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Epidemiology and Prevention |
Is Linked to 12-O-tetradecanoylphorbol-13-acetate-induced Tumor Necrosis Factor-
Ectodomain Shedding and the Development of Metastatic Squamous Cell Carcinoma in Protein Kinase C Transgenic Mice1
Departments of Human Oncology [D. L. W., K. J. N., A. K. V.] and Pathology and Laboratory Medicine [T. D. O.], Medical School, University of Wisconsin, Madison, Wisconsin 53792
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PKC), a Ca2+-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress (
18-fold) PKC protein in basal epidermal cells and cells of the hair follicle develop papilloma-independent metastatic squamous cell carcinoma (mSCC) elicited by 7,12-dimethylbenz(a)anthracene-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion protocol. We now present that PKC transgenic mice elicit elevated serum tumor necrosis factor (TNF)
levels during skin tumor promotion by TPA, and this increase may be linked to the development of mSCC. A single topical application of TPA (5 nmol) to the skin, as early as 2.5 h after treatment, resulted in a significant (P < 0.01) increase (2-fold) in epidermal TNF and more than a 6-fold increase in ectodomain shedding of TNF into the serum of PKC transgenic mice relative to their wild-type littermates. Furthermore, this TPA-stimulated TNF shedding was proportional to the level of expression of PKC in the epidermis. Using the TNF- converting enzyme (TACE) inhibitor, TAPI-1, TPA-stimulated TNF shedding could be completely prevented in PKC transgenic mice and isolated keratinocytes. These results indicate that PKC signal transduction pathways to TPA-stimulated TNF ectodomain shedding are mediated by TACE, a transmembrane metalloprotease. Using the superoxide dismutase mimetic CuDIPs and the glutathione reductase mimetic ebselen, TPA-stimulated TNF shedding from PKC transgenic mice could be completely attenuated, implying the role of reactive oxygen species. Finally, i.p. injection of a TNF synthesis inhibitor, pentoxifylline, during skin tumor promotion completely prevented the development of mSCC in PKC transgenic mice. Taken together, these results indicate that: (a) PKC activation is an initial signal in TPA-induced shedding of TNF from epidermal keratinocytes; (b) PKC-mediated signals to TACE are possibly mediated through reactive oxygen species; and (c) TPA-induced TNF shedding may play a role in the development of mSCC in PKC transgenic mice. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ß I, ß II, and 
) are dependent on PS, DAG, and calcium for their activation. The nPKCs (
, , 
, and 
) retain responsiveness to DAG and PS but do not require calcium for full activation. The aPKCs (
and 
) only require PS for their activation. The members of the PKC family exhibit functional diversity in their roles in the regulation of gene expression, cell growth, differentiation, and apoptosis (10, 11, 12, 13, 14, 15, 16)
. PKC has been well documented as an oncogene (17
, 18)
.
To determine the in vivo functional specificity of PKC in TPA-activated PKC signals to skin tumor multiplicity, we generated transgenic mice that express T7-epitope-tagged PKC in their epidermis. The expression of PKC was directed to the basal cells of the epidermis and cells of the hair follicle using a human cytokeratin14 (K14) promoter (19
, 20)
. This overexpression of PKC in the mouse epidermis resulted in the rapid development of papilloma-independent metastatic squamous cell carcinomas (19
, 20)
. However, the mechanism by which PKC overexpression leads to the development of mSCC remains to be determined. Evidence indicates that the proinflammatory cytokine TNF is linked to skin tumor promotion by TPA (21
, 22)
and UV light (23)
. Experiments using tumor promoters of the okadaic acid class have provided strong evidence that TNF is the central mediator of tumor promotion in the mouse skin. These experiments indicated that TNF shed from the initiated cell or various tissues surrounding the initiated lesion can induce clonal expansion and transformation of initiated cells (21)
. This work led to the development of in vivo mouse models, which have further implicated TNF as the key cytokine for tumor promotion in the mouse skin. Using either the two-stage model of carcinogenesis or UV light, mice deficient for TNF or either of its receptors render the mice resistant to skin tumor formation (22, 23, 24)
.
