Phosphatidylinositol 3-kinase/protein Kinase B Research Articles

Methylglyoxal-Lysine Dimer, an Advanced Glycation End Product, Induces Inflammation via Interaction with RAGE in Mesangial Cells.

Advanced glycation end products (AGEs) and receptor of advanced glycation end products (RAGE) mediate renal function during diabetic and non-diabetic nephropathy development. Methylglyoxal-lysine dimer (MOLD), a typical toxic advanced glycation end product (TAGE), contributes to inflammatory responses during renal diseases. This study determines the effect of MOLD on inflammatory responses in mouse mesangial cells. The murine mesangial cell line SV40 MES 13 is used to assess nuclear factor-kappa B (NF-κB) expression, reactive oxygen species (ROS) production, and mitochondria labeling. The interaction model between RAGE and MOLD is also determined. MOLD treatment of mesangial cells markedly increases RAGE expression and the linkage with V-type Ig domain of RAGE. MOLD induces ROS production and mitochondrial dysfunction. MOLD activates phosphatidylinositol 3-kinase-protein kinase B (PI3KB) and NF-κB signaling pathways. It is confirmed that these changes are reversed when ROS is suppressed. These effects may be regulated through mitogen-activated protein kinases and pro-inflammatory cytokines in circulatory inflammation responses. MOLD plays a major role in nephropathy via ROS production and mitochondrial dysfunction through direct association with RAGE. Further, the NF-kB and PI3K/AKT signaling pathways triggered by ROS mediate the inflammatory response to exacerbate MOLD-induced damages in inflammation-related diabetic and non-diabetic renal diseases.

From Melanoma Development to RNA-Modified Dendritic Cell Vaccines: Highlighting the Lessons From the Past.

Although melanoma remains the deadliest skin cancer, the current treatment has not resulted in the desired outcomes. Unlike chemotherapy, immunotherapy has provided more tolerable approaches and revolutionized cancer therapy. Although dendritic cell-based vaccines have minor side effects, the undesirable response rates of traditional approaches have posed questions about their clinical translation. The immunosuppressive tumor microenvironment can be the underlying reason for their low response rates. Immune checkpoints and indoleamine 2,3-dioxygenase have been implicated in the induction of immunosuppressive tumor microenvironment. Growing evidence indicates that the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/Protein kinase B (PKB) (PI3K/AKT) pathways, as the main oncogenic pathways of melanoma, can upregulate the tumoral immune checkpoints, like programmed death-ligand 1. This study briefly represents the main oncogenic pathways of melanoma and highlights the cross-talk between these oncogenic pathways with indoleamine 2,3-dioxygenase, tumoral immune checkpoints, and myeloid-derived suppressor cells. Moreover, this study sheds light on a novel tumor antigen on melanoma, which has substantial roles in tumoral immune checkpoints expression, indoleamine 2,3-dioxygenase secretion, and stimulating the oncogenic pathways. Finally, this review collects the lessons from the previous unsuccessful trials and integrates their lessons with new approaches in RNA-modified dendritic cell vaccines. Unlike traditional approaches, the advances in single-cell RNA-sequencing techniques and RNA-modified dendritic cell vaccines along with combined therapy of the immune checkpoint inhibitors, indoleamine 2,3-dioxygenase inhibitor, and RNA-modified dendritic cell-based vaccine can overcome these auto-inductive loops and pave the way for developing robust dendritic cell-based vaccines with the most favorable response rate and the least side effects.

