Peroxisome proliferator-activated receptor-beta/delta inhibits epidermal cell proliferation by down-regulation of kinase activity

Recent work has shown that peroxisome proliferator-activated receptor beta (PPARbeta) attenuates cell proliferation and skin carcinogenesis, and this is due in part to regulation of ubiquitin C expression. In these studies, the role of PPARbeta in modulating ubiquitin-dependent protein kinase Calpha (PKCalpha) levels and phosphorylation signaling pathways was evaluated. Intracellular phosphorylation analysis showed that phosphorylated PKCalpha and other kinases were lower in wild-type mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) as compared with PPARbeta-null mouse skin. No differences in expression levels of other PKC isoforms present in skin were observed. Lower ubiquitination of PKCalpha was found in TPA-treated PPARbeta-null skin as compared with wild-type, and inhibition of ubiquitin-dependent proteasome degradation prevented TPA-induced down-regulation of PKCalpha. The activity of PKCalpha and downstream signaling kinases is enhanced, and expression of cyclooxygenase-2 (COX-2) is significantly greater, in PPARbeta-null mouse skin in response to TPA compared with wild-type mouse skin. Inhibition of PKCalpha or COX-2 reduced cell proliferation in TPA-treated PPARbeta-null keratinocytes in a dose-dependent manner, whereas it only slightly influenced cell proliferation in wild-type keratinocytes. Combined, these studies provide strong evidence that PPARbeta attenuates cell proliferation by modulating PKCalpha/Raf1/MEK/ERK activity that may be due in part to reduced ubiquitin-dependent turnover of PKCalpha.     

Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor beta(delta)

To determine the physiological roles of peroxisome proliferator-activated receptor beta (PPARbeta), null mice were constructed by targeted disruption of the ligand binding domain of the murine PPARbeta gene. Homozygous PPARbeta-null term fetuses were smaller than controls, and this phenotype persisted postnatally. Gonadal adipose stores were smaller, and constitutive mRNA levels of CD36 were higher, in PPARbeta-null mice than in controls. In the brain, myelination of the corpus callosum was altered in PPARbeta-null mice. PPARbeta was not required for induction of mRNAs involved in epidermal differentiation induced by O-tetradecanoylphorbol-13-acetate (TPA). The hyperplastic response observed in the epidermis after TPA application was significantly greater in the PPARbeta-null mice than in controls. Inflammation induced by TPA in the skin was lower in wild-type mice fed sulindac than in similarly treated PPARbeta-null mice. These results are the first to provide in vivo evidence of significant roles for PPARbeta in development, myelination of the corpus callosum, lipid metabolism, and epidermal cell proliferation.     

PPARbeta/delta selectively induces differentiation and inhibits cell proliferation

Peroxisome proliferator-activated receptor (PPAR) beta-null mice exhibit exacerbated epithelial cell proliferation and enhanced sensitivity to skin carcinogenesis, suggesting that ligand activation of PPARbeta will inhibit keratinocyte proliferation. By using of a highly specific ligand (GW0742) and the PPARbeta-null mouse model, activation of PPARbeta was found to selectively induce keratinocyte terminal differentiation and inhibit keratinocyte proliferation. Additionally, GW0742 was found to be anti-inflammatory due to inhibition of myeloperoxidase activity, independent of PPARbeta. These data suggest that ligand activation of PPARbeta could be a novel approach to selectively induce differentiation and inhibit cell proliferation, thus representing a new molecular target for the treatment of skin disorders resulting from altered cell proliferation such as psoriasis and cancer.     

