Presence Of TPA Research Articles

Does Ca2+ channel blockade modulate the antidepressant-induced changes in mechanisms of adrenergic transduction?

We investigated how the L-type calcium channel blockade (CCB) with nifedipine affects the cyclic AMP responses to noradrenaline or isoproterenol in cerebral cortical slices from rats receiving antidepressant treatments that induce (electroconvulsive shock, imipramine) or do not induce (amitriptyline) beta-downregulation. To assess the role of protein kinase C (PKC) in receptor crosstalk under CCB conditions, the cyclic AMP responses were tested also in the presence of a PKC activator, TPA. CCB alone induced no changes, but modulated the action of those antidepressants that down regulate the beta-adrenergic system. Chronic ECS and imipramine treatments were differently affected. ECS, under conditions of CCB, down regulated the response to isoproterenol in the presence of TPA, while imipramine ceased to block the TPA-potentiation of cyclic AMP responses. Thus, CCB affects the processes related to the antidepressant-induced changes on the crosstalk between alpha1- and beta-adrenergic receptors, depending on the specific properties of the antidepressant.

The Phorbol Ester 12-O-Tetradecanoylphorbol 13-Acetate Enhances the Heat-induced Stress Response

Induction of heat shock gene expression is mediated by specific heat shock transcription factors (HSFs), but the signaling pathways leading to activation of HSFs are poorly understood. To elucidate whether protein kinase C-responsive signaling pathways could be involved in the regulation of heat shock gene expression, we have examined the effects of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) on the heat-induced stress response in K562 cells. We demonstrate that TPA treatment markedly enhances heat shock gene expression during heat stress, although TPA alone does not induce the heat shock response. This TPA-mediated enhancement can initially be detected as an accelerated acquisition of DNA binding and transcriptional activity of HSF1 resulting in elevated Hsp70 protein concentrations. In the presence of TPA, the attenuation of HSF1 DNA binding activity during continuous exposure to heat shock occurs more rapidly and in concert with the appearance of newly synthesized Hsp70, which supports earlier studies on the autoregulatory role of Hsp70 in deactivation of HSF1. During heat stress, a correlation between the hyperphosphorylation of HSF1 and its transcriptional activity was observed, in both the presence and the absence of TPA. Our results show that the heat-induced stress response can be significantly modulated by activation of protein kinase C-responsive signaling pathways.

Coagulation-Dependent Inhibition of Fibrinolysis: Role of Carboxypeptidase-U and the Premature Lysis of Clots From Hemophilic Plasma

Coagulation is initiated by the binding of factor VIIa to tissue factor, with resultant limited factor IX and X activation and thrombin production. Owing to the feedback inhibition of the factor VIIa/tissue factor complex by tissue factor pathway inhibitor (TFPI), additional factor X activation and thrombin generation must proceed through a pathway involving factors VIII, IX, and XI. Experiments designed to elucidate the requirement for amplified factor Xa and thrombin generation in normal hemostasis show that the resistance of plasma clots to tissue plasminogen activator (tPA)- and urokinase-induced fibrinolysis is related to the extent of thrombin generation. Inhibition of fibrinolysis is mediated in part by plasma carboxypeptidase-U ([CPU] carboxypeptidase-R, procarboxypeptidase-B, thrombin-activatable fibrinolysis inhibitor), a proenzyme that is proteolytically activated by thrombin in a process enhanced dramatically by the cofactor thrombomodulin. A clot induced in factor IX-deficient plasma with limited amounts of tissue factor in the presence of urokinase (100 U/mL) lyses prematurely, and this defect is corrected by supplementation of the deficient plasma with factor IX (5 micrograms/mL) or thrombomodulin (20 ng/mL). These additions enhance the rate and extent of CPU activation: in the case of factor IX, presumably by permitting amplified generation of factor Xa and thrombin, and in the case of thrombomodulin, presumably by increasing the degree of CPU activation produced by the low levels of thrombin generated in the absence of factor IX. Pretreatment of the factor IX-deficient plasma with specific anti-CPU antibodies prevents the increased resistance to fibrinolysis produced by addition of factor IX and thrombomodulin. Likewise, when coagulation is induced by thrombin (2 U/mL) in the presence of tPA (60 U/mL), clots formed from plasmas deficient in factors VIII, IX, X, or XI lyse prematurely unless the missing factor is replaced or thrombomodulin (20 ng/mL) is added.

