Phorbol Ester TPA Research Articles

Repeated stress to the skin amplifies neutrophil infiltration in a keratin 17- and PKCα-dependent manner.

Neutrophils are the first immune cells to reach inflamed sites and contribute to the pathogenesis of chronic inflammatory skin diseases. Yet, little is known about the pattern of neutrophil infiltration in inflamed skin in vivo and the mechanisms mediating their recruitment. Here, we provide insight into the dynamics of neutrophil infiltration in skin in response to acute or repeated inflammatory stress, highlighting a novel keratinocyte- and keratin 17 (K17)-dependent mechanism that regulates neutrophil recruitment to inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12 h and resolves within 24 h. A subsequent TPA treatment or a UVB challenge, when applied 24 h but not 48 h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of chemoattractants by stressed keratinocytes. K17 binds RACK1, a scaffold protein essential for PKCα activity. The N-terminal head domain of K17 is crucial for its association with RACK1 and regulation of PKCα activity. Analysis of RNAseq data reveals a signature consistent with TAR and PKCα activation in inflammatory skin diseases. These findings uncover a novel, keratin-dependent mechanism that amplifies neutrophil recruitment in skin under stress, with direct implications for inflammatory skin disorders.

Keratin 17- and PKCα-dependent amplification of neutrophil influx after repeated stress to skin

Abstract Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their recruitment in inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin and assess whether keratinocyte-derived signals impact neutrophil recruitment. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12h and resolves within 24h. A second TPA treatment or a UVB challenge, when applied at 24h but not 48h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of Protein Kinase C α (PKCα) activity and release of neutrophil chemoattractants by stressed keratinocytes. We show that K17 binds RACK1, an essential scaffolding protein for PKCα activity. Analyses of RNAseq data reveal the presence of a transcriptomic signature consistent with TAR and PKCα activation in chronic inflammatory skin diseases including psoriasis, hidradenitis suppurativa, and atopic dermatitis. These findings uncover a novel, transient, and keratin-dependent mechanism that amplifies neutrophil recruitment to the skin under stress, with direct implications for inflammatory skin disorders.

Radiation Therapy Generates Release of Microvesicle Particles in Keratinocytes

Ionizing radiation (IR) exerts both tissue and systemic effects. However, the exact mechanism by which radiation therapy to skin results in local and systemic effects is incompletely defined. Previously our group has reported that IR of tumors results in the generation of the lipid mediator Platelet-activating factor (PAF) which resulted in systemic immunosuppressive effects via activation of regulatory T cells. Since PAF acting on the PAF receptor (PAFR) has been demonstrated to generate high levels of subcellular microvesicle particles (100-1000nm; MVP), and MVP are known to be able to signal systemically, the current studies seek to define whether IR of keratinocyte cells generates MVP, and to define the role of the PAFR in this process. Thus, we examine MVP release in the human keratinocyte cell line HaCaT. Moreover, the PAFR-dependency of IR-generated MVP is assessed by use of PAFR-positive KBP and PAFR-negative KBM cells. HaCaT cells (human keratinocytes), KBM cells (PAFR-negative), and KBP cells (PAFR-positive) were grown in 10cm dishes and treated with IR at either no treatment 0 Gy (NT), 4 Gy and 10 Gy. IR was delivered utilizing a technology company's medical linear accelerator radiotherapy system set up with dosimetry verified by nanodot optical stimulated luminescence (Landauer, Glenwood, IL). Some cell lines were treated with PAFR agonist N-methyl carbamoyl PAF (CPAF) and a phorbol ester TPA, known inducers of MVP release. A set of cells were treated with only 90% DMSO/10% ethanol vehicle in HBSS with BSA. After treatments, cells were incubated for 4 hours prior to extraction of MVPs. MVPs were isolated through centrifuging at 2000G for 20, supernatant was collected, transferred into different tubes, and centrifuged at 20,000 G for 70 mins. MVP was detected using a NanoSight NS300 instrument. MVP concentrations were recorded, and the data was statistically analyzed using Student's t-test (JMP, Cary, NC). IR treatment of HaCaT cells at various fluences exhibited statistically significant increases in MVP generation when compared to NT. Of note, 4 Gy resulted in the most fluence for MVP release but was not significant. IR treatment of KB cells resulted in MVP release in both KBM and KBP cells at both 4 and 10Gy. Augmented levels of MVP release were noted in KBP over KBM cells with any IR dose suggesting that the presence of the PAFR is involved in MVP release. Testing of inhibitors of the MVP generating enzyme acid sphingomyelinase (aSMase) also revealed involvement of this lipid metabolizing enzyme. The present studies indicate that RT can generates MVP production in epithelial cells. The mechanism for IR-generated MVP appears to involve aSMase and the PAFR. Target cell MVP release may provide a mechanism for RT effects, including the release of cytokines that influence systemic and local inflammation. Elucidation of this novel pathway may provide insights into IR effects on skin along with new therapeutic strategies.

