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Decrease of apoptosis markers during adipogenic differentiation of mesenchymal stem cells from human adipose tissue

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Abstract

Although the proliferation and differentiation of mesenchymal stem cells (MSCs) from adipose tissue (AT) have been widely studied, relatively little information is available on the underlying mechanism of apoptosis during the adipogenic differentiation. Thus, the aim of this study was to analyze how the expression of some apoptotic markers is affected by in vitro expansion during adipogenic differentiation of AT-MSCs. The cultures incubated or not with adipogenic medium were investigated by Western blot at 7, 14, 21, and 28 days for the production of p53, AKT, pAKT, Bax, PDCD4 and PTEN. MSCs were recognized for their immunoreactivity to MSC-specific cell types markers by immunocytochemical procedure. The effectiveness of adipogenic differentiation was assessed by staining with Sudan III and examination of adipogenic markers expression, such as PPAR-γ and FABP, at different time points by Western blot. The adipogenic differentiation medium led to the appearance, after 7 days, of larger rounded cells presenting numerous vacuoles containing lipids in which it was evident a red–orange staining, that increased in size in a time-dependent manner, parallel to an increase of the levels of expression of PPAR-γ and FABP. More than 50 % of human MSCs were fully differentiated into adipocytes within the four-week induction period. The results showed that during adipogenic differentiation of AT-MSCs the PI3K/AKT signaling pathway is activated and that p53, PTEN, PDCD4, and Bax proteins are down-regulated in time-dependent manner. Our data provide new information on the behavior of some apoptotic markers during adipogenic differentiation of AT-MSCs to apply for tissues repair and regeneration.

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References

  1. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7:211–228

    Article  PubMed  CAS  Google Scholar 

  2. Walker MR, Patel KK, Stappenbeck TS (2009) The stem cell niche. J Pathol 217:169–180

    Article  PubMed  CAS  Google Scholar 

  3. Kern S, Eichler H, Stoeve J, Klüter H, Bieback K (2006) Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 24:1294–1301

    Article  PubMed  CAS  Google Scholar 

  4. Tuan RS, Boland G, Tuli R (2003) Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther 5:32–45

    Article  PubMed  CAS  Google Scholar 

  5. Friedenstein AJ, Deriglasova UF, Kulagina NN, Panasuk AF, Rudakowa SF, Luriá EA, Ruadkow IA (1974) Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 2:83–92

    PubMed  CAS  Google Scholar 

  6. Lin G, Garcia M, Ning H, Banie L, Guo YL, Lue TF, Lin CS (2008) Defining stem and progenitor cells within adipose tissue. Stem cells dev 17:1053–1064

    Article  PubMed  CAS  Google Scholar 

  7. Magun R, Gagnon AM, Yaraghi Z, Sorisky A (1998) Expression and regulation of neuronal apoptosis inhibitory protein during adipocyte differentiation. Diabetes 47:1948–1952

    Article  PubMed  CAS  Google Scholar 

  8. Planat-Benard V, Silvestre JS, Cousin B, André M, Nibbelink M, Tamarat R, Clergue M, Manneville C, Saillan-Barreau C, Duriez M, Tedgui A, Levy B, Pénicaud L, Casteilla L (2004) Plasticity of human adipose lineage cells toward endothelial cells. physiological and therapeutic perspectives. Circulation 109:656–663

    Article  PubMed  Google Scholar 

  9. Barry FP, Murphy JM (2004) Mesencymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 36:568–584

    Article  PubMed  CAS  Google Scholar 

  10. Dicker A, Le Blanc K, Aström G, van Harmelen V, Götherström C, Blomqvis L, Arner P, Rydén M (2005) Functional studies of mesenchymal stem cells derived from adult human adipose tissue. Exp Cell Res 308:283–290

    Article  PubMed  CAS  Google Scholar 

  11. Jeon ES, Kang YJ, Song HY, Woo JS, Jung JS, Kim YK, Kim JH (2005) Role of MEK-ERK pathway in sphingosylphosphorylcholine-induced cell death in human adipose tissue-derived mesenchymal stem cells. Biochim Biophys Acta 1734:25–33

