Tumor necrosis factor receptors and cytocidal activity are down-regulated by activators of protein kinase C

Human HeLa cells and murine L(S) cells are highly sensitive to the cytocidal activity of tumor necrosis factor (TNF) when simultaneously treated with the inhibitor of protein synthesis cycloheximide. This cytocidal activity of TNF was inhibited up to 90% in both cell lines after a 15-60-min pretreatment with 3-10 ng/ml of phorbol 12-myristate 13-acetate (PMA). This inhibition was long lasting for HeLa cells but transient for L(S) cells. The protection afforded by PMA was most effective when the cells were pretreated with this phorbol ester, but it decreased when PMA was added together with TNF or after TNF addition. This finding suggested that PMA interfered with one of the early steps in the mechanism of action of TNF. A pretreatment with the calcium ionophore A23187 also reduced the cytocidal activity of TNF in both HeLa and L(S) cells to about the same extent. Treatment of these cells with either PMA or A23187 significantly decreased the binding of 125I-TNF to cell surface receptors. This decrease paralleled the time course and dose-response of the inhibition of cytocidal activity. In addition, treatment of HeLa cells with 1-oleyl-2-acetyl-glycerol (OAG) also induced a rapid loss of TNF binding capacity. Since OAG, PMA, and A23187 are all activators of protein kinase C (Ca2+/phospholipid-dependent enzyme), these results suggest that this kinase is involved in modulation of TNF sensitivity. Furthermore, depletion or inhibition of protein kinase C antagonized PMA-induced effects on TNF cytotoxicity and binding to receptors. Internalization of bound TNF was not significantly affected by PMA treatment, and Scatchard analysis of binding data indicated that PMA decreased TNF receptor binding affinity rather than the number of TNF-binding sites. These findings suggest that protein kinase C may have a physiological role in mediating TNF sensitivity.     

Phosphorylation of T cell membrane proteins by activators of protein kinase C

Activation of the enzyme protein kinase C (PKC) plays an important role in T cell activation. We investigated the phosphorylation of CD2, CD3, CD4, CD5, CD7, CD8, CD28 (Tp44), CD43 (sialophorin, gp115), and LFA-1 after incubation of human PBMC with the (PKC) activator PMA. These proteins were chosen for their role in transmembrane signal transduction (CD2, CD3, CD5, CD28, CD43), cell-cell interaction and adhesion (CD2, CD4, CD8, and LFA-1), or involvement in immunodeficiency states (CD43, CD7). CD5, CD7, CD43, and the alpha-chain of LFA-1 were found to be constitutively phosphorylated. PMA induced rapid hyperphosphorylation of CD5, CD7, and CD43, but not of the LFA-1 alpha-chain, and induced the phosphorylation of CD3, CD4, CD8 and of the LFA-1 beta-chain. PMA did not cause the phosphorylation of CD2 and CD28. PMA-induced phosphorylation was partially inhibited by the PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride. Finally, the T cell activator Con A, which binds to the CD3/TCR complex was shown to induce a profile of protein phosphorylation similar to that observed with PMA. We conclude that PKC-mediated phosphorylation of T cell Ag may represent an important regulatory mechanism that governs the process of T cell activation.     

Downregulation of tumor necrosis factor receptors of macrophages by interferons and interleukin-1. Role of protein kinase C activation

Freshly harvested murine peritoneal macrophages and a line of transformed murine macrophages (RAW) were used in experiments designed to investigate the effect of different interferons (IFN) and interleukin-1 (IL-1) on tumor necrosis factor (TNF) receptors. Low concentrations of IFN-gamma or somewhat higher concentrations of IFN-alpha drastically downregulated the TNF receptors of RAW cells. A similar, but less pronounced, downregulation of TNF receptors was observed in peritoneal macrophages treated with these IFNs. This downregulation could not be accounted for by an induction of TNF secretion. Furthermore, IFN-alpha and gamma interacted synergistically in downregulating TNF receptors of RAW cells. IL-1 also downregulated TNF receptors. When RAW cells were treated with inhibitors of protein kinase C, the downregulation of TNF receptors by IFNs or IL-1 was reversed, and TNF binding increased up to 2-fold over that of untreated cells. Such increase was also observed in RAW cells treated only with the inhibitor of protein kinase C, staurosporine. However, TNF receptors decreased in peritoneal macrophages treated with staurosporine. This finding was explained by activation of macrophages by staurosporine, which induced secretion of TNF. These findings indicate that protein kinase C activity regulates TNF receptors in macrophages.     

