The Phorbol Ester 12-O-tetradecanoylphorbol-13-acetate Research Articles

Potential inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced inflammation, hyperproliferation, and hyperplasiogenic responses by celecoxib in mouse skin

Purpose Skin exposure to noxious agents leads to cutaneous lesion marked by an increase in inflammation, cellular proliferation, and hyperplasiogenic reactions. Studies have demonstrated that these damages breach the skin integrity resulting in the aetiology of various cutaneous disorders like atopic dermatitis, eczema, psoriasis, and development of non-melanoma skin cancer. Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, is an effective treatment for a variety of inflammatory diseases. Its importance in the therapy of skin problems, however, remains under appreciated. Methods We tested efficacy of topically applied celecoxib in mitigating skin inflammation, cellular proliferation, and hyperplasia induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in Swiss albino mice. Results Celecoxib (5 and 10 μmol) markedly reduced TPA (10 nmol) induced prostaglandin E2 (PGE2) production, oedema formation, myeloperoxidase (MPO) activity, and levels of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). It also resulted in a considerable decrease in ornithine decarboxylase (ODC) activity and the incorporation of [3H]-thymidine into DNA. In addition, there was a significant reduction in histoarchitectural abnormalities such as epidermal thickness, number of epidermal cell layers, neutrophil infiltration, intercellular oedema, and vasodilation. Conclusion Our results demonstrate that topical celecoxib can reduce the inflammation, hyperproliferation, and hyperplasiogenic events of skin insults suggesting that it may prove to be a valuable management option for cutaneous lesion and associated illnesses such as atopic dermatitis, eczema, and psoriasis, as well as the emergence of non-melanoma cancer.

Protein kinase C evokes quantal catecholamine release from PC12 cells via activation of L-type Ca2+ channels.

Application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to PC12 cells under resting conditions evoked quantal catecholamine secretion, as detected amperometrically. This effect was not mimicked by 4alpha-phorbol-12,13-didecanoate, another phorbol ester, which is inactive with respect to protein kinase C activation, and was prevented by the protein kinase C inhibitor bisindolylmaleimide. TPA also caused a rise of [Ca(2+)](i) in Fura-2-loaded PC12 cells, and again this was not mimicked by 4alpha-phorbol-12,13-didecanoate and could be blocked by bisindolylmaleimide. TPA-evoked secretion was entirely dependent on extracellular Ca(2+) and was fully abolished by nifedipine, as were TPA-induced rises of [Ca(2+)](i). Resting membrane potential, monitored using perforated patch recordings, was unaffected by TPA. However, a small (6-8 mV) hyperpolarizing shift in the voltage dependence of Ca(2+) channel currents (determined using whole-cell patch clamp recordings) was induced by TPA, and this could be fully prevented by nifedipine. In contrast to results with depolarizing stimuli, which evoke exocytosis because of Ca(2+) influx through N-type channels in these cells, the present results indicate that protein kinase C activation leads directly to quantal catecholamine secretion in the absence of depolarizing stimuli via a selective shift in the activation of L-type Ca(2+) channels.

A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome

Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through invitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 invitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.

Poly-γ-glutamic acid induces apoptosis via reduction of COX-2 expression in TPA-induced HT-29 human colorectal cancer cells.

Poly-γ-glutamic acid (PGA) is one of the bioactive compounds found in cheonggukjang, a fast-fermented soybean paste widely utilized in Korean cooking. PGA is reported to have a number of beneficial health effects, and interestingly, it has been identified as a possible anti-cancer compound through its ability to promote apoptosis in cancer cells, although the precise molecular mechanisms remain unclear. Our findings demonstrate that PGA inhibits the pro-proliferative functions of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a known chemical carcinogen in HT-29 human colorectal cancer cells. This inhibition was accompanied by hallmark apoptotic phenotypes, including DNA fragmentation and the cleavage of poly (ADP-ribose) polymerase (PARP) and caspase 3. In addition, PGA treatment reduced the expression of genes known to be overexpressed in colorectal cancer cells, including cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). Lastly, PGA promoted activation of 5' adenosine monophosphate-activated protein (AMPK) in HT-29 cells. Taken together, our results suggest that PGA treatment enhances apoptosis in colorectal cancer cells, in part by modulating the activity of the COX-2 and AMPK signaling pathways. These anti-cancer functions of PGA make it a promising compound for future study.