    Article  PubMed  CAS  Google Scholar 

  12. Kim YJ, Bae YC, Suh KT, Jung JS (2006) Quercetin, a flavonoid, inhibits proliferation and increase osteogenic differentiation in human adipose stromal cells. Biochem Pharmacol 72:1268–1278

    Article  PubMed  CAS  Google Scholar 

  13. Strissel KJ, Stancheva Z, Miyoshi H, Perfield JW II, DeFuria J, Jick Z, Greenberg AS, Obin MS (2007) Adipocyte death, adipose tissue remodeling, and obesity complications. Diabetes 56:2910–2918

    Article  PubMed  CAS  Google Scholar 

  14. Magun R, Boone DL, Tsang BK, Sorisky A (1998) The effect of adipocyte differentiation on the capacity of 3T3-L1 cells to undergo apoptosis in response to growth factor deprivation. Int J Obes Relat Metab Disord 22:567–571

    Article  PubMed  CAS  Google Scholar 

  15. Niesler CU, Urso B, Prins JB, Siddle K (2000) IGF-1 inhibits apoptosis induced by serum withdrawal, but potentiates TNF-α-induced apoptosis, in 3T3-L1 preadipocytes. J Endocrinol 167:165–174

    Article  PubMed  CAS  Google Scholar 

  16. Gagnon A, Artemenko Y, Crapper T, Sorisky A (2003) Regulation of endogenous SH2 domain-containing inositol 5-phosphatase (SHIP2) in 3T3-L1 and human preadipocytes. J Cell Physiol 197:243–250

    Article  PubMed  CAS  Google Scholar 

  17. Fischer-Posovszky P, Rews D, Horenburg S, Debatin KM, Wabitsch M (2012) Differential function of AKt1 and AKt2 in human adipocytes. Mol Cell Endocrinol 358:135–143

    Article  PubMed  CAS  Google Scholar 

  18. Zebisch K, Voigt V, Wabitsch M, Brandsch M (2012) Protocol for effective differentiation of 3T3-L1 cells to adipocytes. Anal Biochem 425:88–90

    Article  PubMed  CAS  Google Scholar 

  19. Staiger H, Loffler G (1998) The role of PDGF-dependent suppression of apoptosis in differentiating 3T3-L1 preadipocytes. Eur J Cell Biol 77:220–227

    Article  PubMed  CAS  Google Scholar 

  20. Li H, Fong C, Chen Y, Caia G, Yang M (2010) Beta-adrenergic signals regulate adipogenesis of mouse mesenchymal stem cells via cAMP/PKA pathway. Mol Cell Endocrinol 323:201–207

    Article  PubMed  CAS  Google Scholar 

  21. Yu W, Chen Z, Zhang J, Zhang L, Ke H, Huang L, Peng Y, Zhang X, Li S, Lahn BT, Xiang AP (2008) Critical role of phosphoinositide 3-kinase cascade in adipogenesis of human mesenchymal stem cells. Mol Cell Biochem 310:11–18

    Article  PubMed  CAS  Google Scholar 

  22. Aubin D, Gagnon A, Sorisky A (2005) Phosphoinositide 3-kinase is required for human adipocyte differentiation in culture. Int J Obes (Lond) 29:1006–1009

    Article  CAS  Google Scholar 

  23. Kohn AD, Summers SA, Birnbaum MJ, Roth RA (1996) Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271:31372–31378

    Article  PubMed  CAS  Google Scholar 

  24. Magun R, Burgering BM, Coffer PJ, Pardasani D, Lin Y, Chabot J, Sorisky A (1996) Expression of a constitutively activated form of protein kinase B (c-Akt) in 3T3-L1 preadipose cells causes spontaneous differentiation. Endocrinology 137:3590–3593

