Knockdown of integrin b4-induced autophagic cell death associated with P53 in A549 lung adenocarcinoma cells Qiuxia He1,*, Bin Huang1,*, Jing Zhao1, Yun Zhang2, Shangli Zhang1 and Junying Miao1,2
1 Institute of Developmental Biology, School of Life Science, Shandong University, China 2 The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health,
Shandong University Qilu Hospital, China
Keywords autophagy; A549 lung cancer cells; H322 lung cancer cells; integrin b4; p53 protein
Correspondence J. Miao, School of Life Science, Shandong University, 27 Shanda Nan Road, Jinan 250100, China Fax: +86 531 88565610 Tel: +86 531 88364929 E-mail: miaojy@sdu.edu.cn
*These authors contributed equally to this work
Integrin b4 is a tissue-specific protein, but its role in autophagy of lung adenocarcinoma cells is not clear. In this study, we used microtubule- associated protein 1 light chain 3 processing and acridine orange staining to reveal that knockdown of integrin b4 by its specific siRNA induced autophagic cell death in A549 lung cancer cells. Next, we investigated the effects of siRNA-mediated downregulation of integrin b4 on cell death and the level of p53. The proportion of dead cells and level of p53 were significantly increased. Inhibition of autophagy by the inhibitor 3-methyladenine attenuated the cell death induced by integrin b4 knock- down. To further understand the relationship between p53 and integrin b4 in autophagic cell death, we inhibited the expression of integrin b4 by its specific siRNA in p53-mutated H322 lung cancer cells. Knock- down of integrin b4 could not induce autophagic cell death in H322 cells. The data suggest that integrin b4 is implicated in and associated with p53 in autophagy of lung cancer cells.
(Received 18 July 2008, revised 19 September 2008, accepted 22 September 2008)
doi:10.1111/j.1742-4658.2008.06699.x
[7], although it is not expressed in normal type II lung epithelial cells [8]. Therefore, we investigated the role of integrin b4 in A549 cell autophagy in this study.
Autophagy, an evolutionarily conserved process that regulates cell fate, is genetically regulated and is impor- tant in development and normal physiology and in a wide range of diseases [9]. Recent studies have sug- gested that, like apoptosis, autophagy is important in the regulation of cancer development and progression, and in determining the response of tumor cells to anti- cancer therapy [10]. Therefore, in this study, we first investigated the effect of knockdown of integrin b4 on autophagic cell death in A549 lung cancer cells.
Integrin b4, originally identified as a tumor-associated antigen [1], is expressed primarily on the basal surface of most epithelial cells and in most carcinoma cells. This integrin subunit is distinguished from other inte- its long cytoplasmic tail grin subunits because of spanning approximately 1000 amino acids and its important roles in cell signaling [2]. Integrin b4 is implicated in multiple signaling events, such as cancer invasion, cell survival, apoptosis and cell differen- tiation [3,4]. It is a tissue-specific protein [5] and can either mediate a suppressive effect or have a promo- ting effect on epidermal tumor growth depending on the genetic background [6]. The role of integrin b4 in regulating lung adenocarcinoma cell autophagy is not clear. Previously, we found that integrin b4 was strongly expressed in A549 lung adenocarcinoma cells
p53 is a critical mediator of both apoptotic and autophagic cell death, and the activation of p53 in the
Abbreviations 3MA, 3-methyladenine; AO, acridine orange; MAP1-LC3, microtubule-associated protein 1 light chain 3; mTOR, mammalian target of rapamycin.
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apoptosis of carcinoma cells is associated with integrin b4 [11]. Our previous results showed that integrin b4 mediates apoptotic signal transduction by upregulating p53 in human vascular endothelial cells [4,12]. Simi- larly, the level of p53 was increased in the apoptosis of A549 cells induced by safrole oxide [13]. These data suggest that integrin b4 is associated with p53 in apop- tosis, but the relationship between these two factors is not clear for autophagic cell death. In this study, we used H322 lung cancer cells, which contain a mutated p53 gene, as another cell model to study the relation- ship between integrin b4 and p53 in autophagic cell death.
The results of acridine orange (AO) staining showed that the volume of the cellular acidic compartment, a marker of autophagy, was increased, and acidic vesicles were accumulated and dispersed in the cytoplasm (Fig. 2A–D). Furthermore, examination of changes in the level and aggregation of light chain 3 II (LC3-II), a specific marker of autophagy, showed that LC3-II aggregated in A549 cells treated with integrin b4 siRNA (Fig. 3A–D). The results of Western blotting showed that the ratio of LC3-II to LC3-I increased by knock- down of integrin b4 (Fig. 4). Moreover, the increase in the level of LC3-II was inhibited by 3-methyladenine (3MA), a specific inhibitor of autophagy (Fig. 4).
