Ability Of Phorbol 12-myristate 13-acetate Research Articles

Curcumin inhibits agent-induced human neutrophil functions in vitro and lipopolysaccharide-induced neutrophilic infiltration in vivo

Curcumin, extracted from the rhizome of Curcuma longa, is known to possess anti-inflammatory activities. Despite the fact that neutrophils are key player cells in inflammation, the role of curcumin on neutrophil cell biology is not well documented and, in particular, how curcumin can alter primed neutrophils is unknown. In addition, the effect of curcumin on agent-induced neutrophilic inflammation is not well documented. Here, we demonstrated that curcumin inhibited formyl-methionyl-leucyl-phenylalanine (fMLP)- or lipopolysaccharide (LPS)-induced suppression of human neutrophil apoptosis. In addition, we found that curcumin reversed the ability of phorbol myristate acetate (PMA) to induce reactive oxygen species as assessed by flow cytometry using the CM-H2DCF-DA probe. Using an antibody array approach, curcumin was found to inhibit LPS-induced cytokine production, including MIP-1α, MIP-1β, IL-6, IL-8 (CXCL-8) and GRO-α. The inhibitory effect of curcumin on IL-8 production was confirmed by ELISA. Using both an electrophoretic mobility shift assay and a TransFactor p50 NF-κB ELISA, we demonstrated that curcumin inhibited LPS-induced NF-κB activation. In vivo, using the murine air pouch model of acute inflammation, we demonstrated that intraperitoneal administration of curcumin inhibited LPS-induced neutrophilic infiltration in vivo. As assessed by a murine antibody array approach, curcumin was found to decrease the local production of several cytokines/chemokines induced by LPS, including, but not limit to, MIP-1α and MIP-1β. We conclude that curcumin possesses potent modulatory activities on primed or agent-induced human neutrophils in vitro and that it possesses important anti-inflammatory activities in vivo by inhibiting LPS-induced neutrophilic inflammation.

Monoubiquitination of Filamin B Regulates Vascular Endothelial Growth Factor-Mediated Trafficking of Histone Deacetylase 7

Nucleocytoplasmic shuttling of class IIa of histone deacetylases (HDACs) is a key mechanism that controls cell fate and animal development. We have identified the filamin B (FLNB) as a novel HDAC7-interacting protein that is required for temporal and spatial regulation of vascular endothelial growth factor (VEGF)-mediated HDAC7 cytoplasmic sequestration. This interaction occurs in the cytoplasm and requires monoubiquitination of an evolutionarily conserved lysine 1147 (K1147) in the immunoglobulin (Ig)-like repeat 10 (R10) of FLNB and the nuclear localization sequence of HDAC7. Inhibition of protein kinase C (PKC) blocks VEGF-induced ubiquitination of FLNB and its interaction with HDAC7. Small interfering RNA (siRNA) knockdown of FLNB or ubiquitin (Ub) in human primary endothelial cells blocks VEGF-mediated cytoplasmic accumulation of HDAC7, reduces VEGF-induced expression of the HDAC7 target genes Mmp-10 and Nur77, and inhibits VEGF-induced vascular permeability. Using dominant negative mutants and rescue experiments, we demonstrate the functional significance of FLNB K1147 to interfere with the ability of phorbol myristate acetate (PMA) to promote FLNB-mediated cytoplasmic accumulation of HDAC7. Taken together, our data show that VEGF and PKC promote degradation-independent protein ubiquitination of FLNB to control intracellular trafficking of HDAC7.

