Presence Of Phorbol 12-myristate 13-acetate Research Articles

Intercellular Transfer of the Cellular Prion Protein

The cellular prion protein (PrP(C)) is a glycosylphosphatidylinositol (GPI)-anchored protein. We investigated whether PrP(C) can move from one cell to another cell in a cell model. Little PrP(C) transfer was detected when a PrP(C) expressing human neuroblastoma cell line was cultured with the human erythroleukemia cells IA lacking PrP(C). Efficient transfer of PrP(C) was detected with the presence of phorbol 12-myristate 13-acetate, an activator of protein kinase C. Maximum PrP(C) transfer was observed when both donor and recipient cells were activated. Furthermore, PrP(C) transfer required the GPI anchor and direct cell to cell contact. However, intercellular protein transfer is not limited to PrP(C), another GPI-anchored protein, CD90, also transfers from the donor cells to acceptor cells after cellular activation. Therefore, this transfer process is GPI-anchor and cellular activation dependent. These findings suggest that the intercellular transfer of GPI-anchored proteins is a regulated process, and may have implications for the pathogenesis of prion disease.

Generation of C5a by phagocytic cells.

The complement activation product, C5a, is a powerful phlogistic factor. Using antibodies to detect human or rat C5a, incubation at pH 7.4 of human blood neutrophils or rat alveolar macrophages (AMs) with C5 in the presence of phorbol 12-myristate 13-acetate (PMA) led to generation of C5a. Rat AMs activated with lipopolysaccharide also generated C5a from C5. With activated neutrophils, extensive cleavage of C5 occurred, whereas activated macrophages had much more selective proteolytic activity for C5. Peripheral blood human or rat mononuclear cells and rat alveolar epithelial cells when stimulated with phorbol ester all failed to demonstrate an ability to cleave C5, suggesting a specificity of C5 cleavage by phagocytic cells. With rat AMs, C5a generation was time-dependent and was blocked if AMs were pretreated with inhibitors of transcription or protein synthesis (actinomycin D or cycloheximide). Similar treatment of activated human polymorphonuclear leukocytes only partially reduced C5a generation after addition of C5. C5a generated by activated AMs was biologically (chemotactically) active. This generation was sensitive to serine protease inhibitors but not to other classes of inhibitors. These data indicate that phagocytic cells, especially lung macrophages, can generate C5a from C5. In the context of the lung, this may represent an important C5a-generating pathway that is independent of the plasma complement system.

RasGRP4 Is a Novel Ras Activator Isolated from Acute Myeloid Leukemia

Although a number of genetic defects are commonly associated with acute myeloid leukemia (AML), a large percentage of AML cases are cytogenetically normal. This suggests a functional screen for transforming genes is required to identify genetic mutations that are missed by cytogenetic analyses. We utilized a retrovirus-based cDNA expression system to identify transforming genes expressed in cytogenetically normal AML patients. We identified a new member of the Ras guanyl nucleotide-releasing protein (RasGRP) family of Ras guanine nucleotide exchange factors, designating it RasGRP4. Subsequently, cDNA sequences encoding rodent and human RasGRP4 proteins were deposited in GenBank. RasGRP4 contains the same protein domain structure as other members of the RasGRP family, including a Ras exchange motif, a CDC25 homology domain, a C1/diacyglycerol-binding domain, and putative calcium-binding EF hands. We show that expression of RasGRP4 induces anchorage-independent growth of Rat1 fibroblasts. RasGRP4 is a Ras-specific activator and, interestingly, is highly expressed in peripheral blood leukocytes and myeloid cell lines. Unlike other RasGRP proteins, RasGRP4 is not expressed in the brain or in lymphoid cells. We demonstrated that 32D myeloid cells expressing RasGRP4 have elevated levels of activated Ras compared with control cells, and phorbol 12-myristate 13-acetate (PMA) treatment greatly enhanced Ras activation. PMA induced membrane localization of RasGRP4 and 32D cells expressing RasGRP4 were capable of cytokine-independent proliferation in the presence of PMA. We conclude that RasGRP4 is a member of the RasGRP family of Ras guanine nucleotide exchange factors that may play a role in myeloid cell signaling growth regulation pathways that are responsive to diacylglycerol levels.

