Threonine 32 (Thr32) of FoxO3 is critical for TGF-β-induced apoptosis via Bim in hepatocarcinoma cells

doi:10.1007/s13238-014-0121-5

Protein Cell

2015, Vol. 6

Issue (2) : 127-138 https://doi.org/10.1007/s13238-014-0121-5

RESEARCH ARTICLE

Threonine 32 (Thr32) of FoxO3 is critical for TGF-β-induced apoptosis via Bim in hepatocarcinoma cells

Xiangxuan Zhao^1,^2,^3,^*(

),Yong Liu^1,²,Lei Du^1,²,Leya He⁴,Biyun Ni^1,²,Junbo Hu⁴,Dahai Zhu⁵,Quan Chen^1,^2,^6,^*(

)

1. The Joint Laboratory of Apoptosis and Cancer Biology, The State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
3. Department of Radiology, Shengjing Hospital of China Medical University, Shenyang 110004, China
4. Cancer Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430032, China
5. Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences and School of Basic Medicine of Peking Union Medical College, Beijing 100005, China
6. College of Life Science, Nankai University, Tianjin 300071, China

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Abstract

Transforming growth factor- β (TGF- β) exerts apoptotic effects on various types of malignant cells, including liver cancer cells. However, the precise mechanisms by which TGF- β induces apoptosis remain poorly known. In the present study, we have showed that threonine 32 (Thr32) residue of FoxO3 is critical for TGF- β to induce apoptosis via Bim in hepatocarcinoma Hep3B cells. Our data demonstrated that TGF- β induced FoxO3 activation through specific de-phosphorylation at Thr32. TGF- β-activated FoxO3 cooperated with Smad2/3 to mediate Bim up-regulation and apoptosis. FoxO3 (de)phosphorylation at Thr32 was regulated by casein kinase I- ? (CKI- ?). CKI inhibition by small molecule D4476 could abrogate TGF- β-induced FoxO/Smad activation, reverse Bim up-regulation, and block the sequential apoptosis. More importantly, the deregulated levels of CKI- ? and p32FoxO3 were found in human malignant liver tissues. Taken together, our findings suggest that there might be a CKI-FoxO/Smad-Bim engine in which Thr32 of FoxO3 is pivotal for TGF- β-induced apoptosis, making it a potential therapeutic target for liver cancer treatment.

Keywords apoptosis TGF-β FoxO3 casein kinase I-? hepatocarcinoma

Corresponding Author(s): Xiangxuan Zhao,Quan Chen

Issue Date: 05 February 2015

Cite this article:

Xiangxuan Zhao,Yong Liu,Lei Du, et al. Threonine 32 (Thr32) of FoxO3 is critical for TGF-β-induced apoptosis via Bim in hepatocarcinoma cells[J]. Protein Cell, 2015, 6(2): 127-138.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-014-0121-5
https://academic.hep.com.cn/pac/EN/Y2015/V6/I2/127

