1 Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011-1061 and
2 Eppley Institute for Research in Cancer and Allied Diseases and
3 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, 68198-6805, USA
4 Joint first authors
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Abstract |
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Abbreviations: ANOVA, analysis of variance; CCS, corticosterone; DER, dietary energy restriction; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; GR, glucocorticoid receptor; HBSSE, Hank's balanced salt solution containing 0.08% EDTA; IGF-I, insulin-like growth factor I; PKC, protein kinase C; PMSF, phenylmethylsulfonyl fluoride; TPA, 12-O-tetradecanoylphorbol 13-acetate; TRE, TPA response elements.
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Introduction |
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DER may exert an inhibitory effect on the protein kinase C (PKC) signal transduction pathway. Birt et al. have shown that a 40% energy restricted diet significantly inhibits 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced activation of the TPA-responsive PKC isoform (3,4) and inhibits ERK protein levels and activities (5). Whether DER-induced inhibition of c-jun message is mediated through PKC cannot be determined from these results. However, Finkenzellar et al. showed that over-expression of PKC
in NIH3T3 cells stimulates over-expression of c-jun, suggesting a relationship between the
isoform and c-Jun (6). In order to examine whether DER may result in an inhibition of targets downstream from ERK, the present study examined the effect of DER on AP-1DNA binding and expression of c-Jun protein and c-jun mRNA in an in vivo SENCAR mouse skin tumor promotion model.
Since AP-1 activation has been shown to be important in tumor promotion (7) and progression (8,9) in mouse keratinocytes, we hypothesized that DER may inhibit tumor promotion by down-regulating AP-1. Also, considering the in vivo data that (i) the adrenal glands were important for inhibition of TPA-induced papilloma formation by food restriction in the two-stage CD-1 mouse tumorigenesis model (10), (ii) DER was associated with increased concentrations of the glucocorticoid hormone corticosterone (CCS) in mice (11,32) and (iii) glucocorticoid hormones were potent inhibitors of skin tumorigenesis (12,13), we hypothesized that adrenalectomy should reverse the effects of DER on AP-1 and that supplementation of CCS to adrenalectomized mice should prevent this reversal.
Because dietary restriction reduces insulin-like growth factor I (IGF-I) concentrations in p53-deficient mice (17) and because of the evidence that IGF-I mediates the anticarcinogenic effects of caloric restriction (18), this study also examined the effects of DER and CCS on blood plasma IGF-I concentrations in the in vivo SENCAR mouse skin tumorigenesis model system.
Consequences of DER on AP-1 were characterized by measuring DNA-binding ability, c-Jun protein and c-jun message because the AP-1 transcription factor is composed of a Jun protein complexed with another Jun protein or with a Fos protein to form the Jun/Jun or Jun/Fos transcription factor, which acts as a positive or negative regulatory factor through binding to TPA response elements (TRE) on AP-1-responsive genes (14,15). The c-jun gene contains AP-1 sites upstream of the promoter region and c-jun expression was positively auto-regulated through binding of the AP-1 transcription factor to TRE sites on c-jun (16).
Thus, this study examined the time course of effects of DER on c-jun mRNA and c-Jun protein and, in addition, investigated the consequences of TPA, adrenalectomy and CCS supplementation of adrenalectomized mice on AP-1DNA binding and blood plasma IGF-I concentrations in SENCAR mouse epidermis in vivo.
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Materials and methods |
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On the day of killing mice were treated with 3.2 nmol TPA in 200 µl acetone or with 200 µl acetone alone.
Adrenalectomy
Surgeries on shaved mice were performed between 7:00 and 10:00 a.m. to minimize variability in CCS concentrations. The mice were i.p. injected with pentobarbital (75 mg/kg) ~20 min prior to surgery. The anaesthetised mice were swabbed with 70% ethanol and the dorsal skin was cut and the fascia opened to reveal the kidney. The adrenal glands and surrounding fatty tissue were removed and the wound closed with wound clips. After surgery adrenalectomized mice were given 60 µg/ml CCS or saline in ethanol vehicle (final concentration 0.6% in drinking water) and 40% energy restricted diets were begun 2 weeks later. This CCS treatment was previously demonstrated to mimic the CCS elevation observed in DER mice (32).
