Novel role of mitochondrial manganese superoxide dismutase in STAT3 dependent pluripotency of mouse embryonic stem cells

Abstract

Leukemia Inhibitory Factor (LIF)/Signal transducer and activator of transcription 3 (STAT3) signaling pathway maintains the stemness and pluripotency of mouse embryonic stem cells (mESCs). Detailed knowledge on key intermediates in this pathway as well as any parallel pathways is largely missing. We initiated our study by investigating the effect of small molecule Curcumin on various signalling pathways essential for self-renewal. Curcumin sustained the LIF independent self-renewal of mESCs and induced pluripotent stem cells (miPSCs) in a STAT3 activity dependent manner. Gene expression analysis showed LIF/STAT3 and redox signaling components to be majorly modulated. Amongst ROS genes, expression of Manganese Superoxide Dismutase (MnSOD) specifically relied on STAT3 signaling as evidenced by STAT3 inhibition and reporter assay. The silencing of MnSOD, but not Cu-ZnSOD expression, resulted in the loss of mESC pluripotency in presence of LIF, and the overexpression of MnSOD is sufficient for maintaining the expression of pluripotent genes in the absence of STAT3 signaling. Finally, we demonstrate MnSOD to stabilize the turnover of pluripotent proteins at the post-translational level by modulating proteasomal activity. In conclusion, our findings unravel a novel role of STAT3 mediated MnSOD in the self-renewal of mESCs.

Figure 1. Curcumin sustains pluripotency of mESCs in the absence of LIF and feeder.

(a) Cropped gel shows the pluripotent gene expression analysis of mESCs cultured in absence of LIF and in presence of different concentrations of curcumin at passage 1 and passage 15. Alkaline phosphatase staining of mESCs treated with curcumin in the absence of LIF (b) and graph represents (c) the manual counting of the number of differentiated and undifferentiated colonies. (d) Bright field images of mESCs cultured in the absence of LIF and feeders and in the presence of 5 µM Curcumin. Magnification – 10X; Nikon Eclipse TE-2000-S (e) Cropped gel shows the transcript analysis of pluripotent genes of mESCs treated with different concentration of Curcumin and cultured in the absence of LIF and feeders. (f) Immunofluorescent images of pluripotent proteins of mESCs treated with Curcumin and cultured in the presence or absence of LIF. Scale – 10 µm. (g) Expression analysis of the representative germ layer genes from the EBs obtained from the cells cultured in presence or absence of LIF and curcumin till passage 11.

Figure 2. Curcumin sustains self-renewal of iPSCs in absence of LIF.

(a) Flow Cytometric analysis of GFP expression in Oct4-GFP iPSCs cultured for 4–5 days in the absence of LIF and presence or absence of Curcumin. Data is representative of three independent experiments. Graph is represented as mean ± S.E.M. (b) Pluripotent gene expression analysis of Oct4-GFP iPSCs treated with Curcumin and cultured in presence or absence of LIF. (c) Immunofluorescence of Oct4-GFP iPSCs for pluripotent protein gene expression. Expression analysis of OCT4 was indirectly analyzed by measuring the expression of GFP as it is under the control of Oct4 promoter. Scale – 10 µm.

Figure 3. Curcumin maintains the self-renewal of mESCs independent of its proliferative status.

(a) Cell proliferative assay of mESCs treated in the presence and absence of curcumin by trypan blue exclusion method. mESCs cultured in presence and absence of Curcumin for 11 passages with a starting cell number of 80,000 cells and cumulative population doubling calculated. Data is representation of experiments conducted in triplicates, mean ± S.E.M. (b) Cell proliferation analyzed by BrDU incorporation in mESCs treated with Curcumin in absence of LIF. (n = average of 3 independent experiments, mean experiments ± S.E.M, **, p<0.001). (c) Expression analysis of cell cycle proteins in curcumin treated mESCs and compared with mESCs cultured in presence or absence of LIF. (c) Cell cycle analysis of LIF deprived mESCs treated with Curcumin and stained with PI.

Figure 4. Modulation of different ES regulatory pathways by curcumin.

(a) mESCs were cultured till confluency in the presence or absence of LIF and in presence of 5 µM Curcumin and analyzed for the expression levels of various genes belonging to different pathways involved in maintenance of pluripotency in mESCs. The expression levels were densitometrically analyzed and normalized with Gapdh expression and the values are plotted as relative expression levels in comparison with cells cultured in the absence of LIF. (b) qPCR analysis of pluripotent genes and genes involved in LIF/STAT3 pathway of mESCs treated with Curcumin. Data represented as mean ± S.E.M of 3 sets of experiments, *−p<0.05. (c) qPCR analysis of genes involved in oxidative pathway of cells treated with Curcumin. Data represented as mean ± S.E.M of 3 sets of experiments, *− p<0.05; **− p<0.001. (d) Western analysis of MnSOD, STAT3 and pSTAT3 in Curcumin treated and cells cultured in presence or absence of LIF conditions.