The genetic control of chemically and radiation-induced skin tumorigenesis: a study with carcinogenesis-susceptible and carcinogenesis-resistant mice

Outbred carcinogenesis-resistant (Car-R) and carcinogenesis-susceptible (Car-S) mouse lines were generated by phenotypic selection for resistance or susceptibility to two-stage skin carcinogenesis. These two Car mouse lines differ by >100-fold in susceptibility. In the present study, we tested the hypothesis that a subset of genetic loci responsible for susceptibility or resistance to chemical skin tumorigenesis may also be involved in radiation-induced skin tumorigenesis. Skin tumorigenesis was tested in groups of Car-S/R mice after X-ray initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion. We found that ionizing radiation can initiate skin tumors in Car-S mice but not in Car-R mice. In Car-S mice, the most effective radiation doses (6 and 10 Gy given in four fractions) gave a threefold increase in tumor multiplicity and a twofold increase in tumor incidence compared to a TPA-only control group. We performed a molecular analysis of Hras gene mutations in skin tumors of Car-S mice induced by X-ray initiation/TPA promotion or by TPA promotion alone. The most notable difference emerging from the comparison of these mutation patterns is the high incidence ( approximately 50%) of papillomas lacking Hras gene mutations in X-ray-initiated/TPA-promoted papillomas compared to 13% in papillomas induced by TPA alone, suggesting that lack of Hras gene mutations is a consistent feature of radiation-induced papillomas.     

Bezafibrate is a dual ligand for PPARalpha and PPARbeta: studies using null mice

Bezafibrate is a known activator of peroxisome proliferator-activated receptors (PPARs) that can activate both PPARalpha and PPARbeta. To determine the role(s) of these receptors in mediating the biological effects of this chemical, the effect of bezafibrate was examined in PPARalpha-null and PPARbeta-null mice. Wild-type, PPARalpha-null, or PPARbeta-null mice were fed either a control diet or one containing 0.5% bezafibrate for 10 days. Bezafibrate feeding caused a significant increase in liver weight in wild-type and PPARbeta-null mice compared to controls, while liver weight was unchanged in bezafibrate-fed PPARalpha-null mice. Gonadal adipose stores were significantly smaller in wild-type and PPARbeta-null mice fed bezafibrate than in controls, and this effect was not found in similarly fed PPARalpha-null mice. Analysis of liver, white adipose tissue, and intestinal mRNAs showed that bezafibrate caused similar changes of mRNAs encoding lipid metabolizing enzymes in wild-type and PPARbeta-null mice compared to controls. Interestingly, in PPARalpha-null mice, bezafibrate also induced several mRNAs previously thought to be solely controlled by PPARalpha, showing that the effects of this drug are not exclusively modulated by this PPAR isoform. Western blot analysis of liver protein was consistent with changes in mRNA expression showing that the alterations in mRNA expression correlate with protein expression in this tissue. Results from these studies demonstrate that the effect of bezafibrate is mediated in large part by PPARalpha, although some changes in gene expression are dependent on PPARbeta. In contrast to other PPARalpha ligands such as WY-14,643, induction of some target genes by bezafibrate can also be modulated in the absence of a functional PPARalpha.     

Palytoxin: exploiting a novel skin tumor promoter to explore signal transduction and carcinogenesis

Palytoxin is a novel skin tumor promoter, which has been used to help probe the role of different types of signaling mechanisms in carcinogenesis. The multistage mouse skin model indicates that tumor promotion is an early, prolonged, and reversible phase of carcinogenesis. Understanding the molecular mechanisms underlying tumor promotion is therefore important for developing strategies to prevent and treat cancer. Naturally occurring tumor promoters that bind to specific cellular receptors have proven to be useful tools for investigating important biochemical events in multistage carcinogenesis. For example, the identification of protein kinase C as the receptor for the prototypical skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) (also called phorbol 12-myristate 13-acetate, PMA) provided key evidence that tumor promotion involves the aberrant modulation of signaling cascades that govern cell fate and function. The subsequent discovery that palytoxin, a marine toxin isolated from zoanthids (genus Palythoa), is a potent skin tumor promoter yet does not activate protein kinase C indicated that investigating palytoxin action could help reveal new aspects of tumor promotion. Interestingly, the putative receptor for palytoxin is the Na⁺,K⁺-ATPase. This review focuses on palytoxin-stimulated signaling and how palytoxin has been used to investigate alternate biochemical mechanisms by which important targets in carcinogenesis can be modulated. mitogen-activated protein kinase; dual-specificity phosphatase; prostaglandins; sodium, potassium adenosinetriphosphatase     

