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PubMed2007

Insulin-like growth factor-I promotes resistance of bovine preimplantation embryos to heat shock through actions independent of its anti-apoptotic actions requiring PI3K signaling.

For the bovine preimplantation embryo, insulin-like growth factor-I (IGF-I) is a survival factor that blocks the induction of apoptosis and reduces the decrease in development caused by heat shock. The first objective was to determine the signaling pathways whereby IGF-I acts to increase embryo cell number while inhibiting heat-shock induced apoptosis. Exposure of embryos to heat shock reduced cell number and increased percent apoptosis, but IGF-I increased cell number and blocked induction of apoptosis caused by heat shock. Actions of IGF-I to increase cell number were blocked by treatment with the mitogen activated protein kinase kinase (MAPKK) inhibitor PD 98059 whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002 had no effect. Conversely, LY 294002 but not PD 98059 blocked actions of IGF-I to inhibit induction of apoptosis caused by heat shock. The second objective was to determine whether IGF-I blocks effects of heat shock on development to the blastocyst stage by preventing apoptosis. Culture of embryos with IGF-I was effective in blocking the reduction in blastocyst development caused by heat shock-this action occurred even in the presence of LY 294002. Addition of another inhibitor of apoptosis, the caspase-3 inhibitor z-DEVD-fmk, did not mimic the protective effects of IGF-I on blastocyst development. Surprisingly, IGF-I was not effective in blocking the reduction in blastocyst development caused by heat shock when cultured with z-DEVD-fmk. In conclusion, the anti-apoptotic actions of IGF-I require PI3K signaling while actions to promote proliferation require MAPKK signaling. Moreover, actions of IGF-I to allow heat-shocked embryos to continue development to the blastocyst stage are independent of its anti-apoptotic effects.

Animals↗
PubMed2005

Vasoactive intestinal peptide induces neuroendocrine differentiation in the LNCaP prostate cancer cell line through PKA, ERK, and PI3K.

BACKGROUND: Neuroendocrine (NE) differentiation in prostate cancer has been correlated with unfavorable clinical outcome. The mechanisms by which prostate cancer acquires NE properties are poorly understood, but several signaling pathways have been proposed. We have previously observed that vasoactive intestinal peptide (VIP) stimulates cAMP production mainly through VPAC(1) receptor, inducing NE differentiation in LNCaP cells. The aim of this study was to analyze the mechanisms involved in this process. METHODS: Reverse transcriptase (RT)-polymerase chain reaction (PCR), quantitative real-time RT-PCR, Western blotting, and immunocytochemistry were performed. RESULTS: LNCaP cells produce VIP, as demonstrated by RT-PCR and immunocytochemistry. VIP induced NE differentiation of LNCaP cells at a time as short as 1 hr of treatment, and the same occurred with the expression and secretion of neuronal-specific enolase (NSE, a NE differentiation marker). These effects were faster than those exerted by serum-deprivation. VIP induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation and NE differentiation by PKA-dependent and independent pathways, since the PKA inhibitor H89 partially blocked VIP-induced NE differentiation and did not affect ERK1/2 phosphorylation. mitogen-activated protein kinase kinase (MEK) and phosphoinositide 3-kinase (PI3K) appear to be also involved since the inhibitors PD98059 and wortmannin abolished ERK1/2 phosphorylation and decreased NE differentiation induced by VIP. Moreover, VIP activated Ras suggesting the involvement of a Ras-dependent pathway. CONCLUSIONS: VIP behaves as autocrine/paracrine factor in LNCaP cells by inducing NE differentiation through PKA, ERK1/2, and PI3K.

Carcinoma, Neuroendocrine↗
PubMed2006

A PI3K pathway mediates hair cell survival and opposes gentamicin toxicity in neonatal rat organ of Corti.

