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[Cancer Research 65, 7356-7362, August 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Deficiency of Protein Kinase C{alpha} in Mice Results in Impairment of Epidermal Hyperplasia and Enhancement of Tumor Formation in Two-Stage Skin Carcinogenesis

Takeshi Hara1, Yuriko Saito1, Takaaki Hirai1, Kenji Nakamura2, Kazuki Nakao3, Motoya Katsuki4 and Kazuhiro Chida1

1 Graduate School of Agricultural and Life Sciences, University of Tokyo; 2 Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan; 3 RIKEN Center for Developmental Biology, Hyogo, Japan; and 4 National Institute of Basic Biology, Aichi, Japan

Requests for reprints: Kazuhiro Chida, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. Phone: 81-3-5841-8152; Fax: 81-3-5841-8152; E-mail: acchida{at}mail.ecc.u-tokyo.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We generated a mouse strain lacking protein kinase C{alpha} (PKC{alpha}) and evaluated the significance of the enzyme in epithelial hyperplasia and tumor formation. PKC{alpha}-deficient mice exhibited increased susceptibility to tumor formation in two-stage skin carcinogenesis by single application of 7,12-dimethylbenz(a)anthracene (DMBA) for tumor initiation and repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) for tumor promotion. Tumor formation was not enhanced by DMBA or TPA treatment alone, suggesting that PKC{alpha} suppresses tumor promotion. However, the severity of epidermal hyperplasia induced by topical TPA treatment was markedly reduced. In mutant mice, the number of 5-bromo-2'-deoxyuridine–labeled epidermal basal keratinocytes increased 16 to 24 hours after topical TPA treatment as in the case of wild-type mice, but significantly decreased at 36 and 48 hours. Furthermore, the regenerating epithelium induced by skin wound significantly decreased in thickness but was not structurally impaired. The enhanced tumor formation may not be associated with epidermal hyperplasia. The induction levels of epidermal growth factor (EGF) receptor ligands, tumor growth factor {alpha} (TGF-{alpha}), and heparin-binding EGF-like growth factor, in the skin of mutant mice by TPA treatment were significantly lower than those in the skin of wild-type mice. PKC{alpha} may regulate the supply of these EGF receptor ligands in basal keratinocytes, resulting in a reduced epidermal hyperplasia severity in the mutant mice. We propose that PKC{alpha} positively regulates epidermal hyperplasia but negatively regulates tumor formation in two-stage skin carcinogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Protein kinase C (PKC) is a multigene family of phospholipid-dependent serine/threonine kinases that are activated by interactions with the polar head groups of membrane lipids produced by various extracellular stimuli (1, 2). Based on their structural similarities and activation mechanisms, the 10 PKC isotypes have been classified into three groups: conventional PKC ({alpha}, ßI, ßII, and {gamma}), novel PKC ({delta}, {varepsilon}, {eta}, and {theta}), and atypical PKC ({zeta} and {lambda}). Conventional PKC and novel PKC isotypes are activated by diacylglycerol. The presence of multiple PKC isotypes and their tissue-specific expression patterns suggest that different isotypes have different roles in regulating cell functions. The potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) strongly binds to and activates conventional PKC and novel PKC isotypes by replacing diacylglycerol (3), suggesting that these PKC isotypes are directly involved in tumor formation.

Of the PKC isotypes, PKC{alpha} is expressed in almost all cells and is involved in various signal transduction pathways (4). Furthermore, PKC{alpha} seems to function in tumorigenesis. PKC{alpha} overexpression enhances the proliferation and malignant progression of human glioma cells (5, 6) and breast cancer cells (7). PKC{alpha} mediates TPA-induced growth inhibition and morphologic changes in mammary epithelial cells (8, 9). The antisense oligonucleotides of PKC{alpha} inhibit the growth of human lung carcinoma A549, colon carcinoma Colo-205, bladder carcinoma T24 (10), and gastric cancer MKN45 cells (11), and induce apoptosis of glioblastoma multiforme cells (12). On the other hand, selective PKC{alpha} activation induces the apoptosis of gastric cancer cells (13) and prostate cancer LNCaP cells (14). There have been reports on the loss of PKC{alpha} in human tumor cell lines and carcinoma (15–19). The point mutation of the PKC{alpha} gene has been observed in pituitary and thyroid tumor cells (15, 16, 18). Furthermore, PKC{alpha} expression is absent in basal cell carcinomas (19) and adenocarcinomas in APCMIN mice (20). In spite of these, the roles of PKC{alpha} in tumorigenesis remain unclear.

