BBA - Molecular Cell Research (v.1745, #3)

A number of recent studies have provided new insights into the complexity of the endocytic pathways originating at the plasma membrane of mammalian cells. Many of the molecules involved in clathrin coated pit internalization are now well understood but other pathways are less well defined. Caveolae appear to represent a low capacity but highly regulated pathway in a restricted set of tissues in vivo. A third pathway, which is both clathrin- and caveolae-independent, may constitute a specialized high capacity endocytic pathway for lipids and fluid. The relationship of this pathway, if any, to macropinocytosis or to the endocytic pathways of lower eukaryotes remains an interesting open question. Our understanding of the regulatory mechanisms and molecular components involved in this pathway are at a relatively primitive stage. In this review, we will consider some of the characteristics of different endocytic pathways in high and lower eukaryotes and consider some of the common themes in endocytosis. One theme which becomes apparent from comparison of these pathways is that apparently different pathways can share common molecular machinery and that pathways considered to be distinct actually represent similar basic pathways to which additional levels of regulatory complexity have been added.
Keywords: Endocytosis; Lipid raft; Caveolae; Cholesterol; Clathrin;

Signaling via NF-κB in the nervous system by Barbara Kaltschmidt; Darius Widera; Christian Kaltschmidt (287-299).
Nuclear factor kappa B (NF-κB) is an inducible transcription factor present in neurons and glia. Recent genetic models identified a role for NF-κB in neuroprotection against various neurotoxins. Furthermore, genetic evidence for a role in learning and memory is now emerging. This review highlights our current understanding of neuronal NF-κB in response to synaptic transmission and summarizes potential physiological functions of NF-κB in the nervous system. This article contains a listing of NF-κB activators and inhibitors in the nervous system, furthermore specific target genes are discussed. Synaptic NF-κB activated by glutamate and Ca2+ will be presented in the context of retrograde signaling. A controversial role of NF-κB in neurodegenerative diseases will be discussed. A model is proposed explaining this paradox as deregulated physiological NF-κB activity, where novel results are integrated, showing that p65 could be turned from an activator to a repressor of anti-apoptotic genes.
Keywords: Transcription factor; Neuron; Neuroprotection; Memory; Tumor necrosis factor; Calcium; Synapse; Retrograde; Inhibitor; Activator; Target gene; NF-kappaB; IkappaB;

Estrogens are known to induce the expression of the receptor for advanced glycation end products (RAGE). In the current investigation, we examined the effect of three estrogens with different potency for specific estrogen receptors (ER) on RAGE expression in human microvascular endothelial cells (HMEC-1). Of the three estrogens tested, ethinyl estradiol (EE), an estrogen receptor alpha (ERα) agonist, was the strongest inducer of RAGE expression in HMEC-1. By comparison, 17-epiestriol, an estrogen receptor beta (ERβ) agonist and 17-β-E2, an ER agonist that is almost equally potent for ERα and ERβ were less effective in stimulating RAGE expression. We then determined whether the prooxidative and proinflammatory transcription factors Sp1 or NF-κB were downstream modulators of ER-agonists that mediate RAGE expression. The results implicated Sp1 but not NF-κB in estrogen-dependent RAGE expression. We further demonstrated that ERα but not ERβ was responsible for the estrogen-mediated Sp1 activation. In summary, the present investigation demonstrates that a direct interaction of EE-ERα-Sp1 plays a central role in estrogen-induced RAGE expression in HMEC-1.
Keywords: Estrogen; RAGE; ERα; ERβ; NF-κB; Sp1 and microvascular endothelial cell;

Secretory leukocyte protease inhibitor (SLPI), a potent serine protease inhibitor, has been shown to suppress macrophage responses to bacterial lipopolysaccharide (LPS). SLPI contains two topologically superimposable domains. Its C-terminal domain binds and inhibits target proteases. It is not clear whether SLPI's anti-protease function plays a role in the LPS-inhibitory action of SLPI. Four single amino acid substitution mutants of SLPI, M73G, M73F, M73E and M73K, were generated. Wild type SLPI is a potent inhibitor of chymotrypsin and elastase. Mutants M73G and M73F selectively lost inhibitory function towards chymotrypsin and elastase, respectively, whereas mutants M73K and M73E inhibited neither elastase nor chymotrypsin. Macrophage cell lines were established from RAW264.7 cells to stably express each SLPI mutant. Expression of the SLPI protease inhibition mutants suppressed NO and TNF production in response to LPS in a similar fashion as wild type SLPI. Expression of truncated forms of SLPI, containing only its N-terminus or its C-terminus, was similarly sufficient to confer inhibition of LPS responses. Thus, the LPS-inhibitory action of SLPI is independent of its anti-protease function.
Keywords: Macrophage; SLPI mutant; Lipopolysaccharide; Inflammation;

