TAT cell-penetrating peptide modulates inflammatory response and apoptosis in human lung epithelial cells.

Cell-penetrating peptides (CPPs) are commonly used as delivery vehicles for the introduction of a variety of macromolecules into cells. Trans-activator of transcription (TAT) is the most commonly used CPP and, as a delivery vehicle, is assumed to be biologically inert. In this study, we pretreated human lung epithelial cells with TAT prior to stimulation with phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Surprisingly, TAT alone inhibited the production of multiple cytokines induced by PKC activation. Furthermore, PKC activation-induced IκBα degradation was partially reduced by TAT. Moreover, TAT treatment alone induced apoptosis in a dose-dependent manner, influenced expression of several B cell lymphoma 2 (Bcl-2) family members and increased caspase 3 cleavage at a high dose. These findings suggest that TAT as a delivery vehicle should be used cautiously, as it may affect the inflammatory response, as well as signals related to apoptosis.

Age- and diet-related increase in transepithelial colon permeability of Fischer 344 rats.

When transepithelial permeability of rat distal colon is evaluated on the basis of transepithelial electrical resistance, age does not have an effect. Age likewise did not affect the decrease in resistance brought about by phorbol ester exposure. However, age was shown to correlate with increased transepithelial permeability when diffusion of the nonelectrolyte, D-mannitol, was used as an indicator. A phorbol ester-induced increase in transepithelial permeability to D-mannitol was observed to increase with age. Basal permeability to D-mannitol was significantly higher in older rats when the animals were allowed to age on a high-fat diet. Distance from the rectum was shown to be a potential complicating factor in these studies, since distal colon closer to the rectum was observed to have lower transepithelial permeability. The potential effect of such increased leakiness on the increased frequency of colon cancer in older individuals is discussed.

Selenocompounds induce a redox modulation of protein kinase C in the cell, compartmentally independent from cytosolic glutathione: its role in inhibition of tumor promotion.

Since selenite and other redox-active selenocompounds can modify protein kinase C (PKC) in the test tube, we have determined whether or not this redox regulation occurs inside the cell despite having high concentrations of GSH and the role of this regulation in the inhibition of tumor promotion. By using phorbol ester-promoted JB6 epidermal cell transformation assay, the concentrations of selenite, selenocystine, and selenodiglutathione which are optimal for chemopreventive activity were determined. At such concentrations (0.5 to 2 microM) in the cells treated with these agents, only a slight but transient decrease in PKC activity was observed when measured with a low (5 microM), but not with a high (100 microM) concentration of ATP. However, when the cells were serum starved or pretreated with 2-deoxyglucose, there was a pronounced but transient inactivation of PKC when assayed with both low and high concentrations of ATP. The inactivation was reversed in the cell by an endogenous mechanism or by treatment with thiol agents in the test tube. In spite of a substantial (90%) depletion of GSH in the cells by pretreatment with buthionine sulfoximine, there was no further increase in the redox modification of PKC by selenite as well as no change in the inhibitory effect of selenite on the phorbol ester-stimulated induction of ornithine decarboxylase, which is an intermediate marker related to cell transformation. While GSH is known to influence certain actions of selenium, it may not be required to mediate the effects of selenite tested in this study. The water-soluble cytosolic GSH did not interfere with the redox modification of PKC probably due to the shielding of the cysteine-rich region of the enzyme by a weak hydrophobic association with the membrane. Due to the presence of cofactors in the crude cell extracts, PKC was more sensitive to selenite than in the purified form and was inactivated by low concentrations of selenite (IC50 = 0.05 microM). This modification was reversed by thiol agents as well as by NADPH. A protein disulfide reductase, which can regenerate PKC, was present in the homogenate. Conceivably, selenite and other selenocompounds induce a redox modification of cellular PKC, compartmentally independent from the cytosolic GSH, but intimately connected to a NADPH-dependent reductase system, to mediate, at least in part, some of the cancer-preventive actions.

Hyperoxia, unlike phorbol ester, induces glutathione peroxidase through a protein kinase C-independent mechanism.