Isolation of epidermal nuclear proteins
Nuclear proteins were isolated from shaved SENCAR mouse dorsal skin treated with 3.2 nmol TPA or acetone for 4 h using a method modified from that previously described (23). The animal was killed and the skin from each mouse was excised into iced Hank's balanced salt solution (5.4 mM KCl, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 1.3 mM CaCl2) containing 0.08% EDTA (HBSSE). All subsequent steps were performed on ice or at 4°C unless otherwise noted. The fat layer of the dermis was scraped off with a scalpel and the skin was incubated for 45 min at 37°C in HBSSE containing 0.25% trypsin with the fur side up. Trypsinization was stopped by addition of iced HBSSE containing 0.1% trypsin inhibitor. Epidermal cells were scraped from the fur side using a single edge razor, chopped with scissors for 5 min and stirred for 30 min. The epidermal scrapings were filtered through a 100 µm cell strainer (Fisher Scientific, Pittsburgh, PA) and centrifuged at 1850 g for 15 min. The cell pellet was washed with phosphate-buffered saline, resuspended in five times the packed cell volume of 10 mM HEPES, 10 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5 mM dithiothreitol (DTT) and swollen on ice for 10 min. The swollen cells were homogenized with 10 strokes of a Dounce tissue grinder (Fisher Scientific, Pittsburgh, PA) using a type B pestle and centrifuged at 3300 g for 15 min. The nuclear pellet was resuspended with 1x packed nuclear volume of 20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 0.2 mM EDTA, 0.5 mM PMSF and 0.5 mM DTT, placed on an end-over-end rotator for 15 min and centrifuged for 10 min at 25 000 g. The supernatant containing epidermal nuclear proteins was diluted 1:6 with 20 mM HEPES, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF and 0.5 mM DTT and an aliquot was removed for protein quantitation with a bicinchoninic acid BCA kit (Sigma, St Louis, MO).
Electrophoretic mobility shift assay (EMSA)
The AP-1 oligonucleotide probe was labelled by incorporation of [-32P]ATP (>7000 mCi/mmol; ICN, Costa Mesa, CA) into the AP-1 oligonucleotide sequence
5'-CGC TTG ATG AGT CAG CCG GAA-3'
3'-GCG AAC TAC TCA GTC GGC CTT-5'
using a gel shift kit (Promega, Madison, WI) according to the manufacturer's instructions. The DNAprotein binding reaction was performed by incubating 100 000200 000 c.p.m. of probe with 15 µg mouse epidermal nuclear extract and 0.5 µg poly(dI·dC) (Amersham Pharmacia Biotech, Piscataway, NJ) at room temperature for 12 h. AP-1DNA complexes were separated by gel electrophoresis in 1x TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA) on a pre-run 5% polyacrylamide gel. Following electrophoresis, the gel was dried and exposed to a phosphor screen overnight before scanning with a phosphorimager (Molecular Dynamics, Sunnyvale, CA). Radioactivity of shifted bands was quantitated with ImageQuant software (Molecular Dynamics).
Western blot analysis
Whole cell mouse epidermal proteins were extracted as previously described (11) and the protein content of each lysate was determined by the bicinchoninic acid and copper sulfate protein assay (Sigma). Western blot analysis of c-Jun protein was carried out as previously described (11) using a 1:1000 dilution of anti-c-Jun mouse monoclonal and rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and a 1:5000 dilution of anti-mouse and anti-rabbit IgG, conjugated to alkaline phosphatase (Sigma).
Positive immunoreactivity was determined by immunoprecipitating c-Jun protein from epidermal lysates or from proliferating mouse L cells with a rabbit polyclonal antibody and detecting the protein on nitrocellulose membranes with a mouse monoclonal anti-c-Jun antibody. Specificity was also verified by electrophoresis of a commercially available lysate known to contain c-Jun (Transduction Laboratories, Lexington, KY) in the same gel with the experimental lysates.