Regulation of stimulus-induced interleukin-8 gene transcription in human adrenocortical carcinoma cells – Role of AP-1 and NF-κB

Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated Gαq-coupled designer receptor. H295R cells stimulated with either interleukin-1β (IL-1β) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c-Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1β-treated H295R cells required NF-κB while the phorbol ester TPA used both the AP-1 and NF-κB sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NF-κB-responsive reporter genes. While stimulation of Gαq-coupled designer receptors increased the AP-1 activity in the cells, IL-1β specifically stimulated NF-κB-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-κB activities. We conclude that stimulation of Gαq-coupled designer receptors or IL-1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-κB. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription.

Abstract 1831: PKCδ as a novel target for HER2-positive, trastuzumab resistant breast cancer

Abstract Breast cancer is the most common cancer diagnosis and second leading cause of cancer related deaths among women in the United States. This poor prognosis is in part due to the high rates of resistance. In particular, resistance to anti-HER2-based therapy remains a clinical challenge for women with breast cancer suggesting that better treatment strategies are needed. Results demonstrate that resistance to trastuzumab correlates with lower PKC activity compared to trastuzumab sensitive breast cancer cells, suggesting that downregulation of PKC activity may contribute to trastuzumab resistance. The exact mechanism by which PKCs contribute to trastuzumab resistance is not well understood. Here, we hypothesized that activation of PKCs could prevent trastuzumab resistance and thus inhibit the survival and recurrence of HER2+ breast cancer. Our results show that in two distinct trastuzumab-resistant cell lines (HCC1954 and BT474), activation of PKC by the phorbol ester TPA inhibits both proliferation of bulk cells and survival of cancer stem cells (CSC) as assessed by mammosphere formation. Further, treatment using a pharmacological PKC inhibitor (GF109203X) almost completely rescued the anti-proliferative and anti-CSC survival effects of TPA in two distinct trastuzumab-resistant cell lines. Measurement of PKC isoforms at the transcript and protein levels showed that PKCδ was overexpressed in the resistant cell line compared to the sensitive cell line. PKCδ depletion using a siRNA prevented the anti-proliferative and anti-CSC survival effects of TPA, suggesting that PKCδ was the target of TPA activation in trastuzumab resistant cells. TPA-mediated activation of PKCδ was found to cause G2/M cell cycle arrest, almost complete inhibition of AKT phosphorylation at serine 473, and decreased expression of the CSC marker, ALDH1A1. These results suggest that phorbol ester-mediated activation of PKCδ could be a novel therapeutic strategy for HER2+ breast cancer that is resistant to trastuzumab-based therapy. Citation Format: Debra L. Wyatt, Andrei Zlobin, Emily Ma, Mitchell F. Denning, Clodia Osipo. PKCδ as a novel target for HER2-positive, trastuzumab resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1831.

Loxl2 is dispensable for dermal development, homeostasis and tumour stroma formation.