    Article  PubMed  CAS  Google Scholar 

  25. Armstrong JF, Kaufman MH, Harrison DJ, Clarke AR (1995) High-frequency developmental abnormalities in p53-deficient mice. Curr Biol 5:931–936

    Article  PubMed  CAS  Google Scholar 

  26. Choi J, Donehower LA (1999) p53 in embryonic development: maintaining a fine balance. Cell Mol Life Sci 55:38–47

    Article  PubMed  CAS  Google Scholar 

  27. Hall PA, Lane DP (1997) Tumor suppressors: a developing role for p53? Curr Biol 7:R144–R147

    Article  PubMed  CAS  Google Scholar 

  28. Almog N, Rotter V (1997) Involvement of p53 in cell differentiation and development. Biochem Biophys Acta 1333:F1–F27

    PubMed  CAS  Google Scholar 

  29. Zambetti GP, Horwitz EM, Schipani E (2006) Skeletons in the p53 tumor suppressor closet: genetic evidence that p53 blocks bone differentiation and development. J Cell Biol 172:795–797

    Article  PubMed  CAS  Google Scholar 

  30. Constance CM, Morgan JI, Umek RM (1996) C/EBPalpha regulation of the growth-arrest associated gene gadd45. Mol Cell Biol 16:3878–3883

    PubMed  CAS  Google Scholar 

  31. Berberich SJ, Litteral V, Mayo LD, Tabesh D, Morris D (1999) mdm-2 gene amplification in 3T3-L1 preadipocytes. Differentiation 64:205–212

    Article  PubMed  CAS  Google Scholar 

  32. Inoue N, Yamamoto T, Ishikawa M, Watanabe K, Matsuzaka T, Nakagawa Y, Takeuchi Y, Kobayashi K, Takahashi A, Suzuki H, Hasty AH, Toyoshima H, Yamada N, Shimano H (2008) Cyclin-dependent kinase inhibitor, p21WAF1/CIP1, is involved in adipocyte differentiation and hypertrophy, linking to obesity, and insulin resistance. J Biol Chem 283:21220–21229

    Article  PubMed  CAS  Google Scholar 

  33. Hong Y, Cervantes RB, Tichy E, Tischfield JA, Stambrook PJ (2007) Protecting genomic integrity in somatic cells and embryonic stem cells. Mutat Res 614:48–55

    Article  PubMed  CAS  Google Scholar 

  34. Sabbatini P, McCormick F (1999) Phosphoinositide 3-OH kinase (PI3K) and PKB/Akt delay the onset of p53-mediated, transcriptionally dependent apoptosis. J Biol Chem 274:24263–24269

    Article  PubMed  CAS  Google Scholar 

  35. Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310

    Article  PubMed  CAS  Google Scholar 

  36. Gross A, McDonnell JM, Korsmeyer SJ (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13:1899–1911

    Article  PubMed  CAS  Google Scholar 

  37. Castedo M, Ferri KF, Blanco J, Roumier T, Larochette N, Barretina J, Amendola A, Nardacci R, Métivier D, Este JA, Piacentini M, Kroemer G (2001) Human immunodeficiency virus 1 envelope glycoprotein complex-induced apoptosis involves mammalian target of rapamycin/FKBP12-rapamycin-associated protein-mediated p53 phosphorylation. J Exp Med 194:1097–1110

    Article  PubMed  CAS  Google Scholar 

  38. Castedo M, Perfettini JL, Roumier T, Kroemer G (2002) Cyclin dependent kinase-1: linking apoptosis to cell cycle and mitotic catastrophe. Cell Death Differ 9:1287–1293

    Article  PubMed  CAS  Google Scholar 

  39. Marzo I, Brenner C, Zamzami N, Jürgensmeier JM, Susin SA, Vieira HL, Prévost MC, Xie Z, Matsuyama S, Reed JC, Kroemer G (1998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281:2027–2031

