Addition Of Phorbol 12-myristate 13-acetate Research Articles

Acceleration of vascular sprouting from fabricated perfusable vascular-like structures.

Fabrication of vascular networks is essential for engineering three-dimensional thick tissues and organs in the emerging fields of tissue engineering and regenerative medicine. In this study, we describe the fabrication of perfusable vascular-like structures by transferring endothelial cells using an electrochemical reaction as well as acceleration of subsequent endothelial sprouting by two stimuli: phorbol 12-myristate 13-acetate (PMA) and fluidic shear stress. The electrochemical transfer of cells was achieved using an oligopeptide that formed a dense molecular layer on a gold surface and was then electrochemically desorbed from the surface. Human umbilical vein endothelial cells (HUVECs), adhered to gold-coated needles (ϕ600 μm) via the oligopeptide, were transferred to collagen gel along with electrochemical desorption of the molecular layer, resulting in the formation of endothelial cell-lined vascular-like structures. In the following culture, the endothelial cells migrated into the collagen gel and formed branched luminal structures. However, this branching process was strikingly slow (>14 d) and the cell layers on the internal surfaces became disrupted in some regions. To address these issues, we examined the effects of the protein kinase C (PKC) activator, PMA, and shear stress generated by medium flow. Addition of PMA at an optimum concentration significantly accelerated migration, vascular network formation, and its stabilization. Exposure to shear stress reoriented the cells in the direction of the medium flow and further accelerated vascular network formation. Because of the synergistic effects, HUVECs began to sprout as early as 3 d of perfusion culture and neighboring vascular-like structures were bridged within 5 d. Although further investigations of vascular functions need to be performed, this approach may be an effective strategy for rapid fabrication of perfusable microvascular networks when engineering three-dimensional fully vascularized tissues and organs.

Abstract 4066: Glycodelin abolishes PMA-induced migration of MCF-7 breast cancer cells

Abstract Glycodelin is a lipocalin protein mainly expressed in well-differentiated epithelial cells in reproductive tissues. Previously, glycodelin has been shown to induce cell differentiation in endometrial and breast cancer cells. We have used glycodelin-transfected MCF-7 breast cancer cells to study the glycodelin-induced differentiation. Glycodelin expression changed the cellular morphology towards normal, less malignant direction. In addition, glycodelin-producing cells formed significantly smaller tumors in xenograft mice. We have found that the effects of glycodelin are at least partly mediated by repressed PKCδ activity, rendering the cells resistant to activation of phorbol 12-myristate 13-acetate (PMA), a known activator of PKCδ. Here we continued these previous studies by addressing the effect of glycodelin on the invasion and migration of MCF-7 breast cancer cells, and studying the affected signaling route in more detail. Activation of signaling proteins was detected with an antibody array of 43 phospho kinases. Cell migration was studied using wound healing test. The cells were grown on chamber slides until they formed a monolayer and then a scratch was made using a pipet tip. After addition of PMA or DMSO control, the migration of the cells was monitored under microscope. In some of the control cells, PKCδ was down-regulated with RNAi prior the migration assay. For invasion through Matrigel basement membrane preparation, a Boyden chamber assay was used. The cells were grown with and without PMA and the amount of invasive cells was quantified. Phospho-kinase array did not reveal any widespread changes in the amount of phosphorylated signaling molecules between the glycodelin-producing and control cells irrespective of the PMA treatment. However, in addition to PKCδ some changes were found in the levels of phosphoproteins such as p38α and p27. In wound healing test PMA caused a two-fold increase in the migration of the control cells, but had no effect on the glycodelin-producing cells. When the expression of PKCδ was blocked in the control cells using siRNA, the addition of PMA did not increase the migration of the cells. PMA also increased the invasiveness of the cells, but that was detected in both cells clones regardless of glycodelin expression. Without PMA treatment both glycodelin-expressing and control cells behaved similarly in wound healing and invasion tests. In conclusion, glycodelin abolished PMA-induced migration in MCF-7 breast cancer cells, while it did not have any effect on the invasion of the cells. Our results suggest that the effect of glycodelin in migration is mediated by reduced activation of PKCδ. Citation Format: Laura C. Hautala, Riitta Koistinen, Hannu Koistinen. Glycodelin abolishes PMA-induced migration of MCF-7 breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4066. doi:10.1158/1538-7445.AM2014-4066