Gentamicin is well known to promote hair cell death in inner ear, but it also appears to activate opposing pathways that promote hair cell survival. In combination with others, our previous work has indicated that a K-Ras/Rac/JNK pathway is important for hair cell death and an H-Ras/Raf/MEK/Erk pathway is involved in promoting hair cell survival (Battaglia et al., Neuroscience 122(4):1025-1035, 2003). However, these data also suggested that a Ras-independent survival pathway for activation of MEK might be stimulated by gentamicin. To investigate alternatives to the Ras/Raf/MEK/Erk pathway in promoting hair cell survival, cochlear explants were exposed to gentamicin combined with several inhibitors of alternative pathways (LY294002, calphostin C, SH-6, U73122). When exposed to gentamicin with the PI3K inhibitor LY294002 (10, 50 microM), the protein kinase C (PKC) inhibitor calphostin C (50, 100 nM) or the PKB/Akt inhibitor SH-6 (5, 10 microM), hair cell damage was significantly increased compared to gentamicin alone. By Western blotting, strong PKB/Akt activation was observed in the organ of Corti following exposure to 50 microM gentamicin for 6 h. In addition, PKC activation by 12-O-tetradecanoylphorbol-13-acetate protected outer hair cells from gentamicin induced cell death. In contrast, the phospholipase C-gamma (PLCgamma) inhibitor U73122 (2, 5 microM) did not affect hair cell damage when combined with gentamicin. Also, phosphorylation of PLCgamma was not increased in the organ of Corti following gentamicin treatment, as evaluated by Western blot. The results indicate that PI3K promotes hair cell survival via its downstream targets, PKC and PKB/Akt. This suggests that both Ras-dependent and Ras-independent survival pathways are involved during gentamicin exposure. In contrast, PLCgamma activation of PKC does not appear to play a role.

Animals↗
PubMed1993

1H and 15N assignments and secondary structure of the PI3K SH3 domain.

The sequential 1H and 15N assignments of the SH3 domain of human phosphatidyl inositol 3'-kinase (PI3K) were determined by a combination of homonuclear and heteronuclear NMR experiments. With the exception of several protons belonging to lysine and proline residues, all proton and proton-bearing amide nitrogen resonances were assigned. Based on the sequential nuclear Overhauser effects (NOEs), 3JNH-C alpha H coupling constants and locations of slowly exchanging amide protons, we determined that the secondary structures of the protein consists of six beta-strands, two beta-turns and four short helices. Additional long range NOEs indicate that these beta-strands form two antiparallel beta-sheets. The topology of secondary structural elements of the PI3K SH3 domain is similar to those of the SH3 domains from c-Src and alpha-spectrin, suggesting that the SH3 family has a common tertiary structural motif.

Amino Acid Sequence↗
PubMed2005

T cell-to-T cell clustering enhances NF-kappaB activity by a PI3K signal mediated by Cbl-b and Rho.

Full activation of T cells requires the binding of antigen to the T cell receptor and stimulation of the CD28 molecule, a process which typically occurs when T cells bind to an antigen presenting cell. The transcription factor, NF-kappaB, is an integration point for these two signals and its activation is critical for T cell function. Using antibodies to the TCR and CD28 molecules to activate Jurkat T cells, we show that cells that were permitted to aggregate into multi-cellular clusters increased NF-kappaB activity compared to unclustered cells. Inhibition of PI3K signaling with wortmannin decreased the clustering-mediated NF-kappaB signal. Over-expression of a dominant negative form of Cbl-b, an endogenous inhibitor of PI3K, in unclustered cells rescued NF-kappaB activation to the same levels caused by cell clustering. Inhibiting signaling through Rho with dominant negative RhoA abrogated both clustering-mediated and dominant negative Cbl-b-mediated NF-kappaB inactivation, but not TCR/CD28 mediated NF-kappaB activation. Taken together, these results suggest that in addition to pathways stimulated by classical T cell-APC interactions, another signal arising from T cell clustering can enhance activation.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Ischemic postconditioning protects remodeled myocardium via the PI3K-PKB/Akt reperfusion injury salvage kinase pathway.