    Article  PubMed  CAS  Google Scholar 

  40. Oltvai ZN, Milliman CL, Korsmeyer SJ (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609–619

    Article  PubMed  CAS  Google Scholar 

  41. Myers MP, Pass I, Batty IH, Van der Kaay J, Stolarov JP, Hemmings BA, Wigler MH, Downes CP, Tonks NK (1998) The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. Proc Natl Acad Sci USA 95:13513–13518

    Article  PubMed  CAS  Google Scholar 

  42. Wu X, Senechal K, Neshat MS, Whang YE, Sawyers CL (1998) The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc Natl Acad Sci USA 95:15587–15591

    Article  PubMed  CAS  Google Scholar 

  43. Franke TF, Kaplan DR, Cantley LC, Toker A (1997) Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4- bisphosphate. Science 275:665–668

    Article  PubMed  CAS  Google Scholar 

  44. Sun H, Lesche R, Li DM, Liliental J, Zhang H, Gao J, Gavrilova N, Mueller B, Liu X, Wu H (1999) PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc Natl Acad Sci USA 96:6199–6204

    Article  PubMed  CAS  Google Scholar 

  45. Blanco-Aparicio C, Renner O, Leal JFM, Carnero A (2007) PTEN, more than the AKT pathway. Carcinogenesis 28:1379–1386

    Article  PubMed  CAS  Google Scholar 

  46. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC (1998) Regulation of cell death protease caspase-9 by phosphorylation. Science 282:1318–1321

    Article  PubMed  CAS  Google Scholar 

  47. 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 398:630–634

    Article  PubMed  CAS  Google Scholar 

  48. Yilmaz OH, Valdez R, Theisen BK, Guo W, Ferguson DO, Wu H, Morrison SJ (2006) Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 441:475–482

    Article  PubMed  CAS  Google Scholar 

  49. Zhang J, Grindley JC, Yin T, Jayasinghe S, He XC, Ross JT, Haug JS, Rupp D, Porter-Westpfahl KS, Wiedemann LM, Wu H, Li L (2006) PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature 441:518–522

    Article  PubMed  CAS  Google Scholar 

  50. Groszer M, Erickson R, Scripture-Adams DD, Dougherty JD, Le Belle J, Zack JA, Geschwind DH, Liu X, Kornblum HI, Wu H (2006) PTEN negatively regulates neural stem cell self-renewal by modulating G0–G1 cell cycle entry. Proc Natl Acad Sci USA 103:111–116

    Article  PubMed  CAS  Google Scholar 

  51. Lankat-Buttgereit B, Göke R (2009) The tumour suppressor Pdcd4: recent advances in the elucidation of function and regulation. Biol Cell 101:309–317

    Article  PubMed  CAS  Google Scholar 

  52. Mudduluru G, Medved F, Grobholz R, Jost C, Gruber A, Leupold JH, Post S, Jansen A, Colburn NH, Allgayer H (2007) Loss of programmed cell death 4 expression marks adenoma-carcinoma transition, correlates inversely with phosphorylated protein kinase B, and is an independent prognostic factor in resected colorectal cancer. Cancer 110:1697–1707

    Article  PubMed  CAS  Google Scholar 

  53. Dorrello NV, Peschiaroli A, Guardavaccaro D, Colburn NH, Sherman NE, Pagano M (2006) S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science 314:467–471

    Article  PubMed  CAS  Google Scholar 

  54. Caggia S, Libra M, Malaponte G, Cardile V (2011) Modulation of YY1 and p53 expression by transforming growth factor-β3 in prostate cell lines. Cytokine 56:403–410

    Article  PubMed  CAS  Google Scholar 

  55. Sonenberg N, Pause A (2006) Signal transduction. Protein synthesis and oncogenesis meet again. Science 314:428–429

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Venera Cardile.

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Lo Furno, D., Graziano, A.C.E., Caggia, S. et al. Decrease of apoptosis markers during adipogenic differentiation of mesenchymal stem cells from human adipose tissue. Apoptosis 18, 578–588 (2013). https://doi.org/10.1007/s10495-013-0830-x

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