The Novel Function of OCT4B Isoform-265 in Genotoxic Stress§

Authors

  • Yuan Gao,

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China
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  • Jianshu Wei,

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China
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  • Jin Han,

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China
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  • Xia Wang,

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China
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  • Guannan Su,

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China
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  • Yannan Zhao,

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China
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  • Bing Chen,

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China
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  • Zhifeng Xiao,

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China
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  • Jiani Cao,

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China
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  • Jianwu Dai

    Corresponding author
    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China
    • Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 3 Nanyitiao, Zhongguancun, Beijing 100190, People's Republic of China
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    • Telephone: 86-010-82614426; Fax: 86-010-82614426


  • Author contributions: Y.G., J.W., and J.H.: conception and design, acquisition of data, analysis and interpretation of data, and manuscript writing; X.W.: conception and design and analysis and interpretation of data; G.S., Y.Z., B.C., Z.X., and J.C.: analysis and interpretation of data; J.D.: conception and design, financial support, analysis and interpretation of data, and final approval of the version to be published. Y.G., J.W., and J.H. contributed equally to this article.

  • Disclosure of potential conflicts of interest is found at the end of this article.

  • §

    First published online in STEM CELLSEXPRESS January 13, 2012.

Abstract

OCT4 is a key transcription factor in maintaining the pluripotency and self-renewal of embryonic stem cells (ESCs). Human OCT4 gene can generate three mRNA isoforms (OCT4A, OCT4B, and OCT4B1) by alternative splicing and four protein isoforms (OCT4A, OCT4B-265, OCT4B-190, and OCT4B-164) by alternative splicing or alternative translation initiation. OCT4A is a transcription factor responsible for the stemness of ESCs, while the function of OCT4B protein isoforms is still not clear. We have previously reported that OCT4B-190 functioned in cell stress response. Here, we present another product of OCT4 gene, OCT4B-265, which is upregulated under genotoxic stress in stem cells, and it may function in stress response through p53 signaling pathway. This work gives an insight into the novel function of OCT4B protein isoforms and helps us to understand the complex expression patterns and biological functions of OCT4 gene. STEM CELLS2012; 30:665–672

INTRODUCTION

The transcription factor OCT4 (official symbol POU5F1, also known as OCT3, OCT3/4, OTF3, and OTF4) is a main regulator in maintaining the pluripotency and self-renewal of embryonic stem cells (ESCs) [1–4]. It is also an essential factor in generating iPS cells [5–7].

The human OCT4 gene can generate three mRNA isoforms by alternative splicing, termed OCT4A [8], OCT4B [8], and OCT4B1 [9] (Fig. 1). OCT4A mRNA can translate into OCT4A protein. OCT4B mRNA is classically considered to generate a 265-amino-acid protein isoform named OCT4B. In our recent study, we found that OCT4B mRNA could generate three protein isoforms through alternative translation initiation: the 265-amino-acid protein isoform OCT4B-265, the 190-amino-acid protein isoform OCT4B-190, and the 164-amino-acid protein isoform OCT4B-164 [10]. The OCT4B1 mRNA contains a cryptic exon, which contains a TGA stop codon in it. So, OCT4B1 is predicted to generate a potential truncated peptide [9]. Our previous study showed that OCT4B1 could splice into OCT4B and also produced the three protein isoforms: OCT4B-265, OCT4B-190, and OCT4B-164 [11].

Figure 1.

Schematic structures of human OCT4 gene mRNA and protein products. Three mRNA isoforms OCT4A, OCT4B, and OCT4B1 are generated from OCT4 gene. OCT4A mRNA is translated into OCT4A protein, and OCT4B mRNA is translated into three protein isoforms OCT4B-265, OCT4B-190, and OCT4B-164. Abbreviations: CTD: carboxyl terminal domain; NTD: amine terminal domain; POU: POU homeodomains.

