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Suppression of Skin Tumorigenesis in c-Jun NH₂-Terminal Kinase-2-Deficient Mice¹

The Hormel Institute, University of Minnesota, Austin, Minnesota 55912 [N. C., M. N., Q-B. S., W-Y. M., A. M. B., Z. D.]; Department of Pathology, Hilton 11, Mayo Clinic, Rochester, Minnesota 55905 [L. W.]; and Department and Section of Immunology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520 [R. A. F.]

	ABSTRACT

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Previous studies have shown that c-Jun NH₂-terminal kinase (JNK) belongs to the mitogen-activated protein kinase (MAPK) family of signal transduction components that are rapidly initiated and activated by many extracellular stimuli. However, the potential role of JNK in mediating tumor promotion and carcinogenesis is unclear. We show here that in JNK2-deficient (Jnk2^-/-) mice, the multiplicity of papillomas induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) was lower than that in wild-type mice. Papillomas on wild-type mice grew rapidly and were well vascularized compared with Jnk2^-/- mice. After the 12th week of TPA treatment, the mean number of tumors per mouse was 4.13–4.86 in wild-type mice but only 1.13–2.5 in Jnk2^-/- mice. TPA induced phosphorylation of extracellular signal-regulated kinases and activator protein-1 DNA binding activity in wild-type mice, but the phosphorylation of extracellular signal-regulated kinases and activator protein-1 DNA binding were inhibited in Jnk2^-/- mice. These data suggest that JNK2 is critical in the tumor promotion process.

	INTRODUCTION

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The MAPK³ family, comprised of Erks, JNKs, and p38 kinases (1) , activates distinct groups of substrates and is implicated in the regulation of cell proliferation, tumorigenesis, differentiation, and apoptosis (1 , 2) . JNKs (i.e., stress-activated protein kinases) are activated in response to cellular stresses including osmotic shock, UV irradiation, arsenic, and tumor necrosis factor- (2, 3, 4, 5) . The physiological significance of JNK signaling was documented by genetic analysis in Drosophila and mice (6 , 7) . JNKs were shown to phosphorylate c-Jun and increase AP-1 transcription activity (8) . JNKs are implicated in apoptosis (9) and cell proliferation (10) . We reported that JNKs are required for tumor necrosis factor--induced cell transformation (2) . Recently, Jnk2-knockout (Jnk2^-/-) mice were generated, and they developed normally without apparent phenotype expression except deficiency of differentiation of precursor CD4⁺ T cells into effector T helper 1 cells (11) . Because JNK2 enhances AP-1 transcription activity, we hypothesize that JNK2 may mediate the tumor promotion process. Therefore, we used Jnk2^-/- mice to study the mechanism of TPA-induced tumor promotion.

	MATERIALS AND METHODS

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Materials.
DMBA, TPA, aprotinin, and leupeptin were from Sigma Chemical Co. (St. Louis, MO). Antibodies used were rabbit polyclonal anti-phosphorylated Erks (New England BioLabs, Inc., Beverly, MA) and monoclonal antibodies for JNK1 and JNK2 (Santa Cruz Biotechnology Inc., Santa Cruz Biotechnology, CA). Primers for PCR were synthesized by Life Technologies, Inc. (Rockville, MD).

Tumor Induction Experiments.
Jnk2^-/- mice were originally from C57BL/6(B6)-injected D3 ES cells with the construct pJNK2KO (11) . The expression of the endogenous Jnk2 gene was examined by reverse transcription-PCR using total RNA isolated from the thymus, and Jnk2 mRNA was not detected in homozygous Jnk2^-/- mice (11) .

Experimental groups consisted of 29–35 mice, 5 mice/cage. Mice were shaved at 7–8 weeks of age and treated once with 100 µg of DMBA. Two weeks later, tumor growth was promoted by treating with 17 nmol of TPA twice each week for 29 weeks. Visible skin tumors were counted every 2 weeks. The papilloma incidence, expressed as the percentage of animals with one or more papillomas, and the papilloma multiplicity, expressed as the number of papillomas per surviving mouse, were calculated each time tumors were counted.