OBJECTIVE: We tested whether ischemic postconditioning (IPostC) is protective in remodeled myocardium. METHODS: Post-myocardial infarct (MI)-remodeled hearts after permanent coronary artery ligation and one kidney one clip (1K1C) hypertensive hearts of male Wistar rats were exposed to 40 min of ischemia followed by 90 min of reperfusion. IPostC was induced by six cycles of 10 s reperfusion interspersed by 10 s of no-flow ischemia. Activation of reperfusion injury salvage kinases was measured using Western blotting and in vitro kinase activity assays. RESULTS: IPostC prevented myocardial damage in both MI-remodeled and 1K1C hearts, as measured by decreased infarct size and lactate dehydrogenase release, and improved function. The reduction in infarct size and the recovery of left ventricular contractility achieved by IPostC was less in 1K1C hearts, but was unchanged in MI-remodeled hearts when compared to healthy hearts. In contrast, the recovery of inotropy was unaffected in 1K1C hearts, but was less in MI-remodeled hearts. Inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway with LY294002 abolished the protective effects of IPostC on both disease models and healthy hearts. Western blot analysis in conjunction with in vitro kinase activity assays identified protein kinase B (PKB)/Akt but not p42/p44 extracellular-signal regulated kinase 1/2 (ERK1/2) as the predominant kinase in IPostC-mediated cardioprotection in remodeled hearts. IPostC increased phosphorylation of the PKB/Akt downstream targets eNOS, GSK3beta, and p70S6K in remodeled hearts. CONCLUSION: Our results offer evidence that IPostC mediates cardioprotection in the remodeled rat myocardium primarily via activation of the PI3K-PKB/Akt reperfusion injury salvage kinase pathway.

Actins↗
PubMed2006

Hyaluronan induces vascular smooth muscle cell migration through RHAMM-mediated PI3K-dependent Rac activation.

OBJECTIVE: Hyaluronan (HA) is an important constituent of the extracellular matrix and is known to regulate cellular events through binding to CD44 and the receptor for HA-mediated motility (RHAMM). Here we investigated the role of these receptors and the signaling pathways involved in HA-mediated effects in arterial smooth muscle cells (ASMC). METHODS: Effects of high-molecular weight HA (1 to 5 mg/ml) were analyzed in cultured ASMC from rat aorta. RESULTS: HA promoted actin stress fiber and lamellipodia formation and dose-dependently induced ASMC migration without effect on proliferation. Pull-down assay of Rho protein activity indicated that HA activated RhoA and Rac. HA-induced ASMC migration was not affected by the RhoA inhibitor Tat-C3 (10 microg/ml), the Rho kinase inhibitor Y-27632 (10 microM) and blocking anti-CD44 antibody ,but was reduced by the non-selective Rho protein inhibitor simvastatin (10 microM), the Rac inhibitor LT-toxin (1 mug/ml), small interfering RNA (siRNA) targeting Rac and the phosphatidyl inositol 3-kinase (PI3K) inhibitor LY294002 (25 microM), which also blocked HA-induced Rac activation. CD44 knockdown by siRNA inhibited HA-mediated RhoA activation without effect on ASMC migration. In contrast, siRNA targeting RHAMM inhibited both HA-induced migration and Rac activation. CONCLUSIONS: High-molecular weight HA independently activates RhoA and Rac through CD44 and RHAMM, respectively. HA-induced migration depends exclusively on RHAMM-mediated PI3K-dependent Rac activation.

Actins↗
PubMed2006

Inhibitory effect of luteolin on hepatocyte growth factor/scatter factor-induced HepG2 cell invasion involving both MAPK/ERKs and PI3K-Akt pathways.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), and its receptor, the c-Met tyrosine kinase, play roles in cancer invasion and metastasis in a wide variety of tumor cells. Clinical observations suggest that HGF can promote metastasis of hepatoma cells while stimulating tumor invasiveness. We use HGF as an invasive inducer of human hepatoma HepG2 cells to investigate the effect of flavonoids on anti-invasion. In our preliminary study, we investigated the effect of flavonoids including luteolin, quercetin, baicalein, genistein, taxifolin and catechin on HGF-mediated migration and invasion of HepG2 cells. We found that luteolin presented the most potent potential on anti-migration and anti-invasion by Boyden chamber assay. Furthermore, luteolin inhibited HGF-induced cell scattering and cytoskeleton change such as filopodia and lamellipodia was determined by both phase-contrast and fluorescence microscopy studies. In addition, Western blotting and immunoprecipitation were performed to confirm luteolin suppressed the phosphorylation of c-Met, the membrane receptor of HGF, as well as ERK1/2 and Akt, but not JNK1/2, which is activated by HGF. Our investigation demonstrated that luteolin similar to PD98059, which acts as a specific inhibitor of MEK, an up stream kinase regulating ERK1/2, and wortmannin, a PI3K inhibitor, inhibited the invasiveness induced by HGF. In conclusion, the luteolin inhibited HGF-induced HepG2 cell invasion involving both MAPK/ERKs and PI3K-Akt pathways.

