Mutation of lysine 1370 in full-length human alpha2-macroglobulin blocks binding to the low density lipoprotein receptor-related protein-1

alpha2-Macroglobulin (alpha2M) regulates cell physiology by binding to cellular receptors; however, residues that contribute to receptor-binding have not been elucidated in the full-length protein. In alpha2M fragments, expressed in bacteria, Lys(1370) and Lys(1374) are critical for binding to the low density lipoprotein receptor-related protein-1 (LRP-1) and a distinct alpha2M-signaling receptor. We expressed full-length recombinant human alpha2M (r(alpha)2M) and mutants in which Lys(1370) or Lys(1374) was converted to alanine in K-562 cells. The r(alpha)2M species demonstrated intact structure and function, as determined by subunit size, intersubunit disulfide bonds, reaction with trypsin or methylamine, and ability to undergo conformational change. Binding of transforming growth factor-beta1 was unaltered. Mutation of Lys(1370) almost entirely inhibited specific binding of methylamine-activated r(alpha)2M to RAW 264.7 cells. Mutation of Lys(1374) had no effect. Binding of r(alpha)2M to RAW 264.7 cells was blocked by receptor-associated protein, indicating an essential role for LRP-1. These studies demonstrate that a single mutation in full-length r(alpha)2M is sufficient to block binding to LRP-1.     

Identical or overlapping sequences in the primary structure of human alpha(2)-macroglobulin are responsible for the binding of nerve growth factor-beta, platelet-derived growth factor-BB, and transforming growth factor-beta

alpha(2)-Macroglobulin (alpha(2)M) functions as a proteinase inhibitor and as a carrier of diverse growth factors. In this study, we localized binding sites for platelet-derived growth factor-BB (PDGF-BB) and nerve growth factor-beta (NGF-beta) to a linear sequence in the 180-kDa human alpha(2)M subunit which includes amino acids 591-774. A glutathione S-transferase fusion protein containing amino acids 591-774 (FP3) bound PDGF-BB and NGF-beta in ligand blotting assays whereas five other fusion proteins, which collectively include amino acids 99-590 and 775-1451 did not. The K(D) values for PDGF-BB and NGF-beta binding to immobilized FP3 were 300 +/- 40 and 180 +/- 30 nM, respectively; these values were comparable with those determined using methylamine-modified alpha(2)M, suggesting that higher-order alpha(2)M structure is not necessary for PDGF-BB and NGF-beta binding. PDGF-BB and NGF-beta blocked the binding of transforming growth factor-beta1 (TGF-beta1) to FP3. Furthermore, murinoglobulin, which is the only known member of the alpha-macroglobulin family that does not bind TGF-beta, also failed to bind PDGF-BB and NGF-beta. These results support the hypothesis that either a single linear sequence in human alpha(2)M or overlapping sequences are responsible for the binding of TGF-beta, PDGF-BB, and NGF-beta, even though there is minimal sequence identity between these three growth factors. FP3 blocked the binding of PDGF-BB to a purified chimeric protein, in which the extracellular domain of the PDGF beta receptor was fused to the IgG(1) Fc domain, and to PDGF receptors on NIH 3T3 cells. Thus, FP3 may inhibit the activity of PDGF-BB.     

Activated alpha(2)-macroglobulin induces cell proliferation and mitogen-activated protein kinase activation by LRP-1 in the J774 macrophage-derived cell line

The low-density lipoprotein receptor-related protein-1 (LRP-1) is an endocytic receptor of activated forms of the proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M*). It has been proposed that alpha(2)M* and LRP-1 modulate diverse cellular processes, including cell adhesion, proliferation, and migration, which are involved in inflammation and tumor progression. However, relatively little is known about the role of alpha(2)M*/LRP-1 interaction on these processes. In this work, we demonstrate that alpha(2)M* binding to LRP-1 induces cell proliferation and MAPK activation in the J774 macrophage-derived cell line, which were blocked by RAP, an antagonist of LRP-1-binding ligands, and by PD980059, a specific inhibitor for the Mek1-ERK1/2 pathway. In addition, we demonstrate that LPS, a bacterial product that it is known to down-regulate the LRP-1 expression on macrophage, abrogated the signaling activity triggered by alpha(2)M* on LPS-treated J774 cells. These results suggest that alpha(2)M*/LRP-1 interaction constitutes a key role in the macrophage functioning during inflammation and cancer.     

