P48-triggered transmembrane signaling transduction of human monocytes: mobilization of calcium ion and activation of protein kinase c (pkc)

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P48-triggered transmembrane signaling transduction of human monocytes: mobilization of calcium ion and activation of protein kinase c (pkc)"


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ABSTRACT P48 is a cytokine which induces monocyte differentiation and the induction of cytotoxic activity. In this study, the signal transduction events involved in the stimulation of


monocytes with the membrane form of P48 (mP48) were investigated. Monocyte stimulation with mP48 was found to involve the mobilization of intracellular calcium (Ca2+) and the activation and


translocation of PKC from the cytosol to the membrane. Membane P48 induced a rapid rise of intracellular Ca2+ in a dose dependent maner. Similarly, the stimulation of monocytes with P48 was


found to involve the activation and translocation of PKC. The translocation of PKC was rapid (within 0-5 min) yet transient with PKC activity returning to control levels by 8 min. The


functional role of protein kineses in P48 induced TNF secretion was studied using various kinese inhibitors. The PKC inhibitors, H-7 and sphingosine, were found to inhibit P48 induced TNF


secretion with 50% inhibition at 5 _μ_ M. HA1004, which inhibts cyclic nucleotide-dependent kinase (PKA, Ki 1.2 _μ_M), did not inhibit TNF secretion. H-8 (PKA inhibitor) was found to be an


effective inhibitor of TNF secretion only at high concentrations(30 _μ_M). The Calmodulin-dependent kinase inhibitor, W7 (Ki 12 _μ_M) was found to be effective at concentration above 5 _μ_M.


These findings suggest that P48-triggered TNF secretion, involves transmembrane Ca2+ signaling and the subsequent activation of at least two protein kineses, PKC and CaMK. SIMILAR CONTENT


BEING VIEWED BY OTHERS IDENTIFICATION AND ELUCIDATION OF CROSS TALK BETWEEN SLAM FAMILY MEMBER 7 (SLAMF7) AND TOLL-LIKE RECEPTOR (TLR) PATHWAYS IN MONOCYTES AND MACROPHAGES Article Open


access 07 July 2023 27-HYDROXYCHOLESTEROL INDUCES EXPRESSION OF ZONULA OCCLUDENS-1 IN MONOCYTIC CELLS VIA MULTIPLE KINASES PATHWAYS Article Open access 17 May 2022 PACKAGED RELEASE AND


TARGETED DELIVERY OF CYTOKINES BY MIGRASOMES IN CIRCULATION Article Open access 09 December 2024 INTRODUCTION The differentiation of cells along the monocyte-macrophage pathway and the


signals involved in these cells acquiring the ability to kill tumor cells are not fully understood. We have been studing a molecule which appears to be an important member of the cytokine


network involved in the regulation monocyte activation. This cytokine termed P48 was isolated from the human null cell leukemia cell line Reh. It has been purified to homogeneity and found


to be distinct from interferon gamma, colony stimulating factors (CSFs) and TNF alpha and beta1, 2. Functionally, this molecule was identified by this ability to induce human leukemia cells


(HL-60) to differentiate along the monocyte-macrophage pathway and to develop cytotoxic activity1, 2. Using human monocytes, P48 was found to possess potent stimulatory activity inducing the


secretion of TNF and IL-13. Recently, we have shown TNF inducing activity in fixed Reh cells and plasma membrane from Reh cells but not K562 or P815 cells4. Furthermore, P48 was extracted


with Triton X-114, identified by immunopreciptation, and the TNF inducing activity was removed from Reh membrane preperations using an anti-P48 affinity column. Thus, we have identified P48


as an integral membrane protein, which like the secreted moleculae is a potent stimulator of TNF secretion4. Monocytes and its TNF secretion play an important role in immume and in


flammatory responses5, 6, 7, 8, 9. However, the mechnisms by which P48 induces cytokine release from monocyte are not known. The stimulation of Ca2+ influx and the activation of protein