Carcinoma, Hepatocellular↗
PubMed2004

ARAP3 is a PI3K- and rap-regulated GAP for RhoA.

Rho and Arf family small GTPases are well-known regulators of cellular actin dynamics. We recently identified ARAP3, a member of the ARAP family of dual GTPase activating proteins (GAPs) for Arf and Rho family GTPases, in a screen for PtdIns(3,4,5)P(3) binding proteins. PtdIns(3,4,5)P(3) is the lipid product of class I phosphoinositide 3OH-kinases (PI3Ks) and is a signaling molecule used by growth factor receptors and integrins in the regulation of cell dynamics. We report here that as a Rho GAP, ARAP3 prefers RhoA as a substrate and that it can be activated in vitro by the direct binding of Rap proteins to a neighbouring Ras binding domain (RBD). This activation by Rap is GTP dependent and specific for Rap versus other Ras family members. We found no evidence for direct regulation of ARAP3's Rho GAP activity by PtdIns(3,4,5)P(3) in vitro, but PI3K activity was required for activation by Rap in a cellular context, suggesting that PtdIns(3,4,5)P(3)-dependent translocation of ARAP3 to the plasma membrane may be required for further activation by Rap. Our results indicate that ARAP3 is a Rap-effector that plays an important role in mediating PI3K-dependent crosstalk between Ras, Rho, and Arf family small GTPases.

Adaptor Proteins, Signal Transducing↗
PubMed2005

PKC- and PI3K-dependent but ERK-independent proliferation of murine splenic B cells stimulated by chondroitin sulfate B.

High molecular weight polyanions such as dextran sulfate are known to be weak polyclonal activators of murine B cells, but the molecular mechanism of their mitogenic activitiy is not fully elucidated. Although chondroitin sulfate A (CSA), B (CSB) and C (CSC) are highly charged polyanions, little is known about their effects on the proliferation of B cells. In this study, we demonstrated that CSB stimulated proliferation of murine B cells as markedly as did anti-IgM antibody, more markedly than did dextran sulfate and much more markedly than did CSA, CSC, heparin and hyaluronic acid. CSB caused translocation of protein kinase C (PKC) isoform beta from cytosol to membrane fractions and increased phosphorylation of Akt but not phosphorylation of extracellular signal-regulated kinase (ERK) of B cells. CSB-induced B cell proliferation was almost completely blocked by either the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or the PKC inhibitor GF109203X but was not significantly inhibited by the ERK kinase inhibitor PD98059. The mitogenic effect of anti-IgM was significantly inhibited by all the three inhibitors, while the mitogenic effect of LPS was inhibited only by LY294002. These findings indicate that CSB stimulated proliferation of murine B cells more markedly than did dextran sulfate and suggest that PKC and PI3K are crucial but that ERK is less important for the mitogenic activity of CSB, the signaling pathways of which may be at least partly distinct from those of anti-IgM and LPS.

Animals↗
PubMed2006

HSP70 enhances macrophage phagocytosis by interaction with lipid raft-associated TLR-7 and upregulating p38 MAPK and PI3K pathways.