Identification and characterization of the acidic pH binding sites for growth regulatory ligands of low density lipoprotein receptor-related protein-1

The type V TGF-beta receptor (TbetaR-V) plays an important role in growth inhibition by IGFBP-3 and TGF-beta in responsive cells. Unexpectedly, TbetaR-V was recently found to be identical to the LRP-1/alpha(2)M receptor; this has disclosed previously unreported growth regulatory functions of LRP-1. Here we demonstrate that, in addition to expressing LRP-1, all cells examined exhibit low affinity but high density acidic pH binding sites for LRP-1 growth regulatory ligands (TGF-beta(1), IGFBP-3, and alpha(2)M(*)). These sites, like LRP-1, are sensitive to receptor-associated protein and calcium depletion but, unlike LRP-1, are also sensitive to chondroitin sulfate and heparin and capable of directly binding ligands, which do not bind to LRP-1. Annexin VI has been identified as a major membrane-associated protein capable of directly binding alpha(2)M(*) at acidic pH. This is evidenced by: 1) structural and Western blot analyses of the protein purified from bovine liver plasma membranes by alpha(2)M(*) affinity column chromatography at acidic pH, and 2) dot blot analysis of the interaction of annexin VI and (125)I-alpha(2)M(*). Cell surface annexin VI is involved in (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) binding to the acidic pH binding sites and (125)I-alpha(2)M(*) binding to LRP-1 at neutral pH as demonstrated by the sensitivity of cells to pretreatment with anti-annexin VI IgG. Cell surface annexin VI is also capable of mediating internalization and degradation of cell surface-bound (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) at pH 6 and of forming ternary complexes with (125)I-alpha(2)M(*) and LRP-1 at neutral pH as demonstrated by co-immunoprecipitation. Trifluoperazine and fluphenazine, which inhibit ligand binding to the acidic pH binding sites, block degradation after internalization of cell surface-bound (125)I-TGF-beta(1) or (125)I-alpha(2)M(*). These results suggest that cell surface annexin VI may function as an acidic pH binding site or receptor and may also function as a co-receptor with LRP-1 at neutral pH.     

A novel receptor function for the heat shock protein Grp78: silencing of Grp78 gene expression attenuates alpha2M*-induced signalling

The activated proteinase inhibitor alpha2-macroglobulin (alpha2M*) binds to two receptors, the low density lipoprotein receptor-related protein (LRP-1) and the alpha2M* signalling receptor (alpha2MSR). Silencing LRP-1 gene expression in macrophages by RNA interference does not block alpha2M* activation of signalling cascades. We now demonstrate that transfection of macrophages with a double-stranded RNA homologous in sequence to the Grp78 gene markedly decreased induction of inositol 1,4,5-trisphosphate (IP3) and subsequent IP3-dependent elevation of [Ca2+]i induced by alpha2M*. Concomitantly, alpha2M*-induced increase in [3H]thymidine uptake was abolished in these transfected cells. Insulin treatment significantly upregulates alpha2MSR and it also caused a marked increase in Grp78 expression which could be blocked by RNA interference. alpha2M* treatment of cells activates the Ras- and PI 3-kinase-dependent signalling pathways. Suppressing Grp78 expression leads to the loss of these activation events in transfected macrophages. We thus conclude that Grp78 is the alpha2M* signalling receptor.     

