Receptor In HL-60 Cells Research Articles

Genetic and functional modulation by agonist MRS5698 and allosteric enhancer LUF6000 at the native A3 adenosine receptor in HL-60 cells

The A3 adenosine receptor (AR) is an important inflammatory and immunological target. However, the underlying mechanisms are not fully understood. Here, we report the gene regulation in HL-60 cells treated acutely with highly selective A3AR agonist MRS5698, positive allosteric modulator (PAM) LUF6000, or both. Both pro- and anti-inflammatory genes, such as IL-1a, IL-1β, and NFκBIZ, are significantly upregulated. During our observations, LUF6000 alone produced a lesser effect, while the MRS5698 + LUF6000 group demonstrated generally greater effects than MRS5698 alone, consistent with allosteric enhancement. The number of genes up- and down-regulated are similar. Pathway analysis highlighted the critical involvement of signaling molecules, including IL-6 and IL-17. Important upstream regulators include IL-1a, IL-1β, TNF-α, NF-κB, etc. PPAR, which modulates eicosanoid metabolism, was highly downregulated by the A3AR agonist. Considering previous pharmacological results and mathematical modeling, LUF6000’s small enhancement of genetic upregulation suggested that MRS5698 is a nearly full agonist, which we demonstrated in both cAMP and calcium assays. The smaller effect of LUF6000 on MRS5698 in comparison to its effect on Cl-IB-MECA was shown in both HL-60 cells endogenously expressing the human (h) A3AR and in recombinant hA3AR-expressing CHO cells, consistent with its HL-60 cell genetic regulation patterns. In summary, by using both selective agonists and PAM, we identified genes that are closely relevant to immunity and inflammation to be regulated by A3AR in differentiated HL-60 cells, a cell model of neutrophil function. In addition, we demonstrated the previously uncharacterized allosteric signaling-enhancing effect of LUF6000 in cells endogenously expressing the hA3AR.

18α-Glycyrrhetinic Acid Induces Apoptosis of HL-60 Human Leukemia Cells through Caspases- and Mitochondria-Dependent Signaling Pathways.

In this study we investigate the molecular mechanisms of caspases and mitochondria in the extrinsic and intrinsic signal apoptosis pathways in human leukemia HL-60 cells after in vitro exposure to 18α-glycyrrhetinic acid (18α-GA). Cells were exposed to 18α-GA at various concentrations for various time periods and were harvested for flow cytometry total viable cell and apoptotic cell death measurements. Cells treated with 18α-GA significantly inhibited cell proliferation and induced cell apoptosis in a dose-dependent manner, with an IC50 value of 100 μM at 48 h. The cell growth inhibition resulted in induction of apoptosis and decreased the mitochondria membrane potential (ΔΨm) and increased caspase-8, -9 and -3 activities. Furthermore, cytochrome c and AIF were released from mitochondria, as shown by western blotting and confirmed by confocal laser microscopy. Western blotting showed that 18α-GA increased the levels of pro-apoptotic proteins such as Bax and Bid and decreased the anti-apoptotic proteins such as Bcl-2 and Bcl-xl, furthermore, results also showed that 18α-GA increased Fas and Fas-L which are associated with surface death receptor in HL-60 cells. Based on those observations, the present study supports the hypothesis that 18α-GA-induced apoptosis in HL-60 cells involves the activation of the both extrinsic and intrinsic apoptotic pathways.