kinase C following ligand binding with a receptor is a common signal transduction mechanism in a variety of cells10. These mediators have been found to play a major important role in the


cell growth, differentiation, gene expression and secretion1, 2, 10, 11. The interaction of ligand with receptor results in a rapid, transient hydrolysis of cell membrane phosphoinositides


that are cleaved by phospholipases C to generate 1, 2-diacylglycerol (DAG) and 1, 4, 5-trisphosphate (IP3). In turn, these mediators induce Ca2+. mobilization and stimulate the activation


and translocation of PKC. In this study, we have found that mP48 mediates a rapid elevation in intracellular Ca2+ which appears to be coupled to the activation of two independent protein


kinases. Information regarding the transmembrane signal transduction pathways of human monocytes may aid the understanding of the biochemical mechanisms which regulate TNF secretion.


MATERIALS AND METHODS MEDIA AND REAGENTS Culture medium consisted of RPMI 1640 (Cellgro, Mediatech, Washington, DC) supplemented with 2 mM glutamine (GIBCO, Grand Island, NY), 10% (V /V) of


fetal bovine serum (GIBCO), and the antibiotics: penicillin (100 U/ml) and streptomycin (100 U/ml) (GIBCO). Except where otherwise noted, all chemicals were purchansed from Sigma Chemical


Co. (St. Louis, MO). Protein kinase inhibitors (H-7, H-8, HA1004, W-7) were purchased from CALBIOCHEM (La Jolla, CA). PREPARATION OF MONOCYTES Peripheral blood mononucler cells (PBMC) were


isolated from healthy donors by centrifugation over Ficoll-Hypaque (Pharmacia, Piscataway, N J) at 600 g for 20 min. PBMC were washed 3 times, placed in 10 cm petri dishes (3 × 107/dish) in


complete medium and incubated for 60 min at 37°C in an atmosphere of 5% CO2 in air. Nonadherent cells were removed by washing (3 times) with HBSS. The adherent cells were incubated in cold


HBSS (Ca2+ , Mg2+ free, 0.2% EDTA) 20 min and the cells were recovered by gentle scrapping with a rubber policeman. The cells were washed with HBSS, and ultillized in the assays. The cells


were greater than 90% monocytes as assessed by morphology and phagocytosis of latex beads (Dow Diagnostics, IN), with a viability greater than 90%. PREPARATION OF MEMBRANE P48 The isolation


of plasma membrane containing mP48 was previously described in detail12. In brief, Reh cells were hypotonically lysed and the membranes were centrifuged at 175,000 gfor 1 h and washed twice


with 10 mM HEPES, pH 7.0. The pellet was layered onto a 35% sucrose cushion and cetrifuged at 250,000 g for 2 h. Plasma membrane banding at the interface was removed, diluted with 10 mM


HEPES (pH 7.0) and pelleted at 175,000 g for 1h. The palsma membrane pellet was resuspended at 1 mg/ml (total memranre proteins) in 10 mM HEPES (pH 7.0) stored at -70 °C prior to use in the


monocyte stimulation. Plasma membranes from the human chronic mylegenous leukemia cell line, K562, were similarly prepared as control membrane preperation. PKC ASSAY Quantitative analysis of


PKC was carried using a modification of the method of Melton and Kiley13, 14. Monocytes (3 x 106/ ml)were stimulated for various period of time at 37 °C with P48 or PMA. The cells were


suspended in 50/ _μ_l of ice-cold hypotonic lysing buffer (20 mM Tris-HCL, 2 mM EDTA, 1 mM DTT, 10 _μ_g/ml leupeptin and 1 mM PMSF, pH 7.5) and lysed by repeated, rapid aspiration through a


50 _μ_1 Hamilton syringe. Following lysis, the preparation was diluted in 1.0 ml of ice-cold lysing buffer containing 0.33 M sucrose and the cytosolic and membrane fractions were separated


by centrifugation at 100,000 g for 1 h at 4 °C. The membrane pellets were solubilized by sonication for 15 sec. in cold sample buffer containing 1% Triton ×-100 and stored overnight at 4 °C.


