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Keywords:

  • aging;
  • human;
  • longevity gene;
  • neural stem cells;
  • neurogenesis

Summary

  1. Top of page
  2. Summary
  3. Introduction, results, discussion
  4. Acknowledgments
  5. Author contributions
  6. References
  7. Supporting Information

Proteostasis is critical for maintaining cell function and proteome stability may play an important role in human embryonic stem cell (hESC) immortality. Notably, hESC populations exhibit a high assembly of active proteasomes, a key node of the proteostasis network. FOXO4, an insulin/IGF-1 responsive transcription factor, regulates proteasome activity in hESCs. We find that loss of FOXO4 reduces the potential of hESCs to differentiate into neural lineages. Therefore, FOXO4 crosses evolutionary boundaries and links hESC function to invertebrate longevity modulation.


Introduction, results, discussion

  1. Top of page
  2. Summary
  3. Introduction, results, discussion
  4. Acknowledgments
  5. Author contributions
  6. References
  7. Supporting Information

Mammalian embryonic stem cells (ESCs) are unique among all stem cell populations because they do not appear to undergo replicative senescence and, therefore, could be considered immortal in culture (Thomson et al., 1998). The ability to ensure proteostasis is critical for maintaining proper cell function and survival (Powers et al., 2009). Misfolded proteins accumulate as a consequence of mutations and aging or ensue from heat or oxidative stress. These damaged proteins are prone to aggregation and, therefore, must be scavenged. The proteasome has a critical role in terminating damaged proteins. Notably, human embryonic stem cells (hESCs) exhibit a high proteasome activity that is correlated with increased levels of the 19S proteasome subunit PSMD11/RPN-6 and a corresponding increased assembly of 26S/30S proteasomes. This increased proteasome activity is regulated by FOXO4 (Vilchez et al., 2012a). Proteasome inhibition has a profound effect upon hESC function; however, the role of FOXO4 has not been described.

FOXO family transcription factors are the downstream effectors of the insulin/insulin-like growth factor (IGF) signaling (IIS) pathway. IIS reduction results in stress-resistant, long-lived worms (Kenyon et al., 1993), flies (Tatar et al., 2001), and mice (Holzenberger et al., 2003) and correlates with increased longevity in humans (Willcox et al., 2008). Similar to hESCs, proteasome activity and RPN-6 levels are increased in the long-lived C. elegans glp-1 mutant (Vilchez et al., 2012b). In this mutant, increased proteasome activity, rpn-6 expression, and longevity are modulated by the DAF-16/FOXO transcription factor. Therefore, glp-1 long-lived mutant worms and hESCs share a series of features such as increased proteasome activity and its regulation by orthologous FOXO transcription factors. Taken together, these results raise an intriguing question: do the transcription factors that regulate lifespan and stress-resistance in invertebrates control ESC function in mammals? Notably, FOXO transcription factors coordinately regulate diverse pathways to govern adult neural stem cell homeostasis in the mouse brain (Paik et al., 2009; Renault et al., 2009).

As hESCs differentiate, FOXO4 has a corresponding decrease in its expression (Vilchez et al., 2012a). Accordingly, this decrease in FOXO4 expression is reprogrammed from somatic cells to induced pluripotent stem cells (Vilchez et al., 2012a). Prompted by these intriguing results, we reduced FOXO transcription factors in H9 hESCs [for information about knock-down efficiency, see (Vilchez et al., 2012a)] to test whether FOXO was required for proper function of hESCs. We measured the expression levels of several markers of pluripotency in these cells prior to differentiation and found no difference at this stage (Table S1). However, when we forced differentiation into neural lineage (Table S2), we observed profound differences among the FOXO4 shRNA lines that were not present in the control lines: FOXO4 shRNA hESCs had a diminished ability to generate neural rosettes and, accordingly, neural cells (Fig. 1A,B and S1). In contrast, knock-down of FOXO1, FOXO3, or HSF1 did not affect hESCs differentiation into neural cells (Fig. 1A and S1). As a more direct test of molecular changes to these cells, we found that the FOXO4 shRNA lines did not induce expression of neural progenitor markers and proteins involved in neurogenesis to the same extent as control lines after the differentiation treatment. Accordingly, FOXO4 shRNA cells retained pluripotency markers because most of them do not progress through differentiation into neural cells (Fig. 1C–E, S2–S3 and Table S3). The decreased ability to differentiate into the neural lineage by loss of FOXO4 was confirmed in the independent hESC line HUES-6 (Fig. S4). Similar to pluripotency markers, PSMD11 expression decreases when hESCs differentiate into neural cells (Vilchez et al., 2012a). Knock-down of FOXO4 decreases the expression of PSMD11 in hESCs. Due to the diminished ability of neural differentiation of FOXO4 shRNA cells, the levels of PSMD11 in FOXO4 knock-down cells are similar to control cells after subjecting both to neural differentiation treatment (Fig. S5).