Figure 5. STAT3 plays a key role in Curcumin mediated pluripotency of mESCs.

(a) mRNA expression analysis of pluripotent genes in feeder-free culture of Curcumin treated cells exposed to 2 µM JAK inhibitor I for 24 hrs. (b) Transcript analysis of STAT3 inhibitor genes in cells treated with different concentrations of Curcumin. Data represented as mean ± S.E.M of 3 sets of experiments, *- p<0.05. (c) Transcript analysis of redox pathway genes in mESCs treated with curcumin in the presence and absence of 2 µM JAK inhibitor I. (d) Western blot analysis of OCT4, STAT3, pSTAT3 and MnSOD in cells cultured in presence or absence of LIF, Curcumin and JAK inhibitor I. GAPDH was used as a protein loading control (e) The response of MnSOD promoter to LIF signaling was measured by treating mESCs with different concentration of JAK inhibitor I using Luciferase reporter assay. Data represented as mean ± S.E.M of 3 sets of experiments, * denotes p ≤ 0.05. (f) The interaction between STAT3 and MnSOD promoter determined by transfecting different concentrations of pMXs-STAT3 in 293T cells harboring MnSOD Luciferase reporter construct and measuring luciferase expression levels. Data represented as mean ± S.E.M of 3 sets of experiments, * denotes p ≤ 0.05.

Figure 6. MnSOD is essential and sufficient to maintain the expression of pluripotent genes.

(a) RT-PCR analysis of MnSod and Cu-ZnSod in mESCs transfected with ShRNAs specific to MnSod and Cu-ZnSod. (b) Western blot analysis of STAT3, pSTAT3, OCT4, NANOG and MnSOD proteins in mESCs transduced with ShRNAs specific to MnSod and Cu-ZnSod. (c) Bright field image of mESCs lentivirally transduced with Scrambled or MnSOD ShRNA. Magnification – 10X; Nikon Eclipse TE-2000-S. (d) Immuno-fluorescence images of mESCs transduced with scrambled or shRNA specific for MnSOD and cultured for 4 passages. Magnification – 20X. (e) Manual enumeration of the number of differentiated, undifferentiated and partially differentiated mESC colonies transduced with scrambled or MnSOD ShRNA, as observed by alkaline phosphatase staining at passage 4. Data represented as mean of n = 3 experiments. (f) The effect of MnSOD overexpression on the transcript levels of pluripotency genes in mESCs cultured in presence or absence of LIF. (g) Western blot analysis of pluripotent proteins in mESCs overexpressing MnSOD and respective vector controls. (h) Bright field and SSEA1 staining images of mESC cultured either in presence or absence of LIF and with or without MnSOD overexpression. Scale – 10 µm. (i) Transcript analysis of representative genes of three germ layers of EB differentiated mESCs transfected with MnSOD or vector control and cultured in presence or absence of LIF. (j) Transcript analysis of pluripotent genes in mESCs transiently overexpressing MnSOD and treated with 1 µM JAK inhibitor I for 48 hrs. (k) Immunofluorescence imaging of SSEA1 protein expression in mESCs retrovirally transduced with MnSOD and vector control and subsequently treated with 1 µM JAK inhibitor I for 4 passages. Magnification – 20X.

Figure 7. MnSOD maintains pluripotency by modulating protein stability and proteasome activity.

(a) Stability of pluripotent proteins OCT4 and NANOG were measured by Western blot analysis of mESCs transfected with either vector control or MnSOD and cultured in presence or absence of LIF and treated with 50 ug/ml cycloheximide for different time periods. (b) Proteasome activity of mESCs either overexpressing or depleted of MnSOD was measured using cytosolic extract and proteasome specific substrate Suc-LLVY-AMC. Y-axis represents the arbitrary values of proteasomal activity. Data represented as mean ± S.E.M of 3 sets of experiments, **, p ≤ 0.01. (c) Schematic representation of the novel pathway followed by MnSOD in maintaining pluripotency of mESCs. Curcumin, by downregulating SOCS3 enhances STAT3 mediated MnSOD expression and inturn MnSOD increases the stability of pluripotent proteins OCT4 and NANOG by decreasing proteasomal activity.