Insulin receptor substrate (IRS)-1 regulates murine embryonic stem (mES) cells self-renewal

Abstract

Mouse embryonic stem (mES) cells are pluripotent cells that can be propagated in vitro with leukemia inhibitory factor (LIF) and serum. Intracellular signaling by LIF is principally mediated by activation of STAT-3, although additional pathways for self-renewal have been described. Here, we identified a novel role for Insulin receptor substrate-1 (IRS-1) as a critical factor in mES cells self-renewal and differentiation. IRS-1 is expressed and tyrosyl phosphorylated during mES cells self-renewal. Differentiation of mES cells, by LIF withdrawal, is associated with a marked reduction in IRS-1 expression. Targeting of IRS-1 by si-IRS-1 results in a severe reduction of Oct-4 protein expression and alkaline phosphatase activity, markers of undifferentiated mES cells. IRS-1 targeting does not interfere with LIF-induced STAT-3 phosphorylation, but negatively affects protein kinase B (PKB/AKT) and glycogen synthase kinase-3 (GSK-3beta) phosphorylation, which are downstream effectors of the LIF-mediated PI3K signaling cascade. Targeting of IRS-1 also results in a marked down regulation of Id-1 and Id-2 proteins expression, which are important components for self-renewal of ES cells. Conversely, over expression of IRS-1 inhibits mES cell differentiation. Taken together, these results suggest that expression and activity of IRS-1 are critical to the maintenance of the self-renewal program in mES cells.

Fig. 1

mES cells differentiation. A: mES cells were differentiated as EBs at the indicated times following LIF withdrawal. Oct-4 protein expression was detected by Western blot. Grb-2 was used to verify equal loading. Results are representative of three independent experiments. B: Alkaline phosphatase colony assay was performed as described in Materials and Methods. Representative fields of alkaline phosphatase colony assay in mES cells cultured in the presence (left part) or absence (right part) of LIF for 4 days. C: Quantitative analysis of AP colony assay. The number of positive colonies is expressed as percent on total colonies scored and represents the mean ±SD of three separate experiments performed in triplicate. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 2

IRS-1, IGF-IR, and Ins-R expression and phosphorylation in mES cells. A,B: Protein and mRNA expression profiles of IRS-1, IGF-IR, and Ins-R in self-renewing and differentiating ES cells at the indicated time points. Grb-2 and GAPDH were assayed in Western blot and RT-PCR analysis, respectively, to verify equal loading. C,D: ES cells were induced to differentiate. Lysates from cells, at indicated time points from LIF withdrawal, were probed for pY-IRS-1 and pSer-IRS-1 by Western blotting. Grb-2 protein level was used to verify equal loading. E: IGF-IR or Ins-R were immunoprecipitated from ES cells or EBs, and the Western blots probed with a phosphotyrosine antibody. F: mRNA expression of IGF-II and GAPDH by RT-PCR in ES cells and EBs (1 and 4 days). G: mES cells were starved of FBS and LIF for 3 h prior to the addition of LIF (1,000 ng/ml) or IGF-I (50 ng/ml). Lysates were obtained after 30 min, and pY-IRS-1 and IRS-1 visualized by Western blotting.

Fig. 3

IRS-1 targeting inhibits mES cells self-renewal. A: Western blot analysis showing IRS-1 (upper part) and Oct-4 (lower part) proteins expression in parental, control(NT), and IRS-1 targeted ES cells. Grb-2 protein expression was used as loading control. Results are representative of three independent experiments. B: Alkaline phosphatase colony assay showing that IRS-1 targeting affects the morphology of colonies generated after 4 days in medium supplemented with LIF. Representative fields of parental (upper part), control (NT; middle parts) and IRS-1 targeted ES cells (lower parts). C: Same experiment as in (B); AP positive colony numbers are expressed as percent on total number of colonies scored and represents the Mean ±SD of three separate experiments performed in triplicate. D: IRS-1 targeting affects the proliferation of mES cells in LIF containing medium. Total cell number was determined by trypan-blue exclusion after 4 days from cell plating. Results are representative of two independent experiments performed in triplicate. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 4

Effect of IRS-1 targeting on LIF-mediated PI3K depending signals. A: Western blot showing IRS-1 protein expression (upper part), Akt phosphorylation on serine 473 (middle part), and total Akt protein (lower part), in parental, control (NT), and IRS-1 targeted ES cells. B: Same experiment as in (A) to detect phosphorylation of GSK-3β on serine 9, as well as total amount of GSK-3β protein. C: IRS-1 targeting has no effect on LIF-induced STAT-3 phosphorylation. Total amount of STAT3 protein was used as loading control. D: Western blots showing the levels of both Id-1 and Id-2 protein expression. Grb-2 protein levels were used as loading control. Figures are representative of three independent experiments.

Fig. 5

Effect of forced expression and activation of IRS-1 on ES cells differentiation. A: IRS-1 expression in mES cells transduced with a retrovirus carrying mouse IRS-1 cDNA. B: Representative field showing the morphology of colonies generated from ES, ES empty vector, and ES/IRS-1 cells after 4 days from LIF withdrawal and IGF-I supplementation. C: Quantification of AP colony assay at day 5 after LIF withdrawal. The number of positive colonies is expressed as percent on total colonies scored and represents the mean ±SD of three separate experiments performed in triplicate. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 6

Overexpression of IRS-1 maintains Oct-4 protein expression. A: Parental and mES cells with a forced expression of IRS-1 were induced to differentiate as EBs after LIF withdrawal, in the presence of IGF-I. Lysates were separated by SDS–PAGE and probed for the presence of Oct-4 and IRS-1 at the indicated time points. B: Same experiment as previous showing Oct-4 expression level in mES cells infected with the EV. These results are representative of three independent experiments. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 7

Overexpression of IRS-1 maintains ID-1 and ID-2 proteins expression. A,B: Parental and ES/IRS-1 cells were induced to differentiate as EBs after LIF withdrawal, in the presence of IGF-I. Western blot shows the expression of Id-1 and Id-2 proteins at the indicated time points. Row 3 in both pictures indicates the level of IRS-1. Grb-2 protein expression was used as loading control (row 4). Results are representative of three independent experiments.