Induction of histidine decarboxylase activity in mouse skin after application of indole alkaloids,a new class of tumor promoter

The effects of various promoters in two-step carcinogenesis on the induction of histidine decarboxylase in the skin of mice was investigated. The potencies of various phorbol esters in inducing histidine decarboxylase activity were parallel with their tumor-promoting activities. Indole alkaloids such as dihydroteleocidin B and lyngbyatoxin A, which induced ornithine decarboxylase and promoted tumor development in the skin of mice with the same potency as 12-O-tetradecanoylphorbol-13-acetate (TPA), also induced histidine decarboxylase activity. These results suggest that histamine produced by this inducible histidine decarboxylase may play some role in tumor promotion.     

Peroxisome proliferator-activated receptor beta/delta regulates very low density lipoprotein production and catabolism in mice on a Western diet

The results of recent studies using selective agonists for peroxisome proliferator-activated receptor beta (PPARbeta) suggest that this receptor may have a role in regulating levels of serum lipids in animal models of obesity and insulin resistance. To further examine this possibility, serum lipid profiles of mice lacking a functional PPARbeta receptor were determined. PPARbeta-null mice maintained on either normal chow or a 10-week high fat (HF) diet, a condition that has been shown to induce insulin resistance and obesity in mice, have elevated levels of serum triglycerides primarily associated with very low density lipoprotein (VLDL) with no difference in either total cholesterol or phospholipids. Consistent with this finding, PPARbeta-null mice on a HF-diet were shown to have an increased rate of hepatic VLDL production as well as lowered lipoprotein lipase activity in serum compared with wild-type controls. The latter parallels an increase in the hepatic expression of the genes encoding angiopoietin-like proteins 3 and 4 in PPARbeta-null mice on a HF diet, both proteins of which have recently been shown to inhibit lipoprotein lipase (LPL) activity in vivo. Consistent with elevated VLDL production, a marked increase in plasma VLDL apoB48, -E, -AI, and -AII, as well as a sharp depletion of the hepatic lipid stores was also found in PPARbeta-null mice. In addition, PPARbeta-null mice on a HF diet were shown to have increased adiposity, despite lower total body weight. Together, these results indicate a clear role for PPARbeta in regulating levels of serum triglycerides in mice on a high fat Western diet by modulating both VLDL production and LPL-mediated catabolism of VLDL-triglycerides and also suggest a potential therapeutic role for PPARbeta in the improvement of serum lipids in the setting of metabolic syndrome.     

Cholera toxin, a potent inducer of epidermal hyperplasia but with no tumor promoting activity in mouse skin carcinogenesis

Intracutaneous injection of cholera toxin into mice induced epidermal hyperplasia to a greater extent than 12-O-tetradecanoylphorbol-13-acetate. It also induced adenylate cyclase and though weakly, ornithine decarboxylase of the epidermis. Cholera toxin, however, showed no tumor promoting activity in mouse skin carcinogenesis. In the single stage promotion, cholera toxin (50 ng) was injected once a week for 10 weeks into the skin of SENCAR mice initiated with 25 micrograms 7,12-dimethylbenz[a]anthracene, but no tumors developed. In the two-stage promotion test, cholera toxin (10-100 ng) was injected for one or two weeks into the initiated skin and then mezerein (4 micrograms) was applied twice a week for 18 weeks, but the toxin did not increase incidence or numbers of papillomas.     

Biological and molecular aspects of radiation carcinogenesis in mouse skin

The process of mouse skin carcinogenesis can be operationally subdivided into at least three stages which have been termed initiation, promotion, and progression. Ionizing radiation has been found to be a weak initiator of malignant squamous cell carcinomas (SCCs) when radiation was followed by repeated treatments of the skin with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Besides SCCs, ionizing radiation was found to induce, independent of tumor promoters, basal cell carcinomas (BCCs), a tumor histology not normally seen with chemical carcinogens and mouse skin. Fractionated doses of 1 MeV electrons were found to enhance the conversion of chemically induced benign papillomas to malignant SCCs. In addition to the biological studies, questions related to dominant transforming genes and differential gene expression in the radiation-initiated mouse skin tumors have been explored. Distinct non-ras dominant transforming gene(s) have been detected in radiation-initiated, TPA-promoted SCCs. Differences in the expression pattern of tumor-associated genes were seen in comparing chemically to radiation-induced benign and malignant skin tumors. Therefore, ionizing radiation has been shown to be active in the initiation of malignant skin tumors and progression of benign to malignant tumors in the mouse skin. The ability to divide the process of carcinogenesis into multiple stages in the mouse skin model has facilitated mechanistic studies that may elucidate the molecular pathways involved in radiation-versus chemically induced tumor development.     

