Hrvoje Banfić

Acetylcholine and cholecystokinin receptors functionally couple by different G-proteins to phospholipase C in pancreatic acinar cells.

We have studied the involvement of GTP‐binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin‐octapeptide (CCK‐OP) and GTPγS but not carbachol (CCh)‐induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator of adenylyl cyclase, nor the cAMP‐analogue, 8‐bromo cAMP, mimicked the inhibitory effect of cholera toxin on agonist‐induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated Gs with phospholipase C or to an elevation of cAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP‐ribosylated a 40 kDa protein, which was inhibited by CCK‐OP but not by CCh. We conclude from these data that both CCK‐ and muscarinic acetylcholine receptors functionally couple to phospholipase C by two different GTP‐binding proteins.

The role of the nuclear Akt activation and Akt inhibitors in all- trans -retinoic acid-differentiated HL-60 cells

The pharmacological inhibitors of phosphoinositide 3-kinase (PI3K)/Akt pathway have been proposed in the treatment of leukemia based on their antiproliferative effects. However, several studies demonstrated the activation of PI3K in the nuclei of all-trans-retinoic acid (ATRA) – differentiated HL-60 cells, raising the possibility that PI3K/Akt-inhibitors may block antitumor properties of retinoids. The aim of the present study was to investigate the possible activation of nuclear Akt in ATRA-treated cells and to test the effects of Akt-inhibitors on ATRA-mediated differentiation. The Akt-activity was found to be increased in the nuclei and lysates of ATRA-differentiated HL-60 and NB4 cells. The down-modulation of the expression of Akt protein in HL-60 cells using siRNA reduces the CD11b expression in ATRA-treated cells. The treatment of both cell lines with the commercially available Akt inhibitors inhibited the growth of both control and ATRA-treated cells. Akt-inhibitors had no inhibitory effects on ATRA-mediated growth arrest and the expression of CD11b in HL-60 cells, but increased the percentage of control cells expressing CD11b. In contrast, the presence of Akt inhibitors reduced the expression of CD11b in ATRA-treated NB4 cells.

Presence and activation of nuclear phosphoinositide 3-kinase C2β during compensatory liver growth.

Highly purified liver nuclei incorporated radiolabeled phosphate into phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P2, and PtdIns(3,4,5)P3. When nuclei were depleted of their membrane, no radiolabeling of PtdIns(3,4,5)P3 could be detected showing that within the intranuclear region there are no class I phosphoinositide 3-kinases (PI3K)s. In membrane-depleted nuclei harvested 20 h after partial hepatectomy, the incorporation of radiolabel into PtdIns(3)P was observed together with an increase in immunoprecipitable PI3K-C2β activity, which is sensitive to wortmannin (10 nm) and shows strong preference for PtdIns over PtdIns(4)P as a substrate. On Western blots PI3K-C2β revealed a single immunoreactive band of 180 kDa, whereas 20 h after partial hepatectomy gel shift of 18 kDa was noticed, suggesting that observed activation of enzyme is achieved by proteolysis. When intact membrane-depleted nuclei were subjected to short term (20 min) exposure to μ-calpain, similar gel shift together with an increase in PI3K-C2β activity was observed, when compared with the nuclei harvested 20 h after partial hepatectomy. Moreover, the above-mentioned gel shift and increase in PI3K-C2β activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that, in the membrane-depleted nuclei during the compensatory liver growth, there is an increase in PtdIns(3)P formation as a result of PI3K-C2β activation, which may be a calpain-mediated event.

The activation of nuclear phosphoinositide 3-kinase C2β in all-trans-retinoic acid-differentiated HL-60 cells

The activity of nuclear phosphoinositide 3‐kinase C2β (PI3K‐C2β) was investigated in HL‐60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K‐C2β was observed in the nuclei and nuclear envelopes isolated from all‐trans‐retinoic acid (ATRA)‐differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High‐performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P3 with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P2 and PtdIns(4,5)P2. Western blot analysis of the PI3K‐C2β immunoprecipitates with anti‐P‐Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K‐C2β in the nuclei and nuclear envelopes isolated from ATRA‐differentiated cells.

Nuclear phosphoinositide 3-kinase C2β activation during G2/M phase of the cell cycle in HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-calpain in vitro, the similar gel shift and increase in PI3K-C2beta activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2beta but did not prevent the gel shift. When HL-60 cells were released from G(1)/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2beta was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2beta during G(2)/M phase of the cell cycle in HL-60 cells.

