Evidence for coordinate, selective regulation of eicosanoid synthesis in platelet-derived growth factor-stimulated 3T3 fibroblasts and in HL-60 cells induced to differentiate into macrophages or neutrophils

We used Swiss 3T3 fibroblasts stimulated with platelet-derived growth factor and HL-60 cells induced to differentiate into macrophages or neutrophils to study the regulation of prostaglandin and leukotriene synthesis. Addition of platelet-derived growth factor to quiescent 3T3 fibroblasts led within 4 h to a dramatic and preferential increase in prostacyclin synthesis from endoperoxide prostaglandin H2, and microsomal assays showed a strong platelet-derived growth factor-dependent increase in the maximal velocities (Vmax) of both prostaglandin H synthase and prostacyclin synthase. In contrast, addition of phorbol ester to HL-60 cells to induce differentiation into macrophages led within 4 h to a strong and preferential increase in thromboxane synthesis from prostaglandin H2, and microsomal assays disclosed a major rise in Vmax for both prostaglandin H synthase and thromboxane synthase. No comparable changes occurred in HL-60 cells that were differentiating into neutrophils, though upregulation of 5-lipoxygenase pathway enzymes occurred in both differentiation systems. Actinomycin D and cycloheximide prevented the appearance of all of these enzymes of eicosanoid synthesis in all three model systems. Thus, the distinctive patterns of eicosanoid synthesis that are seen in replicating fibroblasts and in differentiating macrophages and neutrophils appear to depend on a coordinate, selective upregulation of several enzymes of eicosanoid biosynthesis that is specific for each cell system.     

Expression of herpes simplex virus ICP47 and human cytomegalovirus US11 prevents recognition of transgene products by CD8(+) cytotoxic T lymphocytes

The in vivo persistence of gene-modified cells may be limited by the development of a host immune response to vector-encoded proteins. Herpesviruses evade cytotoxic T-lymphocyte (CTL) recognition by expressing genes which interfere selectively with presentation of viral antigens by class I major histocompatibility complex (MHC) molecules. Here, we studied the use of retroviral vectors encoding herpes simplex virus ICP47, human cytomegalovirus (HCMV) US3, or HCMV US11 to decrease presentation of viral proteins and transgene products to CD8(+) CTL. Human fibroblasts and T cells transduced to express the ICP47, US3, or US11 genes alone exhibited a decrease in cell surface class I MHC expression. The combination of ICP47 and US11 rendered fibroblasts negative for surface class I MHC and allowed a class I MHC-low population of T cells to be sorted by flow cytometry. Fibroblasts and T cells expressing both ICP47 and US11 were protected from CTL-mediated lysis and failed to stimulate specific memory T-cell responses to transgene products in vitro. Our findings suggest that expression of immunoregulatory viral gene products could be a potential strategy to prolong transgene expression in vivo.     

Control of protein kinase C activity, phorbol ester-induced cytoskeletal remodeling, and cell survival signals by the scaffolding protein SSeCKS/GRAVIN/AKAP12

The product of the SSeCKS/GRAVIN/AKAP12 gene ("SSeCKS") is a major protein kinase (PK) C substrate that exhibits tumor- and metastasis-suppressing activity likely through its ability to scaffold multiple signaling mediators such as PKC, PKA, cyclins, calmodulin, and Src. Although SSeCKS and PKCα bind phosphatidylserine, we demonstrate that phosphatidylserine-independent binding of PKC by SSeCKS is facilitated by two homologous SSeCKS motifs, EG(I/V)(T/S)XWXSFK(K/R)(M/L)VTP(K/R)K(K/R)X(K/R)XXXEXXXE(E/D) (amino acids 592-620 and 741-769). SSeCKS binding to PKCα decreased kinase activity and was dependent on the two PKC-binding motifs. SSeCKS scaffolding of PKC was increased in confluent cell cultures, correlating with significantly increased SSeCKS protein levels and decreased PKCα activity, suggesting a role for SSeCKS in suppressing PKC activation during contact inhibition. SSeCKS-null mouse embryo fibroblasts displayed increased relative basal and phorbol ester (phorbol 12-myristate 13-acetate)-induced PKC activity but were defective in phorbol 12-myristate 13-acetate-induced actin cytoskeletal reorganization and cell shape change; these responses could be rescued by the forced expression of full-length SSeCKS but not by an SSeCKS variant deleted of its PKC-binding domains. Finally, the PKC binding sites in SSeCKS were required to restore cell rounding and/or decreased apoptosis in phorbol ester-treated LNCaP, LNCaP-C4-2, and MAT-LyLu prostate cancer cells. Thus, PKC-mediated remodeling of the actin cytoskeleton is likely regulated by the ability of SSeCKS to control PKC signaling and activity through a direct scaffolding function.     