Lysyl oxidase-like 2 (LOXL2) is a copper-dependent monoamine oxidase that contributes to the remodelling of the extracellular matrix (ECM) by cross linkage of collagen and elastin fibres and has emerged as a potential therapeutic target in cancer and fibrosis. In the skin, LOXL2 is essential for epidermal cell polarity and differentiation. However, its role in the dermis has not been evaluated. We found that Loxl2 is dispensable for mouse dermal development, maturation and homeostasis, yet affects dermal stiffness. Neither loss of Loxl2 nor increased Loxl2 expression affected dermal architecture following treatment with the phorbol ester TPA. Furthermore, Loxl2 expression did not alter the stroma of DMBA-TPA-induced tumours. We conclude that, although Loxl2 is expressed in both dermis and epidermis, its function appears largely confined to the epidermis.

1148 Ethanol augments stimulated microvesicle particle formation and release in a keratinocyte cell line

Microvesicle particles (MVP) are defined as submicron vesicles generated from cellular membranes via exocytosis in an active energy-dependent means. They are irregularly shaped and range from ∼100-1000 nm in size. MVP generation occurs in response to various stimuli, many involving intracellular calcium mobilization. MVP contain a variety of bioactive substances such as proteins, lipids, cytokines, and nucleic acids which can be transferred from parent cell to either nearby or even distant cells. MVP have been hypothesized to play pathological roles in the development of many human disease states. Previous studies by our group suggest that activation of the receptor for the lipid mediator Platelet-activating factor (1-alkyl-2-acetyl-glycerophosphocholine; PAF) results in MVP release in epithelial cell lines, primary keratinocytes and human and murine skin. Because ethanol can augment PAF production in keratinocytes and skin in response to various stressors including ultraviolet B radiation and thermal burn injury, the current studies were designed to test if ethanol can augment MVP production. Human HaCaT keratinocytes were pre-treated with ethanol before stimulation with ultraviolet B radiation, thermal burn injury, the PAF agonist CPAF, or the phorbol ester TPA. MVP released were quantified with a Nanosight particle counter. Ethanol pretreatment augmented both baseline as well as stimulated MVP production in a time-dependent manner. These studies provide a potential mechanism for ethanol-mediated pathologies, namely, via the augmentation of MVP production.

Terpenes and Biological Activities of Euphorbia tortilis

Background: Euphorbia species are known for the diversity of terpenes, and the presence of toxic diterpenoids in Euphorbia species have raised occupational and eco-toxicological concerns. The present study reports the terpene profile of Euphorbia tortilis Rottler ex Ainslie, along with cytotoxicity assay. Method: Triterpenes were isolated from the hexane extract of E. tortilis through column chromatography and were characterized by various spectroscopic techniques. Phorbol esters were detected from the methanol extract of the latex through high performance liquid chromatography-electrospray ionizationquadrupole time of flight tandem mass spectrometry (HPLC-QTOF- MS/MS). Cytotoxicity of the extract was tested against human cervical cancer cell line HeLa and normal cardiac myoblasts cell line H9C2 using MTT assay. Results: The pentacyclic triterpenoids 3β-friedelinol, 3α-friedelinol, friedelin, epi-friedelinyl acetate and taraxerol were isolated and characterized from E. tortilis. Ten phorbol esters belonging to the tigliane family of diterpenoids were detected through HPLC-ESI-QTOF-MS/MS method. The major tumor promoting phorbol ester TPA (phorbol-12-myristate-13-acetate) reported from Euphorbiaceae family could not be detected in E. tortilis, whereas the potential anti HIV and cytotoxic phorbol ester prostratin has been detected. Latex extract was inactive on the normal cell line H9C2 within the concentration range of 10-100μg/ml, while the cell growth inhibition of the extract at 100μg/ml on Hela cell line was negligible (3.06%). Conclusion: Pentacyclic triterpenes were the major compounds isolated and characterized from E. tortilis. HPLC-ESI-QTOF-MS/MS method has been found as an ideal tool for the detection of minor phorbol esters. The terpenoid profile revealed the absence of toxic TPA. Furthermore, the latex extract was non toxic towards normal cell line H9C2. The findings are relevant in the context of toxicological concerns related to Euphorbia members. Keywords: Euphorbia tortilis, triterpene, tigliane diterpenene, phorbol ester, HPLC-ESI-QTOF-MS, cytotoxicity.

