Protein kinase C activation modulates pro- and anti-apoptotic signaling pathways

Activation of protein kinase C (PKC) by TPA in human U937 myeloid leukemia cells is associated with induction of adherence, differentiation, and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these differentiating cells about 25% of U937 cells accumulated in the subG1 phase after TPA treatment. This effect proved to be phorbol ester-specific, since other compounds such as retinoic acid or vitamin D3 failed to induce apoptosis in conjunction with differentiation. Only a specific inhibitor of PKC, GF109203X, but not the broad-spectrum kinase inhibitor staurosporine or a tyrosine kinase inhibitor genistein could reverse the induction of apoptosis. Bryostatin-1, another specific PKC activator with distinct biochemical activity failed to induce apoptosis. Moreover, bryostatin-1 completely abolished the induction of apoptosis in U937 cells even if added 8 hours after TPA treatment. Apart from apoptosis induced by various chemotherapeutic drugs, TPA-related cell death is not mediated by an autocrine Fas-FasL loop and could not be prevented by a blocking antibody to the Fas receptor. However, a 75% reduction in the number of apoptotic cells after TPA stimulation was achieved by preincubation with a blocking antibody to the TNFalpha receptor. Tetrapeptide cleavage assays revealed a four-fold increase in the DEVD-cleavage activity in U937 cells compared to a three-fold increase in TUR cells. Immunoblotting demonstrated that TUR cells did not activate significant levels of caspase-3 or -7, whereas in U937 cells a 20-kDa cleavage product corresponding to activated caspase-3 was detectable after 3 d TPA exposure. Moreover, immunoblots revealed a strongly reduced expression of the adaptor molecule APAF-1, which is required for cytochrome c-dependent activation of caspase-9 and subsequently caspase-3. APAF-1 proved to be inducible after PKC activation with phorbol ester in U937, but not in TUR cells. Thus, APAF-1 expression may, at least in part, be regulated by PKC activity and reduced APAF-1 levels are associated with resistance to various inducers of apoptosis. Furthermore, TPA exposure of U937 cells is associated with increased levels of the pro-apoptotic proteins Bak and Bcl-xs, whereas simultaneously a decline in the Bcl-2 expression was noticable.     

Extracellular-superoxide dismutase expression during monocytic differentiation of U937 cells

Leukemic cell lines, such as U937, THP-1, and HL60 cells, can differentiate into macrophages following exposure to various agents including 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro. It is well known that TPA enhances reactive oxygen species (ROS) generation through the activation of NADPH oxidase (NOX), and ROS act as mediators in TPA signaling. Extracellular-superoxide dismutase (EC-SOD) is a major anti-oxidative enzyme that protects the cells from damaging effects of superoxide. Recently, the reduction of Cu/Zn-SOD and the induction of Mn-SOD by TPA in leukemic cells have been reported; however, the regulation of EC-SOD by TPA remains poorly understood. Here, we explored the regulation of EC-SOD during the monocytic differentiation of U937 cells by TPA. We observed the reduction of EC-SOD and Cu/Zn-SOD, whereas the induction of Mn-SOD during the differentiation of U937 cells. The reduction of EC-SOD and Cu/Zn-SOD was attenuated by pretreatments with GF109203X (an inhibitor of protein kinase C, PKC), diphenyleneiodonium (an inhibitor of NOX), and U0126 (an inhibitor of mitogen-activated protein kinase kinase, MEK/extracellular-signal regulated kinase, ERK). Interestingly, pretreatment with BAY11-7082 (an inhibitor of nuclear factor-κB, NF-κB) suppressed the reduction of Cu/Zn-SOD, but not of EC-SOD. Furthermore, we also determined the involvement of newly synthesized protein and the instability of mRNA in the reduction of EC-SOD. Overall, our results suggest that the expression of EC-SOD is decreased by TPA through intracellular signaling consisting of PKC, NOX-derived ROS and MEK/ERK, but not of NF-κB signaling.     

