Liver HIF-1 alpha induction precedes apoptosis following normothermic ischemia-reperfusion in rats.

Apoptosis plays an important role in ischemia-reperfusion (I-R) injury during liver transplantation. The hypoxia-inducible factor alpha (HIF-1alpha) may trigger liver apoptosis following I-R through the induction of hypoxically regulated genes. The aim of this study was to evaluate the effect of normothermic liver I-R on HIF-1alpha expression and apoptosis in rats. Segmental normothermic ischemia of the liver was induced in rats for 120 minutes. Liver extracts from either ischemic or nonischemic lobes were prepared at 0, 1, 3, and 6 hours after reperfusion. Liver HIF-1alpha protein expression was examined by Western blot analysis. Liver apoptosis was quantified using terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling assay. Normothermic I-R resulted in a significant (P< .05) increase in liver HIF-1alpha protein levels 1 and 3 hours after reperfusion. Liver apoptosis was significantly (P< .005) increased at 3 and 6 hours after reperfusion. In conclusion, normothermic liver I-R leads to increased liver expression of HIF-1alpha and apoptosis.

Alterations in protein tyrosine kinase pathways in rat liver following normothermic ischemia-reperfusion.

The phosphoregulation of signal transduction pathways is a complex series of reactions that modulate the cellular response to ischemia-reperfusion (I-R). The aim of this study was to evaluate the effect of normothermic liver I-R on protein tyrosine phosphorylation, production of angiogenic growth factors, and activation of signal proteins in tyrosine kinase pathways. A segmental normothermic ischemia of the liver was induced in rats by occluding the blood vessels (including the bile duct) to the median and left lateral lobes for 120 minutes. Liver extracts from either ischemic or nonischemic lobes were prepared at 0, 1, 3, and 6 hours after reperfusion. Liver tyrosine phosphorylation of proteins was examined by Western blot analysis, whereas vascular endothelial growth factor (VEGF) mRNA was analyzed by Northern blot. In ischemic liver lobes, VEGF mRNA and total protein levels increased at 1 and 3 hours after reperfusion. Tyrosine phosphorylation of the VEGF receptor Flk-1 and the platelet-derived growth factor receptor (PDGF-R) was increased only at 1 hour after reperfusion, while c-Src tyrosine phosphorylation remained increased at 3 hours and remained up to 6 hours after reperfusion. In conclusion, 1-R led to alterations in protein tyrosine phosphorylation and increased expression of VEGF in rat liver.

Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

BACKGROUND: This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. METHODS: Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. RESULTS: The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. CONCLUSION: Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs.

Surfing the insulin signaling web.

The diverse biological actions of insulin and insulin-like growth factor I (IGF-I) are initiated by binding of the polypeptides to their respective cell surface tyrosine kinase receptors. These activated receptors phosphorylate a series of endogenous substrates on tyrosine, amongst which the insulin receptor substrate (IRS) proteins are the best characterized. Their phosphotyrosine-containing motifs become binding sites for Src homology 2 (SH2) domains on proteins such as SH2 domain-containing protein-tyrosine-phosphatase (SHP)-2/Syp, growth factor receptor bound-2 protein, (Grb-2), and phosphatidyl inositol 3 kinase (PI3 kinase), which participate in activation of specific signaling cascades. However, the IRS molecules are not only platforms for signaling molecules, they also orchestrate the generation of signal specificity, integration of signals induced by several extracellular stimuli, and signal termination and modulation. An extensive review is beyond the scope of the present article, which will be centered on our own contribution and reflect our biases.

Protein kinase C theta and epsilon promote T-cell survival by a rsk-dependent phosphorylation and inactivation of BAD.

Both MAPK and protein kinase C (PKC) signaling pathways promote cell survival and protect against cell death. Here, we show that 12-O-tetradecanoylphorbol-13-acetate (TPA) prevents Fas-induced apoptosis in T lymphocytes. The effect of TPA was specifically abolished by the PKC inhibitor GF109203X and by dominant negative PKCtheta, PKCepsilon, and PKCalpha, suggesting that novel and conventional PKC isoforms mediate phorbol ester action. Moreover, TPA stimulated phosphorylation of BAD at serine 112, an effect abrogated by GF109203X but not by the MEK inhibitor PD98059. Expression of constitutively active PKC increased the phosphorylation of BAD at serine 112 but not at serine 136. Additionally, Fas-mediated cell death was enhanced by overexpression of a catalytically inactive form of p90Rsk (Rsk2-KN). Finally, Rsk2-KN abolished the protective effect of constitutively active PKC and totally blocked phosphorylation of BAD on serine 112. Thus, novel PKCtheta and PKCepsilon rescue T lymphocytes from Fas-mediated apoptosis via a p90Rsk-dependent phosphorylation and inactivation of BAD.