Several reports using transgenic mice have shown the specific functions of PKC{alpha} in the epidermis (21–23). PKC{alpha} overexpression in basal keratinocytes after a single topical TPA treatment leads to severe intraepidermal inflammation and enhances the expressions of inflammatory proteins including tumor necrosis factor-{alpha} (TNF-{alpha}) and cyclooxygenase-2 (COX-2) in the epidermis (21, 23). However, PKC{alpha} overexpression does not induce the differentiation and proliferation of epidermal cells (21, 23) but induces keratinocyte apoptosis in culture (23). In two-stage skin carcinogenesis, which is a useful system for understanding the process of multistep human carcinogenesis, PKC{alpha} overexpression does not affect tumor formation (21, 22). These experiments on gain of functions suggest that PKC{alpha} does not affect epidermal cell proliferation and tumor formation. However, experiments on loss of functions may reveal a potent role of PKC{alpha} in carcinogenesis.

In the present study, to obtain further insight into the roles of PKC{alpha} in tumor formation, we generated PKC{alpha}-deficient mice and did two-stage skin carcinogenesis. Furthermore, we examined epidermal hyperplasia closely associated with the tumor promotion stage. Here, we report that PKC{alpha}-deficient mice are susceptible to skin tumor formation but have a significantly reduced severity of epidermal hyperplasia induced by TPA treatment or wound healing.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of protein kinase C{alpha}–deficient mice. Mouse genomic DNA clones with exons 9 to 13 of the PKC{alpha} gene (Prkca) were isolated as described previously (24). A 5.8 kb NotI-BlnI fragment containing exon 9 and a 3.6 kb BlnI-PstI fragment containing exon 10 were subcloned into the pBluescriptSK(+) vector containing a diphtheria toxin A gene (DT-A; Fig. 1). The loxP-flanked neomycin resistance gene (neor) was inserted into the BlnI site located in intron 9. The third loxP fragment was inserted into the 3.6 kb BlnI-PstI fragment at the XhoI site located in intron 10. The linearized vector was introduced into 129/SvJ-derived CCE embryonic stem cells by electroporation. Genomic DNA from G418-resistant embryonic stem clones was digested with BamHI or EcoRI and hybridized with a 5' or 3' probe. A targeted clone was injected into C57BL/6J blastocysts to generate chimeric mice. Males of these chimeric mice were bred with C57BL/6J. DNA from tail biopsies of the mice was analyzed to confirm the germ line transmission of the mutant allele by a PCR technique using the following primer set: 5'-GTT GAT ATG GAG AGA CCC AGC CCA AAG-3' and 5'-TAT TCC ATG CCC ACG TGC CCA CCA ACA-3' (Ensembl gene ID ENSMUSG50965). The resulting Prkca+/flox mice were bred with CAG-Cre C57BL/6J transgenic mice (25) to generate heterozygous mutant (Prkca+/–) mice with a genetic background of C57BL/6J and 129/SvJ (3:1). The Prkca+/– mice without Cre were selected and interbred to generate homozygous (Prkca–/–), Prkca+/–, and wild-type (Prkca+/+) mice for experiments. All experiments were carried out with strict adherence to guidelines for minimizing distress in experimental animals.



