Vladiana Crljen

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.

Insulin-like growth factor I stimulates phospholipid synthesis in renal cortical slices without production of inositol phosphates

The effects of insulin-like growth factor I (IGF-I) on the metabolism of phospholipids in renal cortical slices were examined using either sodium [ 32 P]orthophosphate or myo -[ 3 H]inositol. IGF-I was found to increase the incorporation of phosphate into phospholipids about 2–3-times above control values, leading to an increase in the concentration of total phospholipid phosphorus of 20% above control value after 1 h of incubation. The increased incorporation of phosphate into phospholipids could be prevented by 10 μM cycloheximide, while with 1 μM TPA (12- O -tetradecanoylphorbol 13-acetate) it could not. Insulin was also found to increase the incorporation of phosphate into phospholipids, but only if its concentration was at least 100-times higher than that of IGF-I. When phospholipids were prelabelled, IGF-I neither decreased the level of 32 P in phospholipids nor stimulated the formation of inositol phosphates. The results show that IGF-I stimulates phospholipid synthesis without production of inositol phosphates in renal cortical slices.

Expression and immunolocalization of metallothioneins MT1, MT2 and MT3 in rat nephron

Rodent kidneys exhibit three isoforms of metallothioneins (MTs), MT1, MT2 and MT3, with poorly characterized localization along the nephron. Here we studied in adult male Wistar rats the renal expression of MTs mRNA by end-point RT-PCR and MT proteins by immunochemical methods The expression pattern of MT1 mRNA was cortex (CO)>outer stripe (OS)=inner stripe (IS)=inner medulla (IM), of MT2 mRNA was IM>CO>IS=OS, and of MT3 mRNA was IM>CO=OS=IM. MT1/2-antibody stained with heterogeneous intensity the cell cytoplasm and nuclei in proximal tubule (PT) and thin ascending limb, whereas MT3-antibody stained weakly the cell cytoplasm in various cortical tubules and strongly the nuclei in all nephron segments. However, the isolated nuclei exhibited an absence of MT1/2 and presence of MT3 protein. In MT1/2-positive PT cells, the intracellular staining appeared diffuse or bipolar, but the isolated brush-border, basolateral and endosomal membranes were devoid of MT1/2 proteins. In the lumen of some PT profiles, the heterogeneously sized MT1/2-rich vesicles were observed, with the limiting membrane positive for NHE3, but negative for V-ATPase, CAIV, and megalin, whereas their interior was positive for CAII and negative for cytoskeleton. They seem to be pinched off from the luminal membrane of MT1/2-rich cells, as also indicated by transmission electron microscopy. We conclude that in male rats, MTs are heterogeneously abundant in the cell cytoplasm and/or nuclei along the nephron. The MT1/2-rich vesicles in the tubule lumen may represent a source of urine MT and membranous material, whereas MT3 in nuclei may handle zink and locally-produced reactive oxygen species.