TNF is a potent proinflammatory cytokine that is produced by a multitude of cell types, including macrophages, lymphocytes, monocytes, fibroblasts, and keratinocytes. This molecule was originally discovered as a cytotoxic cytokine for tumor cells and its ability to cause necrosis of transplanted tumors (24)
. Mature murine TNF consists of 156 amino acids (157 in humans); however, the molecule is translated with a 79 amino acid (76 in humans) long precursor sequence. For TNF to exert its pleiotropic inflammatory responses at distant sites from its synthesis, it must be cleaved from the membrane in a process called ectodomain shedding. A specific enzyme called TACE cleaves pro-TNF in response to extracellular stimuli (25
, 26)
. The cloning of TACE (human and porcine; Refs. 25
and 26
) revealed it to be a member of the "A disintegrin and metalloprotease" or ADAM family of proteins. The TACE protein is a multidomain, type I transmembrane protein that includes a zinc-dependent catalytic domain. The protein is broken into six domains: (a) prodomain; (b) catalytic domain; (c) disintegrin domain; (d) cysteine-rich domain; (e) transmembrane domain; and (f) the cytoplasmic domain. The prodomain contains a cysteine residue that interacts with a zinc molecule in the catalytic domain. This interaction must be displaced for TACE activation and is believed to be mediated by ROS (27)
. On its release, TNF exerts its biological effects by trimerizing and binding to two distinct receptors, TNFR1 and TNFR2. Binding of TNF induces trimerization of each of these receptors, which then recruit several signaling proteins to the cytoplasmic membrane (28)
. With the ability to activate two distinct receptors and recruit different receptor signaling complexes, TNF can regulate a vast array of cellular responses, including cellular inflammation, immunity, cell proliferation, differentiation, and apoptosis.
Here, we present evidence using FVB/N transgenic mice which overexpress PKC in their basal epidermis and cells of the hair follicle: (a) PKC mediates TPA-induced TNF shedding, through the metalloprotease TACE; (b) generation of ROS is perhaps a PKC downstream event in TPA-induced event in TPA-stimulated TNF shedding; and (c) TPA-stimulated ectodomain shedding of TNF may be linked to the development of mSCC.
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MATERIALS AND METHODS |
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PKC Transgenic Mice.
PKC transgenic mice were generated as described (19
, 20)
. Transgenic mice were maintained by mating hemizygous transgenic mice with wild-type FVB/N mice. The mice were housed in groups of two to three in plastic bottom cages in light-, humidity-, and temperature-controlled rooms; food and water were available ad libitum. The animals were kept in a normal rhythm of 12-h light and 12-h dark periods. The transgene was detected by PCR analysis using genomic DNA isolated from 1-cm tail clips.
Generation of PKC-null Mice.
The PKC KO mice were generated and provided by Dr. Michael Leitges. Briefly, the ES cell line used for targeting was E14 from the mouse strain 129/Ola. The embryonic stem cells from 129/Ola were introduced into the blastocyte of C57BL/6. The germ-line chimeras were identified by the presence of agouti coat color in the F1 progeny. These chimeric mice (C57Bl/6/129/Ola) were bred for eight generations for mutant transmission to FVB/N mice for a unified genetic background.
Tumor Induction Experiments.
Mouse skin tumors were induced by the initiation–promotion regimen (19
, 20)
. For mouse skin tumor initiation, a single 100-nmol dose of DMBA in 0.2 ml of acetone was applied topically to the shaved backs of mice. Two weeks after initiation, TPA in 0.2 ml of acetone was applied twice weekly to the skin for the duration of the experiment. Tumor multiplicity was observed every other week. Carcinomas were recorded grossly as downward-invading lesions, which were confirmed histologically by the board-certified anatomical pathologist (T. D. O.).
Cytokine Analysis.
TNF was quantified by ELISA using R&D Systems Mouse TNF-/TNFSF2 Quantikine ELISA Kit (R&D Systems) or the optEIA mouse TNF ELISA kit (PharMingen). Serum was collected by drawing blood from mice, incubating at room temperature for 30 min, followed by centrifuging for 10 min at 5000 rpm. Fresh serum was used for ELISA analysis. For analysis of TNF in the media, media from treated and untreated cells were collected, centrifuged, and analyzed using either the Quantikine ELISA Kit (R&D Systems) or the optEIA mouse TNF ELISA kit (PharMingen). TNF release to the media was normalized to the number of cells plated at the time of collection.
Immunoblotting of PKC Isoforms.
Mice were shaved and depilated before experimentation. The mouse skin was excised and scraped to remove the s.c. fat. The epidermis was removed and homogenized in IP lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 200 µM Na3VO4, 200 µM NaF, and 1 mM EGTA]. The homogenate was centrifuged at 14,000 x g for 30 min at 4°C. One-hundred micrograms of whole cell lysate were fractionated on a 7.5% SDS-polyacrylamide gel. The proteins were transferred to 0.45 µm of Hybond-P polyvinylidene difluoride transfer membrane (Amersham). The membrane was then incubated with the appropriate primary and secondary antibodies, and the detection signal was developed with Amersham’s enhanced chemiluminescence reagent. Rabbit polyclonal antibodies to PKC , , , µ, and 
were used at a 1:1000 dilution to detect the respective PKC isoforms.
