Phorbol Ester Inhibition Research Articles

The Effect of Galangin on the Regulation of Vascular Contractility via the Holoenzyme Reactivation Suppressing ROCK/CPI-17 rather than PKC/CPI-17.

In this study, we investigated the influence of galangin on vascular contractibility and to determine the mechanism underlying the relaxation. Isometric contractions of denuded aortic muscles were recorded and combined with western blot analysis which was performed to measure the phosphorylation of phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) and to evaluate the effect of galangin on the RhoA/ROCK/CPI-17 pathway. Galangin significantly inhibited phorbol ester-, fluoride- and thromboxane mimetic-induced vasoconstrictions regardless of endothelial nitric oxide synthesis, suggesting its direct effect on vascular smooth muscle. Galangin significantly inhibited the fluoride-dependent increase in pMYPT1 and pCPI-17 levels and phorbol 12,13-dibutyrate-dependent increase in pERK1/2 level, suggesting repression of ROCK and MEK activity and subsequent phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that galangin-induced relaxation involves myosin phosphatase reactivation and calcium desensitization, which appears to be mediated by CPI-17 dephosphorylation via not PKC but ROCK inactivation.

Regulation of embryo survival in cattle

Evidence is presented that bovine somatotrophin (bST) treatment of lactating dairy cows enhances both expression of oviductal insulin-like growth factor II (IGF-II) mRNA and endometrial insulin-like growth factor binding protein 3 (IGFBP-3) mRNA between day 3 and day 7 of the oestrous cycle. mRNA encoding growth hormone (GH) receptor in endometrial tissues increased between day 3 and day 7 of the oestrous cycle. The changes induced by bST treatment may contribute to stimulation of embryo development and increase pregnancy rates in lactating dairy cows. Additive effects of bST and rb interferon tau (rbIFN-tau) to inhibit phorbol ester induction of prostaglandin F2alpha secretion in immortalized bovine endometrial cells indicates that there is interplay between their signal transduction pathways. Non-lactating dairy cows were killed at day 17 after oestrus to evaluate the effects of pregnancy status (cyclic versus pregnant) and bST (bST versus control) treatment on endometrial gene expression. Distinctly different mRNA and protein responses were detected between cyclic and pregnant cows that were related to luteolytic-antiluteolytic drive (that is expression of progesterone receptor, oxytocin receptor, oestradiol receptor alpha and prostaglandin GH synthase 2 (PGHS-2)). The bST-induced changes in PGHS-2 protein (+), oxytocin receptor mRNA (+) and oestrogen receptor alpha protein (+) may potentially affect the mechanisms associated with maintenance of pregnancy. Two experiments were conducted to evaluate whether ovarian follicular suppression induced by biodegradable deslorelin implants would reduce either early or late embryo losses. A 450 microg deslorelin implant used to induce ovulation in a timed insemination programme decreased subsequent follicular development and tended to reduce early embryo losses, whereas a 2.1 mg deslorelin implant failed to reduce late embryonic losses when inserted on day 27 of pregnancy.

Pendrin Function and Regulation in Xenopus Oocytes

SLC26A4/PDS mutations cause Pendred Syndrome and non-syndromic deafness. but some aspects of function and regulation of the SLC26A4 polypeptide gene product, pendrin, remain controversial or incompletely understood. We have therefore extended the functional analysis of wildtype and mutant pendrin in Xenopus oocytes, with studies of isotopic flux, electrophysiology, and protein localization. Pendrin mediated electroneutral, pH-insensitive, DIDS-insensitive anion exchange, with extracellular K_(1/2) (in mM) of 1.9 (Cl^-), 1.8 (I^-), and 0.9 (Br^-). The unusual phenotype of Pendred Syndrome mutation E303Q (loss-of-function with normal surface expression) prompted systematic mutagenesis at position 303. Only mutant E303K exhibited loss-of-function unrescued by forced overexpression. Mutant E303C was insensitive to charge modification by methanethiosulfonates. The corresponding mutants SLC26A2 E336Q, SLC26A3 E293Q, and SLC26A6 E298Q exhibited similar loss-of-function phenotypes, with wildtype surface expression also documented for SLC26A2 E336Q. The strong inhibition of wildtype SLC26A2, SLC26A3, and SLC26A6 by phorbol ester contrasts with its modest inhibition of pendrin. Phorbol ester inhibition of SLC26A2, SLC26A3, and SLC26A6 was blocked by coexpressed kinase-dead PKCδ but was without effect on pendrin. Mutation of SLC26A2 serine residues conserved in PKCδ -sensitive SLC26 proteins but absent from pendrin failed to reduce PKCδ sensitivity of SLC26A2 (190).