The diverse transcription and translation products of OCT4 have endowed this gene with multiple expression patterns and functions. During preimplantation of human embryos, OCT4A and OCT4B have shown different temporal and spatial expression patterns. OCT4A is highly expressed in cell nucleus of the blastocysts and compacted embryos, while OCT4B is found in cytoplasm of all cells from the four-cell stage onward [12, 13]. In adult human tissues, both OCT4A and OCT4B mRNA express at low levels in nearly all kinds of tissues, such as heart, liver, islet, kidney, and spleen [8]. Until now, most functional studies about OCT4 focus on OCT4A, which is highly expressed in ESCs and is responsible for maintaining pluripotency [1-4, 14-19]. In recent years, more attentions have been paid to OCT4B, which cannot sustain ESCs' self-renewal [10, 12-14]. OCT4B-190 has been reported to be upregulated after heat shock and oxidative stress treatments in hES and tumor cell lines, and overexpression of OCT4B-190 can resist cell apoptosis induced by heat shock [10]. However, the biological function of other OCT4B isoforms is still unknown. In this study, OCT4B-265 was found to be upregulated under genotoxic stress in hES and human embryonic carcinoma (EC) cell lines. Furthermore, overexpression of OCT4B-265 can promote cell apoptosis in genotoxic stress response.

Genotoxic stress could cause DNA damage and trigger a variety of responding signaling pathways, including cell cycle arrest, DNA repair, and cell apoptosis [20–23]. p53 is one of the most important molecules in DNA damage response, which has multiple functions in cell cycle and apoptosis regulation [21, 24-26]. Here, we show that the response of OCT4B-265 to genotoxic stress is regulated by p53.

MATERIALS AND METHODS

Antibodies and Reagents

OCT4 antibodies: sc-8629 (1:1,000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), sc-5279 (1:1,000, Santa Cruz Biotechnology Inc.), and ab-19857 (1:1,000, Abcam, Cambridge, U.K., http://www.abcam.com); α-tubulin antibody: T5168 (1:5000, Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com); p53 antibody: ab-7757 (1:1,000, Abcam); p53 (phospho Ser15) antibody: ab-1431 (1:1,000, Abcam); sc-8629 blocking peptide (sc-8269P, Santa Cruz Biotechnology Inc.); ab-19857 blocking peptide (ab-20650, Abcam).

Mitomycin C (MMC) (GR-311, Enzo Life Sciences International Inc., Plymouth Meeting, PA, http://www.enzolifesciences.com); doxorubicin (Dox) (GR-319, Enzo Life Sciences International Inc.); tunicamycin (TM) (T8480, Sigma-Aldrich); pifithrin-α (PFT-α) (P4359, Sigma-Aldrich); lactacystin (L6785, Sigma-Aldrich); MG132 (474790, Merck, Darmstadt, Germany, http://www.merck.com).

Cell Culture and Stress Treatment

NTERA-2, MCF-7, 293T, PA-1, and HepG2 cell lines were purchased from Peking Union cell center (ATCC source); RT4 cell line was presented by Professor Ying Jin, Shanghai Jiao Tong University; H9 hES cell line was presented by Professor Lingsong Li, Peking University Health Science Center, Department of Cell Biology, Peking University Stem Cell Research Center.

NTERA-2, MCF-7, and 293T cells were cultured with Dulbecco's modified Eagle's medium (Gibco, Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Thermo Fisher Scientific, Waltham, MA, http://www.thermoscientific.com), 2 mM glutamine (Hyclone, Thermo Fisher Scientific), 1 mM sodium pyruvate (Gibco, Invitrogen), 1% nonessential amino acids (Gibco, Invitrogen), 100 U/ml penicillin (Hyclone, Thermo Fisher Scientific), 100 μg/ml streptomycin (Hyclone, Thermo Fisher Scientific). PA-1 and HepG2 cells were cultured with Minimum Essential Media/Earle's Balanced Salt Solution (MEM/EBSS) medium (Hyclone, Thermo Fisher Scientific), and RT4 cells were cultured with McCOY's 5a medium (Hyclone, Thermo Fisher Scientific), respectively, supplemented with components listed above.

When treating PA-1 or MCF-7 cells, the concentrations of genotoxic or nongenotoxic drugs were 0.1–10 μg/ml MMC, 0.2–1 μg/ml Dox, 1–5 μM TM, or 800 μM hydrogen peroxide (H2O2). The dose of UV light was 10–50 J/m2. After 0–12 hours of treatment, cells were collected. When using PFT-α, lactacystin, and MG132, they were added 12 hours before the stress treatment, and the concentrations were 30, 10, and 20 μM, respectively.