To partially purify PKC, the samples were centrifuged for 8 min at 12,000 rpm and the supernatant from the solubilized membranes as well as the cytosol fractions were applied to 0.6 ml


DE-52 cellulose (Whatman BioSystem Ltd., England) columns equilibrated with hypotonic buffer. Before locading the sample, each column was washed once with 2 ml of sample buffer. Partially


purified PKC was eluted from the column with 1 ml of 100 m_M_ NaCI in hypotonic buffer. PKC activity was determined by measuring the incorporation of (γ- 32p) ATP into histone type-III. The


reaction was carried out in assay buffer (20 mM HEPES, 10 mM MgC12, 0.5 mM Ca2+, 5 mM DTT, 60 _μ_g/ml phosphatidylserine (PS), 6 _μ_g/ml 1,2-diacylglycerol (DAG), and 10 mg /ml lysine-rich


histone III, pH 7.5). Basal kinase activity was measured in the presence of 2 mM EGTA instead of PS DAG, and Ca2+. Reactions were initiated by the addtion of 0.25 _μ_Ci of (γ-32p) ATP


(Specific activity, 6000 Ci/mM, NEN, Boston, MA) and carried out for 10 min at 30 °C The 32p-labeled histone III was isolated by pipetting 20 _μ_l of the reaction mixture onto Whatman P-81


phosphocellulose paper (1.5 × 1.5 cm). The papers were washed with 10 % trichloroacetic acid (TCA), dried, and enumerated by liquid scintillation counting (LS2800, Beckman). Protein was


quantitated using the BCA protein assay (Pierce, Rockford, IL). Results are given as mean ± S.D. of triplicated samples and expressed as pM of 32p incorprated/10 min/mg protein. Specific PKC


activity was caculated as the difference in kinase activity in the presence and absence of Ca2+, DAG, and PS. INTRACELLULAR CA2+ MEASUREMENTS Fura2/AM (Calbiochem, La Jolla, CA) was


prepared as a 10 m_M_ stock solution in DMSO. Monocytes (5 x 106/ml) were incubated with 5 _μ_M Fura 2/AM for 20 min at 37 °C in complete medium. The cells were washed 2 times and


resuspended at 5 x 105 /ml in assay buffer (145 m_M_ NaCl, 5 mM KCl, 1 mM Na2HPO4, 1 mM CaCl2, 0.5 m_M_ MgSO4, 5 m_M_ glucose, 10 m_M_ HEPES, pH 7.4)15. The cells were equilibrated to 37 °C


and transferred to a 1-cm quartz cuvette equipped with a stirring bar in a LS-5B Luminescence Spectrometer (Perkin-Elmer). The excitation (340 and 380 nM) and emission wave lenghts (510


n_M_) were set respectively. Baseline signal was determined by the addition of buffer (control) for 60 s before stimulating the cells, with P48 and A23187. The Ca2+ concentriation was


calculated with the software program Fura 2 (Perkin-Elmer) using a modification of the Grynkiewicz equation16, 17: [Ca2+] = (R-Rmin)/(Rmax-R) × KD × SFB where Rmin is minimum ratio (340/380)


and Rmax is maximum (both derived from the calibration data). KD is the dissociation constant of Fura2 (220). R is the fluorescence ratio at unknown[Ca2+] and SFB is the fluorescence ratio


at the baseline wavelengh (380 nM) in EGTA (40 m_M_ and 0.2% Triton X-100. TNF ASSAY Tumor necrosis factor (TNF) activity in culture supernatants from monocytes was measured as previously


reported9. The murine L929 fibroblasts (CCL 1. American Type Culture Collection, Rockville, MD) were grown to confluency in RPMI 1640 medium. Cells were gently removed using Pancreatin


(GIBCO) and washed medium. Cells genetly removed using Pancreatin (GIBCO) and washed with fresh medium. Cells (2.5 × 104/well) were seeded in 96 well flat bottom plastes (Corning, Corning,


