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[Cancer Research 61, 4084-4091, May 15, 2001]
© 2001 American Association for Cancer Research

Regular Articles

Organ-specific Activation of Activator Protien-1 in Transgenic Mice by 12-O-Tetradecanoylphorbol-13-acetate with Different Administration Methods1

Shuping Zhong, James A. Quealy, Ann M. Bode, Masaaki Nomura, Akira Kaji, Wei-Ya Ma and Zigang Dong2

The Hormel Institute, University of Minnesota [S. Z., A. M. B., M. N., A. K., W-Y. M., Z. D.], and Austin Medical Center, Austin, Minnesota 55912 [J. A. Q.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
12-O-Tetradecanoylphorbol-13-acetate (TPA) is widely used as a tumor promoter with organotropy in skin and esophagus. TPA-induced, organ-specific tumor promotion is not correlated with the distribution of its receptor, protein kinase C (PKC). Using five administration methods (painting, drinking, gavage feeding, i.p. injection, and i.v. injection), we analyzed TPA-stimulated activator protein-1 (AP-1) activity in various organs (liver, kidney, brain, lung, spleen, heart, stomach, colon, esophagus, and skin) from transgenic mice expressing the AP-1 luciferase reporter gene. Topical application of TPA by painting the skin on the back of mice raised AP-1 activity 122.6-fold, and the highest peak of AP-1 activity was at 12 h after administration of TPA. Drinking water containing TPA caused a 25.8-fold induction of AP-1 activity in the skin, whereas gavage feeding with TPA caused a 34.2-fold induction of AP-1 in the skin. Intraperitoneal or i.v. injection of TPA induced a 49.56-fold or 20.4-fold increase in AP-1 activity in the skin, respectively. The highest peaks of AP-1 activity in the skin were at 12 h after drinking, feeding, or injection of TPA. More interesting, in the esophagus, i.p. injection of TPA raised AP-1 activity 13.9-fold, drinking TPA raised AP-1 activity 8.4-fold, and painting with TPA caused a 2.4-fold induction of AP-1 activity. In the colon, i.p. injection of TPA raised AP-1 activity 3.9-fold, drinking TPA induced a 1.2-fold increase in AP-1 activity, but painting with TPA had no effect. AP-1 activity in other organs was not detectable after administration of TPA by painting, drinking, or injection. Phosphorylation of extracellular signal-regulated kinases in the skin increased at 12 h after painting, drinking, or i.p. injection of TPA. In addition, phosphorylation of p38 kinase was raised slightly after TPA administration, but phosphorylation of c-Jun NH2-terminal kinases was not detected at any time point after TPA administration. Similar changes in MAP kinases were also seen in the esophagus after TPA administration. These results indicate that the skin is the most sensitive organ to TPA induction of AP-1 activity. The data suggest that the organ-specific, tumor-promoting effect of TPA may be through AP-1 activation and phosphorylation of ERKs and p38 kinase.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The process of tumor promotion is divided into at least two stages, initiation and promotion (1) . The two stages of carcinogenesis can be initiated on mouse skin with 7,12-dimethylbenz[a]anthracene and promoted with phorbol (2, 3, 4) . In addition, phorbol has been shown to induce mammary carcinogenesis and lymphatic leukemia in Wistar rats after initiation with 7,12-dimethylbenz[a]anthracene (5) . The phorbol diester, TPA,3 is one of the most well-accepted agents for studying the mechanisms of carcinogenesis. TPA also is a potent activator of PKC (6 , 7) . TPA induces several genes, including collagenase, stromelysin, hMT IIA, and SV40, all of which contain a conserved 9-bp motif in their promoter regions. This is known as the TRE and is recognized by AP-1 (8) . The AP-1 complex, a heterodimer consisting of jun and fos multigene family members, is a sequence-specific DNA binding transcription factor that is part of a pathway by which intracellular signals are converted into changes in gene activity (9) . This complex plays a key role in both cell proliferation and cell differentiation (9, 10, 11, 12, 13, 14, 15, 16, 17) and is required for tumor promoter-induced cell transformation (18) .