Effect of phosphoinositide-dependent kinase 1 on protein kinase B translocation and its subsequent activation.

In this report we investigated the function of phosphoinositide-dependent protein kinase 1 (PDK1) in protein kinase B (PKB) activation and translocation to the cell surface. Wild-type and PDK1 mutants were transfected into HeLa cells, and their subcellular localization was analyzed. PDK1 was found to translocate to the plasma membrane in response to insulin, and this process did not require a functional catalytic activity, since a catalytically inactive kinase mutant (Kd) of PDK1 was capable of translocating. The PDK1 presence at the cell surface was shown to be linked to phospholipids and therefore to serum-dependent phosphatidylinositol 3-kinase activity. Using confocal microscopy in HeLa cells we found that PDK1 colocalizes with PKB at the plasma membrane. Further, after cotransfection of PKB and a PDK1 mutant (Mut) unable to translocate to the plasma membrane, PKB was prevented from moving to the cell periphery after insulin stimulation. In response to insulin, a PKB mutant with its PH domain deleted (DeltaPH-PKB) retained the ability to translocate to the plasma membrane when coexpressed with PDK1. Finally, we found that DeltaPH-PKB was highly active independent of insulin stimulation when cotransfected with PDK1 mutants defective in their PH domain. These findings suggest that PDK1 brings PKB to the plasma membrane upon exposure of cells to insulin and that the PH domain of PDK1 acts as a negative regulator of its enzyme activity.

Insulin and insulin-like growth factor-I induce vascular endothelial growth factor mRNA expression via different signaling pathways.

In this study we have investigated the molecular mechanisms of insulin and insulin-like growth factor-I (IGF-I) action on vascular endothelial growth factor (VEGF) gene expression. Treatment with insulin or IGF-I for 4 h increased the abundance of VEGF mRNA in NIH3T3 fibroblasts expressing either the human insulin receptor (NIH-IR) or the human IGF-I receptor (NIH-IGFR) by 6- and 8-fold, respectively. The same elevated levels of mRNA were maintained after 24 h of stimulation with insulin, whereas IGF-I treatment further increased VEGF mRNA expression to 12-fold after 24 h. Pre-incubation with the phosphatidylinositol 3-kinase inhibitor wortmannin abolished the effect of insulin on VEGF mRNA expression in NIH-IR cells but did not modify the IGF-I-induced VEGF mRNA expression in NIH-IGFR cells. Blocking mitogen-activated protein kinase activation with the MEK inhibitor PD98059 abolished the effect of IGF-I on VEGF mRNA expression in NIH-IGFR cells but had no effect on insulin-induced VEGF mRNA expression in NIH-IR cells. Expression of a constitutively active PKB in NIH-IR cells induced the expression of VEGF mRNA, which was not further modified by insulin treatment. We conclude that VEGF induction by insulin and IGF-I occurs via different signaling pathways, the former involving phosphatidylinositol 3-kinase/protein kinase B and the latter involving MEK/mitogen-activated protein kinase.

Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase.

Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.

Involvement of the pleckstrin homology domain in the insulin-stimulated activation of protein kinase B.

Involvement of the pleckstrin homology (PH) domain in the insulin-stimulated activation of protein kinase B (PKB) was investigated in human embryonic kidney 293 cells. Different PKB constructs that contain mutations or deletions in the PH domain were transfected into cells, and the results on the basal and insulin-induced kinase activities were analyzed. Deletion of the entire PH domain (DeltaPH-PKB) did not impair the kinase activity; in contrast, the basal activity was elevated with respect to wild-type PKB. In addition, DeltaPH-PKB was responsive to insulin, and as for wild-type PKB, this was dependent on phosphoinositide 3-kinase. By contrast, a point mutation within the PH domain that impairs phospholipid binding (R25C) resulted in a construct that was not responsive to insulin. However, this defect was overcome by mutations that mimic the phosphorylation state of the active kinase. The increase in the basal activity of DeltaPH-PKB was shown to be due to an elevation in the level of phosphorylation of this construct. In addition, the subcellular localization of DeltaPH-PKB, as determined by both immunofluorescence and fractionation, was predominately cytosolic, and DeltaPH-PKB was present in the plasma membrane at much lower levels compared with wild-type PKB. These data show that phosphorylation is the major factor regulating the activity of PKB and that either removal of the PH domain or binding of phospholipids is required to permit this phosphorylation. In addition, membrane localization does not appear to be required for the activation process, but instead, binding of PKB to membrane phospholipids permits a conformational change in the molecule that allows for phosphorylation.

cAMP stimulates protein kinase B in a Wortmannin-insensitive manner.