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  		Figure 1. Targeted disruption of Prkca. A, targeting strategy. Wild-type allele of Prkca that contains exons 9 to 12 (boxes), a targeting vector, targeted allele, and deleted allele by Cre recombinase are shown. The sites of restriction endonucleases are as follows: B, BamHI; RI, EcoRI. Triangles, loxP sites. The targeting vector contains neor and three loxP sites to disrupt exon 10, which encodes the NH2 terminus of the catalytic domain. B, Southern blot analysis of BamHI- and EcoRI-digested genomic DNAs of embryonic stem clones using 5' and 3' probes, respectively. The BamHI-digested fragments of 12.3 and 8.0 kb were derived from wild-type and targeted alleles, respectively. The EcoRI-digested fragments of 10.1 and 6.6 kb were derived from wild-type and targeted alleles, respectively. C, PCR for genotyping of wild-type, Prkca+/flox, Prkca+/–, and Prkca–/– mice. Amplicons of 893, 2132, and 243 bp were derived from wild-type, targeted, and deleted alleles, respectively. D, immunoblot analysis shows the absence of PKC{alpha} protein in Prkca–/– mice. E, immunoblot analysis shows no change of the expression levels of PKCßII, {delta}, {varepsilon}, {eta}, and {zeta} isotypes in skin of Prkca–/– mice.

 
Immunoblot analysis. Skin or epidermal extracts were subjected to SDS-PAGE, and the separated proteins were electrically blotted onto a nitrocellulose membrane. PKC proteins were detected using antibodies against PKC{alpha}, ßII, {delta}, {varepsilon}, {eta}, and {zeta} (Santa Cruz Biotechnology, Santa Cruz, CA). Antibodies against epidermal growth factor receptor (EGFR) and phosphotyrosine were purchased from Cell Signaling Technology (Beverly, MA) and BD Transduction Laboratories (Lexington, KY), respectively.

Immunohistochemistry. The mice were killed by cervical dislocation and their dorsal skin was removed. The skin was fixed in 4% formaldehyde and embedded in paraffin. For indirect immunochemistry, deparaffinized sections (4 µm) were blocked with 10% normal goat serum and then incubated with antibodies against keratin 1 (K1), keratin 5 (K5), and loricrin (Babco, Richmond, CA) at room temperature for 1 hour. After washing with PBS, the sections were incubated with a goat anti-rabbit IgG antibody conjugated with tetramethyl-rhodamine isothiocyanate. Proliferating cell nuclear antigen (PCNA) was detected using an anti-PCNA antibody conjugated with peroxidase (DAKO Japan, Kyoto, Japan).

Carcinogenesis experiments. Female mice at 6 weeks old were used. All the animals were housed under a 12-hour light/12-hour dark cycle in a controlled atmosphere. Their dorsal skin was shaved 1 week before 7,12-dimethylbenz(a)anthracene (DMBA) treatment, and appropriate chemicals dissolved in 0.2 mL of acetone were applied to the shaved area. Eighteen mice from each group (Table 1) were used for the two-stage carcinogenesis experiment described previously (26). DMBA (100 µg; Sigma, St. Louis, MO) was applied topically as an initiator 1 week before tumor promotion. TPA (10 µg; Sigma) was repeatedly applied twice a week for 20 weeks.


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  		Table 1. Tumor formation in two-stage skin carcinogenesis

 
Epidermal hyperplasia. Mice at 8 weeks old were used. Their dorsal skin was shaved 1 week before TPA treatment. After topical application of TPA (10 µg) to the shaved area, the mice were killed by cervical dislocation and their dorsal skin was removed for histologic examinations. Tissues were fixed in 4% formaldehyde and then embedded in paraffin. Sections (2-4 µm in thickness) were subjected to standard H&E staining.

For wound generation, the mice were anesthetized and their dorsal skins were shaved and washed with 70% ethanol. Two full-thickness excisions were made on the back of each mouse using a punch biopsy instrument of 2 mm inner diameter as previously reported (26).