Regulation of arachidonic acid metabolism, aromatase activity and growth in human breast cancer cells by interleukin-1beta and phorbol ester: dissociation of a mediatory role for prostaglandin E2 in the autocrine control of cell function.

Prostaglandin E2 (PGE2) levels are elevated in malignant human breast tissue. However, the cellular mechanisms regulating this arachidonate metabolism and the autocrine influence PGE2 production may have on breast cancer cell growth and function are unclear. In the present study, we have investigated the effects of 2 putative cyclo-oxygenase inducers, interleukin-1beta (IL-1beta) and the protein kinase C agonist 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on PGE2 production, growth and aromatase activity in the MDA MB 231 breast cancer cell line. TPA stimulated a dose-dependent increase in PGE2 production, inhibited cell growth and stimulated aromatase activity. Although IL-1beta alone had no effect on any of these breast cancer cell functions, the cytokine greatly potentiated PGE2 production in the presence of TPA. Similarly, growth inhibition and aromatase stimulation in response to TPA were both further enhanced by IL-1beta treatment. Indomethacin and dexamethasone both prevented PGE2 production in response to IL-Ibeta and TPA but had no effect on the anti-proliferative action of the cytokine and phorbol ester. While indomethacin had no effect on induction of aromatase activity by IL-1beta and TPA, dexamethasone exhibited a temporally biphasic action. Dexamethasone alone stimulated aromatase activity and demonstrated a permissive action on aromatase stimulation by IL-1beta and TPA. However, pre-treatment of cells with dexamethasone prevented subsequent induction of aromatase activity by IL-1beta and TPA. Our study describes a novel synergistic interaction in response to protein kinase C activation and IL-1beta during the regulation of arachidonate metabolism, cell growth and aromatase activity in human breast cancer cells. We conclude that the cyclooxygenase pathway does not play a mediatory role during the inhibition of cell growth and the induction of aromatase activity by IL-1beta and TPA.

Interleukin-1 beta upregulates tissue-type plasminogen activator in a keratinocyte cell line (HaCaT).

Human keratinocytes synthesize and secrete tissue-type plasminogen activator (tPA). tPA converts the inactive precursor enzyme plasminogen into the trypsin-like proteinase plasmin. tPA is not found in normal epidermis, but in lesional epidermis from patients with a variety of cutaneous diseases, including psoriasis, pemphigus and pemphigoid. The presence of tPA is probably a reaction to the disease process rather than the initiating event in these etiologically and histopathologically diverse lesions. However, the factor(s) that upregulate tPA expression and secretion in keratinocytes have remained largely elusive. We sought to determine whether the inflammatory cytokine interleukin-1 beta (IL-1 beta), which is commonly present in diverse epidermal lesions, influences tPA production. Accordingly, we studied the influence of IL-1 beta on secretion of tPA by cells of the human keratinocyte cell line HaCaT. We found that IL-1 beta increased tPA secretion in these cells. Given the observation that IL-1 beta is a common proinflammatory mediator in cutaneous diseases, our findings may explain the increase in tPA in clinically and etiologically diverse inflammatory epidermal lesions.

Effects of hydroquinone-type and phenolic antioxidants on calcium signals and degranulation of RBL-2H3 cells