C50 mouse keratinocytes, derived by Kulesz-Martin et al. (20) and provided by Dr Jill Pelling (University of Kansas Medical Center, Kansas City, KS), were brought to confluence in Dulbecco's modified Eagle's medium with 5% CO2 in a temperature and humidity controlled environment and serum deprived overnight to inhibit proliferation, as expression of c-Jun has been shown to be inversely related to cellcell contact (21). Whole cell lysates were harvested by scraping confluent cultures into lysis buffer as described above for use as a negative control in western blot analysis. The value of the negative control was also determined by electrophoresis of an irrelevant peptide, IGF-II protein, in the same gel with the immunoprecipitated positive lysates.
RNA and DNA probes
c-jun antisense probes were synthesized from a 250 bp template (Ambion, Austin, TX) and labelled with [-32P]UTP (NEN Corp., Boston, MA) using T7 RNA polymerase (Ambion). AP-1 (c-jun) DNA probes were synthesized from the consensus sequence 5'-CGCTTGATGAGTCAGCCGGAA-3' (Trevigen) and end-labeled with [
-32P]ATP (Amersham, Arlington Heights, IL).
Northern blot analysis
Total cellular RNA was isolated from mouse skin by guanidinium thiocyanate/phenol/chloroform extraction as described by Chomczynski and Sacchi (22). The isolated RNA was electrophoresed in 1.2% agarose gels at 100 V for 3 h, transferred to Nytran membranes (Schleicher & Schuell, Keene, NH) and hybridized to radiolabelled antisense riboprobes against c-jun (Ambion). Hybridization was carried out overnight at 65°C in NorthernMax hybridization buffer containing 50% formamide (Ambion). The hybridized blots were washed in a final wash solution of 0.1x SSC and 0.1% SDS at 5560°C for 20 min. The membranes were scanned with a PhosphorImager (Molecular Dynamics) and exposed to Kodak X-Omat XAR film (Kodak, Rochester, NY) using an intensifying screen.
Blood plasma corticosterone and IGF-I measurements
Mice were killed by decapitation and trunk blood was collected in iced heparinized glass tubes. After isolating blood plasma, radioimmunoassay kits were used to measure CCS concentrations (ICN Biomedical) and IGF-I concentrations (Nichols Institute Diagnostics) in blood plasma following the instructions of the manufacturer.
Statistics
Changes in body weight over time were analyzed by single factor analysis of variance (ANOVA). Analysis of the level of immunoreactive c-Jun protein and c-jun RNA by densitometry proceeded as follows. Bands (at exposures below saturation) corresponding to TPA-induced c-Jun for each diet treatment group at each time point were compared with acetone-treated controls from the same blot. The resulting ratio of values was analyzed for differences between groups by single factor ANOVA and differences between means were tested by t-test. For EMSA the logarithm of the radioactivity of the shifted band was analyzed for differences between groups using ANOVA, as these values were more nearly normally distributed, and differences between means were tested by t-test of the least squares means.
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Results |
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Single factor ANOVA of pooled values across diet and surgery groups showed that at 4 h after treatment with TPA a significant 2-fold induction in AP-1DNA binding was observed over acetone controls (P < 0.0001, n = 68/group). Similarly, single factor ANOVA of pooled values across adrenalectomy, CCS-supplemented and TPA-treated groups showed that DER significantly reduced AP-1DNA binding to half that of ad libitum fed mice (P < 0.0001, n = 68/group). Individual t-test comparisons showed that DER significantly reduced (i) basal and TPA-induced AP-1DNA binding levels (P < 0.002) in sham-operated mice and (ii) basal levels in adrenalectomized groups (P < 0.001) in comparison with ad libitum fed mice (Figure 3). Adrenalectomy significantly increased TPA-induced AP-1DNA binding in DER mice (P < 0.05) relative to sham-operated controls and restored this binding activity in TPA-treated, energy restricted mice to levels not significantly different from that of TPA-treated, ad libitum fed, sham-operated mice (Figure 3
). In ad libitum fed, adrenalectomized mice supplemental CCS reduced basal levels of AP-1DNA binding relative to the vehicle control group (P < 0.005). In DER mice supplementation of adrenalectomized mice with CCS returned basal and TPA-induced AP-1DNA binding to that of sham-operated mice (Figure 3
). Thus, inhibition of AP-1DNA binding by DER in nuclear extracts of SENCAR mouse epidermis treated with acetone or TPA for 4 h was reversed by adrenalectomy and the effects of adrenalectomy on AP-1DNA binding were prevented by supplementation with CCS.