PKC is known as the TPA receptor, and it is mainly distributed in brain, spleen, kidney, and liver (19 , 20) . However, TPA-induced organ tumor promotion is not correlated with the distribution of PKC. Our previous studies showed that TPA induced a high level of AP-1 activity in mouse epidermal JB6 P+ cells (C1 41) and in the skin of transgenic mice expressing the AP-1 luciferase reporter gene (18 , 21) . Inhibition of TPA-induced AP-1 activity also repressed TPA-induced tumor promotion (21 , 22) . Although these studies indicated that TPA strongly induces AP-1 activity when applied directly on the skin on the back of mice and results in tumor formation on the skin and forestomach (2 , 3 , 21 , 23) , whether the organotropy of TPA-induced AP-1 activity exists remains unclear. In this study, we administered TPA by five different routes to investigate the distribution of TPA-induced AP-1 activity, activation of ERKs, JNKs, and p38 kinase in various organs from AP-1 luciferase transgenic mice.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic Mice Expressing the AP-1 Luciferase Reporter Gene.
Transgenic mice carrying the 2x TRE-luciferase reporter were described in previous reports (18 , 21 , 24) . The mice were screened by testing the basal level of AP-1 luciferase activity in the skin. Male and female mice were housed separately in solid-bottom polycarbonated cages on ventilated animal racks (approximately four to five mice/cage) in temperature-, humidity-, and yellow light-controlled conditions. Food and water were available ad libitum, and the dorsal skin of the mice was shaved before the experiment. To determine AP-1 luciferase activity, different organs (liver, kidney, brain, lung, spleen, heart, stomach, colon, esophagus, and skin) were harvested from the transgenic mice after TPA treatments.

Assay of AP-1 Luciferase Activity in Vivo.
The AP-1 luciferase transgenic mice were identified, grouped, and housed as described above. Two weeks after grouping, the basal level of AP-1 luciferase activity was measured by punch skin biopsy. AP-1 luciferase reporter-bearing male and female mice were randomly divided into groups (16–22 mice in each group). Two weeks after the punch biopsy, the mice were treated with TPA by one of five administration methods: topical application by painting TPA on the back of the mice (10 µg of TPA in 200 µl of acetone/mouse); injection of TPA into the intraperitoneum (10 µg of TPA in 200 µl of PBS/mouse); i.v. injection of TPA (10 µg of TPA in 100 µl of PBS/mouse); gavage feeding (10 µg of TPA in 1 ml of PBS/mouse); or addition of TPA to drinking water (0.2 µg/ml) after depriving mice of water for 5 h. Skin biopsies and tissues of equal weight from different organs were harvested in 100 µl of lysis buffer (0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTT, and 2 mM EDTA) for measurement of luciferase activity at the times indicated. AP-1-dependent luciferase activity in the tissue extract was determined using the luciferase assay reagent from Promega and a luminometer from Analytical Luminescence Laboratory (Monolight 2010; Ref. 25 ).

Immunohistochemical Analysis.
For immunohistochemical analysis, the tissues from different organs were harvested and put on dry ice. Frozen sections were produced by using a freezing microtome. Mouse epidermal JB6 P+ 1-1 cells with the 2x TRE-luciferase reporter and frozen slices of different tissues from transgenic luciferase mice were first fixed with 50% acetone and 50% methanol and then blocked with 5% BSA. Primary antibodies of rabbit anti-luciferase (Research Diagnostics, Inc.) and anti-phospho42/44 MAP kinase, phospho-p38 MAP kinase, or phospho-SAPK/JNK (New England Biolabs, Inc.) were incubated with the tissue slices at 37°C for 60 min. The slices were subsequently washed three times (10 min/time) with PBS (58 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl, pH 7.4). The slices were then incubated with the secondary antibody, goat antirabbit IgG conjugated with FITC (Sigma Chemical Co.) at 37°C for 60 min. The slices were finally observed under a Leica DMIRB fluorescence microscope.