Pleiotropic Effects of Chronic Phorbol Ester Treatment to Improve Glucose Transport in Insulin-Resistant Cardiomyocytes.

Stimulation of glucose transport is an important determinant of myocardial susceptibility to ischemia and reperfusion. Stimulation of glucose transport is markedly impaired in cardiomyocytes exposed to free fatty acids (FFA). Deactivation of the Focal Adhesion Kinase (FAK) by FFA contributes to glucose transport impairment, and could be corrected by chronic treatment with the phorbol ester TPA. However, TPA must have effects in addition to FAK reactivation to restore stimulated glucose transport. Chronic treatment with TPA improved basal and stimulated glucose transport in FFA-exposed, but not in control cardiomyocytes. Chronic FFA exposure induced the activation of PKCδ and PKCϵ. TPA markedly downregulated the expression of PKCα, PKCδ, and PKCϵ, suggesting that PKCδ or PKCϵ activation could contribute to inhibition of glucose transport by FFA. Rottlerin, a specific PKCδ inhibitor, improved glucose transport in FFA-exposed cardiomyocytes; and PKCδ was reduced in the particulate fraction of FFA + TPA-exposed cardiomyocytes. TPA also activated Protein Kinase D 1(PKD1) in FFA-exposed cardiomyocytes, as assessed by autophosphorylation of PKD1 on Y916. Pharmaceutical inhibition of PKD1 only partially prevented the improvement of glucose transport by TPA. Chronic TPA treatment also increased basal and stimulated glycolysis and favored accumulation of lipid droplets in FFA-exposed cardiomyocytes. In conclusion, basal and stimulated glucose transport in cardiomyocytes is reduced by chronic FFA exposure, but restored by concomitant treatment with a phorbol ester. The mechanism of action of phorbol esters may involve downregulation of PKCδ, activation of PKD1 and a general switch from fatty acid to glucose metabolism. J. Cell. Biochem. 9999: 4716-4727, 2017. © 2017 Wiley Periodicals, Inc.

Abstract 5105: Exosomes secreted by proinflammatory macrophages modulate breast cancer

Abstract Multiple cell types reside within the tumor microenvironment (TME) including cancer stem cells, stromal cells, and inflammatory cells. Cellular crosstalk is initiated between these cells through a variety of secreted biomolecules such as cytokines, growth factors, and exosomes. The role of inflammation and numerous secretory factors within the TME have been implicated in the development and progression of various cancers including breast. In this study, we wanted to evaluate the effect of macrophages (a major constituent of inflammation) on breast cancer development. Our study focused on macrophage secreted exosomes which are membraneous nanovesicles that contain various miRNA, mRNA, and proteins. We hypothesized that macrophage secreted exosomes will modulate breast cancer phenotype. An in vitro cell culture system was used in this study to examine macrophage exosome treatments on breast cancer cell lines MCF-7 (ER+) and MDA-MB 231 (ER-, PR-, HER2/neu-). Human monocytic THP-1 monocytes were activated with a common phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) and conditioned media (CM) was collected following 48 h. Exosomes were isolated from THP-1 CM via the use of an exosome isolation reagent and standard centrifugation. Breast cancer cells were then treated with THP-1 exosomes and cell morphology, proliferation, MEK/ERK and PI3K/AKT signaling pathways, and cell cycle were evaluated. MCF-7 and MDA-MB 231 exhibited round and spindle-like morphologies as well as a significant decrease in proliferation at 24 h treatments. Furthermore, MDA-MB 231 displayed increased p-ERK, while MCF-7 showed increased p-MEK following Western blot analysis. Both cell lines exhibited decreased p-AKT and p-S6 kinase expression. Cell cycle analysis showed that MCF-7 and MDA-MB 231 arrested at G2/M at 24 h exosome treatments. THP-1 exosome treatments led to a significant increase in MDA-MB 231 senescence as determined by a senescence assay looking at senescence-associated beta-galactosidase. Increased p21 and decreased Cdc2/Cyclin B1 was also observed which indicates a G2/M arrest. We conclude that specific macrophage secreted exosomes may induce breast cancer cell senescence and affect breast cancer progression. Citation Format: Robert Bronislaw Bednarczyk, Neha Y. Tuli, Ghada Ben Rahoma, Rachana Maniyar, Abraham Mittelman, Raj K. Tiwari. Exosomes secreted by proinflammatory macrophages modulate breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5105.