A Molecular Mechanism of Tumor Mediated Suppression of Dendritic Cell Differentiation

AbstractAbstract 1735The immune system plays a key role in preventing and controlling tumor growth. Cancer frequently induces a state of immune suppression in patients mediated, in part, through inhibition of dendritic cell (DC) differentiation. This results in the accumulation of actively immunosuppressive myeloid derived suppressor cells (MDSCs) and a loss of DCs (critical in the induction of T cell mediated immune responses), thus compromising the ability to initiate anti-tumor immune responses. This is mediated by tumor derived factors (TDFs)(e.g. VEGF) that inhibit DC differentiation by driving STAT3 hyperactivation. The downstream target(s) of this STAT3 signaling that inhibits DC differentiation, however, has not been defined. Previous work in our lab has identified protein kinase C βII (PKC βII) as being essential in myeloid progenitor to DC differentiation and demonstrated that PKC βII inhibition (signaling or expression) prevents DC differentiation. We've also found that PKC βII positively regulates its own expression and that, under certain circumstances, the PKCβ promoter is negatively regulated. These observations lead us to hypothesize that TDF activation of Stat3 inhibits DC differentiation by down regulating PKC βII expression. We've previously shown that culture in tumor conditioned media (TCM) decreased PKC βII protein levels and significantly reduced PKC βII mRNA transcript levels in KG1, a myeloid progenitor-like cell line long used to model DC differentiation. We've also previously seen that decreased PKC βII expression following culture in TCM significantly impaired DC differentiation, compared to cells grown in the absence of tumor conditions; however, the mechanism by which Stat3 signaling down regulated PKC βII expression remained unclear. We now show that culture in TCM reduced PKCβ promoter driven transcription 7-fold, compared to cells grown in normal media (p<0.01). Given the previously described importance of Stat3 hyperactivation in tumor-mediated suppression of DC differentiation, and since PKC βII down regulation appears to occur at the promoter level, we examined the role of Stat3 in regulating PKC βII expression. Culture in TCM rapidly (<5 min.) induced Stat3 phosphorylation, an indication of activation. By chromatin immunoprecipitation, we found that TCM treatment induces direct interaction between Stat3 and the PKCβ promoter, suggesting that Stat3 signaling may act to decrease PKC βII expression. To directly test the role of Stat3 signaling in regulation of PKC βII expression, we generated a series of clones stably expressing wild type (WT) or constitutive active (CA) Stat3 constructs in K562, a second DC progenitor-like cell line. We've previously seen that clones stably expressing the CA-Stat3 construct have decreased PKC βII protein levels and significantly decreased PKC βII mRNA levels, compared to the parental cell line and WT-Stat3 clones. We now show that this decrease in PKC βII expression was dependent on constitutive Stat3 signaling, as pharmacologic Stat3 inhibition restored PKC βII expression to levels seen in the parental cell line. Consistent with the proposed model and our previous work, decreased PKC βII expression in clones expressing CA-Stat3 resulted in significantly inhibited phorbol ester driven DC differentiation (p<0.05)(as measured by allogenic T cell proliferation, a key measure of DC differentiation). Interestingly, we've also found that PKC βII antagonizes Stat3 signaling: myeloid progenitor-like cells (KG1a) overexpressing PKC βII do not exhibit Stat3 activation in response to TCM: PKC βII overexpression or activation led to down regulation of the receptors for G-CSF, IL-6, and VEGF, TDFs demonstrated to inhibit myeloid progenitor to DC differentiation. These findings suggest a novel mechanism by which PKC βII negatively regulates the potential of a cell to respond to (tumor derived) inflammatory cytokines. This work demonstrates that tumor driven Stat3 hyperactivation down regulates PKCβ promoter activity, resulting in decreased PKC βII protein levels. In agreement with our previous work, this decrease in PKC βII expression impairs a cell's potential to undergo DC differentiation. Additionally, this work suggests that PKC βII signaling impairs a cell's potential to signal via Stat3 in response to TDFs, perhaps providing an avenue by which to block or reverse tumor mediated suppression of DC differentiation in cancer. Disclosures:No relevant conflicts of interest to declare.

