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B1
Department of Foods and Nutrition, Purdue University, West, Lafayette, IN 47907
2To whom correspondence should be addressed. E-mail: Teegarden{at}cfs.purdue.edu.
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONLITERATURE CITED |
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B (NFB) transcriptional activity was significantly increased 1.8-fold over vehicle controls by 1,25(OH)2D3 after 4 h of treatment. Protein kinase B/AKT, a downstream effector of phosphoinositide 3-kinase (PI3K), was activated 4-fold and 8-fold at 2 and 4 h, respectively, after treatment with 1,25(OH)2D3. Pretreatment with two PI3K inhibitors, LY294002 and wortmannin, abolished the activation of NFB by 1,25(OH)2D3, suggesting that this pathway is essential for NFB transcriptional activation. Additionally, the use of a p-21 activated kinase (PAK1) inhibitory construct (PAKR299) demonstrated that PAK1 was also required for NFB transcriptional activation by 1,25(OH)2D3. Inhibition of NFB activity with transfection of the NFB inhibitory construct (IBAla32) abolished the protective effect of 1,25(OH)2D3 on VES-mediated apoptosis. In summary, NFB transcriptional activation was essential to 1,25(OH)2D3 protection from VES-mediated apoptosis and 1,25(OH)2D3 regulated NFB activity through PI3K and PAK pathways.
KEY WORDS: • 1,25-dihydroxycholecalciferol • p21 activated kinase • nuclear factor B • phosphoinositide-3-kinase • C3H10T1/2 cells
The active form of vitamin D, 1,25-dihydroxycholecalciferol [1,25(OH)2D3],3 is important to the development and the maintenance of bone, the regulation of the immune system, and mineral homeostasis in the body (1). 1,25(OH)2D3 also affects cell-cycle progression, cell growth, differentiation, and the regulation of apoptosis (2–4). Although in some cell lines 1,25(OH)2D3 elicits a pro-apoptotic response (5,6), it also has been shown to protect cells from apoptosis (2,7).
A transcription factor integral to the processes of cell growth, differentiation, and the regulation of apoptosis is nuclear factor B (NFB). The expression of several anti-apoptotic factors, such as BclXL, c-myc, and the inhibitors of apoptosis proteins (IAP) are regulated by NFB. Direct regulation of NFB activity occurs through inhibitory proteins (IB), which bind NFB and sequester it in the cytosol (8). The IBs dissociate from NFB when phosphorylated by IB kinase (IKK), thus allowing nuclear translocation and activation of NFB responsive genes. A number of kinases activate IKK, such as AKT; MEK kinases 1, 2, and 3 (9); and NFB-inducing kinase (NIK) (10). 1,25(OH)2D3 has been shown to increase the activity of PI3K (11), an upstream activator of AKT. AKT activates p-21 activated kinase (PAK1) (12) that has been shown to stimulate the nuclear translocation of the NFB subunit, p65 (13), as well as phosphorylate and activate NIK (14).
1,25(OH)2D3 has been shown to modulate NFB activity in a variety of cell lines. For example, in Jurkat-T cells, 1,25(OH)2D3 treatment resulted in a decrease in the activation of an NFB-CAT reporter gene (15), and in A3 human melanoma cells stably transfected with the IL-8 promoter luciferase construct, 1,25(OH)2D3 inhibited activation of the reporter gene by 50% (16). 1,25(OH)2D3 was also shown to decrease the DNA binding of NFB subunits in human lymphocytes, through inhibition of the expression of the p50 NFB subunit and its precursor, p105 (15). In contrast, 1,25(OH)2D3, in combination with TPA, decreased the expression if IB and increased the concentration of nuclear NFB in NB4 acute promyelocytic leukemia cells (17). However, in this cell line, 1,25(OH)2D3 alone had no effect on NFB. Therefore, 1,25(OH)2D3 can potentially have different effects on the regulation of NFB based on the cell line itself and other intra- and extra-cellular influences, such as the presence or the absence of other NFB-regulating substances.
Vitamin E succinate (VES), a derivative of vitamin E lacking antioxidant properties, is being studied extensively for its chemoprotective and chemotherapeutic potential. VES has been shown to stimulate apoptosis in a wide variety of cell types. Although the mechanisms of apoptotic stimulation are unclear, FAS and FAS ligand have been implicated (18,19). It has also been demonstrated that elevated and prolonged expression of c-jun mRNA and protein is correlated with VES-induced apoptosis (20–24). Thus, VES-mediated apoptosis may be regulated by more than one mechanism.