MAP Kinase Analysis.
TPA was administrated by topical application on the dorsal surface by painting, injection into the intraperitoneum, or ingestion by drinking. At specific time points (2, 12, 24, or 48 h) after administration of TPA, skin or esophagus (equal weights) were harvested from different TPA-treated mice and placed on dry ice. Samples were cut into small pieces, placed on ice, and incubated in 500 µl of SDS lysis buffer [62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% glycerol, and 50 mM DTT] for 60 min. The tissue lysates were sonicated for 20 s and centrifuged at 14,000 rpm in a microcentrifuge at 4°C for 10 min. The supernatant was saved and added into three volumes of acetone and incubated for 10 min on ice. The suspension was centrifuged at 14,000 rpm at 4°C for 10 min, and the pellets were subsequently resuspended in 800 µl of acetone and centrifuged in 14,000 rpm at 4°C for 10 min. The pellets were then dissolved in 200 µl of SDS lysis buffer, and the protein concentration was measured using the Bradford method (26) . After boiling for 5 min, an equal amount of protein from each sample was resolved by 10% SDS-PAGE. The resolved proteins from the skin or esophagus were then transferred to polyvinylidene difluoride membranes for Western blot analysis. Polyvinylidene difluoride membranes were blocked with 5% fat-free milk in PBS for 1 h at room temperature and incubated with specific antibodies for rabbit antiphospho-p42/44 MAP kinase, phospho-p38 MAP kinase, or phospho-SAPK/JNK (New England Biolabs, Inc.) overnight at 4°C to detect phospho-ERKs, phospho-p38 kinase, or phospho-JNKs, respectively. The membranes were then incubated for 4 h at 4°C with the second antibody, rabbit IgG conjugated-alkaline phosphatase. The membranes were developed with chemiluminescence, and the protein-antibody complex was detected using the Storm PhosphorImager 840 (Molecular Dynamics, Inc.). In parallel, non-phospho-ERKs, non-phospho-p38 kinase, and non-phospho-JNKs were detected with the rabbit anti-p42/44 MAP kinase, p38 kinase, or JNKs, respectively (New England Biolabs, Inc.).

Statistical Analysis.
Differences in AP-1 activity were analyzed by using Student’s t test. P < 0.05 was considered significant, and the results are expressed as SE.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AP-1 Transactivation in Different Organs from Transgenic Mice after TPA Treatment.
To investigate whether TPA stimulated AP-1 transactivation in various tissues from transgenic mice, we used one of three methods to administer TPA. TPA was topically applied to the skin by painting, by i.p. injection, or mice were allowed to consume TPA in their drinking water. Twelve h after the administration of TPA, tissues were harvested for analysis of AP-1 luciferase activity. The results showed that AP-1 activity in the skin increased markedly after any of the three TPA administration methods, and skin had the highest level of AP-1 activity compared with other tissues (P < 0.01; Fig. 1Citation ). Additionally, the level of AP-1 activity in skin painted with TPA was significantly higher than that in the skin when TPA was injected or consumed (P < 0.05; Fig. 1Citation ). More interestingly, in the esophagus, injection of TPA raised AP-1 activity 13.9-fold (P < 0.05), drinking TPA raised AP-1 activity 8.4-fold (P < 0.05), whereas painting with TPA caused only a 2.4-fold increase in AP-1 activity. In addition, AP-1 activity in the colon was increased 3.9-fold after administration of TPA by injection, but almost no change was seen when TPA was administrated by painting (1.2-fold) or drinking (1.1-fold). Changes in AP-1 activity in other organs were not detected after administration of TPA by any method (Fig. 1)Citation . The results indicated that the skin is the most sensitive organ for TPA-induction of AP-1 independent of administration method.



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  		Fig. 1. Comparison of AP-1-dependent luciferase activity in different organs from AP-1 luciferase reporter transgenic mice. The AP-1 luciferase transgenic mice were described in "Materials and Methods." Twelve h after the administration of TPA by painting, injection, or drinking, different tissues of the mice were punch biopsied using a biopsy punch (1.5 mm; Acuderm, Inc., Ft. Lauderdale, FL). The tissues of different organs were extracted in 100 µl of lysis buffer overnight at 4°C, and AP-1 activity was measured as described in "Materials and Methods." The different organs from AP-1 transgenic mice and the three administration methods of TPA are indicated. Values are means (n = 4); bars, SE. Significant change in skin after TPA treatment: *, P < 0.001 AP-1 luciferase activity in skin versus other organs after painting with TPA; **, P < 0.05 painting with TPA versus drinking or injection into the intraperitoneum; #, P < 0.05 AP-1 luciferase activity in esophagus versus other organs after administration of TPA by drinking or injection into the intraperitoneum.