Anti-inflammatory effects of Asparagus cochinchinensis extract in acute and chronic cutaneous inflammation

Asparagus cochinchinensis Merrill (Liliaceae) is a traditional herbal medicine used for treatment of various diseases [1, 2]. In the present study, we determine the ability of Asparagus cochinchinensis extract (ACE) to inhibit skin inflammation following exposure to the well-characterized protein kinase C activator and tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) [3]. ACE ameliorate the inflammatory phenotype, leading to substantial reductions in skin thickness (54.3% inhibition) and tissue weight (32.5% inhibition), inflammatory cytokine production, neutrophil-mediated myeloperoxidase activity, and various histopathological indicators (p&lt;0.01). ACE also significantly inhibits phorbol ester–induced irritant contact dermatitis and increases acetic acid–induced vascular permeability (p&lt;0.05). ACE is also effective at reducing inflammatory damage induced by chronic TPA exposure (p&lt;0.01). These results demonstrate that ACE is an effective anti-inflammatory agent in murine phorbol ester–induced dermatitis, and suggest that the compound may have therapeutic potential in a variety of immune-related cutaneous diseases.

Gene induction by glycyrol to apoptosis through endonuclease G in tumor cells and prediction of oncogene function by microarray analysis

Glycyrrhiza uralensis (Leguminosae) has long been known as an antiinflammatory agent for gastric ulcers, arthritis, and rheumatism. The flavonoid glycyrol (GC) (10 microg/ml) isolated from G. uralensis dramatically inhibits phorbol ester (phorbol 12-myristate 13-acetate)-induced nuclear factor (NF)-kappaB-dependent transcriptional activity, as determined by luciferase reporter activity in human kidney epithelial 293T cells. To investigate global gene expression profiling in cells by GC, we performed high-density oligonucleotide microarrays. Our microarray analyses showed that GC inhibited phorbol ester-induced NF-kappaB-dependent transcriptional activity in inflammatory-related gene expression. RT-PCR analysis, based on microarray data, showed that NF-kappaB-dependent genes (such as CCL2, CCL7, CD44, and HSPB8 in addition to NF-kappaB itself) were significantly downregulated by GC. Treatment with GC (10 microg/ml) inhibited I-kappaB degradation induced by phorbol 12-myristate 13-acetate. The microarray data also suggested that GC induces gene expression to p53-dependent apoptosis through endonuclease G, instead of CAD/DFF and AIF/PDCD8, as a downstream-apoptosis factor in human kidney epithelial 293T tumor cells, and induces oncogenes with a suppressor role as an added function.

Pseudopterosins appear to enhance the wound healing process through an adenosine receptor mediated mechanism

Pseudopterosins are marine natural products that inhibit phorbol ester (PMA) induced inflammation, inhibit the release of prostaglandins and leukotrienes from macrophages, and also inhibit degranulation of human PMNs. Pseudopterosins exhibit anti‐inflammatory and wound healing properties in several animal models and have shown significant activity in promoting wound healing at donor sites of patients undergoing autologous tissue transplants. At the cellular level, pseudopterosin activity is blocked by pertussis toxin (PTX) pretreatment, indicating that pseudopterosins act upon a G protein or G protein coupled receptor. One of the pseudopterosins, Iso‐pseudopterosin‐E (Iso‐PsE), has recently been shown to bind the adenosine A2A and A3 receptors expressed in HEK‐293 cells with ED50 values of 13 and 6.6 μM, respectively. In these binding experiments the potency of iso‐PsE is low when compared to the specific adenosine A2A agonist (NECA, ED50 = 49 nM) and A3 agonist (IB‐MECA ED 50 = 1.2 nM), but in physiological and in vivo experiments, iso‐PsE is equipotent, and in some cases more potent than NECA and IB‐MECA. In this report we propose the mechanisms by which iso‐PsE binds to these receptors and contributes to the wound healing process. This research is supported by ARMY grant # W81XWH‐06‐1‐0089.

Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl)

AbstractIn multiple myeloma (MM) protein kinase C (PKC) signaling pathways have been implicated in cell proliferation, survival, and migration. Here we investigated the novel, orally available PKC-inhibitor enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits phorbol ester–induced activation of PKC isoforms, as well as phosphorylation of downstream signaling molecules MARCKS and PKCµ. Importantly, it also inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs), costimulation with fibronectin, vascular endothelial growth factor (VEGF), or interleukin-6 (IL-6), as well as MM patient serum. Consequently, enzastaurin inhibits proliferation, survival, and migration of MM cell lines and MM cells isolated from multidrug-resistant patients and overcomes MM-cell growth triggered by binding to BMSCs and endothelial cells. Importantly, strong synergistic cytotoxicity is observed when enzastaurin is combined with bortezomib and moderate synergistic or additive effects when combined with melphalan or lenalidomide. Finally, tumor growth, survival, and angiogenesis are abrogated by enzastaurin in an in vivo xenograft model of human MM. Our results therefore demonstrate in vitro and in vivo efficacy of the orally available PKC inhibitor enzastaurin in MM and strongly support its clinical evaluation, alone or in combination therapies, to improve outcome in patients with MM.

A Fermented Grain Food Product Extract Ameliorates Tumor Promoter-Induced Skin Oxidative Stress

We demonstrated that a dietary supplement, Antioxidant Biofactor (AOB®), a fermented grain food, inhibits phorbol ester (TPA)-induced inflammatory responses. Pretreatment of mouse skin with ethyl acetate extract of AOB (0.5mg) significantly attenuated the hydrogen peroxide level in mouse skin without inhibition of edema formation. Also, exposure of AOB extract to differentiated HL-60 cells resulted in dose-dependent inhibition of TPA-induced superoxide generation, which was stronger than those of the raw materials of AOB. Our results suggest that AOB is effective for prevention of skin oxidative stress.

High levels of the onco-protein Gfi-1 accelerate T-cell proliferation and inhibit activation induced T-cell death in Jurkat T-cells.

Gfi-1 is a nuclear zinc finger protein with the activity of a transcriptional repressor and the ability to predispose for the development of T-cell lymphoma when expressed constitutively at high levels. Whereas thymic T-cell precursors express endogenous Gfi-1, mature peripheral T-cells lack Gfi-1 but upregulate its expression transiently after antigenic stimulation and activation of Erk1/2 demonstrating a role of Gfi-1 in T-cell activation. Here we show that constitutive expression of Gfi-1 accelerates S phase entry of primary, resting T-cells upon antigenic stimulation. In addition, high level Gfi-1 expression inhibits phorbol ester induced G1 arrest and activation induced cell death in Jurkat T-cells. We demonstrate that these effects of Gfi-1 concur with lower absolute levels and hyperphosphorylation of the pocket protein pRb. Moreover, phorbol ester induced expression of the negative cell cycle regulator p21(WAF1) is blocked in the presence of Gfi-1. These findings suggest that Gfi-1 contributes to T-cell lymphomagenesis by overriding a late G1 cell cycle checkpoint which controls activation induced death and S phase entry of T-cells.

Peroxisome Proliferator-activated Receptor γ Ligands Suppress the Transcriptional Activation of Cyclooxygenase-2: EVIDENCE FOR INVOLVEMENT OF ACTIVATOR PROTEIN-1 AND CREB-BINDING PROTEIN/p300