Abstract 5087: Precise characterization of macrophage secretory exosomes can lead to novel therapeutic approaches

Abstract Multiple cell types interact within the tumor microenvironment (TME) through crosstalk events that are mediated by many biomolecules including cytokines, mRNA, and miRNA-containing exosomes that can influence cancer progression as well as suppression. Inflammation within the TME is a major determinant in tumor progression. Since tumor associated macrophages (TAMs) are a major cellular factor in tumor inflammation, we hypothesized that crosstalk events initiated by macrophage secreted factors such as cytokines and exosomes will modulate breast cancer phenotype. We evaluated the cytokine profile of a human monocytic cell line, THP-1, following stimulation with a phorbol ester TPA (12-O-Tetradecanoylphorbol-13-acetate) in vitro. Activated THP-1 cells exhibited an M1 macrophage phenotype. In addition, we assessed the morphological and cellular growth changes of luminal (T47D, MCF-7) and basal-like (MDA-MB 231) breast cancer cell lines in vitro following treatment with activated THP-1 conditioned medium (CM). Each cell line displayed mesenchymal-like features and decreased cellular growth following activated THP-1 CM treatment. MCF-7 and MDA-MB 231 displayed increased MEK/ERK signaling, while all three cell lines showed increased p-AKT expression. Furthermore, epithelial-like T47D and MCF-7 cells exhibited an increase in EMT transcription factor expression. Moreover, although T47D and MCF-7 did not show drastic changes in E-cadherin expression via western blot analysis, immunofluorescence examination showed a decrease in E-cadherin following activated THP-1 CM treatments emphasizing a possible partial EMT event. Lastly, activated THP-1 exosomes were isolated and used to treat MDA-MB 231 to determine the effects on breast cancer cell proliferation and senescence. MDA-MB 231 exhibited significantly decreased cellular growth and increased senescence following incubation with activated THP-1 exosomes. The variety of secretory factors within the TME as a result of inflammation may contribute to tumor suppression as well as tumor progression via a secretion of cytokines and exosomes. Citation Format: Robert Bronislaw Bednarczyk, Yuki Kitadai, Neha Y. Tuli, Elyse K. Hanly, Ghada Benrahoma, Abraham Mittelman, Raj K. Tiwari. Precise characterization of macrophage secretory exosomes can lead to novel therapeutic approaches. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5087. doi:10.1158/1538-7445.AM2015-5087

Epidermal growth factor receptor inhibition by erlotinib prevents vascular smooth muscle cell and monocyte–macrophage function in vitro

IntroductionVascular smooth muscle cells (VSMCs) and monocyte–macrophages play a central role during the development of chronic allograft injury, which still remains an important challenge in organ transplantation. Inflammation, fibrosis and accelerated arteriosclerosis are typical features for chronic allograft injury. Growth factors participate in cell proliferation, differentiation and migration in this pathological process. ObjectiveHere we studied the role of epidermal growth factor receptor (EGFR) in VSMC and monocyte–macrophage function in vitro. EGFR inhibition by erlotinib, a selective EGF tyrosine kinase inhibitor, was studied in VSMC proliferation and migration as well as monocyte–macrophage proliferation and differentiation. Materials and methodsRat coronary artery SMCs were used for VSMC studies. As a model for monocyte–macrophage proliferation and differentiation human monocytic cell line U937 was used. Phorbol ester TPA was used to induce these cells to differentiate into macrophages. ResultsPlatelet-derived growth factor (PDGF)-B, a known VSMC inducer, caused 2.1-fold stimulation in VSMC proliferation compared to non-stimulated VSMC. Erlotinib prevented this VSMC proliferation in a dose-dependent manner, p<0.001 in all groups compared to controls. PDGF-B stimulation increased VSMC migration to 2.5-fold when compared with non-stimulated cells. Erlotinib decreased VSMC migration dose-dependently and this effect was significant with all doses, p<0.05. Erlotinib inhibited dose-dependently the proliferation of U937 monocytic cells, p<0.001. Erlotinib prevented also TPA-induced macrophage differentiation in a dose-dependent way, p<0.05. DiscussionErlotinib significantly prevents VSMC proliferation and migration in vitro. Erlotinib inhibited also significantly both monocyte proliferation and differentiation. Our data suggest that EGFR inhibition in VSMC and monocyte function has beneficial effects on chronic allograft injury.