비빔밥 재료의 항염증 및 항산화 효과

본 연구에서는 우리나라 전통 음식인 비빔밥의 우수성을 검증하고자 비빔밥에 들어가는 재료인 애호박, 도라지, 숙주, 속대기, 부추, 고사리의 조리 전과 후의 항염증 및 항산화 효과를 조사하였다. 인간유래 비만세포인 HMC-1 (human mast cell)에 70% ethanol로 추출한 조리 전, 후의 다양한 비빔밥 재료의 추출물을 1.0 mg/ml의 농도로 처리한 후, PMA와 A23187로 자극하여 염증반응을 유도하였다. MTT 분석에 의한 세포 생존율 결과, 모든 구에서 82% 이상의 세포 생존율을 보여 조리 전, 후의 비빔밥 재료 추출물에 의한 세포 독성은 나타나지 않았다. HMC-1 비빔밥 재료의 추출물을 0.01, 0.1, 1.0 mg/ml의 농도로 처리한 후, 자극제로 염증반응을 유도하였을 때 그 효과를 TNF-<TEX>${\alpha}$</TEX>, IL-6, IL-8의 분비량으로 알아보았다. 전체적으로 cytokine의 분비가 감소하였는데, 특히 1.0 mg/ml 농도에서 조리 후의 부추 추출물이 TNF-<TEX>${\alpha}$</TEX>, IL-6 분비량을 각각 90%, 93% 저해하였고, 무 추출물은 IL-8 분비량을 85% 저해하여 다양한 비빔밥 재료 중 가장 높은 저해율을 나타냈다. DPPH 분석에 의한 항산화 활성결과, 조리 전, 후의 부추 추출물에서 각각 67.50%, 73.65%로 재료 중 가장 높았으며 전반적으로, 조리함으로써 항산화 활성이 향상되었다. 결론적으로 조리 전보다 조리 후의 재료에서 세 가지의 cytokine을 유의성 있게 억제하였고, 항산화 활성 또한 높게 나타나 비빔밥의 우수성을 검증하는 기초자료로 활용이 가능하다고 사료된다. Bibimbab (mixed rice) is a traditional Korean one-dish meal. This study was carried out to investigate the anti-inflammatory and antioxidative effects of raw and seasoned ingredients used in Bibimbab (Cucurbita moschata P., Platycodon grandiflorum A., Vigna radiata L., Porphyra yezonensis udea, Allium ampeloprasum L., Pterdium aguilinum, Raphanus sativus). Human mast cells (HMC-1) were pretreated with 70% ethanol extracts of Bibimbab and further cultured for an appropriate time after the addition of phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187. Cell viability was determined by an MTT assay. None of the ingredients showed cytotoxic effects at a concentration of 1.0 <TEX>${\mu}g/ml$</TEX>. Anti-inflammatory effects were analyzed at 0.01, 0.1, and 1.0 mg/ml concentrations of the raw, seasoned ingredients of PMA. A23187 stimulated HMC-1. Among the various ingredients, seasoned A. ampeloprasum L. extract showed the highest inhibition of TNF-<TEX>${\alpha}$</TEX> and IL-6 secretion (90% and 93%, respectively) at a concentration of 1.0 mg/ml. The R. sativus extract showed the highest inhibition (85%) of IL-8 secretion. DPPH analysis of the antioxidation properties of the ingredients showed that raw and seasoned A. ampeloprasum extracts exhibited the highest DPPH free radical scavenging activity (67.50 and 73.65%, respectively). These results suggest that seasoned ingredients used in Bibimbab have lower anti-inflammatory effects in relation to TNF-<TEX>${\alpha}$</TEX> and IL-6 secretion than raw ingredients in PMA- and A23187-treated HMC-1. In addition, the seasoned ingredients showed a tendency to increase antioxidative activity. Therefore, the ingredients used in Bibimbab have potential as anti-inflammatory and antioxidation agents.

Abstract LB-514: Contribution of ADAM17 to Kit ligand ectodomain shedding in high-grade gliomas

Abstract Background Malignant anaplastic astrocytoma and glioblastoma are considered to derive from astrocytic precursor cells. They display high vascularization, aggressive growth and invasion into the surrounding brain tissue, and respond poorly to conventional treatments. A disintegrin and metalloproteinases (ADAMs) are membrane-associated metalloproteinases with a complex multi-domain structure. Kit ligand (Kitl), also referred to as stem cell factor (SCF) is synthesized as a membrane-anchored precursor that can be processed to produce the soluble growth factor. As a ligand for Kit receptor tyrosine kinase, Kitl plays important roles in activation of various signalling pathways leading to cell proliferation, differentiation, migration and survival. Previous data has shown that ADAM17 affects Kitl shedding. Hypoxia-induced ADAM17 has been reported to contribute to glioma cell invasion whereas Kitl expression has been demonstrated in glioma cell lines but its function remains unclear. As invasive behaviour is a hallmark of high-grade gliomas, the main aim of our recent study is to investigate the functional role and contribution of ADAM17 to ectodomain shedding of Kitl in these tumors. Methods Quantitative RT-PCR analyses for ADAM17 and Kitl were performed using 36 adult glioma tumor samples including 9 pilocytic astrocytomas WHO grade I (PA I), 9 diffuse astrocytomas WHO grade II (DA II), 9 anaplastic astrocytomas WHO grade III (AA III) and 9 glioblastoma multiforme WHO grade IV (GBM) as well as three (LN-229, LN-308 and U87-MG) glioma cell lines. ADAM17 endogenous expression in glioma cell lines was knocked-down using ADAM17-specific short hairpin RNA (shRNA). The phenotype of the stable transfectants was analyzed by proliferation and migration assays. Immunoblotting and ELISA were performed to detect the Kitl ectodomain released in the cell supernatants. Results We found that ADAM17 and KITLG are overexpressed in high-grade gliomas when compared with low-grade gliomas and normal brain tissue and the higher mRNA levels correlated with the tumor grade. Knock-down of ADAM17 by shRNA significantly reduced glioma cell migration. Moreover shedding of Kitl from control cells (scrambled transfected cells) can be stimulated by addition of phorbol 12-myristate 13-acetate (PMA) together with Ionomycin for 1 hour. In contrast, the PMA/Ionomycin-stimulated Kitl shedding was completely prevented in ADAM17-knock-down cells indicating that ADAM17 is an important PMA/Ionomycin -responsive Kitl sheddase in glioma cells. These results provided evidence for an involvement of ADAM17 in PMA/Ionomycin stimulation of Kitl shedding in glioma cells. Conclusion Our results suggest that ADAM17 in high-grade gliomas effects Kitl ectodomain shedding and may substantially contribute to glioma cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-514. doi:1538-7445.AM2012-LB-514