We previously reported that a hydroquinone-type antioxidant, 2,5-di( tert-butyl)-1,4-hydroquinone (DTBHQ), increases intracellular free Ca 2+ concentration ([Ca 2+] i), causes degranulation together with a protein kinase C activator, phorbol 12-myristate 13-acetate (TPA), and increases antigen-induced degranulation in rat basophilic leukemia (RBL-2H3) cells. In this study, the effects of five hydroquinone-type and phenolic antioxidants (2,5-di( tert-amyl)-1,4-hydroquinone [DTAHQ], 2- tert-butyl-1,4-hydroquinone [MTBHQ], 3,5-di( tert-butyl)-4-hydroxytoluene [BHT], 3,5-di( tert-butyl)-4-hydroxyanisole [DTBHA], and 3- tert-butyl-4-hydroxyanisole [MTBHA]) on [Ca 2+] i and degranulation (β-hexosaminidase release) were examined and compared with that of DTBHQ. DTAHQ (⩾ 3 μM) showed effects similar to those of DTBHQ (10 μM) on [Ca 2+] i elevation, induction of degranulation with TPA, and increase of antigen-induced degranulation. BHT (50 μM) and DTBHA (50 μM) caused [Ca 2+] i elevation and increased degranulation in the presence of TPA or antigen, but their effects were less than those of DTBHQ and DTAHQ. MTBHQ and MTBHA had no effect on [Ca 2+] i and degranulation, even at 50 μM. The degree of Ca 2+ response caused by the compounds correlated well with the increase in degranulation, but not with their antioxidant activity estimated with the first oxidation potential. From these results, it is suggested that the increasing effects of six antioxidants on degranulation in the presence of TPA or antigen were dependent on [Ca 2+] i increase caused by the compounds, probably through their ability to inhibit endoplasmic reticulum Ca 2+-ATPase.

Guinea pig Kurloff (NK-like) cells mediate TNF-dependent cytotoxic activity: analogy with NC effector cells.

Kurloff cells are mononuclear cells possessing a large cytoplasmic inclusion body specific to the guinea pig. In this report, we present strong evidence that Kurloff cells can mediate NC activity against tumor cells in addition to their previously reported NK activity. Using an 18 h 51Cr-release assay we have shown that Kurloff cells were highly effective in killing the TNF-sensitive WEHI 164 target cell line. Lower but significant cytotoxic activity was also observed after only 4 h. However, our results suggest a different mechanism of lysis in the 4 h and 18 h assay. Lysis of WEHI 164 target cells by Kurloff cells in the 4 h assay could be strongly increased in the presence of TPA alone or in combination with ionomycin whereas ionomycin alone was uneffective. In contrast, stimulation of Kurloff cells for 18 h with ionomycin alone or in combination with TPA could induce the release of TNF-like factor(s) as observed by the TNF bioassay using L-929 TNF-sensitive target cells. Release of TNF-like factor(s) could also be induced by stimulation with WEHI 164 target cells. Supernatants of Kurloff cells stimulated for 18 h with TPA + ionomycin were also highly cytotoxic against WEHI 164 target cells, but not against the TNF-resistant P815 target cell line. Pretreatment of these supernatants with antimurine TNF alpha antibodies could almost completely inhibit their cytotoxic activity against WEHI 164 target cells. In contrast, supernatants of Kurloff cells stimulated for only 4 h did not show any TNF-like activity against the L-929 target cell line and were not cytotoxic against WEHI 164 target cells even after 18 h. Taken together, these results suggest that Kurloff cells can mediate NC activity against tumor cells in addition to their previously reported NK activity. By using multiple lytic pathways, these cells may play a crucial role in anti-tumor surveillance and defenses.

The phorbol ester TPA regulates collagen gene expression at the transcriptional level.

Previously, we reported that growth activation of quiescent 3T3-L1 cells by TPA led to a rapid increase of pro-alpha 2 (I) collagen mRNA and protein, while induction of pro-alpha 2 (I) was not observed in VT-1 cells, a line non-mitogenic in the presence of TPA (26). Here, we further examine the expression of pro-alpha 2 (I) collagen during mitogenic stimulation at the molecular level. In addition to pro-alpha 2 (I) mRNA, TPA treatment increased mRNA production of other collagen family members, pro-alpha 1 (I) and pro-alpha 1 (III) although in reduced amounts relative to pro-alpha 2 (I). In contrast to pro-alpha 2 (I), the mRNA expression profiles of several protooncogenes were regulated in both VT-1 and 3T3-L1 cells. Consistent with increased mRNA levels, TPA treated 3T3-L1 cells produced a matrix abundant in collagen type I protein. In vitro nuclear "run-on" transcription assays demonstrated a 4-fold increase in pro-alpha 2 (I) mRNA that was maximal within 10 min of TPA treatment. Using a chloramphenicol-acetyl transferase (CAT) assay, we identified a TPA sensitive domain within the promoter of the COL1A2 gene. These results establish COL1A2 as an early growth responsive gene, and that its regulation is PKC dependent. Additionally, the increased expression of protooncogenes and transin during TPA stimulation of non-mitogenic VT-1 cells indicated that the regulation of these genes is independent of PKC, indicating the existence of multiple regulatory mechanisms amongst early response genes.

