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Cancer Research 67, 6146, July 1, 2007. doi: 10.1158/0008-5472.CAN-07-0867
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Incorporation of an Internal Ribosome Entry Site–Dependent Mechanism in Arsenic-Induced GADD45{alpha} Expression

Qingshan Chang1,3, Deepak Bhatia1,2, Yadong Zhang1,3, Terry Meighan1, Vince Castranova1, Xianglin Shi1,3 and Fei Chen1,2

1 The Health Effects Laboratory Division, National Institute for Occupational Safety and Health; 2 Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, West Virginia; and 3 Institute for Nutritional Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Requests for reprints: Fei Chen, Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, WV 26505. Phone: 304-285-6021; E-mail: LFD3{at}cdc.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that trivalent arsenic (arsenite, As3+) is able to induce GADD45{alpha} expression in human bronchial epithelial cells through activation of c-Jun NH2-terminal kinase and nucleolin-dependent mRNA stabilization. In the present report, we show that As3+ is capable of inducing translation of the GADD45{alpha} protein through a cap-independent, or rather, an internal ribosome entry site (IRES)–dependent mechanism. In growth-arrested cells, As3+ elevated the GADD45{alpha} protein level in a dose- and time-dependent manner which did not correlate with the GADD45{alpha} mRNA expression. Pretreatment of the cells with rapamycin, an inhibitor for the cap-dependent translation machinery through the suppression of mTOR and p70S6 kinase, failed to affect the induction of the GADD45{alpha} protein induced by As3+. Sequence analysis revealed a potential IRES element in the 5'-untranslated region of the GADD45{alpha} mRNA. This IRES element in the 5'-untranslated region of the GADD45{alpha} mRNA is functional in mediating As3+-induced translation of the GADD45{alpha} protein in a dicistronic reporter gene activity assay. Immunoprecipitation and proteomic studies suggest that As3+ impairs the assembly of the cap-dependent initiating complex for general protein translation but increases the association of human elongation factor 2 and human heterogeneous nuclear ribonucleoprotin with this complex. Thus, these results suggest that in growth-arrested cells, As3+ is still capable of inducing GADD45{alpha} expression through an IRES-dependent translational regulation. [Cancer Res 2007;67(13):6146–54]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Growth arrest– and DNA damage–induced gene 45{alpha} (GADD45{alpha}) encodes a small acidic protein that is capable of interacting with a number of important intracellular signaling molecules involved in cell cycle regulation, apoptosis, and immune responses (1). A wide variety of stress inducers, including DNA-damaging reagents, UV radiation, oxidative or osmotic stress, and nutrient deprivation stimulate the expression of GADD45{alpha} mRNA and protein in a manner which may be either p53-dependent or p53-independent (2). The fact that GADD45{alpha} is preferentially localized in the nuclei suggests that the key function of this protein might be the regulation of DNA replication, DNA repair, and cell division. Indeed, the majority of the known GADD45{alpha}-interacting molecules are nuclear functional proteins, such as proliferating cell nuclear antigen (3), cyclin B/CDC2 complex (4), p21cip1 (5), histone (6), aurora-A kinase (7), xeroderma pigmentosum complementation group G (8), and TAFII70 (9). Interaction of GADD45{alpha} with these proteins causes cell cycle arrest, repression of DNA replication or cell apoptosis, thereby providing the cells with time to repair the damaged DNA and prevent the segregation of damaged chromosomes.

Extensive efforts have been made to understand how the transcription of GADD45{alpha} is regulated in cellular responses to extracellular signals. The transcriptional induction of GADD45{alpha} by DNA damage is thought to be dependent on p53 or FoxO3a (10), which antagonizes transcriptional repressors of the GADD45{alpha} gene, such as c-myc (11) and ZBRK (12). Activation of c-Jun NH2-terminal kinase (JNK) may play a key role in mediating p53-independent transcription of GADD45{alpha} in response to other stress signals (13). The influence of posttranscriptional events, mainly the modulation of GADD45{alpha} mRNA stability, has been recognized in recent years. In humans, the bronchial epithelial cell line, BEAS-2B, stimulation of the cells with trivalent arsenic (arsenite, As3+), a general stress inducer, stabilizes GADD45{alpha} mRNA through the inducible association of nucleolin and less potently, HuR, with the GADD45{alpha} mRNA (14). Removal of the RNA destabilization protein AUF1 from the 3-untranslated region (3'-UTR) of the GADD45{alpha} mRNA was considered to be an important mechanism for the enhanced stability of the GADD45{alpha} mRNA in cells treated with genotoxin methyl methanesulfonate (15). Intriguingly, neither nucleolin nor HuR were associated with GADD45{alpha} mRNA in UV- or methyl methanesulfonate–treated HeLa cells (16). It is unknown whether the expression of GADD45{alpha} is subjected to protein translation regulation under certain circumstances.