Sodium butyrate sensitises human pancreatic cancer cells to both the intrinsic and the extrinsic apoptotic pathways by Federica Natoni; Laura Diolordi; Claudio Santoni; Maria Saveria Gilardini Montani (318-329).
Pancreatic cancer is characterised by a highly malignant phenotype with a marked resistance to conventional therapies and to apoptotic activators. Here, we demonstrate that sodium butyrate (NaBt), an inhibitor of histone deacetylases, sensitises human pancreatic cancer cell lines to both mitochondria- and Fas-mediated apoptosis. The analysis of anti-apoptotic and pro-apoptotic members of the Bcl-2 family in untreated pancreatic cancer cell lines shows a generalised low expression of Bcl-2 and a strong expression of Bcl-xL. NaBt treatment results in a marked down-regulation of Bcl-xL expression, mitochondrial membrane depolarization, cytochrome c release from mitochondria, activation of caspase-9 and -3 and apoptosis induction. Furthermore, NaBt sensitises pancreatic cancer cells to Fas-mediated apoptosis as well. In fact, the combined treatment with NaBt and the agonistic antibody anti-Fas (CH11) is able to induce apoptosis at an early time, in which neither NaBt nor CH11 alone induce apoptosis. Down-regulation of FLIP and activation of caspase-8 allow apoptosis to occur. These findings suggest that sodium butyrate could represent a good candidate for the development of new therapeutic strategies aimed at improving chemotherapy and immunotherapy in pancreatic cancer.
Keywords: Apoptosis; Pancreatic cancer; Sodium butyrate; Fas; FLIP; Bcl-2 family;

Phosphatidylinositol 3-phosphate, PtdIns(3)P, is a phosphoinositide which is implicated in regulating membrane trafficking in both mammalian and yeast cells. It also serves as a precursor for the synthesis of phosphatidylinositol 3,5-bisphosphate, PtdIns(3,5)P 2, a phosphoinositide, the exact functions of which remain unknown. In this report, we show that these two phosphoinositides are constitutive lipid components of the ciliate Tetrahymena. Using HPLC analysis, PtdIns(3)P and PtdIns(3,5)P 2 were found to comprise 16% and 30–40% of their relevant phosphoinositide pools, respectively. Treatment of Tetrahymena cells with wortmannin (0.1–10 μM) resulted in the depletion of PtdIns(3)P and PtdIns(3,5)P 2 without any effect on D-4 phosphoinositides. Wortmannin was further used for the investigation of D-3 phosphoinositide involvement in the regulation of lysosomal vesicular trafficking. Incubation of Tetrahymena cells with wortmannin resulted in enhanced secretion of two different lysosomal enzymes without any change in their total activities. Experiments performed with a T. thermophila secretion mutant strain verified that the wortmannin-induced secretion is specific and it is not due to a diversion of lysosomal enzymes to other secretory pathways. Moreover, experiments performed with a phagocytosis-deficient T. thermophila strain showed that a substantial fraction of wortmannin-induced secretion was dependent on the presence of functional phagosomes/phagolysosomes.
Keywords: Tetrahymena; Phosphoinositide 3-kinase; Wortmannin; LY294002; Lysosome; Trafficking;

Proline-rich tyrosine kinase 2 (Pyk2) is activated in neurones following NMDA receptor stimulation via PKC. Pyk2 is involved in hippocampal LTP and acts to potentiate NMDA receptor function. Elevations of intracellular Ca2+ and cAMP levels are key NMDA receptor-dependent triggering events leading to induction of hippocampal LTP. In this study, we compared the ability of A23187 (Ca2+ ionophore) or forskolin (adenylate cyclase activator) to modulate the phosphorylation of Pyk2 in rat hippocampal slices. Using an immunoprecipitation assay, phosphorylated Pyk2 levels were increased following treatment with A23187, levels peaking at around 10 min. Staurosporine, at concentrations inhibiting conventional and novel isoforms of PKC, and chelerythrine, at concentrations inhibiting the atypical PKC isoform PKMξ, were compared for their ability to attenuate the effect of A23187. Exposure of acute hippocampal slices to either chelerythrine or staurosporine completely blocked enhanced phosphorylation of Pyk2 by A23187, suggesting a possible involvement of PKMξ and typical PKCs in Pyk2 activation by Ca2+. In contrast, application of forskolin reduced phosphorylated Pyk2 below basal levels, suggesting that cAMP inhibits Pyk2. These results implicate Ca2+ and multiple forms of PKC in the activation of Pyk2 downstream of NMDA receptors and suggest that cAMP-dependent processes exert a suppressive action on Pyk2.
Keywords: Proline-rich tyrosine kinase; Hippocampus; LTP; Protein kinase C;