Role of protein kinase C signaling in collagen degradation by rabbit corneal fibroblasts cultured in three-dimensional collagen gels.

To understand the mechanism of corneal ulceration by characterizing the intracellular signaling pathways that regulate collagen degradation by corneal fibroblasts cultured in three-dimensional type I collagen gels. Specifically, the potential roles of protein kinase C (PKC) and protein kinase A (PKA) in collagen degradation were investigated. Rabbit corneal fibroblasts were cultured in three-dimensional type I collagen gels for 24 hours in the presence of plasminogen and in the absence or presence of activators or inhibitors of PKC or PKA. Degradation of collagen fibrils was then evaluated by measurement of released hydroxyproline, and the production of matrix metalloproteinases (MMPs) was assessed by gelatin zymography and immunoblot analysis. The PKC activator phorbol 12-myristate 13-acetate (PMA) increased the extent of collagen degradation by corneal fibroblasts in a dose-dependent manner, with the maximal effect apparent at a concentration of 0.1 microM. The inactive analog 4alpha-PMA had no effect on collagen degradation. The PKC inhibitor H-7 reduced the extent of collagen degradation by corneal fibroblasts in the absence or presence of PMA. Phorbol 12-myristate 13-acetate also increased the production of proMMP-1, -3, and -9 by corneal fibroblasts, whereas H-7 inhibited this effect. Neither the PKA activators 8-bromo-cAMP, isobutylmethylxanthine, and forskolin nor the PKA inhibitor HA1004 affected collagen degradation by corneal fibroblasts. These results demonstrate that PKC plays an important role in collagen degradation by corneal fibroblasts in three-dimensional type I collagen gels, whereas PKA does not appear to participate in this process.

Implication of three isoforms of PLA 2 in human T-cell proliferation

We observed that human (Jurkat) T-cells constitutively expressed the mRNA, encoding for the four isoforms of phospholipase A 2 (PLA 2), i.e. two secretory (type IB and type V), and two cytosolic (type IV, Ca 2+-dependent and type VI, Ca 2+-independent). In order to assess whether these PLA 2 isoforms are active, we labeled Jurkat T-cells with [ 3H]arachidonic acid ([ 3H]AA) and determined its release into the extracellular medium in the presence of phorbol 12-myristate 13-acetate (PMA) and ionomycin. The three PLA 2 isoforms seem functional as aristolochic acid and bromoenol lactone (BEL), the respective inhibitors of type IB/type V and type VI PLA 2s, significantly inhibited the release of free [ 3H]AA. On the other hand, arachidonyl trifluoromethyl ketone (AACOCF 3), an inhibitor of type IV PLA 2, failed to curtail significantly the release of free [ 3H]AA into the extracellular medium. We assessed the implication of these PLA 2 isoforms in transcription of the interleukin-2 (IL-2) gene, involved in T-cell proliferation. Hence, aristolochic acid and BEL, but not AACOCF 3, significantly inhibited the PMA and ionomycin-induced induction of mRNA of IL-2. Similarly, aristolochic acid and BEL, but not AACOCF 3, significantly inhibited the PMA and ionomycin-induced secretion of IL-2 in the culture supernatants. Together these results suggest that human Jurkat T-cells possess two secretory and two cytosolic PLA 2 isoforms and only three of them (type IB, type V and type VI) are implicated in T-cell proliferation.

Identification of mechanically induced genes in human monocytic cells by DNA microarrays.

Hypertension is a risk factor for coronary heart disease. Macrophages are critically involved in both atherogenesis and plaque instability. Although macrophages may be subjected to excess mechanical stress in these diseases, the way in which biomechanical forces affect macrophage function remains incompletely defined. To investigate the molecular response to mechanical force in macrophages. We used a DNA microarray with 1056 genes to describe the transcriptional profile of mechanically induced genes in human monocytic THP-1 cells. Mechanical deformation was applied to a thin and transparent membrane on which cells were cultured. After THP-1 cells were pre-incubated in the presence of phorbol 12-myristate 13-acetate (0.2 micromol/l) for 24 h, THP-1 cells attached to the membrane were subjected to biaxial mechanical strain. Interleukin-8 concentrations were determined using an enzyme-linked immunosorbent assay. In DNA microarray analysis, cyclic mechanical strain at 1 Hz induced only three genes more than 2.5-fold at 3 and 6 h in THP-1 cells: prostate apoptosis response-4 (3.0-fold at 3 h, 6.7-fold at 6 h), interleukin-8 (4.3-fold at 6 h) and the immediate-early response gene, IEX-1 (2.6-fold at 6 h). Real-time reverse transcriptase polymerase chain reaction analysis confirmed the amplitude-dependent induction of these three genes. In addition, mechanical strain increased interleukin-8 protein expression. The present study demonstrates that human monocytic cells respond to mechanical deformation with induction of immediate-early and inflammatory genes. These findings suggest that mechanical stress in vivo, such as that associated with hypertension, may play an important part in atherogenesis and instability of coronary artery plaques, through biomechanical effects on vascular macrophages.