RNA-seq analysis reveals TRPC genes to impact an unexpected number of metabolic and regulatory pathways

The seven-member transient receptor potential canonical genes (TRPC1-7) encode cation channels linked to several human diseases. There is little understanding of the participation of each TRPC in each pathology, considering functional redundancy. Also, most of the inhibitors available are not specific. Thus, we developed mice that lack all of the TRPCs and performed a transcriptome analysis in eight tissues. The aim of this research was to address the impact of the absence of all TRPC channels on gene expression. We obtained a total of 4305 differentially expressed genes (DEGs) in at least one tissue where spleen showed the highest number of DEGs (1371). Just 21 genes were modified in all the tissues. Performing a pathway enrichment analysis, we found that many important signaling pathways were modified in more than one tissue, including PI3K (phosphatidylinositol 3-kinase/protein kinase-B) signaling pathway, cytokine-cytokine receptor interaction, extracellular matrix (ECM)-receptor interaction and circadian rhythms. We describe for the first time the changes at the transcriptome level due to the lack of all TRPC proteins in a mouse model and provide a starting point to understand the function of TRPC channels and their possible roles in pathologies.

The hexosamine biosynthesis pathway and O-GlcNAcylation maintain insulin-stimulated PI3K-PKB phosphorylation and tumour cell growth after short-term glucose deprivation.

Glucose provides an essential nutrient source that supports glycolysis and the hexosamine biosynthesis pathway (HBP) to maintain tumour cell growth and survival. Here we investigated if short-term glucose deprivation specifically modulates the phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) cell survival pathway. Insulin-stimulated PKB activation was strongly abrogated in the absence of extracellular glucose as a consequence of the loss of insulin-stimulated PI3K activation and short-term glucose deprivation inhibited subsequent tumour cell growth. Loss of insulin-stimulated PKB signalling and cell growth was rescued by extracellular glucosamine and increased flux through the HBP. Disruption of O-GlcNAc transferase activity, a terminal step in the HBP, implicated O-GlcNAcylation in PKB signalling and cell growth. Glycogenolysis is known to support cell survival during glucose deprivation, and in A549 lung cancer cells its inhibition attenuates PKB activation which is rescued by increased flux through the HBP. Our studies show that rerouting of glycolytic metabolites to the HBP under glucose-restricted conditions maintains PI3K/PKB signalling enabling cell survival and proliferation.

LOX-1 augments oxLDL uptake by lysoPC-stimulated murine macrophages but is not required for oxLDL clearance from plasma

Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macrophages. OxLDL uptake is mainly due to scavenger receptors SR-AI/II and CD36. However, other scavenger receptors such as lectin-like oxLDL receptor-1 (LOX-1) may also play a role. We used mice with targeted inactivation of the LOX-1 gene to define the role of this receptor in the uptake of oxLDL and in activation of survival pathways. There was no difference in uptake or degradation of 125I-oxLDL in unstimulated macrophages from wild-type and LOX-1 knockout mice and no difference in the rate of clearance of oxLDL from plasma in vivo. However, when expression of LOX-1 was induced with lysophosphatidylcholine, oxLDL uptake and degradation increased 2-fold in wild-type macrophages but did not change in LOX-1 knockout macrophages. Macrophages lacking LOX-1 showed the same stimulation of PKB phosphorylation and enhancement of survival by oxLDL as wild-type cells. These data show that LOX-1 does not alter the uptake of oxLDL in unstimulated macrophages and is not essential for the pro-survival effect of oxLDL in these cells. However, LOX-1 expression is highly inducible by lysophosphatidylcholine and pro-inflammatory cytokines, and if that occurred in macrophages within atheromas, LOX-1 could substantially increase oxLDL uptake by lesion macrophages.

Anaesthesia generates neuronal insulin resistance by inducing hypothermia

BackgroundAnaesthesia is commonly employed prior to surgical investigations and to permit icv injections in rodents. Indeed it is standard practise in many studies examining the subsequent actions of hormones and growth factors on the brain. Recent evidence that the basal activity of specific intracellular signalling proteins can be affected by anaesthesia prompted us to examine the effect of anaesthesia not only on the basal activity but also the insulin sensitivity of the major insulin signalling pathways.ResultsWe find that urethane- and ketamine-induced anaesthesia results in rapid activation of the phosphatidylinositol (PI) 3-kinase-protein kinase B (PKB) signalling pathway in the brain, increases tau phosphorylation while at the same time reducing basal activity of the Ras-ERK pathway. Subsequent injection of insulin does not alter the activity of either the PI 3-kinase or ERK signalling pathways, indicating a degree of neuronal molecular insulin resistance. However, if body temperature is maintained during anaesthesia then there is no alteration in the basal activity of these signalling molecules. Subsequent response of both pathways to insulin injection is restored.ConclusionThe data is consistent with a hypothermia related alteration in neuronal signalling following anaesthesia, and emphasises the importance of maintaining the body temperature of rodents when monitoring insulin (or growth factor/neurotrophic agent) action in the brain of anesthetised rodents.

