Inhibition of phorbol ester-induced AP-1–DNA binding, c-Jun protein and c-jun mRNA by dietary energy restriction is reversed by adrenalectomy in SENCAR mouse epidermis

Joseph Przybyszewski1,4, Ann L. Yaktine2,3,4, Ellen Duysen2, Darcy Blackwood2, Weiqun Wang1, Angela Au1 and Diane F. Birt1,2,3,5

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to determine the effects of 40% dietary energy restriction (DER) relative to ad libitum feeding on AP-1–DNA binding and expression of c-Jun protein and c-jun mRNA in SENCAR mouse skin treated with acetone or 12-O-tetradecanoylphorbol 13-acetate (TPA). The role of the glucocorticoid hormone corticosterone (CCS) was investigated by adding CCS or vehicle control to the drinking water of adrenalectomized mice. AP-1–DNA binding, measured by electrophoretic mobility shift assay, showed that TPA treatment for 4 h increased AP-1–DNA binding by 2-fold over acetone controls (P < 0.05) and that DER reduced basal and TPA-induced AP-1–DNA binding in comparison with ad libitum fed groups in sham-operated mice (P < 0.05). TPA treatment increased c-Jun protein levels in control fed mice (4-fold) and in DER mice (2-fold) over basal levels 4 h post-treatment (P < 0.05). Analyzed over all groups, DER reduced c-Jun protein levels (P < 0.01) and this effect was reversed by adrenalectomy. TPA induction of c-jun mRNA was also reduced by DER compared with ad libitum fed mice (P < 0.05). Adrenalectomy and CCS supplementation demonstrated that the effects of DER on AP-1–DNA binding were mediated in part by CCS. Measurement of blood plasma CCS concentrations showed that: (i) DER increased CCS 5-fold over ad libitum fed mice in sham-operated animals (P < 0.05); (ii) adrenalectomy decreased CCS over sham-operated mice (P < 0.05); (iii) TPA treatment had no effect on CCS. Blood plasma IGF-I concentrations were unaffected by CCS modulation or TPA treatment but were decreased by DER compared with ad libitum fed mice (P < 0.05). Thus, dietary energy restriction may inhibit cancer mechanistically by reducing overall AP-1 transcription through a process that is mediated in part by glucocorticoid hormones.

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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our laboratory previously demonstrated that a 40% reduction in dietary caloric intake, compared with ad libitum fed mice, reduced the number of tumors by 50–85% (1) and that this dietary energy restriction (DER) effect occurred during the promotion stage of the SENCAR mouse tumorigenesis model (2). The molecular mechanisms for this preventive dietary effect are not understood.

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 {alpha} 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{alpha} in NIH3T3 cells stimulates over-expression of c-jun, suggesting a relationship between the {alpha} 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-1–DNA 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-1–DNA binding and blood plasma IGF-I concentrations in SENCAR mouse epidermis in vivo.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and diet
Female SENCAR mice, aged 6–8 weeks, were purchased from NIH (Frederick, MD). The mice were housed and fed as previously described (5,19). Ingredients for the diets were obtained from Teklad Premier Laboratory Diets (Madison, WI) and were mixed and pelleted using a California Pellet Mill (Crawfordsville, IN). Control and 40% energy restricted diet pellets were stored at –20°C and were used within 1 month.

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 [{gamma}-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 DNA–protein binding reaction was performed by incubating 100 000–200 000 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 1–2 h. AP-1–DNA 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 cell–cell 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 [{gamma}-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 [{gamma}-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 55–60°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.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Body weights
Body weights for ad libitum and DER mice over 14 weeks of feeding are shown in Figure 1Go. Control mice, fed the AIN 93 formulation ad libitum for 14 weeks of experimental feeding, gained an average of 15% of their body weight and weighed an average of 37.7 ± 2.0 g at week 14. Mice fed the 40% energy restricted diet lost an average of 17% of their body weight during this time and at week 14 they weighed an average of 26.9 ± 1.1 g, significantly less than that seen in control animals (P < 0.01).