Expression of mRNA for Adenosine A1, A2a, A2b, and A3 Receptors in HL-60 Cells: Dependence on Cell Cycle Phases

The present studies investigated changes in expression of mRNA for adenosine A(1), A(2a), A(2b), and A(3) receptors in samples of HL-60 promyelocytic cells differing in the actual presence of cells in various phases of the cell cycle induced by the double thymidine block method. Real-time PCR technique was used for obtaining data on mRNA expression. Statistical analysis of the data revealed that the mRNA expression of adenosine A(1), A(2a), and A(3) receptors is dependent on the cell cycle phase. G(0)/G(1) and G(2)/M phases were characterized by a higher mRNA expression of adenosine A(1) receptors and a lower one of adenosine A(2a) and A(3) receptors whereas the opposite was true for the S phase. Interestingly, expression of mRNA of the adenosine A(2b) receptors was independent on the cell cycle phase. The results indicate the plasticity of mRNA expression of adenosine receptors in the investigated promyelocytic cells and its interaction with physiological mechanisms of the cell cycle.

Isoliquiritigenin Selectively Inhibits H2Histamine Receptor Signaling

Isoliquiritigenin, one of the major constituents of Glycyrrhiza uralensis (licorice), is a natural pigment with a simple chalcone structure 4,2',4'-trihydroxychalcone. In this study, isoliquiritigenin showed selective H(2) histamine receptor (H(2)R) antagonistic effect and remarkably reduced several H(2)R-mediated physiological responses. Preincubation of U937 and HL60 hematopoietic cells with isoliquiritigenin significantly inhibited H(2)R agonist-induced cAMP response in a concentration-dependent manner without affecting the viability of cells. Isoliquiritigenin also blocked the binding affinity of [(3)H]tiotidine to membrane receptors in HL-60 cells. Isoliquiritigenin did not affect the elevation of cAMP levels induced by cholera toxin, forskolin, or isoproterenol, indicating that the action site of isoliquiritigenin is not G(s) protein, effector enzyme, adenylyl cyclase, or beta(2)-adrenoceptor. Isoliquiritigenin affected neither H(1)R-nor H(3)R-mediated signaling. In molecular docking studies, isoliquiritigenin exhibited more favorable interactions with H(2)R than histamine. Isoliquiritigenin prominently inhibited H(2)R selective agonist dimaprit-induced cAMP generation in MKN-45 gastric cancer cell. Moreover, isoliquiritigenin reduced gastric acid secretion and protected gastric mucosal lesion formation in pylorus-ligated rat model. Taken together, the results demonstrate that isoliquiritigenin is an effective H(2)R antagonist and provides the basis for designing novel H(2)R antagonist.

Humic acid induces the expression of ox-LDL receptor in HL-60 cells through activation of PPAR-gamma.

Humic acid induces the expression of ox-LDL receptor in HL-60 cells through activation of PPAR-gamma.

Gene expression of cytokine receptors in HL60 cells exposed to a 50 Hz magnetic field.

The effects of a 50 Hz extremely low frequency (ELF) sinusoidal magnetic field (MF) on the expression of genes relating to cytokine receptors were studied in HL60 cells. Transcription levels of tumor necrosis factor receptor (TNFR) p55 and p75, interleukin-6 receptor-alpha (IL-6Ralpha) and transforming growth factor-beta receptor 1 (TGFbetaR1) were quantified in cells exposed to an intensity of 0.1 or 0.8 mT for periods ranging from 30 min to 72 h. Cells treated with 10 nM of phorbol 12-myristate 13-acetate (PMA) for 8 h served as a positive control. Gene expression values were assessed by the ribonuclease protection assay (RPA) and normalized to those of the noninducible gene GAPDH. The results showed that MF exposure at 0.1 and 0.8 mT for 72 h increased TNFR p75 and IL-6Ralpha mRNA expression in HL60 cells. No significant change in gene expression levels of TNFR p55 and TGFbetaR1 was observed under any of the exposure conditions. In addition, we report here for the first time that IL-6Ralpha mRNA expression can be suppressed by PMA in HL60 cells.

Stimulation of sphingosine-1-phosphate formation by the P2Y(2) receptor in HL-60 cells: Ca(2+) requirement and implication in receptor-mediated Ca(2+) mobilization, but not MAP kinase activation.

