Ritonavir stimulates foam cell formation by activating PKC

Alpha-subtype protein kinase C (PKCα) is closely related to cardiovascular disease. Ritonavir (RTV), which is a human immunodeficiency virus (HIV) protease inhibitor, can induce atherosclerosis in a PKC-dependent manner. However, it remains unclear how RTV acts on PKCα to induce pathological phenotypes. In this study, we obtained mouse peritoneal macrophages from adult female Kunmin mice. The results of Oil Red O staining and immunofluorescence using confocal laser scanning microscope demonstrated that RTV could induce foam cell formation and plasma membrane translocation of PKCα like phorbol-12-myristate-13-acetate (PMA, a PKC activator). Computational modeling also exhibited similar docking of RTV and PMA to PKCα and similar patterns of hydrophobic interaction and hydrogen bond formation. Further in vitro kinase activity studies revealed that RTV could elevate PKC activity. These data provided insight into the PKC-dependent induction of atherosclerosis and useful information for more in-depth toxicity research of HIV protease inhibitor (PI). In addition, western blot analysis proved RTV also up-regulate PKCα expression, which may be related to its influence on estrogen responsiveness in target cells and needs further prove.     

Defective human MutY phosphorylation exists in colorectal cancer cell lines with wild-type MutY alleles

Oxidative DNA damage can generate a variety of cytotoxic DNA lesions such as 8-oxoguanine (8-oxoG), which is one of the most mutagenic bases formed from oxidation of genomic DNA because 8-oxoG can readily mispair with either cytosine or adenine. If unrepaired, further replication of A.8-oxoG mispairs results in C:G to A:T transversions, a form of genomic instability. We reported previously that repair of A.8-oxoG mispairs was defective and that 8-oxoG levels were elevated in several microsatellite stable human colorectal cancer cell lines lacking MutY mutations (human MutY homolog gene, hmyh, MYH MutY homolog protein). In this report, we provide biochemical evidence that the defective repair of A.8-oxoG may be due, at least in part, to defective phosphorylation of the MutY protein in these cell lines. In MutY-defective cell extracts, but not extracts with functional MutY, A.8-oxoG repair was increased by incubation with protein kinases A and C (PKA and PKC) and caesin kinase II. Treatment of these defective cells, but not cells with functional MutY, with phorbol-12-myristate-13-acetate also increased the cellular A.8-oxoG repair activity and decreased the elevated 8-oxoG levels. We show that MutY is serine-phosphorylated in vitro by the action of PKC and in the MutY-defective cells by phorbol-12-myristate-13-acetate but that MutY is already phosphorylated at baseline in proficient cell lines. Finally, using antibody-isolated MutY protein, we show that MutY can be directly phosphorylated by PKC that directly increases the level of MutY catalyzed A.8-oxoG repair.     

Calcium Induces Expression of Cytoplasmic Gelsolin in SH-SY5Y and HEK-293 Cells

Gelsolin plays an important role in the regulation of amyloid beta-protein fibrillogenesis. We report here that calcium ionophore A23187 induces the expression of cytoplasmic gelsolin (c-gelsolin), and that protein kinase C (PKC) is involved in the up-regulation of c-gelsolin. In the presence of calcium, both SH-SY5Y and HEK-293 cells upon treatment with A23187 showed an increase in c-gelsolin expression in a concentration-dependent manner. Calcium-mediated up-regulation of c-gelsolin was inhibited by cycloheximide (a general inhibitor of protein synthesis). When cells were pretreated with staurosporine (an inhibitor of a variety of protein kinases including PKC), the up-regulation of c-gelsolin induced by A23187 was inhibited. Calphostin C, an inhibitor of PKC, blocked the up-regulation of c-gelsolin induced by A23187, while inhibitors of mitogen-activated protein kinases had no effect on c-gelsolin expression. In addition, phorbol-12-myristate-13-acetate, an activator of PKC, up-regulated c-gelsolin expression. These results suggest that calcium mediates up-regulation of c-gelsolin in a PKC-dependent manner.     

