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.     

Tyrosine phosphorylation of I-kappa B kinase alpha/beta by protein kinase C-dependent c-Src activation is involved in TNF-alpha-induced cyclooxygenase-2 expression

The signaling pathway involved in TNF-alpha-induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292 epithelial cells. A protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or a Src kinase inhibitor (PP2) attenuated TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity. TNF-alpha- or TPA-induced I-kappaB kinase (IKK) activation was also blocked by these inhibitors, which reversed I-kappaBalpha degradation. Activation of c-Src and Lyn kinases, two Src family members, was inhibited by the PKC, tyrosine kinase, or Src kinase inhibitors. The dominant-negative c-Src (KM) mutant inhibited induction of COX-2 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKCalpha (PKCalpha A/E) or wild-type c-Src plasmids induced COX-2 promoter activity, and these effects were inhibited by the dominant-negative c-Src (KM), NF-kappaB-inducing kinase (NIK) (KA), or IKKbeta (KM) mutant. The dominant-negative PKCalpha (K/R) or c-Src (KM) mutant failed to block induction of COX-2 promoter activity caused by wild-type NIK overexpression. In coimmunoprecipitation experiments, IKKalpha/beta was found to be associated with c-Src and to be phosphorylated on its tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr(188) and Tyr(199), near the activation loop of IKKbeta, were identified to be crucial for NF-kappaB activation. Substitution of these residues with phenylalanines attenuated COX-2 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways cross-link between c-Src and NIK and converge at IKKalpha/beta, and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate COX-2 expression.     

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.     

Peroxisome proliferator-activated receptor beta (delta)-dependent regulation of ubiquitin C expression contributes to attenuation of skin carcinogenesis

The role of peroxisome proliferator-activated receptor-beta (PPARbeta) in the molecular regulation of skin carcinogenesis was examined. Increased caspase-3 activity associated with apoptosis was found in the skin of wild-type mice after tumor promotion with 12-O-tetradecanoylphorbol-13-acetate, and this effect was diminished in PPARbeta-null mice. The onset of tumor formation, tumor size, and tumor multiplicity induced from a two-stage carcinogen bioassay (7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate) were significantly enhanced in PPARbeta-null mice compared with wild-type mice. To begin to characterize the molecular changes underlying this PPARbeta-dependent phenotype, microarray analysis was performed and a number of differentially regulated gene products were identified including ubiquitin C. Subsequent promoter analysis, reporter gene assays, site-directed mutagenesis, and electrophoretic mobility shift assays provide evidence that PPARbeta regulates ubiquitin C expression, and that ubiquitination of proteins is influenced by PPARbeta. These results strongly suggest that activation of PPARbeta-dependent target genes provides a novel strategy to inhibit tumor promotion and carcinogenesis.     

Ligand activation of peroxisome proliferator-activated receptor-beta/delta(PPARbeta/delta) inhibits cell growth of human N/TERT-1 keratinocytes

The functional role of peroxisome proliferator-activated receptor-beta(PPARbeta; also referred to as PPARdelta) in epidermal cell growth remains controversial. Recent evidence suggests that ligand activation of PPARbeta/delta increases cell growth and inhibits apoptosis in epidermal cells. In contrast, other reports suggest that ligand activation of PPARbeta/delta leads to the induction of terminal differentiation and inhibition of cell growth. In the present study, the effect of the highly specific PPARbeta/delta ligand GW0742 on cell growth was examined using a human keratinocyte cell line (N/TERT-1) and mouse primary keratinocytes. Ligand activation of PPARbeta/delta with GW0742 prevented cell cycle progression from G1 to S phase and attenuated cell proliferation in N/TERT-1 cells. Despite specifically activating PPARbeta/delta as revealed by target gene induction, no changes in PTEN, PDK and ILK expression or downstream phosphorylation of Akt were found in either N/TERT-1 cells or primary keratinocytes. Further, altered cell growth resulting from serum withdrawal and the induction of caspase-3 activity by ultraviolet radiation were unchanged in the absence of PPARbeta/delta expression and/or the presence of GW0742. While no changes in the expression of mRNAs encoding cell cycle control proteins were found in response to GW0742, a significant decrease in the level of ERK phosphorylation was observed. Results from these studies demonstrate that ligand activation of PPARbeta/delta does not lead to an anti-apoptotic effect in either human or mouse keratinocytes, but rather, leads to inhibition of cell growth likely through the induction of terminal differentiation.     

Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin

Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12-O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H(2)O(2) formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-kappaB activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-kappaB signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent.     

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.     

Protein kinase C negatively regulates Akt activity and modifies UVC-induced apoptosis in mouse keratinocytes

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli.     

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.     

Pro/antioxidant Status in Murine Skin Following Topical Exposure to Cumene Hydroperoxide Throughout the Ontogeny of Skin Cancer

Organic peroxides used in the chemical and pharmaceutical industries have a reputation for being potent skin tumor promoters and inducers of epidermal hyperplasia. Their ability to trigger free radical generation is critical for their carcinogenic properties. Short-term in vivo exposure of mouse skin to cumene hydroperoxide (Cum-OOH) causes severe oxidative stress and formation of spin-trapped radical adducts. The present study was designed to determine the effectiveness of Cum-OOH compared to 12-O-tetradecanoylphorbol-13-acetate (TPA) in the induction of tumor promotion in the mouse skin, to identify the involvement of cyclooxygenase-2 (COX-2) in oxidative metabolism of Cum-OOH in keratinocytes, and to evaluate morphological changes and outcomes of oxidative stress in skin of SENCAR mice throughout a two-stage carcinogenesis protocol. Dimethyl-benz[a]anthracene (DMBA)-initiated mice were treated with Cum-OOH (32.8 micro mol) or TPA (8.5 nmol) twice weekly for 20 weeks to promote papilloma formation. Skin carcinoma formed only in DMBA/Cum-OOH-exposed mice. Higher levels of oxidative stress and inflammation (as indicated by the accumulation of peroxidative products, antioxidant depletion, and edema formation) were evident in the DMBA/Cum-OOH group compared to DMBA/TPA treated mice. Exposure of keratinocytes (HaCaT) to Cum-OOH for 18 h resulted in expression of COX-2 and increased levels of PGE(2). Inhibitors of COX-2 efficiently suppressed oxidative stress and enzyme expression in the cells treated with Cum-OOH. These results suggest that COX-2-dependent oxidative metabolism is at least partially involved in Cum-OOH-induced inflammatory responses and thus tumor promotion.     

Flavanones structure-related inhibition on TPA-induced tumor promotion through suppression of extracellular signal-regulated protein kinases: involvement of prostaglandin E2 in anti-promotive process

Biological functions of flavanones have been studied extensively, however, the structure-related activities of flavanones on 12-o-tetradecanoylphorbol 13-acetate (TPA)-induced promotive effects are still unclear. In this study, flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone showed the most significant dose-dependent inhibition on TPA-induced proliferative effects among eight tested flavanones in NIH3T3 cells. TPA-induced mitogen activated protein kinases (MAPK) phosphorylation, ornithine decarboxylase (ODC), c-Jun, and cyclooxygenase 2 (COX-2) protein expressions in a time-dependent manner, and the maximal inductive time point is at 1 h for MAPK phosphorylation and 6 h for others. Flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone showed the dose-dependent inhibition on TPA-stimulated MAPK phosphorylation, COX-2, ODC, c-Jun protein expressions. Induction of, prostaglandin E(2) (PGE(2)) production was detected in TPA-treated NIH3T3 cells, and flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone inhibited significantly PGE(2) production induced by TPA. Addition of PGE(2) reverses the inhibitory activities of flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone on TPA-induced proliferation. And, PD98059, a specific inhibitor of ERKs, inhibited TPA-induced MAPK phosphorylation, accompanied by decreasing COX-2, c-Jun, and ODC protein expression, and showed dose-dependent inhibition on TPA-induced proliferation in cells. These results demonstrated that PGE(2) is an important mediator in TPA-induced proliferation, and MAPK phosphorylation was located at the upstream of COX-2, c-Jun, and ODC gene expressions in TPA-induced responses. Furthermore, flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone (100 microM) suppressed TPA-induced colony formation associated with blocking MAPK phosphorylation, ODC, c-Jun, and COX-2 proteins expression. And, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay showed that flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone did not perform potent anti-radical activities among these eight tested compounds. In conclusion, this study provided molecular evidences to demonstrate that flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone were potent inhibitors on TPA-induced responses without notable cytotoxicity through suppression of PGE(2) production; and anti-radical activity of flavanones was not correlated with preventing the occurrence of tumor promotion. We proposed that blocking TPA-induced intracellular signaling responses might be involved in the anti-promotive mechanism of flavanones.     