Differential NF-κB and MAPK activation underlies fluoride- and TPA-mediated CXCL8 (IL-8) induction in lung epithelial cells

Different toxic agents have a varying potential to induce the production of the proinflammatory chemokine, CXCL8 (interleukin [IL]-8), in lung cells. A critical question is which mechanisms determine the magnitude and persistence of the CXCL8 responses to different stimuli. To approach this, we compared the potential of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and sodium fluoride (NaF) to induce CXCL8 responses in A549 cells, with emphasis on the importance of nuclear factor kappa B (NF-κB)- and mitogen-activated protein kinase (MAPK) signaling. Notably, TPA induced a greater release of CXCL8 than did NaF. Furthermore, TPA induced a strong, rapid, but transient upregulation of CXCL8 messenger (m)RNA, whereas NaF induced a weaker, more delayed, but persistent upregulation. With respect to signaling, TPA led to an early, strong, and relatively transient extracellular signal-regulated kinase (ERK)1/2 phosphorylation, and a less marked and even more transient phosphorylation of c-jun-N-terminal kinases (JNK1/2) and p38. In contrast, NaF elicited a lower, but relatively sustained increase in phosphorylation of ERK1/2, and a marked phosphorylation of p38 and JNK1/2, with the JNK1/2 response as most transient. Only ERK1/2 inhibition affected the TPA response, whereas inhibition of all the three MAPK cascades reduced NaF-induced CXCL8 release. TPA also induced an early, marked phosphorylation/translocation of p65 (NF-κB), whereas NaF induced slower, less pronounced effects on p65. The CXCL8 responses by TPA and NaF were reduced by p65-siRNA. In conclusion, all MAPK cascades were involved in NaF-induced CXCL8 release, whereas only ERK1/2 activation was involved in response to TPA. Furthermore, NF-κB activation appeared to be indispensable for CXCL8 induction. The early response, magnitude, and persistency of MAPK and NF-κB signaling seemed to be critical determinants for the potential to induce CXCL8. These findings underscore that a strong, rapid, and relatively transient activation of ERK1/2 in combination with NF-kB may be sufficient for a strong induction of CXCL8, which may exceed the effects of a more moderate ERK1/2 activation in combination with activation of p38, JNK1/2, and NF-κB.

Keratinocyte-Specific Deletion of the Receptor RAGE Modulates the Kinetics of Skin Inflammation In Vivo

The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor causally related to the pathogenesis of acute and chronic inflammation. In a mouse model of inflammation-driven skin carcinogenesis, RAGE deletion conferred protection from the development of skin tumors due to a severely impaired cutaneous inflammation. Although the impact of RAGE expression in immune cells was shown to be essential for the maintenance of a cutaneous inflammatory reaction, the role of RAGE in keratinocytes remained unsolved. Using mice harboring a keratinocyte-specific deletion of RAGE, we analyzed its role in the regulation of an acute inflammatory response that was induced by topical treatment of the back skin with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We show that RAGE expression in cutaneous keratinocytes modulates the strength and kinetics of acute inflammation and supports the maintenance of epidermal keratinocyte activation. To address the underlying molecular mechanism, we isolated interfollicular epidermis by laser microdissection for gene expression analysis, and identified RAGE as a regulator in the temporal control of TPA-induced epidermal tumor necrosis factor alpha transcript levels. In summary, our data demonstrate that RAGE expression in keratinocytes is critically involved in the perpetuation of acute inflammation and support the central role of RAGE in paracrine communication between keratinocytes and stromal immune cells.