Environmental or occupational exposure to As3+ is associated with a number of human diseases including nerve degeneration, skin hyperkeratinization, birth defects, hepatic cirrhosis, cardiovascular disorders, diabetes, and cancer (17). By binding to sulfhydryl groups, As3+ impairs the function of many proteins, especially glutathione/thioredoxin reductases and protein tyrosine phosphatases which possess vicinal thiols (18). The cumulative effects of this impairment are oxidative stress and kinase activation, including receptor type and non–receptor type tyrosine kinases, JNK, p38, p70S6 kinase (p70S6K), checkpoint kinases, and Akt. Both sustained oxidative stress and aberrant kinase activation have been linked to cell growth and malignant transformation. As3+ is also able to induce ubiquitination or sumoylation and proteasomal degradation of some regulatory proteins, such as CDC25C (19), AML1/MDS1/EVI1 (20), and PML (21). This property of As3+, paradoxically, makes it an effective therapeutic agent for some types of cancers, mainly acute promyelocytic leukemia and lymphomatic malignancy (21). We previously showed that As3+ is a potent inducer for the expression of GADD45{alpha} through activation of JNK (13). We have also provided evidence indicating that As3+ could stabilize GADD45{alpha} mRNA by inducing the association of nucleolin and HuR, two mRNA stabilizing proteins, with GADD45{alpha} mRNA (14). In the present report, we investigated the regulatory role of As3+ on GADD45{alpha} protein translation in growth-arrested cells and showed that As3+ enhanced GADD45{alpha} translation in a cap-independent but internal ribosome entry site (IRES)–dependent manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and Western blotting. The human bronchial epithelial cell line, BEAS-2B, was purchased from American Tissue Culture Collection. Arsenic(III) chloride (As3+) and other chemicals were purchased from Sigma-Aldrich. Antibodies used in Western blotting were purchased from Santa Cruz Biotechnologies. The cell culture, treatment, and Western blotting were done as previously described (14). In some experiments, the cell lysates from the untreated or As3+-treated cells were made using a TRIZOL reagent (Invitrogen) for sequential extraction of total RNA and protein according to the manufacturer's instructions.

Reverse transcription-PCR. The expression levels of GADD45{alpha} and GAPDH mRNAs in the cells were monitored by RT-PCR (reverse transcription-PCR) using the AccessQuick RT-PCR system (Promega) with a temperature scale of 45°C for 50 min for reverse transcription, and 35 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. The RT-PCR was carried out using the primers: GADD45{alpha} (5'-GGAGAGCAGAAGACCGAAA-3', 5'-TCACTGGAACCCATTGATC-3'); GAPDH (5'-CTGAACGGGAAGCTCACTGGCATGGCCTTC-3', 5'-CATGAGGTCCACCACCCTGTTGCTGTAGCC-3').

Dicistronic reporter gene activity assay. The dicistronic reporter construct pRL-HCV-FL was a kind gift from Dr. Richard E. Lloyd at Baylor College of Medicine. This construct contains a Renilla luciferase reporter gene at the upstream 5'-UTR of the hepatitis C virus (HCV) gene, and a Firefly luciferase reporter gene (FL) downstream (22). The 5'-UTR of the HCV in the vector was replaced with the fragments derived from the 5'-UTR of the GADD45{alpha} mRNA. These 5'-UTR fragments were amplified by RT-PCR with the following primers: UTR1-328 (5'-TGATGCGGCCGCTCCAGTGGCTGGTAGGCAGT-3', 5'-TCGATCCGCGGCTGCTCTCCAGCCGAGAAT-3'); UTR2-223 (5'-TGATGCGGCCGCGTGGCTGGTAGGCAGTGG-3', 5'-TCGATCCGCGGGCCTGCTTTCTGCACTCACT-3'); UTR200-318 (5'-TGATGCGGCCGCTGTGAGTGAGTGCAGAAAGCA-3', 5'-TCGATCCGCGGGCCGAGAATTCCTCCAAAGT-3'). The NotI and SacII sites that were artificially added to the primers for cloning purposes were underlined and double-underlined, respectively. The promoterless dicistronic reporter vector, pRL-FL(-P), and the GADD45{alpha} promoter and intron 3 reporter vectors were gifts from Dr. Jian-Ting Zhang at the Indiana University Cancer Center and Dr. Daniel Haber at Massachusetts General Hospital Cancer Center, respectively. Each plasmid DNA was transfected into the cells with LipofectAMINE 2000 (Invitrogen) in the serum-free medium for 24 h. The transfection efficiency for each plasmid was determined by cotransfection of the pcDNA-GFP followed by flow cytometry analysis that showed a similar transfection rate of these vectors. The cells were then incubated with the regular cell culture medium containing 5% fetal bovine serum for an additional 24 h followed by dual-luciferase reporter gene activity assay.