Bromodeoxyuridine labeling. The mice were injected i.p. with BrdUrd in PBS (50 mg/kg body weight). Four hours later, the mice were killed. Their dorsal skin was isolated and subjected to immunohistochemistry using an antibromodeoxyuridine (BrdUrd) monoclonal antibody conjugated with peroxidase (Roche, Mannheim, Germany).

Reverse transcription-PCR analysis. Total RNAs were isolated from the skin samples and reverse-transcribed using an oligo(dT) primer. The resulting cDNA was used as a template for PCR. The following primer sets were used, and the sizes of each amplicon are also indicated: (a) glyceraldehyde-phosphate dehydrogenase (GAPDH), 5'-GTG AAG GTC GGT GTG AAC GGA TTT-3' and 5'-CAC AGT CTT CTG GGT GGC AGT GAT-3' (Genbank accession number M32599, nucleotides 50-604), 555 bp; (b) PKC{alpha}, 5'-TCA CAG ACT TCA ACT TCC TC-3' and 5'-CGA TTT TGA TGT GCC CTT CTG-3' (M25811, 1,262-1,693), 432 bp; (c) tumor growth factor {alpha} (TGF-{alpha}), 5'-CCT CCA GGG CCT CTG AAG TAA GGC ATG-3' and 5'-AAC CTG GGC TAC ACA GCA AGA CCT TGT-3' (U65016, 1,819-2,145), 327 bp; (d) heparin-binding EGF-like growth factor (HB-EGF), 5'-GCT GCC GTC GGT GAT GCT GAA GC-3' and 5'-GAT GAC AAG AAG ACA GAC G-3' (NM010415, 270-795), 526 bp; (e) keratinocyte growth factor (KGF)-1, 5'-ATG CGC AAA TGG ATA CTG ACA CGG-3' and 5'-CTT AGG TTA TTG CCA TAG GAA G-3' (BC052847, 347-932), 586 bp; (f) interleukin (IL)-1{alpha}, 5'-TGG CCA AAG TTC CTG ACT TGT TT-3' and 5'-CAG GTC ATT TAA CCA AGT GGT GCT-3' (X01450, 62-549), 488 bp; (g) IL-1ß, 5'-ATG GCA ACT GTT CCT GAA CTC AAC T-3' and 5'-CAG GAC AGG TAT AGA TTC TTT CCT TT-3' (BC011437, 69-631), 563 bp; (h) granulocyte macrophage colony-stimulating factor (GM-CSF), 5'-ATC AAA GAA GCC CTA AAC CTC CTG-3' and 5'-CTG GCC TGG GCT TCC TCA TT-3' (X03019, 138-473), 336 bp; (i) TGF-ß1, 5'-CGG GGC GAC CTG GGC ACC ATC CAT GAC-3' and 5'-CTG CTC CAC CTT GGG CTT GCG ACC CAC-3' (M13177, 1,082-1,486), 405 bp. The primers for TNF-{alpha} and COX-2 were used as previously described (23).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of protein kinase C{alpha}–deficient mice. As shown in Fig. 1, we constructed a targeting vector in which exon 10 of Prkca was flanked by loxP sites because this exon encodes the ATP-binding site essential for enzymatic activity. Homologous recombinant embryonic stem cell clones were confirmed by Southern blot analysis. Genotyping revealed that mice carrying a floxed Prkca allele (Prkca+/flox mice) and lacking exon 10 of the Prkca allele (Prkca–/– mice) were generated. Prkca–/– and Prkca+/– mice were born at the expected Mendelian frequency and did not show developmental abnormalities. These mutant mice were apparently normal for up to 18 months.

By immunoblot analysis, an 80 kDa> band corresponding to native PKC{alpha} was detected in skin proteins from the wild-type and Prkca+/– mice, but not from the Prkca–/– mice (Fig. 1). In these mutant mice, no accumulation of flanking PKC{alpha} molecules was detected. In addition, the expression levels of other PKCs of the ßII, {delta}, {varepsilon}, {eta}, and {zeta} isotypes in the Prkca–/– mice were not significantly different from those in the wild-type mice.