Rewiring of the Protein Kinase C Beta 2 (PKC βII) and Bcr/Abl Signal Transduction Pathways

Abstract 1673Chronic myeloid leukemia (CML) accounts for 20% of adult leukemias, and is characterized by the presence of the bcr-abl fusion gene. This gene leads to the production of a constitutively active Abl tyrosine kinase, which promiscuously phosphorylates/activates a variety of intracellular signal transduction pathways. The presence of constitutively active Abl is essential for CML blast survival even in advanced disease, and underlies the success that the Bcr/Abl kinase inhibitor imatinib has had. However, resistance to imatinib occurs in a significant number of accelerated phase or blast crisis patients and is a significant clinical obstacle. Instead of trying to inhibit Bcr/Abl signaling, we propose a previously unexplored approach to ‘rewire' kinase signaling pathways to activate a “suicide” prodrug that would not normally be activated by Bcr/Abl. We have previously shown that direct activation of PKCβII by Phorbol 12-myristate 13-acetate (PMA) drives dendritic cell differentiation in both normal and leukemic progenitors, as well as induces apoptosis and growth arrest. PKCβII is a member of the PKC family of serine/threonine kinases and is kept in an inactive state in the cytosol by interactions between its pseudosubstrate and kinase domains; upon activation, the pseudosubstrate domain releases the kinase domain, and PKCβII translocates to the plasma membrane. Substitution of an alanine at position 25 in the pseudosubstrate domain for a phosphomimetic glutamic acid leads to the constitutive activation of PKCβII. Similarly, we hypothesized that substituting the alanine at position 25 for a phosphorylatable tyrosine (A25Y) along with the corresponding Bcr/Abl kinase target motif (Ala-X-X-Ile-Tyr-X-X-Phe/Pro) into the pseudosubstrate domain of PKCβII, would allow the Bcr/Abl tyrosine kinase to activate the PKCβII signaling pathway. Bcr/Abl mediated activation of PKCβII would then lead to the induction of apoptosis, growth inhibition, and differentiation. Using confocal microscopy, we show that following transfection WT-PKCβII is cytoplasmically located in media alone and addition of PMA leads to translocation to the plasma membrane, indicating activation in both Bcr/Abl+ K562 cells, and Bcr/Abl− KG1a cells. However when A25Y-PKCβII constructs are transfected in, A25Y-PKCβII is found at the plasma membrane in K562, but not in KG1a cells in media alone. These observations were then quantified using ImageStream technology, which allows for simultaneous acquisition of both flow cytometric data, and high resolution fluorescent images. Using this technology, we show that A25Y-PKCβII is activated in media alone in K562 cells, and only upon addition of PMA in KG1a cells. Additionally, when Bcr/Abl was stably transfected in KG1a cells, A25Y-PKCβII was able to translocate to the plasma membrane in media alone, indicating activation by Bcr/Abl. Upon activation and translocation to the plasma membrane, PKCβII is rapidly degraded; accordingly, we show that expression of WT PKCβII decreases only by 20% over 72 hours post transfection, whereas expression of A25Y-PKCβII results in an average decrease of 90% over the same 72 hour time course. To test whether activation of A25Y-PKCβII leads to apoptosis and growth arrest, Bcr/Abl+ K562, and Bcr/Abl− KG1a cells were transfected with either WT and A25Y-PKCβII and measured for apoptosis with AnnexinV using Flow Cytometry. We found that A25Y-PKCβII induced a maximum of a 4-fold increase of apoptosis when compared to WT PKCβII in K562 cells; however there was no increase observed in KG1a cells. This work demonstrates that rewiring PKCβII to be inducible by Bcr/Abl is feasible, and that activation of PKCβII by Bcr/Abl induces characteristic translocation to the plasma membrane, and induction of apoptosis. Future work will address whether induction of DC differentiation is maintained in Bcr/Abl activated PKCβII cells, as well as the molecular kinetics of this activation. Disclosures:No relevant conflicts of interest to declare.