Nuclear Protein Analysis.
Gel shift assays were used to detect AP-1 binding activity. Nuclear extracts were prepared as described previously (12) . In brief, the skin tissues were lysed with 500 µl of lysis buffer (50 mM KCl, 0.5% NP40, 25 mM HEPES, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 100 µM DTT). After centrifugation at 14,000 rpm for 1 min, the nuclei were washed with 500 µl of the same buffer but without NP40 and then placed into 200 µl of extraction buffer (500 mM KCl and 10% glycerol with the same concentration of the other reagents as in the lysis buffer). After centrifugation at 14,000 rpm for 5 min, the supernatant fraction was harvested as the nuclear protein extract and stored at -70°C. An AP-1 binding sequence from the human collagenase promoter region, 5'-AGCATGAGTCAGACACCTCTGGC-3', was synthesized and labeled with [³²P]dCTP using the Klenow fragment (Life Science Co., Gaithersburg, MD). Protein concentration was determined using the Modified Lowry Protein Assay (Pierce Chemical), and equal amounts of nuclear protein (3 µg) were added to the DNA binding buffer, which contained 5 x 10⁴ cpm ³²P-labeled oligonucleotide probe, 1.5 µg of poly(deoxyinosinic-deoxycytidylic acid), and 3 µg of BSA. The reaction mixture was incubated on ice for 10 min, followed by incubation at room temperature for 20 min. The DNA-protein complexes were resolved in a 6% nondenaturing acrylamide gel. The gel was dried and scanned using the Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Western Blotting.
Skin was harvested from mice and placed on dry ice. Each sample was 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% SDS, 10% glycerol, and 50 mM DTT] for 60 min. The lysate was sonicated four times, 5 s each, at power 3 and centrifuged at 14,000 rpm in a microcentrifuge at 4°C for 10 min. The supernatant fraction was diluted with three volumes of acetone and left on ice for 10 min. The suspension was centrifuged at 14,000 rpm at 4°C for 10 min, and the pellet was resuspended in 800 µl of acetone and centrifuged at 14,000 rpm at 4°C for 10 min. The pellet was then dissolved in 200 µl of SDS lysis buffer. The protein concentration was measured using the Bradford method (Bio-Rad Laboratories, Hercules, CA).

Samples containing equal amounts of protein were resolved in an 8% SDS-polyacrylamide gel, and proteins were subsequently transferred and analyzed as described previously (13) . Immunoblotting for proteins of Erks and JNKs was carried out using MAPK antibodies against Erks, JNK1, and JNK2 as described previously (13) . Antibody-bound proteins were detected by chemifluorescence (ECF; Amersham Pharmacia Biotech, Piscataway, NJ) and analyzed using the Storm 840 PhosphorImager (Molecular Dynamics).

	RESULTS

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Identification of Phenotype of Genomic DNA and Proteins of Jnk2^-/- Mice.
The genomic DNA phenotype and protein expression of JNK2 in knockout mice were confirmed by PCR and Western blotting, respectively. Results indicated that Jnk2^-/- mice did not express JNK2 protein, whereas expression of JNK1 and Erk were unaffected, agreeing with the results of others (Fig. 1 ; Ref. 11 ). Thus, the knockout of the Jnk2 gene was effective and specific, and mice deficient in JNK2 were used in the present experiments.

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Identification of the targeted disruption of the murine Jnk2 gene. A, identification of genomic DNA of wild-type (WT) and homozygous (-/-) mice by PCR. DNA was isolated from the tail of WT and JNK2-/- mice. The top band (400 bp) corresponds to the wild-type allele (WT), and the bottom band (270 bp) corresponds to the mutant allele (Jnk2-/-). B, Western blot analysis of skin lysates from wild-type (WT) and homozygous knockout (-/-) mice. The blots were probed with an antibody to JNK1, JNK2, or Erks.

View larger version (119K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. External appearance of papillomas. Tumors were initiated by treating once with 100 µg of DMBA, and 2 weeks later, tumors were promoted by treating with 17 nmol of TPA twice per week for 29 weeks. Visible skin tumors were counted once every 1–2 weeks. A, four wild-type mice showing the greatest number of tumors. B, four Jnk2-/- mice showing the greatest number of tumors.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Tumor formation in DMBA/TPA-treated mice. Tumors were counted and measured once every 1–2 weeks. A, papilloma incidence (the percentage of animals having one or more papillomas). B, papilloma multiplicity (the number of papillomas/mouse in mice with tumors). C, tumor size (the percentage of tumors >1.5 mm in diameter was calculated as the number of tumors with a diameter >1.5 mm divided by the total number of tumors).