Histologic examinations revealed a normal architecture of the skin consisting of the dermis, epidermis and appendages in Prkca–/– mice (Fig. 2) and Prkca+/– mice (data not shown). The epidermal localization and expression levels of PCNA as a proliferation marker, and K1, K5, and loricrin as differentiation markers, were not different between the Prkca–/– and wild-type mice, indicating that PKC{alpha} is not essential for epidermal cell proliferation and differentiation under normal conditions in vivo.



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  		Figure 2. Epidermal cell proliferation and differentiation in Prkca–/– mice. The dorsal skin samples of 8-week-old female mice (A) and newborn mice (B and C) are shown. Bar, 50 µm. C, immunohistochemical staining for PCNA, K5, K1, and loricrin. Bar, 50 µm.

 
Tumor formation. To examine the potential effect of PKC{alpha} disruption on tumor formation, we did two-stage skin carcinogenesis in which DMBA was used as an initiator and TPA as a promoter. In the Prkca–/– mice, tumors appeared within 6 weeks of tumor induction and then increased in number continuously (Fig. 3). The incidence of tumor-bearing mice relative to the total number of mice reached 78% and 6.7 tumors/mouse at 20 weeks of tumor promotion (Table 1). In the wild-type mice, tumors appeared at 7 weeks, and the incidence of tumor-bearing mice reached 50% and 1.3 tumors/mouse at 20 weeks. The tumor size of the Prkca–/– mice was similar to that of the wild-type mice (data not shown). All the tumors were papillomas (data not shown). No carcinoma was observed. The mutation of c-Ha-ras at codon 61 was detected in 92% and 94% tumors of the wild-type and Prkca–/– mice, respectively (data not shown). Evidently, the Prkca–/– mice were significantly more susceptible to skin tumor formation than the wild-type mice.



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  		Figure 3. Time-dependent changes of tumor formation in two-stage skin carcinogenesis. Data of the wild-type mice ({circ}; group 1 in Table 1) and Prkca–/– mice ({bullet}; group 2 in Table 1) are shown. Tumors were initiated with DMBA (100 µg) and promoted by repeated topical applications of TPA (10 µg) twice a week for 20 weeks. A, incidence of tumors (percentage of tumor bearers). B, average number of tumors per mouse.

 
DMBA treatment alone without the tumor promotion stage did not induce tumors in Prkca–/– and the wild-type mice (Table 1). Furthermore, repeated treatments with TPA or acetone as a solvent control induced no tumors. These data suggest that PKC{alpha} suppresses skin tumor promotion.

Epidermal hyperplasia. We examined epidermal hyperplasia to determine the potential effect of PKC{alpha} disruption on keratinocyte proliferation. A single topical application of TPA at 10 µg induced the formation of a hyperplastic epidermis consisting of four to six nucleated cell layers of 42 ± 4.3 µm (mean ± SD) thickness on day 2 in the wild-type mice (Fig. 4). The epidermal thickness returned to the basal thickness of 13 ± 1.5 µm within 7 days. In the Prkca+/– mice, epidermal hyperplasia was observed as similar to that in the wild-type mice (data not shown). However, in the Prkca–/– mice, the epidermal cell layers were three to four with a thickness of 29 ± 5.5 µm on day 2. The thickness returned to the basal thickness within 4 days. The effects of TPA on the hyperplasia were dose dependent. However, leukocyte infiltration into the dermis was observed at similar levels in the case of the wild-type mice on days 1 to 4 after TPA treatment.