Protein peptide analysis. Proteins recovered from immunoprecipitation using anti-eIF4G antibody were separated by SDS-PAGE. The protein bands were visualized by silver staining. The excised protein gel bands were subjected to in-gel digestion by adding 50 µL of 50 mmol/L ammonium bicarbonate containing 10 ng/µL of modified trypsin, and then incubated at 37°C overnight. Tryptic peptides were extracted with 100 µL of extraction buffer (0.1% trifluoroacetic acid and 50% acetonitrile; pH 2.5), desalted, and analyzed by online reversed phase nano-LC-MS/MS on a Finnigan LTQ mass spectrometer using a 1-h gradient data-dependent MS/MS collection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
As3+ induces GADD45{alpha} translation in arrested cells. We have previously reported that exposure of the BEAS-2B cells to As3+ resulted in a dose- and time-dependent increase in both GADD45{alpha} mRNA and/or protein when the cells were cultured in a logarithmically growing phase (13, 14, 23, 24). We also frequently observed a variable degree of the GADD45{alpha} mRNA and protein induction by As3+ among the cells under different growing conditions. To determine the source of such variations, we deliberately cultured the cells at two well-defined conditions: logarithmically growing condition and the arrested condition in which the cells reached confluence with a high density (Fig. 1A ). To minimize variations in sampling of total RNA and protein from the cells with or without As3+ treatment, we used the same cells to isolate total RNA and protein sequentially by TRIZOL reagent. The levels of GADD45{alpha} mRNA and protein were determined by RT-PCR and Western blotting, respectively. As previously reported, exposure of the BEAS-2B cells in a growing condition to 20 µmol/L of As3+ resulted in a time-dependent increase in GADD45{alpha} mRNA and protein (Fig. 1B, lanes 1–6). Both mRNA and protein of the GADD45{alpha} were undetectable in the growing cells without As3+ treatment (Fig. 1, lane 1). The peak induction of GADD45{alpha} protein lagged behind the induction of GADD45{alpha} mRNA by As3+ in these cells (compare RT-PCR results with the Western blots in Fig. 1B). To our surprise, a considerable level of GADD45{alpha} mRNA was present in the arrested cells without As3+ treatment (Fig. 1B, lane 7). Despite the expression of the GADD45{alpha} mRNA in these arrested cells without As3+ treatment, the GADD45{alpha} protein was undetectable. Treatment of these arrested cells with As3+ did not increase but rather decreased GADD45{alpha} mRNA expression. In contrast, a time-dependent induction of GADD45{alpha} protein was observed in these cells (Fig. 1B, lanes 9–12) similar to that of growing cells (Fig. 1B, lanes 3–6). Therefore, these data suggest that expression of the GADD45{alpha} mRNA itself is not sufficient for the expression of the GADD45{alpha} protein, and As3+ up-regulates protein translation, but not mRNA transcription of the GADD45{alpha} in arrested cells. It has been generally viewed that the activity of the classic protein translational machinery, cap-dependent translation, is repressed in the cells under growth arrest or stress conditions (25). The longer the exposure of the cells to As3+, the more pronounced is the cellular stress induced. Thus, the time-dependent decrease of GADD45{alpha} mRNA and the increase of GADD45{alpha} protein in the arrested cells in response to As3+ is possibly a result of cellular switch from cap-dependent to IRES-dependent protein translation (discussed later).


Figure 1
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  		Figure 1. As3+ induces GADD45{alpha} mRNA and protein to different degrees in growing cells and arrested cells. A, cell morphology of the BEAS-2B cells in a logarithmic growth phase (left) and in arrested phase (right). B, GADD45{alpha} mRNA and protein in the growing cells and arrested cells treated with 20 µmol/L of As3+ for the indicated times were determined by RT-PCR (top) and Western blotting (bottom), respectively. Left, sizes of DNA markers (M).