In addition to differences in the average number of tumors, we found that tumor size was significantly greater in wild-type mice compared with Jnk2^-/- mice. Although similar from weeks 11 to 16 (Table 2 ; Fig. 3C ), the percentage of tumors >1.5 mm in diameter was greater in wild-type mice than in Jnk2^-/- mice beginning at week 17 until the end of the study (P < 0.003; Table 2 and Fig. 3C ). At 29 weeks, 62% (n = 101) of the total number of tumors (n = 164) in the wild-type group were >1.5 mm, whereas only 46% (n = 26) of the total number of tumors (n = 57) were >1.5 mm in diameter in Jnk2^-/- mice (Fig. 3C ; P < 0.01). These data strongly suggest that deficiency of the Jnk2 gene represses formation and growth of DMBA/TPA-induced skin tumors.

View larger version (102K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Pathology of papillomas. H&E-stained sections of tumors. Top panels, tumors from a wild-type (WT) and a Jnk2-/- mouse at the same level of magnification (x25). Bottom panels, the same sections at a higher magnification (x200).

View larger version (93K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Inhibition of AP-1 DNA binding activity. The dorsal skins of wild-type (WT; n = 5) and Jnk2-/- (n = 5) mice were biopsied, and the sample was incubated in lysis buffer 24 h after treatment with acetone or TPA (17 nmol/mouse). Protein concentration was determined, and equal amounts of protein (3 µg) were loaded in each lane. Sequence-specific AP-1 DNA binding activity was determined by gel shift analysis using a 32P-labeled oligonucleotide containing the AP-1 binding site as described in "Materials and Methods." Each lane shows a skin sample from a different mouse in each treatment group. A competition experiment (AP) using a 10-fold excess of cold AP-1 oligonucleotide indicated that the DNA binding was specific. Representative samples are shown.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. The effect of TPA on the phosphorylation of Erks. The dorsal skins of wild-type (WT; n = 5) and Jnk2-/- (n = 5) mice were biopsied, and the sample was placed in SDS sample buffer 3 h after treatment with acetone or TPA (17 nmol/mouse). The tissue lysates were extracted two times with acetone and suspended in SDS sample buffer again. Equal amounts of protein were analyzed by Western blotting using antibodies against phosphorylated Erks (New England BioLabs, Inc.). Each lane shows representative skin samples from a different mouse in each treatment group.

	DISCUSSION

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Chemically induced skin cancer in mice has three chronological stages, initiation, promotion, and progression (15 , 19) . Tumor initiation is a rapid and irreversible process, whereas promotion is a long-term process that requires chronic exposure to a tumor promoter. A tumor promoter increases proliferation of initiated cells, accelerating cancer progression; however, the exact mechanism of promotion is more complicated (19 , 20) . The role of JNK in mediating carcinogenesis is not clear. Recently, the JNK signaling pathway was found to be constitutively activated in pre-B cells transformed by the leukemogenic oncogene bcr-abl (21) . The expression of JIP-1, a cytoplasmic inhibitor of JNK, markedly inhibits transformation of pre-B cells by bcr-abl (22) . These data provide strong support for the hypothesis that the JNK signaling pathway contributes to malignant transformation of pre-B cells. From the current results, after 10 weeks of TPA treatment, tumors appeared in wild-type and Jnk2 knockout mice at almost the same time, and the incidence of tumors was similar between the two groups until week 17. This suggests that JNK2 deficiency does not affect tumor initiation and the beginning of promotion. However, the growth, external appearance and number of tumors were significantly different between the two groups. Moreover, at the end of the experiment (week 29 after TPA treatment), four tumors in wild-type mice were found to be malignant, whereas none were malignant in JNK2-deficient mice. These data indicate that JNK2 may play a more important role in tumor growth and progression than in tumor initiation.