Rapid dephosphorylation of protein kinase C substrates by protein kinase A activators results from inhibition of diacylglycerol release

The biochemical events encompassing the dephosphorylation of protein kinase C substrates by protein kinase A activators have been investigated in a neurotumor cell line, NCB-20. Treatment of [32P]orthophosphate-labeled cells with protein kinase A activators (e.g. forskolin, dibutyryl cAMP, prostaglandin E1) resulted in an inhibition of protein kinase C activity due to a failure of the protein kinase C complex to translocate into the membrane. Phospholipase C activity, as measured by the synchronous release of diacylglycerol and inositol phosphates (inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1-phosphate) in response to bradykinin, was inhibited up to 50% following exposure to protein kinase A activators. At the same time, phospholipase C-specific inositol phospholipid substrates (phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate) were found to accumulate in NCB-20 cells following treatment with protein kinase A activators. This suggests that phospholipase C may be altered through protein kinase A-mediated protein phosphorylation. Second messenger generation (inositol phosphates, diacylglycerol, and Ca2+) is therefore inhibited through cyclic AMP-mediated shutdown of the inositol lipid cycle at the level of phospholipase C.     

Signal transduction by tumor necrosis factor receptors

Tumor necrosis factor (TNF) is a key mediator in the inflammatory response which is implicated in the onset of a number of diseases. Research on TNF led to the characterization of the largest family of cytokines known until now, the TNF superfamily, which exert their biological effects through the interaction with transmembrane receptors of the TNFR superfamily. TNF itself exerts its biological effects interacting with two different receptors: TNFR1 and TNFR2. TNFR1 presents a death domain on its intracellular region. In contrast to TNFR1, TNFR2 does not have a death domain. Activation of TNFR1 implies the consecutive formation of two different TNF receptor signalling complexes. Complex I controls the expression of antiapoptotic proteins that prevent the triggering of cell death processes, whereas Complex II triggers cell death processes. TNFR2 only signals for antiapoptotic reactions. However, recent evidence indicates that TNFR2 also signals to induce TRAF2 degradation. TRAF2 is a key mediator in signal transduction of both TNFR1 and TNFR2. Thus, this novel signalling pathway has two important implications: on one hand, it represents an auto regulatory loop for TNFR2; on the other hand, when this signal is triggered TNFR1 activity is modified so that antiapoptotic pathways are inhibited and apoptotic reactions are enhanced.     

Lipid activators of protein kinase C

Among the many reported lipid activators of protein kinase C only those of high affinity can be considered true physiological effectors, at present the tumor promoters, e.g., phorbol esters; 1,2-diacyl-sn-glycerols; and phosphatidylinositol 4,5-bisphosphate. Many other compounds (including arachidonic acid) are activators at high, unphysiological concentrations only, and they seem to be sterically unsuited for bonding to the enzyme. Such pseudo-activators possibly act by scrambling the structure of the regulatory moiety of the kinase.     

Direct modulation of Na+ currents by protein kinase C activators in mouse neuroblastoma cells

We investigated the effects of different protein kinase C (PKC) activators on Na⁺ currents using the conventional whole-cell and the inside-out macropatch voltage-clamp techniques in mouse neuroblastoma cells (N1E-115). Two different categories of PKC activators were investigated: the cis-unsaturated fatty acids (CUFAs): oleic (cis-9-octadecenoic), linoleic (cis-9-12-octadecadienoic), and linolenic acid (cis-9-12-15-octadecatrienoic), and, the diacylglycerol (DAG) derivative 1-2-dioctanoyl-sn-glycerol (DOG). These substances caused the following alterations on Na⁺ currents: (i) Na⁺ currents were attenuated as a function of voltage. While DOG attenuated both inward and outward Na⁺ currents in a monotonic and continuous voltage-dependent manner, CUFAs preferentially attenuated inward currents; (ii) the steady-state activation curve of Na⁺ currents shifted to more depolarized voltages; (iii) opposite to the activation curve, the steady-state inactivation curve of Na⁺ channels (h curve) shifted to more hyperpolarized voltages; (iv) the time course of inactivation development was accelerated by PKC activators, while the recovery from inactivation was not affected; (v) substances that inhibit different metabolic pathways (PKC activation, cyclooxygenase, lipooxygenase, and P-450 pathways) did not prevent the effects of PKC activators on Na⁺ currents. One fully saturated fatty acid (octadecanoic acid), a trans-unsaturated fatty acid (trans-9-octadecenoic), and different phorbol esters did not affect Na⁺ currents; (vi) effects of different PKC activators on Na⁺ currents were completely reversible. These observations suggest that PKC activators might interact with Na⁺ channels directly. These direct effects must be taken into consideration in evaluating the overall effect of PKC activation on Na⁺ channels. Moreover, it is likely that this direct interaction could account, at least in part, for the diversity of effects of PKC activators on Na⁺ channels.This work was supported in part by a grant-in-aid from the American Heart Association (National Center).     