Akt-induced promotion of cell-cycle progression at G2/M phase involves upregulation of NF-Y binding activity in PC12 cells

Akt is a key downstream effector of the PI3K signaling pathway and plays a role in cell growth and survival. Expression of a myristoylated constitutively active form of Akt (myr-Akt) in PC12 cells could override cell-growth arrest at G2/M phase and apoptosis that were induced by etoposide treatment. On the other hand, inactivation of Akt by expression of its dominant negative mutant form (km-Akt) inhibited cell proliferation by arresting the cells at G2/M phase. Expression of myr-Akt also led to an increase in the protein and mRNA levels of CDK1 and cyclin B1. Furthermore, EMSA data revealed that expression of myr-Akt promoted the binding of NF-Y to the consensus CCAAT promoter sequence, whereas expression of km-Akt almost completely abolished it. Moreover, the Akt activity was minimal in the cells that were arrested at G2/M phase by nocodazole treatment, but reached to a maximal level as the cells progressed to mitosis and G1 phase upon removal of the drug. Treatment with Akt inhibitors, but not with those of MEK or p70S6K, blocked the release of the cells from the nocodazole-induced G2/M arrest, further revealing that the Akt activity is required for G2/M phase transition. These results suggest that Akt facilitate cell-cycle progression at G2/M phase in PC12 cells and this Akt activity is correlated with upregulation of NF-Y DNA-binding activity and cyclin B1/CDK1 gene expression.     

Possible role of stromal-cell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma

We examined the role of chemokine signaling on the lymph node metastasis of oral squamous cell carcinoma (SCC) using lymph node metastatic (HNt and B88) and nonmetastatic oral SCC cells. Of 13 kinds of chemokine receptors examined, only CXCR4 expression was up-regulated in HNt and B88 cells. CXCR4 ligand, stromal-cell-derived factor-1alpha (SDF-1alpha; CXCL12), induced characteristic calcium fluxes and chemotaxis only in CXCR4-expressing cells. CXCR4 expression in metastatic cancer tissue was significantly higher than that in nonmetastatic cancer tissue or normal gingiva. Although SDF-1alpha was undetectable in either oral SCC or normal epithelial cells, submandibular lymph nodes expressed the SDF-1alpha protein, mainly in the stromal cells, but occasionally in metastatic cancer cells. The conditioned medium from lymphatic stromal cells promoted the chemotaxis of B88 cells, which was blocked by the CXCR4 neutralization. SDF-1alpha rapidly activated extracellular signal-regulated kinase (ERK)1/2 and Akt/protein kinase B (PKB), and their synthetic inhibitors attenuated the chemotaxis by SDF-1alpha. SDF-1alpha also activated Src family kinases (SFKs), and its inhibitor PP1 diminished the SDF-1alpha-induced chemotaxis and activation of both ERK1/2 and Akt/PKB. These results indicate that SDF-1/CXCR4 signaling may be involved in the establishment of lymph node metastasis in oral SCC via activation of both ERK1/2 and Akt/PKB induced by SFKs.     

Potentiation of signal transduction mitogenesis and cellular proliferation upon binding of receptor-recognized forms of alpha2-macroglobulin to 1-LN prostate cancer cells

The alpha2-macroglobulin signalling receptor is upregulated in highly metastatic 1-LN prostate cancer cells. Stimulation of 1-LN cells with activated alpha2-macroglobulin (alpha2M*) caused a two- to threefold increase in [3H]thymidine uptake and cell number. These events require the Ras-dependent MAPK and PI 3-kinase/Akt signalling cascades. Incubation of 1-LN cells with alpha2M* induced Grb2, shc, sos and Raf-1 expression, as well as phosphorylation of MEK 1/2, ERK 1/2, p38 MAPK and JNK. This treatment also increased PI 3-kinase activation, PDK1 expression, Akt phosphorylation and p70s6k phosphorylation. Levels of the early gene products c-fos protein and thymidylate synthase were comparably increased. Exposure of 1-LN cells to alpha2M* significantly raised the levels of phosphorylated CREB by about 15-20 min and phosphorylated p53 by about 60-90 min of incubation. We conclude that the growth regulatory effects of ligating the alpha2M* signalling receptor on 1-LN cells are exerted via the onset and crosstalk between the Ras-dependent MAPK and PI 3-kinase/Akt signalling cascades.     