Characterization of Mechanisms Involved in Secretion of Active Heparanase

Heparanase is an endo-beta-D-glucuronidase involved in extracellular matrix remodeling and degradation and implicated in tumor metastasis, angiogenesis, inflammation, and autoimmunity. The enzyme is synthesized as a latent 65-kDa protein and is processed in the lysosomal compartment to an active 58-kDa heterodimer, where it is stored in a stable form. In contrast, its heparan sulfate substrate is localized extracellularly, suggesting the existence of mechanisms that trigger heparanase secretion. Here we show that secretion of the active enzyme is mediated by the protein kinase A and C pathways. Moreover, secretion of active heparanase was observed upon cell stimulation with physiological concentrations of adenosine, ADP, and ATP, as well as by the noncleavable ATP analogue adenosine 5'-O-(thiotriphosphate). Indeed, heparanase secretion was noted upon cell stimulation with a specific P2Y1 receptor agonist and was inhibited by P2Y receptor antagonists. The kinetics of heparanase secretion resembled the secretion of cathepsin D, a lysosomal enzyme, indicating that the secreted heparanase is of lysosomal origin. We suggest that secretion of active heparanase is initiated by extracellular cues activating the protein kinase A and C signaling pathways. The secreted enzyme(s) then facilitate cell invasion associated with cancer metastasis, angiogenesis, and inflammation.

Regulation of granulosa cell-derived insulin-like growth factor binding proteins (IGFBPs): role for protein kinase-C in the pre- and posttranslational modulation of IGFBP-4 and IGFBP-5.

A growing body of information suggests antigonadotropic and atretogenic roles for granulosa cell-derived insulin-like growth factor binding proteins (IGFBPs) 4 and 5 during ovarian folliculogenesis. Activation of protein kinase-A (PKA) in rat granulosa cells has been shown to modulate the relative expression of IGFBP-4 and -5 transcripts and proteins. In this article, we assess the role of protein kinase-C (PKC) in this regard. Provision of granulosa cells with phorbol 12-myristate 13-acetate (PMA) (but not 4alphaPMA, an inert analogue), a tumor-promoting phorbol ester and an established activator of PKC, was without significant effect on the expression of IGFBP-4 transcripts but resulted in biphasic dose-dependent alterations in IGFBP-5 transcripts and in the accumulation of the IGFBP-4 and -5 proteins. Comparable effects were noted for GnRH, an established PKC agonist. Provision of staurosporine, a potent inhibitor of the catalytic subunit of PKC, produced significant dose-dependent decrements in the relative expression of IGFBP-5 transcripts. Treatment with FSH (presumptively PKA-mediated) markedly attenuated the ability of PMA or GnRH to upregulate the accumulation of the IGFBP-5 (but not IGFBP-4) protein. Taken together, our present findings indicate that the modulation of rat ovarian IGFBP-4 and -5 is PKC as well as PKA dependent and that these two signaling pathways interact in a diametrically opposed and antagonistic fashion.

Inhibition of the activities of matrix metalloproteinases 2, 8, and 9 by chlorhexidine.

Matrix metalloproteinases (MMPs) are a host cell-derived proteolytic enzyme family which plays a major role in tissue-destructive inflammatory diseases such as periodontitis. The aim of the present study was to evaluate the inhibitory effect of chlorhexidine (CHX) on MMP-2 (gelatinase A), MMP-9 (gelatinase B), and MMP-8 (collagenase 2) activity. Heat-denatured type I collagen (gelatin) was incubated with pure human MMP-2 or -9 activated with p-aminophenylmercuric acetate (APMA), and the proteolytic degradation of gelatin was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining. The effect of CHX on MMP-8 activity was also studied with a cellular model addressing the ability of phorbol myristate acetate (PMA)-triggered human peripheral blood neutrophils (polymorphonuclear leukocytes [PMNs]) to degrade native type I collagen. CHX inhibited the activities of both gelatinases (A and B), but MMP-2 appeared to be more sensitive than MMP-9. Adding calcium chloride to the assay mixtures almost completely prevented the inhibition of MMP-9 activity by CHX, while the inhibition of MMP-2 activity could be reversed only when CHX was used at a low concentration. This observation suggests that CHX may act via a cation-chelating mechanism. CHX dose-dependently inhibited collagenolytic activity of MMP-8 released by PMA-triggered PMNs. MMP-8 without APMA activation was inhibited clearly more efficiently than APMA-activated MMP-8. Our study suggests that the direct inhibition of the MMPs' activities by CHX may represent a new valuable effect of this antimicrobial agent and explains, at least in part, the beneficial effects of CHX in the treatment of periodontitis.

Involvement of lipid mediators on cytokine signaling and induction of secretory phospholipase A2 in immortalized astrocytes (DITNC).