Results
Downregulation of integrin b4 by its siRNA induced cell death in A549 cells
Knockdown of integrin b4 induced autophagy in A549 cells
Laser scanning confocal microscopy of A549 cells transfected with non-silencing rhodamine red-labeled siRNA for 24 h revealed a transfection efficiency of approximately 60% (Fig. 1A,B). Therefore, siRNA- mediated gene silencing was effective in these cells.
We examined cell death in A549 cells with downregu- lated integrin b4 using a trypan blue exclusion assay. Cell death was observed after the expression of inte- grin b4 was blocked for 48 h by 40 nm siRNA (Fig. 5). We also performed the immunofluorescent experiment with a negative control of corresponding purified rabbit IgM or IgG antibody. Autofluorescence was not detected in the negative control, as shown in Fig. S1.
Knockdown of integrin b4 increased the level of p53 in A549 cells
To understand the role of integrin b4 in autophagy, we inhibited the expression of integrin b4 by its specific siRNA as described previously [14,15]. By transfecting the cells with 20–80 nm integrin b4-specific siRNA for 48 h, we determined that a 40 nm concentration was appropriate for inhibition (data not shown). As shown in Fig. 1C,D, 40 nm siRNA greatly downregulated the expression of integrin b4.
As p53 is an important factor in autophagy, we exam- ined changes in the level of p53 after knockdown of
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Fig. 1. siRNA-mediated down-regulation of integrin b4 in A549 cells. (A) Fluorescent micrographs of cells transfected with non- silencing rhodamine red-labeled siRNA for 24 h. (B) Phase-contrast micrographs corre- sponding to (A). (C) Western blot analysis of the level of integrin b4. Protein extracts were from untreated cells (normal) and cells treated with 40 nM integrin b4-specific siRNA or scramble control siRNA for 48 h. (D) Western blot analysis of integrin b4 in A549 cells. **P < 0.01 versus the control group (n = 3).
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Fig. 2. Determination of autophagy by acri- dine orange (AO) staining. (A) Cells cultured under normal conditions. (B, C) A549 cells transfected with 40 nM scramble control siRNA or integrin b4 siRNA for 48 h. The cells were stained with 5 lgÆmL)1 AO for 1 min at room temperature, then observed and photographed under a fluorescent microscope. (D) Percentage of cells with acidic vesicles dispersed and increased in number. **P < 0.01 versus control group (n = 3).
integrin b4 in A549 cells, and found that the level of p53 was significantly elevated in cells treated with inte- grin b4 siRNA (Fig. 6A,B).
p53-mutated H322 cells. The level of integrin b4 was depressed effectively in H322 cells by integrin b4 siRNA treatment (Fig. 7A–D). However, the volume of the cellular acidic compartment (Fig. 7E–G) and the level of LC3-II (Fig. 8) did not change.
Knockdown of integrin b4 could not induce autophagic cell death in H322 cells with mutated p53
Discussion
To further understand the role of p53 in integrin b4-mediated autophagic cell death, we investigated the effect of knockdown of integrin b4 on autophagy in
Integrin b4 is an attractive target for anti-angiogenesis and cancer therapy [16]. Agents that are able to dis- rupt integrin b4 signaling can increase the therapeutic
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Fig. 3. Location of MAP1-LC3 in A549 cells. (A–C) Fluorescent micrographs showing the relative intensity and location of MAP1-LC3 in A549 cells. (A) Cells cultured in RPMI- 1640 medium. (B) A549 cells transfected with 40 nM scramble control siRNA for 48 h. (C) A549 cells transfected with 40 nM inte- grin b4 siRNA for 48 h. (D) Relative level of MAP1-LC3 in normal, control and integrin b4 siRNA groups. The values represent the rela- tive fluorescent intensity per cell determined by laser scanning confocal microscopy after treatment for 48 h. *P < 0.05 (n = 3).
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Fig. 4. Induction of MAP1-LC3 processing by knockdown of inte- grin b4. Western blot analysis of MAP1-LC3 in A549 cells treated with 40 nM scramble control siRNA or integrin b4 siRNA for 48 h or in A549 cells pretreated with 10 mM 3MA and then treated with 40 nM scramble control siRNA or 40 nM integrin b4 siRNA for 48 h. The conversion of the LC3-I form to the LC3-II form is shown. *P < 0.05; **P < 0.01 (n = 3).
