Phorbol 12-myristate 13-acetate Treatment Research Articles

Phorbol Myristate Acetate Suppresses Breast Cancer Cell Growth via Down‐regulation of P‐Rex1 Expression

Phosphatidylinositol‐3,4,5‐trisphosphate‐dependent Rac exchange factor 1 (P‐Rex1) is highly overexpressed in estrogen receptor‐positive and ErbB2‐overexpressing human luminal breast tumors. The present study was to investigate the role and mechanism underlying phorbol‐12‐myristate‐13‐acetate (PMA), a protein kinase C (PKC) activator, down‐regulation of P‐Rex1 in breast cancer cells. P‐Rex1 mRNA and protein expression were determined by real‐time PCR and western blot analysis, respectively. Cell growth was analyzed by counting cells number and 5‐bromo‐2′‐deoxyuridine (BrdU) cell proliferation assay. The translocation of PKC was examined by immunofluorescence staining. Our results showed that treatment with PMA caused a time‐ and concentration‐dependent decrease in P‐Rex1 protein levels in breast cancer MCF‐7 and BT‐474 cells with a maximum reduction of 87.2 ± 1.1 and 57.0 ± 8.6%, respectively, at a concentration of 10 ng/ml. Interestingly, either PMA treatment or silence of P‐Rex1 by siRNA inhibits MCF‐7 and BT‐474 cell growth by 60–80%, which can be attenuated by restoring P‐Rex1 expression. MCF‐7 and BT‐474 cells express PMA‐sensitive conventional (α and β) and novel (δ, ɛ and η) PKC isoforms. Immunofluorescence staining analysis of breast cancer cells showed translocation of PKCs from cytosol to the plasma membrane and/or nucleus, an indication of their activation upon PMA stimulation. The PKC general inhibitor Gö6983, but not the conventional PKC isoform inhibitor Gö6976, blocked PMA inhibition of P‐Rex1 expression and breast cancer cell growth. Expression of a constitutively active PKCɛ mutant suppressed P‐Rex1 expression and breast cancer cell growth, which was blocked by Gö6983 treatment. In contrast, expression of wild‐type PKCɛ, wild‐type or constitutively active PKCδ and PKCη had no effect. Together, our data suggest that PMA suppresses breast cancer cell growth via P‐Rex1 down‐regulation and PKCɛ activation plays a key role in this regulation. Our study may provide a novel strategy for development of chemotherapeutic agents for P‐Rex1‐overexpressing breast cancer patients that develop resistance to anti‐estrogen and/or anti‐ErbB2 therapies.Support or Funding InformationSupported by grants from NIH (5P20GM103489 and R01HL116849), Nebraska State LB595 research program and the National Natural Science Foundation of China (31100595 and 31300683).

Phorbol 12-myristate 13-acetate promotes nuclear translocation of hepatic steroid response element binding protein-2

Sterol regulatory element-binding protein (SREBP)-2 is a pivotal transcriptional factor in cholesterol metabolism. Factors interfering with the proper functioning of SREBP-2 potentially alter plasma lipid profiles. Phorbol 12-myristate 13-acetate (PMA), which is a common protein kinase C (PKC) activator, was shown to promote the post-translational processing and nuclear translocation of SREBP-2 in hepatic cells in the current study. Following SREBP-2 translocation, the transcripts of its target genes HMGCR and LDLR were upregulated as demonstrated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Electrophoretic mobility shift assays (EMSA) also demonstrated an induced DNA-binding activity on the sterol response element (SRE) domain under PMA treatment. The increase of activated Srebp-2 without the concurrent induced mRNA expression was also observed in an animal model. As the expression of SREBP-2 was not increased by PMA, the activation of PKC was the focus of investigation. Specific PKC isozyme inhibition and overexpression supported that PKCβ was responsible for the promoting effect. Further studies showed that the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK), but not 5′ adenosine monophosphate-activated protein kinase (AMPK), were the possible downstream signaling proteins of PKCβ. In conclusion, this study illustrated that PKCβ increased SREBP-2 nuclear translocation in a pathway mediated by MEK/ERK and JNK, rather than the one dictated by AMPK. These results revealed a novel signaling target of PKCβ in the liver cells.

PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser(485/491) of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser(485/491) phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser(485/491) phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser(485/491) phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser(491) in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser(491) that plays a negative role in insulin signaling in muscle cells.

Irradiation with narrowband-ultraviolet B suppresses phorbol ester-induced up-regulation of H1 receptor mRNA in HeLa cells

Conclusion These findings suggest that low dose irradiation with 310 nm NB-UVB specifically suppressed the up-regulation of H1R gene expression without inducing apoptosis and that UVB of shorter or longer wavelength than 310 nm NB-UVB had no such effects. Objective To develop a narrowband-ultraviolet B(NB-UVB) phototherapy for allergic rhinitis, this study investigated the effects of irradiation with NB-UVB at wavelength of 310 nm on phorbol-12-myristate-13-acetate (PMA)-induced up-regulation of histamine H1 receptor (H1R) mRNA in HeLa cells. Methods The mRNA levels of H1R in HeLa cells were measured using real-time RT-PCR. Apoptosis were evaluated with DNA fragmentation assay. Results PMA induced a significant increase in H1R mRNA expression in HeLa cells. Irradiation with 305 nm UVB and 310 nm NB-UVB, but not with 315 nm UVB at doses of 200 and 300 mJ/cm2 significantly suppressed PMA-induced up-regulation of H1R mRNA. At a dose of 200 mJ/cm2, irradiation with 305 nm UVB, but not with 310 nm NB-UVB, induced apoptosis, although exposure of the cells to both 305 and 310 nm UVB induced apoptosis at a dose of 300 mJ/cm2 after PMA treatment in HeLa cells. Conversely, irradiation with 315 nm UVB at doses of 200 and 300 mJ/cm2 did not induce apoptosis.

Characterization of Microvesicles Released from Human Red Blood Cells

Background/Aims: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca²⁺ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca²⁺ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca²⁺, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. Conclusion: An increase of intracellular Ca²⁺ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs.

Downregulation of Tissue Factor in Pericytes By a Protein Kinase C-Dependent Mechanism

Tissue factor (TF) is a transmembrane protein in the type II cytokine receptor family that serves as a cofactor for factor VIIa activity, and is essential for normal initiation of hemostasis. Its expression is generally upregulated in the settings of inflammation, immune activation, tissue injury and malignancy. TF has biological functions in addition to its role hemostasis. In malignancy, high expression of TF by tumor cells promotes local angiogenesis. By contrast, we have reported that expression of perivascular TF is downregulated within 24 hours after wounding, and remains low around angiogenic vessels during physiologic wound healing. The mechanism(s) of physiologic downregulation of TF, and its role in physiologic angiogenesis have not been previously characterized. Therefore, the objective of this study was to identify mechanisms contributing to TF downregulation in primary human pericytes. We determined that loss of TF in human pericytes can be recapitulated in vitro by treating pericyte cultures with phorbol 12-myristate 13-acetate (PMA). The time course and degree of TF downregulation over 24 hours, as shown in the figure, is similar to that observed within the first 24 hours in vivo. Since PMA is a potent activator of Protein Kinase C (PKC), we determined the role of PKC in PMA-mediated TF loss by pretreating pericytes with the broad spectrum PKC inhibitor, Go 6983, prior to stimulation with PMA. PKC inhibition eliminated PMA-induced TF loss, supporting the conclusion that the PMA effect occurs through a PKC-dependent mechanism. PKCa has been shown to induce phosphorylation of the TF cytoplasmic tail, which results in budding of TF-rich microvesicles from the plasma membrane. PMA also triggers activation of the cell-surface protease ADAM17. Based on domain homology with other targets, ADAM17 could potentially cleave and release the extracellular domain of TF from the cell surface. To determine if TF loss occurs through its release from the cell by either mechanism we utilized biotin labeling of surface TF, streptavidin pull down, and western blot analysis to assess trafficking of TF into the culture media. Our results show that surface TF significantly decreases within 2 hours of PMA treatment. However, we failed to detect biotinylated TF in the culture media. Isolation of microvesicles from the media followed by western blotting for TF showed that pericytes do release TF-associated microvesicles at a basal rate, but this rate is not affected by PMA. Taken together, these data show that PMA induces downregulation of TF in primary human pericytes through a PKC-dependent mechanism, and that this mechanism does not stimulate release of TF from the cell in either microvesicles or by proteolytic cleavage of the TF extracellular domain. Our working hypothesis is that PMA triggers internalization of surface TF via activation of protein kinase C, leading to its subsequent degradation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evidence that HIV-1 restriction factor SAMHD1 facilitates differentiation of myeloid THP-1 cells.