Rapid phosphorylation of a 27 kDa protein induced by tumor necrosis factor

Tumor necrosis factor (TNF) has been shown to induce the phosphorylation of a 27 kDa protein in a time- and concentration-dependent manner in HeLa D98/AH2, ME 180 and bovine aortic endothelial cells. This phosphorylation could be reproduced by the calcium ionophore, A23187. However, this phosphorylation was not observed in L929 cells, for which TNF is highly cytotoxic, suggesting that it might play a role in actions of TNF other than the induction of cell death.     

Stimulation of protein phosphorylations in frog rod outer segments by protein kinase activators. Suppression of light-induced changes in membrane current and cGMP by protein kinase C activators

Addition of protein kinase C activators to electropermeabilized frog rod photoreceptors enhances the phosphorylation of proteins with molecular masses of 54, 24, 19, 17, 12, and 11 kDa. The latter two correspond to components I and II, which are also phosphorylated by cyclic nucleotide-dependent protein kinase. Stimulation of phosphorylation by the protein kinase C activator oleoylacetylglycerol (OAG) is half-maximal at 7.7 microM OAG and is reduced by the protein kinase C inhibitor H-7. In contrast with earlier observations, no effects of calcium, calmodulin, or insulin on protein phosphorylations are observed. We find evidence for only three protein kinases in rod outer segments: a protein kinase C-like activity, cAMP-dependent protein kinase, and rhodopsin kinase. With the exception of components I and II, the substrate proteins for each kinase are distinct. Treatment of intact rods with OAG decreases the amplitude of the photoresponse and dark levels of cGMP up to 40%, as well as depressing the light-stimulated decrease in cGMP levels. These effects are observed between 0.1 and 1 microM OAG. The data suggest that OAG-sensitive reactions may modulate pathways that support the light response.     

Diacylglycerols as activators of protein kinase C

Diacylglycerols are generated in the membrane as the result of extracellular signals and are able to stimulate the activity of protein kinase C, acting as membrane second messengers. Diacylglycerols are recognized by protein kinases C through the C1 domain and established models propose that they will stabilize the translocation of the protein to the membrane. However, diacylglycerols also act by modulating the physical properties of the membrane, thus favouring the translocation of the enzyme. This is done through alteration of the membrane surface curvature, dehydration of the surface and the separation of phospholipid surface groups. Good correlations have been observed between the physical state of the membrane and protein kinase C activity.     

Structural analogies between protein kinase C activators

Phorbol esters and diacylglycerols activate protein kinase C but specific structural parameters appear to be required for the enzyme activation. We have analyzed the conformation of potent and not potent diacylglycerols and phorbol esters. The orientation of the CH20H group at C3 of 1,2 diolein is remarkably similar to that of the same group at C-20 of 4 beta phorbol didecanoate and crucial for potency in activating the enzyme. Our data suggest that the new conformational approach here described could be used to rationally design specific inhibitors preventing the effects of tumor promoters and to predict the structure of potential tumor promoters.     

Rapid modulation of homing receptors (gp90MEL-14) induced by activators of protein kinase C. Receptor shedding due to accelerated proteolytic cleavage at the cell surface

Lymphocyte entry into lymph nodes and Peyer's patches is initiated by the adhesion of the lymphocytes to specialized postcapillary high endothelial venules (HEV). The binding of lymphocytes to lymph node HEV is mediated by the cell surface receptor gp90MEL-14 (gp90). Previous work has shown that gp90 is down-regulated over a period of days after mitogenic or mixed lymphocyte reaction stimulation of T lymphocytes. In our study, it is shown that stimulation of lymphocytes with activators of protein kinase C (PKC), such as PMA or 1-oleoyl 2-acetyl-glycerol, results in the nearly complete loss of surface expression of gp90 within 1 h. Pretreatment of the cells with H-7 or staurosporine, PKC inhibitors, but not HA1004, a general protein kinase inhibitor, prevents the loss of gp90MEL-14. Within 15 min of stimulation of PKC, a novel form of gp90 can be immunoprecipitated from the supernatant of stimulated cells. Upon deglycosylation, this soluble gp90 polypeptide is shown to be 12 kDa smaller than the cell surface protein. Peptide mapping showed identical patterns for surface and soluble receptor, confirming that the soluble Ag is related to the cell membrane protein. Together, these experiments suggest that activation of PKC results in the proteolytic cleavage of gp90MEL-14, resulting in receptor shedding and the inability of the lymphocytes to adhere to HEV endothelium. Furthermore, because supernatant from unstimulated, normal lymphocytes also contains a small amount of the low Mr form of gp90, cell surface proteolysis may be part of the normal turnover of this receptor glycoprotein. These experiments suggest that PKC may play a role in the regulation of lymphocyte traffic to lymphoid tissues.     