Vector, Small Interfering RNA (siRNA) and Adenovirus Construction

The pQCXIN vector (Clontech Laboratories, Inc., Mountain View, CA, http://www.clontech.com) was used as backbone. Green fluorescent protein (GFP) and OCT4B-265-GFP vectors were constructed as described in our previous work [10]. siRNA of human p53 was purchased from Genepharma Company (Shanghai, People's Republic of China, http://www.genepharma.com) with the sequence: sense 5′-CUACUUCCUGAAAACACGdTdT-3′ and antisense 5′-CGUUGUUUUCAGGAAGUAGdTdT-3′. Adenovirus containing the coding sequence of OCT4B-265 (the start codons of OCT4B-190 and OCT4B-164 were mutated, as described in our previous work) and negative control virus was constructed and purified by SinoGenoMax Company (Beijng, People's Republic of China, http://www.sinogenomax.com).

Cell Transfection

MCF-7 cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. PA-1 and MCF-7 cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol. 293T, PA-1, and MCF-7 cells were infected with adenovirus according to the manufacturer's protocol. Cells were exposed to stress 36 hours after transfection. Detection was performed 10–12 hours after stress.

Western Blot

Cells were lysed with RIPA buffer (Sigma-Aldrich) supplemented with proteinase inhibitor cocktail (Sigma-Aldrich). The lysates were centrifuged at 12,000g for 20 minutes at 4°C. The supernatant was separated through 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) gels and then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with Tris buffered saline with 1% tween-20 (TBST) containing 10% fat-free milk powder and then incubated with primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. Fluorescent signals were detected with the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Thermo Fisher Scientific). The concentration of blocking peptides was 10-fold of primary antibodies. In blocking peptide groups, primary antibodies were preincubated with peptides for 2 hours.

Cell Apoptosis Assay

For each sample, approximately 1 × 106 cells were prepared for apoptosis assay. Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining kit (SiZhengBai, Beijing, People's Republic of China, http://www.4abio.com) and Annexin V-allophycocyanin (APC) and 7-amino-actinomycin D (7-AAD) apoptosis detection kit (Keygen, Nanjing, People's Republic of China, http://www.keygentec.com.cn) were used according to the manufacturer's instructions. The cells were incubated at 37C for 30 minutes and then analyzed on a fluorescence-activated cell sorting (FACS)-LSR (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) equipped with CellQuest (BD Biosciences) software.

Cell Cycle Analysis

For each sample, approximately 1 × 106 cells were collected and fixed with 300 μl phosphate buffer saline (PBS) plus 700 μl 70% ethanol. After being fixed at 4°C overnight, the cells were resuspended with 500 μl PBS containing 50 μg/ml PI and 100 μg/ml RNase A. The cells were incubated at 37°C for 30 minutes and then analyzed on a FACS-LSR (BD Biosciences) equipped with CellQuest (BD Biosciences) software.

Quantitive RT-PCR

Total RNA was isolated with Trizol LS Reagent (Invitrogen). Equal amount of RNA (1 μg) was added to reverse transcription reaction mix (SuperScript III First-Strand Synthesis System, Invitrogen) with oligo-dT primer (Invitrogen). Quantitative polymerase chain reaction (PCR) analysis was performed in triplicate using 1/40 of the reverse transcription reaction product in a Bio-RAD CFX96TM Real-Time system (Bio-Rad Laboratories, Inc., Hercules, CA, http://www.bio-rad.com) with Power SYBR Green RT-PCR Kit (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Cycle threshold values (log2 scale) were obtained and β-actin was used as control for equal cDNA inputs. Relative quantitation of the expression levels of OCT4B isoforms was analyzed by using the 2−ΔΔCT method. The primers for quantitative real-time RT-PCR were shown, respectively, as follows: the OCT4B forward primer 5′-GTTAGGTGGGCAGCTTGGAA-3′ and the reverse primer was 5′-CGTGTGGCCCCAAGGA-3′; the β-actin forward primer was 5′-ACCGAGCGCGGCTACAG-3′ and the reverse primer was 5′-CTTAATGTCACGCACGATTTCC-3′.

Statistical Analysis

Data were presented as means ± SD in this study. Statistical comparisons between data groups were made using Student's t test. *, p < .05 was considered as statistically significant difference.