Keratinocyte Preparation.
Newborn mice were sacrificed by CO2 asphyxiation. The mice were soaked in betadine for 5 min, washed twice with 70% ethanol, and rinsed in water. The skins were removed, placed in a 150-mm dish containing 0.25% trypsin, and incubated at 4°C overnight. The skins were placed in a clean dish dermis side up, and using fine needle tweezers, the epidermis was separated from the dermis. The epidermis was minced with sterile scissors and placed in high calcium solution (Eagles MEM, 1.8 mM Ca2+, Earle’s salts, L-glutamine, 0.25% penicillin/streptomycin, and nonessential amino acids). The separated dermis was rinsed in high calcium solution to isolate any remaining portions of the epidermis. The suspension was filtered through a 100-µm polyester gauge filter and spun at 1000 x g for 3–5 min at 4°C. The supernatant was discarded, and the cells were resuspended in a solution of 1 ml of cold high calcium solution followed by the addition of 6 ml of low calcium solution (Eagles S-MEM, Earle’s salts, L-glutamine, 0.25% penicillin/streptomycin, and nonessential amino acids, 8% Chelex-100-treated FBS, Ca2+ adjusted to 0.05 mM). The cells were plated at 1/2 epidermis per 60-mm dish in 4 ml/dish and incubated for 4–24 h. Once the cells adhered, they were washed three times with sterile PBS and maintained in low calcium solution.
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RESULTS |
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in the Mouse Epidermis Leads to Increased TPA-induced TNF Ectodomain Shedding. has been proposed to be an endogenous tumor promoter of the mouse skin (21
, 22)
. Two independent laboratories have shown that mice null for TNF and their receptors are resistant to mouse skin tumor formation elicited by chemical carcinogenesis (DMBA-TPA, DMBA-OA protocols; Refs. 21
and 22
) or by photocarcinogenesis (29)
. This prompted us to determine the role of TNF in the development of mSCC in PKC transgenic mice. First, we determined if PKC transgenic mice have increased TNF in their serum during skin tumor promotion. In this experiment, the dorsal skins of PKC transgenic mice (n = 3) and their wild-type littermates were shaved and initiated with 100 nmol of DMBA and followed by twice weekly treatments of TPA (5 nmol) for 7 weeks (Fig. 1)
. The serum was collected at 3, 6, 12, 18, 24, 36, and 96 h after the 15th TPA treatment and analyzed by ELISA for TNF. Chronic TPA treatment of PKC transgenic mice led to sustained levels of TNF in the serum at least until 36-h post-TPA, whereas the wild-type littermates had a peak of TNF shed to the serum at 3 h and returned to undetectable levels by 6 h. We further determined the effects of a single application of TPA on TNF shed into the serum in PKC transgenic mice. In this experiment, TPA (5 nmol) in 0.2 ml of acetone or acetone alone was applied to the shaved backs of PKC transgenic mice or their wild-type littermates. Mice were sacrificed at the indicated times, and serum was collected and analyzed by ELISA for TNF. Serum TNF was not detectable in either the acetone-treated PKC transgenic mice or their wild-type littermates (Fig. 2A)
. However, the effect of a single TPA application on serum TNF levels was especially dramatic in PKC transgenic mice. TPA treatment resulted in an elevated level of serum TNF at 2.5, 4, and 6 h after treatment in PKC transgenic mice relative to their wild-type littermates.
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transgenic mice and their wild-type littermates to TPA-stimulated TNF levels in the epidermis is illustrated in Figs. 2, B–D
. TPA-stimulated epidermal TNF levels in wild-type mice at 1, 2.5, 4, 6, 12, and 24 h after TPA treatment was 1.6, 2.6, 1.4, 1.8, 1.3, and 1.5-fold, respectively, above acetone control (Fig. 2B)
. TPA-stimulated epidermal TNF levels in PKC transgenic mice at 1, 2.5, 4, 6, 12, and 24 h after TPA treatment was 1.8, 3.8, 1.6, 1.6, 1.2, and 1-fold, respectively, above acetone control (Fig. 2C)
. TPA treatment appears to elevate epidermal TNF levels in PKC transgenic mice at 2.5 h after treatment relative to their wild-type littermates (Fig. 2D)
. Epidermal TNF levels in acetone treated wild-type and PKC transgenic mice were not significantly different (P > 0.1) at 1, 2.5, 4, 6, and 12 h after treatment (Fig. 2, B and C)
.