Diol lipids are activators of protein kinase C

Diol lipids (dioleoyl- and dioctanoylethylene glycol) at relatively low concentrations (approximately 10 microM) were found to activate significantly protein kinase C in the presence of phosphatidylserine or phosphatidylinositol. Since diol lipids are widespread minor lipid constituents of many cells [(1974) Chem. Ind., 597-604], it has been suggested that they may be involved in the maintaining of basal protein kinase C activity in the absence of external stimuli.     

Regulation of lymphocyte tumor necrosis factor receptors by IL-2

Activated lymphocytes are known to express TNF receptors. The precise stimuli involved in induction and regulation of these receptors have not been elucidated. Our findings demonstrate that IL-2, alone and in serum-free conditions, can trigger and regulate TNF receptor expression on normal lymphocytes. Flow cytometric analyses demonstrated that the receptor was rapidly induced on CD4, CD8, and CD16+ cells after IL-2 stimulation. Receptors increased with culture duration, became maximal between days 5 and 9, and were maintained for 18 to 20 days in the presence of IL-2. By using 125I-TNF and FITC-TNF binding, we present evidence that IL-2 concentration determines the magnitude of lymphoid TNF receptor expression--influencing both the percentage of TNF-positive cells within the population and the number of receptors/cell. Collectively, our results are persuasive for consideration of IL-2 as a central mediator in the regulation of lymphocyte TNF receptors.     

Regulation of VL30 gene expression by activators of protein kinase C

The mouse genome contains a retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenously supplied epidermal growth factor (EGF). Binding of EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity which indirectly lead to activation of protein kinase C. We treated AKR-2B cells, Swiss 3T3 cells, and the 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters and with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoyl phorbol acetate (TPA] increased the level of VL30 expression. Stimulation with either TPA or EGF produced a similar time course of VL30 expression. TPA induced VL30 expression in the EGF-receptorless NR6 cell line, indicating that neither EGF ligand-receptor binding nor phosphorylation of the EGF receptor was required for induction of VL30 expression. Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30 expression was also stimulated by treatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression.     

Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors

The proinflammatory cytokine tumor necrosis factor (TNF) binds two distinct plasma membrane receptors, TNFR1 and TNFR2. We have produced different receptor mutants fused with enhanced green fluorescent protein to study their membrane dynamics by fluorescence correlation spectroscopy (FCS). TNFR1 mutants show diffusion constants of approximately 1.2 × 10^− 9 cm²/s and a broad distribution of diffusion times, which is hardly affected by ligand binding. However, cholesterol depletion enhances their diffusion, suggesting a constitutive affinity to cholesterol rich membrane microdomains. In contrast, TNFR2 and mutants thereof diffuse rather fast (D? = 3.1 × 10^− 9 cm²/s) with a marked reduction after 30 min of TNF treatment (D? = 0.9 × 10^− 9 cm²/s). This reduction cannot be explained by the formation of higher ordered receptor clusters, since the fluorescence intensity of TNF treated receptors indicate the presence of a few receptor molecules per complex only. Together, these data point to a topological segregation of the two TNF receptors in different microcompartments of the plasma membrane independent of the cytoplasmic signaling domains of the receptors.     

Induction of the synthesis of tumor necrosis factor receptors by interferon-gamma

Human HT-29 colon carcinoma and HeLa D98/AH2 and SK-MEL-109 melanoma cells were sensitive to synergistic growth inhibition by concentrations of recombinant human tumor necrosis factor (rTNF) and interferon-gamma (rIFN-gamma) which individually were only slightly inhibitory. We investigated whether this synergism could be explained by the presence of an increased number of TNF receptors in cells treated with rIFN-gamma. These receptors were measured by incubating cells resuspended from monolayers with 125I-rTNF. HT-29 cells treated for a few hours with rIFN-gamma could bind more 125I-rTNF than control untreated cells, but this binding returned to the level of control cells after 24 hr. The treatment with rIFN-gamma did not change the binding affinity of TNF receptors, but increased their number to 1800 per cell from a basal level of about 800 per cell. Inhibitors of RNA synthesis prevented this increase. HT-29 cells were significantly more growth-inhibited when treated first for 6 to 12 hr with rIFN-gamma and then with rTNF, than when treated first with rTNF and then with rIFN-gamma. Untreated HeLa D98/AH2 and SK-MEL-109 cells had 2400 and 9000 receptors per cell, with a KD similar to that of HT-29 cells (approximately 2 X 10(-10)M). A significant increase in TNF receptors after treatment with rIFN-gamma was observed in HeLa D98/AH2, but not in SK-MEL-109 cells. No increase in TNF receptors was detected in cells treated with rIFN-alpha 2. These results indicate that the synergism between rTNF and rIFN-gamma may be due, at least in part, to a transient induction of the synthesis of TNF receptors by rIFN-gamma in cells with a relatively low number of these receptors.