Intracellular Ca2+ potentiates Na+/H+ exchange and cell differentiation induced by phorbol ester in U937 cells

The human cell line U937 differentiates to monocyte macrophage-like cells in response to tumour-promoting phorbol esters. This effect is attributed to activation of protein kinase C. We show here that U937 cell differentiation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) is associated with cytoplasmic alkalinization. Ethyl-isopropyl-amiloride (EIPA), a potent inhibitor of Na+/H+ exchange, blocked both cytoplasmic alkalinization and cell differentiation. Cell acidification by addition of 2-4 mM sodium propionate also blocked TPA-induced U937 cell differentiation. These results suggest that a sustained cell alkalinization mediated by activation of Na+/H+ exchange is essential for TPA-induced differentiation in U937 cells. The increase of cytoplasmic free calcium concentration ([Ca2+]i) by addition of the calcium ionophore ionomycin enhanced TPA-induced alkalinization by increasing the apparent affinity of the Na+/H+ antiporter for intracellular H+. Treatment with ionomycin also potentiated differentiation of U937 cells induced by TPA. This synergism suggests that [Ca2+]i either potentiates the activation of protein kinase C or triggers additional transducing mechanisms. The key events of this interaction occur during the first 30 min of treatment, even though cell differentiation manifests much later.     

12-O-tetradecanoylphorbol-13-acetate-induced apoptosis is mediated by tumor necrosis factor alpha in human monocytic U937 cells.

12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that is known as a tumor promoter, induces differentiation of myeloid cells and suppresses their proliferation. We studied the regulation of apoptosis by TPA in human monocytic cell line U937 cells that lack p53. Untreated U937 cells constitutively underwent apoptosis, and TPA enhanced apoptosis in these cells. Further studies showed that TPA increased production of tumor necrosis factor-alpha (TNFalpha) in U937 cells, and exogenously added TNFalpha induced apoptosis. Moreover, the induction of apoptosis by TPA was blocked by anti-TNFalpha antibody. Similar results were obtained in the myeloblastic cell line KY821 cells. We also found that the induction of apoptosis by TPA was increased in cells overexpressed with TNF receptor 1 but not in control cells. Furthermore, TPA failed to induce the production of TNFalpha and apoptosis in cells with either their protein kinase C or mitogen-activated protein kinase pathway blocked. Our results indicate that TPA induces apoptosis, at least in part, through a pathway that requires endogenous production of TNFalpha in U937 cells. Our data also suggest that the induction of apoptosis by TPA occurs through activation of protein kinase C and mitogen-activated protein kinase and TNFalpha is an autocrine-stimulating factor for the induction of apoptosis in these cells.     

Three activators of protein kinase C, bryostatins, dioleins, and phorbol esters, show differing specificities of action on GH4 pituitary cells

Phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol acetate (TPA) activate the calcium- and phospholipid-dependent protein kinase C and enhance three biological responses (prolactin release, prolactin synthesis, and cell stretching) in GH4C5 rat pituitary cells. We have examined several actions on GH4C5 cells of TPA and two other classes of protein kinase C activators, synthetic cell permeant dioleins and bryostatins isolated from the marine bryozoan Bugula neritina. Bryostatins 1 and 2 (B1 and B2, respectively) competed for [3H]phorbol 12,13-dibutyrate binding to the protein kinase C complex in intact cells nearly equipotently with TPA. B1 and B2, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoylglycerol (Di8) as well as TPA each activated partially purified protein kinase C from GH4C5 cells. B1, B2, and TPA each enhanced the acute release of prolactin from GH4C5 cells to a similar maximal extent. B1, B2, and TPA also enhanced prolactin synthesis. However, B1 and B2 were only partial agonists because they enhanced prolactin synthesis to a lesser maximal extent than did TPA and, given in combination, they reduced TPA-enhanced prolactin synthesis. OAG and Di8 stimulated prolactin release (to a lesser maximal extent than TPA) and did not stimulate prolactin synthesis. Pretreatment with OAG did not reduce TPA-stimulated prolactin release or synthesis. B2 and TPA induced cell stretching in GH4C5 cells, whereas B1, OAG, and Di8 induced little if any stretching. B1, but not B2, given in combination with TPA antagonized TPA-induced stretching but did not reduce thyrotropin-releasing hormone- or epidermal growth factor-induced stretching. We conclude that the bryostatins, phorbol esters, and dioleins bind to the same site on the protein kinase C complex to activate the enzyme, but they alter three biological responses in GH4C5 cells with selectivities and efficacies that differ. We propose that different activators of protein kinase C (such as bryostatins, dioleins, and phorbol esters) may elicit different cellular responses by altering the substrate specificity or activating multiple forms of the kinase.     