Non-melanoma skin cancer in mouse and man

As a frontier organ, skin is exposed to different environmental and/or occupational chemicals which cause cutaneous cancers in experimental animals. In mice, 7,12-dimethylbenz[a]anthrancene (DMBA) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) are frequently used as skin model tumor initiator and promoter, respectively. The sequential administration of DMBA and TPA leads to the appearance of a large number of benign papillomas, of which some convert later into invasive squamous cell carcinomas (SCC). At the molecular level, initiation of carcinogenesis in mouse skin consists in the mutational activation of the Ha-ras oncoprotein. HA-RAS mutations are rare in human SCC, but HA-RAS-mutated tumors appear in melanoma patients treated with B-raf inhibitors, indicating that initiated, HA-RAS-mutated stem cells also reside in human skin. Similarly, UV-induced human SCC show footprint mutations in the tumor suppressor gene TP53 which are also observed in UV-induced mouse SCC. Strong species differences exist with respect to phorbol ester-mediated tumor promotion. While certain mouse strains are very susceptible, other rodent species are much less sensitive. Likewise, humans appear to be much more resistant to phorbol ester-mediated skin toxicity. Papilloma formation as a result of a chemical insult is uncommon in men, questioning the relevance of this preneoplastic lesion for humans. However, skin tumorigenesis in the experimental situation and in humans appears to follow common molecular mechanisms, even though there are species differences in the morphological correlates to the preneoplastic state. Therefore, we recommend not simply labeling them as irrelevant for human risk assessment.

Upregulation of N‐acetylglucosaminyltransferase‐V by heparin‐binding EGF‐like growth factor induces keratinocyte proliferation and epidermal hyperplasia

Oligosaccharide modification by N-acetylglucosaminyltransferase-V (GnT-V), a glycosyltransferase encoded by the Mgat5 gene that catalyses the formation of β1,6 GlcNAc (N-acetylglucosamine) branches on N-glycans, is thought to be associated with cancer growth and metastasis. Overexpression of GnT-V in cancer cells enhances the signalling of growth factors such as epidermal growth factor (EGF) and transforming growth factor-β by increasing galectin-3 binding to polylactosamine structures on receptor N-glycans. We previously demonstrated that transgenic mice overexpressing GnT-V fail to develop spontaneous tumors in any organs, but phenotypes reminiscent of epithelial-to-mesenchymal transition were observed in their skin. However, the biological function of GnT-V in normal skin remained unknown. In this study, we examined the role of GnT-V in keratinocyte proliferation using GnT-V-deficient mice. Proliferation of human keratinocytes was suppressed by treatment with GnT-V siRNA. Mgat5(-/-) mouse keratinocytes also showed impaired cell proliferation through the reduction in EGF receptors on the cell surface. Although the skin of Mgat5(-/-) mice appeared normal, epidermal hyperplasia and proliferation of keratinocytes induced by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) were downregulated in these mutants. Moreover, a dramatic increase in GnT-V expression was observed by treatment with TPA or heparin-binding EGF-like growth factor (HB-EGF) in normal human epidermal keratinocytes. This increase was inhibited by an EGF receptor inhibitor. These results indicate that a high expression of GnT-V in keratinocytes contributes to HB-EGF-mediated epidermal hyperproliferation by inhibiting endocytosis of EGF receptors bearing β1,6 GlcNAc on their N-glycans. Our findings demonstrate a novel role for GnT-V in epidermal homoeostasis, particularly in hyperproliferative conditions.

Rapamycin Is a Potent Inhibitor of Skin Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate

Aberrant activation of phosphoinositide-3-kinase (PI3K)/Akt signaling has been implicated in the development and progression of multiple human cancers. During the process of skin tumor promotion induced by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), activation of epidermal Akt occurs as well as several downstream effectors of Akt, including the activation of mTORC1. Rapamycin, an established mTORC1 inhibitor, was used to further explore the role of mTORC1 signaling in epithelial carcinogenesis, specifically during the tumor promotion stage. Rapamycin blocked TPA-induced activation of mTORC1 as well as several downstream targets. In addition, TPA-induced epidermal hyperproliferation and hyperplasia were inhibited in a dose-dependent manner with topical rapamycin treatments. Immunohistochemical analyses of the skin from mice in this multiple treatment experiment revealed that rapamycin also significantly decreased the number of infiltrating macrophages, T cells, neutrophils, and mast cells seen in the dermis following TPA treatment. Using a two-stage skin carcinogenesis protocol with 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as the promoter, rapamycin (5-200 nmol per mouse given topically 30 minutes prior to TPA) exerted a powerful antipromoting effect, reducing both tumor incidence and tumor multiplicity. Moreover, topical application of rapamycin to existing papillomas induced regression and/or inhibited further growth. Overall, the data indicate that rapamycin is a potent inhibitor of skin tumor promotion and suggest that signaling through mTORC1 contributes significantly to the process of skin tumor promotion. The data also suggest that blocking this pathway either alone or in combination with other agents targeting additional pathways may be an effective strategy for prevention of epithelial carcinogenesis.