Sphingosine-1-phosphate (SPP), produced by sphingosine kinase, has recently been reported to act as an intracellular second messenger for Ca(2+) and mitogenic responses triggered by membrane receptors and as an extracellular ligand for specific SPP receptors. Here, we investigated the signaling pathway leading to SPP production by the G protein-coupled P2Y(2) receptor and its functional implication in human leukemia (HL-60) cells, which do not respond to extracellular SPP. P2Y(2) receptor activation by UTP or ATP resulted in rapid and transient production of SPP, which was insensitive to pertussis toxin and blocked by the sphingosine kinase inhibitor, DL-threo-dihydrosphingosine. Treatment of HL-60 cells with this inhibitor did not affect activation of mitogen-activated protein kinases, but suppressed Ca(2+) mobilization by the P2Y(2) receptor. However, receptor-induced SPP production apparently required an increase in intracellular Ca(2+) concentration, but not Ca(2+) influx, and was mimicked by exposure of cells to Ca(2+) ionophores. Taken together, activation of the P2Y(2) receptor stimulates SPP production in HL-60 cells, a process apparently not required for mitogen-activated protein kinase activation, but most likely representing an amplification system for receptor-mediated Ca(2+) signaling.

Leukotriene receptors in HL-60 cells differentiated into eosinophils, monocytes and neutrophils

The expression of leukotriene B 4 (LTB 4) and leukotriene D 4 (LTD 4) receptors was determined, by binding assay, in HL-60 cells differentiated into the monocyte/macrophage, neutrophil, and eosinophil lineages. Monocyte/macrophage- and neutrophil-differentiated cells developed specific LTB 4 receptors with high affinities ( K d = 1.27 nM and 2.65 nM, respectively) and low affinities ( K d = 26.41 nM and 55.63 nM, respectively). These receptors were functional and specific as indicated by the ability of LTB 4 to elicit an increase in intracellular calcium concentration antagonised by specific antagonists. Eosinophil-differentiated cells developed mainly LTD 4 receptors ( K d = 41.91 nM), and stimulation with LTD 4 induced an increase in intracellular calcium that was antagonised by a specific LTD 4 antagonist. These results show, for the first time, that eosinophil-differentiated HL-60 cells express specific functional LTD 4 receptors. These cells could be used for the study of the actions of peptidoleukotrienes on eosinophils, and for studies on the molecular mechanisms regulating LTD 4 receptor expression.

Induction of Functional Lipoxin A4 Receptors in HL-60 Cells

AbstractThe appearance of [11,12-3H]lipoxin A4 (LXA4) specific binding sites was examined with human acute promyelocytic leukemic cell line 60 (HL-60) cells exposed to either retinoic acid, phorbol 12-myristate 13-acetate (PMA), or dimethyl sulfoxide (DMSO). All three agents induced a threefold to fivefold increase in the expression of specific [11,12-3H]LXA4 binding. Similar results were obtained in parallel with [14,15-3H]leukotriene (LT) B4. For both 3H-ligands, homologous displacement curves were similar and independent of the agent used to induce differentiation. Specific binding of [11,12-3H]LXA4 to differentiated HL-60 cells gave a kd = 0.6 ± 0.3 nmol/L. The appearance of both [11,12-3H]LXA4 and [14,15-3H]LTB4-specific binding sites was inhibited by actinomycin D, and LXA4 binding was sensitive to protease treatment. Specific binding of [11,12-3H]LXA4 was not evident with human platelets, red blood cells (RBCs) or the cultured B-cell (Raji), T-cell (Jurkat) lines save human endothelial cells (kd = 11.0 ± 0.3 nmol/L). The structural specificity of induced [11,12-3H]-LXA4 recognition sites was assessed with LXB4, LTC4, LTB4, and trihydroxyheptanoic methyl ester. Only LTC4, at 3-log molar excess, competed for 3H-LXA4-specific binding with HL-60 cells and gave a 30% reduction. The leukotriene D4 receptor antagonist SKF 104353 was ineffective in blocking [11,12-3H]LXA4-specific binding with HL-60 cells while it competed for specific [11,12-3H]LXA4 binding with endothelial cells where LTD4 binding appears to be virtually identical to that of LXA4 binding. In addition, the LTB4 receptor antagonist ONO 4057 was ineffective at competing for [11,12-3H]LXA4 binding. When phospholipase D activation was monitored in human polymorphonuclear leukocytes (PMN) and HL-60 cells, a correlation was shown between activation and specific 3H-LXA4 binding. LXA4-induced phospholipase D (PLD) activation gave a biphasic concentration-dependent response comprised of at least two components: one phase being islet-activating protein (IAPI-sensitive (LXA4 10–9 mol/L peak activity) and the other was staurosporine-sensitive (LXA4 10−7 mol/L peak activity). Results indicate that HL-60 cells exposed to differentiating agents express [11,12-3H]LXA4 recognition sites also present in PMN. In addition, specific LXA4 recognition sites of myeloid cells can be distinguished by competition binding with SKF 104353 and 3H-LXA4 cross-reactivity with putative LTD4 receptors present on human endothelial cells. Moreover, they provide evidence indicating that binding of LXA4 to its recognition sites confers functional responses.