Neuroprotection by Neurotrophic Factors and Membrane Depolarization Is Regulated by Calmodulin Kinase IV

Neurotrophic factors promote motoneuron (MN) survival through increased intracellular calcium (Ca(2+)) and regulation of the phosphatidylinositol (PI) 3-kinase/protein kinase B (PKB) pathway by calmodulin (CaM). Activation of the PI 3-kinase/PKB pathway is one of the well established mechanisms involved in MN survival. The Ca(2+)/CaM complex interacts with and modulates the functionality of a large number of proteins, including serine/threonine protein kinases such as Ca(2+)/CaM-dependent protein kinases (CaMKs). Using a primary culture of embryonic chicken spinal cord MNs, we investigated the role of CaMKIV in mediating this process. We cloned chicken CaMKIV and demonstrated its expression in purified MNs by means of reverse transcription-PCR, Western blot, and immunofluorescence. Using RNA interference, we show that endogenous CaMKIV mediates cell survival induced by neurotrophic factors or membrane depolarization. The survival effect is independent of CaMKIV kinase activity; however, CaMKIV functionality depends on the presence of Ca(2+)/CaM. Finally, CaMKIV associates to the p85 subunit of PI 3-kinase in a Ca(2+)-dependent manner, suggesting a role in regulating PI 3-kinase/PKB activation.

Upregulation of TGF-β–Induced Tissue Transglutaminase Expression by PI3K-Akt Pathway Activation in Human Subconjunctival Fibroblasts

Excessive scarring in subconjunctival tissues after filtering surgery seems to be characterized by aberrant extracellular matrix (ECM) production, and tissue transglutaminase (tTgase) plays an important role in this process. In the present study, the effects of transforming growth factor (TGF)-beta2 on the expression of tTgase, its activity in subconjunctival fibroblasts and whether the effects of TGF-beta are mediated by prosurvival signaling pathways were examined. Primary subconjunctival fibroblasts treated with TGF-beta2 were examined for the expression of tTgase with Western blot analysis. The modulation of extracellular tTgase activity by TGF-beta2 was measured by both the formation of fibronectin polymers and the ECM protein incorporation of fluorescein cadaverine. The expression of tTgase was analyzed by immunofluorescence staining and Western blot analysis of subconjunctival fibroblasts that were transiently transfected with an Akt dominant negative mutant gene or were treated with an Akt inhibitor or tTgase siRNA. Treatment of subconjunctival fibroblasts with TGF-beta2 caused an increase in activation and expression of tTgase. The effects of TGF-beta stimulation of subconjunctival fibroblasts were twofold, causing both rapid activation of the ERK pathway within minutes of treatment and a more delayed activation of the phosphatidylinositol3-kinase-protein kinase B (PKB)/Akt pathway; however, only Akt activation was necessary for TGF-beta-induced tTgase expression. Transient transfection of subconjunctival fibroblasts with an Akt dominant negative mutant gene, or treatment with an Akt inhibitor (but not with an ERK inhibitor) or tTgase siRNA led to decreased activation and expression of tTgase. TGF-beta2 activated the PI3K-Akt pathway, and this activation was essential for the expression and activity of tTgase in subconjunctival fibroblasts. The results indicate a novel biological function of the PI3K-Akt pathway in subconjunctival fibroblasts. Elevated expression and activity of tTgase may play an important role in the pathogenesis of diseases related to wound healing and fibrogenic reactions in subconjunctival fibroblasts.

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

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. 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. 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. 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.