NY) in complete medium. Following incubation for 18 h, the medium was aspirated and replaced with 100 _μ_l of medium containing 1 _μ_g/ml of actinomycin D sulfate. The monocyte supernatants


to be tested were serially diluted through 8 cells in triplicate. Human TNF-alpha (40 units/ml, interim standard, National Biological Standard Board, NCL), and medium alone were included as


positive and negative controls. The plates were incubated for 18 h at 37 °C and 5% CO2, the cells were rinsed with HBSS, and stained in 50 _μ_l of crystal violet (0.5% in 20% of methanol)


for 10 min. The stained and fixed cells were washed in water and lysed with 33% acetic acid. The uptake of crystal violet was quantitated by absorbance at 592 nM on a microtiter plate reader


(Titertk, Flow Laboratories). TNF activity in the supernatants was determined by comparisons to the TNF standard using the PARLIN program for the statistical analysis of parallel line


bioassays. RESULTS P48-INDUCED INTRACELLULAR CALCIUM MOBILIZATION Calcium mobilization by mP48 was investigated using Fura 2/AM technique. When monocytes were loaded with Fura 2 and


stimulated with mP48, a rapid rise in intracellular Ca2+ was observed. The rise in Ca2+ concentration occurred immediatly upon addtion of P48 and reached a maximum level within 1 to 2 min


and was followed by a gradual decline. The increase of intracellular calcium was not observed with the control membrane preparation isolated from K562 cells (Fig 1). As a positive control,


the ionophore A23187 (50 ng/ml) was found to increase Ca2+ to a greater extent than P48 did. The calcium antagonist, EGTA (40 mM) used to chelate media Ca2+ totally abrogated P48 and


A23187-induced Ca2+ influx in our assay system. The mP48 induced increase in intracellular Ca2+ was dose-dependent in the range of 0.1-10 _μ_g/ml of plasma membrane protein (Tab 1). Because


of a drift in the baseline flurescence over the course of the experiment, the data is presented as net increase in calcium concentration. The data show a net increase from 4-124 n_M_ of


intracellular calcium. These data suggest a role for calcium as an initial event in P48 induced intracelular signalling in monocytes. P48-INDUCED PKC ACTIVITY Next, we sought to determine


whether the increase in calcium results in the activation of PKC. Initially, we measured the subcellular distribution of PKC activity in unstimulated human monocytes and in cells stimulated


with phorbol, 12-myristate, 13-acetate ester (PMA). In unstimulated monocytes, 70 ± 13% of the PKC activity was found in the cytosol while 30 ± 11% was found in the membrane fraction. After


stimulation with PMA (100 n_M_), a significant translocation of PKC from cytosol to the membrane was observed (Fig 2). The stimulation of monocytes with PMA (100 nM) resulted in a 3.4 fold


increase of PKC activity in the membrane (1534 ± 11 to 5219 ± 177 p mol/10 min/mg protein) by 2 min. Concomitant with the increase of PKC activity in membrane, there was a decrease of PKC


activity in cytosol (802 ± 19 to 313 ± 15 p mol/min/mg protein) by 2 min. This translocation was sustained through out the 8 min time course of the experiment. Next, monocytes were


stimulated with P48 and the translocation of PKC was investigated. Fig 3 shows the P48 induced PKC redistribution in monocytes. The stimulation of monocytes with 10 _μ_g/ml of mP48 induced a


decrease cytosolic PKC activity within 2 min, whereas the PKC activity of membrane was significantly increased over the control within the first 5 min. Interestingly, P48 did not alter the


absolute levels of PKC as noted following PMA stimulation. The PKC activity in P48 stimulated monocytes returned to unstimulated levels by 8 min. EFFECT OF INHIBITORS OF PKC ON P48-INDUCED


TNF SECTION. To examine the functional role of PKC in the monocytes response to P48, the effect of protein kinase inhibitors on P48 induced TNF secretion was determined. As shown in Fig 4,


H-7 significantly inhibited mP48 induced TNF secretion in a dose-dependent fashion, compared to medium or HA1004 as a control for the isoquinoline sulfonamide derivative (Fig 4). HA1004 had


no effect on P48-induced TNF secretion event at concentration as high as 15 _μ_M. The effect of another potent PKC inhibitor, sphingosine, on P48 induced TNF secretion was examined.