Human selenium-dependent glutathione peroxidase (GP) is implicated as a mechanism of resistance against oxygen free radicals. The 5' flanking sequence upstream from the coding region of GP contained an oxygen-responsive element termed ORE1 that is responsive to hypoxia, as well as several copies of the activator protein-1 (AP-1)- and AP-1-like-binding sites. In this study, we sought to define the molecular events that lead to GP gene transcription in response to hyperoxia in human umbilical-vein endothelial cells, and asked whether such induction is mimicked and sustained by activation of protein kinase C (PKC) by phorbol esters. Treatment of cells with 100 nM phorbol 12,13-dibutyrate (PdBu) induced a delayed (24-48 h) but significant (2-fold) increase in steady-state GP mRNA levels. Steady-state GP mRNA levels also rose after exposure to 95% O2, again after considerable delay (48-72 h). For both PdBu and oxygen, induction was transcriptionally regulated, as demonstrated by nuclear run-on experiments. The simulations by PdBu and oxygen were additive. In contrast with PdBu, hyperoxia did not stimulate translocation of PKC from the cytosol to the particulate fraction, although the specific activity of both cytosolic and particulate-associated PKC was increased 2-fold in cells exposed to 95% O2 for 5 days. In addition, gel mobility-shift assays using double-stranded tumour-promoting-agent-responsive element (TRE) and nuclear extracts derived from phorbol- and oxygen-treated cells revealed that PdBu, but not hyperoxia, increased AP-1 DNA-binding activity. On the other hand, the up-regulation of GP expression by oxygen could not be accounted for by the ORE1 core sequence, since no specific protein-DNA binding activity could be detected using nuclear extracts from hyperoxic cells and ORE1. Taken together, these results suggest that there may be different molecular mechanisms controlling GP expression. After exposure to PdBu, GP undergoes transcriptional activation via a process that can be readily explained by a classic AP-1 interaction with the TRE sites in the GP promoter. During hyperoxia, GP also undergoes transcriptional activity, but via a process that appears to involve neither TRE nor ORE1.

Deficient tyrosine phosphorylation of c-Cbl and associated proteins in phorbol ester-resistant EL4 mouse thymoma cells.

Two tyrosine phosphoproteins in phorbol ester-sensitive EL4 (S-EL4) mouse thymoma cells have been identified as the p120 c-Cbl protooncogene product and the p85 subunit of phosphatidylinositol 3-kinase. Tyrosine phosphorylation of p120 and p85 increased rapidly after phorbol ester stimulation. Phorbol ester-resistant EL4 (R-EL4) cells expressed comparable amounts of c-Cbl and phosphatidylinositol 3-kinase protein but greatly diminished tyrosine phosphorylation. Co-immunoprecipitation experiments revealed complexes of c-Cbl with p85, and of p85 with the tyrosine kinase Lck in phorbol ester-stimulated S-EL4 but not in unstimulated S-EL4 or in R-EL4 cells. In vitro binding of c-Cbl with Lck SH2 or SH3 domains was detected in both S-EL4 and R-EL4 cells, suggesting that c-Cbl, p85, and Lck may form a ternary complex. In vitro kinase assays revealed phosphorylation of p85 by Lck only in phorbol ester-stimulated S-EL4 cells. Collectively, these results suggest that Cbl-p85 and Lck-p85 complexes may form in unstimulated S-EL4 and R-EL4 cells but were not detected due to absence of tyrosine phosphorylation of p85. Greatly decreased tyrosine phosphorylation of c-Cbl and p85 in the complexes may contribute to the failure of R-EL4 cells to respond to phorbol ester.

Blockade of protein kinase C is involved in the inhibition by cycloheterophyllin of neutrophil superoxide anion generation.