Transcriptional regulation of apolipoprotein A-I expression in Hep G2 cells by phorbol ester

The regulation of apolipoprotein A-I (apo A-I) gene expression by 12- O-tetradecanoylphorbol 13-acetate (TPA) was investigated in the human hepatoma cell line Hep G2. TPA treatment decreased apo A-I mRNA levels in a time-dependent manner, by up to 50% versus control cells within 24 h. Nuclear run-on transcription assays demonstrated a transcriptional effect of TPA. Using transfection analysis with a plasmid construct containing the −1378/+11 apo A-I promoter fused to the secreted placental alkaline phosphatase (SPAP) reporter gene, we showed that the SPAP activity was decreased to 50% when Hep G2 cells were incubated in the presence of TPA. The inhibitory effect of TPA was still maintained when fragment −253 to −4 of apo A-I promoter was linked to the CAT reporter gene. These data indicate that transcriptional modulation of apolipoprotein A-I gene expression following phorbol ester treatment is transduced by gene elements located between −253 and −4 of the apo A-I promoter.

Turnover of glutathione S-transferase alpha mRNAs is accelerated by 12-O-tetradecanoyl phorbol-13-acetate in human hepatoma and colon carcinoma cell lines.

The phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), known to induce murine glutathione S-transferase (GST) Ya, was examined for its effect on the expression of human GST alpha. Unexpectedly, 24-h treatment of the human hepatoma cell line HepG2 with 100 nmol/l TPA caused a decrease of the GST alpha mRNA level to below 5% of controls, i.e. opposite to the known response in the mouse. The level of mRNA for GST Mu was also decreased, but the mRNAs of c-jun and jun-B were elevated after 2 h. The decrease of GST alpha mRNAs was inhibited by staurosporine, suggesting an involvement of protein kinase C. Inhibition of transcription and translation by actinomycin D and cycloheximide also partially inhibited the effect of TPA on the expression of GST alpha. In the presence of actinomycin D, GST alpha mRNA halflife was 14.5 h, compared to 3.5 h in the presence of TPA. The calcium ionophore A23187 caused a loss of GST alpha mRNAs to levels almost as low as those obtained with TPA. The effects of TPA and the calcium ionophore were also observed in CaCo2 colon carcinoma cells. As a consequence of the decrease of mRNA levels, GST alpha protein levels and total GST enzyme activity were also diminished. Also, the morphology of the cells was changed after 3 h exposure to TPA. These data suggest that human GST alpha expression can be regulated at the level of mRNA stability by a pathway involving protein kinase C.

The phorbol ester TPA markedly enhances the binding of calcium to the regulatory domain of protein kinase C beta 1 in the presence of phosphatidylserine.

Activation of protein kinase enzyme activity by Ca2+ and diacylglycerol or phorbol esters is a feature of certain isoforms of protein kinase C (PKC). Although the binding sites of phorbol ester on the regulatory domain of PKC have been extensively studied, little is known about the actual mechanisms of Ca2+ binding and how this leads to enzyme activation. We previously reported that high affinity binding of 45Ca2+ to the regulatory domain of PKC beta 1, expressed as a GST fusion protein in Escherichia coli, is dependent on the presence of phosphatidylserine (PS) or 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study we have used this system to further analyze Ca2+ binding. Using various deletions, we found that different domains in the regulatory domain of PKC beta 1 are involved in TPA-induced Ca2+ binding, depending on whether or not PS was also present in the binding assay. In addition, Ca2+ binding in the presence of TPA alone displayed very different kinetics than Ca2+ binding in the presence of TPA and PS. Scatchard analysis indicated that in the presence of TPA, the Kd value for Ca2+ binding was 51.9 microM. However, in the presence of both TPA and PS, the Kd value dropped to 0.23 microM. These results provide direct evidence that TPA activates certain isoforms of PKC by enhancing PS-dependent Ca2+ binding, thus decreasing the Kd value for Ca2+ binding to a physiological level.