Combined inhibition of the phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways results in synergistic effects in glioblastoma cells

The present study uses cell-based screening assays to assess the anticancer effects of targeting phosphatidylinositol 3-kinase-regulated integrin-linked kinase (ILK) in combination with small-molecule inhibitors of Raf-1 or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK). The objective was to determine if synergistic interactions are achievable through the use of agents targeting two key cell signaling pathways involved in regulating glioblastoma cancer. The phosphatidylinositol 3-kinase/protein kinase B (PKB)/Akt and the Ras/MAPK pathway were targeted for their involvement in cell survival and cell proliferation, respectively. The glioblastoma cell lines U87MG, SF-188, and U251MG were transiently transfected with an antisense oligonucleotide targeting ILK (ILKAS) alone or in combination with the Raf-1 inhibitor GW5074 or with the MEK inhibitor U0126. Dose and combination effects were analyzed by the Chou and Talalay median-effect method and indicated that combinations targeting ILK with either Raf-1 or MEK resulted in a synergistic interaction. Glioblastoma cells transfected with ILKAS exhibited reduced levels of ILK and phosphorylated PKB/Akt on Ser473 but not PKB/Akt on Thr308 as shown by immunoblot analysis. These results were confirmed using glioblastoma cells transfected with ILK small interfering RNA, which also suggested enhanced gene silencing when used in combination with U0126. U87MG glioblastoma cells showed a 90% (P < 0.05) reduction in colony formation in soft agar with exposure to ILKAS in combination with GW5074 compared with control colonies. A substantial increase in Annexin V-positive cells as determined by using fluorescence-activated cell sorting methods were seen in combinations that included ILKAS. Combinations targeting ILK and components of the Ras/MAPK pathway result in synergy and could potentially be more effective against glioblastoma cancer than monotherapy.

Involvement of Lipid Rafts in Impaired fMLP-Stimulated ROS Production of GM-CSF-Primed Neutrophils from Patients with Myelodysplasia.

Neutrophils from MDS patients are generally dysfunctional, resulting in increased susceptibility to bacterial infections. An important bactericidal activity of neutrophils is their production of reactive oxygen species (ROS) upon stimulation with the bacterial product fMLP. ROS production by fMLP can be enhanced by pre-treatment of neutrophils with pro-inflammatory cytokines such as GM-CSF. In MDS patients, this GM-CSF priming of ROS production is severely impaired. Furthermore, activation of the extracellular signal-regulated kinase (ERK1/2) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) signal transduction pathways, involved in ROS production, are impaired in MDS (Fuhler et al, Blood , 101, 2003). In the current study we investigated the involvement of lipid rafts in neutrophil ROS production, and show that treatment of healthy neutrophils with the lipid-raft disrupting agent methyl-b-cyclodextrin (CD) abrogates fMLP-induced ROS production in unprimed cells (mean fluorescence intensity [MFI] of 16±2 vs 4±0.3, n=5, P=0.03) and GM-CSF-primed neutrophils (MFI 50±14 vs 9±1, P=0.04). We further demonstrate that activation of ERK and PKB by fMLP was abolished by CD treatment of unprimed or GM-CSF-primed neutrophils. In contrast, GM-CSF-stimulated phosphorylation of STAT5 was not affected by CD pre-treatment of healthy neutrophils. These results indicate the importance of lipid rafts in fMLP-stimulated ROS production and in activation of signal transduction pathways involved in this process. The expression of the ganglioside GM1 at the plasma membrane of cells has been described to be a marker for the presence of lipid rafts. We analyzed the expression of GM1 on healthy neutrophils by FACS analysis and show that stimulation of cells with fMLP increased the expression of GM1 at the plasma membrane (MFI 102±20 vs 161±38, n=6, P=0.03). Furthermore, pretreatment of neutrophils with GM-CSF further increased the fMLP-induced GM1 expression (MFI 161 ±38 vs 193±36, P=0.03). We next assessed the expression of GM1 at the plasma membrane, in conjunction with ROS production, in 8 MDS patients compared to healthy donors. As described previously, ROS production in response to fMLP was normal in unprimed MDS neutrophils. In contrast, in 6 patients, fMLP-induced ROS production in GM-CSF primed neutrophils was significantly impaired when compared to their healthy controls (mean MFI 128±54 vs 191±72, P=0.03). When investigating the GM1 expression in these patients, we found that although fMLP-triggered GM1 expression was normal in unprimed neutrophils from MDS patients, fMLP-induced GM1 expression on GM-CSF-primed cells was significantly lower on MDS neutrophils compared to their healthy counterpart (mean MFI 174±76 vs 214±78, P=0.03). GM-CSF primed neutrophils from one patient with normal ROS production also exhibited normal GM1 expression. Taken together, these data indicate an involvement of lipid rafts in ROS production, and suggest that in MDS neutrophils, an impaired lipid raft formation in GM-CSF primed cells correlates with impaired fMLP-induced ROS production.