Atrazine represses S100A4 gene expression and TPA-induced motility in HepG2 cells

Atrazine (ATZ) is probably the most widely used herbicide in the world. However there are still many controversies regarding its impacts on human health. Our investigations on the role of pesticides in liver dysfunctions have led us to detect an inhibition of FSP1 expression of 70% at 50μm and around 95% at 500μM of ATZ (p<0.01). This gene encodes the protein S100a4 and is a clinical biomarker of epithelial–mesenchymal transition (EMT), a key step in the metastatic process. Here we investigated the possible effect of ATZ on cell migration and noticed that it prevents the EMT and motility of the HepG2 cells induced by the phorbol ester TPA. ATZ decreases Fak pathway activation but has no effect on the Erk1/2 pathway known to be involved in metastasis in this cell line. These results suggest that ATZ could be involved in cell homeostasis perturbation, potentially through a S100a4-dependant mechanism.

Epidermal stem cells undergo age-associated changes

Epidermal stem cells undergo age-associated changes

OX1 orexin/hypocretin receptor activation of phospholipase D

Orexin receptors potently signal to lipid messenger systems, and our previous studies have suggested that PLD would be one of these. We thus wanted to verify this by direct measurements and clarify the molecular mechanism of the coupling. Orexin receptor-mediated PLD activation was investigated in CHO cells stably expressing human OX(1) orexin receptors using [(14) C]-oleic acid-prelabelling and the transphosphatidylation assay. Orexin stimulation strongly increased PLD activity - even more so than the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate), a highly potent activator of PLD. Both orexin and TPA responses were mediated by PLD1. Orexin-A and -B showed approximately 10-fold difference in potency, and the concentration-response curves were biphasic. Using pharmacological inhibitors and activators, both orexin and TPA were shown to signal to PLD1 via the novel PKC isoform, PKCδ. In contrast, pharmacological or molecular biological inhibitors of Rho family proteins RhoA/B/C, cdc42 and Rac did not inhibit the orexin (or the TPA) response, nor did the molecular biological inhibitors of PKD. In addition, neither cAMP elevation, Gα(i/o) nor Gβγ seemed to play an important role in the orexin response. Stimulation of OX(1) receptors potently activates PLD (probably PLD1) in CHO cells and this is mediated by PKCδ but not other PKC isoforms, PKDs or Rho family G-proteins. At present, the physiological significance of orexin-induced PLD activation is unknown, but this is not the first time we have identified PKCδ in orexin signalling, and thus some specific signalling cascade may exist between orexin receptors and PKCδ.

Induction of cyclooxygenase-2 by phorbol ester TPA modulates chemoresistance by inducing expression and function of drug efflux transporter ABCG2 in breast cancer cell lines