Effect of DER on c-Jun protein levels
The level of c-Jun protein from mouse epidermis following induction with TPA, relative to acetone-treated controls, was determined by western blotting using anti-c-Jun antibodies. The results of a single western blot experiment with one animal used for each treatment are shown in Figure 4. A summary of the results of western blots representing 38 observations of individual animals per treatment group per time point is shown in Figure 5
. Statistical analysis of the data in Figure 5
revealed a significant TPA induction of c-Jun in control fed mice of 3.7-fold over basal acetone-treated mice at 4 h (P < 0.05) and of 3-fold at 6 h (P < 0.01) post-TPA.
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A single factor ANOVA showed that the mean level of c-Jun protein detected in DER groups over time was significantly less than that detected in the control groups (P < 0.01). The level of epidermal c-Jun protein detected in acetone-treated mice at each time point in both diet groups was not significantly different from time 0 controls. These results suggest that TPA induction of c-Jun protein in DER mouse epidermis is diminished compared with the induction seen in control fed animals.
To assess the effect of the adrenal gland on DER-mediated inhibition of c-Jun protein induction, western blotting was performed on epidermal lysates from control fed and DER mice that were sham-operated or adrenalectomized and killed at 0, 1, 3 and 6 h following treatment with acetone or TPA. The results in TPA-treated, adrenalectomized mice, compared with acetone-treated, adrenalectomized mice showed an overall mean pooled value at 1, 3 and 6 h for c-Jun induction of 167 ± 58% in the TPA-treated groups (n = 4 per group) irrespective of diet. Thus, adrenalectomy eliminated the DER reduction in c-Jun protein induction. Single factor ANOVA of pooled values across time showed that values for amount of c-Jun in adrenalectomized mice were significantly less than sham-operated controls (P < 0.01, n = 4 per group). TPA induction values for c-Jun in sham-operated control fed and DER mice were comparable to values seen in intact DER animals (see Figure 5).
Effect of DER on c-jun mRNA levels
The level of c-jun mRNA in mouse skin was determined as the relative density of bands from each diet group at each time point compared with the density of the acetone-treated time 0 control on the same blot. The results of an example northern blot with one animal used for each treatment are shown in Figure 6. Figure 7
shows a summary graph of northern blots representing 36 observations per diet group per time. Note that the percentages of normalized densities shown in Figure 6
are compared with the single acetone-treated control of this figure and may not reflect the average percentages for n = 36, as in Figure 7
.
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Effect of DER and CCS supplementation on CCS and IGF-I concentrations
Blood plasma CCS concentrations, shown in Figure 8, were unaffected by TPA at 4 h after treatment in all groups. DER increased blood plasma CCS concentrations by almost 5-fold over ad libitum fed mice (P < 0.05) in sham-operated animals. This elevation was eliminated in all adrenalectomized animals. In DER mice adrenalectomy significantly decreased CCS concentrations to about one-third that of sham-operated mice (P < 0.05). CCS concentrations of adrenalectomized DER mice supplemented with CCS were not significantly different from adrenalectomized mice, perhaps because mice were killed in the early morning when the mice do not usually drink the supplemented water. This time was chosen specifically because CCS measurements from samples collected from intact mice in the early morning hours were the least variable among those collected at other times throughout the day (32). However, since CCS in adrenalectomized CCS-supplemented mice is dependent on recent fluid intake and mice primarily eat and drink during the dark phase, these data probably do not reflect peak CCS concentrations in the adrenalectomized CCS-supplemented groups. As shown in Figure 9
, blood plasma IGF-I concentrations were unaffected by CCS or TPA at 4 h after treatment but were significantly reduced by DER, on average, to about one-half that of ad libitum fed mice (P < 0.05).