TPA activates protein kinase C and, subsequently, transcription factor AP-1 (23) . The activation of signal transduction pathways leading to stimulation of AP-1 is a common mechanism for tumor promotion (24, 25, 26, 27, 28) . Blocking AP-1 activity prevents TPA-induced cell transformation in JB6 cells and tumor promotion in a mouse skin model (12 , 24 , 29 , 30) . The AP-1 family of transcription factors consists of homodimeric or heterodimeric complexes of c-Jun and c-Fos proteins (31) . A transgenic mouse model overexpressing c-fos developed osteosarcomas and chondrosarcomas (32) , and transgenic mice expressing an oncogenic form of jun developed fibrosarcomas at sites of wound healing (33) . The c-jun knockout mutation is embryonically lethal, whereas c-fos-deficient tumors fail to undergo malignant conversion (34) . Expression of a dominant-negative c-jun (Tam67) blocked tumor promoter-induced AP-1 transactivation and showed a dramatic inhibition of papilloma induction in these transgenic animals (35) . Topical application of perillyl alcohol inhibited UVB-induced AP-1 transactivation and significantly inhibited tumor incidence and multiplicity (36) . All of these data show that components of AP-1 are very important in modulating normal development and carcinogenesis.

AP-1 is one target of MAPK signaling. MAPKs modulate AP-1 activity both by increasing the abundance of AP-1 components and stimulating their activity (37) . JNK has been shown to phosphorylate c-Jun at serine 63 and serine 73 residues, resulting in activation of AP-1 (1 , 18) . The other MAPKs, Erks and p38 kinases, were found to induce c-Fos and c-Jun expression, resulting in increased AP-1 transcriptional activity (38, 39, 40, 41) . Considering that TPA does not induce AP-1 activity in JNK2-deficient mice (Fig. 5) , our present work suggests that the JNK2 pathway may be very important in mediating TPA-induced AP-1 binding activity. However, we found that TPA-induced c-Jun phosphorylation was not different between wild-type and JNK2-deficient mouse skin (data not shown), suggesting that JNK2 deficiency does not affect TPA-induced c-Jun phosphorylation. Because the phosphorylation of Erks induced by TPA was blocked in JNK2-deficient mice (Fig. 6) , the inhibition of Erks phosphorylation may lead to a decrease in AP-1 DNA binding activity. The exact mechanism by which JNK2 mediates Erks phosphorylation is not clear. Because of the lack of direct evidence that JNK2 activates Erks, we suggest that other molecules may mediate the activation of Erks by JNKs. Increasing numbers of additional proteins or protein kinases have been found to be substrates of JNKs. In our laboratory, we reported recently that p90^RSK (42) , histone 3 (43) , and p53⁴ are also substrates of JNKs in vivo and in vitro. Whether these proteins are related to the molecular mechanism of JNK2-mediated carcinogenesis is currently being investigated.

In summary, our studies show that deficiency of the Jnk2 gene inhibits the incidence, size, and number of TPA-promoted tumors. The fact that TPA treatment does not induce AP-1 DNA binding activity in JNK2-deficient mice may be related to the inhibition of Erks phosphorylation. These results strongly support a critical role for JNK2 in the tumor promotion process. The suppression of TPA-induced tumorigenesis in Jnk2 gene-deficient mice may be related to the inhibition of AP-1 DNA binding activity.

	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, Eagles Cancer Telethon Foundation, and NIH Grants CA77646, CA81064, and CA74916. R. A. F. is an investigator of the Howard Hughes Medical Institute.

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

³ The abbreviations used are: MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated protein kinase; JNK, c-Jun NH₂-terminal kinase; AP-1, activator protein-1; TPA, 12-O-tetradecanoylphorbol-13-acetate; DMBA, 7,12-dimethylbenz[a]anthracene.

⁴ Q-B. She and Z. Dong, unpublished data.

Received 10/26/00. Accepted 3/19/01.

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				G. Liu, A. Bode, W.-Y. Ma, S. Sang, C.-T. Ho, and Z. Dong Two Novel Glycosides from the Fruits of Morinda Citrifolia (Noni) Inhibit AP-1 Transactivation and Cell Transformation in the Mouse Epidermal JB6 Cell Line Cancer Res., August 1, 2001; 61(15): 5749 - 5756. [Abstract] [Full Text] [PDF]

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