p38 and EGF receptor kinase-mediated activation of the phosphatidylinositol 3-kinase/Akt pathway is required for Zn2+-induced cyclooxygenase-2 expression

Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus and cell type specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction of COX-2 expression. This study aims to elucidate the role of intracellular signaling pathways in Zn2+-induced COX-2 expression in human bronchial epithelial cells. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) potently block Zn2+-induced COX-2 mRNA and protein expression. Overexpression of adenoviral constructs encoding dominant-negative Akt kinase downstream of PI3K or wild-type phosphatase and tensin homolog deleted on chromosome 10, an important PI3K phosphatase, suppresses COX-2 mRNA expression induced by Zn2+. Zn2+ exposure induces phosphorylation of the tyrosine kinases, including Src and EGF receptor (EGFR), and the p38 mitogen-activated protein kinase. Blockage of these kinases results in inhibition of Zn2+-induced Akt phosphorylation as well as COX-2 protein expression. Overexpression of dominant negative p38 constructs suppresses Zn2+-induced increase in COX-2 promoter activity. In contrast, the c-Jun NH2-terminal kinase and the extracellular signal-regulated kinases have minimal effect on Akt phosphorylation and COX-2 expression. Inhibition of p38, Src, and EGFR kinases with pharmacological inhibitors markedly reduces Akt phosphorylation induced by Zn2+. However, the PI3K inhibitors do not show inhibitory effects on p38, Src, and EGFR. These data suggest that p38 and EGFR kinase-mediated Akt activation is required for Zn2+-induced COX-2 expression and that the PI3K/Akt signaling pathway plays a central role in this event.     

Troglitazone induction of COX-2 expression is dependent on ERK activation in keratinocytes

Cyclooxygenase-2 (COX-2) plays an important role in tumorigenesis of several tissues, including skin. We report here that troglitazone, a thiazolidinedione class of antidiabetic drug, induced COX-2 expression at both the protein and mRNA levels and increased production of prostaglandin E2 (PGE2) in cultured keratinocytes. Troglitazone-induced COX-2 expression in keratinocytes was likely peroxisome proliferator-activated receptor gamma (PPARgamma)-independent. Troglitazone treatment of these cells also resulted in a sustained increase in phosphorylation of ERK. We show that induction of COX-2 by troglitazone was almost completely inhibited by specific inhibitors of ERK activation. These data suggest that troglitazone is capable of inducing COX-2 expression through an ERK-dependent mechanism in mouse skin keratinocytes.     

Identification of Dss1 as a 12-O-tetradecanoylphorbol-13-acetate-responsive gene expressed in keratinocyte progenitor cells,with possible involvement in early skin tumorigenesis

This study identifies genes expressed early in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin carcinogenesis in genetically initiated Tg.AC v-Ha-ras transgenic mice. Keratinocyte progenitor cells from TPA-treated Tg.AC mice were isolated with fluorescence-activated cell sorting and expression was analyzed using cDNA microarray technology. Eleven genes were identified whose expression changed significantly in response to carcinogen treatment. Deleted in split hand/split foot 1 (Dss1) is a gene associated with a heterogeneous limb developmental disorder called split hand/split foot malformation. cDNA microarray expression analysis showed that the mouse homologue of Dss1 is induced by TPA. Dss1 overexpression was detected by Northern blot analysis in early TPA-treated hyperplastic skins and in JB6 Cl 41-5a epidermal cells. Interestingly, Dss1 expression was also shown to be elevated in skin papillomas relative to normal skins, and further increased in squamous cell malignancies. Functional studies by ectopically constitutive expression of Dss1 in JB6 Cl 41-5a preneoplastic cells strongly increased focus formation and proliferation of these cells and enhanced efficiency of neoplastic transformation of the cells in soft agar. These results strongly suggest that Dss1 is a TPA-inducible gene that may play an important role in the early stages of skin carcinogenesis.