In VitroManipulation of Endothelial Progenitor Cell Adhesion to Vascular Endothelium and Extracellular Matrix by the Phorbol Ester PMA

Injection of endothelial progenitor cells (EPCs) into arteries for cell therapy is a promising field in regenerative medicine. However, adhesion of EPCs during capillary passage is restricted, and non-adhering cells are lost into circulation. Here we demonstrate that it is possible to achieve a three- to sevenfold higher rate of EPC adhesion to endothelium and extracellular matrix molecules after short-term activation with phorbol myristate acetate (PMA). In addition, differentiation and toxicity analyses of PMA activated EPCs showed no impact on cell differentiation and negligible impact on cell survival.

αVβ3-integrin expression through ERK activation mediates cell attachment and is necessary for production of tumor necrosis factor alpha in monocytic THP-1 cells stimulated by phorbol myristate acetate

Macrophages play a key role in inflammation. Activated macrophages express adhesion molecules and produce tumor necrosis factor alpha (TNFα). Integrins are the main adhesion molecules that mediate binding to the extracellular matrix and they are involved in intracellular pathways. In the present study, human monocytic THP-1 cell adhesion to uncoated plastic plate was examined to investigate the regulatory mechanism of TNFα secretion. Addition of phorbol myristate acetate (PMA) for THP-1 cell activation induced cell adhesion in parallel with TNFα production. Among the mitogen-activated protein kinase pathways, the protein kinase C (PKC)–extracellular signal-regulated kinase (ERK) pathway was involved in αVβ3-integrin expression and PMA-induced cell adhesion. Flow cytometry and reverse transcription – quantitative polymerase chain reaction analysis revealed increased expression of matrix-binding integrins including integrin-αVβ3. Blockade of αVβ3-integrin by a specific antibody suppressed cell adhesion and TNFα production. These findings indicate that TNFα production from THP-1 cells is PKC–ERK, αVβ3-integrin and adhesion-dependent and its related pathway could be a target for TNFα-related diseases.

158 DEVELOPMENT OF FIELD ASSAY FOR EVALUATION OF WHITE RHINOCEROS NEUTROPHIL FUNCTION AS A STRESS MARKER

Kruger National Park (KNP) is the primary source of translocated white rhinoceroses (Ceratotherium simum) in South Africa. Capture and transport of the rhinoceroses is a highly successful procedure. However, some relocated rhinoceroses present with fertility problems in the 1–2 years post capture. Novel research has shown that one can assess stress using respiratory neutrophil burst levels in several species (Huber et al. 2006 Protoplasma 229, 221–224; Weyts et al. 1998 Dev. Comp. Immunol. 22, 563–572). The hypothesis is that some rhinoceroses respond poorly to stress. The response to stress might be measurable by a neutrophil function (NF) assay, and NF depression might identify adversely stressed animals. The aim was to develop an in-the-bush test for NF and evaluate the changes in neutrophil activity during capture and loading into transportation crates. Neutrophil function was assayed using a portable luminometer (3M Clean-Trace™, 3M, St. Paul, MN, USA) while driving from one capture to the next. For the reaction mixture, 500 μL of PBS was incubated with 10 μL of fresh heparinized blood at 37.6°C. The incubator unit was power by a 230 V convertor in the vehicle. After 5 min of incubation, 100 μL of luminol was added. This sample was read in the luminometer as a blank and then to the sample, 100 μL of 12-O-tetradecanoylphorbol-13-acetate (TPA), also commonly known as phorbol 12-myristate 13-acetate (PMA), a diester of phorbol, was added. The luminescence readings or relative light units (RLU) were read at 2.5 min and then 5 min after PMA addition. All samples were prepared in triplicate. The readings were then taken every 5 min for 65 min. Two samples of blood were evaluated for each rhinoceros: the first sample at capture (anaesthetic induction) and then a second sample after loading of the rhinoceros into the transport crate, 20 to 30 min later. Two response curves were produced for each rhinoceros using the means of the triplicate readings. The curves were then transformed with a trend line and the area under the curve (AUC) was calculated. The 2 AUC for rhinoceros were then compared statistically using Wilcoxon rank regression, with P &lt; 0.05 considered statistically different. Three distinct response curve patters were seen. Sixty-two percent (16/26) of the rhinoceroses had no statistical (P &lt; 0.05) difference between capture and loading samples. In 5 of 26 (19%), the loading AUC was statistically greater than the capture sample, and in 5 of 26 (19%), the capture AUC was statistically greater than the loading sample. In conclusion, NF can be assayed in the field using the blood of wild caught rhinoceroses. The assay is repeatable and can distinguish 3 populations of wild caught rhinoceroses. The hope is that future research will allow us to identify rhinoceroses that have negative stress reactions and to change the capture conditions to make the process less stressful.