Our previous studies demonstrated the ability of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta), to stimulate NFkappaB/DNA binding and synthesis of secretory phospholipase A2 (sPLA2) in immortalized astrocytes (DITNC). In this study, we examined possible involvement of lipid mediators in the cytokine action. Using [14C]serine to label sphingomyelin and ceramide in these cells, subsequent exposure of cells to cytokines did not result in alteration of sphingomyelin/ceramide ratio. Furthermore, neither exogenous sphingomyelinase nor cell-permeable ceramides could stimulate NFkappaB/DNA binding. On the other hand, C-2 ceramide (0.3 microM) as well as other lipid mediators, such as lysophosphatidylcholine and arachidonic acid, were able to elicit a small increase in sPLA2 and potentiate the induction of sPLA2 by TNF-alpha. When DITNC cells were prelabeled with [32P]Pi, an increase in labeled phosphatidic acid (PA) was observed on treatment of cells with IL-1beta (200 U/mL). However, despite the ability of phorbol myristate acetate (PMA) to stimulate phospholipase D (PLD) and synthesis of phosphatidylethanol (PEt) in these cells, PLD activity was not affected by IL-1beta. With the [32P]labeled cells, however, PA-phosphohydrolase inhibitors, such as chlorpromazine and propranolol, could elicit large increases in labeled PA, indicating active PA metabolism in these cells. Cytokines also caused an increase in levels of diacylglycerol (DG) in these cells, although the source of this lipid pool is presently not understood. Taken together, these results provide evidence for the participation of PA and DG in cytokine signaling activity. Furthermore, although cytokines did not cause the release of ceramide, lipid mediators, such as lysophospholipids, and AA could modulate cytokine-mediated induction of sPLA2 in astrocytes.

Protein Kinase Cα Is an Effector of Hexamethylene Bisacetamide-Induced Differentiation of Friend Erythroleukemia Cells

The program of biochemical and molecular events necessary for commitment to erythroid cell differentiation is particularly well characterized in murine Friend erythroleukemia cell lines. Commitment to hemoglobin synthesis in response to a variety of chemical inducers, including hexamethylene bisacetamide and dimethyl sulfoxide is completed by 24 h and proceeds to terminal differentiation by 96 h. Phorbol 12-myristate 13-acetate, a classical tumor promoter phorbol ester that binds to protein kinase C, blocks differentiation in a reversible manner, suggesting an important role for protein kinase C signaling pathways. The classical protein kinase C isoforms α, βI, and βII, play distinct roles in the transduction of proliferative and differentiative signals in human, as well as in murine, erythroleukemia cells. Protein kinase Cα has been implicated in differentiation of human erythroleukemia cells although its translocation to the nucleus has not been observed. Taking advantage of the ability of phorbol 12-myristate 13-acetate to block differentiation in Friend erythroleukemia cells, we determined the localization of the predominant protein kinase C isoforms α and βI during differentiation and in response to their blockade. The ability of phorbol myristate acetate to preferentially diminish protein kinase Cα-protein localization to the nucleus by 24 h and thereby block differentiation induced by hexamethylene bisacetamide was paralleled by the ability of protein kinase Cα antisense transfection to block differentiation. In addition, β-globin transcription, assessed by polymerase chain reaction, was significantly decreased in protein kinase Cα antisense-transfected cells compared to that seen in vector transfected ones. Taken together, these data suggest an important temporal role for nuclear protein kinase Cα localization in Friend erythroleukemia cell differentiation.

Alloantigen-induced unresponsiveness in cord blood T lymphocytes is associated with defective activation of Ras.

