Endocrinol Metab.  2013 Dec;28(4):297-308. 10.3803/EnM.2013.28.4.297.

Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells

• Kim YM
• Song I
• Seo YH
• Yoon G

• KMID: 2169308
• DOI: http://doi.org/10.3803/EnM.2013.28.4.297

Abstract

BACKGROUND
Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence.
METHODS
We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 microM) of deferoxamine (DFO) and H2O2.
RESULTS
In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3alpha (GSK3alpha) and beta corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3alpha and beta also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence.
CONCLUSION
GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.

Keyword

Glycogen synthase kinase 3; Sterol regulatory element binding protein 1; Lipogenesis; Cell aging

MeSH Terms

Aging*
Aminophenols
ATP Citrate (pro-S)-Lyase
Carrier Proteins*
Cell Aging
Deferoxamine
Fatty Acid Synthetase Complex
Glycogen Synthase Kinase 3*
Glycogen Synthase Kinases*
Glycogen Synthase*
Glycogen*
Humans
Lipogenesis*
Liver
Maleimides
Multienzyme Complexes
Oxo-Acid-Lyases
Phosphorylation
RNA, Small Interfering
Sterol Regulatory Element Binding Protein 1
ATP Citrate (pro-S)-Lyase
Aminophenols
Carrier Proteins
Deferoxamine
Fatty Acid Synthetase Complex
Glycogen
Glycogen Synthase
Glycogen Synthase Kinase 3
Glycogen Synthase Kinases
Maleimides
Multienzyme Complexes
Oxo-Acid-Lyases
RNA, Small Interfering
Sterol Regulatory Element Binding Protein 1

Figure

• 

  Fig. 1 Increase in glycogen synthase kinase 3 (GSK3) phosphorylation and mature sterol regulatory element binding protein 1 (SREBP1) in stress-induced senescence. Chang cells were treated with 200 µM deferoxamine (DFO) or H2O2 for the indicated periods. Dimethyl sulfoxide (DMSO) was used as vehicle control (V). (A) Flow cytometric analysis of the cell distribution after treatment of the stressors (200 µM DFO or H2O2) for 3 days. Cell size and cell granularity were analyzed by comparing forward scatter (FSC; R2+R4) and side scatter (SSC; R1+R2). Representative profiles (left) and their analyses for cell granularity (SCC analysis, right upper) and cell size (FSC analysis, right lower) are shown. (B) Representative images of senescence-associated β-galactosidase-positive cell populations are shown. (C) Western blot analysis. Quantitative analyses of the expression levels of SREBP1 mature form are shown in the lower panels. mSREBP1, mature-form SREBP1; pGSK, phosphor-GSK. aP<0.01 vs. V (DMSO control).

• 

  Fig. 2 Glycogen synthase kinase 3 (GSK3) inhibition using SB415286, a specific GSK3 inhibitor, enhances cellular lipogenesis. Chang cells were treated with 7.5 µg/mL SB415286 for the indicated periods. Dimethyl sulfoxide (DMSO) was used as vehicle control (V). (A) Western blot analysis of protein expressions of mature sterol regulatory element binding protein 1 (SREBP1). Quantitative analyses of the expression levels of SREBP1 mature form are shown in the lower panels. (B) Western blot analyses for protein expressions of lipogenic enzymes, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACL), and cardiolipin synthase (CRLS). (C) Cellular lipid profile of SB415286-treated cells was obtained by thin layer chromatography (TLC) as described in Methods. Representative TLC image (Ca) and quantitative estimation (Cb) of cellular lipids extracted from different numbers of cells are shown. Standard lipid mixture (Std) containing 10 µg each was used. Cholesteryl palmitate (CP, 1) belongs to nonpolar storage lipid, and cholesterol (CL, 2), cardiolipin (CA, 3), phosphatidyl choline (PC, 4) and phosphatidyl serine (PS, 5) belong to membrane lipids. 'd' in the x-axis stands for day. mSREBP1, mature-form SREBP1; PA, palmitate; PE, phosphatidyl ethanolamine. aP<0.01 vs. V (DMSO control).