Susi, a Negative Regulator of Drosophila PI3-Kinase

The Phosphatidylinositol-3 kinase/Protein Kinase B (PI3K/PKB) signaling pathway controls growth, metabolism, and lifespan in animals, and deregulation of its activity is associated with diabetes and cancer in humans. Here, we describe Susi, a coiled-coil domain protein that acts as a negative regulator of insulin signaling in Drosophila. Whereas loss of Susi function increases body size, overexpression of Susi reduces growth. We provide genetic evidence that Susi negatively regulates dPI3K activity. Susi directly binds to dP60, the regulatory subunit of dPI3K. Since Susi has no overt similarity to known inhibitors of PI3K/PKB signaling, it defines a novel mechanism by which this signaling cascade is kept in check. The fact that Susi is expressed in a circadian rhythm, with highest levels during the night, suggests that Susi attenuates insulin signaling during the fasting period.

Heme Oxygenase-1 Gene Activation by the NAD(P)H Oxidase Inhibitor 4-(2-Aminoethyl) Benzenesulfonyl Fluoride via a Protein Kinase B, p38-dependent Signaling Pathway in Monocytes

Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme degradation and modulates the inflammatory immune response. Because HO-1 is up-regulated by NAD(P)H oxidase activators such as lipopolysaccharide and 12-O-tetradecanoylphorbol-13-acetate in monocytic cells, we investigated the gene regulation of HO-1 by the chemical NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF). Unexpectedly, AEBSF induced endogenous gene expression and promoter activity of HO-1 in cell cultures of human and mouse monocytes. Inhibition of the phosphatidylinositol 3-kinase/protein kinase B (PKB) pathway by pharmacological inhibitors and cotransfection of an expression vector for a dominant negative mutant of PKB reduced the AEBSF-dependent induction of HO-1 gene transcription. Accordingly, overexpressed constitutively active PKB markedly up-regulated HO-1 promoter activity. AEBSF activated the mitogen-activated protein kinases (MAPK) JNK and p38. Inhibition of p38alpha and p38beta, but not that of JNK or p38gamma and p38delta, prevented the induction of HO-1 gene expression by AEBSF. p38 was stimulated by AEBSF in a PKB-dependent manner as demonstrated by a luciferase assay with a Gal4-CHOP fusion protein. Finally, AEBSF- and PKB-dependent induction of HO-1 promoter activity was reduced by simultaneous mutation of an E-box motif (-47/-42) and a cAMP response element/AP-1 element (-664/-657) of the proximal HO-1 gene promoter. Overexpression of the basic helix-loop-helix transcription factor USF2 and coactivator p300 enhanced the AEBSF-dependent response of the HO-1 promoter. The data suggest that the transcriptional induction of HO-1 gene expression by AEBSF is mediated via activation of a PKB, p38 MAPK signaling pathway.

FSH activates phosphatidylinositol 3-kinase/protein kinase B signaling pathway in 20-day-old Sertoli cells independently of IGF-I.