RESULTS

A 35-kD Protein Band Recognized by OCT4 Antibody Was Upregulated Under Genotoxic Stress

When treating PA-1 cells with MMC, we detected the upregulation of a 35-kD band using OCT4 antibody sc-8629. This band was upregulated after 4–6 hours of treatment and became stronger after 8–12 hours of treatment (Fig. 2A). This upregulated protein band could be induced by as low as 0.1 μg/ml of MMC and showed dose-dependent effect (Fig. 2B). Meanwhile, another genotoxic drug, Dox, could also induce the upregulation of this band at 1.0 μg/ml after 8–12 hours (Fig. 2E). UV irradiation is another inducer of DNA damage. PA-1 cells were subjected to 254 nm UV irradiation with the dose of 10–50 J/m2. After 1–12 hours, the cells were collected for Western blot. As shown in Figure 2C, the 35-kD protein band showed a similar upregulation pattern to MMC treatment after 4–12 hours. PA-1 cells were sensitive to UV irradiation, and 10 J/m2 could induce a strong band. Higher doses did not result in even higher protein level (Fig. 2D).

Figure 2.

A 35-kD OCT4 isoform was upregulated after genotoxic stress in PA-1 cells. (A): Treatment of 5 μg/ml MMC. (B): Treatment of 0.1–10 μg/ml MMC for 10 hours. (C): Irradiation of 10 J/m2 UV. (D): 10–50 J/m2 UV irradiation for 10 hours. (E): Treatment of 0.2, 0.4, or 1.0 μg/ml doxorubicin for 8–12 hours. (F): Treatment of H2O2 or TM. Abbreviations: MMC, mitomycin C; TM, tunicamycin.

On the other hand, cells were treated with nongenotoxic stresses, such as H2O2 and TM. As shown in Figure 2F, 800 μM of H2O2 or 5 μM of TM could not induce the appearance of this 35-kD protein band. The above results indicated that this 35-kD protein band was upregulated only under genotoxic stress.

Identification of This 35-kD Protein Band

Since this protein band could be recognized by OCT4 antibody, it should be one of the OCT4 protein isoforms. To exclude the possibility of nonspecific recognition by antibodies, we used blocking peptides of related antibodies. Sc-8629 and ab-19857 were antibodies against OCT4 C-terminus, and sc-8629P and ab-19857P were their blocking peptides, respectively. As shown in Figure 3A, sc-8629 and ab-19857 could recognize the 35-kD band and their blocking peptides could totally block it. Sc-5279 was an OCT4A N-terminal antibody, and it could not recognize the 35-kD protein band (Fig. 3A), indicating that this possible isoform did not contain the N-terminus specific for OCT4A but contained the C-terminus common for OCT4A and OCT4B [8]. So, this 35-kD protein was considered to be an isoform of OCT4B.

Figure 3.

Identification of this protein isoform as OCT4B-265. (A): Blocking peptides of OCT4 antibodies could block this band. Also, the N-terminal antibody of OCT4A could not recognize this band. (B): Proteasome inhibitor lactacystin or MG132 could not inhibit this isoform from upregulation after MMC treatment and could induce the expression of this protein isoform under normal conditions. MMC treatment in (A) and (B) was 5 μg/ml for 10 hours. (C): Overexpression of OCT4B-265 showed a band at the same position with the protein isoform induced by genotoxic stress. Abbreviations: MMC, mitomycin C; PA-1 +, PA-1 cells were treated with MMC for 12 hours; UV: cells were subjected to 10 J/m2 of UV irradiation after transfection; vB: virus expressing OCT4B-265, vN: negative control of empty virus.

Also, in order to exclude the possibility that this isoform was the product of OCT4A degradation, we used proteasome inhibitor lactacystin and MG132 to inhibit protein degradation. Approximately, 10 μM lactacystin or 20 μM MG132 was added to cell culturing medium 12 hours before the genotoxic stress treatment. As shown in Figure 3B, lactacystin or MG132 did not abolish the upregulation of this isoform after MMC treatment. Besides, the proteasome inhibitor (lactacystin or MG132) itself can induce this isoform without genotoxic treatment. These results indicated that the appearance of this protein isoform was not due to protein degradation. It also suggested that this isoform might undergo degradation under normal conditions, and it accumulated under genotoxic stress conditions.