Presence of different phospholipase C isoforms in the nucleus and their activation during compensatory liver growth

Phospholipase C (PLC) was purified from the membrane‐depleted rat liver nuclei. About 60% of the total PLC‐activity corresponded to β1b isoform, 30% to PLC‐γ1 and less than 10% to PLC‐δ1. PLC‐β1b and ‐γ1 were found in the nuclear matrix, while PLC‐δ1 was detected in the chromatin. Two peaks of an increase in the total PLC‐activity were detected occurring at 6 and 20 h after partial hepatectomy. An early increase in PLC‐β1b activity in the nuclear matrix was associated with serine phosphorylation of the enzyme, while the later increase paralleled the increase in the amount of protein. The increase in the PLC‐γ1 activity measured at 6 and 20 h after partial hepatectomy was associated with tyrosine phosphorylation of the enzyme. The activity of PLC‐δ1 and the amount of the protein found in the chromatin was increased only at 20 h after partial hepatectomy.

Short-term and long-term stimulation of Na+−H+ exchange in cortical brush-border membranes during compensatory growth of the rat kidney

The effect of unilateral nephrectomy on Na+−H+ exchange in rat renal cortical brush-border membrane vesicles (BBMV) was studied by the method of acridine orange fluorescence quenching. The exchanger activity in BBMV from remnant kidney increased rapidly by 70–75% within first 30 min following uninephrectomy. Only a slight further increase was found in later stages of renal growth, i.e. 30 min to 7 days following uninephrectomy. The changes in antiporter activity were restricted toVmax, whereas theKm for Na+ was similar in control and compensatory growing kidney. The increase of Na+−H+ exchange at 15 min was not affected by actinomycin D in vivo, whereas the increase at 48 h was completely abolished indicating that protein synthesis could be involved in the late, but not in the initial stimulation of renal Na+−H+ exchange. The late, but not the initial stimulations of Na+−H+ exchange were associated with elevated activities of cortical (Na++K+)-ATPase indicating that changes in antiporter activity precede those in the (Na++K+)-pump. The early stimulation of Na+−H+ exchange in BBMV in one kidney was induced also by the occlusion of blood flow through the contralateral kidney for 15 min, without removing it. Thirty min after the occlusion was removed and the reflow established, the Na+−H+ exchange in BBMV from the intact kidney decreased to the control values. The observed modulations in renal Na+−H+ exchanger may be regulated by phosphorylation-dephosphorylation events. In support, the concentration of a well known protein kinase C activator, 1,2-diacylglycerol, in the cortical tissue of the remnant kidney increased up to 100% within 5 min following unilateral nephrectomy and preceded the increase in Na+−H+ exchange. The early stimulation of Na+−H+ exchange may be a “trigger” in initiating the kidney growth.

Different roles of protein kinase C alpha and delta isoforms in the regulation of neutral sphingomyelinase activity in HL-60 cells.

The signalling mechanisms responsible for the hydrolysis of sphingomyelin mediated by 1,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)] and interferon gamma (IFN-gamma) in HL-60 cells were investigated. IFN-gamma was found to increase selectively the activity of cytosolic, Mg(2+)-independent, neutral sphingomyelinase. The treatment of HL-60 cells with the combination of 1,25(OH)(2)D(3) and IFN-gamma had an additive effect on sphingomyelin hydrolysis, ceramide release and the activity of cytosolic, Mg(2+)-independent, neutral sphingomyelinase. The pretreatment of HL-60 cells with staurosporine, chelerythrine chloride and bisindolylmaleimide abolished the activity of sphingomyelinase in response to 1,25(OH)(2)D(3) and IFN-gamma. Calphostin C, which acts on the regulatory site of protein kinase C (PKC), and Gö 6976, a selective inhibitor of Ca(2+)-dependent PKC isoforms, inhibited the effect of 1,25(OH)(2)D(3) but had no effect on the IFN-gamma-mediated increase in activity of sphingomyelinase. Isoform-specific antibodies were used to deplete different PKC isoforms from cytosol before the treatment of the cytosolic fraction with 1,25(OH)(2)D(3), arachidonic acid (AA) and PMA. The depletion of PKC isoforms beta(1), beta(2), epsilon, eta, mu, zeta and lambda had no effect on the activation of sphingomyelinase induced by 1,25(OH)(2)D(3) or by AA. The depletion of PKC alpha from the cytosol completely abolished the effect of 1,25(OH)(2)D(3) on sphingomyelinase activity but had no effect on the AA-induced activity of sphingomyelinase. PMA had no effect on the activity of sphingomyelinase in either untreated or alpha-depleted cytosol but significantly increased the activity of sphingomyelinase when added to cytosol depleted of PKC delta. Moreover, PMA inhibited the effect of 1,25(OH)(2)D(3) on sphingomyelinase activation but the inhibitory effect was abolished by prior depletion of PKC delta from the cytosol. These studies demonstrate that 1,25(OH)(2)D(3)-induced activation of sphingomyelinase is mediated by PKC alpha. Furthermore, PKC delta had an inhibitory effect on sphingomyelinase, suggesting that the difference between the 1,25(OH)(2)D(3)- and PMA-mediated effects on sphingomyelin turnover depends on the specific regulation of the PKC alpha and PKC delta isoforms.