• 

  Fig. 3 Glycogen synthase kinase 3 (GSK3) inhibition induces senescence of Chang cell, accompanying an increase in cellular membranous organellar mass. Chang cells were treated with 7.5 µg/mL SB415286 (SB) for the indicated periods. Dimethyl sulfoxide (DMSO) was used as vehicle control (V). (A) Subcellular organellar masses were estimated by comparing the fluorescence intensities with flow cytometry after staining cells with organelle-specific dyes as described in Methods. Representative images of the fluorescence stained cells are shown in the lower panels. (B) Cell size and cell granularity were analyzed by comparing forward scatter (FSC; R2+R4) and side scatter (SSC; R1+R2). (Ba) Quantitative analyses for cell size (FSC analysis, left) and cell granularity (SSC analysis, right). (Bb) Representative cell distribution profiles are shown. (C) Quantitative analysis for senescence-associated β-galactosidase-positive cell populations (left) and representative images (right) are shown. ER, endoplasmic reticulum; BODIPY, boron-dipyrromethene. aP<0.01 vs. V (DMSO control).

• 

  Fig. 4 Glycogen synthase kinase 3 (GSK3) knockdown induces senescence, accompanying an increase of mature-form sterol regulatory element binding protein 1 (mSREBP1) expression. Chang cells were transfected with si-GSK3α, si-GSK3β, and both si-GSK3α/β for 3 days. (A) Western blot analysis (Aa) and quantitative analysis (Ab) of mSREBP1 (mature form) levels. (B) (Ba) Cell growth rate. (Bb) Representative images (upper) and quantitative analysis of senescence-associated β-galactosidase-positive cell populations (lower) are shown. (C) Quantitative analyses for cell size (Ca, forward scatter; FSC analysis, left) and cell granularity (Cb, side scatter; SSC analysis, right). aP<0.05; bP<0.01 vs. negative control (NC).

• 

  Fig. 5 Sterol regulatory element binding protein 1 (SREBP1)-mediated lipogenesis is the key event of glycogen synthase kinase 3 (GSK3) inhibition-induced senescence. Quantitative analysis of senescence-associated β-galactosidase-positive cell populations was used to evaluate the extent of senescence. (A) Chang cells (1×104) seeded on 6-well plates were transfected with siRNAs for SREBP1 and then treated with 7.5 µg/mL SB415286 for 4 days. (B) Chang cells were transfected with siRNAs for ATP citrate lyase (ACL) and then treated with 7.5 µg/mL SB415286 for 4 days. (C) Chang cells were pretreated with the indicated concentrations of cerulenin (Cer; Sigma C2389) and C75 (Sigma 5490) for 24 hours and then further treated with 7.5 µg/mL SB415286 for 4 days. siRNA with a random sequence was used as the negative control (NC), and dimethyl sulfoxide was used as the vehicle control (V). aP<0.05; bP<0.01 vs. NC or V.

Reference

1. Campisi J, Kim SH, Lim CS, Rubio M. Cellular senescence, cancer and aging: the telomere connection. Exp Gerontol. 2001; 36:1619–1637. PMID: 11672984.

2. Cristofalo VJ, Kritchevsky D. Cell size and nucleic acid content in the diploid human cell line WI-38 during aging. Med Exp Int J Exp Med. 1969; 19:313–320. PMID: 5408801.

3. Greenberg SB, Grove GL, Cristofalo VJ. Cell size in aging monolayer cultures. In Vitro. 1977; 13:297–300. PMID: 326658.

4. Hwang ES, Yoon G, Kang HT. A comparative analysis of the cell biology of senescence and aging. Cell Mol Life Sci. 2009; 66:2503–2524. PMID: 19421842.