1	Akagi M, Yasui W, Akama Y, Yokozaki H, Tahara H, Haruma K, Kajiyama G, Tahara E (1996) Inhibition of cell growth by transforming growth factor β1 is associated with p53-independent induction of p21 in gastric carcinoma cells. Jpn J Cancer Res 87: 377-384 https://doi.org/10.1111/j.1349-7006.1996.tb00233.x
2	Biggs WH III, Cavenee WK, Arden KC (2001) Identification and characterization of members of the FKHR (FOX O) subclass of winged-helix transcription factors in the mouse. Mamm Genome 12: 416-425 https://doi.org/10.1007/s003350020002
3	<?Pub Caret1?> 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 https://doi.org/10.1016/S0092-8674(00)80595-4
4	Chen Q, Gong B, Mahmoud-Ahmed AS, Zhou A, Hsi ED, Hussein M, Almasan A (2001) Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferon-induced apoptosis in multiple myeloma. Blood 98: 2183-2192 https://doi.org/10.1182/blood.V98.7.2183
5	Conery AR, Cao Y, Thompson EA, Townsend CM Jr, Ko TC, Luo K (2004) Akt interacts directly with Smad3 to regulate the sensitivity to TGF-β induced apoptosis. Nat Cell Biol 6: 366-372 https://doi.org/10.1038/ncb1117
6	Derynck R, Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature 425: 577-584 https://doi.org/10.1038/nature02006
7	Engel ME, Datta PK, Moses HL (1998) RhoB is stabilized by transforming growth factor β and antagonizes transcriptional activation. J Biol Chem 273: 9921-9926 https://doi.org/10.1074/jbc.273.16.9921
8	Enroth S, Andersson R, Bysani M, Wallerman O, Termen S, Tuch BB, De La Vega FM, Heldin CH, Moustakas A, Komorowski J, Wadelius C (2014) Nucleosome regulatory dynamics in response to TGF-β. Nucleic Acids Res 42: 6921-6934 https://doi.org/10.1093/nar/gku326
9	Fish KJ, Cegielska A, Getman ME, Landes GM, Virshup DM (1995) Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family. J Biol Chem 270: 14875-14883 https://doi.org/10.1074/jbc.270.25.14875
10	Graves PR, Haas DW, Hagedorn CH, Paoli-Roach AA, Roach PJ (1993) Molecular cloning, expression, and characterization of a 49-kilodalton casein kinase I isoform from rat testis. J Biol Chem 268: 6394-6401
11	Green DR, Reed JC (1998) Mitochondria and apoptosis. Science 281: 1309-1312 https://doi.org/10.1126/science.281.5381.1309
12	Hagenbuchner J, Kuznetsov A, Hermann M, Hausott B, Obexer P, Ausserlechner MJ (2012) FOXO3-induced reactive oxygen species are regulated by BCL2L11 (Bim) and SESN3. J Cell Sci 125: 1191-1203 https://doi.org/10.1242/jcs.092098
13	Higaki M, Shimokado K (1999) Phosphatidylinositol 3-kinase is required for growth factor-induced amino acid uptake by vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 19: 2127-2132 https://doi.org/10.1161/01.ATV.19.9.2127
14	Hua X, Liu X, Ansari DO, Lodish HF (1998) Synergistic cooperation of TFE3 and smad proteins in TGF-β -induced transcription of the plasminogen activator inhibitor-1 gene. Genes Dev 12: 3084-3095 https://doi.org/10.1101/gad.12.19.3084
15	Huynh H, Nguyen TT, Chan E, Tran E (2003) Inhibition of ErbB-2 and ErbB-3 expression by quercetin prevents transforming growth factor alpha (TGF-alpha)- and epidermal growth factor (EGF)-induced human PC-3 prostate cancer cell proliferation. Int J Oncol 23: 821-829
16	Karube K, Nakagawa M, Tsuzuki S, Takeuchi I, Honma K, Nakashima Y, Shimizu N, Ko YH, Morishima Y, Ohshima K, Nakamura S, Seto M (2011) Identification of FOXO3 and PRDM1 as tumorsuppressor gene candidates in NK-cell neoplasms by genomic and functional analyses. Blood 118: 3195-3204 https://doi.org/10.1182/blood-2011-04-346890
17	Kato M, Yuan H, Xu ZG, Lanting L, Li SL, Wang M, Hu MC, Reddy MA, Natarajan R (2006) Role of the Akt/FoxO3a pathway in TGF- β 1-mediated mesangial cell dysfunction: a novel mechanism related to diabetic kidney disease. J Am Soc Nephrol 17: 3325-3335 https://doi.org/10.1681/ASN.2006070754
18	Knippschild U, Gocht A, Wolff S, Huber N, Lohler J, Stoter M (2005) The casein kinase 1 family: participation in multiple cellular processes in eukaryotes. Cell Signal 17: 675-689 https://doi.org/10.1016/j.cellsig.2004.12.011
19	Kops GJ, Burgering BM (1999) Forkhead transcription factors: new insights into protein kinase B (c-akt) signaling. J Mol Med (Berl)77: 656-665 https://doi.org/10.1007/s001099900050
20	Liao XD, Tang AH, Chen Q, Jin HJ, Wu CH, Chen LY, Wang SQ (2003) Role of Ca2+ signaling in initiation of stretch-induced apoptosis in neonatal heart cells. Biochem Biophys Res Commun 310: 405-411 https://doi.org/10.1016/j.bbrc.2003.09.023
21	Massague J, Weis-Garcia F(1996) Serine/threonine kinase receptors: mediators of transforming growth factor β family signals. Cancer Surv 27: 41-64
22	Massague J, Seoane J, Wotton D (2005) Smad transcription factors. Genes Dev 19: 2783-2810 https://doi.org/10.1101/gad.1350705
23	Miyazono K (2000) TGF-beta/SMAD signaling and its involvement in tumor progression. Biol Pharm Bull 23: 1125-1130 https://doi.org/10.1248/bpb.23.1125
24	Naka K, Hoshii T, Muraguchi T, Tadokoro Y, Ooshio T, Kondo Y, Nakao S, Motoyama N, Hirao A (2010) TGF-β-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia. Nature 463: 676-680 https://doi.org/10.1038/nature08734