image

Figure 1. FOXO4 is required for human embryonic stem cells (hESCs) differentiation into neural cells. (A, B) Graph represents the percentage of embryoid bodies (hEBs) containing neural progenitor cells (NPCs) relative to GFP (OE) overexpressing stable control cells. FOXO4 shRNA H9 hESCs made into embryoid bodies (hEBs) have a diminished ability to generate rosettes and neural cells. (A) n = 20, P = 2.6*10−23. (B) GFP (n = 7–9), P < 0.001. (C, D) After culturing in neural differentiation media, FOXO4 shRNA cells show decreased expression in neural markers (n = 12) and maintain high expression of pluripotency markers (n = 7) compared with GFP OE cells [*(P < 0.05), **(P < 0.01), ***(P < 0.001)]. (E) Immunocytochemistry after neural differentiation assay. β-III-tubulin, OCT4, and DAPI staining were used as markers of neurogenesis, pluripotency, and nuclei, respectively. Scale bar represents 100 μm. (F) Graph represents the percentage of hEBs containing MAP2-positive cells (n = 3 independent experiments). GFP OE clone 1 vs. FOXO4 shRNA (P < 0.0005), GFP OE clone 1 vs. GFP OE clone 3 (P = 0.93), GFP OE clone 1 vs. 3′UTR FOXO4 shRNA 3 (P < 0.05), GFP OE clone 1 vs. 3′UTR FOXO4 shRNA 3 + FOXO4 AAA (OE) (P = 0.41), 3′UTR FOXO4 shRNA 3 vs. 3′UTR FOXO4 shRNA 3 + FOXO4 AAA (OE) (P < 0.05). All the graphs represent mean ± SEM. All statistical comparisons were made by Student's t-test for unpaired samples.

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FOXO4 transcriptional activity is inhibited by phosphorylation (Kops et al., 1999). FOXO4 contains three putative phosphorylation sites for Akt/protein kinase B, Thr32, Ser197, and Ser262 (Yang et al., 2002). Once FOXO4 is dephosphorylated, it translocates to the nucleus and induces target gene expression (Matsuzaki et al., 2005). Ectopic expression of constitutively active FOXO4 triple alanine mutant (FOXO4 AAA) ameliorated the low ability of FOXO4 shRNA hESCs to differentiate into neural cells [Fig. 1F, S6. For information about ectopic expression levels of FOXO4 AAA, see (Vilchez et al., 2012a)].

Intrigued by the finding that reduction of FOXO4, but not other FOXO family members affected the differentiation of hESCs into neural cells, we asked whether differentiation into other cell lineages might also be affected. We found that FOXO4 shRNA hESCs were able to properly differentiate into trophoblasts and keratinocytes (Fig. 2A,B, S7–S8 and Table S4). Notably, we observed that after the corresponding differentiation process, FOXO4 shRNA hESCs showed increased levels of trophoblast or keratinocytes markers compared with control cells (Fig. 2A,B and Table S4). Moreover, we were able to generate fibroblast-resembling cells from FOXO4 shRNA hESCs after the fibroblast differentiation treatment (Fig. 2C, S9 and Table S5). Intrigued by the specific impact of loss of FOXO4 over neural commitment, we measured the expression levels of several specific differentiation markers in these cells prior to differentiation treatment (Table S6). Notably, we found a decrease in the expression of PAX6, an ectodermal transcription factor necessary for neurogenesis, whereas we observed increased levels of specific endodermal markers (GATA4, AFP). These results suggest that loss of FOXO4 could predispose hESCs toward differentiation of specific cell lines in the detriment of neurogenesis.