Previous results from our laboratory demonstrated that 1,25(OH)2D3 inhibits VES-mediated apoptosis in untransformed C3H10T1/2 mouse fibroblast cells but not C3H10T1/2 cells transfected with the Harvey ras oncogene (7). The untransformed C3H10T1/2 and Harvey ras-transformed C3H10T1/2 cell, as defined by growth characteristics, are components of a model for multistage carcinogenesis (25). The parent C3H10T1/2 represents untransformed cells with growth characteristics similar to primary cells (26). It is important to understand the mechanism of 1,25(OH)2D3 regulation of apoptosis in untransformed as well as transformed cell lines to determine its effects during normal growth, as well as during cancer progression. The focus of this study was to determine whether 1,25(OH)2D3 regulates apoptosis in untransformed C3H10T1/2 cells through the activation of NFB, and to determine the roles of PAK1 and PI3K in NFB regulation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONLITERATURE CITED |
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B (Ala32) mutant plasmid was obtained from Dr. E. Taparowsky (Purdue University). This mutant construct has a serine to alanine mutation that inhibits its phosphorylation, as described by Brockman et al. (27). The pRL-CMV and pRL-TK luciferase internal control expression vectors were from Promega. The pNFB-luc reporter plasmid was obtained from Stratagene. This plasmid contains the luciferase reporter gene driven by a TATA box joined to tandem repeats of NFB binding elements (Stratagene Technical Manual cat. # 219077). The PAKR299, containing an Nru 1 site at amino acid 299, was a gift from Dr. J. Field (University of Pennsylvania School of Medicine). This construct is a kinase deficient PAK-1 mutant as described in Sells et al. (28) and Tang et al. (23). Cell culture. C3H10T1/2 (CCL-226) clone 8, murine fibroblast cells were used in all experiments. Cells were grown in DMEM with 10% FBS, 100 kU/L penicillin, and 0.1 g/L streptomycin in a humidified environment at 37°C with 5% CO2. Cells were maintained in linear growth and were used below passage 23.
Reporter assays.
C3H10T1/2 cells were plated in 60-mm dishes at a density of 70,000 cells per dish and were transiently transfected with 0.5 µg pRL-CMV or 0.2 µg pRL-TK, 1 µg pNFB-luciferase, or 0.5 µg PAK1 mutant using the Lipofectamine Plus system (Life Technologies, Gibco-BRL). At 18 h posttransfection, cells were treated with vehicle control (0.3% ethanol), 100 mg/L TNF
(as a positive control), or 1,25(OH)2D3 for indicated time points. Luciferase activity was assayed via the Dual Luciferase Assay (Promega) on a Lumat LB 9501 luminometer (Perkin Elmer Life Sciences) and was normalized for transfection efficiency using the cotransfected Renilla expression vector. Data are expressed as relative luciferase units with treated samples over vehicle controls.
Assessment of apoptosis.
Apoptosis was assessed using the Cell Death Detection ELISAPLUS Assay (Boehringer Mannheim). This assay is a photometric enzyme-linked immunoassay that quantitatively measures the internucleosomal degradation of DNA, which occurs during apoptosis. Specifically, the assay detects histone associated mono- and oligonucleosomes, which are indicators of apoptosis. C3H10T1/2 cells were plated in 60-mm dishes at a density of 70,000 cells per dish and were transiently transfected with 2 µg of the IB(Ala32) mutant plasmid by the Lipofectamine Plus method. Cells were treated with vehicle control (100% ethanol; 0.3% final concentration), 30 mg/L VES, or a combination of 30 mg/L VES and 100 nmol/L 1,25(OH)2D3 for indicated times. After treatments, nonadherent cells were collected and pelleted at 200 x g for 10 min. The supernatant was discarded; the cell pellet was washed with cold CMF-PBS and was re-centrifuged. Adherent cells were washed with cold CMF-PBS (137 mmol/L sodium chloride, 1.5 mmol/L potassium phosphate, 7.2 mmol/L sodium phosphate, 2.7 mmol/L potassium chloride, pH 7.4), trypsinized, collected, and combined with nonadherent cells into a total of 1 mL DMEM. Both live and dead cells were then counted via trypan blue exclusion (Pierce), and an equal number of cells were added to the microtiter plate for all treatment groups, and apoptosis assay was performed according to the manufacturer’s instructions. Data are expressed as absorbance at 405 nm of each sample over vehicle controls.