TPA-induced TNF Ectodomain Shedding Is Linked to the Level of Expression of PKC in the Mouse Skin.
We performed a series of experiments (Figs. 3
and 4
) to determine whether PKC expression level and activity in the mouse epidermis correlate with TNF shed into the serum in PKC transgenic mice. First, we compared two different PKC transgenic mouse lines 224 and 215, which express PKC 8- and 18-fold over wild type, respectively. As shown in Fig. 3
, TPA-induced epidermal shedding of TNF into the serum was directly proportional to the level of PKC in the mouse epidermis (Fig. 3
, inset).
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shedding are shown in Fig. 4
. In these experiments (Fig. 4)
, the dorsal skins of PKC transgenic and their wild-type littermates (n = 4) were shaved, and then 30 min before TPA (5 nmol) treatment, 3 µmol PC or 0.6 µmol of Bis I in acetone or acetone alone was applied to the shaved backs of PKC transgenic mice and their wild-type littermates. The mice were sacrificed 2.5 h after TPA treatment, and serum TNF levels were measured. PC treatment completely blocked the shedding of TNF into the serum in PKC transgenic mice (Fig. 4A)
. Bis I also completely blocked shedding of TNF into the serum of PKC transgenic mice (Fig. 4B)
. The detection of serum TNF in the wild-type was below the minimum level of detection by ELISA.
The role of PKC in TPA-induced TNF in mouse primary keratinocytes was further evaluated using PKC KO mice. PKC KO mice were generated with the LacZNeo cassette (Fig. 5A)
by interrupting the initiating codon. The genetic background used was 129/Ola and C57BL/6 strains. These mice (C57Bl/6/129/Ola) were bred for eight generations for mutant transmission to FVB/N for a unified genetic background. Western blot analysis of epidermal extract from the dorsal skin indicated a lack of PKC protein in the PKC KO FVB/N mouse epidermis, whereas the heterozygote mice contained less protein than wild-type mice (Fig. 5A
, inset and B). The possibility was explored that loss of PKC may result in compensatory alterations in the level of expression of other PKC isoforms (Fig. 5B)
. The levels of expression of various PKC isoforms in epidermal extracts from untreated wild-type mice and mice heterozygous and homozygous null at the PKC allele were determined by immmunoblot analysis. Using actin as an equal loading control, it appears that none of the other PKC isoforms was either lost or dramatically elevated. To determine the role of PKC in TPA-induced TNF shedding, keratinocytes from PKC KO FVB/N mice and their wild-type littermates were prepared. The keratinocytes were treated with either the vehicle ethanol or 100 nM TPA for 24 h, and TNF levels in the media were determined. As shown in Fig. 5C
, both ethanol and TPA-induced TNF release in the media at 24-h post-treatment was significantly (P < 0.01) reduced in PKC-null primary keratinocytes relative to the wild-type keratinocytes.
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Ectodomain Shedding in PKC Transgenic Mice. transgenic mice with either chronic or single application of 5 nmol TPA led to increased levels of TNF into the serum, these experiments did not provide clues about the source of TNF. We determined whether mouse keratinocytes are the primary source of TNF release into the serum. In these experiments (Fig. 6)
, we isolated primary keratinocytes from 1-day-old PKC transgenic pups (line 215) and their wild-type littermates. The cells were plated and then treated with 100 nM TPA in ethanol or ethanol alone. The media were collected and analyzed for TNF. TNF levels were normalized to the number of cells plated and expressed as picograms of TNF per million cells. Fig. 6A
illustrates that PKC transgenic mice had a consistent significant increase (P < 0.001) in TNF in the media relative to the wild-type keratinocytes after TPA stimulation. To link TPA-induced TNF shedding in PKC primary keratinocytes to TACE activity, we used a TACE inhibitor called TAPI-1. TACE is a transmembrane metalloprotease that cleaves the membrane bound precursor of TNF to its soluble form (25
, 26)
. The specificity of TAPI-1 toward TACE inhibition as well as dosage required to inhibit TACE both in vivo and in vitro are reported by Mohler et al. (30)
. Primary keratinocytes isolated from PKC transgenic mice (line 215) when treated with TAPI-1 (133 µM) in conjunction with 100 nM TPA for 24 h significantly (P < 0.001) prevented shedding of TNF in the media (Fig. 6B)
. It is noteworthy that although TAPI-1 treatment caused the prevention of TPA-induced release of TNF in the media, this prevention was accompanied by increased accumulation of TNF in the primary keratinocytes (Fig. 6C)
. To determine whether TAPI-1 could prevent release of TNF in PKC transgenic mice in vivo, the dorsal skin of PKC transgenic mice and their wild-type littermates were shaved and then injected s.c. with 1.5 mg of TAPI-1 before treatment with TPA (5 nmol). The mice were sacrificed 2.5 h after TPA treatment, and serum was collected for analysis of TNF level. As shown in Fig. 6D
, TPA-induced TNF shedding from the intact mouse skin in vivo was completely prevented when treated with TAPI-1.
