Human amniotic fluid stem cells (AFSC) with multilineage differentiation potential are novel source for cell therapy. However, in vitro expansion leads to senescence affecting differentiation and proliferative capacities. Reactive oxygen species (ROS) have been involved in the regulation of stem cell pluripotency, proliferation, and differentiation. Redox-regulated signal transduction is coordinated by spatially controlled production of ROS within subcellular compartments. NAD(P)H oxidase family, in particular Nox4, has been known to produce ROS in the nucleus; however, the mechanisms and the meaning of this function remain largely unknown. In the present study, we show that Nox4 nuclear expression (nNox4) increases during culture passages up to cell cycle arrest and the serum starvation causes the same effect. With the decrease of Nox4 activity, obtained with plumbagin, a decline of nuclear ROS production and of DNA damage occurs. Moreover, plumbagin exposure reduces the binding between nNox4 and nucleoskeleton components, as Matrin 3. The same effect was observed also for the binding with phospho-ERK, although nuclear ERK and P-ERK are unchanged. Taken together, we suggest that nNox4 regulation may have important pathophysiologic effects in stem cell proliferation through modulation of nuclear signaling and DNA damage. 1. Introduction Stem cells are characterized by a high capacity of self-renewal and differentiation. Through self-renewal, stem cells maintain the homeostasis of a stem cell pool; through differentiation, stem cells can give rise to terminal cells with diverse morphology and functions [1]. In tissues, most stem cells are in the quiescent state, and they are protected by special microenvironments (niches) [2]. The quiescence of stem cells may prevent the accumulation of DNA replication errors [3] and may facilitate resistance to many stressors [4]. The intracellular ROS level is a critical factor that regulates the quiescent status of mesenchymal stem cells (MSC) [5]. Similar to the low partial pressure of oxygen, low levels of ROS in niches are important for the stemness of MSC [6]. However, in vitro expansion of stem cells implies normoxic culture condition. Indeed, MSC proliferative and colony formation capacity is significantly increased in normoxia. However, MSC expanded under normoxia show a threefold to fourfold increase in senescence, suggesting that hypoxia prevents oxidative stress-induced senescence and preserves MSC long-term self-renewal [7]. Accumulation of ROS is a common occurrence in senescent cells. Studies have shown
References
[1]
X. Shi, Y. Zhang, J. Zheng, and J. Pan, “Reactive oxygen species in cancer stem cells,” Antioxidants and Redox Signaling, vol. 16, no. 11, pp. 1215–1228, 2012.
[2]
S. J. Morrison and A. C. Spradling, “Stem cells and niches: mechanisms that promote stem cell maintenance throughout life,” Cell, vol. 132, no. 4, pp. 598–611, 2008.
[3]
F. Arai, A. Hirao, M. Ohmura et al., “Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche,” Cell, vol. 118, no. 2, pp. 149–161, 2004.
[4]
K. Ito, R. Bernardi, A. Morotti et al., “PML targeting eradicates quiescent leukaemia-initiating cells,” Nature, vol. 453, no. 7198, pp. 1072–1078, 2008.
[5]
Z. Tothova, R. Kollipara, B. J. Huntly et al., “FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress,” Cell, vol. 128, no. 2, pp. 325–339, 2007.
[6]
Y.-Y. Jang and S. J. Sharkis, “A low level of reactive oxygen species selects for primitive hematopoietic stem cells that may reside in the low-oxygenic niche,” Blood, vol. 110, no. 8, pp. 3056–3063, 2007.
[7]
G. Pattappa, S. D. Thorpe, N. C. Jegard, H. K. Heywood, J. D. de Bruijn, and D. A. Lee, “Continuous and uninterrupted oxygen tension influences the colony formation and oxidative metabolism of human mesenchymal stem cells,” Tissue Engineering C, vol. 19, no. 1, pp. 68–79, 2013.
[8]
J. Dai, X. Zhu, M. C. Yoder, Y. Wu, and R. W. Colman, “Cleaved high-molecular-weight kininogen accelerates the onset of endothelial progenitor cell senescence by induction of reactive oxygen species,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 31, no. 4, pp. 883–889, 2011.