Induction of functional lipoxin A4 receptors in HL-60 cells

The appearance of [11,12–3H]lipoxin A4 (LXA4) specific binding sites was examined with human acute promyelocytic leukemic cell line 60 (HL- 60) cells exposed to either retinoic acid, phorbol 12-myristate 13- acetate (PMA), or dimethyl sulfoxide (DMSO). All three agents induced a threefold to fivefold increase in the expression of specific [11,12- 3H]LXA4 binding. Similar results were obtained in parallel with [14,15- 3H]leukotriene (LT) B4. For both 3H-ligands, homologous displacement curves were similar and independent of the agent used to induce differentiation. Specific binding of [11,12–3H]LXA4 to differentiated HL-60 cells gave a kd = 0.6 +/- 0.3 nmol/L. The appearance of both [11,12–3H]LXA4 and [14,15–3H]LTB4-specific binding sites was inhibited by actinomycin D, and LXA4 binding was sensitive to protease treatment. Specific binding of [11,12–3H]LXA4 was not evident with human platelets, red blood cells (RBCs) or the cultured B-cell (Raji), T-cell (Jurkat) lines save human endothelial cells (kd = 11.0 +/- 0.3 nmol/L). The structural specificity of induced [11,12–3H]-LXA4 recognition sites was assessed with LXB4, LTC4, LTB4, and trihydroxyhepatanoic methyl ester. Only LTC4, at 3-log molar excess, competed for 3H-LXA4-specific binding with HL-60 cells and gave a 30% reduction. The leukotriene D4 receptor antagonist SKF 104353 was ineffective in blocking [11,12- 3H]LXA4-specific binding with HL-60 cells while it competed for specific [11,12–3H]LXA4 binding with endothelial cells where LTD4 binding appears to be virtually identical to that of LXA4 binding. In addition, the LTB4 receptor antagonist ONO 4057 was ineffective at competing for [11,12–3H]LXA4 binding. When phospholipase D activation was monitored in human polymorphonuclear leukocytes (PMN) and HL-60 cells, a correlation was shown between activation and specific 3H-LXA4 binding. LXA4-induced phospholipase D (PLD) activation gave a biphasic concentration-dependent response comprised of at least two components: one phase being islet-activating protein (IAP)-sensitive (LXA4 10(-9) mol/L peak activity) and the other was staurosporine-sensitive (LXA4 10(-7) mol/L peak activity). Results indicate that HL-60 cells exposed to differentiating agents express [11,12–3H]LXA4 recognition sites also present in PMN. In addition, specific LXA4 recognition sites of myeloid cells can be distinguished by competition binding with SKF 104353 and 3H-LXA4 cross-reactivity with putative LTD4 receptors present on human endothelial cells. Moreover, they provide evidence indicating that binding of LXA4 to its recognition sites confers functional responses.