Arachidonic acid,protein kinase C activators and bud formation in Hydra vulgaris

1. The effect of the protein kinase C (PKC) activators verrucosin B (VB), 1,2-sn-dioctanoylglycerol (diC₈) and phorbol-12-myristate-13-acetate (PMA), of arachidonic acid (AA) and of substances interfering with its release, re-uptake and metabolism was studied in Hydra vulgaris.2. All PKC activators potently inhibited bud formation, VB and PMA being 10,000 × more potent than diC⁸. VB effect was maximal already after 10 min incubation with hydra and persisted at 24 hr incubations.3. AA and substances inhibiting its re-uptake from cell membrane or its metabolism also inhibited bud formation, whereas oleyl-oxyethyl-phosphorylcholine (OOPC), an inhibitor of phospholipase A₂, potently induced bud formation.4. The findings described herein suggest a role for both PKC activation and AA in the inhibition of bud formation in H. vulgaris.     

Activation of protein kinase C inhibits human keratinocyte migration

The involvement of protein kinase C (PKC) in epidermal growth factor (EGF)-induced human keratinocyte migration was studied with the phagokinetic assay. It was concluded that PKC activation does not mediate, but rather inhibits, EGF-induced keratinocyte migration. The following experimental observations support these conclusions: 1) The PKC inhibitor H-7 did not inhibit EGF-induced migration but instead led to a modest enhancement. 2) PKC activators such as phorbol-12-myristate-13-acetate (PMA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoly-sn-glycerol inhibited migration, but biologically inactive 4α-PMA had no effect. 3) PMA did not inhibit keratinocyte attachment and spreading but blocked migration almost immediately after addition. 4) Migration of PKC-depleted cells, which were produced by prolonged treatment with PDBu, was enhanced similarly to normal cells by EGF. 5) PKC-depleted cells were not susceptible to the inhibitory effects of phorbol esters on migration. Additional experiments, in which cells were preactivated with EGF, suggested that PKC inhibits the EGF effect at a post-receptor level. The inhibitory effect of PKC on keratinocyte migration was not restricted to EGF-induced migration; PKC activation also inhibited keratinocyte migration induced by bovine pituitary extract, insulin, insulin-like growth factor-1, and keratinocyte growth factor. © 1993 Wiley-Liss, Inc.     

A continuous fluorescence assay for protein kinase C

A 6-acryloyl-2-dimethylaminonapthalene (acrylodan)-labeled 25-amino acid peptide (acrylodan-CKK-KKRFSFKKSFKLSGFSFKKNKK-COO-), containing the protein kinase C (PKC) phosphorylation sites of brain myristoylated alanine-rich kinase C substrate protein, undergoes a 20% fluorescence decrease when it is phosphorylated by phospholipid/calcium-dependent protein kinase (PKC). This fluorescence decrease is dependent on the presence of PKC, calcium (half-maximal stimulation at pCa = 6.2), phosphatidylserine, diacylglycerol, or phorbol-12-myristate-13-acetate (half-maximal stimulation at 2 nM) and ATP, and correlates well (r = 0.997) with [32P]phosphate incorporation into the peptide. This fluorescence assay allows detection of 0.02 nM PKC, while similar concentrations of cyclic AMP-dependent or type II calmodulin-dependent protein kinases produced no change in peptide fluorescence. The method can be used to assay purified PKC as well as activity in crude brain homogenates. Incubation of PKC with staurosporine inhibits the fluorescence decrease with an IC50 of 2 nM. Thus the fluorescence decrease that occurs in the acrylodan-peptide provides a continuous fluorescence assay for PKC activity.     

Direct interaction of all-trans-retinoic acid with protein kinase C (PKC). Implications for PKC signaling and cancer therapy

Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [(3)H]atRA into PKCalpha, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCalpha, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca(2+). At pharmacological concentrations (10 micrometer), atRA decreased PKCalpha activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca(2+). These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCalpha and prevent PKCalpha activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.     

