Karl Maly

Protein Kinase C θ Affects Ca2+ Mobilization and NFAT Activation in Primary Mouse T Cells

Protein kinase C (PKC)θ is an established component of the immunological synapse and has been implicated in the control of AP-1 and NF-κB. To study the physiological function of PKCθ, we used gene targeting to generate a PKCθ null allele in mice. Consistently, interleukin 2 production and T cell proliferative responses were strongly reduced in PKCθ-deficient T cells. Surprisingly, however, we demonstrate that after CD3/CD28 engagement, deficiency of PKCθ primarily abrogates NFAT transactivation. In contrast, NF-κB activation was only partially reduced. This NFAT transactivation defect appears to be secondary to reduced inositol 1,4,5-trisphosphate generation and intracellular Ca2+ mobilization. Our finding suggests that PKCθ plays a critical and nonredundant role in T cell receptor–induced NFAT activation.

Critical Role of Calcium- dependent Epidermal Growth Factor Receptor Transactivation in PC12 Cell Membrane Depolarization and Bradykinin Signaling

PC12 cells respond to a variety of external stimuli such as growth factors, neurotransmitters, and membrane depolarization by activating the Ras/mitogen-activated protein kinase pathway. Here we demonstrate that both depolarization-induced calcium influx and treatment with bradykinin stimulate tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Using a tetracycline-controlled expression system in conjunction with a dominant-negative EGFR mutant, we demonstrate that depolarization and bradykinin triggered signals involve EGFR function upstream of SHC and MAP kinase. Furthermore, bradykinin-stimulated EGFR transactivation is critically dependent on the presence of extracellular calcium, and when triggered by ionophore treatment, calcium influx is already sufficient to induce EGFR tyrosine phosphorylation. Taken together, our results establish calcium-dependent EGFR transactivation as a signaling mechanism mediating activation of the Ras/mitogen-activated protein kinase pathway in neuronal cell types.

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Ficks law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

Altered cell volume regulation in ras oncogene expressing NIH fibroblasts

Expression of the Ha-ras oncogene has been reported to stimulate the dimethylamiloride sensitive Na+/H+ exchanger and Na+, K+, 2Cl− cotransport, both transport systems which are involved in cell volume regulation. The present study has been performed to test for an influence of ras oncogene expression on cell volume regulation in NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ ras). As controls served NIH 3T3 fibroblasts not expressing the ras oncogene (− ras). In isotonic extracellular fluid, the cell volume of + ras cells (2.70±0.08 pl) is significantly greater than the cell volume of −ras cells (2.04±0.10 pl). Both, + ras and − ras cells exhibit a regulatory cell volume increase in hypertonic extracellular fluid and a regulatory cell volume decrease in hypotonic extracellular fluid. The regulatory cell volume decrease is inhibited by 1 mmol/l quinidine and barium, the regulatory cell volume increase is inhibited in − ras and + ras cells by dimethyl-amiloride (100 μmol/l) and, only in + ras cells, by furosemide (100 μmol/l) and bumetanide (10 μmol/l). In conclusion, expression of the ras oncogene leads to a shift of the set point for cell volume regulation to greater cell volumes, which may contribute to the activation of the Na+/H+ exchanger and Na+, K+, 2Cl− cotransport.

The effect of hypoosmolarity on the electrical properties of Madin Darby canine kidney cells