 
Induction of GADD45{alpha} protein by As3+ is through a cap-independent mechanism. More than 90% of the cellular proteins are translated in eukaryotes in a manner that depends on the m7GpppN 5'-cap structure of mRNA, which recruits cap-binding protein eIF4E along with the ribosomal preinitiation complex (26). A successful assembly of the translational initiation complex requires the activation of mTOR kinase, which phosphorylates 4E-BP and p70S6 kinase (p70S6K). Thus, functional interruption of mTOR will inhibit cap-dependent protein translation. To determine whether As3+ affects the cap-dependent translational machinery for the expression of GADD45{alpha} protein in arrested cells, we pretreated the cells with 60 or 300 nmol/L of rapamycin (a specific inhibitor of mTOR kinase) overnight, followed by exposure of the cells to 20 µmol/L of As3+ for an additional 6 h. Both concentrations of rapamycin exhibited potent inhibition of the activation of p70S6K as evidenced by the disappearance of the phosphorylated form of the p70S6K in a Western blotting experiment using an antibody specific for the phosphorylated p70S6K on serine389 (Fig. 2A, lanes 7–10, middle ). However, rapamycin showed no inhibition on the expression of the GADD45{alpha} protein induced by As3+ (Fig. 2A, lanes 9 and 10, top). To determine the effect of the newly synthesized mRNA on As3+-induced GADD45{alpha} protein expression, we also preincubated the cells with 4 or 8 µmol/L of actinomycin D overnight to diminish mRNA synthesis. As expected, both concentrations of actinomycin D showed no effect on the activation of p70S6K, but abolished induction of the GADD45{alpha} protein by As3+, indicating that the newly synthesized GADD45{alpha} mRNA is required for protein synthesis (Fig. 2A, lanes 5 and 6). Based on these results, we concluded that As3+ induces GADD45{alpha} protein translation through a cap-independent mechanism requiring GADD45{alpha} mRNA synthesis.


Figure 2
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  		Figure 2. As3+ induced translation of the GADD45{alpha} protein is cap-independent. A, BEAS-2B cells were pretreated overnight with actinomycin D (Act. D, 4 or 8 µmol/L), or rapamycin (60 or 300 nmol/L) and then treated with 20 µmol/L of As3+ for an additional 6 h. The expression of the GADD45{alpha} protein, phosphor-p70S6K and total p70S6K were determined on the same membrane by sequential Western blotting using the indicated antibodies. B, the BEAS-2B cells were pretreated overnight with 100 nmol/L of rapamycin and then treated with taxol (2 or 10 µmol/L) for 6 h, or UV radiation (2 or 4 kJ/m2). The levels of GADD45{alpha}, phosphor-p70S6K, and total p70S6K were determined as in (A).

 
To investigate whether this cap-independent mechanism is only specific for cellular response to As3+ or is a general response for other forms of stress signal–induced GADD45{alpha} expression, we next stimulated the cells with taxol or UV radiation. The induction of GADD45{alpha} by taxol, a microtubule-stabilizing drug, and UV had been previously shown to be dependent on p53- or JNK-mediated transcription of the GADD45{alpha} gene in some cancer cell lines (27–29). Both taxol (2 or 10 µmol/L) and UV (2 or 4 kJ/m2) induced an appreciable elevation of the GADD45{alpha} protein in a roughly dose-dependent manner (Fig. 2B). The dosages of taxol and UV were predetermined based on the minimal cytotoxic response of the tested cells. Pretreatment of the cells with 100 nmol/L of rapamycin showed no remarkable effect on taxol- or UV-induced GADD45{alpha} protein, despite the fact that this concentration of rapamycin abrogated p70S6K activation completely (Fig. 2B). Thus, these data suggest that a cap-independent translational mechanism of the GADD45{alpha} protein is involved not only in cellular response to As3+ but to other stress signals from microtubule interference (taxol) and DNA damage (UV) as well. Blockage of the cap-dependent translational machinery by rapamycin has no effect on the induction of the GADD45{alpha} protein using these inducers.

GADD45{alpha} mRNA contains an IRES. Less than 10% of cellular proteins were translated in a cap-independent but IRES-dependent mechanism (30). Because rapamycin was unable to inhibit As3+- as well as UV- or taxol-induced GADD45{alpha} protein translation in the arrested cells, we believe that an IRES-dependent mechanism must be involved in this case. The IRES-dependent pathway translates proteins when the cells are in growth-arrested, apoptotic, mitotic, or extreme stress conditions in which the cap-dependent translational machinery is stalled. In apoptotic and mitotic cells, this translational mechanism is considered to be important for the expression of some functional proteins for the completion of apoptotic and mitotic processes, despite a substantial reduction in the rate of general protein synthesis (31). The mRNAs subjected to cap-independent but IRES-dependent translation usually contain IRES element(s) in the 5'-UTR. To determine whether the human GADD45{alpha} mRNA contains a potential IRES element responsible for the As3+-induced GADD45{alpha} translation, we first analyzed the 5'-UTR region of the GADD45{alpha} mRNA using an online version of UTRScan program.4 After inputting the 295nt 5'-UTR along with an additional 25nt of the open reading frame, a potential IRES element from 209 to 300 nt was identified (Fig. 3A ). The sequence was derived from Genbank NM_001924.