Conventional protein kinase C isoforms mediate neuroprotection induced by phorbol ester and estrogen

Rapid signal transduction pathways play a prominent role in mediating neuroprotective actions of estrogen in the CNS. We have previously shown that estrogen-induced neuroprotection of primary cerebrocortical neurons from beta-amyloid peptide (Abeta) toxicity depends on activation of protein kinase C (PKC). PKC activation with phorbol-12-myristate-13-acetate (PMA) also provides neuroprotection in this paradigm. Because the PKC family includes several isoforms that have opposing roles in regulating cell survival, we sought to identify which PKC isoforms contribute to neuroprotection induced by PMA and estrogen. We detected protein expression of multiple PKC isoforms in primary neuron cultures, including conventional (alpha, betaI, betaII), novel (delta, epsilon, theta) and atypical (zeta, iota/lambda) PKC. Using a panel of isoform-specific peptide inhibitors and activators, we find that novel and atypical PKC isoforms do not participate in the mechanism of either PMA or estrogen neuroprotection. In contrast, a selective peptide activator of conventional PKC isoforms provides dose-dependent neuroprotection against Abeta toxicity. In addition, peptide inhibitors of conventional, betaI, or betaII PKC isoforms significantly reduce protection afforded by PMA or 17beta-estradiol. Taken together, these data provide evidence that conventional PKC isoforms mediate phorbol ester and estrogen neuroprotection of cultured neurons challenged by Abeta toxicity.     

Modulation of endothelial autacoid release by protein kinase C: Feedback inhition or non-specific attenuation of receptor-dependent cell activation?

Receptor-mediated elevations of intracellular Ca²⁺ in endothelial cells may be controlled by a negative feedback mechanism through activation of protein kinase C (PKC). To test this hypothesis, we studied the effects of an activation or inhibition of PKC on the release of nitric oxide (NO) and prostacyclin (PGI₂) from cultured bovine and porcine aortic endothelial cells (EC). Preincubation with the PKC activators phorbol-12-myristate-13-acetate (PMA) (3-300 nM) or 1-oleyl-2-acetyl-glycerol (OAG) (30 μM) significantly attenuated the release of NO and PGI₂ from EC stimulated with bradykinin (0.3–30 nM), whereas phorbol-12, 13-didecanoate (PDD) (30–300 nM), which does not activate PKC, had no effect. UCN-01 (10 nM), a specific PKC inhibitor, significantly augmented the bradykinin-stimulated release of NO from EC. These effects were correlated with a reduced (PMA) or enhanced (UCN-01) elevation of intracellular Ca²⁺ in response to bradykinin in both types of EC. Neither the PKC activators nor the inhibitor had any effect on resting intracellular Ca²⁺ or basal endothelial autacoid release. Several isoforms of PKC (namely PKC_α, PKC_δ, PKC_?, and PKC_ζ) were detected in bovine, human, and porcine EC by immunoblotting analysis with isotype-specific anti-PKC antibodies, which, except PKC_?, were predominantly located in the cytosol. Incubation of bovine EC with PMA elicited a significant increase in membrane-bound PKC_α immunoreactivity, whereas there was no translocation of PKC_α from the cytosolic to the membrane fraction with bradykinin. As determined by histone phosphorylation, PKC activity was similarly reduced in the cytosol, but increased in the membrane fraction of bovine EC exposed to PMA, whereas bradykinin had no significant effect. These findings indicate that endothelial autacoid release can be modulated by activators and inhibitors of PKC. However, stimulation of EC with bradykinin does not lead to a detectable activation of PKC, suggesting that PKC does not exert a negative feedback in the signal transduction pathway of this receptor-dependent agonist. © 1993 Wiley-Liss, Inc.     