Human Sin1 contains Ras-binding and pleckstrin homology domains and suppresses Ras signalling

Human Sin1 (SAPK-interacting protein 1) is a member of a conserved family of orthologous proteins that have an essential role in signal transduction in yeast and Dictyostelium. This study demonstrates that most Sin1 orthologues contain both a Raf-like Ras-binding domain (RBD) and a pleckstrin homology (PH) domain. These domains are functional in the human Sin1 protein, with the PH domain involved in lipid and membrane binding by Sin1, and the RBD able to bind activated H-and K-Ras. Sin1 and Ras co-immunoprecipitated and co-localised, showing that these proteins associate with each other in vivo. Overexpression of Sin1 inhibited the activation of ERK, Akt and JNK signalling pathways by Ras. In contrast, siRNA knockdown of endogenous Sin1 protein expression in HEK293 cells enhanced the activation of ERK1/2 by Ras. These data suggest that Sin1 is a mammalian Ras-inhibitor.     

Induction of neurite extension and survival in pheochromocytoma cells by the Rit GTPase

The Rit, Rin, and Ric proteins comprise a distinct and evolutionarily conserved subfamily of the Ras-like small G-proteins. Although these proteins share the majority of core effector domain residues with Ras, recent studies suggest that Rit uses novel effector pathways to regulate NIH3T3 cell proliferation and transformation, while the functions of Rin and Ric remain largely unknown. Since we demonstrate that Rit is expressed in neurons, we investigated the role of Rit signaling in promoting the differentiation and survival of pheochromocytoma cells. In this study, we show that expression of constitutively active Rit (RitL79) in PC6 cells results in neuronal differentiation, characterized by the elaboration of an extensive network of neurite-like processes that are morphologically distinct from those mediated by the expression of oncogenic Ras. Although activated Rit fails to stimulate mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways in COS cells, RitL79 induced the phosphorylation of ERK1/2 in PC6 cells. We also find that Rit-mediated effects on neurite outgrowth can be blocked by co-expression of dominant-negative mutants of C-Raf1 or mitogen-activated protein kinase kinase 1 (MEK1). Moreover, expression of dominant-negative Rit is sufficient to inhibit NGF-induced neurite outgrowth. Expression of active Rit inhibits growth factor-withdrawal mediated apoptosis of PC6 cells, but does not induce phosphorylation of Akt/protein kinase B, suggesting that survival does not utilize the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Instead, pharmacological inhibitors of MEK block Rit-stimulated cell survival. Taken together, these studies suggest that Rit represents a distinct regulatory protein, capable of mediating differentiation and cell survival in PC6 cells using a MEK-dependent signaling pathway to achieve its effects.     

Activation and cross-talk between Akt, NF-kappaB, and unfolded protein response signaling in 1-LN prostate cancer cells consequent to ligation of cell surface-associated GRP78

Binding of activated forms of the proteinase inhibitor alpha2-macroglobulin (alpha2M*) to cell surface-associated GRP78 on 1-LN human prostate cancer cells causes their proliferation. We have now examined the interplay between Akt activation, regulation of apoptosis, the unfolded protein response, and activation of NF-kappaB in alpha2M*-induced proliferation of 1-LN cells. Exposure of cells to alpha2M* (50 pM) induced phosphatidylinositol 3-kinase-dependent activation of Akt by phosphorylation at Thr-308 and Ser-473 with a concomitant 60-80% increase in Akt-associated kinase activity. ERK1/2 and p38 MAPK were also activated, but there was only a marginal effect on JNK activation. Treatment of 1-LN cells with alpha2M* down-regulated apoptosis and promoted NF-kappaB activation as shown by increases of Bcl-2, p-Bad(Ser-136), p-FOXO1(Ser-253), p-GSK3beta(Ser-9), XIAP, NF-kappaB, cyclin D1, GADD45beta, p-ASK1(Ser-83), and TRAF2 in a time of incubation-dependent manner. alpha2M* treatment of 1-LN cells, however, showed no increase in the activation of caspase -3, -9, or -12. Under these conditions, we observed increased unfolded protein response signaling as evidenced by elevated levels of GRP78, IRE1alpha, XBP-1, ATF4, ATF6, p-PERK, p-eIF2alpha, and GADD34 and reduced levels of GADD153. Silencing of GRP78 gene expression by RNAi suppressed activation of Akt(Thr-308), Akt(Ser-473), and IkappaB kinase alpha kinase. The effects of alpha2M* on the NF-kappaB activation, antiapoptotic signaling, unfolded protein response signaling, and proapoptotic signaling were also reversed by this treatment. In conclusion, alpha2M* promotes cellular proliferation of 1-LN prostate cancer cells by activating MAPK and Akt-dependent signaling, down-regulating apoptotic signaling, and activating unfolded protein response signaling.     