Enhanced papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene.

We have studied the induction of papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene and in their nontransgenic littermates. The transgenic animals displayed a basal epidermal ornithine decarboxylase activity that was nearly 20 times higher than in their nontransgenic littermates. A single topical application of 12-O-tetradecanoylphorbol-13-acetate induced a much more profound and longer-lasting increase in transgene-derived ornithine decarboxylase activity in comparison with the endogenous enzyme activity. Initiation of skin tumorigenesis with a single topical application of dimethylbenz[a]antracene followed by twice-weekly application of 12-O-tetradecanoylphorbol-13-acetate resulted in the appearance of first papillomas both in nontransgenic and transgenic animals by week 7. However, after 11 weeks of 12-O-tetradecanoylphorbol-13-acetate application, the number of papillomas per animal was almost 100% higher in the transgenic animals than in their nontransgenic littermates. These results indicate that an overexpression of epidermal ornithine decarboxylase confers a growth advantage on skin tumors in vivo.     

Mice deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis.

Given the associations between chronic inflammation and epithelial cancer, we studied susceptibility to skin carcinogenesis in mice deficient for the pro-inflammatory cytokine TNF-alpha (refs. 5,6). TNF-alpha(-/-) mice were resistant to development of benign and malignant skin tumors, whether induced by initiation with DMBA and promotion with TPA or by repeated dosing with DMBA. TNF-alpha(-/-) mice developed 5-10% the number of tumors developed by wild-type mice during initiation/promotion and 25% of those in wild-type mice after repeated carcinogen treatment. TNF-alpha could influence tumor and stromal cells during tumor development. The early stages of TPA promotion are characterized by keratinocyte hyperproliferation and inflammation. These were diminished in TNF-alpha(-/-) mice. TNF-alpha was extensively induced in the epidermis, but not the dermis, in TPA-treated wild-type skin, indicating that dermal inflammation is controlled by keratinocyte TNF-alpha production. Deletion of a TNF-alpha inducible chemokine also conferred some resistance to skin tumor development. TNF-alpha has little influence on later stages of carcinogenesis, as tumors in wild-type and TNF-alpha(-/-) mice had similar rates of malignant progression. These data provide evidence that a pro-inflammatory cytokine is required for de novo carcinogenesis and that TNF-alpha is important to the early stages of tumor promotion. Strategies that neutralize TNF-alpha production may be useful in cancer treatment and prevention.     

Effect of Onosma echioides on DMBA/croton oil mediated carcinogenic response, hyperproliferation and oxidative damage in murine skin

The current study unveils the effect of O. echioides extract on two-stage skin carcinogenesis and on tumor promoter induced markers and oxidative stress in Swiss mice. Treatment of dorsal shaven cutaneous portions of the mice with single topical application of benzoyl peroxide (BPO) followed by exposure to ultraviolet B (UVB) radiation induced significant oxidative stress and elevated the marker parameters of tumor promotion. Similar effects were observed with 12-O-tetradecanoyl phorbol-13-acetate (TPA) treatment. Pretreatment of O. echioides extract (5 mg & 10 mg/Kg b.wt) in both the studies with BPO+UVB and TPA restored the levels of reduced glutathione (GSH) and cellular protective enzymes (p < 0.05). Concomitantly, malondialdehyde (MDA) formation and hydrogen peroxide (H2O2) content were also reduced significantly (p < 0.05) at both the doses. The promotion parameters tested (ornithine decarboxylase activity and DNA synthesis) were also significantly suppressed (p < 0.05). Thereafter, we proceeded with studies on mouse skin carcinogenesis. After ten days of 7,12-dimethylbenz(a)anthracene (DMBA) treatment, twice-weekly applications of croton oil for 20 weeks resulted in 100% incidence of tumors in the animals. However, O. echioides showed reduction in the number of tumors/ mouse and percentage of tumor bearing mice at the end of the study. The study was further histologically confirmed. The protective activity of the plant might be due to the two major constituents (alkannins and shikonins) present in the plant. O. echioides is thus, proposed to be helpful in prevention of experimental skin carcinogenesis.     