Regulation of interleukin 6 production in a human gastric epithelial cell line MKN-28

Interleukin (IL-) 6 is closely related to gastrointestinal diseases. The question of whether gastric epithelial cell contributes to IL-6 production remains undefined. We aim to evaluate the regulatory pathway of IL-6 expression in gastric epithelial cells, by using different inflammatory cytokines, endotoxin, or protein kinase modulators. IL-6 was measured by ELISA. Phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187, TNF-alpha, IL-1beta, oncostatin M (OSM) but not lipopolysaccharide stimulated IL-6 production from gastric epithelial cell line MKN-28. Blocking protein tyrosine kinase (PTK) activation by herbimycin A or genistein, or blocking NF-kappaB activation by pyrrolidinedithiocarbamate, reduced the IL-6 expression induced by TNF-alpha, IL-1beta and OSM. Dexamethasone mimicked this effect. Protein kinase (PK) C inhibitor only reduced the PMA and OSM induced IL-6 production. Both inhibitors and activators for PKA and G-protein as well as IL-10 had no effects on IL-6 expression. These results indicate that inflammatory cytokines are crucial for IL-6 regulation in gastric epithelial cells. The IL-6 signal pathway is mediated through PTK, NF-kappaB, and also involve PKC, intracellular calcium and sensitive to dexamethasone, but is not related to PKA, G-protein and IL-10.     

Protein kinase C alpha requirement in the activation of p38 mitogen-activated protein kinase, which is linked to the induction of tumor necrosis factor alpha in lipopolysaccharide-stimulated microglia

Activated microglia have been suggested to produce a cytotoxic cytokine, tumor necrosis factor alpha (TNF alpha), in many pathological brains. Thus, determining the molecular mechanism of this induction and suppression has been the focus of a great deal of research. Using lipopolysaccharide (LPS) as an experimental inducer of TNF alpha, we investigated the regulatory mechanism by which TNFalpha is induced or suppressed in microglia. We found that LPS-induced TNF alpha is suppressed by pretreatment with the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580. Similar suppression was achieved by pretreatment with specific protein kinase C (PKC) inhibitors, G?6976, myristoylated pseudosubstrate (20-28), and bisindolylmaleimide. These results suggest that PKC alpha activity as well as p38MAPK activity is associated with TNF alpha induction in LPS-stimulated microglia. The requirement of PKC alpha in LPS-dependent TNFalpha induction was verified in PKC alpha-downregulated microglia which could be induced by phorbol-12-myristate-13-acetate pretreatment. Simultaneously, PKC alpha was found to be requisite for the activation of p38MAPK in LPS-stimulated microglia. In addition, the PKC alpha levels in the LPS-stimulated microglia were observed to decrease in response to the p38MAPK inhibitor, indicating that the PKC alpha levels are regulated by the p38MAPK activity. We therefore concluded that PKC alpha and p38MAPK are interactively linked to the signaling cascade inducing TNFalpha in LPS-stimulated microglia, and that in this cascade, PKC alpha is requisite for the activation of p38MAPK, leading to the induction of TNF alpha.     

Activation of protein kinases A and C promoted proliferation of chicken primordial germ cells

Many growth factors or cytokines regulate cell proliferation via different intracellular signaling pathways. The mechanisms remained quite unclear in avian primordial germ cells (PGCs). In the present study, two major protein kinases, PKA and PKC, were investigated to be involved in signal transduction of PGC proliferation. PGCs were isolated from genital ridge of 3.5-day chicken embryos and primary culture was performed with 5% fetal calf serum (FCS)-supplemented medium 199. After culture for 24 h, PGCs were subcultured on chicken embryonic fibroblast feeder (CEF) and the cells were characterized by histochemical stainings of alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) reagent as well as immunocytochemical stainings of c-kit and stage-specific embryonic antigen-1 (SSEA-I). In addition, cells were challenged with adenylate cyclase activator forskolin (FRSK) and PKC activator phorbol-12-myristate-13-acetate (PMA) alone or in combinations with PKA inhibitor H(89) and PKC inhibitor H(7), respectively. Results showed that subcultured PGCs on CEF displayed positive histochemical and immunocytochemical stainings for ALP, PAS, c-kit and SSEA-I and manifested intensive proliferating activity by colony formation. Downstream activation of PKA by FRSK (10(-7) to 10(-5)M) significantly promoted the proliferation of PGCs by increasing colony number (ALP-stained) in a dose-dependant manner. PMA (10(-8)M) also increased PGC colony number (P<0.05). However, the proliferating effects elicited by FRSK or PMA could be inhibited by the respective protein kinase inhibitor H(89) or H(7). Therefore, the above results suggest that activation of intracellular protein kinases A and C by external factors may promote proliferation of cultured PGCs and PKA represents the most likely mediator of PGC proliferation in embryonic chickens.     