Comparative effects of polymyxin B, phorbol ester and bryostatin on protein phosphorylation, protein kinase C translocation, phospholipid metabolism and differentiation of HL60 cells

The effects of protein kinase C (PKC) inhibitor polymyxin B (PMB) and PKC activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin on intact HL60 cells were examined. It was found that each of the three agents exhibited similar effects on phosphorylation of certain endogenous proteins, PKC translocation from cytoplasm to plasma membrane and formation of CDP-choline. TPA, however, was the only agent that stimulated phosphatidylcholine formation. Differentiation of HL60 cells was potently induced by TPA; in comparison bryostatin was a relatively weaker inducer and PMB was without effect. The data indicated that the effects of the PKC inhibitor PMB on intact cells could not be predicted by its in vitro activity, and that certain TPA-dependent but PKC-independent reactions might be crucial in HL60 cell differentiation.     

Decreased sulfation of cellular chondroitin sulfate in response to activators of protein kinase C

The sulfation of cellular chondroitin sulfate in human promyelocytic leukemia HL60 cells was inhibited by a number of phorbol diesters, which concurrently induced differentiation into monocytic cells. Inhibition was dependent on concentration, and was 90% complete at 10 nM 12-0-tetradecanoylphorbol-13-acetate (TPA), the most active ester. Maximal effects were seen within 2-4 hours following initiation of treatment. The degree of inhibition observed correlated well with the ability of the esters to induce differentiation, and with their reported affinity for a "receptor", identified as protein kinase C associated with certain lipids. Chondroitin sulfation was also inhibited in cells treated with sn-1,2-dioctanoylglycerol, a lipid which is considered to be an endogenous activator of protein kinase C. Our findings therefore indicate that monocytic differentiation of HL60 cells occurs subsequent to reduced glycosaminoglycan sulfation via activation of the calcium-activated, phospholipid-dependent protein kinase.     

Dissociation of c-fos induction from macrophage differentiation in human myeloid leukemic cell lines.

Treatment of five human myeloid leukemic cell lines (KG1, ML3, HL-60, U-937, and HEL) with TPA was followed by macrophage differentiation and was accompanied by an early and transient increase in the mRNA level of c-fos proto-oncogene. The induction of c-fos was also observed in human cell lines K562 and K-Gla that did not respond to TPA with terminal macrophage differentiation. The treatment of HL-60 and U-937 cell lines with 1-oleoyl-2-acetylglycerol, a synthetic analog of diacylglycerol that, like TPA, stimulates protein kinase C activity, was followed by early and transient induction of c-fos mRNA in the absence of terminal macrophage differentiation. Finally, treatment of HL-60 with TPA in the presence of retinal, an inhibitor of protein kinase C, drastically reduced the induction of c-fos mRNA but had no effect on the terminal macrophage differentiation that is induced in this cell line by TPA. These results indicate that the induction of c-fos and terminal macrophage differentiation in response to TPA treatment can be dissociated in the in vitro models provided by human myeloid leukemic cell lines. Moreover, these findings suggest that the induction of c-fos is not only insufficient but may also be unnecessary for the differentiation along the monocyte-macrophage pathway.     

Decrease in CuZn-superoxide dismutase mRNA level during differentiation of human monocytic and promyelotic leukemia cells

Change in the level of CuZn-superoxide dismutase (SOD) mRNA was examined using a molecular probe during differentiation of human monocytic leukemia U937 cells or promyelotic leukemia HL-60 cells induced by either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or dimethylsulfoxide (DMSO). CuZn-SOD mRNA levels were found to decrease during the course of differentiation, and this response is specific for differentiation, since the treatment of human B cell leukemia cells or normal diploid fibroblasts with TPA failed to have any effect on the level of CuZn-SOD mRNA. The activity of CuZn-SOD in U937 cells also decreased during differentiation, but following that of the CuZn-SOD mRNA level. The expression of the CuZn-SOD gene is thus concluded to diminish during the differentiation of HL-60 and U937 cells.     