Protein kinase Calpha is a calpain target in cultured embryonic muscle cells.

Previously we isolated a micro-calpain/PKCalpha complex from skeletal muscle which suggested tight interactions between the Ca2+-dependent protease and the kinase in this tissue. Our previous studies also underlined the involvement of ubiquitous calpains in muscular fusion and differentiation. In order to precise the relationships between PKCalpha and ubiquitous calpains in muscle cells, the expression of these two enzymes was first examined during myogenesis of embryonic myoblasts in culture. Our results show that calpains and PKCalpha are both present in myotubes and essentially localized in the cytosolic compartment. Moreover, calpains were mainly present after 40 h of cell differentiation concomitantly with a depletion of PKCalpha content in the particulate fraction and the appearance of PKMalpha fragment. These results suggest a possible calpain dependent down-regulation process of PKCalpha in our model at the time of intense fusion. In our experimental conditions phorbol myristate acetate (PMA) induced a rapid depletion of PKCalpha in the cytosolic fraction and its translocation toward the particulate fraction. Long term exposure of myotubes in the presence of PMA induced down-regulation of PKCalpha, this process being partially blocked by calpain inhibitors (CS peptide and inhibitor II) and antisense oligonucleotides for the two major ubiquitous calpain isoforms (m- and micro-calpains). Taken together, our findings argue for an involvement of calpains in the differentiation of embryonic myoblasts by limited proteolytic cleavage of PKCalpha.

CD8+T cell-mediated enhancement of tumour necrosis factor-alpha (TNF-α) production and HIV-1 LTR-driven gene expression in human monocytic cells is pertussis toxin-sensitive

HIV replication and LTR-mediated gene expression can be modulated by CD8+ T cells in a cell type-dependent manner. We have previously shown that supernatants of activated CD8+ T cells of HIV-infected individuals greatly enhanced p24 levels in human macrophages infected with NSI or SI primary isolates of HIV-1. Here we have examined the effect of culture with CD8+ T cell supernatants on HIV-1 LTR-mediated gene expression in monocytic cells. CD8+ T cell supernatants enhanced LTR-mediated gene expression in U38 cells activated with Tat in the absence or presence of phorbol myristate acetate (PMA) and ionomycin or TNF-alpha. Further, enhancement of LTR-mediated gene expression and virus replication in U38 cells and U1 cells, respectively, was pertussis toxin-sensitive. The enhancement of gene expression and virus replication was associated with increased levels of TNF-alpha and was significantly abrogated by antibody to TNF-alpha. In contrast, the suppression of LTR-mediated gene expression by CD8+ T cell supernatants in Jurkat T cells was not pertussis toxin-sensitive and TNF-alpha levels were not affected. These results demonstrate that factors produced by CD8+ T cells utilize different cellular pathways to mediate their effects on HIV transcription and replication in different cell types.

Cytokines play an aetiopathogenetic role in fibromyalgia: a hypothesis and pilot study.