Melatonin activates PKC-alpha but not PKC-epsilon in N1E-115 cells.

Previous reports have revealed that calmodulin antagonism by melatonin is followed by microtubule enlargements and neurite outgrowths in neuroblastoma N1E-115 cells. In addition, activation of protein kinase C (PKC) by this neurohormone is also followed by increased vimentin phosphorylation, and reorganization of vimentin intermediate filaments (IFs) in N1E-115 cells. In this work, we further characterize the activation of PKC by melatonin in neuroblastoma N1E-115 cells. We studied the Ca(2+)-dependent effects of melatonin on PKC activity and distribution of PKC-alpha in isolated N1E-115 cell IFs. Also, the effects of melatonin on PKC-alpha translocation in comparison to PKC-epsilon, were studied in intact N1E-115 cells. The results showed that both melatonin and the PKC agonist phorbol-12-myristate-13-acetate increased PKC activity in isolated IFs. The effects of the hormone were Ca(2+)-dependent, while those caused by the phorbol ester were produced with or without Ca(2+). Also, in isolated in situ IFs, the hormone changed the distribution of PKC-alpha. In intact N1E-115 cells, melatonin elicited PKC-alpha translocation and no changes were detected in PKC-epsilon. Phorbol-12-myristate-13-acetate modified the subcellular distribution of both PKC isoforms. The results showed that melatonin selectively activates the Ca(2+)-dependent alpha isoform of PKC and suggest that PKC-alpha activation by melatonin underlies IF rearrangements and participates in neurite formation in N1E-115 cells.     

Schistosoma mansoni: evidence for protein kinase-C-like modulation of muscle activity

The phorbol esters, phorbol-12,13-dibutyrate, phorbol-12-myristate-13-acetate, phorbol-12,13-didecanoate, and phorbol-12,13-diacetate, as well as mezerin at concentrations as low as 10 nM produce a spastic paralysis of the schistosome musculature. The action of these protein kinase-C activators is dependent on the sites of esterification and is stereo-specific since phorbol-13,20-diacetate, phorbol-12,13,20-triacetate, 20-oxo, 20-deoxy-beta-phorbol-12,13-dibutyrate, alpha-phorbol-12,13-didecanoate, and alpha-phorbol are inactive. A phospholipid and phorbol ester-dependent protein kinase is identified. This kinase is stimulated by all of the phorbol esters that increase muscle tone but is not stimulated by phorbol esters that do not affect muscle tone. A high affinity, stereo-specific phorbol ester receptor is identified. Dose-response curves of phorbol-12,13-dibutyrate-induced muscle tension and -stimulated kinase activity and receptor binding indicate that these responses are mediated by the same system. These results indicate that protein kinase-C-like enzyme may play an important role in modulating activity of the schistosome musculature.     

Alpha interferon suppresses virion but not soluble human immunodeficiency virus antigen production in chronically infected T-lymphocytic cells.