 
Time-dependent AP-1 Activity in Skin after TPA Treatment.
To investigate whether the TPA-induced change in AP-1 activity was time dependent, we compared AP-1 activity in the skin at various times after the five kinds of TPA treatment. The time course showed that AP-1 activity sharply increased from 2 to 12 h after all methods of TPA administration and then decreased markedly by 24 and 48 h (Fig. 2)Citation . The highest peak of AP-1 activity was at 12 h after any method of treatment of TPA. Five kinds of treatment with TPA all significantly increased AP-1 activity, compared with the control groups (P < 0.05; Fig. 2Citation ), but topical application of TPA by painting on the skin markedly raised AP-1 activity, compared with other treatments with TPA (Table 1)Citation . These results showed that AP-1 activity in skin of AP-1 transgenic mice was strongly induced after any of the five methods of TPA administration, and that TPA-induced AP-1 activity was time dependent.



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  		Fig. 2. Time course of AP-1-dependent luciferase activity in skin after different administration modes of TPA. The AP-1 luciferase transgenic mice were described in "Materials and Methods." At different times (2, 6, 12, 24, or 48 h) after administration of TPA by painting, injection, or drinking, the dorsal skin of the mice was punch biopsied using a biopsy punch (1.5 mm; Acuderm, Inc.). The biopsies were extracted in 100 µl of lysis buffer overnight at 4°C and measured as described in "Materials and Methods." The methods of administration and times are indicated. Values are means (n = 22). Significant change in skin after TPA treatment: #, P < 0.001 painting with TPA versus control; **, P < 0.01 TPA injection into the intraperitoneum versus control; *, P < 0.05 gavage feeding, drinking, or i.v. injection of TPA versus control.

 

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  		Table 1 Maximal changes in AP-1 activity in skin at 12 h following different administrations with TPA

 
Dose-dependent AP-1 Activity in Skin after Painting with TPA.
Above results show that TPA-induced AP-1 activity was time dependent. To investigate whether AP-1 activity induced by TPA is dose dependent, we painted the skin on the back of AP-1 transgenic mice with different amounts of TPA, and then after 12 h, we took biopsies for detection of TPA-induced AP-1 luciferase activity. Painting with 2.5 µg of TPA significantly increased AP-1 activity (22.9-fold) in skin, compared with the untreated group (P < 0.05; Fig. 3Citation ). The AP-1 activity continued to rise with an increasing amount of TPA (P < 0.005; Fig. 3Citation ). These results clearly indicated that TPA-induced AP-1 activity was dose dependent.



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  		Fig. 3. Dose dependence of AP-1 luciferase activity in skin after painting with different amounts of TPA. The AP-1 luciferase transgenic mice were described in "Materials and Methods." Twelve h after the administration of TPA (2.5, 5, 10, or 20 µg of TPA/200 µl of acetone/mouse) by painting it on the back of the mouse’s skin, the dorsal skin of the mice was punch biopsied using a biopsy punch (1.5 mm; Acuderm, Inc.). The skin was extracted in 100 µl of lysis buffer overnight at 4°C, and AP-1 activity was measured as described in "Materials and Methods." The different amounts of TPA administered are indicated. Values are means (n = 17); bars, SE. Significant change in the skin after TPA treatment: *, P < 0.05 painting with TPA (2.5 µg/mouse) versus control; **, P < 0.005 painting with TPA (5, 10, or 20 µg/mouse) versus control.