ATP-binding cassette transporter, sub-family G, member 2 (ABCG2/BCRP) is a xenobiotic transporter and also regulates serum uric acid levels as a urate transporter. We have shown that the severity of ABCG2 dysfunction can be estimated by simple genotyping of two dysfunctional variants, Q126X (rs72552713) and Q141K (rs2231142). This genotyping method is widely accepted for the risk analysis of hyperuricemia/gout, but there is no report on ethnic differences in ABCG2 dysfunctions. Here, we estimated ABCG2 dysfunctions by its genotype combination (Q126X and Q141K) and compared them in three different ethnic groups (500 Japanese, 200 Caucasians and 100 African-Americans). The minor allele frequencies of Q126X and Q141K in Japanese (0.025 and 0.275, respectively) were significantly higher than those in Caucasians (0.005 and 0.085, respectively) and African-Americans (0 and 0.090, respectively). Additionally, the rates of mild, moderate and severe ABCG2 dysfunctions in Japanese (35.4%, 12.4% and 1.6%, respectively) were higher than those in Caucasians (14.0%, 2.5% and 0%, respectively) and African-Americans (14.0%, 2.0% and 0%, respectively). Because ABCG2 dysfunctional diplotypes were commonly observed in both Caucasians (16.5%) and African-Americans (16.0%), the genotyping of the two ABCG2 dysfunctional variants is useful for evaluating individual differences in the ABCG2 dysfunction which affect the pharmacokinetics of substrate drugs and hyperuricemia risk in all three ethnic groups.

Cholinergic drugs inhibit in vitro megakaryopoiesis via the alpha7-nicotinic acetylcholine receptor

Besides thrombopoietin several additional factors (i.e neurotransmitters and receptors) are known to be involved in the regulation of megakaryopoiesis at different stages. Recently, we identified functional α7 nicotinic acetylcholine receptors (nAChRα7) on platelets and megakaryocytic precursors. In platelets nAChRα7 form functional Ca2+ channels and are involved in fibrinogen receptor activation and aggregation. Here, we investigated the impact of nAChRα7 on the differentiation of the human megakaryoblastic cell line MEG-01. In vitro differentiation of MEG-01 cells was induced by the phorbol ester TPA for 5 days in the absence or presence of nicotine or the nAChRα7-selective antagonist methyllycaconitine (MLA), and this was monitored by the expression of the megakaryocytic antigens CD41 and CD61. In the presence of the cholinergic drugs (nicotine or MLA) CD41 and CD61 expression was significantly reduced, both at RNA and protein level. We postulate that the nAChRα7 receptor is involved in megakaryopoietic signal transduction and gene regulation. This could affect the generation of platelets in vivo and contribute to the development of novel therapeutic drugs that regulate platelet formation.

B7-H1 Overexpression Regulates Epithelial–Mesenchymal Transition and Accelerates Carcinogenesis in Skin

B7-H1 (CD274) is a T-cell coinhibitory molecule that is also often induced on human carcinoma cells, where its expression has been implicated in immune escape. Under inflammatory conditions, B7-H1 is also inducible in normal epithelial cells but little is known about its involvement in conversion of normal cells to tumor cells. Here, we show that skin-specific expression of B7-H1 accelerates inflammatory carcinogenesis in a methylcholantrene (MCA)-induced model of squamous cell carcinoma (SCC). Inflammatory responses induced by MCA or phorbol ester TPA were clearly inhibited in B7-H1 transgenic mice (B7-H1tg mice). Antibody-mediated blockade of either B7-H1 or the related molecule PD-1 revealed that their ability to limit inflammation relied on ligand interactions made by B7-H1 or PD-1. Skin keratinocytes derived from B7-H1tg mice exhibited constitutive reduction of E-cadherin, and SCC induced in B7-H1tg mice also showed loss of E-cadherin along with elevated expression of the transcription factors Slug and Twist that drive epithelial-mesenchymal transition (EMT). Our results indicate that upregulation of B7-H1 in skin epithelial cells promotes EMT and accelerates carcinogenesis, revealing insights into the significance of B7-H1 overexpression on solid tumor cells and hinting at a close relationship between EMT and immune escape signaling pathways in cancer.