Tumor necrosis factor α stimulates sphingomyelinase through the 55 kDa receptor in HL-60 cells

Tumor necrosis factor α (TNFα) stimulated rapid (seconds) hydrolysis of sphingomyelin in HL-60 cells, formation of phosphocholine (PCho) and a decrease in choline. The response to TNFα was concentration dependent with a maximal effect at 3–10 nM. The monoclonal antibody (mAb). htr-9, which behaves as an agonist at the 55 kDa subtype of the TNF receptor, also stimulated sphingomyelin hydrolysis in intact cells. In contrast, the mAb, utr-1, which behaves as an antagonist at the 75 kDa receptor subtype, had no effect on sphingomyelin hydrolysis either on its own or in the presence of TNFα. In addition, htr-9 or TNFα stimulated hydrolysis of sphingomyelin in a membrane fraction of HL-60 cells. These results are consistent with a role of sphinpomyelin hydrolysis as an early event in the signalling mechanism or TNFα, and suggest that this pathway is activated through the 55 kDa subtype of the TNF receptor.

Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells.

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.

Expression of the types A and B tumor necrosis factor (TNF) receptors is independently regulated, and both receptors mediate activation of the transcription factor NF-kappa B. TNF alpha is not needed for induction of a biological effect via TNF receptors.

The expression and biological function of the types A and B tumor necrosis factor (TNF) receptors were studied using three cell types. SW480T, HEp2, and HL60 cells had, respectively, mainly the type A, only the type B, and roughly similar amounts of both receptors. Dibutyric cAMP treatment induced a 3-6-fold increase in the amount of the type A receptor in HL60 cells without affecting the amount of the type B receptor. Expression of both receptors can thus be regulated independently. HEp2 and human umbilical vein endothelial cells only showed the type B receptor, and expression of the type A receptor could not be induced in these cells. HL60 cells showed, upon Scatchard analysis, a single binding site for TNF alpha, and its Kd may correspond to that of the type A receptor. The approximately 7-fold lower affinity of TNF alpha binding to the type B receptor of HL60 cells was only detected after blocking all TNF alpha binding to the type A receptor. Both the types A and B receptors mediated TNF alpha-induced activation of the transcription factor NF-kappa B. The agonistic antibody htr9 to the type B receptor also activated NF-kappa B. Thus, signal transduction via the type B receptor may only require interaction with the receptor's extracellular domain.

The receptors for ATP and fMetLeuPhe are independently coupled to phospholipases C and A2 via G-protein(s). Relationship between phospholipase C and A2 activation and exocytosis in HL60 cells and human neutrophils

The relationship between phospholipase A2 and C activation and secretion was investigated in intact human neutrophils and differentiated HL60 cells. Activation by either ATP or fMetLeuPhe leads to [3H]arachidonic acid release into the external medium from prelabelled cells. This response was inhibited when the cells were pretreated with pertussis toxin. When the [3H]arachidonic acid-labelled cells were stimulated with fMetLeuPhe, ATP or Ca2+ ionophore A23187, and the lipids analysed by t.l.c., the increase in free fatty acid was accompanied by decreases in label from phosphatidylinositol and phosphatidylcholine. Moreover, incorporation of label into triacylglycerol and to a lesser extent phosphatidylethanolamine was evident. Activation of secretion was evident with ATP and fMetLeuPhe but not with A23187. The pharmacological specificity of the ATP receptor in HL60 cells was investigated by measuring secretion of beta-glucuronidase, formation of inositol phosphatases and release of [3H]arachidonic acid. External addition of ATP, UTP, ITP, adenosine 5'-[gamma-thio]triphosphate (ATP[S]), adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p), XTP, CTP, GTP, 8-bromo-ATP and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) to intact HL60 cells stimulated inositol phosphate production, but only the first five nucleotides were effective at stimulating secretion or [3H]arachidonic acid release. In human neutrophils, addition of ATP, ITP, UTP and ATP[S] also stimulated secretion from specific and azurophilic granules, and this was accompanied by increases in cytosolic Ca2+ and in [3H]arachidonic acid release. The addition of phorbol 12-myristate 13-acetate (PMA; 1 nM) prior to the addition of either fMetLeuPhe or ATP led to inhibition of phospholipase C activity. In contrast, this had no effect on phospholipase A2 activation, whilst secretion was potentiated. Phospholipase A2 activation by either agonist was dependent on an intact cell metabolism, as was secretion. It is concluded that (1) activation of phospholipase C does not always lead to activation of phospholipase A2, (2) phospholipase A2 is coupled to the receptor independently of phospholipase C via a pertussis-toxin-sensitive G-protein and (3) for secretion to take place, the receptor has to activate both phospholipases C and A2.