Sphingosine was found to inhibit TNF secretion at similar concentrations as H-7 (Fig 5). In monocytes treated with various concentrations of H-8, a potent PKA inhibitor (Fig 6), H-8 reduced


P48-induced TNF secretion only at higher concentrations (50% inhibition at 30 _μ_ M). Our data clearly suggests the involvement of Ca2+ mobilization and Ca2+-phosphlipid-dependent PKC


activation in P48-induced TNF secretion by monocytes. In order to investigate the possibility that other calcium dependent enzymes are involved, we utilized W-7,


N-(6-aminohexyl)-5-chloro-l-naphthalene-sulfonamide hydrochloride, an inhibitor of calmodulin-dependent kinase (CaMk). When monocytes were treated with of W-7 (1-30 _μ_ _M_), P48-induced TNF


production was significantly reduced in a W7 dose-dependent fashion. The 50% inhibition of P48 induced TNF secretion by W7 was observed at 5 _μ_ _M_, with complete inhibition at 30 _μ_M


(Fig 7). These results suggest that both PKC and CaMk may be involved in the P48 induced signal transduction. DISCUSSION We have previously identified a novel cytokine involed in monocyte


differentiation and the induction of cytotoxic activity1, 2, 3, 4. We have identified P48 in the supernatant and membrane of the null cell line Reh. Both the secreted protein and the


membrane moleculae have been shown to be potent stimulators of TNF secretion by human monocytes3, 4. In the present study, we have examined the signal transduction pathways involved in P48


induced TNF secretion using the membrane form of P48. We have found that mP48 is a potent stimulator of Ca2+ mobilization and the subsequent activation and translocation of PKC activity. The


mobilization of intracellular Ca2+ and the stimulation of a Ca2+-phospholipid dependent PKC has been implicated in a common signal transduction pathway involving hydrolysis of


phosphotidylionstitol. Stimulation of this pathway by receptor ligand bining results in the rapid hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2) and subsequent generation of DAG


and IP3. These second mediators have been implicated in Ca2+ fluxes (IP3), as well as the activation of PKC. The subcellular redistribution of PKC from the cytosol or a loosely associated


membrane compartment to a more integral membrane protein has been demonstrated. This translocation event is thought to be necessary for PKC activation by Ca2+ and DAG in vivo18. The


translocation of PKC induced by P48 was found to be transient, within 0-5 min and returned to control levels by 8 min. This was found to be substantially different from the response to PMA


which was prolonged and did not return to control levels. The difference between PMA and P48 may be explained by the fact that PMK is metabolized very slowly and prolonged exposure to PMA


results in the degradation of cellular PKC19, 20. Thus it is would be expected to induce a protracted stimulation of PKC21. However, in repeated experiments using monocytes stimulated with


P48 or PMA, we found that TNF secretion was induced only by P48 and not by PMA (data not shown). Numerous investigators have reported that PMA stimulates the induction of TNF messenger RNA,


however, few reports demonstrate the secretion of TNF activity after PMA treatment. Thus, it appeares that although PMA stimulates PKC activation and TNF messenger RNA production, it dose


not represent an functional signal for secretion of the protein. In order to further investigate the functional role of PKC in P48-induced TNF secretion, we have used various kinase


inhibitors. A number of synthetic isoquinoline sulfonamide derivative are available which differentially inhibit protein kinase due to different binding affinities for the various enzymes.