Abstract 3820: Inhibition of M1 macrophages may aid the conversion process to M2, which may aid tumor survival and growth

Abstract Macrophages perform a variety of functions, from phagocytosis to the repair and remodeling of damaged tissues. Two phenotypes have been proposed: M1 and M2. The M1 macrophage performs phagocytosis of tumor cells and releases pro-inflammatory cytokines. The M2 macrophage acts as a support cell by promoting the healing of damaged cells by angiogenesis and tissue remodeling. The transition from M1 to M2 may be facilitated by tumor environments. To further study the relationship between cancer and macrophages we developed a live-cell migration assay, which measures cancer's ability to recruit macrophages. We hypothesize that cancer cell lines have varied abilities to recruit macrophages and convert them into M2, and that this ability corresponds to the aggressiveness of the cell line. Macrophages were derived from the human mononuclear phagocyte cell line U937, after addition of phorbol 12-myristate 13-acetate (PMA). These U937 cells were incubated with three adenocarcinomas of varying aggressivity. The adenocarcinomas were MDA-MB-435, MDA-MB-231, and MCF7. The U937 cells were separated from the three cell lines by 3.0 µm trans-permeable polycarbonate membranes (Corning Inc.) which allowed the U937 cells to migrate to the cell lines. After 1 hour non-migratory U937 cells were isolated and counted by means of a hemocytometer. The control without cancer caused 73.86% of U937 cells to remain, MDA-MB-435 69.28% (N=24, P=0.29), MCF7 61.09% (N=24, P&amp;lt;0.01), and MDA-MB-231 55.83% (N=24, P&amp;lt;0.01). This data was then compared with expression levels of macrophage Migration Inhibition Factor (MIF) analyzed from Real Time PCR data. These samples consisted of monocytes, from human blood, which were co-incubated with the cell lines for 48 hours. The cells were separated by 0.4 µm trans-permeable polycarbonate membranes (Corning Inc.) to prevent cells from coming into contact and allowing signal exchange. The RT-PCR data correlated with the migration data. The amount of MIF increased 7.56 fold, (N=9) in MDA-MB-435 when compared to controls, 3.13 fold for MCF7 (N=9), and 1.33 fold for MDA- MB-231 (N=9). This data suggests that the more aggressive the cancer the more MIF emitted and the greater the potential to inhibit macrophages. Previous studies have shown Interleukin-10, (IL-10), IL-6, IL-4, and prostaglandin F2 (PGF2) to be released from tumors and inhibit the cytotoxic activity of macrophages. This exposure to IL-10 has been suggested to convert M1 macrophages to a M2 phenotype. The migration inhibition observed from this study might be the initial step in the conversion process from M1 to M2. 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 3820.

Induction of ANGPTL4 expression in human airway smooth muscle cells by PMA through activation of PKC and MAPK pathways

In this study, we demonstrate that protein kinase C (PKC) activators, including phorbol-12-myristate-13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DOG), and platelet-derived growth factor alpha are potent inducers of angiopoietin-like protein 4 (ANGPTL4) expression in several normal lung cell types and carcinoma cell lines. In human airway smooth muscle (HASM) cells induction of ANGPTL4 expression is observed as early as 2 h after the addition of PMA. PMA also increases the level of ANGPTL4 protein released in the medium. PKC inhibitors Ro31-8820 and Gö6983 greatly inhibit the induction of ANGPTL4 mRNA by PMA suggesting that this up-regulation involves activation of PKC. Knockdown of several PKCs by corresponding siRNAs suggest a role for PKCalpha. PMA does not activate MAPK p38 and p38 inhibitors have little effect on the induction of ANGPTL4 indicating that p38 is not involved in the regulation of ANGPTL4 by PMA. In contrast, treatment of HASM by PMA induces phosphorylation and activation of Ra, MEK1/2, ERK1/2, JNK, Elk-1, and c-Jun. The Ras inhibitor manumycin A, the MEK1/2 inhibitor U0126, and the JNK inhibitor SP600125, greatly reduce the increase in ANGPTL4 expression by PMA. Knockdown of MEK1/2 and JNK1/2 expression by corresponding siRNAs inhibits the induction of ANGPTL4. Our observations suggest that the induction of ANGPTL4 by PMA in HASM involves the activation of PKC, ERK, and JNK pathways. This induction may play a role in tissue remodeling during lung injury and be implicated in several lung pathologies.