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Fig. 1. Body weights of SENCAR mice. Values are mean (± SD) total body weights analyzed by single factor ANOVA in SENCAR mice maintained on experimental diet for 14 weeks. At 14 weeks of experimental diet administration body weights were significantly higher in control than in DER mice (P < 0.01); n = 14 per treatment group.

 
Effect of DER and CCS on AP-1–DNA binding
The AP-1–DNA binding ability of nuclear extracts from ad libitum fed and DER mouse epidermis treated with acetone or TPA for 4 h was assessed by EMSA. This experiment addressed whether DER would result in decreased AP-1–DNA binding and if this effect would be lost in adrenalectomized mice and if it would be restored with CCS supplementation. Results from an example EMSA experiment are shown in Figure 2Go and the results for 6–8 observations per treatment group are shown graphically in Figure 3Go.



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Fig. 2. An example image of EMSA of AP-1–DNA binding in mouse epidermal nuclear extracts. The EMSA binding reaction consisted of 15 µg epidermal nuclear extract incubated with 0.5 µg poly(dI·dC) and ~100 000 c.p.m. radiolabelled AP-1 probe and was separated on a 5% polyacrylamide gel in 1x TBE. Lanes 1–12 each contain epidermal nuclear extract from one mouse in each of the 12 experimental groups loaded in order to compare the effects of acetone versus TPA; lane 13, radiolabeled AP-1 oligonucleotide probe + poly(dI·dC); lane 14, TPA nuclear extract with 100-fold excess of unlabelled AP-1 oligonucleotide probe; lane 15, TPA nuclear extract with 100-fold excess of unlabeled non-specific AP-2 oligonucleotide. AL, ad libitum fed; DER, dietary energy restricted; CCS, corticosterone supplemented.

 


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Fig. 3. Effects of TPA, CCS and dietary energy restriction on AP-1–DNA binding. Values for radioactivity of the AP-1–DNA shifted band from EMSA analysis of nuclear extracts from SENCAR mouse skin at 4 h after treatment with 3.2 nmol TPA or acetone are arbitrary pixel units and expressed as the means (± SE) for n = 6–8 per group as derived from ANOVA on natural log transformed data. Two data points from the AL/adx/TPA group were eliminated as outliers from the normal distribution of data on a plot of residual versus predicted values. Values that do not share italicized letters are significantly different by t-test of the least squares means following ANOVA (P < 0.05). AL, ad libitum fed; DER, dietary energy restricted; sham, sham operated; adx, adrenalectomized; ccs, 60 µg/ml corticosterone in ethanol in the drinking water.

 
To verify the specificity of DNA binding, competition experiments were performed using an excess of unlabeled AP-1 oligonucleotide, which bound to the labeled AP-1 site and nearly eliminated the radioactivity of the shifted band (Figure 2Go, lane 14), and an excess of unlabeled non-specific AP-2 oligonucleotide, which did not bind the AP-1 site and had little or no effect on the radioactivity of the shifted band (Figure 2Go, lane 15).

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-1–DNA binding was observed over acetone controls (P < 0.0001, n = 6–8/group). Similarly, single factor ANOVA of pooled values across adrenalectomy, CCS-supplemented and TPA-treated groups showed that DER significantly reduced AP-1–DNA binding to half that of ad libitum fed mice (P < 0.0001, n = 6–8/group). Individual t-test comparisons showed that DER significantly reduced (i) basal and TPA-induced AP-1–DNA 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 3Go). Adrenalectomy significantly increased TPA-induced AP-1–DNA 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 3Go). In ad libitum fed, adrenalectomized mice supplemental CCS reduced basal levels of AP-1–DNA 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-1–DNA binding to that of sham-operated mice (Figure 3Go). Thus, inhibition of AP-1–DNA 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-1–DNA 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 4Go. A summary of the results of western blots representing 3–8 observations of individual animals per treatment group per time point is shown in Figure 5Go. Statistical analysis of the data in Figure 5Go 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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Fig. 4. An example image of a western blot of TPA-induced immunoreactive c-Jun protein in epidermal lysates prepared from SENCAR mouse skin. C, ad libitum fed; ER, dietary energy restricted. Positive control lysates were immunoprecipitated with anti-c-Jun polyclonal antibody and detected with anti-c-Jun monoclonal antibody. Negative control lysates were prepared from confluent cultures of serum-starved C-50 mouse keratinocytes. 39 kDa indicates migration of the c-Jun protein band.