According to the molecular weight of OCT4B isoforms, 35 kD for OCT4B-265, 23 kD for OCT4B-190, and 20 kD for OCT4B-164, this 35-kD isoform should be OCT4B-265. 293T and PA-1 cells were transfected with virus containing OCT4B-265 coding sequence, while empty virus containing no target gene was transfected as negative controls. As shown in Figure 3C, 293T cells transfected with empty virus showed no band, and cells transfected with OCT4B-265 virus showed a clear band at 35 kD, which had the same molecular weight with the band induced by genotoxic stress in PA-1 cells. Similar experiments were repeated in PA-1 cells (Fig. 3C).

The Translation and the Upregulation of OCT4B-265 Under Genotoxic Stress Was Specific in Stem Cells

Since OCT4B mRNA is widely expressed in different kinds of cells and tissues [8, 12, 13], we wondered if OCT4B-265 responded to genotoxic stress in other cell types besides PA-1 cells. Human ES H9 cells and another EC cell line NTERA-2 were treated with MMC or Dox. Western blot results showed that OCT4B-265 was also upregulated in hES H9 and NTERA-2 cells (Fig. 4A). However, in MCF-7, 293T, HepG2, and RT4 cell lines, even higher doses (20–50 μg/ml) of MMC treatment could not induce the upregulation of OCT4B-265 protein level (Fig. 4B). In the tumor cell lines above, the OCT4B mRNA is reported to express in similar level to that in ESCs or EC cells [8, 10]. Whereas, we found that the proteasome inhibitor cannot induce the band of OCT4B-265 in the Western blotting assay (Fig. 4D), which is different from the case in PA-1 cells. Then we detected the change of mRNA expression level of OCT4B under genotoxic stress. As shown in Figure 4C, the mRNA expression level of OCT4B did not significantly change after MMC treatment in PA-1 cells.

This interesting phenomenon suggested that the translation of OCT4B-265 and the response of OCT4B-265 to genotoxic stress might be specific in hES and EC cells that owned stemness. Although OCT4B mRNA could be detected in a variety of cells, there might be some special translation regulation toward OCT4B in cells that owned stemness.

Functional Study of OCT4B-265 Under Genotoxic Stress

GFP or OCT4B-265-GFP fusion protein expression plasmid was transfected into MCF-7 cells. The cells were subjected to 10 μg/ml MMC 36 hours after transfection. Subcellular localization of GFP or OCT4B-265-GFP was observed with fluorescent microscope 10 hours after MMC treatment. Cell nucleus was labeled with Hoechst 33342. As shown in Figure 5A, both proteins were diffusely localized in cells under normal conditions. However, OCT4B-265-GFP tended to aggregate into granules in nucleus after MMC treatment, while the localization of GFP did not change. The subcellular localization change of OCT4B-265 suggested that it might have certain functions in genotoxic stress response.

Figure 4.

OCT4B-265 was upregulated after genotoxic stress in stem cells. (A): Human embryonic stem cells H9, PA-1, and NTERA-2 had this phenomenon. (B): OCT4B-265 was not expressed in MCF-7, 293T, HepG2, or RT4 cells after genotoxic stress. MMC in this figure was treatment with 5 μg/ml MMC for 10 hours. (C): The OCT4B mRNA of PA-1 cells treated with MMC for 0 and 12 hours was measured by quantitative PCR assay, and the result is presented by fold change relative to the mRNA level of 0-hour group. (D): The proteasomal inhibitor MG132 cannot induce the expression of OCT4B-265 protein in MCF-7, 293T, HepG2, or RT4 cells. Abbreviations: MMC, mitomycin C; PCR, polymerase chain reaction.

Figure 5.

Functional study of OCT4B-265. (A): OCT4B-265 formed granules and relocalized in cell nucleus after MMC treatment. Magnification: ×200; green: GFP; blue: Hoechst 33342. (B): Overexpression of OCT4B-265 in MCF-7. (C): Overexpression of OCT4B-265 promoted cell apoptosis after 5 lg/ml of MMC treatment for 12 hours. The apoptotic data are acquired as percentage changes to the average data of untreated cells of respective group. *p<0.05. (D): Overexpression of OCT4B-265 did not affect the percentage of G1-, G2/M-, or S-phase cells in cell cycle analysis. “c” meant treatment of 2 μg/ml of MMC for 12 hours. Abbreviations: GFP, green fluorescent protein; MMC, mitomycin C; MOI, number of virus particles per cell; V-null, empty virus control.