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Ectodomain Shedding in PKC Transgenic Mice. shedding in PKC transgenic mice, we used inhibitors of the generation of ROS (CuDIPS and ebeslen). CuDIPS is a superoxide dismutase biomimetic (32)
and depletes superoxide anions, whereas ebselen is a Glutathione peroxidase mimetic and depletes lipid hydroperoxides (33)
. The dorsal skins of PKC transgenic mice and their wild-type littermates (n = 4) were shaved, and then 30 min before TPA treatment (5 nmol), mice were treated with either 2 µmol of CuDips in 0.2 ml of acetone or acetone alone or 810 nmol of ebselen in 0.2 ml of acetone or acetone alone. The mice were sacrificed 2.5 h after TPA treatment, and serum was collected and analyzed for TNF in the serum. Fig. 7
shows that both antioxidants completely prevented TNF ectodomain shedding in PKC transgenic mice.
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Synthesis Using Pentoxifylline Completely Prevents the Development of mSCC. shedding in the development of mSCC in PKC transgenic mice, we performed tumor promotion experiments using pentoxifylline, the TNF synthesis inhibitor. In this experiment, PKC transgenic mice and their wild-type littermates (n = 25) were shaved and initiated with 100 nmol of DMBA in 0.2 ml of acetone. One week later, mice were injected i.p. with either 0.2 ml of sterile PBS or pentoxifylline (50 µg/gram body weight) in 0.2 ml of sterile PBS 30 min before the application of 5 nmol TPA to the dorsal skin twice weekly during the entire duration of the experiment. To ascertain that pentoxifylline inhibited the shedding of TNF into the serum during the skin tumor promotion protocol, mice were collected (n = 4) 2.5 h after the last TPA treatment and assayed for serum TNF (Fig. 8A)
. After 18 weeks of tumor promotion, wild-type mice had an average papilloma burden of 8.92 ± 0.913, whereas wild-type mice treated with pentoxifylline had a significant reduction in their average papilloma burden to 5.32 ± 0.579 (P < 0.005; Fig. 8B
). A portion (40%; 10 of 25) of the PKC transgenic mice receiving PBS injections had at least one carcinoma after 18 weeks of tumor promotion. However, none (0 of 25) of the PKC transgenic mice receiving pentoxifylline injections before TPA treatment developed mSCC (Fig. 8C)
. Representative groups of mice are depicted in Fig. 8D
. Mice in this tumor induction experiment were checked daily for any toxic side effects associated with pentoxifylline. Pentoxifylline treatment did not affect the body weight gains, and survival in each treatment group varied from 90 to 100%.
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DISCUSSION |
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is a calcium-independent, TPA/DAG-activated serine/threonine kinase. PKC participates in the regulation of diverse cellular functions, including gene expression (34, 35, 36)
, neoplastic transformation (17
, 18)
, cell adhesion (37)
, mitogenicity (38
, 39)
, and cellular motility (40)
. There is now direct evidence that PKC may mediate its oncogenic properties by directly activating the classic mitogenic signaling pathway involving Ras and Raf-1 kinase (38
, 39
, 41, 42, 43)
. Alternatively, TGFß family members have been proposed to be, in part, responsible for the downstream effects of PKC (36)
. Rat-6 fibroblasts which overexpress PKC have been shown to secrete active forms of TGFß 2 and TGFß 3 in conjunction with a yet unidentified mitogen, indicating that growth-stimulating autocrine/paracrine loops may be involved in the oncogenic activity of PKC (36)
. In addition to being able to transform rodent cells, gene transfer experiments have shown that PKC regulates tumor invasion in Matrigel assays and nude mice (44)
. Evidence indicates that the actin binding site in the C1 domain of PKC may be critical for invasion but not necessary for tumorigenicity, implying that PKC must interact with the actin cytoskeleton to enhance metastatic potential (44)
. This interaction was shown to stimulate polymerization and extensions of actin protrusions used for penetration of the surrounding tissue (44)
. We found PKC mediates the development of mSCC when overexpressed in the mouse skin elicited by the initiation (DMBA), promotion (TPA) protocol (19
, 20)
. We now present that TNF may be a downstream messenger in PKC signaling network to the development of mSCC.