To measure soluble factors having a possible role in fibromyalgia (FM) and compare the profiles of patients with recent onset of the syndrome with patients with chronic FM. The production of cytokines, cytokine-related molecules, and a CXC chemokine, interleukin (IL)-8, was examined. Fifty-six patients with FM (23 with <2 yr and 33 with >2 yr of symptoms) were compared with age- and sex-matched healthy controls. Cytokines and cytokine-related molecules were measured in sera and in supernatants of peripheral blood mononuclear cells (PBMC) that were incubated with and without lectins and phorbol myristate acetate (PMA). No differences between FMS and controls were found by measuring IL-1beta, IL-2, IL-10, serum IL-2 receptor (sIL-2R), interferon gamma (IFN-gamma), and tumour necrosis factor alpha (TNF-alpha). Levels of IL-1R antibody (IL-1Ra) and IL-8 were significantly higher in sera, and IL-1Ra and IL-6 were significantly higher in stimulated and unstimulated FM PBMC compared with controls. Serum IL-6 levels were comparable to those in controls, but were elevated in supernatants of in vitro-activated PBMC derived from patients with >2 yr of symptoms. In the presence of PMA, there were additional increases in IL-1Ra, IL-8 and IL-6 over control values. In patients with FM we found increases over time in serum levels and/or PBMC-stimulated activity of soluble factors whose release is stimulated by substance P. Because IL-8 promotes sympathetic pain and IL-6 induces hyperalgesia, fatigue and depression, it is hypothesized that they may play a role in modulating FM symptoms.

Inhibition of Neutrophil Spreading during Adhesion to Fibronectin Reveals Formation of Long Tubulovesicular Cell Extensions (Cytonemes)

Human neutrophils developed long thin tubulovesicular extensions (cytonemes) upon adhesion to fibronectin-coated substrata, when spreading was blocked. We observed extension formation when neutrophils were plated to fibronectin-coated substrata in Na+-free extracellular medium or in the presence of drugs capable of inhibiting spreading: 4-bromophenacyl bromide, N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and cytochalasin D. Addition of Na+ ions or washing of inhibitors restored neutrophil spreading. Phase-contrast and scanning electron microscopy revealed two types of extensions: (1) highly dynamic, flexible tubulovesicular extensions with unattached tips 0.2–0.4 μm in diameter, which can achieve 70–80 μm in length during 20 min, and (2) thinner straight extensions with flattened tips, which were formed in the presence of phorbol 12-myristate 13-acetate and connected cells to substratum or to the neighboring cells several cell diameters away. The latter may have derived from the former through tension after attachment of the tips. Spreading and extension formation may represent two states of the cell adhesive and communicative mechanism.

Effects of different durations of exercise on macrophage functions in mice.

The effects of differing durations of daily exercise on macrophage functions in mice were studied. Male ICR mice aged 4 wk were divided into five groups: a nonexercise group (control) and four exercise groups with differing daily exercise durations of 15--120 min (Exr groups). The exercise applied was 5 days/wk treadmill running at 13 m/min for 12 wk. The potentiation of the phagocytosis function of the reticuloendothelial system and the glucose consumption of peritoneal macrophages in the Exr 30, 60, and 120 groups were significantly higher than those in the control group. Superoxide anion production of peritoneal macrophages in both the absence and the presence of phorbol 12-myristate 13-acetate in the Exr 60 and 120 groups was significantly higher than that in the control group. The acid phosphatase and beta-glucuronidase activities of peritoneal macrophages in the Exr 30, 60, and 120 groups were significantly increased. These results suggest that treadmill running exercise for at least 30 min/day (30--120 min) effectively enhances macrophage functions in mice. These data provide preliminary evidence indicating that chronic exercise-induced increases in phagocytic activity exhibit a dose-dependent relationship with exercise duration.

The effect of sodium nitrite on some parameters of the immune system

The effect of orally administered sublethal doses of 25, 50 and 100 mg/kg of sodium nitrite in drinking water ad lib. for 21 days on the immune response of Balb/c mice was investigated. The immunological parameters were examined at three phases: 1 day (phase A), 1 week (phase B) and 3 weeks (phase C) after the end of exposure to sodium nitrite. A significant decrease in dose-dependent manner was obtained in the following tests: lymphocyte percentages, concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced lymphocyte proliferation assessed by the colorimetric MTT method, natural killer (NK) cell activity against WEHI-164 target cells, as well as IgM and IgG titers against injected sheep erythrocytes. Maximum suppressions were obtained in phase A after treatment with sodium nitrite at 100 mg/kg including lymphocyte count (17.5%), Con A-induced lymphocyte proliferation (40.1%), LPS-induced lymphocyte proliferation (31.4%), IL-2-stimulated NK cell activity (59.2%), unstimulated NK cell activity (59.6%), IgM titer (57.5%) and IgG titer (61.1%). On the other hand, a significant dose-dependent increase in neutrophil count (71.3%) in phase A and phagocytic activation (133%) in the first two phases was obtained using the nitroblue tetrazolium (NBT) assay in the presence of phorbol myristate acetate (PMA). It was found that the immunosuppressive effect of sodium nitrite is reversible after cessation of exposure.