Limited mutations in full-length tetrameric human alpha2-macroglobulin abrogate binding of platelet-derived growth factor-BB and transforming growth factor-beta1

alpha2-Macroglobulin (alpha2M) inhibits diverse extracellular proteases, binds growth factors such as platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1), and carries beta-amyloid peptide. alpha2M may also trigger cell signaling by binding to the low density lipoprotein receptor-related protein (LRP-1) and/or other cell surface receptors. Based on studies with recombinant alpha2M fragments expressed in bacteria and synthetic peptides, we previously localized a growth factor-binding site near the center of the alpha2M subunit. However, because intact alpha2M forms a hollow cylinder structure, an alternative model for growth factor binding involves nonspecific entrapment within the alpha2M core. To distinguish between these two models, we engineered mutations in the putative growth factor binding sequence of full-length alpha2M. These mutations did not perturb the tetrameric structure of alpha2M, reaction with proteases, the thiol ester bonds, or binding to LRP-1. A single mutation (E730R) completely blocked binding of platelet-derived growth factor-BB to intact alpha2M. E730R did not alter TGF-beta1 binding; however, this mutation in combination with mutations at Glu714 and Asp719 eliminated the increase in TGF-beta1 binding associated with alpha2M conformational change. These studies demonstrate that growth factor binding to intact alpha2M is specific, involving a defined region of the alpha2M subunit. The exact sequences required for binding different growth factors may be non-identical, mimicking the model of the bait region in which different proteases target adjacent and sometimes overlapping sequences.     

Differential functions of PKC-delta and PKC-zeta in cisplatin response of normal and transformed thyroid cells

We investigated the effects of cisplatin (cisPt) in normal PC Cl3 and in transformed and tumourigenic PC E1Araf cells. cisPt cytotoxicity was higher in PC Cl3 than in PC E1Araf cells. In both cell lines, cisPt provoked the ERK1/2 phosphorylation; this was unaltered by G?6976, a conventional PKC inhibitor, whilst it was blocked by low doses (0.1 microM) or high doses (10 microM) of GF109203X, an inhibitor of all PKC isozymes, in PC Cl3 and in PC E1Araf cells, respectively. In PC E1Araf, the cisPt-provoked ERK phosphorylation was also blocked by the use of a myristoylated PKC-zeta pseudosubstrate peptide. Conversely, in PC Cl3 the cisPt-provoked ERK phosphorylation was blocked by the use of rottlerin, a PKC-delta inhibitor. Results show that cisPt activates both PKC (the -delta and the -zeta isozymes in PC Cl3 and in PC E1Araf cells, respectively) and ERK in association with prolonged survival of thyroid cell lines.     

CXCL13 mediates prostate cancer cell proliferation through JNK signalling and invasion through ERK activation

Objectives: The focus of this study was to determine the dedicator of cytokinesis 2 (DOCK2), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase-1 (JNK) and Akt signals involved in CXCL13-mediated prostate cancer (PCa) cell invasion and proliferation. Materials and methods: Androgen-sensitive (LNCaP), hormone-refractory (PC3) cells and normal cells (RWPE-1) were used to determine CXCL13-mediated PCa cell invasion and proliferation. Immuno-blotting, fast activated cell-based (FACE) ELISA, caspase activity, cell invasion and proliferation assays were performed to ascertain some of the signalling events involved in PCa cell proliferation and invasion. Results: Unlike androgen-sensitive LNCaP cells, we report for the first time that the hormone-refractory cell line, PC3, expresses DOCK2. CXCL13-mediated LNCaP and PC3 cell invasion was regulated by Akt and ERK1/2 activation in a DOCK2-independent fashion. CXCL13 also promoted LNCaP cell proliferation in a JNK-dependent fashion even in the absence of DOCK2. In contrast, CXCL13 induced PC3 cell proliferation through JNK activation, which required DOCK2. Conclusions: Our results show CXCL13-mediated PCa cell invasion requires Akt and ERK1/2 activation and suggests a new role for DOCK2 in proliferation of hormone-refractory CXCR5-positive PCa cells.     