Human umbilical cord blood T lymphocytes (CBTL) respond to primary allostimulation but they do not proliferate upon rechallenge with alloantigen. Using PKH-26-labeled cells created a proliferative block that was observed only in CBTL that have divided during primary stimulation (PKH-26(dim)) but not in unstimulated (PKH-26(bright)) CBTL. CBTL's secondary unresponsiveness resembles anergy and can be overcome by treatment with phorbol myristate acetate (PMA) and ionomycin or by high doses (50-100 units/ml) of interleukin 2. Addition of interleukin 2 to the primary cultures does not prevent the induction of secondary unresponsiveness. Defective Ras activation is detected in PKH-26(dim) CBTL during secondary response to alloantigen or after antibody-mediated T cell receptor stimulation whereas Ras is activated and proliferation is induced in CBTL during primary alloantigenic stimulation. Upon stimulation with PMA plus ionomycin, PMA plus alloantigen, but not alloantigen plus ionomycin, Ras is activated in PKH-26(dim) CBTL, and the block in proliferation is overcome. Correction of PKH-26(dim) CBTL's proliferative defect correlates with PMA-induced Ras activation, suggesting a defect in the signaling pathway leading to Ras. Ras-independent signals, necessary but not sufficient to induce PKH-26(dim) CBTL proliferation, are provided by alloantigen exposure, as evident by the ability of PMA plus alloantigen but not PMA alone to overcome the proliferative block. Functional signal transduction through CD28 in PKH-26(dim) CBTL is supported by detectable CD28-mediated PI-3 kinase activation after PKH-26(dim) CBTL's exposure to alloantigen or CD28 cross-linking. These results suggest that defective activation of Ras plays a key role in PKH-26(dim) CBTL's secondary unresponsiveness and point to a defect along the T cell receptor rather than the CD28 signaling pathway.

Dependence of intracellular signaling and neurosecretion on phospholipase D activation in immortalized gonadotropin-releasing hormone neurons.

The excitability of gonadotropin-releasing hormone (GnRH) neurons is essential for episodic neuropeptide release, but the mechanism by which electrical activity controls GnRH secretion is not well characterized. The role of phospholipase D (PLD) in mediating the activity-dependent secretory pathway was investigated in immortalized GT1 neurons, which both secrete GnRH and express GnRH receptors. Activation of these Ca2+-mobilizing receptors was associated with transient hyperpolarization of GT1 cells, followed by sustained firing of action potentials. This was accompanied by an increase in PLD activity, as indicated by elevated phosphatidylethanol (PEt) production. GnRH-induced PEt production was reduced by inhibition of phospholipase C-dependent phosphoinositide hydrolysis by U73122 and neomycin, suggesting that signaling from phospholipase C led to activation of PLD. The intermediate role of protein kinase C (PKC) in this process was indicated by the ability of phorbol 12-myristate 13-acetate to induce time- and dose-dependent increases in PEt and diacylglycerol, but not inositol trisphosphate, and by reduction of GnRH-induced PEt accumulation in PKC-depleted cells. Consistent with the role of action potential-driven Ca2+ entry in this process, agonist-induced PLD activity was also reduced by nifedipine and low extracellular Ca2+. Inhibition of the PLD pathway by ethanol and propranolol reduced diacylglycerol production and caused a concomitant fall in GnRH release. These data indicate that voltage-gated Ca2+ entry and PKC act in an independent but cooperative manner to regulate PLD activity, which contributes to the secretory response in GT1 cells. Thus, the electrical activity of the GnRH-secreting neuron participates in the functional coupling between GnRH receptors and PLD pathway.

Reduction of angiotensin II-induced activation of mitogen-activated protein kinase in cardiac hypertrophy.

Mitogen-activated protein (MAP) kinases play a role in cell growth and are activated in the heart by cardiac stretch and various growth factors, but their role in signal transduction pathways once the heart has undergone hypertrophy is uncertain. To investigate the regulation of MAP kinases in the heart in response to angiotensin II (ang II), once cardiac hypertrophy has become established, ventricular and skeletal muscle explants were studied from Dahl S salt-sensitive and Dahl R salt-resistant rats that were on a high (6% NaCl) salt supplement in their diet. Cardiac hypertrophy was produced in the Dahl S but not R rat through NaCl-induced hypertension. MAP kinases were assayed by myelin protein phosphotransferase activity in MonoQ fractions of cell extracts. Ang II increased MAP kinases mainly in extracts from nonhypertrophic ventricles of Dahl R rats on a high-salt diet. Immunoblots revealed predominantly p44ERK1 with lower amounts p42MAPK in rat ventricle, and no apparent changes with hypertrophy. In hypertrophied hearts, ang II-induced MAP kinase activity was less markedly increased and more rapidly fell to baseline levels in comparison to the response in nonhypertrophied hearts. Prolonged ang II exposure did not produce the same effect on MAP kinase activity in ventricles from Dahl S rats on a low-salt diet, or skeletal muscle from salt-fed Dahl R and S rats. The ability of phorbol myristate acetate to simulate MAP kinase and ang II to simulate translocation of protein kinase C from the cytosole to the membrane was similarly compromised in hypertrophied ventricles. These results are consistent with a disturbance in the regulation of cell-signalling pathways in cardiac hypertrophy in which the MAP kinase response to ang II is dramatically altered.