Figure 3
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  		Figure 3. The 5'-UTR of the GADD45{alpha} mRNA contains an IRES element. A, the 5'-UTR of the GADD45a mRNA was analyzed by the UTRScan program. http://www.ba.itb.cnr.it/BIG/UTRScan/Underlined sequence, the computer-identified IRES element. The ATG translation start code was highlighted. B, schematic illustration of the dicistronic reporter vectors used in IRES activity assay. C, IRES activity was determined in the BEAS-2B cells transfected with the indicated dicistronic vectors for 48 h and then cultured for an additional 12 h in the absence or presence of 20 µmol/L of As3+. D, dose-dependent induction of the GADD45{alpha} IRES activity by As3+ in the cells transfected with the indicated dicistronic vectors harboring the indicated 5'-UTR region from the GADD45{alpha} mRNA. E, dose-dependent induction of the HCV IRES activity by As3+ in the cells transfected with the dicistronic vector containing an established IRES element from HCV promoter. F, the increased ratio of FL versus RL is not due to inhibition of the Renilla luciferase translation by As3+. Points, means of four repeats; bars, SD.

 
To verify the IRES activity of the 5'-UTR from the GADD45{alpha} mRNA, we next inserted the 5'-UTR and its derivatives into the pRL-FL dicistronic vector that contains the Renilla luciferase (RL) reporter gene and the Firefly luciferase reporter gene (Fig. 3B). The expression of the RL is cap-dependent, whereas the expression of the FL is dependent on the presence of the IRES element between these two reporter genes in this dicistronic vector (22). The IRES activity was determined by the ratio of FL versus RL. After transient transfection of the BEAS-2B cells with these vectors for 48 h, the cells were treated with 20 µmol/L of As3+ for an additional 12 h. Figure 3C showed relative basal (control) and As3+-induced IRES activities in the cells transfected with the indicated dicistronic vectors. The pRF vector that did not contain any IRES elements was used as a control vector in this experiment. As depicted in this figure, the cells transfected with vectors containing either HCV IRES or GADD45{alpha} IRES showed a notable increase in As3+-induced IRES activity relative to the cells transfected with the pRF vector. A marginal increase of the IRES-like activity was observed in the cells transfected with the pRF vector and treated with As3+, which was very likely a result of ribosomal read-through due to shortening of the space between the two cistrons by removal of the IRES element (data not shown). As3+ is able to increase the IRES activity of the whole 5'-UTR (UTR1-328) and the UTR200-318 of the GADD45{alpha} mRNA in which an IRES element was identified by the UTRScan program. Interestingly, the UTR2-223 region also exhibited As3+-induced IRES activity, indicating the possible presence of a cryptic IRES element in this region, or that the region from 200 to 223 is the key element for the IRES activity in the GADD45{alpha} mRNA. The induction of IRES activity in the 5'-UTR region from GADD45{alpha} mRNA using As3+ is also dose-dependent. An induction plateau occurred at the As3+ dose range from 5 to 20 µmol/L (Fig. 3D).

The ability of As3+ to stimulate IRES activity was further confirmed in the cells transfected with pRL-HCV-FL dicistronic vector, which contains a well-documented HCV IRES element. In a single dose test, As3+ induced a 2- to 3-fold increase of the HCV IRES activity (Fig. 3C). A dose-dependent increase in the HCV IRES activity was observed when the cells were treated with As3+ at a concentration ranging from 0 to 30 µmol/L (Fig. 3D). The elevated FL/RL ratio obtained in the presence of As3+ was not a result of reduced RL translation because As3+ did not inhibit the RL activity (Fig. 3E). Taken together, all of these data clearly suggest that As3+ is capable of inducing IRES-dependent protein translation.

The IRES activity of the GADD45{alpha} 5'-UTR is not a result of cryptic promoter activity. It has been reported that some so-called IRES activities were actually the result of a cryptic promoter element in the 5'-UTR of the given mRNAs (32). To differentiate between the IRES activity and the cryptic promoter activity of the 5'-UTR derived from the GADD45{alpha} mRNA, we first constructed monocistronic vectors by inserting the 5'-UTR of the GADD45{alpha} mRNA to the promoterless pGL3 basic vector (Fig. 4A ). We also used the pGL3-based GADD45{alpha} promoter reporter vector as a positive control. As previously reported (14), As3+ induced GADD45{alpha} promoter activity marginally (Fig. 4B). UTR2-223 showed a notable increase in the luciferase activity relative to other 5'-UTR constructs in the cells without any treatment, indicative of the cryptic promoter activity in this region. However, this cryptic promoter activity was not induced, but was instead inhibited by As3+. There was no detectable cryptic promoter activity in UTR200-318, which showed the highest IRES activity induced by As3+ as indicated in Fig. 3C, in either the control cells or in the As3+-treated cells (Fig. 4B). We also tested the potential cryptic promoter activity of the 5'-UTR derived from GADD45{alpha} mRNA in a promoterless pRL-FL dicistronic vector. Again, although UTR1-328 and UTR2-223 showed cryptic promoter activity in the control cells, this activity was not induced, but rather, was inhibited by As3+ (data not shown). Similar to the monocistronic reporter vector, the UTR200-318 showed no basal or inducible cryptic promoter activity in this promoterless dicistronic reporter system (data not shown).