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Discussion |
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Our investigation clearly demonstrated that DER significantly reduced AP-1DNA binding (Figure 3) and c-Jun protein levels (Figure 5
) in TPA-treated mouse epidermis. Both c-Jun protein levels and AP-1DNA binding were found to be ~2-fold greater in ad libitum fed mice than in DER mice at 4 h after TPA. This suggests that the decrease in AP-1DNA binding activity in DER mice may be a consequence of lower levels of c-Jun protein. To determine whether the effect of DER on c-Jun protein was a consequence or a cause of the reduction in AP-1DNA binding, we measured TPA-induced c-jun mRNA levels in response to DER. This was important since AP-1 transcriptionally regulates the c-jun gene. DER significantly blocked c-jun mRNA induction as early as 1h following TPA treatment, while TPA induction of AP-1DNA binding required 4 h treatment. Thus, the DER reduction in mRNA may have caused the subsequent decreases in c-Jun protein and AP-1DNA binding. Furthermore, unlike the results with c-jun mRNA and c-Jun protein, DER reduced the basal (acetone-treated) level of AP-1DNA binding without affecting the ratio of TPA-induced AP-1DNA binding to acetone-treated AP-1DNA binding levels, suggesting that DER may act at both the transcriptional and post-translational levels to down-regulate AP-1.
Supporting Pashko's finding that glucocorticoid hormones are important in DER inhibition of tumorigenesis (10), we showed that adrenalectomy reversed the effect of DER on c-Jun protein and AP-1DNA binding and that DER significantly increased circulating CCS concentrations above those observed in ad libitum fed mice. Furthermore, supplementation of adrenalectomized mice with CCS resulted in reversion of AP-1DNA binding to near that of ad libitum fed, sham-operated mice. Previous experiments from this laboratory assessed the potential role of DER-related increases in glucocorticoid hormone and potential activation of the glucocorticoid receptor (GR) (11). However, these studies did not provide evidence of an increase in activated GR that could interact with and inhibit AP-1DNA binding activity (11). Unfortunately, only indirect measures of GR activation could be used in the animal model employed and thus an endogenous GRAP-1 interaction may still be present and could contribute to the overall effect seen in DER mice of a reduction in AP-1DNA binding.
Inhibition of c-jun and AP-1 induction by DER and CCS are expected to be important in the prevention of tumorigenesis by DER. In fact, results of a current study indicate that DER decreased the incidence of papillomas and carcinomas and, while adrenalectomy reversed this DER effect, the loss of inhibition by DER in adrenalectomized mice was re-established upon supplementation with CCS (unpublished data). This, together with the presented data, supports a role for CCS and AP-1 in the mechanism of DER inhibition of carcinogenesis in vivo.
The fact that adrenalectomy with or without supplementation with CCS did not eliminate differences between DER mice and controls with respect to AP-1DNA binding indicates that other factors contribute to the DER effects observed. One such factor may be IGF-I, which has been shown to be nutritionally regulated and reduced by underfeeding (for reviews see refs 27,28). IGF-I has also been shown to modulate diet restriction-induced inhibition of mononuclear cell leukaemia in rats (29) and dietary restriction-induced inhibition of bladder cancer progression in p53-deficient mice (17). Supporting the potential importance of nutritional regulation of IGF-I in cancer, we have shown that IGF-I was significantly decreased by DER and unaffected by TPA treatment or glucocorticoid modulation in SENCAR mouse epidermis.
Since heterodimers of the various isoforms of Jun and/or Fos comprise the AP-1 transcription factor, the effect of DER and/or adrenalectomy on tumor promoting events may be influenced by the other constituent proteins of AP-1 (i.e. Jun D, Jun B, c-Fos, Fos B, Fos D, Fra-1 and Fra-2). Reports supporting this include: (i) induction of c-fos mRNA by hepatic regeneration was decreased by reduction of caloric intake in Fischer rats (30) and (ii) Jun B, Jun D and Fos B were identified as the major AP-1 components in mouse keratinocytes treated with the tumor promoter okadaic acid (31). Thus, investigations of the effect of DER on the levels and phosphorylation of AP-1 constituent proteins are in progress.
Taken together, our data suggest that DER may reduce skin cancer by inhibiting AP-1 transcriptional activation, in part through a glucocorticoid-mediated mechanism. Further examination of the mechanisms of the in vivo DER effect on AP-1 is warranted.
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Notes |
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Acknowledgments |
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References |
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