The protein kinase C activator PMA modulates LPS lethality in normal mice and protects against LPS lethality in D-galactosamine-sensitized mice

Pretreatment for 5 h with 10 μg phorbol-12-myristate-13-acetate (PMA), a well established activator of protein kinase C (PKC) in many kinds of cells including macrophages, was found to either (a) delay, or (b) potentiate, lethal endotoxin shock in mice, depending upon the dose of LPS. The latter occurred despite a marked attenuation (>90%) of the TNFα response to LPS. In mice sensitized with D-galactosamine the same PMA pretreatment offered protection against challenge from either LPS or TNFα. This protection, coupled with the ability of PMA to reduce serum TNFα while increasing serum corticosterone in response to LPS, adds in vivo support for a possible role for PKC activation in early endotoxin tolerance. A phorbol ester (4α-phorbol) that is not a PKC activator was found ineffective. PMA produced a prompt and profound decrease in body temperature which reached a nadir at 3 h. However, the protective effect produced by PMA was not dependent upon a decrease in body temperature per se, but was dependent upon administration of PMA at the same time or prior to LPS.

A role for protein phosphorylation in modulating Ca2+ elevation in rabbit platelets treated with thapsigargin.

The effect of modifying protein kinase and phosphatase activity on Ca2+ influx induced by inhibition of Ca(2+)-ATPase activity has been investigated in rabbit platelets. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) or inhibition of phosphatase type 1/2A (PP1/2A) activity with calyculin A caused a dose-dependent inhibition of cytosolic Ca2+ elevation in thapsigargin (Tg)-treated platelets and decreased Ca2+ influx into platelets at a time when Ca2+ channels had already been opened by pretreatment of cells with Tg. In addition, both activation of PKC and inhibition of PP1/2A activity caused a dose-dependent inhibition of bivalent cation (Mn2+) influx (acting as a surrogate for Ca2+ influx) in Tg-treated platelets. Inhibition of cyclo-oxygenase activity caused a small decrease in [Ca2+]i elevation in Tg-treated platelets, but had no effect on the ability of PMA or calyculin A to inhibit Tg-induced [Ca2+]i elevation Unexpectedly, PMA inhibited Tg-induced [Ca2+]i elevation in the absence of extracellular Ca2+, and in agreement calyculin A decreased [Ca2+]i elevation almost to basal levels. The results from this study were confirmed with another Ca(2+)-ATPase inhibitor, namely 2,5-di(tert-butyl)hydroquinone (tBHQ). These findings therefore suggest that modification of phosphorylation of target protein(s) on serine/threonine amino acid residues plays a role in the regulation of both Ca2+ influx and in the filling state of the intracellular Ca2+ pool in platelets treated with Tg.

Regulation of follicle-stimulating hormone receptor messenger ribonucleic acid levels in cultured rat granulosa cells