Identification of the Rage-dependent gene regulatory network in a mouse model of skin inflammation

BackgroundIn the past, molecular mechanisms that drive the initiation of an inflammatory response have been studied intensively. However, corresponding mechanisms that sustain the expression of inflammatory response genes and hence contribute to the establishment of chronic disorders remain poorly understood. Recently, we provided genetic evidence that signaling via the receptor for advanced glycation end products (Rage) drives the strength and maintenance of an inflammatory reaction. In order to decipher the mode of Rage function on gene transcription levels during inflammation, we applied global gene expression profiling on time-resolved samples of mouse back skin, which had been treated with the phorbol ester TPA, a potent inducer of skin inflammation.ResultsRanking of TPA-regulated genes according to their time average mean and peak expression and superimposition of data sets from wild-type (wt) and Rage-deficient mice revealed that Rage signaling is not essential for initial changes in TPA-induced transcription, but absolutely required for sustained alterations in transcript levels. Next, we used a data set of differentially expressed genes between TPA-treated wt and Rage-deficient skin and performed computational analysis of their proximal promoter regions. We found a highly significant enrichment for several transcription factor binding sites (TFBS) leading to the prediction that corresponding transcription factors, such as Sp1, Tcfap2, E2f, Myc and Egr, are regulated by Rage signaling. Accordingly, we could confirm aberrant expression and regulation of members of the E2f protein family in epidermal keratinocytes of Rage-deficient mice.ConclusionsIn summary, our data support the model that engagement of Rage converts a transient cellular stimulation into sustained cellular dysfunction and highlight a novel role of the Rb-E2f pathway in Rage-dependent inflammation during pathological conditions.

Abstract 1347: Immune escape mediator IDO is a crucial defining element of ‘cancer-associated’ inflammation in the tumor microenvironment

Abstract Chronic inflammation promotes cancer susceptibility and progression but a precise genetic definition of ‘cancer-associated’ inflammation’ has been elusive. We report that dysregulation of the immune escape mediator indoleamine 2,3-dioxygenase (IDO) is selectively required for carcinogenesis driven by chronic inflammatory stimuli, but not tumor formation per se, thereby linking IDO relatively more tightly to chronic inflammatory disease. IDO is a tryptophan catabolic enzyme that mediates immune tolerance in physiological processes such as pregnancy and pathophysiological processes such as cancer. Here our findings define IDO as a component of the peculiar inflammatory state that contributes to a supportive tumor microenvironment. In the well-established two-stage model of inflammatory skin carcinogenesis, initiated by ras-activating carcinogen DMBA and promoted by pro-inflammatory phorbol ester TPA, we found that genetic deletion of IDO abolished papilloma formation and progression to carcinoma. IDO deletion did not alter the histological phenotype elicited by TPA in skin, but it did abolish generation of a class of IDO+ regulatory antigen-presenting cells within skin-draining lymph nodes as previously reported (1). Intriguingly, carcinogenesis studies conducted in bone marrow chimeras in wild-type or IDO null animals argued that the supportive action of IDO was required mainly in non-immune host cells. In stark contrast to these findings, IDO deletion had no discernable effect on the efficiency of complete skin carcinogenesis (chronic DMBA treatment alone), or on induction and progression of breast carcinomas that were initiated by i.p. DMBA treatment and promoted by progesterone delivery. Thus, IDO contributed to carcinogenesis chiefly through its ability to support a peculiar inflammatory state engendered by TPA that is associated with tumor formation and progression. Two other lines of evidence furthered these findings. First, mice that were deficient in the IDO regulatory tumor suppressor gene Bin1 exhibited a greater susceptibility to two-stage inflammatory skin carcinogenesis, as expected, and the benefits of Bin1 loss to tumor formation relied on IDO dysregulation, based on their reversal by IDO inhibition. Second, we determined that ethyl pyruvate, a simple anti-inflammatory agent known to dampen ‘danger signaling’ and sepsis-like conditions in inflammed tissues, could mediate profound antitumor effects that relied upon an inhibition of IDO expression. Together, our results offered multiple lines of genetic and pharmacological evidence that IDO function is integral to establishing the peculiar inflammatory state that drives cancer, and that genetic pathways of immune escape and ‘cancer-associated’ inflammation may closely overlap. (1) Muller et al. PNAS 105, 17073 (2008). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1347.