We investigated whether peroxisome proliferator-activated receptor gamma (PPARgamma) ligands (ciglitazone, troglitazone, and 15-deoxy-Delta(12,14) prostaglandin J(2)) inhibited cyclooxygenase-2 (COX-2) induction in human epithelial cells. Ligands of PPARgamma inhibited phorbol ester (phorbol 12-myristate 13-acetate, PMA)-mediated induction of COX-2 and prostaglandin E(2) synthesis. Nuclear run-offs revealed increased rates of COX-2 transcription after treatment with PMA, an effect that was inhibited by PPARgamma ligands. PMA-mediated induction of COX-2 promoter activity was inhibited by PPARgamma ligands; this suppressive effect was prevented by overexpressing a dominant negative form of PPARgamma or a PPAR response element decoy oligonucleotide. The stimulatory effects of PMA were mediated by a cyclic AMP response element in the COX-2 promoter. Treatment with PMA increased activator protein-1 (AP-1) activity and the binding of c-Jun, c-Fos, and ATF-2 to the cyclic AMP response element, effects that were blocked by PPARgamma ligands. These findings raised questions about the mechanism underlying the anti-AP-1 effect of PPARgamma ligands. The induction of c-Jun by PMA was blocked by PPARgamma ligands. Overexpression of either c-Jun or CREB-binding protein/p300 partially relieved the suppressive effect of PPARgamma ligands. When CREB-binding protein and c-Jun were overexpressed together, the ability of PPARgamma ligands to suppress PMA-mediated induction of COX-2 promoter activity was essentially abrogated. Bisphenol A diglycidyl ether, a compound that binds to PPARgamma but lacks the ability to activate transcription, also inhibited PMA-mediated induction of AP-1 activity and COX-2. Taken together, these findings are likely to be important for understanding the anti-inflammatory and anti-cancer properties of PPARgamma ligands.

Tachykinin activation of human monocytes from patients with rheumatoid arthritis: in vitro and ex-vivo effects of cyclosporin A

Three types of tachykinin receptors, namely NK1, NK2and NK3, are known to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), respectively. We previously demonstrated that NK1and NK2receptors are present on human monocytes, SP and NKA inducing superoxide anion production and tumor necrosis factor- alpha (TNF-α) mRNA expression. NK2receptor stimulation also triggered an enhanced respiratory burst in monocytes isolated from rheumatoid arthritis (RA) patients. This study was aimed to evaluate the in vitro and ex-vivo effects of cyclosporin A (CsA) on tachykinins-evoked TNF-α release from monocytes of healthy donors and RA patients. CsA (100ng/ml) potently inhibited phorbol ester- and tachykinin-evoked TNF-α secretion. In RA patients treated with CsA (SandimmunRNeoralR) 2.5mg/kg/die, a significant time-dependent reduction in TNF-α secretion from monocytes was measured. This may contribute to the CsA therapeutic activity in RA.

Tamoxifen inhibits phorbol ester stimulated osteoclastic bone resorption: An effect mediated by calmodulin

Tamoxifen inhibits bone resorption by disrupting calmodulin-dependent processes. Since tamoxifen inhibits protein kinase C in other cells, we compared the effects of tamoxifen and the phorbol ester, phorbol myristate acetate, on osteoclast activity. Phorbol esters stimulate bone resorption and calmodulin levels four-fold (k0.5 = 0.1-0.3 microM). In contrast, tamoxifen inhibited osteoclast activity approximately 60% with an IC50 of 1.5 microM, had no apparent effect on protein kinase C activity in whole-cell lysates, and reduced protein kinase C alpha recovered by immunoprecipitation 75%. Phorbol esters stimulated resorption in a time-dependent manner that was closely correlated with a similar-fold increase in calmodulin. Protein kinase C alpha, beta, delta, epsilon, and zeta were all down-regulated in response to phorbol ester treatment. Tamoxifen and trifluoperazine inhibited PMA-dependent increases in bone resorption and calmodulin by 85 +/- 10%. Down-regulation of protein kinase C isoforms by phorbol esters suggests that the observed increases in bone resorption and calmodulin levels are most likely due to a mechanism independent of protein kinase C and dependent on calmodulin. In conclusion, the data suggest that protein kinase C negatively regulates calmodulin expression and support the hypothesis that the effects of both phorbol esters and tamoxifen on osteoclast activity is mediated by calmodulin.

Activator Protein-1 Mediates Induced but not Basal Epidermal Growth Factor Receptor Gene Expression