Keloid fibroblasts are refractory to inhibition of DNA synthesis by phorbol esters. Altered response is accompanied by reduced sensitivity to prostaglandin E2 and altered down-regulation of phorbol ester binding sites.

To investigate abnormal growth regulation in keloid fibroblasts, responses to phorbol esters were examined. Treatment of quiescent cultures with phorbol 12-myristate 13-acetate (PMA) blocked a normally occurring (20-24 h) peak of serum-stimulated thymidine incorporation in normal and keloid cells. In keloid fibroblasts PMA induced a delayed peak of DNA synthesis. When indomethacin was added with PMA the delayed peak appeared in normal fibroblasts. The ED50 for inhibition of the 20-24-h peak was 1 nM, whereas the delayed peak required a 50-fold-higher PMA concentration. In both cell types PMA induced prostaglandin E2 (PGE2) synthesis, and exogenous PGE2 caused 50% inhibition of the 20-24-h peak. When PMA and indomethacin were added with PGE2 the delayed peak was inhibited 90% in normal fibroblasts, whereas inhibition of keloid cells was the same as with PGE2 alone. Normal and keloid fibroblasts had the same number of phorbol ester binding sites. However, in normal cells, phorbol 12,13-dibutyrate bound with greater affinity, and down-regulation of phorbol ester binding occurred to a greater extent. These findings suggest that altered expression of protein kinase C isozymes or another molecule that binds phorbol esters may play a role in abnormal growth regulation of keloid cells.     

Inhibition of cell communication between Balb/c 3T3 cells by tumor promoters and protection by cAMP

The effect of phorbol ester tumor promoters on the communication between individual cells in confluent culture was studied using a fluorescent dye transfer method. Cell-cell communication between mouse Balb/c 3T3 cells and between Chinese hamster V79 cells was inhibited almost completely by tumor-promoting phorbol esters, but not by nonpromoting derivatives; the effect was reversed upon removal of the promoter. Intercellular communication between Balb/c 3T3 cells, but not Chinese hamster V79 cells, was increased significantly in the presence of dbcAMP and caffeine, and these compounds counteracted the effects of tumor promoters. Inhibition of cell communication by phorbol esters appears to be receptor-mediated, since specific binding of 3H-phorbol-12,13-dibutyrate to Balb/c 3T3 cells was inhibited only by compounds that also inhibit intercellular dye transfer. A study with cycloheximide suggests that the reversible inhibition of intercellular communication by phorbol esters may not need de novo protein synthesis, while upregulation of communication by cAMP requires protein synthesis.     

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.     