Abstract 2729: Proteomic analysis reveals a network of inflammatory genes in epidermis associated with skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice

Abstract Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with tumor promotion susceptibility by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a proteomic approach of two-dimensional (2-D) gel electrophoresis and mass spectrometry was used. In these experiments, we examined epidermal protein lysates and identified proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following TPA treatment. Among 19 differentially expressed proteins identified using this methodology were two calcium-binding proteins, S100A8 and S100A9. Their differential expression was further examined and validated by one or more of the following methods: i) one-dimensional (1-D) Western blot analysis; ii) 2-D Western blot analysis; iii) immunohistochemical analysis; and iv) quantitative real-time PCR. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially up-regulated in epidermis of DBA/2 vs C57BL/6 mice following topical treatment with two other tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that S100A8/A9 could be linked to several inflammatory networks. Further analyses revealed significantly increased expression of several inflammation-related genes including TNF-α, NFκB and IL-22 in epidermis of TPA-treated DBA/2 mice. Follow-up studies confirmed that these three inflammation related genes were upregulated in epidermis of TPA-treated DBA/2 mice compared to similarly treated C57BL/6 mice. These data suggest that differential expression of inflammation related genes in epidermis contributes to TPA-induced inflammation and skin tumor promotion susceptibility in DBA/2 mice. Taken together, our present data provide further insight into potential molecular mechanisms for the differential susceptibility of DBA/2 and C57BL/6 mice in terms of both inflammation and skin tumor promotion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2729. doi:10.1158/1538-7445.AM2011-2729

The Carboxyl-terminal Fragment of Pro-HB-EGF Reverses Bcl6-mediated Gene Repression

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family, is synthesized as a type I transmembrane precursor (pro-HB-EGF). Ectodomain shedding of pro-HB-EGF yields an amino-terminal soluble ligand of EGF receptor (HB-EGF) and a carboxyl-terminal fragment (HB-EGF-CTF) consisting of the transmembrane and cytoplasmic domains. We previously showed that the HB-EGF-CTF translocates from the plasma membrane to the nucleus and plays a role as a signaling molecule. Immunoprecipitation showed that HB-EGF-CTF can associate with Bcl6, a transcriptional repressor in mammalian cells. A glutathione S-transferase pulldown assay revealed that HB-EGF-CTF interacted efficiently with zinc fingers 4-6 of Bcl6. A luciferase reporter assay showed that the nuclear translocation of HB-EGF-CTF following shedding reversed transcriptional repression of cyclin D2 by Bcl6. Additionally, the level of cyclin D2 protein increased and Bcl6 interaction with the cyclin D2 promoter decreased in parallel with the shedding of pro-HB-EGF at all endogenous levels. These findings suggest that HB-EGF-CTF is a potent regulator of gene expression via its interaction with the transcriptional repressor Bcl6.

Up-regulation of tyrosine hydroxylase gene transcription by tetradecanoylphorbol acetate is mediated by the transcription factors Elk-1 and Egr-1

Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of catecholamines. Expression of the tyrosine hydroxylase gene is regulated at the transcriptional level by extracellular signaling molecules, including EGF, NGF, and glucocorticoids. We have analyzed the stimulation of tyrosine hydroxylase gene transcription by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in noradrenergic locus coeruleus-like CATH.a cells and observed a striking enhancement of the transcriptional activation potential of the ternary complex factor Elk-1, a key transcriptional regulator of serum response elementdriven gene transcription. Likewise, TPA strongly upregulated the biosynthesis of the transcription factor Egr-1 via distal serum response elements within the Egr-1 5'-flanking region. Subsequently, Egr-1 responsive transcriptional activation was observed. Overexpression of Egr-1 was sufficient to activate transcription of a tyrosine hydroxylase promoter/reporter gene, corroborating the view that the tyrosine hydroxylase gene is a target gene of Egr-1. Expression of dominant-negative mutants of Elk-1 or Egr-1 impaired TPA-induced stimulation of a tyrosine hydroxylase promoter/reporter gene transcription. In contrast, dominant-negative mutants of the transcription factors ATF2, ATF4, CREB, c-Jun and C/EBP did not change TPAinduced tyrosine hydroxylase promoter activity, indicating that these proteins are not part of the TPA-mediated signaling cascade directed towards the tyrosine hydroxylase gene. from Annual Meeting of the Study Group Neurochemistry. International Conference of the Gesellschaft fur Biochemie und Molekularbiologie 2006 (GBM 2006): Molecular pathways in health and disease of the nervous system Witten, Germany. 28–30 September 2006