The epidermal growth factor receptor (EGFR) is expressed at different levels in many cell types and found overexpressed in many cancers. EGFR expression is increased or decreased in response to extracellular stimuli. We examined the effect of increased c-Jun expression on EGFR promoter activity. We used DNAse I foot-printing analysis to determine the binding of activator protein 1 (AP-1) to the promoter region. We also used cotransfection experiments and western blotting analysis to determine the effect of AP-1 family members on EGFR expression. AP-1 was able to bind to at least seven sites in the EGFR promoter region. Cotransfection of MCF-7 cells with a c-Jun expression vector and the EGFR promoter reporter resulted in a 7-fold increase in promoter activity. JunB, but not c-fos, also enhanced the EGFR promoter activity. An A-Fos-dominant negative shown to inhibit Jun-dependent transactivation was able to prevent c-Jun induction of the promoter activity, but only slightly decreased the basal activity of the promoter. Furthermore, the A-Fos dominant negative was able to inhibit phorbol ester induction of the EGFR promoter. Examination of EGFR expression of MCF-7 stable cell lines that overexpress c-Jun revealed an increase in EGFR expression. Additionally, a cisplatin-resistant cell line, A2780/CP70, which has an increase in AP-1 activity compared with the parental cell line, A2780, was found to have an increase in EGFR level. These results indicate that AP-1 can act to increase the expression of EGFR and may play a role in upregulation of EGFR in cancer cells.

Tamoxifen inhibits phorbol ester stimulated osteoclastic bone resorption: An effect mediated by calmodulin

Tamoxifen inhibits phorbol ester stimulated osteoclastic bone resorption: An effect mediated by calmodulin

Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors through attenuation of endothelial superoxide anion formation.

Three 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), atorvastatin, pravastatin, and cerivastatin, inhibited phorbol ester-stimulated superoxide anion (O(2)(-)) formation in endothelium-intact segments of the rat aorta in a time- and concentration-dependent manner (maximum inhibition of 70% after 18 hours at 1 to 10 micromol/L). The HMG-CoA reductase product mevalonic acid (400 micromol/L) reversed the inhibitory effect of the HCRIs, which, conversely, was mimicked by inactivation of p21 Rac with Clostridium sordellii lethal toxin but not by inactivation of p21 Rho with Clostridium botulinum exoenzyme (C3). A mevalonate-sensitive inhibition of phorbol ester-stimulated O(2)(-) formation by atorvastatin was also observed in porcine cultured endothelial cells and in a murine macrophage cell line. In the rat aorta, no effect of the HCRIs on protein kinase C, NADPH oxidase, or superoxide dismutase (SOD) activity and expression was detected, whereas that of endothelial nitric oxide (NO) synthase was enhanced approximately 2-fold. Moreover, exposure of the segments to atorvastatin resulted in a significant improvement of endothelium-dependent NO-mediated relaxation, and this effect was abolished in the presence of SOD. Taken together, these findings suggest that in addition to augmenting endothelial NO synthesis, HCRIs inhibit endothelial O(2)(-) formation by preventing the isoprenylation of p21 Rac, which is critical for the assembly of NADPH oxidase after activation of protein kinase C. The resulting shift in the balance between NO and O(2)(-) in the endothelium improves endothelial function even in healthy blood vessels and therefore may provide a reasonable explanation for the beneficial effects of HCRIs in patients with coronary heart disease in addition to or as an alternative to the reduction in serum LDL cholesterol.

INDUCTION OF AP-1 BINDING TO INTRON 1 OF SP-A1 AND SP-A2 IS IMPLICATED IN THE PHORBOL ESTER INHIBITION OF HUMAN SP-A PROMOTER ACTIVITY

A deletional analysis of the SP-A1 promoter in NCI-H441 cells was performed to identify potential cis-acting elements involved in phorbol ester mediated repression of human SP-A transcription. The phorbol ester TPA reduced SP-A1 and SP-A2 promoter activity to approximately 35% to 45% compared to that of control cells. The inhibitory effect of TPA was significantly reduced upon removal of the region + 64/+ 394 relative to the SP-A1 transcription start site. Using NCI-H441nuclear proteins, electromobility shift assay analysis showed that the intron region + 309/+ 329 of SP-A1 and the corresponding region of SP-A2 formed sequence-specific DNA/protein complexes that were induced by TPA exposure. The region + 318/+ 324 of SP-A1 contains sequences similar to a consensus AP-1 binding site, TGACTGA (TCACTGA for SP-A2), which when mutated (TGAGAGT) prevented the formation of the TPA-induced DNA/protein comple. The TPA-induced complex was supershifted in the presence of antibody against the Jun family of proteins, but not the Fos family of proteins. These results suggest that the binding of AP-1 or an AP-1 like factor to the rst intron of SP-A1 and SP-A2 may be involved in the phorbol ester inhibition of human SP-A gene expression.