In order to study the function of OCT4B-265, we transfected MCF-7 cells with OCT4B-265 and compared the cell cycle and cell apoptosis with control group. As shown in Figure 5B, OCT4B-265 was successfully overexpressed in MCF-7 cells. Cells were treated with 5 μg/ml of MMC for 12 hours and then subjected to apoptosis assay. The results showed that, the percentage of apoptotic cells increased from 21.64% ± 0.79% in control group to 26.38% ± 1.38% in OCT4B-265 group (both groups subtracted their percentages of apoptotic cells under normal conditions, respectively) (Fig. 5C, Supporting Information Fig. S1A). Cell apoptosis is a protective mechanism in the condition of severe DNA damage, preventing mutated DNA from passing down to daughter cells. When the damage is repairable, cells undergo cell cycle arrest and DNA repair [27–29]. So, MCF-7 cells were treated with lower concentration (2 μg/ml) of MMC for 12 hours, which would not cause significant apoptosis. Cell cycle was detected using flow cytometry after PI staining. As shown in Figure 5D and Supporting Information Figure S1B, overexpression of OCT4B-265 did not change the percentage of G1-, G2/M-, or S-phase cells both under normal conditions and genotoxic stress. These results suggested that OCT4B-265 might not affect cell cycle under mild genotoxic stress, while it could promote cell apoptosis under severe genotoxic stress.

p53 Was Involved in OCT4B-265's Response to Genotoxic Stress

p53 is an important regulator in DNA damage response [30, 31]. It is reported that p53 could be activated by various kinds of genotoxic stress [32, 33] and promote apoptosis under severe DNA damage conditions [34]. So, we tested whether p53 was involved in OCT4B-265's response to genotoxic stress.

As shown in Figure 6A, PA-1 cells were collected at indicated time after MMC treatment. Ser15 site of p53 was phosphorylated after 30 minutes of treatment and lasted for at least 6 hours. PA-1 cells were incubated with p53 inhibitor PFT-α in advance and then treated with MMC. As shown in Figure 6B, PFT-α could abolish the phosphorylation of p53 after MMC treatment, and the upregulation of OCT4B-265 protein level also disappeared. Similar results were found in the PA-1 cells preincubated with p53 siRNA. The phosphorylation of p53 induced by MMC was almost suppressed, and the inducement of OCT4B-265 was also abolished (Fig. 4C). Furthermore, MCF-7 cells overexpressing OCT4B-265 and control group were preincubated with PFT-α or p53 siRNA, then treated with MMC, and subjected to apoptosis assay. The results showed that both PFT-α and p53 siRNA could abolish the apoptosis promotion effect of OCT4B-265 (Fig. 6D, 6E). These results indicated that the response of OCT4B-265 to genotoxic stress was dependent on p53 and p53 was involved in OCT4B-265's response to genotoxic stress.

Figure 6.

OCT4B-265 functioned through p53. (A): Phosphorylation of p53 Ser 15 and total p53 was upregulated after MMC treatment. (B): PFT-α could inhibit phospho-p53 and total p53. In PFT-α group, MMC could not upregulate OCT4B-265. (C): p53 siRNA could inhibit the phospho-p53 and total p53. In the p53 siRNA group, MMC could not upregulate OCT4B-265. (D): The apoptosis promoting effect of OCT4B-265 was abolished by PFT-α. (E): The apoptosis promoting effect of OCT4B-265 was abolished by p53 siRNA. Abbreviations: MMC, mitomycin C; PFT α, pifithrin-α; siRNA, small interfering RNA.

DISCUSSION

Human OCT4 gene can generate three mRNA products and four protein products [8, 10]. Distinguishing and studying the different products of OCT4 are very important to illuminate the complex biological functions of this gene. In this research, we found OCT4B-265, one of the protein products of OCT4B mRNA, could be upregulated under genotoxic stress. Overexpressing OCT4B-265 could promote cell apoptosis under genotoxic stress, and this function was dependent on p53.