[9]
P. J. Ho, M. L. Yen, B. C. Tang, C. T. Chen, and B. L. Yen, “H2O2 accumulation mediates differentiation capacity alteration, but not proliferative decline, in senescent human fetal mesenchymal stem cells,” Antioxid Redox Signal, vol. 18, no. 15, pp. 1895–1905, 2013.
[10]
T. Kietzmann, “Intracellular redox compartments: mechanisms and significances,” Antioxidants & Redox Signaling, vol. 13, no. 4, pp. 395–398, 2010.
[11]
Y.-M. Go and D. P. Jones, “Redox control systems in the nucleus: mechanisms and functions,” Antioxidants & Redox Signaling, vol. 13, no. 4, pp. 489–509, 2010.
[12]
R.-M. Liu, J. Choi, J.-H. Wu et al., “Oxidative modification of nuclear mitogen-activated protein kinase phosphatase 1 is involved in transforming growth factor β1-induced expression of plasminogen activator inhibitor 1 in fibroblasts,” The Journal of Biological Chemistry, vol. 285, no. 21, pp. 16239–16247, 2010.
[13]
K. Chen, M. T. Kirber, H. Xiao, Y. Yang, and J. F. Keaney Jr., “Regulation of ROS signal transduction by NADPH oxidase 4 localization,” The Journal of Cell Biology, vol. 181, no. 7, pp. 1129–1139, 2008.
[14]
R. K. Ambasta, P. Kumar, K. K. Griendling, H. H. Schmidt, R. Busse, and R. P. Brandes, “Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase,” The Journal of Biological Chemistry, vol. 279, no. 44, pp. 45935–45941, 2004.
[15]
K. Block, Y. Gorin, and H. E. Abboud, “Subcellular localization of Nox4 and regulation in diabetes,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 34, pp. 14385–14390, 2009.
[16]
L. L. Hilenski, R. E. Clempus, M. T. Quinn, J. D. Lambeth, and K. K. Griendling, “Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 24, no. 4, pp. 677–683, 2004.
[17]
Y.-M. Lee, B.-J. Kim, Y.-S. Chun et al., “NOX4 as an oxygen sensor to regulate TASK-1 activity,” Cellular Signalling, vol. 18, no. 4, pp. 499–507, 2006.
[18]
J. Kuroda, K. Nakagawa, T. Yamasaki et al., “The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells,” Genes to Cells, vol. 10, no. 12, pp. 1139–1151, 2005.
[19]
P. Wang, F. Tang, R. Li et al., “Contribution of different Nox homologues to cardiac remodeling in two-kidney two-clip renovascular hypertensive rats: effect of valsartan,” Pharmacological Research, vol. 55, no. 5, pp. 408–417, 2007.
[20]
S. I. Dikalov, A. E. Dikalova, A. T. Bikineyeva, H. H. Schmidt, D. G. Harrison, and K. K. Griendling, “Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production,” Free Radical Biology and Medicine, vol. 45, no. 9, pp. 1340–1351, 2008.
[21]
S. Pendyala, I. A. Gorshkova, P. V. Usatyuk et al., “Role of Nox4 and Nox2 in hyperoxia-induced reactive oxygen species generation and migration of human lung endothelial cells,” Antioxidants and Redox Signaling, vol. 11, no. 4, pp. 747–764, 2009.
[22]
A. N. Lyle, N. N. Deshpande, Y. Taniyama et al., “Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells,” Circulation Research, vol. 105, no. 3, pp. 249–259, 2009.
[23]
G. Gordillo, H. Fang, H. Park, and S. Roy, “Nox-4-dependent nuclear H2O2 drives DNA oxidation resulting in 8-OHdG as urinary biomarker and hemangioendothelioma formation,” Antioxidants and Redox Signaling, vol. 12, no. 8, pp. 933–943, 2010.
[24]
N. S. De Mochel, S. Seronello, S. H. Wang et al., “Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection,” Hepatology, vol. 52, no. 1, pp. 47–59, 2010.
[25]
U. Weyemi and C. Dupuy, “The emerging role of ROS-generating NADPH oxidase NOX4 in DNA-damage responses,” Mutation Research, vol. 751, no. 2, pp. 77–81, 2012.