25	Nass SJ, Li M, Amundadottir LT, Furth PA, Dickson RB (1996) Role for Bcl-xL in the regulation of apoptosis by EGF and TGF-β in c-myc overexpressing mammary epithelial cells. Biochem Biophys Res Commun 227: 248-256 https://doi.org/10.1006/bbrc.1996.1497
26	Ni YG, Wang N, Cao DJ, Sachan N, Morris DJ, Gerard RD, Kuro O, Rothermel BA, Hill JA (2007) FoxO transcription factors activate Akt and attenuate insulin signaling in heart by inhibiting protein phosphatases. Proc Natl Acad Sci U S A 104: 20517-20522 https://doi.org/10.1073/pnas.0610290104
27	Park JT, Kato M, Yuan H, Castro N, Lanting L, Wang M, Natarajan R (2013) FOG2 protein down-regulation by transforming growth factor-β1-induced microRNA-200b/c leads to Akt kinase activation and glomerular mesangial hypertrophy related to diabetic nephropathy. J Biol Chem 288: 22469-22480 https://doi.org/10.1074/jbc.M113.453043
28	Philips N, McFadden K (2004) Inhibition of transforming growth factor-β and matrix metalloproteinases by estrogen and prolactin in breast cancer cells. Cancer Lett 206: 63-68 https://doi.org/10.1016/j.canlet.2003.10.019
29	Qi W, Weber CR, Wasland K, Roy H, Wali R, Joshi S, Savkovic SD (2011a) Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells. Am J Physiol Gastrointest Liver Physiol 300: G264-G272 https://doi.org/10.1152/ajpgi.00416.2010
30	Qi W, Weber CR, Wasland K, Savkovic SD (2011b) Genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of epidermal growth factor on tumor suppressor FOXO3 activity. BMC Cancer 11: 219 https://doi.org/10.1186/1471-2407-11-219
31	Ramjaun AR, Tomlinson S, Eddaoudi A, Downward J (2007) Upregulation of two BH3-only proteins, Bmf and Bim, during TGF beta-induced apoptosis. Oncogene 26: 970-981 https://doi.org/10.1038/sj.onc.1209852
32	Remy I, Montmarquette A, Michnick SW (2004) PKB/Akt modulates TGF-β signalling through a direct interaction with Smad3. Nat Cell Biol 6: 358-365 https://doi.org/10.1038/ncb1113
33	Renard E, Chadjichristos C, Kypriotou M, Beauchef G, Bordat P, Dompmartin A, Widom RL, Boumediene K, Pujol JP, Galera P (2008) Chondroitin sulphate decreases collagen synthesis in normal and scleroderma fibroblasts through a Smad-independent TGF-β pathway—implication of C-Krox and Sp1. J Cell Mol Med 12: 2836-2847 https://doi.org/10.1111/j.1582-4934.2008.00287.x
34	Renault VM, Thekkat PU, Hoang KL, White JL, Brady CA, Kenzelmann BD, Venturelli OS, Johnson TM, Oskoui PR, Xuan Z, Santo EE, Zhang MQ, Vogel H, Attardi LD, Brunet A (2011) The pro-longevity gene FoxO3 is a direct target of the p53 tumor suppressor. Oncogene 30: 3207-3221 https://doi.org/10.1038/onc.2011.35
35	Runyan CE, Liu Z, Schnaper HW (2012) Phosphatidylinositol 3-kinase and Rab5 GTPase inversely regulate the Smad anchor for receptor activation (SARA) protein independently of transforming growth factor-β 1. J Biol Chem 287: 35815-35824 https://doi.org/10.1074/jbc.M112.380493
36	Seoane J, Le HV, Massague J (2002) Myc suppression of the p21 (Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature 419: 729-734 https://doi.org/10.1038/nature01119
37	Torregroza I, Evans T (2006) Tid1 is a Smad-binding protein that can modulate Smad7 activity in developing embryos. Biochem J 393: 311-320 https://doi.org/10.1042/BJ20050785
38	Tremblay ML, Giguere V (2008) Phosphatases at the heart of FoxO metabolic control. Cell Metab 7: 101 -103 https://doi.org/10.1016/j.cmet.2008.01.004
39	Tuazon PT, Traugh JA (1991) Casein kinase I and II–multipotential serine protein kinases: structure, function, and regulation. Adv Second Messenger Phosphoprotein Res 23: 123-164
40	Valderrama-Carvajal H, Cocolakis E, Lacerte A, Lee EH, Krystal G, Ali S, Lebrun JJ (2002) Activin/TGF-β induce apoptosis through Smad-dependent expression of the lipid phosphatase SHIP. Nat Cell Biol 4: 963-969 https://doi.org/10.1038/ncb885
41	Vignais ML (2000) Ski and SnoN: antagonistic proteins of TGF-β signaling. Bull Cancer 87: 135-137
42	Vogt PK, Jiang H, Aoki M (2005) Triple layer control: phosphorylation, acetylation and ubiquitination of FOXO proteins. Cell Cycle 4: 908-913 https://doi.org/10.4161/cc.4.7.1796
43	Waddell DS, Liberati NT, Guo X, Frederick JP, Wang XF (2004) Casein kinase Iepsilon plays a functional role in the transforming growth factor-β signaling pathway. J Biol Chem 279: 29236-29246 https://doi.org/10.1074/jbc.M400880200
44	Wildey GM, Howe PH (2009) Runx1 is a co-activator with FOXO3 to mediate transforming growth factor β (TGF-β )-induced Bim transcription in hepatic cells. J Biol Chem 284: 20227-20239 https://doi.org/10.1074/jbc.M109.027201
45	Yamamura Y, Hua X, Bergelson S, Lodish HF (2000) Critical role of Smads and AP-1 complex in transforming growth factor-β -dependent apoptosis. J Biol Chem 275: 36295-36302 https://doi.org/10.1074/jbc.M006023200
46	Yan L, Lavin VA, Moser LR, Cui Q, Kanies C, Yang E (2008) PP2A regulates the pro-apoptotic activity of FOXO1. J Biol Chem 283: 7411-7420 https://doi.org/10.1074/jbc.M708083200

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