The gonadotropin FSH plays a key role in the control of Sertoli cell function. The FSH molecular mechanism of action is best recognized for its stimulation of the adenylyl cyclase/cAMP pathway. However, other signaling events have also been demonstrated in Sertoli cells. We have recently presented evidence that FSH can stimulate the phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) pathway in 20-day-old Sertoli cells. At the same time, it was proposed that in 8-day-old Sertoli cells the effects of FSH on phosphorylated PKB (P-PKB) levels can be explained by a combination of increased secretion of endogenous IGF-I, decreased IGF-binding protein-3 (IGFBP-3) production, and a synergistic action of FSH on IGF-I-dependent PI3K activation. The aim of the present study was to determine whether the effect of FSH on 20-day-old Sertoli cells is mediated by IGF-I secretion. Twenty-day-old rat Sertoli cell cultures were used. FSH stimulation produced a time-dependent increment in P-PKB levels reaching maximal values in 60-min incubations. IGF-I stimulation was also time-dependent reaching maximal values in 15-min incubations. On the other hand, stimulation of the cultures with FSH showed time-dependent inhibition in phosphorylated mitogen-activated protein kinase (P-MAPK) levels. In sharp contrast, stimulation of the cultures with IGF-I showed time-dependent increments in P-MAPK levels reaching maximal stimulus in 15-min incubations. In order to rule out an IGF-I action on FSH stimulation of P-PKB levels, the effect of a specific IGF-I antibody on the ability of both hormones to increase P-PKB levels was evaluated. As expected, the antibody inhibited IGF-I stimulation of P-PKB levels. However, simultaneous addition of an IGF-I antibody with FSH did not modify the ability of the hormone to increase P-PKB levels. The next set of experiments intended to analyze the relevance of a PI3K/PKB pathway to two biological responses of Sertoli cells to FSH and IGF-I. The PI3K inhibitor, wortmannin, dose-dependently decreased FSH-stimulated lactate and transferrin production. On the other hand, wortmannin was not able to modify the ability of IGF-I to stimulate these metabolic events. In addition, the analysis of the participation of a MAPK pathway in IGF-I regulation of Sertoli cell biological responses showed that the MAPK kinase inhibitors, PD98059 and U0126, decreased IGF-I-stimulated transferrin secretion while not modifying IGF-I-stimulated lactate levels. In summary, results obtained so far support the hypothesis that FSH action on P-PKB levels and Sertoli cell metabolism in 20-day-old animals is not mediated by autocrine regulation of an IGF-I/ IGFBP-3 axis as previously proposed in 8-day-old Sertoli cells.

Assessment of the roles of mitogen-activated protein kinase and phosphatidyl inositol 3-kinase/protein kinase B pathways in the basic fibroblast growth factor regulation of Sertoli cell function.

Basic fibroblast growth factor (bFGF) belongs to the large set of intratesticular regulators that provide the fine tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of the present study was to determine the participation of mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase/protein kinase B (PI3K/PKB) pathways in bFGF regulation of Sertoli cell function. Twenty-day-old rat Sertoli cell cultures were used. Stimulation of the cultures with bFGF showed a time-dependent increment in phosphorylated MAPK and PKB levels that reached maximal values in 5-min incubations. MAPK kinase inhibitors U0126 (U) and PD98059 (PD) and a PI3K inhibitor wortmannin (W) were able to block the stimulatory effects of bFGF on phosphorylated MAPK and PKB levels respectively. The participation of MAPK- and PI3K/PKB-signaling pathways in the regulation by bFGF of two well-known Sertoli cell-differentiated functions, lactate and transferrin production, was next explored. As for lactate production, PD and W did not modify the ability of bFGF to stimulate lactate production. However, a combination of PD and W partially impaired the increase in lactate production elicited by bFGF. The participation of MAPK- and PI3K/PKB-signaling pathways in the regulation by bFGF of glucose uptake and lactate dehydrogenase (LDH) activity was also analysed. In this respect, it was observed that W markedly decreased basal and bFGF-stimulated glucose uptake and that U and PD did not modify it. On the other hand, U and PD decreased the stimulation of LDH activity by bFGF whereas W did not modify it. As for transferrin production, while both MAPK kinase inhibitors partially decreased the ability of bFGF to stimulate transferrin secretion, the PI3K inhibitor did not modify it. In summary, the results demonstrated that bFGF stimulates MAPK- and PI3K/PKB-dependent pathways in rat Sertoli cells. Moreover, these results showed that while bFGF utilizes the MAPK pathway to regulate transferrin production and LDH activity, it uses the PI3K/PKB pathway to regulate glucose transport into the cell.