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  		Figure 4. Epidermal hyperplasia induced by TPA treatment or wound healing. A, the dorsal skin of 8-week-old female mice after topical treatment with 10 µg of TPA was excised and stained with H&E. The histologic results 1 and 2 days after TPA treatment are shown. Bar, 50 µm. B, the epidermal thickness (µm) after TPA treatment was measured at eight independent regions of the epidermis of the wild-type ({circ}) and Prkca–/– mice ({bullet}). Points, mean. The epidermal thicknesses of Prkca–/– mice 36, 48, and 72 hours after treatment with 10 µg of TPA were significantly reduced (P < 0.01, t test). Bars, SD (n = 5). C, dependency of TPA dosage on induction of epidermal hyperplasia at 48 hours. The epidermal thicknesses (points) of Prkca–/– mice were significantly reduced by treatment with 4 µg (P < 0.02) but not with 2 µg (P > 0.05) of TPA. Bars, SD (n = 4). D, epidermal hyperplasia induced by wound healing. Histology of wound site 3 days after the induction of a 2-mm-diameter wound with a punch biopsy instrument. Bar, 100 µm.

 
Furthermore, epidermal hyperplasia induced by wound healing was examined. Three days after inducing the punched injury on the dorsal skin, the wild-type mice showed eight or more cell layers in their regenerating epidermis (Fig. 4D). In contrast, the Prkca–/– mice significantly showed a thinner regenerating epidermis of approximately four cell layers. Leukocyte infiltration into the regenerating dermis at the healing site in the Prkca–/– mice was similar to that observed in the wild-type mice. The period required for complete healing in the Prkca–/– mice was ~13 days, which was almost the same as that in the wild-type mice.

DNA synthesis in basal cells in epidermis. We did BrdUrd labeling to examine basal keratinocyte DNA synthesis, which is synchronously induced at least thrice after topical TPA treatment (27). In the wild-type mice, the percentage of BrdUrd-labeled basal cells was 3.9%. After the TPA treatment, it increased to 23.2% at 16 hours, reached its maximum at 55.0% at 20 hours, and then decreased (Fig. 5). Then, the percentage of BrdUrd-labeled cells in the wild-type mice increased to 42.6 ± 6.2% at 36 hours and 30.6 ± 6.6% at 48 hours, indicating that DNA synthesis was synchronously induced thrice by the topical TPA treatment. In the Prkca–/– mice, the numbers of BrdUrd-labeled cells were almost the same as those in the wild-type mice at 0 and 16 to 24 hours. However, at 36 and 48 hours, the Prkca–/– mice showed significant decreases in the number of BrdUrd-labeled cells to 22.1 ± 6.0% and 14.0 ± 2.2% (P < 0.005), respectively. These data suggest that PKC{alpha} controls the second and subsequent cycles but not the first cycle of DNA synthesis induced by TPA in basal keratinocytes.



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  		Figure 5. Effects on basal keratinocyte DNA synthesis after topical TPA treatment. Four hours after BrdUrd injection, the mice were killed and their dorsal skin was isolated. A, arrows indicate BrdUrd-labeled cells. Counterstaining was done using hematoxylin. Bar, 50 µm. B, percentages of BrdUrd-labeled basal cells in the wild-type ({circ}) and Prkca–/– mice ({bullet}) 20, 36, and 48 hours after TPA treatment. Bars, SD (n = 4).

 
Expression levels of cytokines and inflammation-related proteins in skin after 12-O-tetradecanoylphorbol-13-acetate treatment. We examined the expression levels of cytokines that influence keratinocyte proliferation (28–30) and inflammation-related proteins in the skin after TPA treatment. The expressions of TGF-{alpha} and HB-EGF were slightly detected in the non–TPA-treated wild-type and Prkca–/– mice (Fig. 6). Three hours after TPA treatment, the expressions of TGF-{alpha} and HB-EGF were induced strongly in wild-type mice, but weakly in Prkca–/– mice. However, the expression levels of TNF-{alpha}, IL-1ß (Fig. 6), KGF-1, GM-CSF, IL-1{alpha}, and COX-2 did not differ between the Prkca–/– and wild-type mice (data not shown). EGF expression was not detected in the skin of mice (data not shown). Furthermore, tyrosine phosphorylation of an epidermal 175 kDa protein, which an anti-EGFR antibody recognizes, was stimulated 1 and 24 hours after TPA treatment of skin in the wild-type mice (Fig. 6). In Prkca–/– mice, the stimulation was observed at 1 hour but weakly at 24 hours. These data suggest that PKC{alpha} regulates the induction of TGF-{alpha} and HB-EGF.