Evidence that activation of a common G-protein by receptors for leukotriene B4 and N-formylmethionyl-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms

Differentiated HL-60 cells were found to respond to the chemoattractants leukotriene B4 (LTB4) and N-formylmethionyl-leucyl-phenylalanine (FMLP), in a manner similar to neutrophils. Membranes of myeloid differentiated HL-60 cells were used (a) to examine the ability of LTB4 receptors to interact with a guanine-nucleotide-binding protein (G-protein), and (b) to compare this G-protein with that which is coupled to the FMLP receptor. LTB4 stimulated a dose-dependent increase in GTP hydrolysis and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) binding, demonstrating that LTB4 receptors on HL-60 cells are coupled to a G-protein. Both pertussis toxin and cholera toxin inhibited stimulation of GTPase activity and GTP[S] binding by either LTB4 or FMLP, indicating that both receptors are coupled to a G-protein containing a 40 kDa alpha-subunit. That the two receptors share a common G-protein was shown by FMLP enhancement of cholera-toxin-induced inhibition of GTPase activity stimulated by either FMLP or LTB4. However, LTB4 did not enhance cholera-toxin-induced inhibition of GTPase activity, suggesting that the receptors interacted differently with this G-protein. This difference was confirmed by showing that FMLP, but not LTB4, stimulated receptor-specific [32P]ADP-ribosylation of the 40 kDa alpha-subunit. Concentrations of LTB4 and FMLP which produced maximal responses produced enhanced stimulation in both assays. This additive effect was not abolished by inactivation of up to 80% of G-protein activity by N-ethylmaleimide or cholera toxin. We conclude that LTB4 and FMLP receptors in HL-60 cells are coupled to a common G-protein. The receptor--G-protein interaction is different for the two receptors, and G-proteins not coupled to both receptors may account for the additive response.

Uptake of retinyl ester in HL-60 cells via the low-density-lipoprotein-receptor pathway.

Newly absorbed retinol is transported in association with chylomicrons and their remnants. In addition, after intake of high doses of retinol, significant amounts are also found in low-density lipoprotein (LDL). As both chylomicron remnants and LDL may be taken up by cells via the LDL receptor, and retinoids inhibit proliferation of some leukaemic cells, we have studied the uptake of retinol in leukaemic cells via the LDL-receptor pathway. HL-60 cells contain saturable binding sites for LDL. The binding of LDL to its receptor has a dissociation constant of about 3.2 x 10(-9) M, and the number of receptors per cell was calculated to be about 2700. Uptake of 125I-LDL by HL-60 cells was increased 2-fold by preincubating the cells with mevinolin. The presence of specific receptors for LDL on HL-60 cells was further confirmed by the finding that exogenous LDL cholesterol was able to up-regulate the ACAT (acyl-CoA: cholesterol acyltransferase) activity of HL-60 cells. We then tested the uptake of retinyl ester in leukaemic cells via the LDL-receptor pathway. HL-60 cells were incubated with LDL or chylomicron remnants labelled with [3H]retinyl palmitate. Uptake of retinyl ester associated with both LDL and chylomicron remnants was observed. Furthermore, the presence of excess LDL decreased the uptake by 75-100%, supporting the hypothesis that the uptake of retinyl ester occurred via the LDL receptor in HL-60 cells.