Apigenin and LY294002 prolong EGF-stimulated ERK1/2 activation in PC12 cells but are unable to induce full differentiation

In rat pheochromocytoma cell line (PC12) cells, initial epidermal growth factor (EGF)-stimulated extracellular signal-regulated protein kinases 1/2 (ERK1/2) phosphorylation was similar to that promoted by nerve growth factor (NGF), but declined rapidly. Pre-treatment with apigenin or LY294002 sustained EGF-stimulated ERK1/2 phosphorylation whereas wortmannin partially blocked initial ERK1/2 phosphorylation. Changes in ERK1/2 phosphorylation correlated with alterations in p90 ribosomal S6 kinase activity. Wortmannin, LY294002 and apigenin totally blocked growth factor-induced protein kinase B phosphorylation. However, none of them potentiated Raf activation, which was in fact decreased by LY290042 and wortmannin. The sustained EGF-induced ERK1/2 activation promoted by apigenin was not sufficient to commit PC12 cells to differentiate, which was achieved by stimulation with NGF, either alone or in the presence of apigenin.     

GM1 Ganglioside Activates ERK1/2 and Akt Downstream of Trk Tyrosine Kinase and Protects PC12 Cells Against Hydrogen Peroxide Toxicity

Ganglioside GM1 at micro- and nanomolar concentrations was shown to increase the viability of pheochromocytoma PC12 cells exposed to hydrogen peroxide and diminish the accumulation of reactive oxygen species and oxidative inactivation of Na⁺,K⁺-ATPase, the effects of micromolar GM1 being more pronounced than those of nanomolar GM1. These effects of GM1 were abolished by Trk receptor tyrosine kinase inhibitor and diminished by MEK1/2, phosphoinositide 3-kinase and protein kinase C inhibitors. Hydrogen peroxide activates Trk tyrosine kinase; Akt and ERK1/2 are activated downstream of this protein kinase. GM1 was found to activate Trk receptor tyrosine kinase in PC12 cells. GM1 (100 nM and 10 µM) increased the basal activity of Akt, but did not change Akt activity in cells exposed to hydrogen peroxide. Basal ERK1/2 activity in PC12 cells was increased by GM1 at a concentration of 10 µM, but not at nanomolar concentrations. Activation of ERK1/2 by hydrogen peroxide was enhanced by GM1 at a concentration of 10 µM and to a lesser extent at a concentration of 100 nM. Thus, the protective and metabolic effects of GM1 ganglioside on PC12 cells exposed to hydrogen peroxide appear to depend on the activation of Trk receptor tyrosine kinase and downstream activation of Akt and ERK1/2.     

Regulation of Rac1 activation by the low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1-deficient MEFs demonstrated increased Rac1 activation compared with LRP-1-expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR-/- MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK- cells. Untreated uPAR+/+ MEFs demonstrated substantially increased Rac1 activation compared with uPAR-/- MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signal-regulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked.     

A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation

Activation of phosphatidylinositol 3-kinase (PI3-K) is considered to be a key event upon stimulation of cells with growth factors. Akt is known to be a downstream target of PI3-K when it is activated by nerve growth factor (NGF). NGF induces cell differentiation of PC12 cells as indicated by neurite outgrowth. In order to investigate the role of PI3-K/Akt in NGF-induced differentiation of PC12 cells, we generated cells ectopically expressing constitutively activated (CA), wild type (WT) and dominant negative (DN) forms of Akt. NGF-induced neurite outgrowth was greatly accelerated in the cells expressing CA-Akt, and dramatically inhibited in those expressing DN-Akt. Pre-treatment with an Akt inhibitor, ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine], inhibited NGF-induced Akt phosphorylation as well as neurite outgrowth but did not markedly affect the activities of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The PI3-K inhibitors wortmannin and LY294002 blocked NGF-induced Akt phosphorylation as well as neurite outgrowth. These results indicate that PI3-K/Akt is a positive regulator of NGF-induced neuronal differentiation in PC12 cells.