The acquisition of follicle-stimulating hormone (FSH) receptors during follicogenesis is believed to be a key event in the subsequent development of the follicle. We have examined the effect of FSH on FSH receptor mRNA in cultured rat granulosa cells by means of FSH receptor cRNA probe. Northern blot analysis indicated the existence of two predominant FSH receptor mRNA transcripts of approximately 5.5 and 2.4 kb in total RNA prepared from rat granulosa cells. Treatment of granulosa cell culture with FSH resulted in tentative suppression of FSH receptor mRNA level 2–6 h after treatment, with subsequent recovery at 24 h. Culture of granulosa cells for 6 h in the presence of increasing concentration of FSH resulted in a dose-dependent decrease in FSH receptor mRNA with a maximal suppression about 50% of control observed in response to 100 ng/ml FSH. We could not detect a similar effect on FSH receptor mRNA by 8-brom-adenosine 3,5-cyclic monophosphate (8-Br-cAMP; 0.2 mM) which showed continuous stimulation on FSH receptor mRNA during a similar time course. In this system, therefore, this transient down-regulation of FSH mRNA was not mediated by the cAMP pathway. Since the inhibitory effect of follistatin on activin-induced FSH binding to rat granulosa cells had been investigated, we studied the action of follistatin on the levels of activin-induced FSH receptor mRNA in rat granulosa cell culture. Although follistatin alone had no detectable effect on FSH receptor mRNA levels, co-culture of the cells with follistatin (0–200 ng/ml) and activin (100 ng/ml) caused a significant reduction in the mRNA levels of activin-induced FSH receptor in a dose-dependent manner. In this same culture system, we have recognized the ability of phorbol 12-myristate 13-acetate to reduce the level of FSH receptor mRNA in a dose-dependent manner. Moreover, PMA inhibited the effect of 8-Br-cAMP (0.2 mM) on increasing FSH receptor mRNA level in a dose-dependent manner.

Influence of acridine tracer dyes on neutrophil function

A study was performed to elucidate the effect of two commonly used fluorescent dyes in in vivo microscopic studies, acridine orange (AO) and acridine red (AR), on the ability of phorbol myristate acetate (PMA)- or formyl peptide (fMLP)-stimulated human neutrophils to adhere to a bovine serum albumin matrix and to generate superoxide anions (SOX). Unlabeled stimulated human neutrophils showed 36 +/- 9% (PMA, 10(-7) M) and 11 +/- 7% (fMLP, 10(-7) M) adherence to the matrix. This adhesion was CD18 dependent as evidence by 98% and 92% reduction, respectively, when the anti-CD18 antibody IB4 was included. A dose-dependent inhibition of stimulated human neutrophil adhesion was evident after 30 min of in vitro dye labeling and the EC50 was approximately 70 micrograms/ml (AR) and 145 micrograms/ml (AO). SOX generation by PMA-stimulated neutrophils was unaffected up to 100 micrograms/ml AR and AO but was reduced by 40-60% at higher doses. Rabbit neutrophils labeled in vivo or in vitro with 100 micrograms/ml AR exhibited 41% and 61% lower SOX generation, respectively. The study indicate that neutrophil function, in terms of ability to adhere to a BSA matrix using CD11/CD18 integrins and to generate SOX upon stimulation, is reduced depending on the dose and choice of fluorescent dye. Caution should be exercised when using these compounds at high concentrations in studies of PMN function.

Role of ceramide-activated protein phosphatase in ceramide-mediated signal transduction.

Extracellular agonists such as tumor necrosis factor-alpha (TNF-alpha) activate the sphingomyelin cycle leading to the generation of ceramide. Ceramide has been suggested as an important mediator of the effect of TNF-alpha on growth inhibition, c-myc down-regulation, apoptosis, and the activation of the nuclear factor kappa B. Although there is no clearly defined intracellular target for ceramide activity, previous studies have demonstrated the existence of a ceramide-activated protein phosphatase (CAPP) in vitro. Since c-myc is an early downstream cellular target for TNF-alpha, we examined the role of ceramide and CAPP in c-myc down-regulation. In intact HL-60 cells ceramide induced down-regulation of c-myc RNA levels. C2-ceramide was active at 1-10 microM and caused 40-80% inhibition of c-myc RNA levels at 30-120 min of treatment. In nuclear run-on studies, C2-ceramide induced a block to transcription elongation of the c-myc transcript without affecting transcription through the first exon. Therefore, ceramide appeared to inhibit c-myc expression via a mechanism identical with that of TNF-alpha. HL-60 cells contained CAPP which was inhibited by okadaic acid (0.1-10 nm). CAPP in HL-60 cells was activated by D-erythro-ceramide but not D-erythro-dihydroceramide. The specificity of activation of CAPP by ceramide in vitro was matched by a similar specificity of c-myc down-regulation in cells. Moreover, okadaic acid inhibited the effects of ceramide and TNF-alpha on c-myc down-regulation. On the other hand, okadaic acid did not inhibit the ability of phorbol 12-myristate 13-acetate to down-regulate c-myc, demonstrating the existence of at least two distinct pathways in the regulation of c-myc expression. These results demonstrate that CAPP is important for ceramide-induced down-regulation of c-myc in myeloid leukemia cells. The implications of these findings in further delineating a sphingomyelin signaling pathway with important anti-proliferative effects are discussed.