Fig. 6. Knockdown of integrin b4 increased the level of p53 in A549 cells. (A) Western blot analysis of the level of p53. Protein extracts were from the untreated cells (normal) or cells treated with 40 nM integrin b4 siRNA or scramble control siRNA for 48 h. (B) Western blot analysis of the level of p53. *P < 0.05 (n = 3).
A recent report showed that adhesion of primary prostate basal epithelial cells to LM5-rich matrix is required to maintain autophagy. Signaling through a3b1, and to a lesser extent a6b4, is required for auto- phagy [17]. Our data suggested that integrin b4 is a key factor in autophagy in A549 lung cancer cells. Our results further support the view that the functions of integrin b4 are cell-type-specific [18].
integrin b4 was
efficacy of existing targeted therapies for ErbB2-posi- tive human breast cancers and vascular endothelial growth factor-driven retinal neovascularization [16]. However, whether integrin b4 signaling promotes tumorigenesis only in the mammary gland or also in other tissues is not clear. Depending on the genetic background, integrin b4 can either mediate a suppres- sive effect or have a promoting effect on epidermal tumor growth, which suggests tissue-specific function of integrin b4 in the control of tumor growth [6]. Pre- strongly viously, we found that expressed in A549 lung adenocarcinoma cells [7], but its role in these cells was not clear. Here, we report that knockdown of integrin b4 induces autophagic cell death in A549 cells, which suggests the importance of this integrin in the control of autophagy.
p53 is known to function in the apoptosis of various types of carcinoma cells in association with integrin b4 [11]. Our previous results showed that integrin b4 med- transduction by upregulating iated apoptotic signal p53 in human vascular endothelial cells [4,12]. Simi- larly, apoptosis was provoked by safrole oxide, with accumulation of p53 protein in A549 cells, which express wild-type p53 [13]. These data suggest that integrin b4 is associated with p53 during apoptosis. Recently, the function of p53 in autophagy has been described [19], but the relationship between integrin b4 and p53 in autophagy is not clear. We found that inte- grin b4-specific siRNA induced autophagic cell death in wild-type p53 A549 cells, but not in p53-mutated H322 cells, which suggests that integrin b4-regulated autophagy is associated with p53 in lung adenocarci- noma cells.
Fig. 5. Knockdown of integrin b4 induced cell death in A549 cells. Cell death rate after treatment with integrin b4-specific siRNA for the indicated times determined using a trypan blue exclusion assay. *P < 0.05, **P < 0.01 versus control group (n = 3).
p53 is a sequence-specific transcription factor and a central signal integrator of stresses such as DNA dam- age, hypoxia and oncogene activation [20]. Although p53 can be activated by a number of stress signals, few cell-surface receptors that can activate p53 and trigger response have been a p53-dependent autophagic
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Fig. 7. Knockdown of integrin b4 did not induce autophagic cell death in p53-mutated H322 cells. (A–D) Fluorescent micrographs showing the relative intensity and location of integrin b4 in H322 cells. (A) Cells transfected with 40 nM scramble control siRNA for 48 h. (B) The merged image of (A) and its corresponding phase-contrast graph. (C) Cells transfected with 40 nM integrin b4 siRNA for 48 h. (D) The merged image of (C) and its corresponding phase-contrast graph. (E, F) H322 cells transfected with 40 nM scramble control siRNA (E) or in- tegrin b4 siRNA (F) for 48 h. The cells were stained with 5 lgÆmL)1 AO for 1 min at room temperature, then observed and photographed under a fluorescent microscope. (G) Percentage of cells in which acidic vesicles were dispersed and increased in number. #P > 0.05 versus control group (n = 3).
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Fig. 8. Location and relative levels of MAP1-LC3 in H322 cells. (A–C) Fluorescent micrographs showing the relative intensity and location of MAP1-LC3 in H322 cells. (A) Cells cultured in RPMI-1640 medium. (B) Cells transfected with 40 nM scramble control siRNA for 48 h. (C) Cells transfected with 40 nM integrin b4 siRNA for 48 h. (D) The relative levels of MAP1-LC3 in normal, control and integrin b4 siRNA groups. The values represent the relative fluorescent intensity per cell determined by laser scan- ning confocal microscopy after treatment for 48 h. #P > 0.05 versus control (n = 3).
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described. Here, we demonstrated that integrin b4 is associated with the upregulation of p53 in A549 lung cancer cell autophagy.