The present study has been performed to test for the effect of hypotonic extracellular fluid on the electrical properties of Madin Darby canine kidney (MDCK)-cells. The volume of suspended MDCK-cell is 1,892±16 fl (n=8) in isotonic (298.7 mosmol/l) extracellular fluid. Exposure of the cells to hypotonic (230.7 mosmol/l) extracellular fluid is followed by cellular swelling to 2,269±18 fl (n=4) and subsequent volume regulatory decrease to 2,052±22 fl (n=4) within 512 s. Volume regulatory decrease is abolished by quinidine (1 mmol/l) and by lipoxygenase inhibitor nordihydroguaiaretic acid (50 μmol/l). The potential difference across the cell membrane averages −53.6±0.9 mV (n=49) in isotonic extracellular perfusates. Reduction of extracellular osmolarity depolarizes the cell membrane by +25.7±0.8 mV (n=67), reduces the apparent potassium selectivity of the cell membrane, from 0.55±0.07 (n=9) to 0.09±0.01 (n=26), and increases the apparent chloride selectivity from close to zero to 0.34±0.02 (n=21). Potassium channel blocker barium (1 mmol/l) depolarizes the cell membrane by +15.2±1.1 mV (n=13). In the presence of barium, reduction of extracellular osmolarity leads to a further depolarization by +14.0±1.4 mV (n=12). Addition of chloride channel blocker anthracene-9-COOH (1 mmol/l) leads to a hyperpolarization of the cell membrane by −6.7±2.2 mV (n=11). In the presence of anthracene-9-COOH, reduction of the extracellular osmolarity leads to a depolarization by +22.4±1.7 mV (n=11). Application of 1 mmol/l quinidine depolarizes the cell membrane to −6.6±0.5 mV (n=8) and virtually abolishes the effect of reduced extracellular osmolarity on cell membrane potential. Nordihydroguaiaretic acid (50 μmol/l), a substance known to inhibit lipoxygenase, increases steady state cell membrane potential in isotonic extracellular fluid to −58.8±1.8 mV (n=10) and blunts the depolarizing effect of hypotonic extracellular fluid (+5.4±1.5 mV,n=10). In conclusion, exposure of MDCK-cells to hypotonic media depolarizes the cell membrane by activation of a conductive pathway, which is insensitive to both barium and anthracene-9-COOH. The conductive pathway is possibly activated by leucotrienes.

Synergistic enhancement of the antiproliferative activity ofcis-diamminedichloroplatinum(II) by the ether lipid analogue BM41440, an inhibitor of protein kinase C

The new phospholipid analogue 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine inhibits the phospholipid-calcium-dependent protein kinase, partially purified from Walker carcinoma cells with a Ki value of 0.56 μM. The compound inhibits the phorbol ester stimulated phosphorylation of the ribosomal protein S6 indicating that the depression of Ca2+-phospholipid-dependent protein kinase by the alkyl phospholipid also occurs in intact cells. The dose effect curve for the inhibition of cell proliferation by 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine in Walker cells exhibits a close correlation to the dose effect curve for the depression of Ca2+-phospholipid-dependent protein kinase activity. Although alternative mechanisms cannot be excluded, the data suggest that the growth inhibitory activity of 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine correlates with the inhibition of Ca2+-phospholipid-dependent protein kinase. The antiproliferative activity of 3-hexadecylmercapto-2-methoxymethyl-propyl-1-phosphocholine is synergistically enhanced bycis-diamminedichloroplatinum(II).

Critical role of protein kinase C α and calcium in growth factor induced activation of the Na+/H+ exchanger NHE1

The ubiquitously expressed Na+/H+ exchanger (NHE1) plays an important role in the regulation of the intracellular pH. Induction of NHE activity by phorbol esters and inhibition of growth factor‐mediated stimulation of the NHE by protein kinase C (PKC) inhibitors suggest an implication of PKCs in the regulation of the NHE. Expression of PKC isotype‐specific dominant negative and constitutively active mutants or downregulation of PKC by isotype‐specific antisense oligonucleotides revealed that stimulation by epidermal growth factor (EGF) or phorbol ester of the NHE in NIH3T3 cells is a PKCα‐specific effect. Elevation of cytoplasmic calcium by a Ca2+ ionophore or thapsigargin causes a growth factor‐independent stimulation of the NHE predominantly mediated by calcium/calmodulin kinase II. It is concluded that in NIH3T3 cells overexpressing the EGF receptor (EGFR6 cells), EGF requires cPKCα for the activation of the NHE, while calcium/calmodulin‐dependent kinases are essential in thapsigargin induced stimulation of the NHE.

Differential Ca2+ Signaling Induced by Activation of the Epidermal Growth Factor and Nerve Growth Factor Receptors