image

Figure 2. FOXO shRNA human embryonic stem cells can differentiate into trophoblasts, keratinocytes, and fibroblasts. (A) Graph (relative expression to GFP OE cells) represents the mean ± SEM (n = 8). After culturing in trophoblast differentiation media, FOXO4 shRNA cells show increased expression in trophoblast markers compared with GFP cells [CD9 (P < 0.05), CGB (P < 0.01), GATA2 (P < 0.01), GATA3 (P < 0.001), GCM (P < 0.001), HEY1 (P < 0.01), MSX2 (P < 0.001), PAEP (P < 0.001), TFAP2 (P < 0.001)]. (B) Graphs (relative expression to GFP OE cells) represent the mean ± SEM (n = 4). After culturing in keratinocyte differentiation media, FOXO4 shRNA cells show increased expression in keratinocyte markers compared with GFP OE cells [P-value: *(P < 0.05), **(P < 0.01), ***(P < 0.001)]. (C) Graph (relative expression to GFP OE cells) represent the mean ± SEM (n = 8). After culturing in fibroblast differentiation media, FOXO4 shRNA cells show similar expression of both vimentin and P4H1 compared with GFP OE cells [vimentin (P = 0.37), P4H1 (P < 0.12)]. ACTA2 (P < 0.05), desmin (P < 0.05), FSP1 (P < 0.01), Col1a1 (P < 0.01), and FAP (P < 0.05) levels are decreased in FOXO4 shRNA cells. In contrast, Col6a2 levels are increased (P < 0.05). All statistical comparisons were made by Student's t-test for unpaired samples.

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Therefore, FOXO4 is specifically critical for the differentiation of hESCs into neural cells, and it will be fascinating to understand how this regulation is achieved. As FOXO3 is essential for the maintenance of adult neural stem cells in the mouse (Renault et al., 2009), it is intriguing to speculate that FOXO4 may be required for embryonic neural development and that FOXO3 is later required for adult neural self-renewal. Accordingly, FOXO4 levels decrease during differentiation whereas FOXO3 levels increases, especially in the transition from hESCs to neural cells (Vilchez et al., 2012a). FOXO6, the most recently discovered FOXO isoform in mammals, is highly expressed in adult hippocampus and plays an important role in synaptic function during memory consolidation (Salih et al., 2012). As FOXO6 becomes highly expressed in neurons compared with hESCs and NPCs (Vilchez et al., 2012a), we hypothesized that this transcription factor acquires a prominent role in neuronal function after embryonic neural development.

It will be of particular interest to identify genes regulated by FOXO4 in hESCs. The downstream genes regulated by DAF-16 and its mammalian homologs suggest that FOXO4 may have a pivotal role in the characteristics that define stem cell biology, such as genome stability and self-renewal (Ramaswamy et al., 2002; Tran et al., 2002; Kenyon, 2005). In conclusion, our findings may trigger new advances in the field of hESC differentiation and cell reprogramming and open new possibilities for cell therapy by modulation of FOXO4.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction, results, discussion
  4. Acknowledgments
  5. Author contributions
  6. References
  7. Supporting Information

We thank Alexander Stauffer and Veronica Modesto for their help with cell culture.

Author contributions

  1. Top of page
  2. Summary
  3. Introduction, results, discussion
  4. Acknowledgments
  5. Author contributions
  6. References
  7. Supporting Information

D.V. and A.D. planned and supervised the project. D.V performed the experiments, data analysis, and interpretation. L.B. performed neural differentiation assays and contributed to other assays. M.L. performed cell culturing. C.M., I.M., and C.T. performed biochemistry experiments and contributed to other assays. B.S., L.P., and E.M. generated lentiviral constructs. W.T.B. and F.H.G. helped to supervise the project. The manuscript was written by D.V. and A.D. and edited by L.B., I.M., C.M., W.T.B., and F.H.G.

