ivan A.
AU - Shamsi, Farnaz
AU - Yi, Lian
AU - Swensen, Adam C.
AU - Tseng, Yu Hua
AU - Qian, Wei Jun
AU - Kulkarni, Rohit N.
N1 - Funding Information:
We thank G. Mostoslavsky PhD (Boston University) for the kind gift of STEMCCA lentiviral plasmids. We thank J.Hu for technical assistance. Authors acknowledge Joslin Diabetes Center iPS Core Facility (DRC, NIH DK036836). MKG is supported by JDRF advanced postdoctoral fellowship grant 3-APF-2017-393-A-N. DFJ was supported by the Portuguese Foundation for Science and Technology ? FCT (SFRH/BD/51699/2011), Albert Ronald Travel Fellowship 2015, 2016 FLAD R&D@PhD Internship Grant. RNK is supported by the HSCI and NIH grants R01 DK67536 and R01 DK103215. WQ is supported by NIH grants UC4 DK104167 and DP3 DK110844. Y-HT is supported by NIH grants R01DK077097 and R01DK102898. FS is supported by American Diabetes Association Fellowship #1-18-PDF-169. Part of the experimental work was performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the DOE and located at Pacific Northwest National Laboratory, which is operated by Battelle Memorial Institute for the DOE under Contract DE-AC05-76RL0 1830. All authors declare no financial conflict of interest.
Funding Information:
We thank G. Mostoslavsky PhD (Boston University) for the kind gift of STEMCCA lentiviral plasmids. We thank J.Hu for technical assistance. Authors acknowledge Joslin Diabetes Center iPS Core Facility (DRC, NIH DK036836). MKG is supported by JDRF advanced postdoctoral fellowship grant 3-APF-2017-393-A-N . DFJ was supported by the Portuguese Foundation for Science and Technology – FCT ( SFRH/BD/51699/2011 ), Albert Ronald Travel Fellowship 2015, 2016 FLAD R&D@PhD Internship Grant. RNK is supported by the HSCI and NIH grants R01 DK67536 and R01 DK103215 . WQ is supported by NIH grants UC4 DK104167 and DP3 DK110844 . Y-HT is supported by NIH grants R01DK077097 and R01DK102898 . FS is supported by American Diabetes Association Fellowship #1-18-PDF-169 . Part of the experimental work was performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the DOE and located at Pacific Northwest National Laboratory, which is operated by Battelle Memorial Institute for the DOE under Contract DE-AC05-76RL0 1830. All authors declare no financial conflict of interest.
Publisher Copyright:
© 2018 The Authors
PY - 2018/12
Y1 - 2018/12
N2 - 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.
AB - 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.
KW - Adipocyte
KW - Beta cells
KW - Insulin receptor signaling
KW - Lineage differentiation
KW - Neurons
KW - Phosphoproteomics
KW - Pluripotency
KW - Reprogramming
KW - Stem cells
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U2 - 10.1016/j.molmet.2018.09.003
DO - 10.1016/j.molmet.2018.09.003
M3 - Article
C2 - 30316806
AN - SCOPUS:85054573660
VL - 18
SP - 153
EP - 163
JO - Molecular Metabolism
JF - Molecular Metabolism
SN - 2212-8778
ER -