 
Phosphorylation of MAP Kinases in the Skin after Painting with TPA.
MAP kinases, including ERKs, p38 kinase, and JNKs, are mediators in the regulation of transcription factor AP-1 (21 , 27, 28, 29) . MAP kinases are proline-directed serine/threonine kinases that are activated by dual phosphorylation of threonine and tyrosine residues in response to a wide array of extracellular stimuli. MAP kinases are mediators of signal transduction from the cell surface to the nucleus, and AP-1 family proteins are the target molecules of these kinases. We extracted protein from the skin and esophagus for Western blotting to investigate the relationship between AP-1 activity and MAP kinase activation by TPA. In the skin, phosphorylation of ERKs increased markedly (P < 0.05; Fig. 4, A and DCitation ), whereas induction of phosphorylation of p38 kinases was not significant (P > 0.05; Fig. 4BCitation ) and phosphorylation of JNKs was not detectable (Fig. 4C)Citation . The change in MAPKs phosphorylation was independent of the method of TPA administration. A time course study revealed that phosphorylation of ERKs increased in a time-dependent manner with the highest level (P < 0.05; Fig. 5, A and DCitation ) occurring 12 h after administration of TPA by painting. Phosphorylation of p38 kinase was not significantly increased after painting with TPA (P > 0.05; Fig. 5, B and ECitation ), and phosphorylation of JNKs was not detected at any time point (Fig. 5C)Citation . In addition, we also analyzed changes in MAP kinases in the esophagus after administration of TPA by painting, and results showed that phosphorylation of ERKs in the esophagus increased at 12 h (P < 0.05) after TPA administration (Fig. 6, A and D)Citation . Phosphorylation of p38 in the esophagus increased markedly from 2 to 48 h with a biphasic response after painting with TPA (P < 0.05; Fig. 6, B and ECitation ), but no change was observed in the phosphorylation of JNKs (Fig. 6C)Citation . The first peak of p38 phosphorylation occurred at 2 h, and another occurred at 48 h. The induction of p38 phosphorylation at 2 h may be a direct effect of TPA, whereas the second peak at 48 h may be the result of secondary events caused by TPA-induced growth factors. In addition, we also compared levels of phospho-ERKs and phospho-p38 kinase in liver and kidney to levels in skin after painting with TPA. We found no increases in phospho-ERKs or phospho-p38 kinase in liver or kidney after TPA painting (Fig. 7)Citation . These results suggested that the tumor promoter, TPA, is organ specific and that phosphorylation of ERKs increases after painting with TPA, corresponding with AP-1 activity in skin and esophagus. In addition, the phosphorylation of p38 kinase displays a similar pattern, but phosphorylation of JNKs does not appear to be related. These data suggest that AP-1, ERKs, and p38 kinase may be involved in TPA-stimulated tumor promotion in the skin and esophagus.



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  		Fig. 4. Level of phosphorylated MAP kinases from skin at 12 h after administration of TPA by topical painting, drinking, or injection into the intraperitoneum. Skin proteins were extracted and resolved as described in "Materials and Methods." Phosphorylated (P-) ERK, p38 kinase, and JNKs were detected with rabbit antiphospho-p42/44 MAP kinase, antiphospho-p38 MAP kinase, and antiphospho-SAPK/JNK (New England Biolabs, Inc.), respectively. Total nonphosphorylated (NP-) ERKs, p38, and JNKs were detected with rabbit anti-p42/44 MAP kinase, anti-p38 kinase, and anti-JNKs (New England Biolabs, Inc.), respectively. A, P-ERKs and NP-ERKs in the skin. B, P-p38 and NP-p38 kinase in the skin. C, P-JNKs and NP-JNKs in the skin. D, fold increase of P-ERKs. Values are means (n = 3); bars, SE. Significant change in skin after TPA treatment: *, P < 0.05 TPA drinking, i.p. injection, or painting versus control; C1 and C2, control; D1 and D2, drinking water with TPA; I1 and I2, TPA injection into intraperitoneum; P1 and P2, painting with TPA. Positive control of phospho-JNKs is mouse epidermal JB6 cells after UVB irradiation (4 kJ/m2). Arrows, positions of P-ERK1/2 and NP-ERK1/2, P-p38 and NP-p38 kinase, and P-JNKs and NP-JNKs. P, phosphorylated; NP, nonphosphorylated.