TNF levels are chronically elevated in the serum during mouse skin tumor promotion (Fig. 1)
. TNF is a highly regulated molecule, and the source of increased circulating levels during TPA promotion in PKC transgenic mice can be explained. Experiments using radiolabelled TNF revealed that TNF is cleared from the serum with a half-life of 6–7 min (45)
. This suggests that PKC transgenic mice maintain the serum levels by chronic release of this molecule. In this context, the effects of TPA on TNF level in mouse epidermis are noteworthy. TPA treatment resulted in a 2-fold increase in epidermal TNF, whereas a 6-fold increase in serum TNF in PKC transgenic mice as compared with their wild-type littermates. TPA treatment resulted in 2-fold increase in epidermal TNF mRNA levels in PKC transgenic mice relative to their wild-type littermates (data not shown). This may account for the TPA-induced increase (2-fold) in epidermal TNF levels. An alternative interpretation for the TPA-induced epidermal rise in TNF may be attributable to increased shedding of TNF from the epidermal keratinocytes to the serum, its clearance from the blood, and subsequent concentration in the skin. Experiments reported previously using radiolabelled TNF indicated that 30% of serum TNF concentrated to the skin. Additional data to support that PKC affects ectodomain shedding are supported by the results illustrated in Figs. 3
and 4
. The release of TNF to the blood was directly proportional to the level of the transgene expressed in the skin of PKC transgenic mice. Furthermore, primary keratinocytes from PKC transgenic mice were more sensitive than keratinocytes from wild-type littermates to TPA-induced TNF release. However, TPA-induced TNF release was not completely prevented in PKC-null keratinocytes, indicating that there may be functional overlap in the regulation of TNF release (Fig. 5)
.
TACE is the key metalloprotease that catalyzes the shedding of the proform of TNF into its mature soluble form (25
, 26)
. To determine whether PKC mediates the shedding of TNF from epidermal keratinocytes via TACE, we inhibited TACE activity using the TACE inhibitor TAPI-1 (25)
. This blockade both in intact mouse skin in vivo and cultured primary keratinocytes in vitro (Fig. 6)
indicated that TPA-mediated shedding of TNF in PKC transgenic mice is regulated through increased TACE activity. The TACE molecule is a member of the ADAM family (a disintegrin and metalloprotease domain). This enzyme is synthesized initially in a latent form which contains a highly conserved inhibitory prodomain (25
, 26) . It has been shown that this prodomain inhibits TACE catalytic activity by interaction between the thiol group from a cysteine residue in the prodomain and a zinc molecule in the catalytic domain (46
, 47)
. Disruption of this cysteine-zinc bond results in conformational changes, resulting in an active molecule. Furthermore, it has been shown that ROS and nitrogen radicals can oxidize this zinc thiol bond and thus create an active enzyme (48, 49, 50, 51)
. This activation mediated through ROS has been shown for TACE (27)
. To explore the role of ROS in PKC-mediated TNF shedding, we used the antioxidants CuDIPs and ebselen, an superoxide dismutase and glutathione peroxidase mimetic, respectively (Fig. 7)
. Both of these ROS scavengers completely prevented the release of TNF in PKC transgenic mice. Taken together, these results indicate that overexpression of PKC may result in generation of ROS leading to an activation of TACE by oxidation of the zinc thiol cysteine bond.