5. De Priester W, Van Manen R, Knook DL. Lysosomal activity in the aging rat liver: II. morphometry of acid phosphatase positive dense bodies. Mech Ageing Dev. 1984; 26:205–216. PMID: 6482519.

6. Lipetz J, Cristofalo VJ. Ultrastructural changes accompanying the aging of human diploid cells in culture. J Ultrastruct Res. 1972; 39:43–56. PMID: 5017037.

7. Yoon YS, Yoon DS, Lim IK, Yoon SH, Chung HY, Rojo M, Malka F, Jou MJ, Martinou JC, Yoon G. Formation of elongated giant mitochondria in DFO-induced cellular senescence: involvement of enhanced fusion process through modulation of Fis1. J Cell Physiol. 2006; 209:468–480. PMID: 16883569.

8. Goldstein S, Moerman EJ, Porter K. High-voltage electron microscopy of human diploid fibroblasts during ageing in vitro. Morphometric analysis of mitochondria. Exp Cell Res. 1984; 154:101–111. PMID: 6468521.

9. Schmucker DL, Sachs H. Quantifying dense bodies and lipofuscin during aging: a morphologist's perspective. Arch Gerontol Geriatr. 2002; 34:249–261. PMID: 14764327.

10. Kim YM, Shin HT, Seo YH, Byun HO, Yoon SH, Lee IK, Hyun DH, Chung HY, Yoon G. Sterol regulatory element-binding protein (SREBP)-1-mediated lipogenesis is involved in cell senescence. J Biol Chem. 2010; 285:29069–29077. PMID: 20615871.

11. Horton JD, Shah NA, Warrington JA, Anderson NN, Park SW, Brown MS, Goldstein JL. Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes. Proc Natl Acad Sci U S A. 2003; 100:12027–12032. PMID: 14512514.

12. Demoulin JB, Ericsson J, Kallin A, Rorsman C, Ronnstrand L, Heldin CH. Platelet-derived growth factor stimulates membrane lipid synthesis through activation of phosphatidylinositol 3-kinase and sterol regulatory element-binding proteins. J Biol Chem. 2004; 279:35392–35402. PMID: 15213220.

13. Porstmann T, Santos CR, Lewis C, Griffiths B, Schulze A. A new player in the orchestra of cell growth: SREBP activity is regulated by mTORC1 and contributes to the regulation of cell and organ size. Biochem Soc Trans. 2009; 37(Pt 1):278–283. PMID: 19143646.

14. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest. 2002; 109:1125–1131. PMID: 11994399.

15. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 1995; 378:785–789. PMID: 8524413.

16. Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur J Biochem. 1980; 107:519–527. PMID: 6249596.

17. Forde JE, Dale TC. Glycogen synthase kinase 3: a key regulator of cellular fate. Cell Mol Life Sci. 2007; 64:1930–1944. PMID: 17530463.

18. Rylatt DB, Aitken A, Bilham T, Condon GD, Embi N, Cohen P. Glycogen synthase from rabbit skeletal muscle. Amino acid sequence at the sites phosphorylated by glycogen synthase kinase-3, and extension of the N-terminal sequence containing the site phosphorylated by phosphorylase kinase. Eur J Biochem. 1980; 107:529–537. PMID: 6772446.

19. Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, Klevernic I, Arthur JS, Alessi DR, Cohen P. The selectivity of protein kinase inhibitors: a further update. Biochem J. 2007; 408:297–315. PMID: 17850214.

20. Huang W, Chang HY, Fei T, Wu H, Chen YG. GSK3 beta mediates suppression of cyclin D2 expression by tumor suppressor PTEN. Oncogene. 2007; 26:2471–2482. PMID: 17043650.