Cycloheterophyllin, a prenylflavone, inhibited the superoxide anion (O2-) generation from formylmethionyl-leucyl-phenylalanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-stimulated rat neutrophils in a concentration-dependent manner with IC50 values of 47.0 +/- 5.0 and 1.7 +/- 0.4 microM, respectively. Cycloheterophyllin had no effect on O2- generation in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation. Cycloheterophyllin exerted a concentration-dependent inhibition of neutrophil cytosolic protein kinase C (PKC) and rat brain PKC, but had no effect on porcine heart protein kinase A (PKA). Unlike staurosporine, cycloheterophyllin did not affect the trypsin-treated rat brain PKC. [3H]Phorbol 12,13-dibutyrate ([3H]PDB) binding to neutrophil cytosolic PKC was significantly suppressed by cycloheterophyllin. However, cycloheterophyllin had negligible effect on the PMA-induced membrane translocation of PKC-beta and PKC-delta in neutrophils. Moreover, the fMLP-induced [Ca2+]i elevation and inositol trisphosphate (IP3) formation of neutrophils were not affected by cycloheterophyllin at concentrations which significantly suppressed the O2- generation. In cell-free system, addition of arachidonate (AA) into the mixture of cytosol and membrane fractions of the resting neutrophils to make NADPH oxidase assembly and activation. Cycloheterophyllin had no effect on O2- generation in AA-activated cell-free system. These results suggest that the suppression of PKC activity through the interaction with the regulatory region of PKC is involved in the inhibition by cycloheterophyllin of the O2- generation in rat neutrophils.

Effects of substances affecting protein kinase C on histamine-evoked stimulation of cyclic AMP formation in chick cerebral cortex.

In membrane preparation of chick cerebral cortex, HA (histamine) did not affect both basal and forskolin-stimulated adenylyl cyclase activity. However, in slices of chick cerebral cortex prelabeled with [3H]adenine, HA, as well as 2-methylHA, 4-methylHA, and N alpha-methylHA, potently increased [3H]cyclic AMP accumulation in a concentration-dependent manner. The stimulatory effect of the HA-ergic compounds on cyclic AMP formation was antagonized by HA H2-receptor blockers (aminopotentidine, ranitidine), and by chelerythrine (50 microM), a potent and selective inhibitor of PKC (protein kinase C). Of the two other tested PKC inhibitors H-7 (100 microM) significantly reduced the HA action, while the effect of staurosporine (1 microM) did not reach the level of statistical significance. Preincubation of chick cerebral cortical slices with a PKC activator, PDB (4 beta-phorbol 12,13-dibutyrate, 1 microM), markedly enhanced the accumulation of cyclic AMP evoked by HA, 2-methylHA, 4-methylHA and N alpha-methylHA. 4 beta-Phorbol, inactive on PKC, was ineffective. A possible role of PKC in the regulation of HA-induced cyclic AMP synthesis in chick cerebrum has been discussed.

Phorbol esters impair endothelium-dependent and independent relaxation in rat aortic rings.

1. This study examined the ability of various nitro-vasodilators, 8-bromo cyclic guanosine 3':5' monophosphate (8-BrcGMP) and forskolin to relax rings of rat thoracic aorta pre-contracted with either noradrenaline (0.1 microM) or the protein kinase C activators, phorbol 12,13-dibutyrate (PDB, 0.1 microM) or phorbol 12-myristate 13-acetate (PMA, 0.5 microM). 2. In noradrenaline pre-contracted rings, acetylcholine (10 nM-10 microM), sodium nitroprusside (1 nM-0.5 microM), the calcium ionophore A23187 (10 nM-10 microM) and 8-BrcGMP (10 mM) totally reversed the smooth muscle contraction. In PDB-contracted aortic rings acetylcholine, sodium nitroprusside and 8-BrcGMP-induced relaxation was reduced compared to that in noradrenaline-contracted aortic rings, but A23187 and forskolin-induced relaxations were unaffected. Both acetylcholine and A23187-induced relaxations in PDB-contracted rings were abolished in the presence of the nitric oxide synthesis inhibitor N omega-nitro-L-arginine (NOLA, 100 microM). 3. Acetylcholine and sodium nitroprusside were even less potent in their ability to relax PMA-contracted aortic rings compared with noradrenaline and PDB-contracted rings. A23187-induced relaxation was also inhibited in PMA-contracted rings. 4. These results show that protein kinase C activation reduces the ability of agents which liberate nitric oxide to induce smooth muscle relaxation, and also inhibits the biochemical pathways which are subsequently activated by nitric oxide and lead to vascular smooth muscle relaxation.

Phorbol ester-induced ventricular fibrillation in the Langendorff-perfused rabbit heart: antagonism by staurosporine and glibenclamide.