Regulation by phorbol esters of the glycine transporter (GLYT1) in glioblastoma cells

The high-affinity glycine transporter in neurons and glial cells is the primary means of inactivating synaptic glycine. The effects of 12- O-tetradecanoylphorbol ester (IPA), a potent activator of protein kinase C (PKC), on the high-affinity Na +-dependent glycine transport were investigated in C6 cells, a cell line of glial origin. Incubation of C6 cells with TPA led to concentration- and time-dependent decrease in the glycine transport that could be completely suppressed by the addition of the PKC inhibitor staurosporine. The TPA effect could be mimicked by oleoylacetylglycerol and exogenous phospholipase C. Northern and Western blot analysis indicate that C6 cells express the GLYTI glycine transporter. Incubation of COS cells transiently transfected with a full-length clone of the GLYT1 transporter in the presence of TPA, produces a decrease in glycine uptake.

Glycosaminoglycans enhance phorbol ester-induced proteolytic activity and angiogenesis in vitro.

It has been reported that endothelial cells suspended in three-dimensional type I collagen gels can be induced to undergo tube formation by 12-o-tetradecanoyl phorbol 13-acetate (TPA). In this report, we show that TPA-induced endothelial cell tube formation can be further enhanced by the addition of other matrix components in the collagen gels. In the presence of TPA, both high molecular weight hyaluronate and chondroitin sulfate elicit a dose-dependent stimulation of tube formation. The enhanced tube formation appears to be due to an increase in the number of cells undergoing morphogenesis as the average length per tube is not obviously increased. Concomitant with the increased cell morphogenesis, there is an increase in proteolytic activity secreted by the cells. Treatment of cells with cycloheximide suppresses hyaluronate- and chondroitin sulfate-enhanced cell morphogenesis and proteolytic activity suggesting that new protein synthesis, perhaps proteases, is necessary for endothelial cell morphogenesis. The possible role of the production of proteolytic activity in endothelial cell tube formation is discussed.

Activation of the ζ-subspecies of PKC in the normal human melanocytes grown in the presence of TPA

Activation of the ζ-subspecies of PKC in the normal human melanocytes grown in the presence of TPA

Influence of protein kinase C activation by 4β-phorbol ester or 1-oleoyl-2-acetylglycerol on disaturated phosphatidylcholine synthesis and secretion, and protein phosphorylation in differentiating fetal rabbit type II alveolar cells

Undifferentiated type II alveolar cells were isolated from the fetal rabbit lung on the 24th gestational day, grown in vitro for 2–3 days, and used to test the hypothesis that activation of protein kinase C by 4β-phorbol ester (TPA) or the diacylglycerol analogue, sn-1-oleoyl-2-acetylglycerol (OAG), stimulates disaturated phosphatidylcholine (DSPC) synthesis and secretion. To measure secretion, cells were prelabelled with [ 3H]choline in serum-free medium or medium with 10% carbon-stripped fetal bovine serum for 24 h. The radiolabel was removed and TPA (10 −6–10 −9 M) or OAG (125, 250 or 500 μM) was incubated with the cells for 2 h. The medium was removed and filtered. Fresh medium with the same compound was added for an additional 16 h. To measure synthesis, cells were incubated with [ 3H]choline and concurrently TPA or OAG was added. Cells were removed at 2 or 18 h. After 2 h at concentrations of 10 −8 M, TPA augmented the release of 3H-labelled DSPC, the major component of the surfactant, by cells incubated in serum-free medium. In the presence of carbon-stripped fetal bovine serum, TPA (10 −7 and 10 −6 M) induced release of DSPC. The incorporation of [ 3H]choline into intracellular DSPC was increased after 2 or 18 h in fetal alveolar cells exposed to TPA at 10 −9 M or higher. OAG also significantly stimulated the release of labelled DSPC after 2 h at all concentrations tested. In contrast, OAG-exposed cells displayed a reduction of [ 3H]choline incorporation into cellular DSPC. Characterization of radioactive material released by prelabelled fetal type II cells showed that phorbol ester stimulation increased the release of material which co-migrated with adult rabbit lung lamellar bodies on a sucrose gradient. Electrophoretic examination of [γ- 32P]ATP phosphorylation sites in fetal type II cells showed that TPA and OAG induced an increase in phosphorylation of a group of proteins with apparent molecular masses of 45, 50 and 55 kDa. Addition of phosphatidylserine to the incubations produced substantial increases in the phosphorylation of these proteins, particularly in the presence of TPA. Fetal type II cells also displayed a phosphorylation product with an apparent molecular mass of 97 kDa. This protein as well as two high-molecular-mass products appeared to be particular to cells incubated with TPA plus phosphatidylserine and may in part account for the different action of TPA compared to OAG with regard to synthesis and secretion of DSPC by the fetal type II cells.

