Abstract
We examined the effects of cadmium chloride (CdCl2) exposure on the phosphorylation and function of the forkhead box class O (FOXO) transcription factor FOXO3a in HK-2 human renal proximal tubular cells. Phosphorylation of FOXO3a (at Thr32 and Ser253) and its upstream kinase, Akt (at Thr308 and Ser473) were markedly increased following exposure to 10 or 20 μM CdCl2. Treatment with wortmannin (500 nM), an inhibitor of phosphoinositide-3-kinase (PI3K), suppressed CdCl2-induced phosphorylation of Akt and FOXO3a at their Akt phosphorylation sites. CdCl2-induced phosphorylation of FOXO3a was markedly suppressed by the epidermal growth factor receptor inhibitor, AG1478 (1 μM), the Ca2+/calmodulin-dependent kinase II inhibitor, KN-93 (10 μM), and the Src inhibitor, PP2 (10 μM), but only partially suppressed by the insulin-like growth factor-1 receptor inhibitor, PPP (2.5 μM). Furthermore, the p38 inhibitor, SB203580 (20 μM), suppressed CdCl2-induced phosphorylation of Akt and FOXO3a, suggesting possible cross-talk between p38 mitogen-activated protein kinase and Akt. Although phosphorylation of FOXO3a was associated with reduced levels of nuclear FOXO3a, this change in cellular localization was transient. Silencing of FOXO3a expression using short interfering RNA suppressed CdCl2-induced cellular damage and accumulation of cytoplasmic nucleosomes in HK-2 cells. These results show that cadmium exposure induces phosphorylation of FOXO3a through the PI3K/Akt signaling pathway and suggest that FOXO3a phosphorylation (inactivation) transiently promotes survival of HK-2 cells. Phosphorylation of FOXO3a by the PI3K/Akt pathway may regulate cell fate in proximal tubules exposed to cadmium.
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References
Asada S, Daitoku H, Matsuzaki H, Saito T, Sudo T, Mukai H, Iwashita S, Kako K, Kishi T, Kasuya Y, Fukamizu A (2007) Mitogen-activated protein kinases, Erk and p38, phosphorylate and regulate Foxo1. Cell Signal 19:519–527
Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868
Burgering BMT (2008) A brief introduction to FOXOlogy. Oncogene 27:2258–2262
Chen L, Xu B, Liu L, Luo Y, Zhou H, Chen W, Shen T, Han X, Kontos CD, Huang S (2011) Cadmium induction of reactive oxygen species activates the mTOR pathway, leading to neuronal cell death. Free Radic Biol Med 50:624–632
Dansen TB, Burgering BMT (2008) Unravelling the tumor-suppressive functions of FOXO proteins. Trends Cell Biol 18:421–429
Essaghir A, Dif N, Marbehant CY, Coffer PJ, Demoulin J-B (2009) The transcription of FOXO genes is stimulated by FOXO3 and repressed by growth factors. J Biol Chem 284:10334–10342
Fu Z, Tindall DJ (2008) FOXOs, cancer and regulation of apoptosis. Oncogene 27:2312–2319
Hamada T, Tanimoto A, Sasaguri Y (1997) Apoptosis induced by cadmium. Apoptosis 2:359–367
Hasegawa K, Wakino S, Yoshioka K, Tatematsu S, Hara Y, Minakuchi H, Washida N, Tokuyama H, Hayashi K, Itoh H (2008) Sirt1 protects against oxidative stress-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression. Biochem Biophys Res Commun 372:51–56
Iwatsuki M, Inageda K, Matsuoka M (2011) Cadmium induces phosphorylation and stabilization of c-Fos in HK-2 renal proximal tubular cells. Toxicol Appl Pharmacol 251:209–216
Komoike Y, Inamura H, Matsuoka M (2012) Effects of salubrinal on cadmium-induced apoptosis in HK-2 human renal proximal tubular cells. Arch Toxicol 86:37–44