Figure 4
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  		Figure 4. As3+-induced IRES activity does not result from a cryptic promoter in the 5'-UTR of the GADD45{alpha} mRNA. A, schematic illustration of the monocistronic luciferase reporter vectors. The indicated regions of the GADD45{alpha} 5'-UTR were inserted into the promoterless pGL3 basic vector. The GADD45{alpha} promoter was used as a positive control. B, luciferase activity was determined in the cells transfected with these vectors in (A) and cultured in the absence or presence of 20 µmol/L of As3+ for 12 h (right). Columns, means of four repeats; bars, SD.

 
As3+ impairs the assembly of the cap-dependent preinitiation complex. Accumulating evidence suggests that the rate of protein synthesis is reduced in cells under growth arrest or stress conditions (33). To obtain further insight into the translational regulation of As3+ on protein synthesis, coimmunoprecipitation was then done using antibodies against eIF4G, a general component of the cap-binding preinitiation complex for protein translation. Although the As3+-treated cells showed a similar level of total eIF4E to the control cells (Fig. 5A, input ), an appreciable decrease in the interaction between eIF4G and eIF4E was noted in the cells treated overnight with 20 µmol/L of As3+ (Fig. 5A, top). It is well-known that the association of eIF4E with eIF4G is essential for the further assembly of the translational initiation complex (26). Perturbation in the interaction between eIF4G and eIF4E by As3+, therefore, must influence the general translation of the cellular proteins. Indeed, analysis of the total protein synthesis by running the protein samples in SDS-PAGE followed by Coomassie blue staining showed a reduction of protein synthesis in As3+-treated cells (Fig. 5B, filled bars, right). As3+ induced a mild increase of the proteins with a molecular weight >200 kDa and <30 kDa (Fig. 5B, open bars, right), which possibly resulted from cap-independent, but rather, IRES-dependent translation.


Figure 5
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  		Figure 5. As3+ impairs the assembly of the cap-dependent translational initiation complex. A, BEAS-2B cells cultured in the absence or presence of 20 µmol/L of As3+ for 12 h and then subjected to immunoprecipitation using antibody against eIF4G. The immunoprecipitated complexes were separated on 14% SDS-PAGE gel and were sequentially immunoblotted with antibodies for eIF4G and eIF4E. Total cell lysates were used as input to determine the level of eIF4E. B, total cellular proteins were separated on 14% SDS-PAGE gel and stained by Coomassie blue. Left, molecular weights (M, kDa). Right, open and filled bars indicate increased and decreased expression, respectively, of proteins induced by 20 µmol/L of As3+ for 12 h. C, the complexes from eIF4G immunoprecipitation of the control and As3+-treated cells were separated on 14% SDS-PAGE gel and monitored by silver staining. Left, molecular weights (M, kDa). Right, number of protein bands that were affected by As3+ treatment. *, positions of the protein bands that were excised for in-gel digestion followed by reversed phase nano-LC-MS/MS analysis. D, representative peptide fragmentation spectrum for bands 1 to 4 from (C).