Collagenase expression in transgenic mouse skin causes hyperkeratosis and acanthosis and increases susceptibility to tumorigenesis.

In a series of transgenic mice, the human tissue collagenase gene was expressed in the suprabasal layer of the skin epidermis. Visually, the mice had dry and scaly skin which upon histological analysis revealed acanthosis, hyperkeratosis, and epidermal hyperplasia. At the ultrastructural level, intercellular granular materials were absent in the transgenic skin epidermis but contact was maintained through the intact desmosomes. Despite a diversity of underlying etiologies, similar morphological hyperproliferative changes in the epidermis are observed in the human skin diseases of lamellar ichthyosis, atopic dermatitis, and psoriasis. Subsequent experiments demonstrate that when the transgenic mouse skin was treated once with an initiator (7,12-dimethyl-benz[a]anthracene) and then twice weekly with a promoter (12-O-tetradecanoylphorbol-13-acetate), there was a marked increase in tumor incidence among transgenic mice compared with that among control littermates. These experiments demonstrate that by overexpressing the highly specific proteolytic enzyme collagenase, a cascade of events leading to profound morphological changes which augment the sensitivity of the skin towards carcinogenesis is initiated in the epidermis.     

Only a subset of 12-O-tetradecanoylphorbol-13-acetate-promoted mouse skin papillomas are promotable by benzoyl peroxide

The two-stage skin carcinogenesis model of initiation and promotion in Carcinogenesis-susceptible (Car-S) mice has been used to investigate the pathways of promotional activity of 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester tumor promoter, and benzoyl peroxide (BzPo), a free radical-generating compound. To test whether distinct populations of 9,10-dimethyl-1,2-benzanthracene (DMBA)-initiated epidermal keratinocytes are responsive to the two promoters, tandem experiments were performed. DMBA-initiated Car-S mice were promoted twice weekly with maximal promoting doses of TPA or BzPo. When the number of papillomas/mouse reached a plateau, promotion in the TPA and BzPo groups was switched to BzPo or TPA, respectively, until achievement of a new plateau. Mice promoted with BzPo developed 11.0 +/- 1.3 papillomas/mouse and subsequent TPA promotion induced 13.8 additional papillomas, for a total of 24.8 +/- 2.1 papillomas/mouse. TPA-promoted mice developed 23.3 +/- 1.1 papillomas/mouse, and subsequent BzPo promotion for 91 days did not promote additional papillomas. Our results show a less than additive tumor response after sequential promotion with BzPo and TPA, or vice versa, indicating that the pathways of promotional activity of TPA and BzPo are interacting. While the final papilloma yield was similar at the end of the two tandem promotion experiments independently of promoter sequence, the percentage of mice developing carcinomas was significantly higher in mice that were promoted with BzPo in the first stage. No significant differences in the frequency and type of c-Ha-ras mutations were observed in TPA- and BzPo-promoted tumors, suggesting that promotion of DMBA-initiated cells by BzPo requires introduction of additional molecular alterations compared to TPA.     

Antitumor agents 220. Antitumor-promoting effects of cimigenol and related compounds on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis

Cimigenol (1) and 39 related compounds were screened as potential antitumor promoters by examining the ability of the compounds to inhibit Epstein-Barr virus early antigen (EBV-EA) activation (induced by 12-O-tetradecanoylphorbol-13-acetate) in Raji cells. Structure-activity relationship analysis indicated that compound 1 showed the highest activity and also exhibited significant inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. These data suggest that 1 and the related compounds might be valuable anti-tumor promoters.     