BackgroundSAMHD1 counteracts HIV-1 or HIV-2/SIVsmm that lacks Vpx by depleting the intracellular pool of nucleotides in myeloid cells and CD4+ quiescent T cells, thereby inhibiting the synthesis of retroviral DNA by reverse transcriptase. Depletion of nucleotides has been shown to underline the establishment of quiescence in certain cellular systems. These observations led us to investigate whether SAMHD1 could control the transition between proliferation and quiescence using the THP-1 cell model.FindingsThe entry of dividing THP-1 myeloid cells into a non-dividing differentiated state was monitored after addition of phorbol-12-myristate-13-acetate (PMA), an inducer of differentiation. Under PMA treatment, cells overexpressing SAMHD1 display stronger and faster adhesion to their support, compared to cells expressing a catalytically inactive form of SAMHD1, or cells depleted of SAMHD1, which appear less differentiated. After PMA removal, cells overexpressing SAMHD1 maintain low levels of cyclin A, in contrast to other cell lines. Interestingly, SAMHD1 overexpression slightly increases cell adhesion even in the absence of the differentiation inducer PMA. Finally, we found that levels of SAMHD1 are reduced in proliferating primary CD4+ T cells after T cell receptor activation, suggesting that SAMHD1 may also be involved in the transition from a quiescent state to a dividing state in primary T cells.ConclusionsAltogether, we provide evidence that SAMHD1 may facilitate some aspects of THP-1 cell differentiation. Restriction of HIV-1 by SAMHD1 may rely upon its ability to modify cell cycle parameters, in addition to the direct inhibition of reverse transcription.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0425-y) contains supplementary material, which is available to authorized users.

Pathways related to PMA-differentiated THP1 human monocytic leukemia cells revealed by RNA-Seq.

Previous analyses have reported that the human monocytic cell line THP1 can be differentiated into cells with macrophage-like characteristics by phorbol 12-myristate 13-acetate (PMA). However, little is known about the mechanism responsible for regulating this differentiation process. Here, we performed high-throughput RNA-Seq analysis to investigate the genes differently expressed in THP1 cells treated with and without PMA and examined those that may be responsible for the PMA-induced differentiation of monocytes into macrophages. We found 3,000 genes to be differentially expressed after PMA treatment. Gene ontology analysis revealed that genes related to cellular processes and regulation of biological processes were significantly enriched. KEGG analysis also demonstrated that the differentially expressed genes (DEGs) were significantly enriched in the PI3K/AKT signaling pathway and phagosome pathway. Importantly, we reveal an important role of the PI3K/AKT pathway in PMA-induced THP1 cell differentiation. The identified DEGs and pathways may facilitate further study of the detailed molecular mechanisms of THP1 differentiation. Thus, our results provide numerous potential therapeutic targets for modulation of the differentiation of this disease.