Influence of the fungal NF-κB inhibitor panepoxydone on inflammatory gene expression in MonoMac6 cells

The fungal secondary metabolite panepoxydone has been recently described as an inhibitor of NF-κB activation which is a pivotal regulator of the inflammatory and immune response. These findings have led to propose that panepoxydone may be useful as anti-inflammatory agent. In this study we investigated for the first time the effects of panepoxydone on inflammatory gene expression in the monocytic cell line MonoMac6, stimulated with lipopolysaccharide (LPS) and the phorbolester 12- O-tetradecanoylphorbol-13-acetate (TPA). DNA microarray analysis of 110 human genes known to be strongly regulated during inflammation, combined with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) revealed that low micromolar concentrations (12–24 μM) of panepoxydone strongly inhibited the expression of thirty-three NF-κB dependent pro-inflammatory genes such as the chemokines CCL3, CCL4, CCL8; CXCL8, CXCL10, CXCL20, the cytokines IL-1, IL-6, TNF-α, pro-inflammatory enzymes like COX-2, and components of the REL/ NF-κB/ IκB family without significant effects on the expression of house-keeping genes. Panepoxydone strongly inhibited hTNF-α, IL-8 and NF-κB promoter activity in LPS/TPA stimulated MonoMac6 cells with IC 50 values of 0.5–1 μg/ml by blocking the phosphorylation of IκB and subsequent binding of the activated NF-κB transcription factor to the DNA. From our data, panepoxydone may serve as lead structure for the development of transcription-based inhibitors of pro-inflammatory gene expression.

A Novel Aspartic Proteinase-Like Gene Expressed in Stratified Epithelia and Squamous Cell Carcinoma of the Skin

Homeostasis of stratified epithelia, such as the epidermis of the skin, is a sophisticated process that represents a tightly controlled balance between proliferation and differentiation. Alterations of this balance are associated with common human diseases including cancer. Here, we report the cloning of a novel cDNA sequence, from mouse back skin, that is induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and codes for a hitherto unknown aspartic proteinase-like protein (Taps). Taps represents a potential AP-1 target gene because TPA-induced expression in epidermal keratinocytes critically depends on c-Fos, and co-treatment with dexamethasone, a potent inhibitor of AP-1-mediated gene regulation, resulted in impaired activation of Taps expression. Taps mRNA and protein are restricted to stratified epithelia in mouse embryos and adult tissues, implicating a crucial role for this aspartic proteinase-like gene in differentiation and homeostasis of multilayered epithelia. During chemically induced carcinogenesis, transient elevation of Taps mRNA and protein levels was detected in benign skin tumors. However, its expression is negatively associated with dedifferentiation and malignant progression in squamous cell carcinomas of the skin. Similar expression was observed in squamous skin tumors of patients, suggesting that detection of Taps levels represents a novel strategy to discriminate the progression state of squamous skin cancers.

Up‐regulation of tyrosine hydroxylase gene transcription by tetradecanoylphorbol acetate is mediated by the transcription factors Ets‐like protein‐1 (Elk‐1) and Egr‐1

Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of catecholamines. Expression of the tyrosine hydroxylase gene is regulated at the transcriptional level by extracellular signalling molecules, including epidermal growth factor (EGF), nerve growth factor (NGF) and glucocorticoids. We have analysed the stimulation of tyrosine hydroxylase gene transcription by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in noradrenergic locus coeruleus-like CATH.a cells and observed a striking enhancement of the transcriptional activation potential of the ternary complex factor Ets-like protein-1 (Elk-1), a key transcriptional regulator of serum response element-driven gene transcription. Likewise, TPA strongly up-regulated the biosynthesis of the transcription factor Egr-1 via distal serum response elements within the Egr-1 5'-flanking region. Subsequently, enhancement of the transcriptional activation potential of Egr-1 was observed. Overexpression of Egr-1 was sufficient to activate transcription of a tyrosine hydroxylase promoter/reporter gene, corroborating the view that the tyrosine hydroxylase gene is a target gene of Egr-1. Expression of dominant-negative mutants of Elk-1 or Egr-1 impaired TPA-induced stimulation of a tyrosine hydroxylase promoter/reporter gene transcription. In contrast, dominant-negative mutants of the transcription factors activating transcription factor (ATF)-2, ATF4, cAMP response element-binding protein, c-Jun and CCAAT/enhancer binding protein (C/EBP) did not change TPA-induced tyrosine hydroxylase promoter activity, indicating that these proteins are not part of the TPA-mediated signalling cascade directed towards the tyrosine hydroxylase gene.