Alpha interferon (IFN-alpha) is effective in preventing the release of human immunodeficiency virus (HIV) from chronically infected T-lymphocytic (ACH-2) and promonocytic (U1) cell lines stimulated with the phorbol ester phorbol-12-myristate-13 acetate (PMA). In the present study, we observed that together with particle production, shedding of HIV antigen (p24gag) occurs in the T-cell line ACH-2 both constitutively and after stimulation with PMA. IFN-alpha, although effective in suppressing the release of HIV particles, did not inhibit shedding of p24gag into the culture supernatants of either unstimulated or PMA-stimulated cells. These observations may be of relevance in the evaluation of the in vivo efficacy of IFN-alpha treatment of HIV-infected individuals as determined by levels of p24 antigen in plasma.     

PKC-ERK通路在热损伤诱导单核细胞凋亡中的作用

应用流式细胞检测术、Western印迹、激酶活性测定等技术,检测PKC与ERK在热损伤诱导单核细胞株Raw264.7细胞凋亡中的作用。结果显示热损伤导致PKC短暂激活,PKC激活剂佛波脂(PMA)与热损伤联合作用导致PKC持续活化;并且PKC的持续激活抑制热损伤诱导的Raw264.7细胞凋亡,而PKC的抑制可促进细胞凋亡;ERK活性检测显示热损伤抑制ERK磷酸化,而PMA激活ERK磷酸化活化,并且这种激活作用通过PKC;进一步细胞凋亡检测显示ERK抑制剂PD098059可解除PMA对热损伤诱导Raw264.7细胞凋亡的抑制作用,从而提示PKC通过ERK负调控热损伤诱导的Raw264.7细胞凋亡。     

Effect of Modulators of Protein Kinase C Activity on Ca2+ Transport in Retinal Rod Microsomes

The effect of modulators of protein kinase C (PKC) activity on Ca²⁺ translocation in retinal rod microsomes was studied. It is shown that PKC activators (phorbol 12-myristate-13-acetate (PMA) and diacylglycerol (DAG)) and inhibitors (chelerythrine chloride, polymyxin B, and phloretin) stimulate and inhibit ATP-dependent Ca²⁺ uptake in retinal rod microsomes, respectively. This effect is apparently due to an influence of PKC on Ca-ATPase contained in these vesicular structures. It was found that PKC inhibitors (chelerythrine chloride, polymyxin B, and phloretin) and activators (PMA and DAG) potentiate Ca²⁺ release from Ca²⁺ -loaded retinal rod microsomes. Specific and nonspecific mechanisms of Ca-release stimulation by the modulators of PKC activity are discussed.     

Changes in the K562 cell sensitivity to nonspecific lysis by human and rat leukocytes under the influence of sodium butyrate, dimethyl sulfoxide and phorbol-12-myristate-13-acetate

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to non-specific lysis (non-restricted with respect to antigens of the major histocompatibility complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.     

Exposure to sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristine-13-acetate alters sensitivity of K562 cells to nonspecific lysis by human or rat leukocytes

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to nonspecific lysis (nonrestricted with respect to antigens of the major histocompatibilty complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.     

Inhibition of protein kinase CbetaII increases glucose uptake in 3T3-L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane

The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the rate of glucose uptake in many different cell systems. We attempted to investigate the mechanism via which PMA stimulates glucose transport in 3T3-L1 adipocytes in more detail. We observed a good correlation between the rate of disappearance of PKCbetaII during prolonged PMA treatment and the increase in glucose uptake. Moreover, inhibition of PKCbetaII with a specific myristoylated PKCbetaC2-4 peptide inhibitor significantly increased the rate of glucose transport. Western blot analysis demonstrated that both PMA treatment and incubation with the myristoylated PKCbetaC2-4 pseudosubstrate resulted in more glucose transporter (GLUT)-1 but not GLUT-4 at the plasma membrane. To our knowledge, we are the first to demonstrate that inactivation of PKC, most likely PKCbetaII, elevates glucose uptake in 3T3-L1 adipocytes. The observation that PKCbetaII influences the rate of glucose uptake through manipulation of GLUT-1 expression levels at the plasma membrane might reveal a yet unidentified regulatory mechanism involved in glucose homeostasis.