Treatment with protein kinase C activator is effective for improvement of male pronucleus formation and further embryonic development of sperm-injected oocytes in pigs

To assist the process of oocyte activation, which is essential for promotion of fertilization events, i.e., resumption of meiosis, extrusion of the second polar body and formation of the pronucleus (PN), artificial stimuli such as an electrical pulse have been applied to porcine oocytes after injection of sperm. However, the efficiency of fertilization and embryonic development remains low. It is well known that in vertebrates, inactivation of mitogen-activated protein (MAP) kinase is required for oocyte activation. We have hypothesized that even after electrical stimulation of sperm-injected oocytes, MAP kinase may not be inactivated. As it has been reported that MAP kinase activity is regulated by protein kinase C, we examined the effectiveness of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for improvement of fertilization and embryonic development of sperm-injected porcine oocytes. First, we examined the concentrations (0, 0.01, 0.1, 1, and 10 μM) and durations (0, 1, 3, 5 hours) of PMA treatment that were efficient for the extrusion of two polar bodies and formation of two PNs (2PB+2PN) and embryonic development. When the sperm-injected oocytes were treated with 0.01-μM PMA for 3 hours after electrical stimulation, the rates of 2PB+2PN and embryonic development were higher than those in the other treatment groups. We then examined the effect of PMA treatment (0.01 μM, 3 hours) on MAP kinase activity. Unexpectedly, after electrical stimulation, the activity remained low until PN formation, irrespective of whether or not the oocytes had been treated with PMA. On the other hand, transformation of the injected sperm nucleus into the male PN was accelerated after the PMA treatment. Our present results suggest that the low efficiency of fertilization and embryonic development in sperm-injected oocytes is not due to high activity of MAP kinase but due to poor transformation of the injected sperm nucleus into the male PN. Furthermore, a combination of electrical stimulation and PMA is a fairly effective artificial protocol for promoting 2PB+2PN and embryonic development in sperm-injected porcine oocytes.

Synergism between phorbol myristate acetate and calcium ionophore in inducing proliferation of in vitro γ-irradiated murine lymphocytes.

The objective of this study was to analyze the in vitro effects of γ-irradiation (0-5 Gy) on lymphocyte proliferation in animals sensitive to radiation as BALB/c mice. Lymphocytes were irradiated and underwent different treatments: quiescent cells were cultured with calcium ionophore A23187 (5 min or 48 h) with or without phorbol myristate acetate (PMA); lymphocytes (control cells or incubated with A23187 and PMA) were also cultured with four mitogens that are specific to the different subpopulations to determine the degree of inhibition of the response to radiation. Results obtained indicated that in quiescent cells, A23187 and PMA treatment had a mitogenic effect, which peaked with long A23187 treatment (48 h); synergism was further demonstrated between both drugs and was enhanced with higher ionizing radiation doses. However, in both irradiated and non-irradiated mitogen-stimulated cells, A23187 (48 h) and PMA had a strong inhibitory effect on cell proliferation. In conclusion these results indicate that irradiated BALB/c mice lymphocytes respond to treatment with A23187 and PMA more actively than controls. Inhibition of the post-exposure mitogen-induced proliferative response and the synergic effect between A23187 and PMA also suggest altered PKC activation mechanisms in cell membranes. Comparing with previous studies with in vivo irradiated mice, the effects of IR in vitro were less intense.

Inhibition of TRPC6 by protein kinase C isoforms in cultured human podocytes.

Transient receptor potential canonical-6 (TRPC6) ion channels, expressed at high levels in podocytes of the filtration barrier, are recently implicated in the pathogenesis of various forms of proteinuric kidney diseases. Indeed, inherited or acquired up-regulation of TRPC6 activities are suggested to play a role in podocytopathies. Yet, we possess limited information about the regulation of TRPC6 in human podocytes. Therefore, in this study, we aimed at defining how the protein kinase C (PKC) system, one of the key intracellular signalling pathways, regulates TRPC6 function and expression. On human differentiated podocytes, we identified the molecular expressions of both TRPC6 and several PKC isoforms. We also showed that TRPC6 channels are functional since the TRPC6 activator 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced Ca(2+) -influx to the cells. By assessing the regulatory roles of the PKCs, we found that inhibitors of the endogenous activities of classical and novel PKC isoforms markedly augmented TRPC6 activities. In contrast, activation of the PKC system by phorbol 12-myristate 13-acetate (PMA) exerted inhibitory actions on TRPC6 and suppressed its expression. Importantly, PMA treatment markedly down-regulated the expression levels of PKCα, PKCβ, and PKCη reflecting their activation. Taken together, these results indicate that the PKC system exhibits a 'tonic' inhibition on TRPC6 activity in human podocytes suggesting that pathological conditions altering the expression and/or activation patterns of podocyte-expressed PKCs may influence TRPC6 activity and hence podocyte functions. Therefore, it is proposed that targeted manipulation of certain PKC isoforms might be beneficial in certain proteinuric kidney diseases with altered TRPC6 functions.

ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells

Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation.

The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity.

Multifactorial mechanisms comprising countless cellular factors and virus-encoded transactivators regulate the transcription of HIV-1 (HIV). Since poly(ADP-ribose) polymerase 1 (PARP-1) regulates numerous genes through its interaction with various transcription factors, inhibition of PARP-1 has surfaced recently as a powerful anti-inflammatory tool. We suggest a novel tactic to diminish HIV replication via PARP-1 inhibition in an in vitro model system, exploiting human primary monocyte-derived macrophages (MDM). PARP-1 inhibition was capable to lessen HIV replication in MDM by 60–80% after 7 days infection. Tat, tumor necrosis factor α (TNFα), and phorbol 12-myristate 13-acetate (PMA) are known triggers of the Long Terminal Repeat (LTR), which can switch virus replication. Tat overexpression in MDM transfected with an LTR reporter plasmid resulted in a 4.2-fold increase in LTR activation; PARP inhibition caused 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85–95%). PARP inhibition in MDM exhibited 90% diminution in NFκB activity (known to mediate TNFα- and PMA-induced HIV LTR activation). Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These discoveries suggest that inactivation of PARP suppresses HIV replication in MDM by via attenuation of LTR activation, NFκB suppression and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide an effective approach to management of HIV infection.

Abstract 3915: Inhibition of cdc2 is linked to PMA-induced cell differentiation

Abstract The critical role of cdc2 (CDK1) in G2-M transition of the cell cycle control in mammalian cells has been well documented. Recent studies have also demonstrated that cdc2 is capable of driving G1 progress and G1-S transition via association with multiple interphase cyclins and through the interactions with Rb proteins. However, whether this pluripotent CDK regulates cell differentiation is unclear. Thus, the effect of phorbol 12-myristate 13-acetate (PMA) on cell differentiation and the expression of cdc2 in a human myeloid leukemia cell line, TF-1a, was investigated. When TF-1a cells were treated with 10−5, 10−6, and 10−7 PMA for 48 and 72h, they showed marked (≥50%) macrophage-like changes, evidenced by significant decrease in nucleus/cytoplasm ratio and increase (1-3 folds) in the expression of IL-1β, a macrophage marker. PMA treatment also caused time-dependent inhibition of cdc2 in both cytosol and nucleus of the cells, with a maximal inhibition being observed by 48 and 72h, which paralleled with the cell differentiation course. In contrast, there was no significant cell differentiation and inhibition of cdc2 being observed in control human myeloid leukemia TF-1 cells treated with PMA. Previous studies suggest that prolonged activation of MAPK pathway is linked to cell differentiation. PMA treatment also rapidly induced phosphorylation of MAPK kinases (MEK and ERK), which persisted for 24h, after which phosphorylated MEK and ERK were returned to base level, while the expression of cdc2 was still significantly downregulated, as compared with the control cells treated with DMSO. Pretreatment of TF-1a cells with sense cdc2 oligo nucleotides partially inhibited PMA-induced differentiation and upregulation of IL-1β. Taken together, our data suggest that MAPK pathway may be responsible for initiation of cell differentiation whereas inhibition of cdc2 is required for late differentiation of TF-1a cells in response to PMA stimulation. Whether activation of the MAPK pathway inhibits expression of cdc2 is currently under investigation (Supported by Faculty Incentive Grant and NIH-NIGMS MBRS RISE: R25 GM059244-13, Barry University). Note: This abstract was not presented at the meeting. Citation Format: Daria Vasilyeva, Pairat Dolinsky, Shashana Fielder, Alice Nakasone, Gerhild Packert, Xiaotang Hu. Inhibition of cdc2 is linked to PMA-induced cell differentiation. [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 3915. doi:10.1158/1538-7445.AM2015-3915