Differential effects of bryostatin 1 and 12-O-tetradecanoylphorbol-13-acetate on the regulation and activation of RasGRP1 in mouse epidermal keratinocytes

The antitumor agent bryostatin 1 and the tumor-promoting phorbol esters function as structural mimetics of the second lipid messenger diacylglycerol (DAG) by binding to the C1 domain of DAG receptors. However, bryostatin 1 and the phorbol esters often differ in their cellular actions. In mouse skin, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter, whereas bryostatin 1 lacks this activity and antagonizes the tumor-promoting effects of TPA. Although protein kinase C mediates many of the effects of DAG on skin, the exact mechanisms responsible for the biology of bryostatin 1 and TPA in the epidermis have not been elucidated. We recently reported that the novel DAG receptor RasGRP1 is expressed in mouse keratinocytes and mediates TPA-induced Ras activation. This finding prompted us to examine the regulation of RasGRP1 by bryostatin 1. We found that whereas TPA induced translocation of RasGRP1 to both the plasma and internal membranes of the keratinocytes, bryostatin 1 recruited RasGRP1 only to internal membranes and the nuclear envelope. In addition, TPA led to a concentration-dependent down-regulation of RasGRP1, whereas bryostatin 1 failed to induce full RasGRP1 down-regulation. Interestingly, bryostatin 1 was less effective than TPA at activating Ras. The results presented here suggest the possibility that a differential modulation of RasGRP1 by bryostatin 1 compared with TPA could participate in the disparate responses of the epidermal cells to both DAG analogues. This result may have implications in the understanding of the antitumor effects of bryostatin 1 in the skin.

Na+/K+/Cl− cotransporter activates MAP‐kinase cascade downstream to protein kinase C, and upstream to MEK

In this study, we demonstrated that the specific inhibitors of the Na+/K+/Cl- cotransporter (NKCC1), bumetanide and furosemide, inhibited extracellular regulated kinase (ERK) phosphorylation in Balb/c 3T3 fibroblasts, stimulated with a variety of mitogens. In addition to fibroblast growth factor (FGF) shown before, the various mitogens tested in the present study (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), insulin, thrombin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA)). Enter, the Ras/Raf/MEK/ERK cascade via different growth factors receptors and through one of the two main routes. The results of the present study provide evidence that have led us to conclude that the target protein which is controlled by the Na+/K+/Cl- cotransporter, is downstream of tyrosine kinase receptors, as well as of the G-protein-coupled receptor (GPCR). Several additional lines of evidence supported the above conclusion: (i) furosemide inhibits phosphorylation of MAPK kinase (MEK) induced by receptor tyrosine kinase (RTK) ligands, such as PDGF, FGF, and EGF. (ii) Furosemide also inhibited ERK phosphorylation, induced by thrombin, a GPCR. (iii) Furosemide inhibited MEK and ERK phosphorylation even when ERK phosphorylation was induced by direct activation of protein kinase C (PKC) by TPA, which bypasses early steps of the mitogenic cascade. In addition, we found that furosemide did not affect PKC phosphorylation induced directly by TPA. Taken together, the results of the present study indicate that the signal transduction protein, controlled by the Na+/K+/Cl- cotransporter, must be downstream of the PKC, and at/or upstream to MEK in the Ras/Raf/MEK/ERK cascade.