The mechanisms of p53-dependent
because of its exceptionally large cytoplasmic domain [28]. In essence, the mTOR pathway may be an impor- tant mediator of the relationship between integrin b4 and p53 in autophagy, which will be substantiated experimentally in our next study.
In summary, the results of this study show that knockdown of integrin b4 induces autophagic cell death in A549 cells but not H322 cells, and suggest that integrin b4 is a key factor associated with p53 in autophagy of lung adenocarcinoma cells.
Experimental procedures
Reagents
RPMI-1640 was obtained from Gibco Co. (Carlsbad, CA, USA). Bovine calf serum (BCS) was obtained from Beijing DingGuo Biotechnology Co. (Beijing, China). 3MA was pur- chased from Sigma Chemical Co. (St Louis, MO, USA). 3MA was dissolved directly into the culture medium, divided into aliquots and stored at )20 (cid:2)C until use. In the studies involving 3MA, drug-free medium was exchanged for medium containing 0 or 10 mm 3MA 2 h prior to RNA interference. Antibodies to integrin b4, microtubule-associ- ated protein 1 light chain 3 (MAP1-LC3) and b-actin (sc-9090, sc-28266 and sc-47778, respectively) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
induction of incompletely understood, but are autophagy are still thought to involve both transcription-independent functions, e.g. AMP-activated protein kinase activa- tion, and transcription-dependent functions, e.g. upreg- rapamycin the mammalian target of ulation of (mTOR) inhibitor phosphatase and tensin homolog and the tuberous sclerosis complex, or the p53-regu- lated autophagy and cell death gene damage-regulated autophagy modulator [21]. Recent observations have indicated that, once activated, p53 also inhibits mTOR activity and upregulates autophagy [21]. In cancer cells with mutated p53, mTOR activation resulting from abnormal p53 activity may contribute to the lower autophagic capacities of the malignant cells [22]. The direct downstream modulator of p53 in autophagy is not well known. Crighton et al. identified the cell death gene damage-regulated autophagy modulator (DRAM) as a p53 target mediating induction of auto- phagy [11]. In our study, the upregulation of p53 was minimal, but p53 probably performed a role in auto- phagy through its downstream elements to amplify the pro-autophagy signal. This hypothesis needs to be tested in subsequent experiments.
Cell cultures
A549 and H322 lung cancer cells were cultured in RPMI- 1640 medium supplemented with 10% v ⁄ v BCS at 37 (cid:2)C in 5% CO2. The cells were seeded at 6.25 · 103Æcm2 in plates or dishes.
Immunofluorescence assay
The immunofluorescence assay was performed as previously described [12]. The value for relative fluorescent intensity per cell was the total value of the sample in the zoom scan divided by the total number of cells (at least 200 cells) in the same scan.
Western blot assay
Western blot analysis was performed as described previ- ously [13]. The relative quantity of proteins was analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA) and normalized to that of b-actin.
Autophagy detection by acridine orange staining
The volume of the cellular acidic compartment, as a marker of autophagy, was visualized by AO staining [29,30]. After
Consistent with a report by Edick et al. [17], a recent report showed that incubation of attached mammary epithelial cells with function-blocking antibody against integrin b1 is sufficient to induce autophagy [23]. The supposed pathways linking loss of integrin engagement at the cell surface to the autophagy machinery were grouped into three categories: growth-factor and nutri- ent-sensing, energy-sensing, and integrated stress- response pathways [24]. For example, the epidermal growth factor receptor is downregulated upon loss of integrin binding in multiple epithelial cell types [25]. Downregulation of epidermal growth factor receptor or nutrient sensors on the cell surface leads to inactivation of the mammalian mTOR pathway, the archetypal inhibitor of autophagy. Accordingly, mTOR downregu- lation is also observed following extracellular matrix detachment [26]. Focal adhesion kinase, a crucial com- ponent of adhesion-mediated signaling, can phosphory- late TSC2, an upstream mTOR regulator, suppressing its activity and maintaining mTOR activation [26]. These data suggest that the growth-factor and nutrient- sensing pathways may provide a more direct link between the loss of integrin function and autophagy. Integrin b4 has functions that are distinct from those of integrin b1 in epidermal growth and differentiation [27], and is an important integrin in cell survival signaling
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2 Tsuruta D, Hopkinson SB, Lane KD, Werner ME,
Cryns VL & Jones JC (2003) Crucial role of the speci- ficity-determining loop of the integrin beta4 subunit in the binding of cells to laminin-5 and outside-in signal transduction. J Biol Chem 278, 38707–38714.
treatment, cells were stained with 5 lgÆmL)1 AO for 1 min at room temperature. Fluorescent micrographs were taken using an Olympus (Tokyo, Japan) BH-2 fluorescence micro- scope. Acidic vesicles were stained red by AO. Autophagy was quantified as the mean number of cells displaying intense red staining in five fields (containing at least 40 cells per field) for each experimental condition.