 


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Fig. 5. Expression of epidermal c-Jun protein over time from TPA-treated, ad libitum fed and DER mice. Band density was measured by scanning densitometry with TPA-treated control and DER densities expressed as a percentage of acetone-treated, ad libitum fed controls. Values are mean (± SEM) immunoreactive c-Jun protein, detected at specified time points by western blot analysis of mouse epidermal lysates. The ratio of the density of DER bands compared with acetone controls was tested by single factor ANOVA and t-test. *, Values that are significantly different from acetone controls (P < 0.05), n = 3–8 observations per treatment group at each time point.

 
The onset of c-Jun protein induction by TPA agrees with the findings of Kennard et al. of a maximum c-Jun induction of ~4-fold over basal levels between 4 and 6 h in TPA-treated, tumor promotion-sensitive (SSIN and SENCAR) and tumor promotion-resistant (C57BL/6) mouse strains (24). In contrast, in the epidermis of DER mice c-Jun protein was only moderately increased and was significantly different from acetone-treated controls only at 4 h after TPA treatment (P < 0.05, Figure 5Go).

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 5Go).

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 6Go. Figure 7Go shows a summary graph of northern blots representing 3–6 observations per diet group per time. Note that the percentages of normalized densities shown in Figure 6Go are compared with the single acetone-treated control of this figure and may not reflect the average percentages for n = 3–6, as in Figure 7Go.



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Fig. 6. An example image of a northern blot of c-jun mRNA in total RNA from TPA-treated, dietary energy restricted SENCAR mouse epidermis. Each lane contained the mRNA from one mouse fed either ad libitum (Control) or dietary energy restricted (ER) and treated with acetone (0 h) or 3.2 nmol TPA for 1 or 4 h. The upper panel shows the c-jun mRNA bands and the lower panel shows the corresponding rRNA bands used as a loading control. The table shows the densities of the c-jun bands normalized to those of the 18S loading control and a comparison of these densities to that of the single 0 h acetone-treated control expressed as a percentage.

 


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Fig. 7. Effects of DER on TPA induction of c-jun mRNA detected by northern blot analysis of total RNA from SENCAR mouse skin. Band density (normalized to rRNA bands) was measured with a PhosphorImager and expressed as a percentage of acetone-treated, ad libitum fed controls. Values are means (± SEM) of TPA-induced c-jun mRNA, with the dotted line indicating 100% of the acetone-treated, ad libitum fed control. The ratio of the density of DER bands compared with controls was tested by single factor ANOVA and by t-test. Values that do not share italicized letters are significantly different (P < 0.05); n = 4, 5 and 5 for control and n = 3, 6 and 6 for DER groups at 0, 1–3 and 4–6 h, respectively.

 
Induction of c-jun mRNA was significantly increased at 1–3 h (P < 0.01) in control fed mice by t-test. In DER mice, however, statistically significant induction was not observed. A single factor ANOVA used to test an overall effect of diet across time showed that c-jun mRNA levels were significantly different between control and DER mice over time (P < 0.05). c-jun mRNA levels returned to near basal levels by 4–6 h following TPA treatment in both groups. The observed increase in c-jun mRNA in SENCAR mouse skin from TPA-treated control animals was consistent with findings in phorbol ester-treated, tumor promotion-sensitive (SSIN and SENCAR) and tumor promotion-resistant (C57BL/6) mouse strains (24).