Enhancement of TPA-induced growth inhibition and apoptosis in myeloid leukemia cells by BAY 11-7082, an NF-κB inhibitor

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent stimulator of differentiation and apoptosis in myeloid leukemia cells. In the present study, we investigated the role of the transcription factor NF-kappaB in TPA-induced growth inhibition and apoptosis in the myeloid leukemia HL-60 cell line and its TPA-resistant cell variant HL-525. Unlike the parental cell line, HL-525 cells are protein kinase C (PKC)-beta deficient and resistant to TPA-induced differentiation and apoptosis. We found that treatment of HL-60 cells with TPA resulted in a concentration-dependent growth inhibition and an increase in apoptotic cells. TPA only had a small effect on growth and apoptosis in HL-525 cells. Treatment of HL-60 cells with TPA (0.64-3.2 nM) caused a rapid activation of NF-kappaB as determined by electrophoresis mobility shift assay (EMSA) and immunocytochemistry. Although the basal level of NF-kappaB activity was low in HL-60 cells, TPA-resistant HL-525 cells had a high basal level of NF-kappaB activity. Treatment of HL-525 cells with higher concentrations of TPA (16-80 nM) resulted in a further increase in NF-kappaB activity. (E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY 11-7082; BAY), which inhibits IkappaB alpha phosphorylation and thus decreases NF-kappaB activation, was found to decrease TPA-induced nuclear translocation of NF-kappaB. Furthermore, BAY enhanced TPA-induced growth inhibition and apoptosis in both HL-60 and HL-525 cells. Results from the present study indicate that inhibition of NF-kappaB by BAY was associated with enhanced TPA-induced growth inhibition and apoptosis in human myeloid leukemia cells. TPA in combination with pharmacological inhibitors of NF-kappaB may improve the therapeutic efficacy of TPA and overcome the resistance to TPA in some myeloid leukemia patients.

Differential gene expression in epidermis of mice sensitive and resistant to phorbol ester skin tumor promotion

Previous data from two-stage carcinogenesis studies in mouse skin demonstrated that genetic control of susceptibility to skin tumor promotion by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), in crosses between susceptible DBA/2J and resistant C57BL/6J mice is a multigenic trait. Utilizing a cDNA microarray approach, we compared global gene expression profiles in the epidermis of these two mouse strains treated with TPA or vehicle (acetone). Gene expression in the epidermis was analyzed after the treatment to identify global effects of TPA, as well as potential candidate genes that modify susceptibility to skin tumor promotion. DBA/2J and C57BL/6J mice were treated topically four times with 3.4 nmol TPA or acetone over a 2-wk period, and RNA was extracted from epidermis 6 h after the final treatment. Labeled cDNA generated from each group was hybridized to commercial cDNA microarrays (Agilent) containing more than 8000 targets. More than 450 genes were significantly influenced, directly or indirectly, by TPA treatment in the epidermis of either strain. Notably, 44 genes exhibited differential expression between the tumor promotion sensitive and resistant mouse strains. Several genes that were differentially expressed in DBA/2J versus C57BL/6J epidermis after TPA treatment were located in chromosomal regions linked to TPA promotion susceptibility. Three genes, Gsta4, Nmes1 (MGC58382), and Serpinb2, located within promotion susceptibility loci Psl1 (chr 9), Psl2 (chr 2), and Psl3 (chr 1), respectively, were identified in this analysis as potential candidates for modifiers of susceptibility to skin tumor promotion by TPA.

C-Fos-Dependent Induction of the Small Ras-Related GTPase Rab11a in Skin Carcinogenesis

Malignant transformation of mouse skin by tumor promoters and chemical carcinogens, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process leading to the formation of squamous cell carcinomas. It has been shown that mice lacking the AP-1 family member c-Fos exhibit an impaired transition from benign to malignant skin tumors. Here, we demonstrate enhanced expression of the small Ras-related GTPase Rab11a after short-term TPA treatment of mouse back skin. Expression of Rab11a in vivo and in vitro critically depended on c-Fos, because TPA application to the back skin of c-Fos-deficient mice and to mouse embryonic fibroblasts did not induce Rab11a mRNA or protein expression. Moreover, dexamethasone, which is a potent inhibitor of AP-1-mediated transactivation that exhibits anti-inflammatory and anti-tumor promoting activities, inhibited TPA-induced expression of Rab11a. Within the Rab11a gene promoter, we identified a functional AP-1 binding element that exhibited elevated c-Fos binding activity after TPA treatment of keratinocytes. Enhanced expression was not restricted to chemically induced mouse skin tumors but was also found in tumor specimens derived from patients with epithelial skin tumors. These data identify Rab11a as a novel, tumor-associated c-Fos/AP-1 target and may point to an as yet unrecognized function of Rab11a in the development of skin cancer.