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  		Figure 6. Expressions of cytokines and inflammation-related proteins in mouse skin after TPA treatment. A, total RNAs were isolated from skin 0, 3, 6, 16, and 24 hours after topical application of TPA at 10 µg. The expression levels were examined by reverse transcription-PCR analysis. TGF-{alpha}, HB-EGF, TNF-{alpha}, and IL-1ß are shown. GAPDH is used as internal control. B, expression levels relative to GAPDH in the wild-type ({circ}) and Prkca–/– mice ({bullet}) after TPA treatment. Bars, SD (n = 4). C, immunoblot analysis shows inhibition of tyrosine-phosphorylation (pTyr) of 175 kDa EGFR (arrows) in epidermis of Prkca–/– mice after TPA treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The loss of PKC{alpha} caused by the deletion of exon 10 in Prkca has no effect on the viability, development, and fertility of a mouse with the main genetic background of C57BL/6J. These findings are consistent with reports on other PKC{alpha}-deficient mice in which exon 2 (in mice of the 129 background; ref. 31) and exon 9 (32) are disrupted. Under normal conditions, the architecture of the skin, including the epidermis and appendages, and cell proliferation and differentiation in the epidermis are not altered by the loss of PKC{alpha}. However, the PKC{alpha}-deficient mouse shows a significant reduction in the severity of epidermal hyperplasia induced by topical TPA treatment or wound generation. Furthermore, the mutant mice show the enhancement of tumor formation, but not of malignancy, in two-stage skin carcinogenesis. Thus, the PKC{alpha}-deficient mouse can be a useful model for the understanding of the mechanisms underlying the induction of epithelial hyperplasia and tumor formation.

In mouse skin, TPA induces the synchronous proliferation of basal keratinocytes, resulting in epidermal hyperplasia (27, 33). The reduction in the severity of epidermal hyperplasia in the PKC{alpha}-deficient mouse is caused by the inhibition of the second and subsequent cycles of DNA synthesis in basal keratinocytes induced by TPA treatment; the first cycle of DNA synthesis is not affected. There are many reports on the roles of PKC{alpha} in keratinocyte proliferation and differentiation in vitro and in vivo (21, 23, 34–38). PKC{alpha} overexpression does not affect the proliferation and differentiation of primary human keratinocytes (35) and PKC{alpha}-transgenic mouse keratinocytes (21, 23). In contrast, PKC{alpha} activation triggers irreversible cell cycle withdrawal in human keratinocytes (37, 38). PKC{alpha} is necessary for the expression of some epidermal differentiation markers, such as loricrin and filaggrin, but not K1 and keratin 14 (34, 36). The apoptosis of PKC{alpha}-transgenic mouse keratinocytes in culture is induced by TPA treatment or UV irradiation (23). These suggest that PKC{alpha} does not stimulate epidermal cell proliferation but influences epidermal cell differentiation or apoptosis in some cases. However, based on our findings, PKC{alpha} regulates the proliferation of epidermal basal keratinocytes in vivo, possibly associated with the induction of TGF-{alpha} and HB-EGF by TPA treatment.