BACKGROUND: Exogenous Heat Shock Protein-70 (HSP70), a product of necrotic cell death, binds the lipid raft microdomains of macrophages and, within minutes, stimulates the phagocytosis and presentation of internalized antigens. The aim of this study was to identify (a) the receptor on the lipid raft microdomain that interacts with HSP70 and (b) the subsequent signaling pathways that mediate HSP70-enhanced phagocytosis. METHODS: Cells including RAW264.7, bone-marrow-derived macrophages from TLR7-/- mice or controls and cells subjected to genetic methods reducing the mRNA expression of TLR7 were used to examine the interaction of HSP70 with TLR7. The effect of HSP70-TLR7 interaction on phagocytosis was assessed using phagocytosis assays described earlier. RESULTS: HSP70 binds Toll-like receptor-7 (TLR7) on the lipid raft microdomain of macrophage plasma membrane. Subsequent signaling is mediated through phosphoinositide 3-kinase (PI3K) and the up-regulation of the p38 MAP kinase pathways, both known activators of the phagocytic mechanisms. Reduced expression of TLR7 either via short interfering RNA for TLR7 (siRNA-TLR7) or using bone-marrow derived macrophages from TLR7-/- mice show that, as macrophages lose expression of TLR7, their ability to mediate HSP70-induced phagocytosis undergoes a corresponding diminution. Similarly, disruption of lipid rafts or blocking HSP70-TLR7-interaction or treatment with wortmannin and SB203580, inhibitors of PI3K or p38 MAPK, respectively, abrogates HSP70-induced macrophage phagocytosis. CONCLUSIONS: The interaction of HSP70 and LR-associated TLR7, two phylogenetically conserved molecules, activates a rapid, membrane-bound signaling pathway that enhances phagocytosis, a vital innate defense mechanism. This study elucidates critical mechanistic elements that mediate HSP70-enhanced phagocytosis by macrophages.

Animals↗
PubMed2005

Angiotensin II augmented migration and invasion of choriocarcinoma cells involves PI3K activation through the AT1 receptor.

While angiotensin II (Ang II) has been shown to inhibit migration of extravillous trophoblasts via plasminogen activator inhibitor-1 (PAI-1) activation, it has remained unclear whether it stimulates or inhibits malignant behavior of choriocarcinoma cells. Since we previously found an involvement of the renin-angiotensin system (RAS) in the proliferative potential in choriocarcinoma cells (BeWo), mediated via the Ang II type 1 receptor (AT1R), in the present study we investigated the effects of Ang II on choriocarcinoma cell migration/invasion in vitro using Transwell cell culture chambers. Ang II (10(-8)M) promoted migration and invasion by a choriocarcinoma cell line and augmented random cell mobility on checkerboard analysis. Immunoblotting showed Ang II to activate the phosphorylation of FAK and Akt in BeWo cells. Furthermore Ang II effects on cell migration were abolished by a selective AT1R antagonist and a phosphatidylinositol 3-kinase (PI3K) inhibitor. The present results suggest that Ang II-induced migration and invasion of choriocarcinoma cells probably involves PI3K following binding to the AT1R.

Angiotensin II↗
PubMed2005

Radioresistance of K-Ras mutated human tumor cells is mediated through EGFR-dependent activation of PI3K-AKT pathway.

BACKGROUND AND PURPOSE: In the context of EGFR-targeting strategies we investigated autocrine/paracrine factors leading to in vitro radioresistance of K-Ras mutated tumor cells through activation of EGFR mediated signal transduction. PATIENTS AND METHODS: Ras mutated (Rasmt) and normal Ras (Raswt) presenting human tumor cell lines were used to analyze the potential of conditioned media (CM) of both cell types to mediate radioresistance and to activate EGFR-signaling cascades. Therefore, clonogenic assays as well as SDS-PAGE combined with immunoblotting was performed. Additionally, Ras-mutated cells were transfected with K-Ras-siRNA to investigate, how downregulation of mutated K-Ras affects secretion of EGFR-ligands, stimulation of EGFR-signaling and modulation of radiation response. RESULTS: TGFalpha, Amphiregulin (ARG) and CM from Rasmt cells (Rasmt-CM) resulted in an increased clonogenic survival of irradiated Raswt cells. Both, EGFR ligands as well as Rasmt-CM led to a strong phosphorylation of EGFR and activation of downstream pathways, i.e. PI3K-AKT. However, neutralization of TGFalpha or ARG in Rasmt-CM led to a marked reduction of P-AKT. Furthermore, Rasmt-CM from K-Ras-siRNA transfected Rasmt-cells markedly inhibited phosphorylation of AKT in Raswt cells and enhanced radiation sensitivity of A549 cells transfected with the siRNA. CONCLUSION: The data suggest that constitutively upregulated autocrine/paracrine secretion of EGF receptor ligands, especially ARG from K-Ras mutated cells, mediates radioresistance in Rasmt-cells through stimulation of EGFR-PI3K-AKT pathway.