21. Shakoori A, Ougolkov A, Yu ZW, Zhang B, Modarressi MH, Billadeau DD, Mai M, Takahashi Y, Minamoto T. Deregulated GSK3beta activity in colorectal cancer: its association with tumor cell survival and proliferation. Biochem Biophys Res Commun. 2005; 334:1365–1373. PMID: 16043125.

22. Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, Woodgett JR. Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. Nature. 2000; 406:86–90. PMID: 10894547.

23. Seo YH, Jung HJ, Shin HT, Kim YM, Yim H, Chung HY, Lim IK, Yoon G. Enhanced glycogenesis is involved in cellular senescence via GSK3/GS modulation. Aging Cell. 2008; 7:894–907. PMID: 18782348.

24. Bartke A. Mutations prolong life in flies: implications for aging in mammals. Trends Endocrinol Metab. 2001; 12:233–234. PMID: 11445429.

25. Holzenberger M, Dupont J, Ducos B, Leneuve P, Geloen A, Even PC, Cervera P, Le Bouc Y. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature. 2003; 421:182–187. PMID: 12483226.

26. Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G. daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science. 1997; 277:942–946. PMID: 9252323.

27. Tatar M, Kopelman A, Epstein D, Tu MP, Yin CM, Garofalo RS. A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science. 2001; 292:107–110. PMID: 11292875.

28. Tissenbaum HA, Ruvkun G. An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans. Genetics. 1998; 148:703–717. PMID: 9504918.

29. Punga T, Bengoechea-Alonso MT, Ericsson J. Phosphorylation and ubiquitination of the transcription factor sterol regulatory element-binding protein-1 in response to DNA binding. J Biol Chem. 2006; 281:25278–25286. PMID: 16825193.

30. Yoon YS, Byun HO, Cho H, Kim BK, Yoon G. Complex II defect via down-regulation of iron-sulfur subunit induces mitochondrial dysfunction and cell cycle delay in iron chelation-induced senescence-associated growth arrest. J Biol Chem. 2003; 278:51577–51586. PMID: 14512425.

31. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957; 226:497–509. PMID: 13428781.

32. Kupke IR, Zeugner S. Quantitative high-performance thin-layer chromatography of lipids in plasma and liver homogenates after direct application of 0.5-microliter samples to the silica-gel layer. J Chromatogr. 1978; 146:261–271. PMID: 212444.

33. Coghlan MP, Culbert AA, Cross DA, Corcoran SL, Yates JW, Pearce NJ, Rausch OL, Murphy GJ, Carter PS, Roxbee Cox L, Mills D, Brown MJ, Haigh D, Ward RW, Smith DG, Murray KJ, Reith AD, Holder JC. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol. 2000; 7:793–803. PMID: 11033082.

34. Omura S. The antibiotic cerulenin, a novel tool for biochemistry as an inhibitor of fatty acid synthesis. Bacteriol Rev. 1976; 40:681–697. PMID: 791237.

35. Pizer ES, Chrest FJ, DiGiuseppe JA, Han WF. Pharmacological inhibitors of mammalian fatty acid synthase suppress DNA replication and induce apoptosis in tumor cell lines. Cancer Res. 1998; 58:4611–4615. PMID: 9788612.

36. Byun HO, Jung HJ, Seo YH, Lee YK, Hwang SC, Hwang ES, Yoon G. GSK3 inactivation is involved in mitochondrial complex IV defect in transforming growth factor (TGF) β1-induced senescence. Exp Cell Res. 2012; 318:1808–1819. PMID: 22652454.

37. Dokken BB, Sloniger JA, Henriksen EJ. Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle. Am J Physiol Endocrinol Metab. 2005; 288:E1188–E1194. PMID: 15671078.

38. Kenyon C. The plasticity of aging: insights from long-lived mutants. Cell. 2005; 120:449–460. PMID: 15734678.

39. Miyauchi H, Minamino T, Tateno K, Kunieda T, Toko H, Komuro I. Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway. EMBO J. 2004; 23:212–220. PMID: 14713953.