H-7 is the most potent inhibitor of PKC (Ki 6 _μM_) while, H-8 and HA1004 are much less potent than H-722, 23. Sphingosine is a widely used, naturally occuring inhibitor of PKC activation24,


25, 26. The treatment of human monocytes with the PKC inhibitors, H-7 and sphingosine, blocked P48 induced TNF secretion. The PKA inhibitor, H-8 (Ki=1.2 _μ_M) was found to inhibit P48


induced TNF secretion only at higher concentrations (15-30 _μ_M). Since the Ki of H-8 for PKC is in this range27, it suggests that the inhibition we observed with H-8 is due to inhibition of


PKC rather than PKA. Additionally, HA1004 had no inhibitory activity on P48 induced TNF secretion further suggesting that PKC and not PKA is involed in P48 induced TNF secretion. An


interesting observation in our studies was that W-7, an inhibitor of calmodulin-dependent kinase, significantly blocked P48 induced TNF secretion. In six experiments, we found that W-7


inhibited P48-induced TNF production in a dose dependent manner. W-7 inhibits CaMK with a Ki=12 _μ_M, while it inhibits PKC with a Ki=110 _μ_M28. Therefore, it appears that the inhibitory


activity of W-7 is due to the inhibition of CaMK and not PKC. It has been reported that LPS induced TNF-alpha mRNA in mouse macrophages can be blocked by a PKC inhibitor, H-7, but not W-729.


One possible explanation for thr difference in our data is that they used a high dose (10 _μ_g/ ml) of LPS to treat the macrophages. In our hands, W-7 can block TNF secretion in monocytes


induced with optimal concentrations of LPS (100 ng /ml) but not in monocytes stimulated with 10 _μ_g/ml of LPS (data not shown). Thus, the inhibition of TNF secretion by W-7 was dependent on


the concentration of LPS used for inducing of TNF production, these results suggest that the biochemical pathways involved in P48 induced TNF secretion involve multiple kinase activies.


Recently, it has been suggested that transmembrane signals involved in the stimulation of cytokine secretion by monocytes may involve different pathways. Taniguchi et al., have found that


H-7 and W-7 inhibit pertussis toxin PT induced IL-1 production in human monocytes is involved the different pathways: both calmodulin and PKC-dependent processed are necessary30. On the


other hand, CaMK has been shown its functional new aspects, such as regulates a number of secretory responses31, since it phosphorylates myosin light chain (MLC-8), may also phosphorylate


other substrates that alter the activation of c-fos, c-myb, c-myc and lymphokine genes as well as their receptor genes32. The future determination of the role of PKC and CaMK for P48-induced


late events, such as the activation of the regulatory DNA-binding protein, gene expression and TNF secretion, should lead to an elucidation of P48-triggered multiple transmembrane signal


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_Immunology Today_ 1988; 9: 222–9. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The authors are grateful for the reagents and the excellent Ca2+ measurement provided by


Dr. D. Beezhold. We also thank Dr. D. Kostyol for the support of p48. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Shanghai Institute of Cell Biology, Chinese Academy of Sciences,


Shanghai, 200031, China ZL Chang & Re Hall * Dept. of Medicine, Director of Div. of Hematology /Oncology, Univ. of Tennessee Medical Center, Knoxville, 37920, TN Re Hall Authors * ZL


Chang View author publications You can also search for this author inPubMed Google Scholar * Re Hall View author publications You can also search for this author inPubMed Google Scholar


ADDITIONAL INFORMATION *Dedicated to Professor Zhen YAO's 80th Birthday. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Chang, Z., Hall, R.


P48-Triggered transmembrane signaling transduction of human monocytes: mobilization of calcium ion and activation of protein kinase C (PKC). _Cell Res_ 5, 101–114 (1995).


https://doi.org/10.1038/cr.1995.10 Download citation * Received: 20 April 1995 * Revised: 26 May 1995 * Accepted: 01 June 1995 * Issue Date: 01 June 1995 * DOI:


https://doi.org/10.1038/cr.1995.10 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently


available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * P48 monocyte differentiation inducing factor * Signal


transduction * Ca+ mobilization * PKC activation * TNF secretion


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