Tetradecanoyl phorbol acetate-induced microtubule reorganization is required for sustained mitogen-activated protein kinase activation and morphological differentiation of U937 cells.

Investigation of 12-tetradecanoyl phorbol 13-acetate (TPA)-resistant U937 cell clones has demonstrated that the normal sustained p42 mitogen-activated protein kinase (p42MAPK) activation produced by TPA treatment is absent. This is shown to be due to the inability of TPA to maintain activation of MAP/extracellular signal-regulated kinase kinase (MEK) and cRaf1. A direct relationship between sustained p42MAPK activation and differentiation is provided by the demonstration that blockade of MEK activation by PD098059 prevents TPA-induced morphological differentiation of wild type U937 cells. Using TPA-resistant clones, an involvement of microtubule reorganization and granule release is demonstrated by the ability of the microtubule depolymerizing agent nocodazole, to promote sustained p42MAPK activation in the presence of TPA. This response correlates with the lack of TPA-induced microtubule reorganization in these clones and the ability of nocodazole to partially bypass resistance to TPA. The results demonstrate a causal link between protein kinase C-dependent microtubule reorganization, sustained p42MAPK activation, and the induction of differentiation in U937 cells.     

Effect of phorbol esters on protein kinase C-zeta.

Protein kinase C-zeta (PKC-zeta) is a member of the protein kinase C gene family which using in vitro preparations has been described as being resistant to activation by phorbol esters. PKC-zeta was found to be expressed in several cell types as an 80-kDa protein. In vitro translation of a full-length PKC-zeta construct also yielded as a primary translation product an 80-kDa protein. In the U937 cell, PKC-zeta was slightly more abundant in the cytosol than in the particulate fraction. Acute exposure of U937 cells to tetradecanoyl-phorbol-13-acetate (TPA), phorbol dibutyrate, mezerin, or diacylglycerol derivatives did not induce translocation of this isoform to the particulate fraction. Chronic exposure to 1 microM TPA failed to translocate or down-regulate PKC-zeta in U937, HL-60, COS, or HeLa-fibroblast fusion cells. To examine whether PKC-zeta was activated by TPA, PKC activity was evaluated in COS cells transiently over-expressing this isoform. In non-transfected cells, two peaks of phospholipid- and TPA-dependent kinase activity were observed. Eluting at a lower salt concentration was a peak of activity associated with PKC-alpha. PKC-zeta eluted with the second peak of activity and at a higher salt concentration. In transfected cells which expressed PKC-zeta at 4-10-fold over endogenous levels, there was only a slight increase in activity associated with the second peak. The activity and quantity of PKC-zeta did not strictly correlate. Treatment with TPA under conditions that did not alter PKC-zeta content abolished detection of the second peak of PKC activity eluting from the Mono Q column. Thus, PKC-zeta does not translocate or down-regulate in response to phorbol esters or diacylglycerol derivatives. However, for reasons discussed these studies do not resolve the issue of whether this isoform is activated by TPA.     

Phorbol diester-induced phosphorylation of nuclear matrix proteins in HL60 promyelocytes. Possible role in differentiation studied by cationic detergent gel electrophoresis

Immortal HL60 promyelocytes are induced to differentiate to mortal adherent cells by a variety of agents which activate protein kinase C, including 12-O-tetradecanoylphorbol 13-acetate (TPA). In order to investigate the mechanism of this effect, we incubated HL60 cells with [32P]orthophosphate with or without TPA and extracted their proteins with the cationic detergent benzyldimethyl-n-hexadecylammonium chloride prior to electrophoresis in a discontinuous polyacrylamide gel system in the first dimension. In this system, proteins migrate toward the cathode as a function of their molecular weight, and they are separated from other radioactive components which can obscure the pattern of protein phosphorylation on sodium dodecyl sulfate (SDS) gels. SDS gel electrophoresis was used in the second dimension, resulting in the clear resolution of a large number of proteins. TPA caused many changes in the pattern of protein phosphorylation in intact cells. Two proteins which prominently increased their incorporation of 32P were investigated in particular, and they were both found to be retained in the nuclear matrix following successive extraction of cells with Triton, digestion with DNase and RNase, and extraction with 2 M NaCl. These proteins migrated with apparent molecular weights of 80,000 and 33,000 on SDS gels, and are designated NP80 and NP33, respectively. NP80 was half-maximally phosphorylated after 7 min exposure to TPA, and half-maximally phosphorylated by 10 nM TPA. NP80 co-migrated with a faint Coomassie Blue-stained protein, and NP33 co-migrated with a more prominent protein. Several proteins incorporated less 32P when the cells were exposed to TPA, including one which was extracted from nuclei with the core histones and which co-migrated with histone H2A. Further study will be needed to determine whether the differentiation of HL60 induced by TPA is mediated via phosphorylation of these nuclear matrix proteins.     