Activation of inositol phospholipid breakdown in HL60 cells by P2-purinergic receptors for extracellular ATP. Evidence for mediation by both pertussis toxin-sensitive and pertussis toxin-insensitive mechanisms.

The mechanisms whereby P2-purinergic receptors for extracellular ATP are coupled to the inositol phospholipid-signaling system were studied in the HL60 human promyelocytic leukemia cell line. Brief pretreatment of either undifferentiated or differentiated HL60 cells with various activators of protein kinase C Ca2+/phospholipid-dependent enzyme (e.g. phorbol myristate acetate) produced a 50-fold decrease in the potency of extracellular ATP to induce mobilization of intracellular Ca2+. The ATP-induced increase in rate of inositol trisphosphate (InsP3) accumulation in these 4-beta-phorbol 12-myristate-13-acetate-treated cells was characterized by a 40% decrease in the maximal rate of InsP3 accumulation. Incubation of the cells with NaF also induced mobilization of the same Ca2+ stores released in response to extracellular ATP; this provided indirect evidence that the transmembrane signaling actions of P2-purinergic receptors may be mediated by GTP-binding regulatory proteins. This latter possibility was further supported by the finding that treatment of either undifferentiated or differentiated HL60 cells with pertussis toxin produced a significant, but partial, inhibition of ATP-induced signaling actions. These included: 1) a 60-70% decrease in the maximum rate of InsP3 accumulation, and 2) a 1.5 log unit increase in the half-maximally effective [ATP] required for mobilization of intracellular Ca2+. In cells treated with both pertussis toxin and 4-beta-phorbol 12-myristate-13-acetate, there was an 80% decrease in maximal rate of ATP-induced InsP3 accumulation and near-complete inhibition of ATP-induced Ca2+ mobilization. Significantly, the residual, pertussis toxin-insensitive portion of ATP-induced signaling was observed in the same samples of differentiated HL60 cells wherein pertussis toxin treatment produced complete abolition of InsP3 accumulation and Ca2+ mobilization in response to occupation of chemotactic peptide receptors. These results indicate that the activation of inositol phospholipid breakdown by P2-purinergic receptors in HL60 cells may be mediated by both pertussis toxin-sensitive and toxin-insensitive mechanisms; this suggests that these myeloid progenitor cells may express two distinct types of GTP-binding proteins coupled to phospholipase C.

Induction of monocytic differentiation by calcitriol (1,25-dihydroxyvitamin D 3) in the human promyelocytic leukemic cell line (HL-60) in serum-free medium