Amphiregulin↗
PubMed2005

Targeted RNA interference of PI3K pathway components sensitizes colon cancer cells to TNF-related apoptosis-inducing ligand (TRAIL).

BACKGROUND: The phosphoinositide 3-kinase (PI3K/Akt) pathway transduces signals initiated from growth factors. Previously, we identified an important role for PI3K/Akt in colon cancer progression. The purpose of this study was to determine (1) whether short interfering RNA (siRNA) directed to PI3K/Akt components can render colon cancer cells sensitive to treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and (2) the cellular mechanisms contributing to the enhanced sensitivity. METHODS: Human colon cancer cells KM20 and KM12C (both TRAIL resistant) were transfected with siRNA directed against the PI3K p85alpha regulatory subunit Akt1 or nontargeting control sequence and then treated with TRAIL (100 ng/mL) or vehicle. A ribonuclease protection assay was performed to assess changes in TRAIL receptor expression. Protein was extracted and analyzed by Western blot for expression of cleavage of TRAIL receptors (death receptor (DR) 4 and 5), caspase-3, caspase-8, and BID. Apoptosis was measured by enzyme-linked immunosorbent assay of DNA fragmentation. RESULTS: Combination treatment with p85alpha or Akt1 siRNA and TRAIL increased apoptosis in KM20 and KM12C cells, compared with TRAIL alone; these results were corroborated further by complete inhibition of apoptosis by Z-acetyl-Asp-Glu-Val-Asp-(DEVD)-fmk, a caspase-3 inhibitor. Furthermore, siRNA-mediated PI3K pathway inhibition resulted in increased expression of the TRAIL death receptors 4 and 5. CONCLUSIONS: Inhibition of PI3K/Akt by RNA interference sensitizes resistant colon cancer cells to TRAIL-induced cell death through the induction of TRAIL receptors and activation of caspase-3 and caspase-8. Agents that selectively target the PI3K/Akt pathway may enhance the effects of chemotherapeutic agents and provide novel adjuvant treatment for selected colon cancers.

Apoptosis↗
PubMed2003

17alpha-estradiol-induced VEGF-A expression in rat pituitary tumor cells is mediated through ER independent but PI3K-Akt dependent signaling pathway.

17alpha-E(2), a weak estrogen exhibited both agonistic and antagonistic effects, and caused a time- and dose-dependent induction of VEGF-A mRNA expression in GH3 rat pituitary tumor cells. This effect was unaffected by the presence of the pure estrogen receptor antagonist ICI 182,780 but was specifically blocked by a protein synthesis inhibitor puromycin. Inhibition of phosphatidylinositol-3 kinase (PI3K) activity by wortmannin decreased the effect of 17alpha-E(2) on VEGF-A mRNA expression. This inhibitor also blocked the increase in phosphorylation of Akt induced by exposure to 17alpha-E(2). In contrast, exposure to the MAP kinase inhibitor, U0126, had no impact on 17alpha-E(2)-induced VEGF-A mRNA expression. Taken together, these studies indicate that like potent estrogens 17alpha-E(2) up-regulates VEGF-A mRNA expression in estrogen responsive GH3 rat pituitary tumor cells, but this induction is not mediated through a classical estrogen receptor pathway. PI3K-Akt signaling pathway is required for the induction of VEGF-A mRNA in GH3 cells by 17alpha-E(2).

Androstadienes↗
PubMed2003

Calcium regulates the PI3K-Akt pathway in stretched osteoblasts.