Immunologic detection of proteins. C3H10T1/2 cells were plated in 100-mm dishes at a density of 105,000 cells per dish. Cells were treated with vehicle control (100% ethanol; 0.3% final concentration), 100 nmol/L 1,25(OH)2D3, 30 mg/L VES, or a combination of 30 mg/L VES and 100 nmol/L 1,25(OH)2D3 up to 24 h. After treatment, cells were rinsed with cold CMF-PBS. Cells were scraped and harvested into buffer (25 mmol/L HEPES, 150 mmol/L sodium chloride, 1% Triton, 5 mmol/L EDTA) containing protease inhibitor cocktail and lysed on ice for 15 min. Cell lysis was confirmed by trypan blue staining. Cells were then centrifuged at 500 x g, at 4°C for 15 min, and the supernatant was collected for analysis. The BCA protein assay (Pierce) was used to determine the protein concentration of each sample. Proteins were separated by SDS-PAGE, using 20 µg of protein per well. Proteins were then transferred to nitrocellulose membranes and were probed with phospho- or nonphospho-specific antibodies to AKT at a 1:1000 dilution. Bands were visualized via chemiluminescence using horseradish peroxidase-conjugated anti-mouse IgG (Imgenex). Bands were quantified using Biorad Quantity One software in conjunction with the Biorad Fluor S MultiImager system.
Statistical analysis.
Data were analyzed by either Student’s t test or one-way ANOVA, followed by Dunnett’s Multiple Range test ( = 0.05) with Statistical Analysis Software (SAS) as appropriate.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONLITERATURE CITED |
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B by 1,25(OH)2D3 in C3H10T1/2 cells.
To determine whether NFB is activated in response to 1,25(OH)2D3 treatment, NFB-luciferase activity was measured. Treatment with 1,25(OH)2D3 resulted in a significant 1.78-fold increase in NFB-luciferase reporter activity over vehicle controls at 4- and 0.86-fold at 6 h (Fig. 1). There was no induction of NFB-luciferase in vehicle controls at any time point (data not shown). Because of the significant activation of NFB-luciferase at 4 h, this time point was chosen for further studies.
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B through PI3K C3H10T1/2 cells.
Protein kinase B (AKT), a downstream effector of PI3K, has been shown to activate NFB. In the present study, 1,25(OH)2D3 treatment of C3H10T1/2 cells resulted in a significant increase in AKT activation at 2 h (4-fold) and 4 h (8-fold) compared with time zero (Fig. 2A,B), suggesting an increase in PI3K activity. The PI3K inhibitors LY294002 (Fig. 2C) or wortmannin (Fig. 2D), in separate experiments, prevented NFB-luciferase activation by 1,25(OH)2D3. These results demonstrate a role for the PI3K pathway in NFB transcriptional activation by 1,25(OH)2D3 in C3H10T1/2 cells.
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B in C3H10T1/2 cells.
PAK-1 has been shown to increase the transcriptional activity of NFB regulated genes by several mechanisms (13,14). Because 1,25(OH)2D3 increases PI3K activity and PI3K activates PAK-1, the role of PAK-1 in 1,25(OH)2D3 activation of NFB was explored. Transfection with PAKR299 resulted in the inhibition of 1,25(OH)2D3 mediated increase in NFB transcriptional activity (Fig. 3). These results demonstrate that NFB activation by 1,25(OH)2D3 in C3H10T1/2 cells requires PAK-1 activity.
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B inhibitory construct (IBAla32) prevented activation of NFB-luciferase activity when cotransfected in C3H10T1/2 cells (data not shown). In mock-transfected cells, 1,25(OH)2D3 significantly inhibited VES-mediated apoptosis by 60% compared with cells treated with VES alone as assessed by ELISA (Fig. 4). However, there was no difference between apoptosis in cells treated with VES or the combination of VES and 1,25(OH)2D3 in cells transfected with the IBAla32 mutant plasmid. These results demonstrate that the NFB transcriptional activation is required for 1,25(OH)2D3 inhibition of apoptosis in C3H10T1/2 cells.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONLITERATURE CITED |
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B. In this study, we investigated transcriptional activation of NFB by 1,25(OH)2D3, its role in the inhibition of apoptosis and the role of PI3K and PAK-1 in NFB regulation. These results show that 1,25(OH)2D3 activates NFB transcription in C3H10T1/2 cells and that this activation is essential for protection from VES-mediated apoptosis.