Differential susceptibility of morphologically TPA-resistant and -sensitive Balb/c 3T3 variants to TPA-induced cell transformation: relationship to induction of membrane ruffling.

To investigate the responsiveness to phorbol ester-mediated malignant cell transformation of Balb/c 3T3 variant clones that were morphologically phorbol ester-sensitive and -resistant, we used an in vitro two-stage transformation assay in which cells were treated with 0.1 microgram/ml of MCA as an initiator and subsequently with 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. The morphologically TPA-sensitive variant, TR5, and the parent cells showed a relatively low sensitivity to TPA-induced cell transformation, whereas the morphologically TPA-resistant variant, TR4 cells, exhibited 50- to 100-fold higher sensitivity to phorbol ester-induced cell transformation than the parent or the TR5 cells. We investigated the effects of TPA on protein kinase C activity, 80 kDa PKC substrate phosphorylation, the organization of actin stress fibers, DNA synthesis, and anchorage-independent growth in the three cell clones. They showed similar responses to these biological and biochemical events, indicating that these PKC-mediated events may not be the causes of the differential responsiveness of the variant cells to TPA-induced cell transformation. We further examined their responsiveness to growth factor-mediated and spontaneous induction of membrane ruffling. When these cells were stimulated by PDGF in their growing phase, membrane ruffling was rapidly induced in the three cells. However, the PDGF-mediated membrane ruffling was completely suppressed in parent and TR5 but not TR4 cells in the confluent, contact-inhibited (steady-state) growth phase. Similar responses were observed by other growth factors such as insulin, IGF-I, acidic or basic FGF. In addition, when the cells were cultured beyond confluence in the presence of TPA, spontaneous membrane ruffling was induced continuously up to termination of culture in TR4 but not in parent and TR5 cells. These results suggest that the deficiency in cell contact-mediated inhibition of membrane ruffling may be responsible for hypersensitivity of TR4 cells to TPA-induced cell transformation.

12- O-Tetradecanoylphorbol 13-Acetate Stimulates Human T-Lymphocyte Adherence to the Fibronectin RGD Domain and the Laminin IKVAV Domain

In order for T cells to exit the circulatory system, these cells must attach to extracellular matrix proteins. We have used 12- O-tetradecanoylphorbol 13-acetate (TPA) to study the ability of human T cells to adhere to fibronectin or laminin or to specific domains on these extracellular matrix proteins. Both primary human T-lymphocytes and a T-cell line (H-9) adhered and spread well on solid-phase fibronectin and laminin in the presence of TPA, with maximum activity at 3 hr of treatment. Furthermore, attachment of both cell populations to fibronectin was inhibited using a soluble RGD-containing synthetic peptide or by pretreating the fibronectin with antibodies that block the RGD domain. A synthetic peptide from the CSI alternatively spliced region of fibronectin did not inhibit attachment to fibronectin. The H-9 cells also attached to the laminin A chain IKVAV-containing synthetic peptide, but not to the laminin-derived YIGSR- or RGD-containing sequences. Immunoprecipitation of 32 P-labeled H-9 cells with antibodies to the β1 integrin subunit demonstrated phosphorylation of an α integrin subunit after treatment with TPA. These data demonstrate that TPA activates T-cell adherence to laminin and to fibronectin via specific sites on each protein and that this adhesion may be associated with integrin phosphorylation.