Zymosan and PMA activate the immune responses of Mutz3-derived dendritic cells synergistically

Beta-glucan (β-glucan) including zymosan has been known as a super food because of its multifunctional activities, such as the enhancement of immune responses. To study the functional mechanism of β-glucan in immune stimulation, the effect of zymosan on dendritic cell (DC) was investigated by monitoring the production of TNF-α, a pro-inflammatory cytokine. DC was differentiated from Mutz-3, a human acute myeloid leukemia cell line, by cytokine treatment and characterized. DC-specific cell surface markers were increased during the differentiation. Especially, Dectin-1, a β-glucan receptor, was upregulated during DC differentiation, and mediated zymosan-induced TNF-α production, which was inhibited by silencing of dectin-1. Zymosan exhibited synergistic effect with other immune stimuli such as lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator. Simultaneous treatment of zymosan and PMA enhanced the nuclear translocation of NF-κB subunits, p50 and p65, mediating the increase of TNF-α production. Bay 11-7082, an NF-κB inhibitor, blocked morphological changes and TNF-α production induced by zymosan and/or PMA treatment. Western blot analysis has showed zymosan-Dectin-1 pathway mediated destructive phosphorylation of inhibitor of NF-κB (IκB) kinase α subunit (IKKα) in IKK complexes, while PMA-PKC pathway regulated selective phosphorylation and degradation of IKKβ. Simultaneous phosphorylation of separate IKK subunits by co-treatment of zymosan and PMA resulted in cooperative activation of NF-κB and TNF-α production.

Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence.

Superoxide, a naturally produced reactive oxygen species (ROS) in the human body, is involved in many pathological and physiological signaling processes. However, if superoxide formation is left unregulated, overproduction can lead to oxidative damage to important biomolecules, such as DNA, lipids, and proteins. Superoxide can also lead to the formation of peroxynitrite, an extremely hazardous substance, through its reaction with endogenously produced nitric oxide. Despite its importance, quantitative information regarding superoxide production is difficult to obtain due to its high reactivity and low concentrations in vivo. MitoHE, a fluorescent probe that specifically reacts with superoxide, was used in conjunction with microchip electrophoresis (ME) and laser-induced fluorescence (LIF) detection to investigate changes in superoxide production by RAW 264.7 macrophage cells following stimulation with phorbol 12-myristate 13-acetate (PMA). Stimulation was performed in the presence and absence of the superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC) and 2-metoxyestradiol (2-ME). The addition of these inhibitors resulted in an increase in the amount of superoxide specific product (2-OH-MitoE(+)) from 0.08 ± 0.01 fmol (0.17 ± 0.03 mM) in native cells to 1.26 ± 0.06 fmol (2.5 ± 0.1 mM) after PMA treatment. This corresponds to an approximately 15-fold increase in intracellular concentration per cell. Furthermore, the addition of 3-morpholino-sydnonimine (SIN-1) to the cells during incubation resulted in the production of 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE(+) per cell on average. These results demonstrate that indirect superoxide detection coupled with the use of SOD inhibitors and a separation method is a viable method to discriminate the 2-OH-MitoE(+) signal from possible interferences.

The expression of SLAMF7 levels in malignant B cells: a novel therapeutic pathway for patients with CLL

Signalling lymphocyte activation molecule (SLAM) F7 is found on the surface of some immune cells including B-lymphocytes. Its activation leads to the proliferation or differentiation of immune cells. The objectives of the study were to measure SLAMF7 expression levels on B-CLL cells, and to upregulate the expression of SLAMF7 with phorbol myristate acetate (PMA) and Bryostatin. The levels of expression of SLAMF7 receptors of B-CLL cells from patients were measured; using immunofluorescence, flow cytometry, confocal microscopy and reverse transcriptase polymerase chain reaction (RT-PCR). The effects of treatments with PMA and Bryostatin were determined from different patients. Different levels of SLAMF7 expression were found to be associated with B-CLL cells from different patients. PMA treatment of B-CLL cells showed more positive SLAMF7 staining with the majority of the extracted B-CLL cell cases, while less positive results were associated with Bryostatin treatment. The study has shown that both PMA and Bryostatin could upregulate SLAMF7. Successful modulation of SLAMF7 expression may provide a novel target for the treatment of patients with CLL. Keywords : SLAMF7, RT PCR, Elotuzumab, Bryostatin, PMA

Activation of protein kinase C by phorbol ester increases red blood cell scramblase activity and external phosphatidylserine.