It was reported that OCT4B mRNA is widely expressed in various kinds of cells and tissues, and the expression level of OCT4B is not exceptionally high in stem cells [35–38]. However, the upregulation of OCT4B-265 only happened in hES and EC cells according to our research (Fig. 4B). The mRNA level of OCT4B did not obviously change under genotoxic stress (Fig. 4C), and the protein level of OCT4B-265 was upregulated after proteasome inhibitor treatment (Fig. 3B). These results suggested that the upregulation of OCT4B-265 after genotoxic stress was due to the regulation on protein level. OCT4B-265 might be degraded by proteasome under normal conditions, and the degradation might be inhibited under genotoxic stress due to some possible modification on protein. However, the expression of OCT4B-265 in tumor cell lines which do not own stemness seemed to be regulated differently. The proteasome inhibitor MG132 cannot induce the expression of OCT4B-265 protein (Fig. 4D). This result indicated that OCT4B-265 protein may not be translated in tumor cell lines, so that the response to the genotoxic stress of OCT4B may not be detected in tumor cell lines. Thus, these results suggested that in ESCs and EC cells, there might be special mechanisms regulating OCT4B-265's protein level expression, which needs further study.

Indeed, OCT4B-265 loss of function experiment is useful in elucidating the OCT4B-265 function. However, we can hardly carry it out. Because OCT4B-265 is encoded by OCT4B mRNA and this mRNA can be translated into three protein isoforms, one is OCT4B-265 by 5′-cap-dependent translation, and the other two are protein isoforms which have 190aa and 164aa, respectively, by IRES-dependent translation [10]. If OCT4B-265 is knocked down, the OCT4B-190 and OCT4B-164 would be also silenced. The OCT4B-190 is also expressed in hES and PA-1 cells and functions upon stresses [10]. Thus, we can hardly figure out whether the “loss of function” results are caused by the knockdown of OCT4B-265 or by the knockdown of all the three proteins.

In our previous work, OCT4B-190 was found to be upregulated and antagonize cell apoptosis after heat shock [10]. Compared with OCT4B-190, OCT4B-265 did not respond to nongenotoxic stress such as heat shock. These two protein isoforms of OCT4B had different responding conditions and contrary effects on cell apoptosis. This interesting phenomenon might be attributed to the different influence of genotoxic and nongenotoxic stress on cells. Genotoxic stress causes DNA damage, and it potentially causes DNA mutation and even tumor genesis [39–41], while nongenotoxic stress does not. Cells respond to DNA damage in two aspects: DNA repair or cell apoptosis. When the damage is “repairable,” cells may undergo cell cycle arrest to gain more time for DNA repair; while the damage is not repairable, cells may undergo apoptosis to prevent the damaged DNA from passing down [20, 28, 29]. So OCT4B-265's promotion of cell apoptosis after genotoxic stress is also a protective mechanism for the whole organism.

p53 can regulate cell apoptosis and cell cycle, such as G1/S checkpoint and G2/M checkpoint during DNA damage [30, 31, 42-44]. The downstream molecules of p53 are different in cell apoptosis and cell cycle regulating pathways. For example, p53 can activate waf1, proliferating cell nuclear antigen (PCNA), and BRCA1 in cell cycle control [42, 45, 46]. In cell apoptosis regulation, p53 activates apoptosis relating proteins such as Bcl-2 and Bax [47–50]. In our study, OCT4B-265's function was dependent on p53, and it did not affect cell cycle but increased cell apoptosis, suggesting that OCT4B-265 may participate in the p53 pathway in cell apoptosis regulation.

This research indicates that OCT4B-265 is an isoform related to genotoxic stress in ESCs and EC cells. Besides the stemness-maintaining function, OCT4 gene also has significant functions in cell stress response. At least two isoforms of OCT4B, OCT4B-190 and OCT4B-265, respond to different kinds of cell stress. The expression and function of another OCT4B isoform OCT4B-164 still need further investigation. The growing interest in the study of OCT4 in ESCs, iPS cells, adult stem cells, and cancer cells requires more detailed illustration of the different functions of the four OCT4 protein isoforms.

CONCLUSION

We have identified that OCT4B-265, one of the OCT4 protein isoforms, was upregulated under genotoxic stress. What is more, the response to genotoxic stress was only detected in stem cells. Under genotoxic stress, OCT4B-265 was found to promote the cell apoptosis, and this function was dependent on p53. This work gives an insight into the novel function of OCT4B protein isoforms and helps us to understand the complex expression patterns and biological functions of OCT4 gene.

Acknowledgements

This work was supported by grants from National Natural Science Foundation of China (90919042), and the Ministry of Science and Technology of China (2011CB965001, 2011 CB710905).

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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