The hepatocyte growth factor/Met pathway controls proliferation and apoptosis in multiple myeloma.

The evolution of multiple myeloma (MM) depends on complex signals from the bone marrow (BM) microenvironment, supporting the proliferation and survival of malignant plasma cells. An interesting candidate signal is hepatocyte growth factor/scatter factor (HGF), since its receptor Met is expressed on MM cells, while HGF is produced by BM stromal cells and by some MM cell lines, enabling para- or autocrine interaction. To explore this hypothesis, we studied the biological effects of HGF stimulation on MM cell lines and on primary MMs. We observed that Met is expressed by the majority of MM cell lines and by approximately half of the primary plasma cell neoplasms tested. Stimulation of MM cells with HGF led to the activation of the RAS/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) pathways, signaling routes that have been implicated in the regulation of cell proliferation and survival. Indeed, functional studies demonstrated that HGF has strong proliferative and anti-apoptotic effects on both MM cell lines and primary MM cells. Furthermore, by applying specific signal-transduction inhibitors, we demonstrated that MEK is required for HGF-induced proliferation, whereas activation of PI3K is required for both HGF-induced proliferation and for rescue of MM cells from apoptosis. Taken together, our data indicate that HGF is a potent myeloma growth and survival factor and suggest that the HGF/Met pathway is a potential therapeutic target in MM.

Cannabinoids Protect Astrocytes from Ceramide-induced Apoptosis through the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway

Cannabinoids, the active components of marijuana and their endogenous counterparts, exert many of their actions on the central nervous system by binding to the CB(1) cannabinoid receptor. Different studies have shown that cannabinoids can protect neural cells from different insults. However, those studies have been performed in neurons, whereas no attention has been focused on glial cells. Here we used the pro-apoptotic lipid ceramide to induce apoptosis in astrocytes, and we studied the protective effect exerted by cannabinoids. Results show the following: (i) cannabinoids rescue primary astrocytes from C(2)-ceramide-induced apoptosis in a dose- and time-dependent manner; (ii) triggering of this anti-apoptotic signal depends on the phosphatidylinositol 3-kinase/protein kinase B pathway; (iii) ERK and its downstream target p90 ribosomal S6 kinase might be also involved in the protective effect of cannabinoids; and (iv) cannabinoids protect astrocytes from the cytotoxic effects of focal C(2)-ceramide administration in vivo. In summary, results show that cannabinoids protect astrocytes from ceramide-induced apoptosis via stimulation of the phosphatidylinositol 3-kinase/protein kinase B pathway. These findings constitute the first evidence for an "astroprotective" role of cannabinoids.

SHIP Negatively Regulates IgE + Antigen-Induced IL-6 Production in Mast Cells by Inhibiting NF-κB Activity