Effect of DER and CCS supplementation on CCS and IGF-I concentrations
Blood plasma CCS concentrations, shown in Figure 8Go, 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 9Go, 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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Fig. 8. Effect of adrenalectomy on CCS blood plasma concentrations in dietary energy restricted SENCAR mice. CCS concentrations were measured using a radioimmunoassay kit (ICN Biomedicals) from blood collected between 7:00 and 10:00 a.m. Because acetone and TPA had no significant effect on CCS concentrations, values across these groups are pooled and expressed as the means (± SE) for n = 13–14 per group as derived from ANOVA on natural log transformed data. Values that do not share italicized letters are significantly different by t-test of the least squares means following ANOVA (P < 0.05). Abbreviations are as in Figure 3Go.

 


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Fig. 9. Effect of dietary energy restriction on IGF-I blood plasma concentrations in SENCAR mice. IGF-I concentrations were measured using a radioimmunoassay kit (Nichols Institute Diagnostics). Because acetone and TPA had no significant effect on IGF-I concentrations, values across these groups are pooled and expressed as the means (± SE) for n = 13–14 per group as derived from ANOVA on natural log transformed data. Values that do not share italicized letters are significantly different by t-test of the least squares means following ANOVA (P < 0.05). Abbreviations are as in Figure 3Go.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This is the first report to examine the importance of AP-1 in the mechanism of dietary energy restriction inhibition of carcinogenesis in an in vivo mouse skin model system. To identify potential targets downstream of ERK, the effect of DER on AP-1–DNA binding was examined in nuclear extracts from mouse epidermal tissue. Because ERK activation was necessary for AP-1 activation in vitro (25) and lack of AP-1 activation and cell transformation correlated with low ERK levels in a cultured cell tumor promotion model system (26), investigation of AP-1 in an in vivo model of carcinogenesis was likely to be especially informative.

Our investigation clearly demonstrated that DER significantly reduced AP-1–DNA binding (Figure 3Go) and c-Jun protein levels (Figure 5Go) in TPA-treated mouse epidermis. Both c-Jun protein levels and AP-1–DNA 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-1–DNA 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-1–DNA 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-1–DNA binding required 4 h treatment. Thus, the DER reduction in mRNA may have caused the subsequent decreases in c-Jun protein and AP-1–DNA binding. Furthermore, unlike the results with c-jun mRNA and c-Jun protein, DER reduced the basal (acetone-treated) level of AP-1–DNA binding without affecting the ratio of TPA-induced AP-1–DNA binding to acetone-treated AP-1–DNA 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-1–DNA 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-1–DNA 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-1–DNA binding activity (11). Unfortunately, only indirect measures of GR activation could be used in the animal model employed and thus an endogenous GR–AP-1 interaction may still be present and could contribute to the overall effect seen in DER mice of a reduction in AP-1–DNA 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-1–DNA 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.


    Notes
 
5 To whom correspondence should be addressed at: Department of Food Science and Human Nutrition, Iowa State University, 2312 Food Sciences Building, Ames, IA 50011-1061, USA Email: dbirt{at}iastate.edu Back