 


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  		Fig. 5. Level of phosphorylated MAP kinases in skin after painting with TPA. Skin proteins were extracted and resolved as described in "Materials and Methods." ERKs, p38 kinases, and JNKs were detected with specific antibodies. A, P-ERKs and NP-ERKs in the skin. B, P-p38 and NP-p38 kinase in the skin. C, P-JNKs and NP-JNKs in the skin. Positive control of phospho-JNKs is mouse epidermal JB6 cells after UVB irradiation (4 kJ/m2). D, the counts incorporated into P-ERKs in A were quantified using a Storm PhosphorImager 840 (Molecular Dynamics, Inc.), and the results are represented graphically. Values are the mean (n = 7) from different groups of AP-1 luciferase reporter transgenic mice after administration of TPA at different times; bars, SE. Significant change in skin after TPA treatment: *, P < 0.05 TPA painting versus control. E, the counts incorporated in P-p38 kinase in B. Arrows, positions of P-ERK1/2 and NP-ERK1/2, P-p38 and NP-p38 kinase, and P-JNKs and NP-JNKs. Time after TPA treatment is indicated. P, phosphorylated; NP, nonphosphorylated.

 


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  		Fig. 6. Level of phosphorylated MAP kinases in esophagus after painting with TPA. Esophageal proteins were extracted and resolved as described in "Materials and Methods." Phosphorylation of ERKs, p38 kinase, and JNKs was detected with specific antibodies. A, P-ERKS and NP-ERKs in the esophagus. B, P-p38 and NP-p38 kinase in the esophagus. C, P-JNKs and NP-JNKs in the esophagus. Positive control of phospho-JNKs is mouse epidermal JB6 cells after UVB irradiation (4 kJ/m2). D, the counts incorporated into P-ERKs in A were quantified using a Storm PhosphorImager 840 (Molecular Dynamics, Inc.), and the results are represented graphically. Values represent the mean (n = 3) from the different groups of AP-1 luciferase reporter transgenic mice after administration of TPA at different times; bars, SE. Significant change in esophagus after TPA treatment: *, P < 0.05 TPA painting versus control. E, the counts incorporated in P-p38 kinase in B. Arrows, positions of P-ERK1/2 and NP-ERK1/2, P-p38 and NP-p38 kinases, and P-JNKs and NP-JNKs. Time after TPA treatment is indicated. P, phosphorylated; NP, nonphosphorylated.

 


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  		Fig. 7. Level of phosphorylated ERKS and p38 kinases in different tissues after painting with TPA. Liver, kidney, and skin proteins were extracted and resolved as described in "Materials and Methods." Phospho-ERK and p38 were detected with rabbit antiphospho-p42/44 MAP kinase and phospho-p38 MAP kinase, respectively (New England Biolabs, Inc.). K’, kidney without TPA painting; K, kidney after painting with TPA; L, liver after painting with TPA; Skin, after painting with TPA (A and B) or UVB irradiation (C and D). A, phospho-ERK1/2 in liver, kidney, and skin (12 h). B, phospho-p38 in liver, kidney, and skin (12 h). C, phospho-ERK1/2 in kidney and skin (12 h). D, phospho-p38 in kidney and skin (12 h). Arrows, positions of phospho-ERK1/2 and p38, respectively. P, phosphorylated; NP, nonphosphorylated.