Generation of choline for acetylcholine synthesis by phospholipase D isoforms

Dedication This article is dedicated to the memory of Sue Kim Hanson, a graduate student in the department of Pathology and Laboratory Medicine at Boston University School of Medicine, who perished in the terrorist attacks of September 11, 2001.BackgroundIn cholinergic neurons, the hydrolysis of phosphatidylcholine (PC) by a phospholipase D (PLD)-type enzyme generates some of the precursor choline used for the synthesis of the neurotransmitter acetylcholine (ACh). We sought to determine the molecular identity of the relevant PLD using murine basal forebrain cholinergic SN56 cells in which the expression and activity of the two PLD isoforms, PLD1 and PLD2, were experimentally modified. ACh levels were examined in cells incubated in a choline-free medium, to ensure that their ACh was synthesized entirely from intracellular choline.ResultsPLD2, but not PLD1, mRNA and protein were detected in these cells and endogenous PLD activity and ACh synthesis were stimulated by phorbol 12-myristate 13-acetate (PMA). Introduction of a PLD2 antisense oligonucleotide into the cells reduced PLD2 mRNA and protein expression by approximately 30%. The PLD2 antisense oligomer similarly reduced basal- and PMA-stimulated PLD activity and ACh levels. Overexpression of mouse PLD2 by transient transfection increased basal- (by 74%) and PMA-stimulated (by 3.2-fold) PLD activity. Moreover, PLD2 transfection increased ACh levels by 26% in the absence of PMA and by 2.1-fold in the presence of PMA. Overexpression of human PLD1 by transient transfection increased PLD activity by 4.6-fold and ACh synthesis by 2.3-fold in the presence of PMA as compared to controls.ConclusionsThese data identify PLD2 as the endogenous enzyme that hydrolyzes PC to generate choline for ACh synthesis in cholinergic cells, and indicate that in a model system choline generated by PLD1 may also be used for this purpose.

Biomechanical Regulation of Human Monocyte/Macrophage Molecular Function

When the monocyte infiltrates a tissue, adhesion to the extracellular matrix provides structural anchors, and the cell may be deformed through these attachments. To test the hypothesis that human monocytes/macrophages are mechanically responsive, we studied the effects of small cyclic mechanical deformations on cultured human monocytes/macrophages. When monocytes/macrophages were subjected to 4% strain at 1 Hz for 24 hours, neither matrix metalloproteinase (MMP)-1 nor MMP-3 was induced; however, in the presence of phorbol myristate acetate, strain augmented MMP-1 expression by 5.1 +/- 0.7-fold (P < 0.05) and MMP-3 expression by 1. 6 +/- 0.1-fold (P < 0.05). In contrast, MMP-9 expression was not changed by mechanical strain in the presence or absence of phorbol myristate acetate. Deformation rapidly induced the immediate early response genes c-fos and c-jun. In addition, mechanical deformation induced the transcription factor PU.1, an ets family member that is essential in monocyte differentiation, as well as mRNA for the M-CSF receptor. These studies demonstrate that human monocytes/macrophages respond to mechanical deformation with selective augmentation of MMPs, induction of immediate early genes, and induction of the M-CSF receptor. In addition to enhancing the proteolytic activity of macrophages within repairing tissues, cellular deformation within tissues may play a role in monocyte differentiation.