 
To determine whether As3+ affects the association of other proteins with the translational initiation complex, we analyzed the protein complexes from eIF4G immunoprecipitation in SDS-PAGE and silver staining. Although the majority of protein bands are equally presented between the immunoprecipitated complexes from the control cells and the cells treated with 20 µmol/L of As3+ overnight, changes in a few bands in the As3+-treated cells did occur (Fig. 5C). Bands 1 and 2 were increased, whereas bands 3 and 4 were decreased by As3+ treatment. To identify these changed bands, the gel slices were excised (Fig. 5C, asterisk) and subjected to in-gel digestion by trypsin to generate peptides with different lengths based on the distribution of tryptic cleavage sites (arginine and lysine). The digested products were analyzed by reversed phase nano-LC-MS/MS on a Finnigan LTQ mass spectrometer. This analysis identified 8 peptides from various regions of the human elongation factor 2 (eEF-2) for band 1, 6 peptides of human heterogeneous nuclear ribonucleoprotin M (hnRNP M) for band 2, 10 peptides of protein disulfide-isomerase A3 (PDIA3) along with 46 peptides of vimentin for band 3, and 7 peptides of eIF4A-I for band 4. Figure 5D shows the representative fragmentation spectrum for bands 1 to 4 from Fig. 5C. The eEF-2 is a key factor for efficient peptide elongation during protein translation (34). The increased association of eEF-2 with eIF4G in the As3+-treated cells might be a reflection of reciprocal functional antagonism or coordination between cap-dependent and IRES-dependent protein translations through sharing or competing common cofactors. Some hnRNP family members had been previously implicated in direct binding to the IRES elements (31, 35). Thus, the increased association of hnRNP M with translational initiation complex by As3+ is suggestive of As3+ regulation of IRES-dependent translation. The role of PDIA3 might be mainly to order proper folding of the newly translated proteins. A decrease in PDIA3 association with the initiation complex by As3+, thus, would likely result in improper protein folding. The most important finding in this analysis is the nearly complete depletion of eIF4A from the translational initiation complex by As3+. eIF4A is an essential subunit of the eIF4F complex for cap-dependent initiation, which possesses RNA helicase activity to unwind secondary structures within the 5'-UTR of mRNA (36). Taken together, these data show that As3+ treatment favors IRES-dependent translation by impairing the assembly of the cap-dependent translational complex.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings presented here provide the first evidence that As3+ is able to induce translation of the GADD45{alpha} protein in an IRES-dependent manner in the cells under growth-arrested conditions. The 5'-UTR of the GADD45{alpha} mRNA contains an IRES element, the activity of which can be induced by As3+ as evidenced in a dicistronic reporter gene activity. Although the 5'-terminal 200 nt 5'-UTR of the GADD45{alpha} mRNA possibly possesses a cryptic promoter activity under basal conditions, As3+ was unable to induce this cryptic promoter activity in an analysis using either a monocistronic vector or a promoterless dicistronic vector. The IRES-dependent induction of the GADD45{alpha} protein was further supported by the fact that As3+ impairs the assembly of the cap-dependent translational initiation complex by decreasing the association of eIF4G with both eIF4E and eIF4A. This impairment causes a reduction of the global protein translation. As3+ has previously been shown to be capable of regulating transcription and stabilization of the GADD45{alpha} mRNA. The effect of As3+ on IRES-dependent translation of the GADD45{alpha} protein suggested an additional layer of regulation for the expression and function of the GADD45{alpha} protein in cellular responses to As3+.

A wide spectrum of extracellular signals could induce rapid elevation of GADD45{alpha} (1). Remarkable progress has been made during the past decade on the regulation and functional aspects of GADD45{alpha} protein in cellular responses to DNA damage and a variety of stress inducers. However, the answer to how the expression of the GADD45{alpha} is regulated when the cells are under extremely stressed conditions in which the major transcriptional and translational machineries are severely impaired, is still elusive. It was assumed that cells can use an alternative approach to synthesize proteins with the remaining trace amount mRNAs when the canonical cap-dependent protein translation process is compromised either by dephosphorylation of 4E-BP1 or cleavage of eIF4G (37, 38). When the cells are in a state of mitosis, quiescence, differentiation, or hypoxia, the phosphorylated free 4E-BP1 is dephosphorylated, possibly by protein phosphatase 2A, leading to association with and inhibition of eIF4E. Consequently, cap-dependent protein translation is drastically reduced (25, 39). A hallmark of cell apoptosis is the activation of initiating and executing caspases. It has been shown that caspases are highly capable of cleaving cap-dependent translational initiation factors such as eIF4B, eIF3, eIF2a, eIF4GI, and eIF4GII, leading to global repression of protein translation (33). However, a subset of mRNAs, <10% of total mRNAs, manages to escape this translational depression and maintains translation through a cap-independent, or rather, IRES-dependent translation. Considering the fact that GADD45{alpha} was induced mostly by the signals leading to cell cycle arrest or apoptosis, it is very likely that the expression of GADD45{alpha} protein is achieved via IRES-mediated translation.