[26]
G. Lattanzi, S. Marmiroli, A. Facchini, and N. M. Maraldi, “Nuclear damages and oxidative stress: new perspectives for laminopathies,” European Journal of Histochemistry, vol. 56, no. 4, article e45, 2012.
[27]
N. Anilkumar, G. S. Jose, I. Sawyer et al., “A 28-kDa splice variant of NADPH oxidase-4 is nuclear-localized and involved in redox signaling in vascular cells,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 33, no. 4, pp. e104–e112, 2013.
[28]
P. De Coppi, G. Bartsch Jr., M. M. Siddiqui et al., “Isolation of amniotic stem cell lines with potential for therapy,” Nature Biotechnology, vol. 25, no. 1, pp. 100–106, 2007.
[29]
T. Maraldi, M. Riccio, A. Pisciotta et al., “Human amniotic fluid-derived and dental pulp-derived stem cells seeded into collagen scaffold repair critical-size bone defects promoting vascularization,” Stem Cell Research & Therapy, vol. 4, no. 3, article 53, 2013.
[30]
G. Carnevale, M. Riccio, A. Pisciotta et al., “In vitro differentiation into insulin-producing β-cells of stem cells isolated from human amniotic fluid and dental pulp,” Digestive and Liver Disease, vol. 45, no. 8, pp. 669–676, 2013.
[31]
A. Trounson, “A fluid means of stem cell generation,” Nature Biotechnology, vol. 25, no. 1, pp. 62–63, 2007.
[32]
Y. Ding, Z.-J. Chen, S. Liu, D. Che, M. Vetter, and C.-H. Chang, “Inhibition of Nox-4 activity by plumbagin, a plant-derived bioactive naphthoquinone,” Journal of Pharmacy and Pharmacology, vol. 57, no. 1, pp. 111–116, 2005.
[33]
M. Zavatti, E. Resca, L. Bertoni et al., “Ferutinin promotes proliferation and osteoblastic differentiation in human amniotic fluid and dental pulp stem cells,” Life Sciences, vol. 92, no. 20-21, pp. 993–1003, 2013.
[34]
T. Maraldi, M. Riccio, L. Zambonin, M. Vinceti, A. De Pol, and G. Hakim, “Low levels of selenium compounds are selectively toxic for a human neuron cell line through ROS/RNS increase and apoptotic process activation,” NeuroToxicology, vol. 32, no. 2, pp. 180–187, 2011.
[35]
T. Maraldi, J. Bertacchini, M. Benincasa et al., “Reverse-phase protein microarrays (RPPA) as a diagnostic and therapeutic guide in multidrug resistant leukemia,” International Journal of Oncology, vol. 38, no. 2, pp. 427–435, 2011.
[36]
V. Cenni, J. Bertacchini, F. Beretti et al., “Lamin A Ser404 Is a nuclear target of akt phosphorylation in C2C12 cells,” Journal of Proteome Research, vol. 7, no. 11, pp. 4727–4735, 2008.
[37]
V. Cenni, A. Bavelloni, F. Beretti et al., “Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H2O2,” Molecular Biology of the Cell, vol. 22, no. 16, pp. 2946–2956, 2011.
[38]
C. J. Hanson, M. D. Bootman, C. W. Distelhorst, T. Maraldi, and H. L. Roderick, “The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect,” Cell Calcium, vol. 44, no. 3, pp. 243–258, 2008.
[39]
E. Resca, M. Zavatti, L. Bertoni et al., “Enrichment in c-Kit+ enhances mesodermal and neural differentiation of human chorionic placental cells,” Placenta, vol. 34, no. 7, pp. 526–535, 2013.
[40]
A. Pisciotta, M. Riccio, G. Carnevale et al., “Human serum promotes osteogenic differentiation of human dental pulp stem cells in vitro and in vivo,” PLoS One, vol. 7, no. 11, Article ID e50542, 2012.
[41]
M. Riccio, E. Resca, T. Maraldi et al., “Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures,” European Journal of Histochemistry, vol. 54, no. 4, article 46, 2010.
[42]
B. C. Dickinson, Y. Tang, Z. Chang, and C. J. Chang, “A nuclear-localized fluorescent hydrogen peroxide probe for monitoring sirtuin-mediated oxidative stress responses in vivo,” Chemistry and Biology, vol. 18, no. 8, pp. 943–948, 2011.