The finding that the inhibition of TPA-induced TNF shedding completely prevents the development of mSCC indicates that TNF is perhaps the key downstream component of PKC signaling pathway to the development of mSCC (Fig. 8)
. In this experiment (Fig. 8)
, we used a pharmacological inhibitor pentoxifylline to inhibit the synthesis of TNF in vivo. Pentoxifylline is a methylxanthine derivative that has been used for >20 years to treat patients with peripheral vascular disease (21
, 22)
. A generally accepted mechanism of action for pentoxifylline is the inhibition of phosphodiesterases, leading to increased intracellular levels of cAMP which negatively regulate the synthesis of TNF (52)
. Inhibiting the synthesis and thus release of TNF from epidermal keratinocytes in PKC transgenic mice completely prevented the development mSCC (Fig. 8)
. A previous study using pentoxifylline has shown that it decreases cutaneous inflammation and has decreased the DMBA/TPA-induced papilloma formation (53)
. This suggests a role of inflammation in the development of papillomas in mice, and increased release of TNF in PKC transgenic mice may have dramatic effects on s.c. inflammation and thus lead to the rapid develop of mSCC with interactions of inflammatory cells in the epidermis. Because pentoxifylline is not a specific inhibitor of TNF, the results illustrated in Fig. 8, B–D
should be interpreted with caution. Pentoxifylline caused inhibition of the development of mSCC in PKC transgenic mice may be attributable to the effect of pentoxifylline on epidermal cAMP levels. Increased epidermal cAMP levels have been shown to inhibit carcinoma formation in the mouse skin (54)
. To prove the link of TNF to the PKC signaling to the development of mSCC, additional experiments using TNF KO mice crossed with PKC transgenic mice are warranted. Furthermore, PKC signaling to the development of carcinomas via TNF may involve the role of the AP-1 family member of transcription factors (e.g., c-fos) and p53 (55, 56, 57)
.
In summary, the results indicate that: (a) PKC is an initial signal in TPA-induced TNF ectodomain shedding; (b) keratinocytes appears to be the primary source of TPA-stimulated TNF shedding in PKC transgenic mice; (c) PKC may regulate TNF processing in epidermal keratinocytes through ROS and the membrane bound metalloprotease TACE; and (d) blocking of TNF shedding in PKC transgenic mice by inhibiting its synthesis can completely prevent the development of mSCC in the mouse skin. We conclude that TNF may be a useful biomarker for the prognosis of squamous cell carcinoma of the skin and that intervention by neutralizing TNF may be considered for therapy of mSCC.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH Grant CA35368. 
2 To whom requests for reprints should be addressed, at Department of Human Oncology, Medical School, University of Wisconsin, Madison, WI 53792.
3 The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; PS, phosphatidylserine; cAMP, cyclic AMP; TNF, tumor necrosis factor; ROS, reactive oxygen species; TACE, tumor necrosis factor converting enzyme; TAPI-1, tumor necrosis factor processing inhibitor-1; TGF, transforming growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; PC, palmitoylcarnitine; KO, knockout; CuDIPS, Copper (II; 3,5-diisopropyl-salicylate)2; Bis I, Bisindolylmaleimide; DMBA, 7,12-dimethylbenz(a)anthracene; OA, okadaic acid.
Received 4/10/03. Revised 6/28/03. Accepted 7/14/03.
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REFERENCES |
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in their epidermis exhibit reduced papilloma burden but enhanced carcinoma formation after tumor promotion. Cancer Res., 60: 595-602, 2000. transgenic mice: a unique model for metastatic squamous cell carcinoma. Cancer Res., 61: 808-812, 2001.) in tumor promotion as revealed by TNF--deficient mice. Cancer Res., 59: 4516-4518, 1999.This article has been cited by other articles:
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 M. Meier, J. Menne, J.-K. Park, M. Holtz, F. Gueler, T. Kirsch, M. Schiffer, M. Mengel, C. Lindschau, M. Leitges, et al. Deletion of Protein Kinase C-{varepsilon} Signaling Pathway Induces Glomerulosclerosis and Tubulointerstitial Fibrosis In Vivo J. Am. Soc. Nephrol., April 1, 2007; 18(4): 1190 - 1198. [Abstract] [Full Text] [PDF]
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M. H. Aziz, H. T. Manoharan, and A. K. Verma Protein Kinase C{varepsilon}, which Sensitizes Skin to Sun's UV Radiation-Induced Cutaneous Damage and Development of Squamous Cell Carcinomas, Associates with Stat3 Cancer Res., February 1, 2007; 67(3): 1385 - 1394. [Abstract] [Full Text] [PDF]