The IRES-dependent translation was initially discovered in the viral gene translation of picornaviruses in host cells (40). The mRNAs of these viruses are naturally uncapped at their 5'-end. More importantly, many of these viruses produce proteases to proteolytically deplete cap-binding complex to shut off protein translation of the host cells completely during their infection. However, the viral proteins are still efficiently translated in the infected cells. Further studies led to the discovery of the viral IRES element in the 5'-UTR of viral mRNA which mediates viral protein translation by recruiting IRES binding and/or recognition ribosomal proteins and other RNA-binding proteins from the infected host cells (41). Since then, a growing list of mammalian cell mRNAs have been found to possess an IRES element, especially for those containing a relatively long 5'-UTR and function as important regulators for cell growth and stress responses (30). These mRNAs include c-myc (42), p53 (43), vascular endothelial growth factor (44), fibroblast growth factor 2 (45), platelet-derived growth factor B/c-sis (46), hypoxia-inducible factor-1 (47), death-associated protein 5 (33), apoptotic protease activating factor (48), X-linked inhibitor of apoptosis (31), and others. The IRES-dependent translation of the growth factors confers a survival advantage of the cells under hypoxia or stressed conditions, such as solid tumor cells undergoing chemotherapy. On the other hand, cells use the IRES-dependent translational machinery to express proteins functioning in apoptosis or cell cycle arrest to eliminate genetically damaged cells, or prevent accumulation of genomic damage. GADD45{alpha} has been implicated in cell cycle regulation and DNA damage repair. Thus, the IRES-dependent translation of the GADD45{alpha} protein might be theoretically beneficial for protecting normal cells from malignant transformation under extreme stress conditions. The final outcome of the IRES-dependent expression of the GADD45{alpha} protein in either normal cells or tumor cells, however, might be largely dependent on the integrity of other apoptotic and DNA repair machineries which act in concert with GADD45{alpha}. In addition, the fate of a cell following GADD45{alpha} induction is also dependent on whether the expressed GADD45{alpha} can be sufficient to override the tumorigenic signals that are induced simultaneously or asynchronously by the treatments.

Because IRES-dependent protein translation occurred mostly in the cells under circumstances of genotoxic stress, apoptosis, nutrient starvation, mitosis, or growth arrest, it is conceivable that manipulating the IRES pathway would alter the growth or survival status of the cell. Evidence supporting this notion is mainly from studies that show mutations of IRES elements or functional impairment of IRES binding proteins, the so-called IRES trans-acting factors in cancers or diseases associated with cancer susceptibility (49, 50). In cell lines derived from patients with neoplasia multiple myeloma, a single C to U transition in the IRES region of c-myc mRNA was found to be responsible for the enhanced IRES activity and the consequent increased expression of c-myc protein (49). The increase of c-myc protein obviously provides the cells with a growth advantage and elevated oncogenic potentials. On the other hand, a defect in the IRES machinery due to a genetic deficiency in a gene encoding pseudouridine synthase, dyskerin, has been linked to cancer predisposition, a result of decreased IRES-mediated expression of antiapoptotic protein, p27 (50). Currently, IRES-mediated protein translation has been implicated in the expression of both prosurvival and antisurvival factors. An interesting question to be asked is, therefore, whether different IRES machineries present in given cells mediate the expressions of proteins with unique functions in context with the type and duration of extracellular signals.

In summary, our data support the model that GADD45{alpha} protein translation is up-regulated by As3+, as well as UV radiation or taxol treatment, through the IRES element identified in the 5'-UTR of the GADD45{alpha} mRNA. Treatment of the cells with As3+, and possibly other stress inducers, compromises the function of the cap-dependent translational machinery, and thereafter favors IRES-dependent protein translation. The IRES-dependent translation of the GADD45{alpha} protein may not be limited to the cells in severe stress conditions, such as growth arrest in this study, but also in the cells with mild environmental insults. The regulation of the GADD45{alpha} protein on transcriptional, mRNA stability, and translational levels might be important to ensure that cells produce sufficient amounts of the GADD45{alpha} protein to reduce the possibility of improper chromosomal segregation and cell division.


   Acknowledgments
 
Grant support: Intramural research grant from the National Institute for Occupational Safety and Health/Centers for Disease Control and Prevention (F. Chen), partial support from the National Science Foundation of China (30471980, Q. Chang and Y. Zhang), and NIH 7RO1CA119028-02 (X. Shi).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Richard E. Lloyd at Baylor College of Medicine (Houston, TX), Jian-Ting Zhang at the Indiana University Cancer Center (Indianapolis, IN), and Daniel Haber at Massachusetts General Hospital Cancer Center (Boston, MA) for the dicistronic vectors and GADD45{alpha} promoter reporter constructs, respectively. We also thank Dr. Kristy J. Brown at CTL Bio Services (Rockville, MD) for her technical assistance in peptide analyses using reversed phase nano-LC-MS/MS.


   Footnotes
 
Note: Q. Chang, D. Bhatia, and Y. Zhang made equal contributions to this report.

Disclaimer: The opinions expressed in this article are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention.

4 http://www.ba.itb.cnr.it/BIG/UTRScan/ Back

Received 3/ 5/07. Revised 4/19/07. Accepted 4/25/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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