Different endothelin receptor subtypes are involved in phospholipid signalling in the proximal tubule of rat kidney

Phospholipid signalling mediated by endothelin (ET) receptor subtypes was studied in the rat proximal tubule. In freshly isolated proximal tubule cells, ET-1, ET-2 and sarafotoxin S6c (S6c) evoked an increase in 1,2-diacylglycerol (DAG), inositol 1,4,5-trisphosphate (InsP3 and phosphocholine (PCho), suggesting stimulation of both phosphatidyl-inositol 4,5-bisphosphate- and phosphatidyl-choline-specific phospholipase C (PLC), while ET-3 increased only DAG and PCho, presumably via phosphatidyl-choline-dependent PLC. Renal cortical slices were also stimulated by the above-mentioned agonists, followed by isolation of either brush border (BBM) or basolateral (BLM) membranes for which mass measurements of inositol lipids and DAG were performed. In BBM, DAG increased in response to ET-1, ET-2 and ET-3, and was followed by protein kinase C (PKC) translocation to the BBM, while in BLM, DAG formation and translocation of PKC were observed only in response to ET-3, suggesting spatial segregation of signalling systems between two membane domains of proximal tubule cells. Tyrphostine, pertussis toxin (PTX) or cholera toxin (CTX) did not influence ET-mediated signalling in either of the membranes, suggesting involvement of PTX- and CTX-insensitive G-protein-mediated stimulation of PLCβ by ET receptors. ET-dependent stimulation of PLC in BBM and BLM was used as a tool to examine the presence of different ET receptor subtypes in these two cell membrane domains. BQ123, an inhibitor of ETA receptors, did not prevent ET-1-mediated signalling in BBM, but an ETA,B antagonist, bosentan, inhibited ET-3-mediated signalling in BBM. In addition, an ETB agonist, S6c, stimulated PLC in BBM. Neither BQ123 nor bosentan inhibited ET-3 signalling in BLM. Therefore, these data strongly suggest the presence of ETB receptors coupled to phosphatidyl-inositol 4,5-bisphosphate- and phosphatidyl-choline-dependent PLC in BBM and ETC receptors linked to phosphatidyl-choline-dependent PLC in BLM.

Combined Inhibition of PI3K and mTOR Exerts Synergistic Antiproliferative Effect, but Diminishes Differentiative Properties of Rapamycin in Acute Myeloid Leukemia Cells

A novel strategy has been suggested to enhance rapamycin-based cancer therapy through combining mammalian target of rapamycin (mTOR)-inhibitors with an inhibitor of the phosphatydilinositol 3-kinase PI3K/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. However, recent study demonstrated the potentiating effect of rapamycin on all-trans-retinoic acid (ATRA)-mediated differentiation of acute myelogenous leukemia (AML) cells, prompting us to investigate the effects of longitudinal inhibition of PI3K/Akt/mTOR signaling pathway on both proliferation and differentiative capacity of AML. In NB4, HL-60, U937 and K562 cell lines, rapamycin exerted minimal antiproliferative effects, and combining PI3K inhibitor LY 294002 and rapamycin inhibited proliferation more than LY 294002 alone. Rapamycin potentiated differentiation of ATRA-treated NB4 cells, but the combination of rapamycin and LY 294002 inhibited the expression of CD11b in both ATRA- and phorbol myristate acetate (PMA)-stimulated cells more than PI3K inhibitor alone. These results demonstrate that, although the combination of PI3K inhibitor and rapamycin is more effective in inhibiting proliferation of AML, the concomitant inhibition of PI3K and mTOR by LY 294002 and rapamycin has more inhibitory effects on ATRA-mediated differentiation than the presence of PI3K-inhibitor alone, and diminishes positive effects of rapamycin on leukemia cell differentiation.