 
Distribution of AP-1 Luciferase Activity in Skin.
The above results clearly demonstrate that TPA-induced AP-1-dependent luciferase activity was highest in the skin compared with other organs. Immunohistochemical staining of the skin revealed that the epidermis was stained by anti-luciferase antibody conjugated with FITC, and luciferase activity was found in the cytoplasm (Fig. 8, A–Q)Citation , compared with the staining of mouse epidermal JB6 P+ 1-1 cells after UVB irradiation (Fig. 8U)Citation . DNA staining with Hoechst 33258 also indicated that the nuclei did not display the luciferase signal (Fig. 8V)Citation , confirming that luciferase activity was localized in the cytoplasm. AP-1-dependent luciferase activity in the skin was barely detectable before TPA painting (Fig. 8A)Citation but markedly increased at 2 h after TPA administration (Fig. 8E)Citation , and the highest peak was observed at 12 h (Fig. 8I)Citation but then gradually diminished (Fig. 8, M and Q)Citation . Staining for phosphorylated ERKs, p38, and JNKs in the skin after painting with TPA showed that phospho-ERKs increased slightly at 2 and 12 h (Fig. 8, F and J)Citation and then decreased (Fig. 8, N and R)Citation . However, no change was observed in the level of phospho-p38 kinase (Fig. 8, C–S)Citation or phospho-JNKs (Fig. 8, D–T)Citation . Staining of mouse epidermal JB6 P+ 1-1 cells after UVB irradiation indicated that the phosphorylated ERKs were located in the nucleus (Fig. 8W)Citation , because the phosphorylated ERKs signal completely matched the DNA staining by Hoechst 33258 (Fig. 8X)Citation . In addition, some nonspecific staining of AP-1-dependent luciferase (Fig. 8, M and Q)Citation or phosphorylated JNKs (Fig. 8, H and L)Citation in hair follicles was observed, compared with control staining (data not shown). The results clearly indicated that AP-1 luciferase activity after administration of TPA by painting is time dependent.



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  		Fig. 8. AP-1-dependent luciferase activity in skin of transgenic mice. AP-1-dependent luciferase activity was stained with FITC in skin slices as described in "Materials and Methods." A–D, no TPA. E–H, 2 h after TPA treatment. I–L, 12 h after TPA treatment. M–P, 24 h after TPA treatment. Q–T, 48 h after TPA treatment. A, E, I, M, and Q, the staining indicating AP-1-dependent luciferase activity in the skin. B, F, J, N, and R, the staining indicating phosphorylation of ERKs in the skin. C, G, K, O, and S, the staining indicating phosphorylation of p38 kinase in the skin. D, H, L, P, and T, the staining indicating phosphorylation of JNKs in the skin. x300. Luciferase activity (U) and phosphorylated ERKs (W) were stained in JB6 P+ 1-1 cells after UVB irradiation. V and X, DNA staining with Hoechst 33258, as compared with the same views of U and W. Arrows, nonspecific staining of hair follicles, which reacted with FITC (without primary antibodies) and show positive staining.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we provide the first experimental evidence that AP-1 activity in skin after administration of TPA is markedly higher than in any other organ. Importantly, TPA-induced AP-1 activation was independent of the method of TPA administration and was time and dose dependent. Expression of phosphorylated ERKs in skin also increased after administration of TPA. In addition, phosphorylated p38 kinase was slightly increased after TPA administration, but phosphorylation of JNKs was not detectable at any time point. Similar changes in AP-1 activity and phosphorylation of MAP kinases were also seen in the esophagus after TPA administration.

TPA is a potent tumor promoter and exhibits many biological effects by inducing altered gene expression, a process that involves the activation of PKC (8 , 30) . TPA induces the transcription of cellular proto-oncogenes, including c-fos, c-myc, and c-sis (31, 32, 33, 34) , resulting in the loss of growth control. Other targets for TPA are the collagenase and stromelysin genes (35 , 36) , which may have a role in tumor invasiveness, metastasis, and angiogenesis (37 , 38) . Although TPA has been widely used in tumor promotion studies, the molecular mechanisms of TPA-induced skin tumor promotion remain unclear. In vitro, TPA directly stimulates Ca2+-activated, phospholipid-dependent PKC (39) . However, brain, spleen, liver, and kidney express high levels of PKC (19 , 20) , but TPA does not induce tumor promotion in these organs. TPA induces tumor promotion in skin (2) , forestomach (23) , mammary tissue, and lymphocytes (5) . However, some carcinogens induce tumors of certain organs independent of the application routes (40) . In the present report, we used AP-1 transgenic mice to investigate the organotropy of TPA-induced AP-1 activity. Topical application of TPA by painting (10 µg/mouse) strongly induced AP-1 activity in skin (Figs. 1Citation and 2Citation ), whereas administration of TPA by drinking, gavage feeding, and i.p. or i.v. injection also all markedly stimulated AP-1 activity in skin (Fig. 2)Citation . These results showed that regardless of how TPA was administered, AP-1 activity increased markedly in the skin compared with all other tissues. This may suggest that TPA targets mainly AP-1 proteins located in the skin. The fold-induction of AP-1 activity induced by painting TPA on the skin of AP-1 transgenic mice was higher than any of the other TPA administration methods. A dose-response study of AP-1 activity induced by TPA further supported this finding (Fig. 3)Citation . Our previous study showed that AP-1-dependent luciferase activity was high in liver, kidney, brain, lung, heart, and skin of 1-day-old, AP-1 transgenic mice (41) . AP-1-dependent luciferase activity also is high in esophagus and colon after TPA treatment (Fig. 1)Citation . We, therefore, conclude that luciferase activity is stable in all of the examined organs in AP-1 transgenic mice.