Externalization of phosphatidylserine (PS) is thought to contribute to sickle cell disease (SCD) pathophysiology. The red blood cell (RBC) aminophospholipid translocase (APLT) mediates the transport of PS from the outer to the inner RBC membrane leaflet to maintain an asymmetric distribution of PL, while phospholipid scramblase (PLSCR) equilibrates PL across the RBC membrane, promoting PS externalization. We previously identified an association between PS externalization level and PLSCR activity in sickle RBC under basal conditions. Other studies showed that activation of protein kinase C (PKC) by PMA (phorbol-12-myristate-13-acetate) causes increased external PS on RBC. Therefore, we hypothesized that PMA-activated PKC stimulates PLSCR activity in RBC and thereby contributes to increased PS externalization. In the current studies, we show that PMA treatment causes immediate and variable PLSCR activation and subsequent PS externalization in control and sickle RBC. While TfR+ sickle reticulocytes display some endogenous PLSCR activity, we observed a robust activation of PLSCR in sickle reticulocytes treated with PMA. The PKC inhibitor, chelerythrine (Chel), significantly inhibited PMA-dependent PLSCR activation and PS externalization. Chel also inhibited endogenous PLSCR activity in sickle reticulocytes. These data provide evidence that PKC mediates PS externalization in RBC through activation of PLSCR.

Effects of PMA (PHORBOL-12-MYRISTATE-13-ACETATE) on the Developing Rodent Brain.

Perinatal infections have a negative impact on brain development. However, the underlying mechanisms leading to neurological impairment are not completely understood and reliable models of inflammation are urgently needed. Using phorbol-myristate-acetate as an activator of inflammation, we investigated the effect on the developing rodent brain. Neonatal rats and mice deficient in IL-18 or IRAK-4 were exposed to PMA. Brains were assessed for regulation of pro- and anti-inflammatory cytokines and cell death 24 hrs, 7 and 14 days after treatment. PMA induced an inflammatory response and caused widespread neurodegeneration in the brains of 3- and 7-day-old rats. In contrast, 14-day-old rats were resistant to the neurotoxic effect of PMA. Histological evaluation at the age of 14 and 21 days revealed a destruction of the cortical microstructure with decreased numerical density of neuronal cells. Mice deficient in IL-18 or IRAK-4 were protected against PMA induced brain injury. PMA treatment during a vulnerable period can alter brain development. IL-18 and IRAK-4 appear to be important for the development of PMA induced injury.

Ets and GATA transcription factors play a critical role in PMA-mediated repression of the ckβ promoter via the protein kinase C signaling pathway.

BackgroundCholine kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ckα and ckβ, which produce three isoforms, CKα1, CKα2, and CKβ. Previous studies have associated ckβ with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckβ has never been elucidated.Methodology/Principal FindingsIn this report, the distal promoter region of the ckβ gene was characterized. Mutational analysis of the promoter sequence and electrophoretic mobility shift assays (EMSA) showed that Ets and GATA transcription factors were essential for the repression of ckβ promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckβ promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements. PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated. EMSA further revealed that PMA treatment increased the binding of both Ets and GATA transcription factors to their respective DNA elements. The PMA-mediated repressive effect was abolished by chronic PMA treatment and by treatment with the PKC inhibitor PKC412, but not the PKC inhibitor Go 6983, suggesting PKCε or PKCη as the PKC isozyme involved in the PMA-mediated repression of ckβ promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCε as the isozyme that mediated the PMA repression of ckβ promoter.Conclusion/SignificanceThese results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.