To our knowledge, this is the first report of 1,25(OH)2D3 alone increasing the transcriptional activity of NFB. A similar report by Berry et al. (17) showed that 1,25(OH)2D3 induced phosphorylation of the IB proteins in NB4 leukemia cells; however, a decrease in IB protein expression required cotreatment with TPA. The authors of this report suggested that although 1,25(OH)2D3 induced IB phosphorylation in NB4 cells, degradation of the IB protein required the action of TPA. Our study results differ from those of Berry’s group in that activation of NFB transcription was achieved by 1,25(OH)2D3 alone in C3H10T1/2 murine fibroblast cells. Cell-cycle regulatory proteins that have been shown to have NFB binding sites in their promoters include p27/KIP1 (30), cyclin D1 (31), and c-myc (32). 1,25(OH)2D3 has been shown to regulate the cell cycle via these 3 proteins (33,34). Therefore, it is possible that the protection from apoptosis by 1,25(OH)2D3 observed in the C3H10T1/2 cell line could be mediated through an inhibition of proliferation involving NFB regulation of p27/KIP1, cyclin D1, and/or c-myc.
Previous results in our laboratory demonstrate the anti-apoptotic response of C3H10T1/2 cells occurs in the untransformed C3H10T1/2 cells but not in cells of the same lineage that have been transfected with the Harvey ras oncogene (7). In the ras-transfected C3H10T1/2 cells (7) and in keratinocytes (35), the classic nuclear vitamin D receptor (nVDR) transcriptional response to 1,25(OH)2D3 is attenuated; therefore, signals through this pathway may not be functional in cells through all stages of carcinogenesis. Alternatively, 1,25(OH)2D3 also acts through a rapid signaling receptor, the putative membrane associated rapid response receptor, and the signals elicited by 1,25(OH)2D3 in this manner may involve cross-talk with the nVDR. Dependent on the functionality of the receptors for 1,25(OH)2D3 in different cell models, differential responses are likely to be elicited. It is important to understand the mechanism underlying the regulation of NFB by 1,25(OH)2D3 in different cell types to clarify the circumstances that elicit differential responses.
Both PI3K and PAK-1 activities are required for activation of NFB by 1,25(OH)2D3 in C3H10T1/2 cells. These results are similar to other investigations that demonstrate a dependence of NFB transcriptional activation on PI3K. Results of several studies have shown that TNF-induced IKK phosphorylation requires PI3K and AKT (36–39). IKK activates NFB through the phosphorylation of IB inhibitory proteins and also through the direct phosphorylation of the p65 subunit of NFB, which enhances its association with coactivators of transcription. Similarly, PAK1 has been shown to stimulate the nuclear translocation of the NFB subunit, p65 (13), as well as phosphorylate and activate NIK (14) and may be the mechanism through which 1,25(OH)2D3 regulates NFB activity in C3H10T1/2 cells. Further research is needed to clarify the interactive signaling pathways of PI3K and PAK1 in response to 1,25(OH)2D3.
Thus, 1,25(OH)2D3 regulation of NFB transcriptional activity is required for protection of C3H10T1/2 cells from VES-mediated apoptosis. The mechanism by which 1,25(OH)2D3 regulates NFB transcriptional activity requires both the PI3K and PAK1 pathways in this cell line. Understanding these mechanisms will lead to clarification of the role of 1,25(OH)2D3 in the regulation of apoptosis during different stages of carcinogenesis.
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FOOTNOTES |
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3 Abbreviations used: 1,25(OH)2D3, 1,25-dihydroxycholecalciferol; IAP, inhibitors of apoptosis; IKK, IB kinase; NFB, nuclear factor B; NIK, NFB inducing kinase; nVDR, nuclear vitamin D receptor; PAK-1, p21 activated kinase; PI3K, phosphatidylinositol 3-kinase; TPA, phorbol 12-myristate 13-acetate; VES, vitamin E succinate.
Manuscript received 24 May 2004. Initial review completed 22 June 2004. Revision accepted 26 August 2004.
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