Selective nature of phorbol 12-myristate 13-acetate-induced neoplastic transformation in NIH 3T3 cells.

The ability of phorbol 12-myristate 13-acetate (PMA) at 0.02 microgram/ml to induce neoplastic transformation in NIH 3T3 cells is highly dependent on the culture conditions. Optimal transformation, indicated by the saturation density and extent of focus formation in transferred cultures raised under standard conditions, was observed when the original cells were grown in 2% calf serum (CS) and exposed continually to PMA for at least 4 weeks before transfer into the assay. Transformation of stationary cultures in 10% CS occurred later and to a lesser degree than in 2% CS. The same cells subjected to thrice-weekly transfer in 2% or 10% CS at low cell density so that they were in a constant state of exponential growth exhibited no evidence of transformation in response to PMA. This strong condition-dependence of PMA-enhanced transformation is indicative of a selection process similar to that described for spontaneous transformation. In both cases, transformation is promoted by inhibiting multiplication and prevented by maximizing multiplication. Therefore, it has the earmarks of an epigenetic rather than a mutational process and requires phenotypic rather than genotypic variation to supply the states for selection. The concept of "progressive state selection," originally proposed to account for spontaneous transformation, can also account for PMA-enhanced transformation.

Activation of β1 Integrin Fibronectin Receptors on HL60 Cells After Granulocytic Differentiation

AbstractPhorbol esters upregulate the functional affinity of β1 integrin receptors for fibronectin on human neutrophils and other leukocytes. We investigated the ability of phorbol myristate acetate (PMA) to stimulate the human promyelocyte cell line HL-60 to adhere to fibronectin, either in its undifferentiated state (HL60) or after dimethylsulfoxide-in-duced differentiation along the granulocytic pathway (dHL60). PMA stimulated little adherence of undifferentiated HL60 to fibronectin or to the 120-kD chymotryptic cell-binding domain (CBD) of fibronectin. In contrast, PMA stimulated dHL60 cells to rapidly adhere to both fibronect-in- and to CBD-coated plastic. PMA-stimulated dHL60 adherence to fibronectin was largely mediated by both α4β1 and α5β1, whereas PMA-stimulated dHL60 adherence to CBD was largely mediated by α5β1. There was little contribution from β2 integrins to PMA-stimulated dHL60 adherence to fibronectin or CBD. The inability of undifferentiated HL60 to adhere to fibronectin and CBD did not result from lack of expression of α4β1 or α5β1 because HL60 and dHL60 express similar amounts of both α4β1, and α5β1, on their surface. In addition, 1 mmol/LMn2+ induced similar amounts of α5β1-dependent adherence of both HL60 and dHL60, showing that α5β1 on undifferentiated HL60 is capable of binding to its ligand. These data suggest that activation of protein kinase C cannot functionally upregulate these β1 integrins on undifferentiated HL60 cells. The development of PMA-stimulated β1-dependent adherence after granulocytic differentiation of HL60 cells suggests that the differentiated HL60 cell is a useful model for investigating functional coupling of protein kinase C to β1 integrin in myeloid cells.

