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Akt Is Involved in the Inhibition of Cell Proliferation by EGF

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Akt Is Involved in the Inhibition of Cell Proliferation by EGF EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 39, No. 4, 491-498, August 2007 Akt is involved in the inhibition of cell proliferation by EGF Soung Hoo Jeon1, Woo-Jeong Jeong1, proliferation and embryonic axis formation (Zeng et Jae-Young Cho1, Kee-Ho Lee2 al., 1997). Axin is a multi-domain scaffold protein and Kang-Yell Choi1,3 that associates directly with β-catenin, GSK3β, and phosphatase PP2A (Hsu et al., 1999). Axin is 1 implicated in down-regulation of Wnt/β-catenin Department of Biotechnology signaling (Ikeda et al., 1998; Itoh et al., 1998; Yonsei University Sakanaka et al., 1998; Kikuchi et al., 2006). There Seoul 120-752, Korea 2 are two vertebrate Axins (Axin 1 and Axin 2). Axin1 Laboratory of Molecular Oncology is constitutively expressed, but Axin 2 is induced Korea Institute of Radiological and Medical Sciences by active Wnt signaling and acts therefore in a Seoul 139-706, Korea negative feedback loop (Yan et al., 2001; Jho et 3 Corresponding author: Tel, 82-2-2123-2887; al., 2002; Lustig et al., 2002). Overexpressed Axin Fax, 82-2-362-7265; E-mail, [email protected] destabilizes β-catenin (Behrens et al., 1998; Hart et al., 1998; Ikeda et al.,1998; Kishida et al., 1998; Accepted 28 May 2007 Nakamura et al., 1998; Sakanaka et al., 1998; Yamamoto et al., 1998). Loss of Axin results in nu- Abbreviations: APC, adenomatos polyposis coli; BrdU, bromode- clear accumulation of β-catenin followed by forma- oxyuridine; DAPI, 4'6-diamidino-2-phenylindole; PI3K, phosphatidyl tion of the β-catenin-TCF transcriptional complex inositol 3-kinase involving transcriptional activation of its target genes (Sakanaka et al., 1998). Mutations of Axin have been reported in hepatocellular carcinoma Abstract (Clevers, 2000; Satoh et al., 2000). However, the role and mechanism of Axin in the proliferation and Axin is a negative regulator of the Wnt/β-catenin path- transformation of cells are poorly understood. way and is involved in the regulation of axis formation The PI3 kinase (PI3K)-Akt signaling pathway is and proliferation. Involvement of Axin in the regulation involved in various cellular processes including of other signaling pathways is poorly understood. In growth, metabolism, reproduction and life span this study, we investigated the involvement of Akt in (Gao et al., 2004; Kim et al., 2004; Phung et al., growth regulation by Axin in L929 fibroblasts stimu- 2006; Stoeltzing et al., 2006). Akt/PKB, a serine/ lated by EGF. Akt activity was increased by EGF treat- threonine kinase, is an intermediate signaling com- ment and Ras activation, respectively. Both the EGF- ponent of the PI3K-Akt pathway, and is involved in and Ras-induced Akt activations were abolished by the transmission of insulin or Ras signaling (She- Axin induction, as revealed by both Western blot and pherd et al., 1998; Toker and Yoeli-Lerner, 2006). Aberrant regulation of Akt signaling is frequently immunocytochemical analyses. The proliferation and observed in several human cancers, and Akt plays Akt activation induced by EGF were decreased by Axin an important role in cancer progression (Toker and induction, and the effects of EGF were abolished by Yoeli-Lerner, 2006; Vega et al., 2006). treatment of an Akt-specific inhibitor. Therefore, Axin The ERK pathway is one of the major trans- inhibits EGF-induced proliferation of L929 fibroblasts forming signaling pathways, and the cross-talk be- by blocking Akt activation. tween the ERK and the Wnt/β-catenin pathways was identified in several of our previous experi- Keywords: Axin protein; epidermal growth factor; ments (Yun et al., 2005; Park et al., 2006; Jeon et 1-phosphatidylinositol 3-kinase; proto-oncogene pro- al., 2007). The ERK pathway is immediately teins c-akt; Wnt proteins activated by recombinant Wnt3a (Yun et al., 2005), and is also regulated by adenomatos polyposis coli (APC), the negative regulator of the Wnt/β-catenin Introduction pathway (Park et al., 2006). Although both the Wnt/β-catenin pathway and the PI3K/Akt pathway Axin is a negative regulator in the canonical Wnt/ are involved in tumor progression, the relationship β-catenin signaling pathway, and was first identi- between these two pathways is poorly understood. fied as the product of the fused locus in mice (Zeng The role of Axin in anti-proliferation might be et al., 1997). Axin is involved in the regulation of acquired by inhibition of the Wnt/β-catenin pathway 492 Exp. Mol. Med. Vol. 39(4), 491-498, 2007 (Behrens et al., 1998; Hart et al., 1998; Ikeda et al., bitor (20 µM) was pre-treated for 1.5 h before EGF 1998; Kishida et al., 1998; Lustig and Behrens, treatment. For