[43]
A. R. Lippert, G. C. Van De Bittner, and C. J. Chang, “Boronate oxidation as a bioorthogonal reaction approach for studying the chemistry of hydrogen peroxide in living systems,” Accounts of Chemical Research, vol. 44, no. 9, pp. 793–804, 2011.
[44]
V. S. Lin, B. C. Dickinson, and C. J. Chang, “Boronate-based fluorescent probes: imaging hydrogen peroxide in living systems,” Methods in Enzymology, vol. 526, pp. 19–43, chapter 2 in Hydrogen Peroxide and Cell Signaling, Part A, 2013.
[45]
B. C. Dickinson and C. J. Chang, “Chemistry and biology of reactive oxygen species in signaling or stress responses,” Nature Chemical Biology, vol. 7, no. 8, pp. 504–511, 2011.
[46]
S. Altenh？fer, P. W. Kleikers, K. A. Radermacher et al., “The NOX toolbox: validating the role of NADPH oxidases in physiology and disease,” Cellular and Molecular Life Sciences, vol. 69, no. 14, pp. 2327–2343, 2012.
[47]
K. D. Pendergrass, T. M. Gwathmey, R. D. Michalek, J. M. Grayson, and M. C. Chappell, “The angiotensin II-AT1 receptor stimulates reactive oxygen species within the cell nucleus,” Biochemical and Biophysical Research Communications, vol. 384, no. 2, pp. 149–154, 2009.
[48]
N. Y. Spencer, Z. Yan, R. L. Boudreau et al., “Control of hepatic nuclear superoxide production by glucose 6-phosphate dehydrogenase and NADPH oxidase-4,” The Journal of Biological Chemistry, vol. 286, no. 11, pp. 8977–8987, 2011.
[49]
R.-F. Wu, Z. Ma, Z. Liu, and L. S. Terada, “Nox4-derived H2O2 mediates endoplasmic reticulum signaling through local ras activation,” Molecular and Cellular Biology, vol. 30, no. 14, pp. 3553–3568, 2010.
[50]
J. Chen, J.-K. Chen, and R. C. Harris, “Angiotensin II induces epithelial-to-mesenchymal transition in renal epithelial cells through reactive oxygen species/Src/caveolin-mediated activation of an epidermal growth factor receptor-extracellular signal-regulated kinase signaling pathway,” Molecular and Cellular Biology, vol. 32, no. 5, pp. 981–991, 2012.
[51]
K. J. Schmitz, H. Lang, J. Wohlschlaeger et al., “AKT and ERK1/2 signaling in intrahepatic cholangiocarcinoma,” World Journal of Gastroenterology, vol. 13, no. 48, pp. 6470–6477, 2007.
[52]
J. D. Lambeth, “NOX enzymes and the biology of reactive oxygen,” Nature Reviews Immunology, vol. 4, no. 3, pp. 181–189, 2004.
[53]
D. I. Brown and K. K. Griendling, “Nox proteins in signal transduction,” Free Radical Biology and Medicine, vol. 47, no. 9, pp. 1239–1253, 2009.
[54]
J. Miku？a-Pietrasik, A. Kuczmarska, B. Rubi？ et al., “Resveratrol delays replicative senescence of human mesothelial cells via mobilization of antioxidative and DNA repair mechanisms,” Free Radical Biology and Medicine, vol. 52, no. 11-12, pp. 2234–2245, 2012.
[55]
P. J. Cook, B. G. Ju, F. Telese, X. Wang, C. K. Glass, and M. G. Rosenfeld, “Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions,” Nature, vol. 458, no. 7238, pp. 591–596, 2009.
[56]
M. J. Zeitz, K. S. Malyavantham, B. Seifert, and R. Berezney, “Matrin 3: chromosomal distribution and protein interactions,” Journal of Cellular Biochemistry, vol. 108, no. 1, pp. 125–133, 2009.
[57]
G. M. Bokoch, B. Diebold, J.-S. Kim, and D. Gianni, “Emerging evidence for the importance of phosphorylation in the regulation of NADPH oxidases,” Antioxidants and Redox Signaling, vol. 11, no. 10, pp. 2429–2441, 2009.