3 Lipscomb EA & Mercurio AM (2005) Mobilization and activation of a signaling competent alpha6beta4integrin underlies its contribution to carcinoma progression. Cancer Metastasis Rev 24, 413–423.
RNA interference (RNAi)
4 Zhao J, Miao JY, Zhao BX & Zhang SL (2005) Upre- gulating of Fas, integrin beta4 and P53 and depressing of PC-PLC activity and ROS level in VEC apoptosis by safrole oxide. FEBS Lett 579, 5809–5813.
5 Bachelder RE, Marchetti A & Falcioni R (1999) Activa- tion of p53 function in carcinoma cells by the alpha6- beta4 integrin. J Biol Chem 274, 20733–20737.
6 Raymond K, Kreft M, Song JY, Janssen H & Sonnen- berg A (2007) Dual role of alpha6beta4 integrin in epidermal tumor growth: tumor-suppressive versus tumor-promoting function. Mol Biol Cell 18, 4210–4221. 7 Du AY, Zhao BX, Miao JY, Yin DL & Zhang SL RNAi was performed as described previously [14] using specific integrin b4 siRNA, a pool of three target-specific, 20–25 nt siRNAs (sc-35678; Santa Cruz Biotechnology) [15]. RNAi experiments were performed as described in the manufacturer’s protocols. A549 cells or H322 cells were transfected with 20–80 nm integrin b4 siRNA using RNAi- Fect(cid:3) transfection reagent (Qiagen, Hilden, Germany). Then we monitored the effect of gene silencing using a Western blot assay. To evaluate siRNA-mediated gene silence, rhodamine red-labeled siRNA (Qiagen) and scram- ble siRNA (Santa Cruz Biotechnology, CA, USA) were used as controls.
(2006) Safrole oxide induces apoptosis by up-regulating Fas and FasL instead of integrin beta4 in A549 human lung cancer cells. Bioorg Med Chem 14, 2438–2445.
Cell death assay
8 Coraux C, Delplanque A, Hinnrasky J, Peault B, Puchelle E & Gaillard D (1998) Distribution of integrins during human fetal lung development. J Histochem Cytochem 46, 803–810. 9 Maiuri MC, Zalckvar E, Kimchi A & Kroemer G After treatment, A549 cells were harvested by trypsiniza- tion, mixed in a 1 : 1 ratio (v ⁄ v) with 0.4% trypan blue dye, and counted by phase-contrast microscopy. Cells that excluded trypan blue dye were considered viable.
Statistical analysis
(2007) Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8, 741–752.
statistical 10 Lerena C, Calligaris SD & Colombo MI (2008) Auto- phagy: for better or for worse, in good times or in bad times. Curr Mol Med 8, 92–101.
spss software (version 11.5; SPSS Inc., Chicago, IL, USA) was used for calculations. All values are expressed as means ± SE. One-way ANOVA was used and differences at P < 0.05 are considered statistically significant.
Acknowledgements
11 Crighton D, Wilkinson S, O’Prey J, Syed N, Smith P, Harrison PR, Gasco M, Garrone O, Crook T & Ryan KM (2006) DRAM, a p53-induced modulator of auto- phagy, is critical for apoptosis. Cell 126, 121–134. 12 Zhang L, Zhu XS, Zhao BX, Zhao J, Zhang Y, Zhang SL & Miao JY (2008) A novel isochroman derivative inhibited apoptosis in vascular endothelial cells through depressing the levels of integrin beta4, p53 and ROS. Vascul Pharmacol 48, 63–69.
13 Du A, Zhang SL, Miao JY & Zhao BX (2004) Safrole oxide induces apoptosis in A549 human lung cancer cells. Exp Lung Res 30, 419–429.
This study was supported by the National Natural Sci- ence Foundation of China (grant number 90813022), the National 973 Research Project (grant number 2006CB503803) and the Natural Science Foundation of Shandong Province (grant numbers Z2006D02 and Y2005B12). The authors declare that they have no conflict of interest.
14 Lv X, Su L, Yin DL, Sun CH, Zhao J, Zhang SL &
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The following supplementary material is available: Fig. S1. Autofluorescence in A549 cells treated with 40 nm integrin b4 siRNA for 48 h was not detected in the negative control.
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