The effect of calcitriol on the induction of differentiation in human promyelocytic leukemic cell line (HL-60) cultured in serum-free chemically defined medium (SFM) was investigated. The utilization of SFM containing RPMI-1640 basal medium supplemented with insulin (5 μg/ml), transferrin (5 μg/ml), sodium selenite (5 ng/ml), and bovine serum albumin (0.5 mg/ml) allowed a more precise examination of the cellular/molecular mechanism of calcitriol's action in HL-60 cell differentiation without interference of components present in serum. HL-60 cells grown in SFM were induced to differentiate into monocytes/macrophages by calcitriol as indicated by induction of differentiation-associated biological and biochemical parameters: chemiluminescent (CL) responsiveness, lysozyme activity, nonspecific esterase, expression of cell surface antigens, and reduced proliferation. The exposure of HL-60 cells in SFM to calcitriol (from 10 −10 to 10 −8 M) resulted in dose-dependent induction of these parameters, which was similar to those obtained with cells grown in 10% fetal calf serum containing medium (10% SCM). However, calcitriol was 5-fold more potent for HL-60 cells cultured in SFM than those cultured in 10% SCM as indicated by shifts in dose-response curves for induction of CL responsiveness and lysozyme activity. The effect of calcitriol on the proliferation and acquisition of several monocyte-associated cell surface antigens was also more sensitive for HL-60 cells cultured in SFM than for cells grown in 10% SCM. We characterized and quantitated calcitriol receptors in HL-60 cells cultured in SFM in comparison to those in 10% SCM after exposing intact cells to radiolabeled calcitriol. Cells cultured in either SFM or 10% SCM exhibited calcitriol receptors that migrated at 3.4S as a single peak on sucrose gradients and elicited inherent DNA binding ability. There was essentially no difference in the apparent dissociation constants ( K d ) nor in the number of calcitriol binding sites per HL-60 cell, that is ~6.0 × 10 −11 M and ~3000 binding sites/cell respectively. It is concluded that culturing HL-60 cells in SFM results in full expression of calcitriol-induced phenotypic changes excluding the possibility that such changes result from the indirect effect of calcitriol mediated by identified and/ or unidentified components present in serum.

Regulation of HLA class II antigen expression: intracellular signaling molecules responsible for the regulation by IFN-gamma and cross-linking of Fc receptors in HL-60 cells.

The cross-linking of Fc receptors (FcR) on HL-60 cells inhibited the ability of recombinant IFN-gamma to induce HLA class II antigens. This appeared to be correlated with intracellular mRNA level. HL-60 lacked detectable HLA class II mRNA. IFN-gamma led to appearance of these transcripts, which were canceled by the cross-linking of FcR. Therefore, experiments were designed to investigate the intracellular signaling molecules regulating the appearance of HLA class II molecules or transcripts. The expression of HLA class II antigen induced by IFN-gamma was blocked by a calmodulin antagonist, W-7, but not by a protein kinase C (PKC) inhibitor, H-7. Furthermore, a direct activator of PKC, phorbol myristate acetate, was not able to induce the HLA class II antigen expression. These results suggest that IFN-gamma induces HLA class II antigens on HL-60 cells via a calcium-calmodulin pathway and not via a PKC pathway. Calmodulin is activated by a transient rise in the cytosolic free calcium. In fact, the measurement of calcium influx into HL-60 cells showed that a remarkable and time-dependent calcium accumulation was caused by IFN-gamma, and that depletion of Ca2+ from culture medium resulted in failure of IFN-gamma to induce class II antigen expression. Furthermore, calcium ionophore, A23187, by itself induced HLA class II antigen expression. These results suggest that IFN-gamma stimulates calcium influx and activates the calmodulin branch of the calcium messenger system, resulting in the induction of class II antigen expression on HL-60 cells. On the other hand, cross-linking of FcR elicited the accumulation of intracellular cAMP, which appeared to suppress the IFN-gamma-induced calcium influx, resulting in annulling HLA class II antigen-inducing activity of IFN-gamma. These intracellular events of HL-60 regulate the expression of HLA class II transcripts and molecules.

Association of phorbol ester-induced hyperphosphorylation and reversible regulation of transferrin membrane receptors in HL60 cells.

Phorbol diesters are tumor-promoting agents that cause differentiation of HL60 human leukemic cells and concomitantly alter surface transferrin-receptor expression [Rovera, G., Ferreo, D., Pagliardi, G. L., Vartikar, J., Pessano, S., Bottero, L., Abraham, S. & Lebman, D. (1982) Ann. N.Y. Acad. Sci. 397, 211-220]. Transferrin-receptor regulation is shown here to result from a rapid and reversible internalization process that is temporally associated with reversible increased phosphorylation (hyperphosphorylation) of the transferrin receptor. Such a reversible mechanism involving regulation of these surface proteins could result in the rapid generation of an early signal for HL60 cellular differentiation.