P90 Ribosomal S6 Kinase and p70 Ribosomal S6 Kinase Link Phosphorylation of the Eukaryotic Chaperonin Containing TCP-1 to Growth Factor, Insulin, and Nutrient Signaling

Chaperonin containing TCP-1 (CCT) is a large multisubunit complex that mediates protein folding in eukaryotic cells. CCT participates in the folding of newly synthesized polypeptides, including actin, tubulin, and several cell cycle regulators; therefore, CCT plays an important role in cytoskeletal organization and cell division. Here we identify the chaperonin CCT as a novel physiological substrate for p90 ribosomal S6 kinase (RSK) and p70 ribosomal S6 kinase (S6K). RSK phosphorylates the beta subunit of CCT in response to tumor promoters or growth factors that activate the Ras-mitogen-activated protein kinase (MAPK) pathway. CCTbeta Ser-260 was identified as the RSK site by mass spectrometry and confirmed by site-directed mutagenesis. RSK-dependent Ser-260 phosphorylation was sensitive to the MEK inhibitor UO126 and the RSK inhibitor BID-1870. Insulin weakly activates RSK but strongly activates the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway and utilizes S6K to regulate CCTbeta phosphorylation. Thus, the Ras-MAPK and PI3K-mTOR pathways converge on CCTbeta Ser-260 phosphorylation in response to multiple agonists in various mammalian cells. We also show that RNA interference-mediated knockdown of endogenous CCTbeta causes impaired cell proliferation that can be rescued with ectopically expressed murine CCTbeta wild-type or phosphomimetic mutant S260D, but not the phosphorylation-deficient mutant S260A. Although the molecular mechanism of CCTbeta regulation remains unclear, our findings demonstrate a link between oncogene and growth factor signaling and chaperonin CCT-mediated cellular activities.

The effects of pentoxifylline on the generation of reactive oxygen species and lipid peroxidation in human spermatozoa.

The aims of this study were to compare the in vitro effects of 3.6 mM and 7.2 mM pentoxifylline on the ability of spermatozoa to generate reactive oxygen species (ROS) and on lipid peroxidation (LPO). Semen samples were obtained from 10 asthenozoospermic men who had been previously identified as producing ROS after addition of Phorbol 12-myristate 13-acetate (PMA) during the screening of patients attending with male factor infertility. Spermatozoa were prepared by a swim-up technique from unprocessed semen and divided into 3 aliquots. To the control aliquot [A] an equal volume of BWW medium was added. To aliquots B and C an equal volume of BWW medium containing pentoxifylline was added to obtain final concentrations of 3.6 and 7.2 mM, respectively. ROS production was measured from peak luminescence (mV 10(-7) sperm) using a lucigenin chemiluminescent probe. LPO was also measured in the medium surrounding the spermatozoa after 30 min exposure to pentoxifylline using the thiobarbituric acid (TBA) assay for malondialdehyde (MDA). The reduction in ROS production was significantly greater in the samples exposed to 7.2 mM pentoxifylline as compared with the control and 3.6 mM pentoxifylline samples. There was no significant difference in peak luminescence between control and 3.6 mM pentoxifylline specimens. Both concentrations of pentoxifylline caused comparable reductions in MDA concentration in the medium (P < 0.05) surrounding the spermatozoa compared with control after 30 min exposure. Extracellular ROS generation may damage surrounding healthy spermatozoa. These findings suggest that higher concentrations of pentoxifylline are protective against ROS release in susceptible spermatozoa and may also reduce collateral LPO.

Effects of phorbol 12-myristate 13-acetate on potassium transport in the red blood cells of frog Rana temporaria

Phorbol 12-myristate 13-acetate (PMA), a stimulator of PKC, was examined for its influence on K(+) ((86)Rb) influx in the frog erythrocyte. PMA, 0.1 microM, was found to accelerate ouabain-sensitive K(+) influx, which was suppressed by 73% with 1 mM amiloride, indicating secondary activation of the Na(+)-K(+)-pump due to stimulation of Na(+ )/H(+) exchange. PMA-induced stimulation of the sodium pump was completely inhibited with 1 microM staurosporine and by approximately 50% with 20 microM chelerythrine. In contrast to Na(+)-K(+)-pump, an activity of Cl(-)-dependent K(+) transport (K-Cl cotransport, KCC), calculated as the difference between K(+) influxes in Cl(-) and NO(3) (-)-media, was substantially decreased under the influence of PMA. Staurosporine fully restored the PMA-induced inhibition of KCC, whereas chelerythrine did not exert any influence. Osmotic swelling of the frog erythrocytes was accompanied by approximately twofold stimulation of KCC. Swelling-activated KCC was inhibited by approximately 50 and approximately 83% in the presence of PMA and genistein, respectively, but not chelerythrine. Exposure of the frog erythrocytes to 5 mM fluoride (F(-)) also reduced the KCC activity in isotonic and hypotonic media, with maximal suppression of K(+) influx in both media being observed upon simultaneous addition of PMA and F(-). Furosemide and [(dihydronindenyl)oxy] alkanoic acid inhibited the K(+) influx in both the media by approximately 50-60%. The results obtained show both the direct and indirect effects of PMA on the K(+) transport in frog erythrocytes and a complicated picture of KCC regulation in frog erythrocytes with involvement of PKC, tyrosine kinase and protein phosphatase.

Phosphorylation of Endothelial Nitric-oxide Synthase Regulates Superoxide Generation from the Enzyme

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O(2)* generation are unknown. Therefore, we phosphorylated recombinant BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O(2)* generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O(2)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O(2)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.