Involvement of protein kinase C in taxol-induced polyploidization in a cultured sarcoma cell line

Taxol was found to inhibit the proliferation and to induce the polyploidization of cultured methylcholanthrene-induced sarcoma cells (Meth-A cells). To investigate whether protein kinase C is involved in taxol-induced polyploidization, phorbol 12-myristate 13-acetate (PMA), which regulates the activity of protein kinase C, was used along with taxol to treat the cells. We found that PMA did not interfere with the proliferation and did not induce polyploidization by itself. However, at low concentration, taxol, which by itself did not induce polyploidization, clearly induced polyploidization in the presence of PMA. To explore the mechanism by which PMA potentiates polyploidization, the levels of the G1 checkpoint-related proteins cyclin E and cdk2, and those of the G2 checkpoint-related proteins cyclin B and cdc2 were determined by flow cytometry. We found that both G1 and G2 checkpoint-related proteins increased during the induction of polyploidization. To verify the relationship between protein kinase C and tubulin polymerization, flow cytometry was used to determine the total content of tubulin protein, and morphological observation was used to examine spindle organization. PMA did not affect the taxol-induced increase in tubulin protein, but markedly potentiated taxol-induced spindle disorganization. These findings suggest that protein kinase C plays an important role in regulating the induction of polyploidization in Meth-A cells.

Nuclear factor-kappaB and caspases co-operatively regulate the activation and apoptosis of human macrophages.

Accumulating evidence suggests that macrophages function as major effector cells in the pathological process of various human diseases. We examined here the role of nuclear factor-kappaB (NF-kappaB) and caspases in the regulation of activation and apoptosis of macrophages. Activation of the human monoblastic leukaemia cell line, U937, by phorbol 12-myristate 13-acetate (PMA) increased the expression of CD14/CD86, and cytokine production. PMA stimulation also increased the expression of both pro-caspase-8 and pro-caspase-3 in U937, but not apoptosis or intracellular caspase-3 activity. PMA also increased the expression of X-chromosome-linked inhibitor of apoptosis protein (XIAP) in U937, suggesting an inhibitory action for XIAP on the caspase cascade in PMA-stimulated U937. Electrophoretic mobility shift assay (EMSA) showed a significant increase of nuclear NF-kappaB activity in PMA-stimulated U937. When a potent NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), was added to U937 cell culture in the presence of PMA, apoptosis was triggered by activation of caspase-3, which was induced by caspase-8 activation. XIAP expression was markedly suppressed in PMA-treated U937 in the presence of PDTC. The inhibitors of caspase-8 and caspase-3 mostly inhibited apoptosis of U937 treated with PMA in the presence of PDTC. Furthermore, a phenotype of U937 treated with PMA and PDTC in the presence of caspase inhibitor was almost identical to that of unstimulated U937. Our results suggest that the signalling pathways involved in the activation and apoptosis of human macrophages could be co-operatively regulated by the use of NF-kappaB and caspase inhibitors, thus enabling the control of macrophage function and number.

Parathyroid hormone inhibits collagen synthesis and the activity of rat col1a1 transgenes mainly by a cAMP-mediated pathway in mouse calvariae.

We examined the effect of parathyroid hormone and various signaling molecules on collagen synthesis and chloramphenicol acetyltransferase activity in cultured transgenic mouse calvariae carrying fusion genes of the rat Col1a1 promoter and the chloramphenicol acetyltransferase reporter. After 48 h of culture, parathyroid hormone, forskolin, dibutyryl cAMP, 8-bromo cAMP, and phorbol myristate acetate inhibited transgene activity, while the calcium ionophore ionomycin had no effect. Pretreatment of calvariae with the phosphodiesterase inhibitor isobutylmethylxanthine potentiated the inhibitory effect of 1 nM parathyroid hormone on transgene activity and collagen synthesis. Parathyroid hormone further inhibited transgene activity and collagen synthesis in the presence of phorbol myristate acetate. Parathyroid hormone inhibition of transgene activity and collagen synthesis was not affected by indomethacin or interleukin-6. After 48 h of culture, parathyroid hormone inhibited chloramphenicol acetyltransferase activity by 50-85% in cultured calvariae carrying transgenes having progressive 5' upstream deletions of promoter DNA down to -1683 bp. These data show that the inhibitory effect of parathyroid hormone on Col1a1 expression in mouse calvariae is mediated mainly by the cAMP signaling pathway. Prostaglandins and IL-6 are not local mediators of the parathyroid hormone response in this model. Finally, regions of the Col1a1 promoter downstream of -1683 bp are sufficient for parathyroid hormone inhibition of the Col1a1 promoter.