Evidence That Protein Kinase Cε Mediates Phorbol Ester Inhibition of Calphostin C- and Tumor Necrosis Factor-α-induced Apoptosis in U937 Histiocytic Lymphoma Cells

Protein kinase C (PKC) activators, such as the tumor-promoting phorbol esters, have been reported to protect several cell lines from apoptosis induced by a variety of agents. Recent evidence suggests that PKCepsilon is involved in protection of cardiac myocytes from hypoxia-induced cell death (Gray, M. O., Karliner, J. S., and Mochly-Rosen, D. (1997) J. Biol. Chem. 272, 30945-30951). We investigated the protective effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on U937 histiocytic lymphoma cells induced to undergo apoptosis by tumor necrosis factor-alpha (TNF-alpha) or by the specific PKC inhibitor calphostin C. U937 cells were transiently permeabilized with a peptide (epsilonV1-2) derived from the V1 region of PKCepsilon that has been reported to specifically block translocation of PKCepsilon. The epsilonV1-2 peptide blocked the inhibitory effect of TPA on both TNF-alpha- and calphostin C-induced apoptosis. A scrambled version of epsilonV1-2 and a peptide reported to inhibit PKCbeta translocation (betaC2-4) had no effect on the ability of TPA to inhibit apoptosis. These results suggest that PKCepsilon is required for the protective effect of TPA in TNF-alpha- and calphostin C-induced apoptosis. Furthermore, calphostin C reduced membrane-associated PKCepsilon activity and immunoreactivity, suggesting that PKCepsilon may play an important role in leukemic cell survival.

Resveratrol Inhibits Cyclooxygenase-2 Transcription and Activity in Phorbol Ester-treated Human Mammary Epithelial Cells

We determined whether resveratrol, a phenolic antioxidant found in grapes and other food products, inhibited phorbol ester (PMA)-mediated induction of COX-2 in human mammary and oral epithelial cells. Treatment of cells with PMA induces COX-2 and causes a marked increase in the production of prostaglandin E2. These effects were inhibited by resveratrol. Resveratrol suppressed PMA-mediated increases in COX-2 mRNA and protein. Nuclear run-offs revealed increased rates of COX-2 transcription after treatment with PMA, an effect that was inhibited by resveratrol. PMA caused about a 6-fold increase in COX-2 promoter activity, which was suppressed by resveratrol. Transient transfections utilizing COX-2 promoter deletion constructs and COX-2 promoter constructs, in which specific enhancer elements were mutagenized, indicated that the effects of PMA and resveratrol were mediated via a cyclic AMP response element. Resveratrol inhibited PMA-mediated activation of protein kinase C. Overexpressing protein kinase C-alpha, ERK1, and c-Jun led to 4.7-, 5.1-, and 4-fold increases in COX-2 promoter activity, respectively. These effects also were inhibited by resveratrol. Resveratrol blocked PMA-dependent activation of AP-1-mediated gene expression. In addition to the above effects on gene expression, we found that resveratrol also directly inhibited the activity of COX-2. These data are likely to be important for understanding the anti-cancer and anti-inflammatory properties of resveratrol.

Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors.

1. The rat CCK(A) and CCK(B) receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon. 2. Spectrofluorophotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCK(A) cells, measured as an increase in Fura-2 fluorescence emission ratio, 1000 fold more potently than its non-sulphated form (CCK-8-NS) (EC50 values of 0.19 nM and 0.18 microM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK(B) cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCK(A) receptor agonist JMV-180 increased [Ca2+]i only in CCK(A) cells. Likewise, pentagastrin increased [Ca2+]i only in CCK(B) cells. Finally, CCK-8-S-induced Ca2+ signalling through the CCK(A) receptor was most potently inhibited by the CCK(A) receptor antagonist L364,718, whereas the CCK(B) receptor antagonist L365,260 was more potent in CCK(B) cells. 3. Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK(A) cells. By contrast, none of these agonists increased cyclicAMP in CCK(B) cells. 4. Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca2+ mobilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK(A) cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-alpha-phorbol 12-myristate 13-acetate. 5. During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKC-alpha, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6. This study demonstrates that following expression in CHO cells (i) both CCK(A) and CCK(B) receptors are coupled to Ca2+ mobilization, (ii) only CCK(A) receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.