Identification of ERK and JNK as signaling mediators on protein kinase C activation in cultured granulosa cells

PKC signaling is critical for follicular development and the induction of ovulatory genes including Pgr, Prkg2, and Cyp11a1 (SCC). We investigated PKC signaling mechanisms in the JC-410 porcine granulosa cell line stably expressing an SCC-luciferase reporter gene containing 2kb of the porcine SCC promoter. Addition of phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C, induced the promoter approximately 6-fold over the basal levels in 4h. This effect was predominantly mediated by the PKC beta and delta isoforms. PMA-mediated induction of the SCC promoter was sensitive to inhibition of ERK1/2 or JNK. Inhibition of p38 MAP kinase or Src tyrosine kinase did not alter the PMA-mediated inducibility of the promoter. SCC promoter induction in response to PMA treatment required basal EGF-receptor activity, but did not involve ectodomain shedding. Western blot analyses using phospho-specific antibodies showed that PMA treatment of JC-410 cells induced phosphorylation of MEK1/2, ERK1/2, and its downstream target p90 RSK at 15min. We also documented the rapid phosphorylation of JNK1/2 in response to PMA treatment. Phosphorylation of ERK and JNK was robust and sustained in contrast to activation of PKA and EGF-receptor signaling in these cells. Pretreatment of JC-410 granulosa cells with IGF-1 had a synergistic effect on PMA-mediated induction of the SCC promoter. We demonstrated the importance of PMA activation of ERK signaling and the synergism with IGF-1 by showing similar responses for Prkg2 expression in primary granulosa cells. In conclusion, our studies demonstrated PMA activation of ERK and JNK signaling which is relevant in the regulation of gene expression during follicular development, ovulation, and luteinization.

Acidosis, oxygen, and interference with mitochondrial permeability transition pore formation in the early minutes of reperfusion are critical to postconditioning’s success

Repetitive cycles of reflow/reocclusion in the initial 2 min following release of a prolonged coronary occlusion, i.e., ischemic postconditioning (IPoC), salvages ischemic myocardium. We have proposed that the intermittent ischemia prevents formation of mitochondrial permeability transition pores (MPTP) by maintaining an acidic myocardial pH for several minutes until survival kinases can be activated. To determine other requisites of IPoC, isolated rabbit hearts were subjected to 30 min of regional myocardial ischemia and 120 min of reperfusion. Infarct size was determined by staining with triphenyltetrazolium chloride. During the first 2 min of reperfusion the perfusate was either at pH 7.4 following equilibration with 95% O(2)/5% CO(2), pH 6.9 following equilibration with 80% N(2)/20% CO(2), or pH 7.8 following equilibration with 100% O(2). Whereas acidic, oxygenated perfusate for the first 2 min of reperfusion was cardioprotective, protection was lost when acidic perfusate was hypoxic. However, the acidic, hypoxic hearts could be rescued by addition of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, to the perfusate. Therefore, both low pH and restoration of oxygenation are necessary for protection, and the signaling step requiring combined oxygen and H(+) must be upstream of PKC. To gain further insight into the mechanism of IPoC, the latter was effected with 6 cycles of 10-s reperfusion/10-s reocclusion. Its protective effect was abrogated by either making the oxygenated perfusate alkaline during the reperfusion phases or making the reperfusion buffer hypoxic. Presumably the repeated coronary occlusions during IPoC keep myocardial pH low while the resupply of oxygen during the intermittent reperfusion provides fuel for the redox signaling that acts to prevent MPTP formation even after restoration of normal myocardial pH. Hearts treated simultaneously with IPoC and alkaline perfusate could not be rescued by addition to the perfusate of either PMA or SB216763 which inhibits GSK-3beta, the putative last cytoplasmic signaling step in the signal transduction cascade leading to MPTP inhibition. Yet cyclosporin A which also inhibits MPTP formation does rescue hearts made alkaline during IPoC. In view of prior studies in which the ROS scavenger N-2-mercaptopropionyl glycine aborts IPoC's protection, our data reveal that IPoC's reperfusion periods are needed to support redox signaling rather than improve metabolism. The low pH, on the other hand, is equally necessary and seems to suppress MPTP directly rather than through upstream signaling.

Production of the Soluble Form of KIT, s-KIT, Abolishes Stem Cell Factor-Induced Melanogenesis in Human Melanocytes

The signaling of stem cell factor (SCF) and its receptor KIT (membrane-bound KIT; m-KIT) plays an important role in melanocyte development, survival, proliferation, and melanogenesis. It has been demonstrated in other systems that a soluble form of m-KIT released from the cell surface (s-KIT) regulates SCF signaling, although there have been no reports pertaining to the existence and the biological role of s-KIT in melanocytes. In this study, we therefore examined the involvement of s-KIT in melanogenesis. Western blotting analysis revealed that treatment with phorbol 12-myristate-13-acetate (PMA) or 4-aminophenylmercuric acetate (APMA) induced s-KIT production in cultured human melanocytes. Inhibitors of tumor necrosis factor-alpha-converting enzyme (TACE) and metalloproteinases (MMPs) muted this release of s-KIT into the media. Human recombinant s-KIT added to melanocytes inhibited SCF-induced phosphorylation of m-KIT, resulting in suppression of SCF-induced melanogenesis. Additionally, APMA-induced s-KIT production abolished SCF-induced melanogenesis as effectively as a KIT-neutralizing antibody. Concomitantly, APMA and TACE inhibitors significantly decreased and increased melanin synthesis, respectively, in an in vitro skin model. Taken together, these findings provided an insight into the elaborate mechanism of SCF/m-KIT signaling in human melanocytes and suggested that production of s-KIT contributes to the regulation of human skin pigmentation. Journal of Investigative Dermatology (2008) 128, 1763-1772; doi:10.1038/jid.2008.9; published online 31 January 2008.