Progressive iron overload enhances chemically mediated tumor promotion in murine skin

Iron overload has been shown to enhance chemically mediated cutaneous tumor promotion in animals. However, the majority of these animal studies have used high concentrations of iron before initiating tumor development. The current study was designed to evaluate the effect of small doses of iron on the promotion stage of chemically mediated cutaneous carcinogenesis. We found an increased tumor response in mice initiated with dimethylbenz(a)anthracene (DMBA) when iron at the dose levels of 0.5, 1.0, and 1.5mg/mouse was injected (intramuscularly) once a week into mice at the promotion stage of skin carcinogenesis, employing 12-O-tetradecanoyl phorbol-13-acetate (TPA)/benzoyl peroxide (BPO) as tumor promoter. The appearance of first papilloma and the number of tumors/mouse were recorded weekly. When compared to the control (non-iron-treated) group, the iron-treated groups showed an augmented incidence of tumors and number of tumors/mouse. In iron-treated mice, tumors appeared earlier than in the control group. TPA/BPO treatment resulted in a significant decrease in the activities of antioxidant enzymes and depletion in the level of epidermal reduced glutathione (GSH). TPA treatment in non-iron-treated mice resulted in approximately 20-40% decrease in GSH level and in the activities of antioxidant enzymes, whereas 1.5-mg iron treatment along with TPA treatment resulted in about approximately 30-70% decrease in GSH level and in the activities of antioxidant enzymes. Similarly, treatment of iron along with BPO treatment resulted in a dose-dependent higher depletion of GSH and the antioxidant enzymes as compared to non-iron-treated animals treated with BPO. Further, TPA/BPO-mediated induction in ornithine decarboxylase activity and [3H]thymidine incorporation in cutaneous DNA was approx two- to threefold higher in mice treated with iron as compared to non-iron-treated mice. Cutaneous lipid peroxidation and iron levels were also higher in mice treated with iron as compared to non-iron-treated mice. These data suggest that progressive iron overload can enhance the tumor promotion ability of TPA/BPO in DMBA-initiated murine skin.     

Ethanol specifically decreases peroxisome proliferator activated receptor beta in B12 oligodendrocyte-like cells

Peroxisome proliferator activated receptors (PPARs) are nuclear receptors that control important genes involved in lipid metabolism. Their role in nerve cells is uncertain, although anomalous myelination of the corpus callosum has been described in the PPARbeta-null mouse, and abnormalities of this tissue have been documented in fetal alcohol syndrome in humans. We report here that ethanol treatment of B12 oligodendrocyte-like cells induces a concentration- and time-dependent decrease in the mRNA and protein levels of PPARbeta, with no effect on PPARalpha or PPARgamma. The effect on PPARbeta is seen as an increase in mRNA degradation, as assessed by run-off assays, due to a significant decrease in PPARbeta mRNA half-life, with no observed changes in intracellular localization. Our results suggest a possible link between PPARbeta function and ethanol-induced abnormal myelination in oligodendrocytes.     

Transgenic overexpression of transforming growth factor alpha bypasses the need for c-Ha-ras mutations in mouse skin tumorigenesis.

The induction of skin papillomas in mice can be divided into two different stages. Chemical initiation frequently elicits mutations in the Ha-ras gene, leading to the constitutive activation of ras. The second step, promotion, involves repetitive topical application of phorbol esters or wounding, leading to epidermal hyperproliferation and papilloma formation. We have found that overexpression of transforming growth factor alpha (TGF-alpha) in the basal epidermal layer of transgenic mice yielded papillomas directly upon wounding or 12-O-tetradecanoylphorbol-13-acetate treatment without the need for an initiator. Moreover, papillomas from TGF-alpha mice did not exhibit mutations in the Ha-ras gene. Interestingly, TGF-alpha acted synergistically with 12-O-tetradecanoylphorbol-13-acetate to enhance epidermal hyperproliferation. Our results demonstrate a central role for TGF-alpha overexpression in tumorigenesis and provide an important animal model for the study of skin tumorigenesis.