References

  1. Top of page
  2. Summary
  3. Introduction, results, discussion
  4. Acknowledgments
  5. Author contributions
  6. References
  7. Supporting Information
  • Holzenberger M, Dupont J, Ducos B, Leneuve P, Geloen A, Even PC, Cervera P, Le Bouc Y (2003) IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature 421, 182187.
  • Kenyon C (2005) The plasticity of aging: insights from long-lived mutants. Cell 120, 449460.
  • Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R (1993) A C. elegans mutant that lives twice as long as wild type. Nature 366, 461464.
  • Kops GJ, de Ruiter ND, De Vries-Smits AM, Powell DR, Bos JL, Burgering BM (1999) Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 398, 630634.
  • Matsuzaki H, Ichino A, Hayashi T, Yamamoto T, Kikkawa U (2005) Regulation of intracellular localization and transcriptional activity of FOXO4 by protein kinase B through phosphorylation at the motif sites conserved among the FOXO family. J. Biochem. 138, 485491.
  • Paik JH, Ding Z, Narurkar R, Ramkissoon S, Muller F, Kamoun WS, Chae SS, Zheng H, Ying H, Mahoney J, Hiller D, Jiang S, Protopopov A, Wong WH, Chin L, Ligon KL, DePinho RA (2009) FoxOs cooperatively regulate diverse pathways governing neural stem cell homeostasis. Cell Stem Cell 5, 540553.
  • Powers ET, Morimoto RI, Dillin A, Kelly JW, Balch WE (2009) Biological and chemical approaches to diseases of proteostasis deficiency. Annu. Rev. Biochem. 78, 959991.
  • Ramaswamy S, Nakamura N, Sansal I, Bergeron L, Sellers WR (2002) A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR. Cancer Cell 2, 8191.
  • Renault VM, Rafalski VA, Morgan AA, Salih DA, Brett JO, Webb AE, Villeda SA, Thekkat PU, Guillerey C, Denko NC, Palmer TD, Butte AJ, Brunet A (2009) FoxO3 regulates neural stem cell homeostasis. Cell Stem Cell 5, 527539.
  • Salih DA, Rashid AJ, Colas D, de la Torre-Ubieta L, Zhu RP, Morgan AA, Santo EE, Ucar D, Devarajan K, Cole CJ, Madison DV, Shamloo M, Butte AJ, Bonni A, Josselyn SA, Brunet A (2012) FoxO6 regulates memory consolidation and synaptic function. Genes Dev. 26, 27802801.
  • Tatar M, Kopelman A, Epstein D, Tu MP, Yin CM, Garofalo RS (2001) A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science 292, 107110.
  • Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282, 11451147.
  • Tran H, Brunet A, Grenier JM, Datta SR, Fornace AJ Jr, DiStefano PS, Chiang LW, Greenberg ME (2002) DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science 296, 530534.
  • Vilchez D, Boyer L, Morantte I, Lutz M, Merkwirth C, Joyce D, Spencer B, Page L, Masliah E, Berggren TW, Gage FH, Dillin A (2012a) Increased proteasome activity in human embryonic stem cells is regulated by PSMD11. Nature 489, 304308.
  • Vilchez D, Morantte I, Liu Z, Douglas PM, Merkwirth C, Rodrigues AP, Manning G, Dillin A (2012b) RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature 489, 263268.
  • Willcox BJ, Donlon TA, He Q, Chen R, Grove JS, Yano K, Masaki KH, Willcox DC, Rodriguez B, Curb JD (2008) FOXO3A genotype is strongly associated with human longevity. Proc. Natl Acad. Sci. USA 105, 1398713992.
  • Yang Z, Whelan J, Babb R, Bowen BR (2002) An mRNA splice variant of the AFX gene with altered transcriptional activity. J. Biol. Chem. 277, 80688075.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction, results, discussion
  4. Acknowledgments
  5. Author contributions
  6. References
  7. Supporting Information
FilenameFormatSizeDescription
acel12067-sup-0001-DataS1-FigS1-S9-TableS1-S7.pdfapplication/PDF19151K

Data S1 Methods.

Fig. S1 Impaired neural differentiation of FOX4 shRNA H9 hESCs.

Fig. S2 FOXO4 shRNA H9 hESCs have a diminished ability to differentiate into neural cells.

Fig. S3 FOXO4 shRNA H9 hESCs have a diminished ability to differentiate into neural cells (II).

Fig. S4 FOXO4 shRNA HUES-6 hESCs have a diminished ability to differentiate into neural cells.

Fig. S5 No significant differences in PSMD11 mRNA levels between GFP OE and FOXO4 shRNA cells after neural differentiation treatment.

Fig. S6 Ectopic expression of FOXO4 AAA ameliorates the decrease ability of 3′UTR FOXO4 shRNA hESCs to differentiate into neural cells.

Fig. S7 Trophoblast differentiation.

Fig. S8 Keratinocyte differentiation.

Fig. S9 Fibroblast differentiation.

Table S1 Pluripotency marker levels in FOXO4 shRNA hESCs.

Table S3 Neural differentiation of hESCs.

Table S3 FOXO4 shRNA hESCs have a diminished ability to differentiate into neural cells.

Table S4 Trophoblast differentiation of stable FOXO4 shRNA hESCs.

Table S5 Fibroblast differentiation of stable FOXO4 shRNA hESCs.

Table S6 Expression of specific differentiation markers for ectodermal (PAX6), mesodermal (MSX1), endodermal (GATA4, GATA6, AFP) and trophectodermal (Cdx2) germ layers in hESCs prior to differentiation treatment.

Table S7 Sequences cloned to generate shRNA expressing lentiviral vectors.

acel12067-sup-0002-TableS8.xlsxapplication/msexcel12KTabls S8 List of primers used for qPCR assays

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