Using a paced Lagendorff-perfused rabbit heart paradigm, we investigated the role of protein kinase C (PKC) in the development of ventricular fibrillation (VF) in hearts subjected to hypoxia (12 min) and re-oxygenation (40 min). We studied the effect of putative activators and inhibitors of PKC on the incidence of VF. Hearts exposed to 4 beta-phorbol,12,13-dibutyrate (PDBu), isophorbol or the membrane permeant diacylglycerol analog, 1-oleoyl-2-acetyl-rac-glycerol (OAG), during the prehypoxic phase had an increased incidence of VF during the hypoxic and reoxygenation periods. The incidence of VF was 90%, 83% and 75% in hearts exposed to PDBu, isophorbol and OAG, respectively (P < 0.05 vs control). Perfusion of hearts with PDBu was associated with a significant increase in the membrane fraction of cardiac PKC activity. In the presence of the inactive phorbol ester 4 alpha-phorbol didecanoate, the incidence of VF was 17% (P > 0.05 vs control). PKC activators were profibrillatory at concentrations that did not affect cardiac function: neither left ventricular developed pressure nor coronary perfusion pressure were affected. The effect of PDBu was antagonized by staurosporine: the incidence of VF was 17% in PDBu+staurosporine treated hearts (P < 0.05 vs control). To further study the profibrillatory effect of PDBu, hearts were exposed to PDBu in the presence of the ATP-dependent potassium channel antagonist glibenclamide. The latter prevented PDBu-induced VF. The results show that under the conditions employed, PDBu-induced activation of PKC induces redistribution of PKC activity and is associated with the development of VF.

Toxicity of phorbol esters for human epithelial cells expressing a mutant ras oncogene.

Phorbol esters and related compounds provide a promising source of potential anticancer agents. The mechanism of their toxicity, however, is unclear, and interpretation has been complicated by the conflicting responses exhibited by different transformed cell lines. Previously we showed that in primary thyroid follicular cells, expression of mutant p21ras conferred a striking sensitivity to the toxic effects of phorbol esters. We have now extended this work using a thyroid cell line with an inducible mutant ras gene to exclude the possibility that this result was a trivial consequence of the marked growth stimulation induced in these cells by mutant p21ras. Furthermore, by assessing the action of a panel of phorbol esters and a potential chemotherapeutic agent, bryostatin, we demonstrated that this phenomenon was only a function of biologically active phorbol esters. These results provide a molecular rationale for the development of phorbol ester analogues as chemotherapeutic agents.

Uncoupling of phospholipase C from receptor regulation of [Ca2+]i in T84 colonic cells by prolonged exposure to phorbol dibutyrate.

The T84 colonic cell line, a cultured Cl- secretory cell, elevates intracellular free Ca2+ [( Ca2+]i) in a concentration-dependent manner when exposed to carbachol or histamine. As determined with a fluorescence microscope imaging system, exposure of T84 cells to 100 microM carbachol or histamine resulted in an immediate [Ca2+]i rise of approximately 50-80 nM in all cells. Preincubation of monolayers for 1 h or longer with 0.4 microM phorbol 12,13-dibutyrate (PDB) reduced the number of cells which responded to histamine or carbachol and reduced the magnitude of the increase in the responding cells. This effect reached its maximum after 2 h and persisted for at least 24 h of PDB incubation. Binding of quinuclidinyl benzilate, a cholinergic receptor antagonist, indicated that down-regulation of external receptors was not an explanation for this effect. Examination of phospholipase C activity in T84 cell membranes showed increased basal activity in PDB-treated compared with control cells. Measurement of inositol phosphates generated by intact cells using myo-[3H]inositol incorporation or receptor binding assays showed that 2 h of incubation with PDB elevated basal levels of inositol 1,4,5-trisphosphate and prevented any further carbachol-induced generation of inositol trisphosphate. Probably as a consequence, both total cell calcium and Ca2+ ionophore-releasable calcium were decreased after 2 h of PDB incubation. Membrane-associated protein kinase C activity was elevated after a 2 h exposure to PDB but was below the level of detection after 24 h with PDB. Protein kinase C antagonists neither duplicated nor blocked the uncoupling of carbachol receptors induced by long term treatment with PDB. The results suggest that prolonged PDB incubation caused uncoupling and elevation of phospholipase C activity from cholinergic and histaminergic receptor regulation resulting in increased basal levels of inositol 1,4,5-trisphosphate. Protein kinase C apparently is not involved directly in the mechanism that leads to these effects.