Effect of Β-Sitosterol, a Plant Sterol, on Growth, Protein Phosphatase 2A, and Phospholipase D in LNCaP Cells

Previous work from this laboratory suggests an activation of the sphingomyelin cycle as a mechanism for growth inhibition with the incorporation of &Beta-sitosterol (SIT) into human prostate cancer LNCaP cells. In the present study we examined two key enzymes that have been shown to play a role in the sphingomyelin cycle. Dietary sterols (SIT and cholesterol) were compared for their effect on LNCaP cell growth, phospholipase D (PLD) activity, and protein phosphatase 2A (PP 2A) activity and expression. PP 2A has been suggested as a direct in vitro target of ceramide action on cell growth and apoptosis. Ceramide also inhibits phorbol myristate acetate-stimulated PLD. SIT (16 μM) increased PP 2A activity by 50% compared with cholesterol treatment in LNCaP prostate cells; however, SIT did not alter protein levels of PP 2A. There was an increase in PLD activity in the presence of phorbol myristate acetate in cells supplemented with 16 μM SIT compared with those supplemented with cholesterol after five days of treatment. The present study suggests that the activation of PP 2A added support to the role of the activation of the sphingomyelin cycle by SIT treatment. However, the increase in PLD activity, which was modest but significant, with SIT supplementation suggests that this pathway may be modulated by other mechanisms. This includes the incorporation of SIT into cell membranes that may alter fluidity and, thus, influence the activation of membrane-bound enzymes such as PLD.

Regulation of MMP-9 (type IV collagenase) production and invasiveness in gliomas by the extracellular signal-regulated kinase and jun amino-terminal kinase signaling cascades.

Our previous studies have shown that MMP-9 levels are significantly elevated during the progression of human gliomas. In the current study, we examined the role of JNK- and ERK-dependent signaling modules in the regulation of MMP-9 production and the invasive behavior of the human glioblastoma cell line SNB19, in which JNK/ERK1 is constitutively activated. SNB19 cells that were transfected with dominant-negative JNK, MEKK, and ERK1 expression vectors showed reduced MMP-9 promoter activity. In addition, conditioned medium collected from SNB19 cells transfected with these expression vectors showed diminished MMP-9 activity in the presence of phorbol myristate acetate, as determined by gelatin zymography. The cotransfection of SNB19 cells with kinase-deficient c-raf also diminished MMP-9 promoter activity. Further, in the presence of a specific inhibitor of MEKK (PD098059), the Matrigel invasion assay showed the invasiveness of dominant-negative SNB19 cells transfected with dominant-negative JNK1 or ERK1 to be remarkably reduced. In conclusion, our studies showed for the first time that MMP-9 production and the invasive behavior of SNB 19 cells are regulated by JNK- and ERK-dependent signaling modules and that interfering with either of the pathways reduces invasiveness.

HEMOGLOBIN POTENTIATES THE PRODUCTION OF REACTIVE OXYGEN SPECIES BY ALVEOLAR MACROPHAGES

The objectives of this investigation were (1) to determine the effects of hemoglobin on the production of reactive oxygen species by activated rat alveolar macrophages, (2) to determine a possible mechanism for these effects, and (3) to determine which part of the hemoglobin molecule is responsible for these effects. Production of reactive oxygen species by phorbol myristate acetate (PMA)-stimulated cells was assessed by measuring luminol-enhanced chemiluminescence (CL). Hemoglobin enhances PMA-stimulated CL in a dose-dependent manner. The effect is maximal at 0.5 1.0mu M hemoglobin where PMA-induced CL is increased by approximately 20-fold. Superoxide anion release from PMA stimulated cells is not affected by hemoglobin. However, the hemoglobin-induced enhancement of PMA stimulated CL is inhibited by superoxide dismutase, catalase, dimethylthiourea, or deferoxamine. These results suggest that hydroxyl radical may be formed from hydrogen peroxide which is derived from superoxide anion. Measurements of electron spin resonance spectra following spin trapping of radicals verify that hydroxyl radicals are produced by the cells in the presence of PMA and hemoglobin. The hemoglobin effects appear to require iron in a protoporphyrin complex, because hemin stimulates PMA induced CL, whereas neither ferrous nor ferric iron has any effect. These findings taken together suggest that hemoglobin can act as a biological Fenton reagent to enhance the production of reactive oxygen species from alveolar macrophages and potentially contribute to lung damage during leakage of blood into the alveolar spaces.