    Acknowledgments
 
The authors appreciate the help of Kari Jovaag and Jill Yoder in the statistical analyses of the data. This research was supported by the American Institute for Cancer Research (97B039 and 95B126-REV), the American Cancer Society (SIG-16) and the National Institutes of Health (R01 CA77451-01 and P30 CA36727). Journal Paper no. J-19255 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project no. IOW 03360, and supported by Hatch Act and State of Iowa funds.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Birt,D.F., Pinch,H.J., Barnett,T., Phan,A. and Dimitroff,K. (1993) Inhibition of skin tumor promotion by restriction of fat and carbohydrate calories in SENCAR mice. Cancer Res., 53, 27–31.[Abstract]
  2. Birt,D.F., Pelling,J.C., White,L.T., Dimitroff,K. and Barnett,T. (1991) Influence of diet and calorie restriction on the initiation and promotion of skin carcinogenesis in the SENCAR mouse model. Cancer Res., 51, 1851–1854.[Abstract]
  3. Birt,D.F., Copenhaver,J., Pelling,J.C. and Anderson,J. (1994) Dietary energy restriction and fat modulation of protein kinase C isoenzymes and phorbol ester binding in SENCAR mouse epidermis. Carcinogenesis, 15, 2727–2732.[Abstract]
  4. Nair,S.C., Toshkov,I.A., Yaktine,A.L., Barnett,T.D., Chaney,W.G. and Birt,D.F. (1995) Dietary energy restriction-induced modulation of protein kinase C zeta isozyme in the hamster pancreas. Mol. Carcinog., 14, 10–15.[Medline]
  5. Liu,Y., Duysen,E., Yaktine,A.L., Au,A., Wang,W. and Birt,D.F. (2001) Dietary energy restriction inhibits ERK but not JNK or p38 activity in the epidermis of SENCAR mice. Carcinogenesis, 22, 607–612.[Abstract/Full Text]
  6. Finkenzellar,G., Marme,D. and Hug,H. (1992) Inducible over expression of human protein kinase C alpha in NIH 3T3 fibroblasts results in growth abnormalities. Cell. Signal., 4, 163–177.[Medline]
  7. Dong,Z., Birrer,M.J., Watts,R.G., Matrisian,L.M. and Colburn,N.H. (1994) Blocking of tumor promoter-induced AP-1 activity inhibits induced transformation in JB6 mouse epidermal cells. Proc. Natl Acad. Sci. USA, 91, 609–613.[Abstract]
  8. Dong,Z., Crawford,H.C., Lavrovsky,V., Taub,D., Watts,R., Matrisian,L.M. and Colburn,N.H. (1997) A dominant negative mutant of jun blocking 12-O-tetradecanoylphorbol-13-actetate-induced invasion in mouse keratinocytes. Mol. Carcinog., 19, 204–212.[Medline]
  9. Li,J.J., Rhim,J.S., Schlegel,R., Vousden,K.H. and Colburn,N.H. (1998) Expression of dominant negative Jun inhibits elevated AP-1 and NF-kappaB transactivation and suppresses anchorage independent growth of HPV immortalized human keratinocytes. Oncogene, 16, 2711–2721.[Medline]
  10. Pashko,L.L. and Schwartz,A.G. (1992) Reversal of food restriction-induced inhibition of mouse skin tumor promotion by adrenalectomy. Carcinogenesis, 13, 1925–1928.[Abstract]
  11. Yaktine,A.L., Vaughn,R., Blackwood,D., Duysen,E. and Birt,D.F. (1998) Dietary energy restriction in the SENCAR mouse: elevation of glucocorticoid hormone concentrations but no change in distribution of glucocorticoid receptor in epidermal cells. Mol. Carcinog., 21, 62–69.[Medline]
  12. Schwartz,J.A., Viaje,A. and Slaga,T.J. (1977) Fluocinolone acetonide: a potent inhibitor of mouse skin tumor promotion and epidermal DNA synthesis. Chem. Biol. Interact., 17, 331–347.[Medline]
  13. Slaga,T.J., Lichti,U., Elgjo,K. and Yuspa,S.H. (1978) Effects of tumor promoters and steroidal anti-inflammatory agents on skin of newborn mice in vivo and in vitro. J. Natl Cancer Inst., 60, 425–431.[Medline]
  14. Vogt,P.K. and Bos,T.J. (1990) jun: oncogene and transcription factor. Adv. Cancer Res., 55, 1–34.[Medline]
  15. Whitmarsh,A.J. and Davis,R.J. (1996) Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J. Mol. Med., 74, 589–607.[Medline]