AP-1 is a transcription factor comprised of Jun and Fos family heterodimers that bind to a consensus cis element found in the transcriptional promoter region of a number of genes, the expression of which is induced by tumor promoters (42 , 43) . AP-1 was activated by ERKs (44, 45, 46, 47) , p38 kinase (48, 49, 50, 51, 52) , and JNKs (53, 54, 55) . The critical importance of AP-1 activity in cellular transformation by tumor promoters and/or oncogenes has been reported extensively (43 , 56, 57, 58, 59, 60) . Transgenic mice overexpressing c-Fos developed osteosarcomas and chondrosarcomas, suggesting that aberrant expression of the c-fos gene can promote neoplastic transformation in bone tissue (61) . c-Fos participates directly in regulating gene expression by using the c-Jun protein as an anchor to bind to TREs within the regulatory regions of specific target genes (9) . Knockout of the c-fos gene inhibits malignant progression of skin tumors (62) , and inhibitors of AP-1 block tumor promotion in AP-1 luciferase transgenic mice (21 , 22) . These data show that skin carcinogenesis is associated closely with AP-1 activation, implying that TPA-induced AP-1 activity may be related to skin carcinogenesis. Therefore, to study the upstream signal transduction of AP-1 induced by TPA is important. Our results showed that the level of phospho-ERKs increased significantly at 12 h (P < 0.05) after administration of TPA by painting, drinking, or injection into the intraperitoneum (Fig. 4)Citation . This implies that AP-1 activity in skin after administration of TPA may mainly be mediated by phospho-ERKs. PI-3 kinase is upstream of ERKs and p38 kinase. TPA was shown to markedly stimulate PI-3 kinase activity and increase phospho-ERKs and phospho-p38 kinase, leading to activation of AP-1 in JB6 Cl 41 cells (63 , 64) . Whether TPA-induced AP-1 activity is mediated by PI-3 kinase in AP-1 transgenic mouse is under investigation in this laboratory.

In summary, we have provided evidence that AP-1 activation is a possible molecular mechanism of TPA-induced skin tumor promotion. TPA displays a specific affinity for the skin and esophagus, resulting in increased AP-1 expression and ERKs and p38 kinase phosphorylation in these tissues. Skin is the most sensitive organ to TPA induction of AP-1, and the activation of AP-1 was independent of the route of TPA administration. AP-1 activity was mainly mediated by phosphorylation of ERKs and p38 kinase in the skin and esophagus. Our experiments suggest that AP-1, ERKs, and p38 kinase may play an important role in TPA-induced skin and esophageal carcinogenesis.


   ACKNOWLEDGMENTS
 
We thank Andria Hansen and Jeanne Ruble for secretarial assistance.


   FOOTNOTES
 
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.

1 Supported by The Hormel Foundation and Grants CA77646, CA74916, and CA81064 from the National Cancer Institute. Back

2 To whom requests for reprints should be addressed, at The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912. Phone: (507) 437-9640; Fax: (507) 437-9606; E-mail: zgdong{at}smig.net Back

3 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, TPA response element; PKC, protein kinase C; AP-1, activator protein-1; SAPK, stress-activated protein kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated protein kinase; MAP, mitogen-activated protein. Back

Received 3/28/00. Accepted 3/19/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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