Regulation of Glucocorticoid Receptor (GR) mRNA and protein levels by phorbol ester in MCF-7 cells. Mechanism of GR mRNA induction and decay

Treatment of MCF-7 cells with the phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA) (10 −7 M) was associated with a time-dependent increase in specific binding of [ 3H]dexamethasone (34.8 ± 4.6 fmol/mg protein after9 h of TPA treatment compared with 16.0 ± 2.3 fmol/mg protein in control cells) as well as a transient induction in the level of glucocorticoid receptor (GR) mRNA (4- to 8-fold stimulation after 2–3 h, followed by a decline towards the control value after 6h). In the presence of the transcription inhibitor actinomycin D (AMD) 5.0 μg/ml) the TPA-dependent induction of GR mRNA was completely abolished, and GR mRNA showed a gradual decline with a half-life of 2–3 h. In contrast , treatment with TPA and the protein synthesis inhibitor cycloheximide (50 μM) resulted in a superinduction of GR mRNA ( > 50-fold after 6 h). Inhibition of transcription by AMD after 3 h of TPA treatment was associated with a decay of GR mRNA with a half-life of 2–3 h, which is identical to that observed in non-treated cells. We conclude that the increase in GR mRNA in the presence of TPA is dependent on ongoing transcription, whereas the rate by which GR transcripts are degraded, is not altered by TPA.

Modulation of murine and human interferon-γ receptor expression by their ligands or phorbol ester

IFN-γ receptor expression on murine leukaemic L1210-cells has been studied. With the help of a transfected cell-line expressing the heterologous human receptor it was possible to discern receptor-specific properties like internalization from those regulating their expression on the surface. Recombinant IFN-γ binds specifically to its homologous receptor at 4°C and is rapidly internalized at physiologic temperatures. For this effect to occur, ligand binding to its receptor at 37°C is necessary and sufficient. This notion is confirmed since a reduction in the number of heterologous human IFN-γ receptors on the murine cell surface occurred exclusively after treatment with human IFN-γ. Even weak doses of ligand, insufficient to occupy all receptors, led to a pronounced disappearance of binding sites. However, both receptors are simultaneously up-regulated in the presence of TPA, indicating a separate pathway which is not species-specific. Our findings imply that similar elements of the intracellular signal transduction machinery are involved in the control of MuIFN-γ and HuIFN-γ receptor expression. The results indicate also that factors involved in binding, internalization, and regulation of receptor gene expression are not species-specific.

Ca2+-ATPase Inhibitors Induce Interleukin-2 Synthesis and T Cell Proliferation

In Jurkat cells, the three Ca2+-ATPase blockers, thapsigargin, cyclopiazonic acid, and di-teributylhydroquinone (DtBuHQ) induced both a release of Ca2+ from intracellular stores and a Ca2+ influx. In contrast to CD3 mAb, the Ca2+-ATPase inhibitors did not induce the formation of inositol trisphosphate from the hydrolysis of phosphatidylinositides. Emptying intracellular Ca2+ stores was accompanied by a decrease of phosphatidylserine (PtdSer) synthesis as previously observed in PHA- or CD3 mAb-treated Jurkat cells. In the presence of a phorbol ester able to activate protein kinase C, TPA, the three Ca2+-ATPase inhibitors induced Jurkat cells to synthesize large amounts of interleukin-2 demonstrating that early signal transduction mechanisms can be bypassed by Ca2+-ATPase inhibitors. In purified human peripheral blood T lymphocytes, the same inhibitors induced moderate if any cytosolic Ca2+ rise, in the absence of external calcium. Nevertheless analysis of PtdSer synthesis suggested that intracellular stores were efficiently depleted by DtBuHQ and cyclopiazonic acid but not by thapsigargin. In contrast, the three compounds induced similar Ca2+ influx. However, in the presence of TPA, cyclopiazonic acid and DtBuHQ induce highly purified T cells to proliferate while thapsigargin did not, suggesting that the status of internal Ca2+ store may have a decisive role in T cell activation.