Mechanical loading plays a vital role in maintaining bone architecture. The process by which osteoblasts convert mechanical signals into biochemical responses leading to bone remodeling is not fully understood. The earliest cellular response detected in mechanically stimulated osteoblasts is an increase in intracellular calcium concentration ([Ca(2+)](i)). In this study, we used the clonal mouse osteoblast cell line MC3T3-E1 to show that uniaxial cyclic stretch induces: (1) an immediate increase in [Ca(2+)](i), and (2) the phosphorylation of critical osteoblast proteins that are implicated in cell proliferation, gene regulation, and cell survival. Our data suggest that cyclic stretch activates the phosphoinositide 3-kinase (PI3K) pathway including: PI3K, Akt, FKHR, and AFX. Moreover, cyclic stretch also causes the phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase. Attenuation in the level of phosphorylation of these proteins was observed by stretching cells in Ca(2+)-free medium, using intra- (BAPTA-AM) and extracellular (BAPTA) calcium chelators, or gadolinium, suggesting that influx of extracellular calcium plays a significant role in the early response of osteoblasts to mechanical stimuli.

Animals↗
PubMed2003

Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K, Akt and eNOS.

Attenuation of reperfusion injury by growth factors has recently been linked to recruitment of phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt), a pathway also linked to the phosphorylation of eNOS by bradykinin. We, therefore, hypothesised that bradykinin would limit infarct size when given as an adjunct to reperfusion. Using an isolated perfused mouse heart model of ischaemia/reperfusion injury, we show that 100 nmol/l bradykinin, administered upon reperfusion, attenuates infarct size (32 +/- 2% to 22 +/- 2%, P < 0.01). This protection was abrogated by concomitant administration of the PI3K inhibitor, wortmannin (100 nmol/l), whereas wortmannin alone had no impact upon infarct size (31 +/- 3% and 30 +/- 1%, respectively). In eNOS knockout hearts, bradykinin was not seen to be protective (31 +/- 2% versus 32 +/- 2%), yet knockout hearts could be rescued with the nitric oxide donor, S-nitroso-N-acetyl penicillamine (SNAP) (1 micromol/l) (17 +/- 4%, P < 0.01). Using western blot analysis, we show that bradykinin administration results in rapid, robust phosphorylation of both Akt and eNOS, greater than that seen in control hearts upon reperfusion (Akt/eNOS phosphorylation: 68 +/- 7/122 +/- 29 AU versus 32 +/- 5/47 +/- 10 AU respectively, P < 0.01). This pattern of Akt phosphorylation was mimicked in the absence of eNOS, whereas Akt phosphorylation was inhibited by wortmannin. Exogenous nitric oxide administration had no impact upon Akt phosphorylation. Therefore, we demonstrate that exogenous bradykinin, administered at reperfusion, limits infarct size with concomitant rapid phosphorylation of Akt and eNOS, and that this protection is dependent upon the presence of eNOS. These results may open new avenues for research into clinical limitation of reperfusion injury following acute myocardial infarction.

Androstadienes↗
PubMed2004

A cAMP-activated pathway, including PKA and PI3K, regulates neuronal differentiation.

Neuronal differentiation is a complex process in which many different signalling pathways may be involved. An increase in the intracellular levels of cyclic AMP (cAMP) has been shown to induce neuronal differentiation and also to cooperate with NGF to induce PC12 neurite outgrowth in a Ras-dependent manner. However, the neuritogenic activities associated with cAMP are still not well understood. The purpose of this study was to investigate the potential neuritogenic activities mediated by cAMP. For this purpose, we used the human neuroblastoma cell line SH-SY5Y. These neuroblastoma cells respond to cAMP by forming neurite-like extensions. We tried to identify some essential pathways involved in the cAMP-induced neurite elongation of these cells. Our results indicated that PKA is transiently activated in this elongation model. When we blocked PKA activity, elongation did not take place. Similarly, PI3K also plays an essential role because when we blocked this kinase activity, there was no neurite elongation. Indeed, over-expression of the p110-catalytic subunit or an activating form of the p85-regulatory subunit (p65) is able to induce some degree of neurite extension. Moreover, our results showed that when elongation is initiated, PI3K is still essential for maintenance of the neuronal morphology, whereas PKA or MAPK (ERKs or p38) activation does not appear to be necessary during this process.

Cell Differentiation↗