Activation of protein kinase B (PKB) by growth factors has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). Here, we show that agents which raise intracellular cAMP can also stimulate PKB. However, this effect is not sensitive to wortmannin, indicating that it is PI3-kinase independent. This activation does not appear to result from direct phosphorylation by protein kinase A (PKA) since GST-PKB is not an effective PKA substrate. In addition, the activation pathway of PKB by cAMP seems to be linked to that of growth factors, albeit downstream of PI3-kinase. Evidence for this is that a constitutive active PKB, T308D, S473D, containing activating mutations in the serine and threonine residues which are phosphorylated subsequent to PI3-kinase activation, cannot be further stimulated by cAMP elevations. Hence, these data suggest that, in addition to growth factors, cAMP can also lead to activation of PKB. This cAMP stimulatory action appears to require phosphorylation of T308 and S473, and hence would indicate that cAMP modulates the phosphorylation event of these PKB regulatory sites.

[Long-term assessment of bone loss in patients treated with GnRH agonists]. / Valutazione a lungo termine della perdita calcica ossea nelle pazienti trattate con GnRH Agonisti.

GnRH Analogues therapy of estrogen-dependent gynaecological diseases aims at suppression of physiologic ciclic ovaric function producing a hypogonadotropic condition. The first consequence of GnRH Analogues administration is hypoestrogenism; this condition permits the disease regression but, on the other, it causes a negative impact on bone metabolism particularly for prolonged therapeutic schemes (6 months). This study has evaluated the faculty of bone mass protection combining GnRH Analogues therapy with Ipriflavone that stimulates, both "in vivo" and "in vitro", Osteoblastic cells activity. The result of this study showed a significant bone loss in patients treated with GnRH Analogues only. The Ipriflavone association prevented bone loss.

Bone mass and long-term monophasic oral contraceptive treatment in young women.

A prospective study has been designed to investigate bone metabolism in young women taking an oral monophasic contraceptive formulation (ethinylestradiol 20 micrograms + desogestrel 0.150 mg) over 5 years. Healthy women (n = 200) between 19 and 22 years of age were divided into two groups. Group A received oral contraception, Group B did not receive any treatment. All the subjects underwent a bone mass density (BMD) evaluation at spinal level L2-L4 with Dexa (Norland XR-26) and a measurement of the serum alkaline phosphatase levels and urinary excretion of OH-proline at baseline and every 12 months over 5 years. Our results demonstrated that Group A did not show any significant BMD change after 5 years of oral contraceptive treatment, while Group B demonstrated a significant increase (p < 0.01) in the bone mass content at the end of the time of observation (+7.8% after 5 years). No significant changes were found in serum alkaline phosphatase levels and in urinary excretion of OH-proline at the end of the study in comparison with basal levels in both groups. Our data suggested that long-term treatment with an oral monophasic contraceptive formulation (ethinylestradiol 20 micrograms + desogestrel 0.150 mg) did not modify the BMD but prevented the occurrence of the physiologic peak of bone mass in young women.

PIP: Between June 1988 and March 1989 in Italy, health workers enrolled 200 women aged 19-23 attending obstetric-gynecology clinics in and around Pavia into a five-year study of the effects of an oral contraceptive (OC) with 20 mcg ethinyl estradiol and 0.15 mg desogestrel on bone mass. They were able to follow 76 of the 100 women using the OC and 71 of the 100 women using no OC for five years. Health workers conducted a bone mass density (BMD) evaluation at spinal level L2-L4 with Dexa (Norland XR-26). They measured serum alkaline phosphatase levels and urinary excretion of hydroxyproline (OH-proline) at baseline and every 12 months for five years. Neither urinary excretion of OH-proline levels nor alkaline phosphatase levels differed significantly in the two groups during the five years from baseline levels. Over the five years, the BMD of OC users did not change significantly while the BMD of the controls increased 7.8% from baseline (p 0.01). These findings suggest that this monophasic OC prevented or delayed the physiologic peak bone mass. Even though the low dosage of ethinyl estradiol (20 mcg) may have contributed to the prevention of bone mass loss, it could not achieve the peak bone mass. More studies are needed to understand the effect of long-term OC treatment on bone mineral content.