Lehtinen MK, Yuan Z, Boag PR, Yang Y, Villén J, Becker EBE, DiBacco S, de la Iglesia N, Gygi S, Blackwell TK, Bonni A (2006) A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span. Cell 125:987–1001
Liu Z, Yu X, Shaikh ZA (2008) Rapid activation of ERK1/2 and AKT in human breast cancer cells by cadmium. Toxicol Appl Pharmacol 228:286–294
Liu J, Qu W, Kadiiska MB (2009) Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicol Appl Pharmacol 238:209–214
Matsuoka M, Call KM (1995) Cadmium-induced expression of immediate early genes in LLC-PK1 cells. Kidney Int 48:383–389
Nakamura T, Sakamoto K (2008) Forkhead transcription factor FOXO subfamily is essential for reactive oxygen species-induced apoptosis. Mol Cell Endocrinol 281:47–55
Nordberg GF, Nogawa K, Nordberg M, Friberg LT (2007) Cadmium. In: Nordberg GF, Fowler BA, Nordberg M, Friberg LT (eds) Handbook on the toxicology of metals, 3rd edn. Academic Press, Burlington, pp 445–486
Park S, Ahn J-Y, Lim M-J, Kim M-H, Yun Y-S, Jeong G, Song J-Y (2010) Sustained expression of NADPH oxidase 4 by p38 MAPK-Akt signaling potentiates radiation-induced differentiation of lung fibroblasts. J Mol Med 88:807–816
Raman M, Chen W, Cobb MH (2007) Differential regulation and properties of MAPKs. Oncogene 26:3100–3112
Son Y-O, Wang L, Poyil P, Budhraja A, Hitron JA, Zhang Z, Lee J-C, Shi X (2012) Cadmium induces carcinogenesis in BEAS-2B cells through ROS-dependent activation of PI3K/AKT/GSK-3β/β-catenin signaling. Toxicol Appl Pharmacol 264:153–160
Sunayama J, Tsuruta F, Masuyama N, Gotoh Y (2005) JNK antagonizes Akt-mediated survival signals by phosphorylating 14-3-3. J Cell Biol 170:295–304
Sunters A, Madureira PA, Pomeranz KM, Aubert M, Brosens JJ, Cook SJ, Burgering BMT, Coombes RC, Lam EW-F (2006) Paclitaxel-induced nuclear translocation of FOXO3a in breast cancer cells is mediated by c-Jun NH2-terminal kinase and Akt. Cancer Res 66:212–220
Tzivion G, Dobson M, Ramakrishnan G (2011) FoxO transcription factors; Regulation by AKT and 14-3-3 proteins. Biochim Biophys Acta 1813:1938–1945
van der Vos KE, Coffer PJ (2011) The extending network of FOXO transcriptional target genes. Antioxid Redox Signal 14:579–592
Wang X, Liu JZ, Hu JX, Wu H, Li YL, Chen HL, Bai H, Hai CX (2011) ROS-activated p38 MAPK/ERK-Akt cascade plays a central role in palmitic acid-stimulated hepatocyte proliferation. Free Radic Biol Med 51:539–551
Wang X, Chen WR, Xing D (2012) A pathway from JNK through decreased ERK and Akt activities for FOXO3a nuclear translocation in response to UV irradiation. J Cell Physiol 227:1168–1178
Xiao W, Liu Y, Templeton DM (2009) Pleiotropic effects of cadmium in mesangial cells. Toxicol Appl Pharmacol 238:315–326
Zhang X, Tang N, Hadden TJ, Rishi AK (2011) Akt, FoxO and regulation of apoptosis. Biochim Biophys Acta 1813:1978–1986
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This study was supported by the Department Fund of the Tokyo Women’s Medical University School of Medicine.
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Fujiki, K., Inamura, H. & Matsuoka, M. Phosphorylation of FOXO3a by PI3K/Akt pathway in HK-2 renal proximal tubular epithelial cells exposed to cadmium. Arch Toxicol 87, 2119–2127 (2013). https://doi.org/10.1007/s00204-013-1077-6
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DOI: https://doi.org/10.1007/s00204-013-1077-6