TPA treatment induces TGF-{alpha} in human keratinocytes (39, 40) and colon carcinoma cell lines (41), and HB-EGF in rat aortic smooth muscle cells (42). In mouse skin, the induction of TGF-{alpha} and HB-EGF by tumor promoters correlates with sustenance of the proliferation of basal keratinocytes (29, 43, 44). TPA-induced PKC activation is closely associated with EGFR transactivation (45–47). EGFR activation is necessary for the ectodomain shedding of pro–TGF-{alpha} (48) and pro–HB-EGF (49) by metalloproteases. The soluble forms of these EGFR ligands bind to and activate EGFR, following the phosphorylation and activation of Erk (47). These processes may be associated with the first cycle of DNA synthesis in basal keratinocytes induced by TPA treatment. The overexpression of PKC{delta}, but not PKC{alpha}, induces the shedding of pro–HB-EGF by MDC9/ADAM9 metalloprotease (49). After the TPA treatment, the extent of Erk phosphorylation in keratinocytes in the culture and skin in vivo was not significantly different between the PKC{alpha} mutant and wild-type mice (data not shown). Taken together, PKC{alpha} may function in the induction of EGFR ligands but not in EGFR transactivation in keratinocytes. The decrease in the expression levels of TGF-{alpha} and HB-EGF by TPA treatment possibly inhibits the second and subsequent cycles of DNA synthesis in epidermal basal keratinocytes in vivo.

Epidermal hyperplasia is closely associated with skin inflammation (50). Some reports suggest a relationship between immunologic responses and PKC{alpha} activation (21, 23, 51, 52). However, PKC{alpha} disruption does not influence acute skin inflammation induced by TPA treatment. The discrepancy in the roles of PKC{alpha} in inflammation might have been due to the difference in experiments done (i.e., experiments on loss and gain of functions of the enzyme). PKC{alpha} can be regulated in intracellular localization, activation, and substrate accession by some proteins, e.g., PICK1 (53) and PDK1 (54, 55). These proteins may not completely regulate PKC{alpha} overexpression. Conversely, the loss of PKC{alpha} may disturb the states of such proteins. As another possibility, the genetic background of the mice used may be a major source of the discrepancy.

PKC{alpha} deficiency enhances skin tumor formation. However, PKC{alpha} overexpression does not influence tumor formation (21, 22). There are some reports on skin tumor formation in transgenic and knockout mice induced by other PKC isotypes that are activated by TPA and expressed in the epidermis (22, 26, 56, 57). Transgenic mice that overexpress PKC{delta} or PKC{varepsilon} in the basal keratinocytes of their epidermis are resistant to TPA-induced tumor formation but sensitive to epidermal hyperplasia induced by TPA treatment (22, 56, 57). PKC{eta} disruption enhances tumor formation in the promotion stage and prolongs epidermal hyperplasia (26). Thus, conventional PKC and novel PKC isotypes in the epidermis may not enhance but inhibit tumor formation. However, there are different roles of PKC isotypes in epidermal hyperplasia. From the viewpoint of PKC activation, tumor promotion may not be associated with epidermal hyperplasia induced by TPA treatment. As a functional mechanism, it was hypothesized that the down-regulation of epidermal PKCs is associated with tumor promotion and induces papilloma growth (58–60). PKC{alpha} is down-regulated by repeated TPA applications on the skin, and its expression level is low in two-stage skin carcinogenesis (21). Our results partially support this hypothesis. However, there is evidence against the association between PKC down-regulation and skin tumor promotion (61).

Our findings suggest that PKC{alpha} suppresses tumor formation in some tissues, including the skin, in accordance with previous reports (15, 16, 18–20). If PKC{alpha} suppresses tumor formation, it can also affect cell cycle regulation, apoptosis, DNA repair, cell movement, or the attachment of the extracellular matrix. However, further studies are necessary to elucidate the roles of PKC{alpha} in tumor formation and malignant conversion at the molecular and cellular levels.


   Acknowledgments
 
Grant support: Grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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.

We thank Dr. M. Seiki (Institute of Medical Science, University of Tokyo) and Dr. H. Nakayama (Department of Veterinary Medical Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo) for helpful discussions, Dr. T. Tezuka (Institute of Medical Science, University of Tokyo) for the gift of EGFR, and Dr. J. Miyazaki (Graduate School of Medicine, Osaka University, Osaka, Japan) for the gift of CAG-Cre mice.

Received 11/29/04. Revised 4/11/05. Accepted 5/25/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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