Ras activation, L929-Axin-GFP cells 2003). However, involvement of Axin in regulations were infected with H-ras retrovirus before Axin of other transforming pathways is poorly un- induction with 0.5 µg/ml of Dox for 0-4 h. To knock derstood. We recently identified a role of Axin in down Axin, cells were transfected with pAxin- the regulation of the Ras-ERK pathway and sub- siRNA (Jeon et al., 2007) using LipofectAMINE sequent proliferation (Jeon et al., 2007). Currently, transfection reagent according to the manufacturer involvement of Axin in the regulation of the PI3K- (Invitrogen)’s instructions, followed by further incu- Akt signaling pathway is unknown. bation of the cells for 48 h (Figure 1B). In the present study, we investigated the role of Axin in the inhibition of EGF- or Ras-induced pro- liferation via Akt. Akt was activated by treatment of Western blot analysis EGF or by overexpression of active Ras, indicating In preparation of the proteins, attached cells were that Ras is a mediator of Akt activation by EGF rinsed twice with ice-cold PBS, harvested and then signaling. The Akt activation by either Ras or EGF lysed directly in Laemmli SDS sample buffer (Park was abolished by doxycyclin (Dox)-induced in- et al., 2004). The samples were boiled and sub- duction of Axin. The Akt regulations by Axin were jected to 5-10% SDS PAGE followed by Western further confirmed by inhibitions of EGF- and Ras- blot analysis using anti-Axin, -β-catenin, -p-Akt, p- induced nuclear localization of Akt by Axin. We ERK, or α-tubulin antibody followed by matching identified inhibitions of the EGF- or Ras-induced HRP conjugated secondary antibody. The protein proliferation and PI3K-Akt pathway activation by bands were visualized by enhanced chemilumines- Akt inhibitor. These results indicate that Axin inhi- cence (Amersham Pharmacia, Uppsala, Sweden) bits EGF- or Ras-induced cell proliferation at least according to the manufacturer’s instructions. partly through inhibition of the Akt pathway. Immunocytochemistry and bromodeoxyuridine Materials and Methods incorporation L929 cells were grown on glass cover slips at a Reagents density of 2 × 104 cells/well and induced with DMEM and FBS, both antibiotics, were purchased 1 µg/ml Dox for 24 h. In the cases required, the from Invitrogen (Grand Island, NY). ERK, phos- cells were treated with 10 ng/ml of EGF for 30 min or infected with retroviral H-ras for 24 h. The cells pho-ERK (p-ERK), p-Akt, Akt, α-tubulin and β- catenin antibodies were obtained from Santa Cruz were then incubated with anti-p-Akt antibody at a 1:200 dilution for 1 h and then treated with goat Biotechnology (Santa Cruz, CA). HRP-conjugated TM anti-mouse IgG antibodies were acquired from Cell anti-rabbit-Rhodamine Red -X-conjugated secon- dary antibody at a 1:1,000 dilution for 1 h, and Signaling Biotechnology (Beverly, MA) and α- tubulin antibodies were purchased from Oncogene washed 5 times with PBS. GFP-Axin was visua- Research Products (San Diego, CA). An enhanced lized as GFP by confocal microscopic analysis. chemiluminescence (ECL) system was obtained DAPI (Boehringer Mannheim, Mannheim, Ger- from Amersham Pharmacia (Uppsala, Sweden). many) was then treated at a final concentration of Akt inhibitor was acquired from Calbiochem (EMD 0.1 µg/ml in PBS for 10 min, and the cells were Biosciences, Germany). H-ras retrovirus was extensively washed with PBS and mounted for obtained from Dr. S. J. Lee at the Korean Institute photography using a Radiance 2000/MP, multi- of Radiological and Medical Sciences (Seoul, photon imaging system (Bio-Rad, Herculus, CA). Korea). EGF and Dox were purchased from Sigma Each experiment was performed at least three Aldrich (St. Louis, MO). times. Cells, and induction or knock down of Axin FACS analysis for measurement of bromodeoxyuridine incorporation L929-Axin-GFP cells (Jeon et al., 2007) were grown in DMEM supplemented with 10% FBS, For BrdU incorporation, L929-Axin-GFP cells were seeded in 6-well plates at 5 × 104 cells per well. G418 (200 µg/ml), streptomycin (100 µg/ml), and penicillin G sodium (100 µg/ml) and incubated at The cells were treated with 0.5 µg/ml of Dox for o Axin induction and/or Akt inhibitor with or without 37 C in 5% CO2. Axin was induced by 0.5 µg/ml of Dox for 24 h and/or stimulated with 20 ng/ml of EGF. BrdU was added to achieve a final concen- EGF for 30 min before cell harvest. The Akt inhi- tration of 20 µM for 8 h before harvesting the cells. Akt in EGF-induced cell proliferation 493 Figure 1. Effects of Axin on EGF- or Ras-induced Akt activation. (A) L929-Axin-GFP cells were grown in DMEM supplemented with 10% heat-inactivated FBS, G418 (200 µg/ml), streptomycin (100 µg/ml), and penicillin G so- o dium (100 µg/ml) in 5% CO2 at 37 C. Axin was induced by 0.5 µg/ml of Dox treatment for 48 h.
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