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 S. Balasubramanian, L. Zhu, and R. L. Eckert Apigenin Inhibition of Involucrin Gene Expression Is Associated with a Specific Reduction in Phosphorylation of Protein Kinase C{delta} Tyr311 J. Biol. Chem., November 24, 2006; 281(47): 36162 - 36172. [Abstract] [Full Text] [PDF]
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 K.-P. Xu, J. Yin, and F.-S. X. Yu SRC-family tyrosine kinases in wound- and ligand-induced epidermal growth factor receptor activation in human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci., July 1, 2006; 47(7): 2832 - 2839. [Abstract] [Full Text] [PDF]
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 E. Lessmann, M. Leitges, and M. Huber A redundant role for PKC-{varepsilon} in mast cell signaling and effector function Int. Immunol., May 1, 2006; 18(5): 767 - 773. [Abstract] [Full Text] [PDF]
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M. H. Aziz, D. L. Wheeler, B. Bhamb, and A. K. Verma Protein Kinase C {delta} Overexpressing Transgenic Mice Are Resistant to Chemically but not to UV Radiation-Induced Development of Squamous Cell Carcinomas: A Possible Link to Specific Cytokines and Cyclooxygenase-2 Cancer Res., January 15, 2006; 66(2): 713 - 722. [Abstract] [Full Text] [PDF]
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A. M. Gonzalez-Guerrico and M. G. Kazanietz Phorbol Ester-induced Apoptosis in Prostate Cancer Cells via Autocrine Activation of the Extrinsic Apoptotic Cascade: A KEY ROLE FOR PROTEIN KINASE C{delta} J. Biol. Chem., November 25, 2005; 280(47): 38982 - 38991. [Abstract] [Full Text] [PDF]
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F. Chu, J. M. Koomen, R. Kobayashi, and C. A. O'Brian Identification of an Inactivating Cysteine Switch in Protein Kinase C{varepsilon}, a Rational Target for the Design of Protein Kinase C{varepsilon}-Inhibitory Cancer Therapeutics Cancer Res., November 15, 2005; 65(22): 10478 - 10485. [Abstract] [Full Text] [PDF]
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 Y. Li, D. L. Wheeler, W. Alters, L. Chaiswing, A. K. Verma, and T. D. Oberley Early Epidermal Destruction with Subsequent Epidermal Hyperplasia Is a Unique Feature of the Papilloma-Independent Squamous Cell Carcinoma Phenotype in PKC{varepsilon} Overexpressing Transgenic Mice Toxicol Pathol, October 1, 2005; 33(6): 684 - 694. [Abstract] [Full Text] [PDF]
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B. Worden, X. P. Yang, T. L. Lee, L. Bagain, N. T. Yeh, J. G. Cohen, C. Van Waes, and Z. Chen Hepatocyte Growth Factor/Scatter Factor Differentially Regulates Expression of Proangiogenic Factors through Egr-1 in Head and Neck Squamous Cell Carcinoma Cancer Res., August 15, 2005; 65(16): 7071 - 7080. [Abstract] [Full Text] [PDF]
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 G. Klein, A. Schaefer, D. Hilfiker-Kleiner, D. Oppermann, P. Shukla, A. Quint, E. Podewski, A. Hilfiker, F. Schroder, M. Leitges, et al. Increased Collagen Deposition and Diastolic Dysfunction but Preserved Myocardial Hypertrophy After Pressure Overload in Mice Lacking PKC{epsilon} Circ. Res., April 15, 2005; 96(7): 748 - 755. [Abstract] [Full Text] [PDF]
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 D. L. Wheeler, P. J. Reddig, K. J. Ness, C. P. Leith, T. D. Oberley, and A. K. Verma Overexpression of Protein Kinase C-{epsilon} in the Mouse Epidermis Leads to a Spontaneous Myeloproliferative-Like Disease Am. J. Pathol., January 1, 2005; 166(1): 117 - 126. [Abstract] [Full Text] [PDF]
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D. L. Wheeler, K. E. Martin, K. J. Ness, Y. Li, N. E. Dreckschmidt, M. Wartman, H. N. Ananthaswamy, D. L. Mitchell, and A. K. Verma Protein Kinase C {epsilon} Is an Endogenous Photosensitizer That Enhances Ultraviolet Radiation-Induced Cutaneous Damage and Development of Squamous Cell Carcinomas1 Cancer Res., November 1, 2004; 64(21): 7756 - 7765. [Abstract] [Full Text] [PDF]
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 C. D. Woodworth, E. Michael, L. Smith, K. Vijayachandra, A. Glick, H. Hennings, and S. H. Yuspa Strain-dependent differences in malignant conversion of mouse skin tumors is an inherent property of the epidermal keratinocyte Carcinogenesis, September 1, 2004; 25(9): 1771 - 1778. [Abstract] [Full Text] [PDF]
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S. Naus, M. Richter, D. Wildeboer, M. Moss, M. Schachner, and J. W. Bartsch Ectodomain Shedding of the Neural Recognition Molecule CHL1 by the Metalloprotease-disintegrin ADAM8 Promotes Neurite Outgrowth and Suppresses Neuronal Cell Death J. Biol. Chem., April 16, 2004; 279(16): 16083 - 16090. [Abstract] [Full Text] [PDF]
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