Activation of beta 1 integrin fibronectin receptors on HL60 cells after granulocytic differentiation

Phorbol esters upregulate the functional affinity of beta 1 integrin receptors for fibronectin on human neutrophils and other leukocytes. We investigated the ability of phorbol myristate acetate (PMA) to stimulate the human promyelocytic cell line HL-60 to adhere to fibronectin, either in its undifferentiated state (HL60) or after dimethylsulfoxide-induced differentiation along the granulocytic pathway (dHL60). PMA stimulated little adherence of undifferentiated HL60 to fibronectin or to the 120-kD chymotryptic cell-binding domain (CBD) of fibronectin. In contrast, PMA stimulated dHL60 cells to rapidly adhere to both fibronectin- and to CBD-coated plastic. PMA-stimulated dHL60 adherence to fibronectin was largely mediated by both alpha 4 beta 1 and alpha 5 beta 1, whereas PMA-stimulated dHL60 adherence to CBD was largely mediated by alpha 5 beta 1. There was little contribution from beta 2 integrins to PMA-stimulated dHL60 adherence to fibronectin or CBD. The inability of undifferentiated HL60 to adhere to fibronectin and CBD did not result from lack of expression of alpha 4 beta 1 or alpha 5 beta 1 because HL60 and dHL60 express similar amounts of both alpha 4 beta 1 and alpha 5 beta 1 on their surface. In addition, 1 mmol/L Mn2+ induced similar amounts of alpha 5 beta 1-dependent adherence of both HL60 and dHL60, showing that alpha 5 beta 1 on undifferentiated HL60 is capable of binding to its ligand. These data suggest that activation of protein kinase C cannot functionally upregulate these beta 1 integrins on undifferentiated HL60 cells. The development of PMA-stimulated beta 1-dependent adherence after granulocytic differentiation of HL60 cells suggests that the differentiated HL60 cell is a useful model for investigating functional coupling of protein kinase C to beta 1 integrin in myeloid cells.

Mechanism of the action of angiotensin-converting enzyme inhibitors on agonist-induced Ca2+ influx.

To evaluate the direct effects of the angiotensin-converting enzyme (ACE) inhibitors, captopril, enalaprilat, enalapril (a prodrug without therapeutically significant ACE inhibitory effect) and ramiprilat, on cellular calcium metabolism, the cytosolic free calcium concentration was measured in cultured rat vascular smooth muscle cells using the fluorescent dye, fura-2. Preincubation with captopril, enalaprilat, enalapril, or ramiprilat for 40 min significantly reduced the angiotensin II-induced transplasma membrane calcium influx but did not influence the angiotension II-induced calcium release from internal stores. Captopril and ramiprilat also inhibited arginine vasopressin, but not the thapsigargin-, norepinephrine-, or the BayK 8644-induced changes in cytosolic calcium. Phorbol 12-myristate 13-acetate pretreatment for 30 s caused an increase in the angiotensin II-induced rise in cytosolic calcium. Although both captopril and verapamil reduced responses to angiotensin II to similar extents, only verapamil blocked the ability of phorbol 12-myristate 13-acetate to enhance responses to angiotensin II. It is concluded that ACE inhibitors modulate the effects of some but not all agonist-induced transplasma membrane calcium influx.

Identification of a protein transiently phosphorylated by activators of endothelial cell function as the heat-shock protein HSP27. A possible role for protein kinase C.

Several agonists of endothelial cell function (thrombin, histamine, dioctanoylglycerol, phorbol 12-myristate 13-acetate, interleukin-1) have previously been shown to enhance the level of phosphorylation of an undefined 29,000-M(r) protein (P29). Comparison of this protein with other phosphoproteins suggested that it may be related to the mammalian heat-shock protein HSP27. Immunoprecipitation and immunoblot analysis with antibodies specific for human HSP27 demonstrated that P29 was immunochemically identical with HSP27. Further characterization of agonist-induced phosphorylation of HSP27 indicated that phosphorylation occurred exclusively on serine residues, and phosphopeptide analysis of tryptic- and chymotryptic-cleavage products demonstrated that the phosphopeptides generated were identical for each agonist and okadaic acid. Down-regulation of protein kinase C-alpha by prolonged treatment with phorbol esters eliminated the ability of phorbol 12-myristate 13-acetate, dioctanoylglycerol, thrombin and histamine to phosphorylate HSP27 above background levels and deceased interleukin-1-stimulated HSP27 phosphorylation by 60%. These data suggest that the various agonists employed stimulate HSP27 phosphorylation through similar mechanisms and that protein kinase C is probably involved.