Macrophage glycolipid receptors for human migration inhibitory factor (MIF): differentiated HL-60 cells exhibit MIF responsiveness and express surface glycolipids which both bind MIF and convert nonresponsive cells to responsiveness

The human promyelocytic leukemia line HL-60 when treated with a phorbol diester (TPA) differentiates into cells (HL60-TPA) that respond to human migration inhibitory factor (MIF). Unresponsive HL-60 cells became responsive to MIF when preincubated with a glycolipid-enriched preparation extracted from HL60-TPA cells, human monocytes, human macrophage-like (U937) cell line, or with the purified glycolipid receptor for MIF from guinea pig peritoneal macrophages. Human blood monocytes exhibited an increased response to MIF when preincubated with glycolipids from HL60-TPA and U937 cells but not from HL-60 cells. Finally, glycolipids from HL60-TPA cells but not from HL-60 cells were able to reversibly bind MIF when covalently coupled to agarose. These studies suggest that TPA induces the differentiation of HL-60 cells into MIF-responsive cells through the expression of a glycolipid receptor for MIF.     

Effect of phorbol esters on cytosolic protein kinase C content and activity in the human monoblastoid U937 cell

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulates the human monoblastoid U937 cell to differentiate into a mature monocyte/macrophage-like cell. Since TPA may produce cellular responses by activating protein kinase C, the effects of TPA on kinase activity in the U937 cell were investigated. Brief exposures (less than or equal to 60 min) to TPA dramatically diminished protein kinase C-dependent phosphorylation of histone and endogenous substrates. However, using a peptide substrate corresponding to residues 720-737 of protein kinase C-epsilon, Ca2(+)-, phospholipid-, and diacylglycerol-dependent kinase activity was reduced only modestly after exposure to TPA. This phospholipid-dependent kinase activity coeluted on DEAE chromatography with protein kinase C. Examination of cytosolic protein kinase C content by Western blot analysis demonstrated a moderate decline in kinase content after TPA treatment. The decline was due primarily to loss of an 80-kDa species with preservation of a 76-kDa protein. The immunoreactive 76-kDa protein observed after TPA treatment comigrated on DEAE chromatography with the kinase activity phosphorylating the protein kinase C-epsilon peptide and had an elution profile similar to protein kinase C derived from untreated cells. Using antisera recognizing the catalytic and regulatory domains of the kinase, no evidence for proteolytic degradation of protein kinase C was observed. Although incubation of extracts from vehicle and TPA-treated cells inhibited the activity of partially purified protein kinase C, the degree of inhibition was similar in the two extracts. These findings suggest that TPA markedly diminishes protein kinase C-dependent phosphorylation of histone and endogenous substrates in part by altering kinase substrate specificity. These observations provide evidence for a novel post-translational process that can modulate protein kinase C-dependent phosphorylation.     

Activation of distinct protein kinase C isozymes by phorbol esters: correlation with induction of interleukin 1 beta gene expression

Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., & Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1 beta (IL-1 beta) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 X 10(-8) M TPA. Higher concentrations of TPA, which partially or totally depleted protein kinase C levels in the cells (10(-9)-2 X 10(-5) M), had an inhibitory effect on IL-1 beta mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1 beta mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 X 10(-8) M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the alpha, beta, and gamma isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC alpha was particulate, and PKC beta was cytosolic, while there was no detectable PKC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)