Down-regulation of protein kinase C and of an endogenous 80-kDa substrate in transformed fibroblasts

Subconfluent cultures of NIH-3T3 fibroblasts transformed by the Ha-ras, Ki-v-ras, v-src, and v-fms oncogene proteins all possess elevated steady-state levels of diacylglycerol, the endogenous activator of protein kinase C, as compared to the nontransformed parental lines. These oncogene-transformed fibroblasts also exhibit a significantly decreased level of cellular protein kinase C activity as measured by four different criteria: phorbol ester-stimulated phosphorylation of an endogenous 80-kilodalton (80 kDa) substrate; phorbol ester-stimulated changes in 86Rb uptake; enzymatic assay; and [3H]phorbol ester binding. In all cases, the transformed cells demonstrated an attenuated response to phorbol ester addition and a lower phorbol ester binding capacity as compared to the parental lines. Western analysis of the endogenous 80-kDa substrate of protein kinase C revealed a significantly lower level of this protein in the transformed cells than in the untransformed controls, and this decrease could be mimicked in parental cells by long-term incubation with phorbol esters, suggesting that the level of the 80-kDa protein is regulated by the state of activation of protein kinase C. These effects do not appear to be nonspecific responses to autocrine secretions by the transformed cells. They may represent an unsuccessful attempt by the transformed cells to negatively modulate the constitutive proliferative signals generated by the oncogene products.     

Translocation of diacylglycerol kinase from the cytosol to the membrane in phorbol ester-treated Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent Swiss 3T3 fibroblasts. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on membrane-bound diacylglycerol kinase in 3T3 cells. When phorbol ester is added to 3T3 membranes in the presence of ATP, Mg2+, and Ca2+, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, also suggesting that the translocation of DAG kinase is regulated primarily by substrate concentration.     

Phorbol ester-induced translocation of diacylglycerol kinase from the cytosol to the membrane in Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-sn-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent, starved Swiss 3T3 fibroblasts. We utilized exogenous dioleoylglycerol as substrate for the kinase. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C (Ca2+/phospholipid-dependent enzyme) by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on Swiss 3T3 membrane-bound diacylglycerol kinase nor does it directly effect cytosolic diacylglycerol kinase. When phorbol ester is added to Swiss 3T3 membranes in the presence of ATP, magnesium, and calcium, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Reconstitution studies show that the soluble rat brain diacylglycerol kinase binds to diacylglycerol-enriched membranes, produced by treatment of red cell ghosts with phospholipase C or calcium, suggesting that cytosolic diacylglycerol kinase may be capable of translocation to the membrane in response to elevated substrate concentration in the intact cell. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, by 5 min, also suggesting that the translocation of diacylglycerol kinase activity is regulated primarily by substrate concentration.     

Protein kinase C-dependent upregulation of N-cadherin expression by phorbol ester in human calvaria osteoblasts

Cell-cell adhesion mediated by cadherins is believed to play an essential role in the control of cell differentiation and tissue formation. Our recent studies indicate that N-cadherin is involved in human osteoblast differentiation. However, the signalling molecules that regulate cadherins in osteoblasts are not known. We tested the possibility that N-cadherin expression and function may be regulated by direct activation of protein kinase C (PKC) in human osteoblasts. Treatment of immortalized human neonatal calvaria (IHNC) cells with phorbol 12,13-dibutyrate (100 nM) transiently increased PKC activity. RT-PCR analysis showed that transient treatment with phorbol ester transiently increased N-cadherin mRNA levels at 4-12 h. Western blot analysis showed that N-cadherin protein levels were increased by phorbol ester at 24-48 h, and this was confirmed by immunocytochemical analysis. In contrast, E-cadherin expression was not affected. Transient treatment of IHNC cells with phorbol ester increased cell-cell aggregation, which was suppressed by neutralizing N-cadherin antibody, showing that the increased N-cadherin induced by phorbol ester was functional. Finally, phorbol ester dose-dependently increased alkaline phosphatase activity, an early marker of osteoblast differentiation. This effect was comparable to the promoting effect of BMP-2, a potent activator of osteoblast differentiation. These data show that direct activation of PKC by phorbol ester increases N-cadherin expression and function, and promotes ALP activity in human calvaria osteoblasts, which provides a signaling mechanism by which N-cadherin is regulated and suggests a role for PKC in N-cadherin-mediated control of human osteoblast differentiation.