  16. Angel,P., Allegretto,E.A., Okino,S.T., Hattori,K., Boyle,W.J., Hunter,T. and Karin,M. (1988) Oncogene jun encodes a sequence-specific trans-activator similar to AP-1. Nature, 332, 166–171.[Medline]
  17. Dunn,S.E., Kari,F.W., French,J., Leininger,J.R., Travlos,G., Wilson,R. and Barrett,J.C. (1997) Dietary restriction reduces insulin-like growth factor I concentrations, which modulates apoptosis, cell proliferation and tumor progression in p53-deficient mice. Cancer Res., 57, 4667–4672.[Abstract]
  18. Kari,F.W., Dunn,S.E., French,J.E. and Barrett,J.C. (1999) Roles for insulin-like growth factor-1 in mediating the anti-carcinogenic effects of caloric restriction. J. Nutr. Health Aging, 3, 92–101.[Medline]
  19. Reeves,P.G., Nielsen,F.H. and Fahey,G.C. (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr., 111, 208–218.
  20. Kulesz-Martin,M., Kilkenny,A.E., Holbrook,K.A., Digernes,V. and Yuspa,S.H. (1983) Properties of carcinogen altered mouse epidermal cells resistant to calcium-induced terminal differentiation. Carcinogenesis, 1, 1367–1377.
  21. Lu,Y.-P., Chang,R.L., Lou,Y.-R., Huang,M.-T., Newmark,H.L., Reuhl,K.R. and Conney,A.H. (1994) Effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate- and ultraviolet B light-induced expression of c-jun and c-Fos in JB6 cells and in mouse epidermis. Carcinogenesis, 15, 2363–2370.[Abstract]
  22. Chomczynski,P. and Sacchi,N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156–159.[Medline]
  23. Abmayr,S.M. and Workman,J.L. (1993) Preparation of nuclear and cytoplasmic extracts from mammalian cells. In Ausubel,F.M. et al. (eds) Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY, Vol. 2, pp. 12.1.1–12.1.9.
  24. Kennard,M.D., Kang,D.-C., Montgomery,R.L. and Butler,A.P. (1995) Expression of epidermal ornithine decarboxylase and nuclear proto-oncogenes in phorbol ester tumor promotion-sensitive and -resistant mice. Mol. Carcinog., 12, 14–22.[Medline]
  25. Frost,J.A., Geppert,T.D., Cobb,M.H. and Feramisco,J.R. (1994) A requirement for extracellular signal-regulated kinase (ERK) function in the activation of AP-1 by Ha-ras, phorbol 12-myristate 13-acetate and serum. Proc. Natl Acad. Sci. USA, 91, 3844–3848.[Abstract]
  26. Huang,C., Ma,W.Y., Young,M.R., Colburn,N. and Dong,Z. (1998) Shortage of mitogen-activated protein kinase is responsible for resistance to AP-1 transactivation and transformation in mouse JB6 cells. Proc. Natl Acad. Sci. USA, 95, 156–161.[Abstract/Full Text]
  27. Straus,D.S. (1994) Nutritional regulation of hormones and growth factors that control mammalian growth. FASEB J., 8, 6–12.[Abstract]
  28. Thissen,J.-P., Ketelslegers,J.-M. and Underwood,L.E. (1994) Nutritional regulation of the insulin-like growth factors. Endocr. Rev., 15, 80–101.[Abstract]
  29. Hursting,S.D., Switzer,B.R., French,J.E. and Kari,F.W. (1993) The growth hormone:insulin-like growth factor 1 axis is a mediator of diet restriction-induced inhibition of mononuclear cell leukemia in Fischer rats. Cancer Res., 53, 2750–2757.[Abstract]
  30. Himeno,Y., Engelman,R.W. and Good,R.A. (1992) Influence of caloric restriction on oncogene expression and DNA synthesis during liver regeneration. Proc. Natl Acad. Sci. USA, 89, 5497–5501.[Abstract]
  31. Rosenberger,S.F. and Bowden,G.T. (1996) Okadaic acid stimulated TRE binding activity in a papilloma producing mouse keratinocytes cell line involves increased AP-1 expression. Oncogene, 12, 2301–2308.[Medline]
  32. Birt,D.F., Duysen,E., Wang,W. and Yaktine,A. (2001) Corticosterone supplementation reduced selective protein kinase C isoform expression in the epidermis of adrenalectomized mice. Cancer Epidemiol. Biomarkers Prev., accepted for publication.
Received March 8, 2001; revised May 14, 2001; accepted June 1, 2001.