We demonstrate in this study that IgE + Ag-induced proinflammatory cytokine production is substantially higher in Src homology-2-containing inositol 5'-phosphatase (SHIP)(-/-) than in SHIP(+/+) bone marrow-derived mast cells (BMMCs). Focusing on IL-6, we found that the repression of IL-6 mRNA and protein production in SHIP(+/+) BMMCs requires the enzymatic activity of SHIP, because SHIP(-/-) BMMCs expressing wild-type, but not phosphatase-deficient (D675G), SHIP revert the IgE + Ag-induced increase in IL-6 mRNA and protein down to levels seen in SHIP(+/+) BMMCs. Comparing the activation of various signaling pathways to determine which ones might be responsible for the elevated IL-6 production in SHIP(-/-) BMMCs, we found the phosphatidylinositol 3-kinase/protein kinase B (PKB), extracellular signal-related kinase (Erk), p38, c-Jun N-terminal kinase, and protein kinase C (PKC) pathways are all elevated in IgE + Ag-induced SHIP(-/-) cells. Moreover, inhibitor studies suggested that all these pathways play an essential role in IL-6 production. Looking downstream, we found that IgE + Ag-induced IL-6 production is dependent on the activity of NF-kappa B and that I kappa B phosphorylation/degradation and NF-kappa B translocation, DNA binding and transactivation are much higher in SHIP(-/-) BMMCs. Interestingly, using various pathway inhibitors, it appears that the phosphatidylinositol 3-kinase/PKB and PKC pathways elevate IL-6 mRNA synthesis, at least in part, by enhancing the phosphorylation of I kappa B and NF-kappa B DNA binding while the Erk and p38 pathways enhance IL-6 mRNA synthesis by increasing the transactivation potential of NF-kappa B. Taken together, our data are consistent with a model in which SHIP negatively regulates NF-kappa B activity and IL-6 synthesis by reducing IgE + Ag-induced phosphatidylinositol-3,4,5-trisphosphate levels and thus PKB, PKC, Erk, and p38 activation.

Lithium inhibits caspase 3 activation and dephosphorylation of PKB and GSK3 induced by K+ deprivation in cerebellar granule cells.

Lithium protects cerebellar granule cells from apoptosis induced by low potassium, and also from other apoptotic stimuli. However, the precise mechanism by which this occurs is not understood. When cerebellar granule cells were switched to low potassium medium, the activation of caspase 3 was detected within 6 h, suggesting a role of caspase 3 in mediating apoptosis under conditions of low potassium. In the same conditions, lithium (5 mM) inhibited the activation of caspase 3 induced by low potassium. As lithium did not inhibit caspase 3 activity in vitro, these results suggest that this ion inhibits an upstream component that is required for caspase 3 activation. Lithium is known to inhibit a kinase termed glycogen sythase kinase 3 (GSK3), which is implicated in the survival pathway of phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB). Here we demonstrate that low potassium in the absence of lithium induces the dephosphorylation, and therefore the activation, of GSK3. However, when lithium was present, GSK3 remained phosphorylated at the same level as observed under conditions of high potassium. Low potassium induced the dephosphorylation and inactivation of PKB, whereas when lithium was present PKB was not dephosphorylated. Our results allow us to propose a new hypothesis about the action mechanism of lithium, this ion could inhibit a serine-threonine phosphatase induced by potassium deprivation.

The Pyridinyl Imidazole Inhibitor SB203580 Blocks Phosphoinositide-dependent Protein Kinase Activity, Protein Kinase B Phosphorylation, and Retinoblastoma Hyperphosphorylation in Interleukin-2-stimulated T Cells Independently of p38 Mitogen-activated Protein Kinase

Pyridinyl imidazole inhibitors, particularly SB203580, have been widely used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase (p38/HOG/SAPKII) in a wide array of biological systems. Studies by this group and others have shown that SB203580 can have antiproliferative activity on cytokine-activated lymphocytes. However, we recently reported that the antiproliferative effects of SB203580 were unrelated to p38 MAP kinase activity. This present study now shows that SB203580 can inhibit the key cell cycle event of retinoblastoma protein phosphorylation in interleukin-2-stimulated T cells. Studies on the proximal regulator of this event, the phosphatidylinositol 3-kinase/protein kinase B (PKB)(Akt/Rac) kinase pathway, showed that SB203580 blocked the phosphorylation and activation of PKB by inhibiting the PKB kinase, phosphoinositide-dependent protein kinase 1. The concentrations of SB203580 required to block PKB phosphorylation (IC(50) 3-5 microM) are only approximately 10-fold higher than those required to inhibit p38 MAP kinase (IC(50) 0.3-0.5 microM). These data define a new activity for this drug and would suggest that extreme caution should be taken when interpreting data where SB203580 has been used at concentrations above 1-2 microM.