Eosinophils adhere to and stimulate replication of lung fibroblasts ‘in vitro’

Eosinophils have been implicated in several disorders associated with the development of fibrosis. This led us to investigate the interactions between eosinophils and fibroblasts in vitro. Adhesion between purified guinea pig peritoneal eosinophils and monolayers of human fetal lung fibroblasts was assessed using the rose bengal dye staining assay. Fibroblast replication was assessed using a colorimetric assay based upon the uptake and subsequent release of methylene blue. Addition of phorbol myristate acetate induced a rapid, time-dependent increase in eosinophil adhesion (127% and 328% over basal adhesion after 10 and 30 min, respectively). Phorbol myristate acetate-induced adhesion was inhibited by the peptides RGDS and GRGDS (48% and 42%, respectively using 1 mM peptide) and by nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism (46% inhibition at 15 microM). In addition, 24 h culture of fibroblast monolayers with interleukin 1 alpha (IL-1 alpha) or tumour necrosis factor alpha (TNF alpha) resulted in enhanced adhesion (10 U/ml IL-1 alpha stimulated adhesion by 55% of control, 500 U/ml TNF alpha by 75% of control). Conditioned media from cultured eosinophils stimulated fibroblast replication in a time-dependent fashion with maximal stimulation at 3 h. In contrast, media from guinea pig peritoneal macrophages in culture did not show such an effect. This study indicates that eosinophils are capable of both adhering to and releasing mitogens for fibroblasts in vitro. These observations suggest that eosinophils have the capacity to play a role in the development of fibrosis in disorders where they have been shown to be present.

Defective expression of early activation genes in cartilage-hair hypoplasia (CHH) with severe combined immunodeficiency (SCID).

Cartilage-hair hypoplasia (CHH) is an autosomal recessive disease of unknown etiology characterized by metaphyseal dysostosis, unpigmented hair, and defective cellular immunity. We studied peripheral blood mononuclear cells (PBMC) of a boy with CHH and combined immunodeficiency in an attempt to characterize further the immune defect in this disease. Stimulation of his PBMC with mitogens was associated with severely depressed IL-2 and interferon-gamma (IFN-gamma) synthesis and IL-2 receptor alpha-chain (IL-2R alpha) expression and resulted in poor lymphocyte proliferation that was only modestly upregulated by the addition of recombinant IL-2 (rIL-2). The defective proliferation and lymphokine synthesis were not corrected by the addition of phorbol myristate acetate (PMA) and ionomycin, agents that bypass receptor-mediated signalling, indicative of a distal abnormality. Importantly, the levels of mRNA encoding c-myc, IL-2R alpha, IL-2 and IFN-gamma were markedly decreased in patient lymphocytes stimulated with PMA+ionomycin as compared to control lymphocytes. The defect in the expression of these early activation genes was selective in that induction by mitogens of mRNA encoding other early activation gene products such as c-fos and c-jun was not impaired. These results suggest that the underlying defect in this patient and perhaps others with CHH may be an abnormality in a component of intracellular signalling pathways or in a trans-acting factor which regulates the expression of a selected number of early activation genes.

Defective functional response to membrane stimuli in lymphocytes from patients with benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is a local disturbance in the prostate that may involve an inflammatory infiltrate predominantly composed of activated lymphocytes and macrophages. The activation and proliferative response of T lymphocytes to different mitogenic signals has been analysed in peripheral blood mononuclear cells (PBMC) from 45 patients with BPH and 55 healthy controls. The PBMC obtained from the patients showed a significant specific impairment in proliferation, CD25 expression and IL-2 production in response to stimulation with lectins (phytohaemagglutinin (PHA), concanavalin A (Con A)), that was not corrected by the addition of IL-2 or of phorbol esters (phorbol myristate acetate (PMA)). Also, the CD28 response was defective in patient PBMC. Activation with anti-CD3 or anti-CD2 MoAbs was normal, but the addition of PMA to these stimuli provoked a significant defective response. Only the use of transmembrane stimuli (PMA and ionomycin) elicited responses similar to those found in the control group. The results indicate that peripheral T lymphocytes from BPH patients show a functional impairment that is mainly explained by an alteration of membrane signals (PHA, CD28) and is distal to protein kinase C (PKC) activation.