Stimulation by epidermal growth factor (EGF) of NIH3T3 cells overexpressing the EGF receptor (EGFR) results in a release of Ca2+ from internal stores. Ca2+ release is followed by an influx of extracellular calcium which can be recorded by the influx of the calcium surrogate Mn2+. Both Ca2+ release and Mn2+/Ca2+ influx are inhibited by expression of the dominant negative Asn17-Ras mutant and abrogated by microinjected neutralizing anti-Ras antibody Y13-259, whereas microinjection of the anti-Ras antibody Y13-238 which does not interact with the effector binding domain of Ras is without any effect on the EGF-induced Ca2+ transient. Neither Asn17-Ha-Ras nor the Y13-259 antibody interferes with the thapsigargin-induced Mn2+/Ca2+ influx. The nerve growth factor receptor (Trk)-mediated Ca2+ transient was found to be unaffected by the dominant negative Ras mutant or microinjected neutralizing anti-Ras antibodies. Substitution of the phospholipase Cγ1 (PLCγ1) binding site of the EGFR by the PLCγ binding domain of Trk renders the EGFR-induced Ca2+ influx insensitive to the expression of Asn17-Ha-Ras, whereas the Ca2+ signal induced by Trk carrying the PLC binding site of EGFR is Ras-dependent and abrogated by the dominant negative Ras mutant. It is concluded that the Ca2+ transient induced by the activated EGFR, not, however, the Ca2+ transient elicited by the activated NGFR/Trk, is a Ras-mediated phenomenon and that the role of Ras in regulating EGFR-induced Ca2+ influx depends on the structure of the PLCγ binding domain.

Constituents of autocrine IL-6 loops in myeloma cell lines and their targeting for suppression of neoplastic growth by antibody strategies

We examined the constitution and biological relevance of an autocrine IL‐6/IL‐6‐receptor (r) loop in 7 multiple myeloma and plasma‐cell leukemia lines in order to determine its biological role and potential therapeutic impact on antibody strategies. The expression and constitution of the IL‐6r [i.e. membrane‐bound gp‐80, soluble (s)gp‐55 and the gp‐130 IL‐6 signal‐transducing element (str)], the binding capacity of the membrane‐associated receptor(s) for IL‐6, the production and secretion of IL‐6 by neoplastic plasma cells, and the effect of IL‐6 on tumor‐cell proliferation were investigated. In the U‐266 cell line, the growth‐inhibitory effects of antibodies (Abs) against IL‐6 and the IL‐6‐binding subunit of its receptor were compared with each other. From our results the following conclusions may be drawn: (i) Substantial differences in the quantificative assembly of the IL‐6r constituents and in the response to recombinant (r) human (h) IL‐6 became evident in the 7 myeloma cell lines. (ii) The components of an autocrine IL‐6 loop may be regulated in an independent and, in the case of IL‐6 and sgp‐55, probably counteractive manner. (iii) The level of endogenous IL‐6 and the reservoir of recruitable sgp‐55 were important for the response to exogenous rhIL‐6. (iv) Apart from IL‐6, other growth factors are important for the propagation of myeloma cells but at least some of them exert their effect through an IL‐6‐dependent pathway. Their growth‐promoting activity, as well as that of IL‐6, may be successfully targeted by immunological means, with Abs against the IL‐6r being more efficient than those against the ligand.

Role of the Na+/H+ antiport in the regulation of the internal pH of Ehrlich ascites tumor cells in culture

SummaryEhrlich ascites tumor cells contain a Na+ uptake system, which is activated by internal protons and is inhibited by amiloride with an IC50 of 25 μm and by dimethylamiloride with an IC50 of 0.6 μm at 1mm external Na+. Decrease of external Na+ or addition of amiloride is followed by a decrease of internal pH. Taken together, these findings suggest the presence of an operative Na+/H+ antiport system, which is involved in the regulation of internal pH. We cannot find a significant contribution of a proton pump activated by glycolysis to the pH gradient. At an external pH between 7.0 and 7.6, quiescent cells are more alkaline than exponentially growing cells (0.1 to 0.17 units). Accordingly, an increase of the affinity of the Na+/H+ antiport for internal protons in quiescent cells is demonstrated by the following findings: 1. The internal pH, at which the half-maximal activation of the amiloride-sensitive Na+ uptake occurs, is shifted from 6.85 to 7.1 at 1mm external Na+. 2. The threshold value of external pH, below which a pronounced effect of amiloride on steadystate internal pH is observed, is shifted from 7.0 in growing to 7.5 in quiescent cells at physiological Na+ concentrations. Therefore, we conclude that quiescent Ehrlich ascites tumor cells raise their internal pH by increasing the affinity of their Na+/H+ antiporter to internal protons. The Na+/H+ antiport cannot be activated further by addition of serum growth factors to quiescent cells. All experiments were performed at bicarbonate concentrations in the medium which do not exceed 0.5mm. The data are discussed in view of existing models of mitogenic activity of transitory pH changes.