In atherosclerosis, abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role to form fibroproliferative lesions and platelet-derived growth factor (PDGF)-BB is one of the most potent chemoattractants and proliferative factors for VSMCs. Taurine, sulfur-containing β-amino acid, has been considered to prevent the development of atherosclerosis, although the molecular mechanism remains obscure. Previously, we demonstrated that taurine significantly suppressed PDGF-BB-induced cell proliferation, DNA synthesis, immediate-early gene expressions and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in VSMCs. The present study was aimed at elucidating the precise molecular mechanism of taurine in PDGF-BB signaling pathway. We showed that taurine significantly suppressed PDGF-BB-induced phosphorylation of PDGF-β receptor and activation of its downstream signaling molecules such as Ras, MAPK/ERK kinase (MEK)1/2 and Akt. Because taurine did not attenuate phorbol 12-myristate 13-acetate (PMA)-induced PDGF-β receptor-independent ERK1/2 phosphorylation, we further investigated the suppressive mechanism of taurine in PDGF-β receptor level. Although taurine did not directly affect PDGF receptor autophosphorylation in vitro, taurine promoted PDGF-β  receptor dephosphorylation and restored PDGF-BB-induced suppression of protein tyrosine phosphatase (PTPase) activity. Taken together, we propose that taurine could prevent or delay the progression of atherosclerosis by PTPase-mediated suppression of PDGF-β receptor phosphorylation, and by decreasing the activation of its downstream signaling molecules in VSMCs.
Keywords: Atherosclerosis; Platelet-derived growth factor; Platelet-derived growth factor receptor; Protein tyrosine phosphatase; Taurine; Vascular smooth muscle cell;

The atherosclerotic plaque is an inflammatory site where macrophage cells are exposed to cytotoxic oxidised low density lipoprotein (oxLDL). Interferon-γ released from T-cells results in macrophage synthesis of 7,8-dihydroneopterin which has antioxidant and cytoprotective activity. Using the human derived monocyte-like U937 and THP-1 cell lines, we examined whether 7,8-dihydroneopterin could inhibit the cytotoxic effect of oxLDL. In U937 cells, oxLDL caused a dramatic loss of cellular glutathione and caspase independent cell death associated with phosphatidylserine exposure on the plasma membrane. 7,8-Dihydroneopterin completely blocked the cytotoxic effect of oxLDL. In contrast, oxLDL initiated THP-1 cell apoptosis with reduction in cellular thiols, caspase-3 activation and plasma membrane phosphatidylserine exposure. 7,8-Dihydroneopterin was unable to alter these processes or restore the THP-1 cellular thiol content. 7,8-Dihydroneopterin did provide some protection to both THP-1 cells and U937 cells from AAPH derived peroxyl radicals. The preincubation of oxLDL with 7,8-dihydroneopterin did not reduce cytotoxicity, suggesting that 7,8-dihydroneopterin may be acting in U937 cells by scavenging intracellular oxidants generated by the oxLDL. The data show that μM levels of 7,8-dihydroneopterin may prevent oxLDL mediated cellular death within atherosclerotic plaques.
Keywords: Neopterin; Oxidised-low-density-lipoprotein; Macrophage; Caspase; Apoptosis; Glutathione;

β-catenin signaling and regulation of cyclin D1 promoter in NRK-49F cells transformed by down-regulation of the tumor suppressor lysyl oxidase by Monia Giampuzzi; Roberta Oleggini; Chris Albanese; Richard Pestell; Armando Di Donato (370-381).
Lysyl oxidase is the enzyme that is essential for collagen and elastin cross-linking. Previous investigations showed that lysyl oxidase is down-regulated in many human tumors and ras-transformed cells. Recently, we proved that antisense down-regulation of lysyl oxidase in NRK-49F cells induced phenotypic changes and oncogenic transformation, characterized by p21ras activation and β-catenin/cyclin D1 up-regulation. In the present paper, we examined β-catenin intracellular distribution and its association with E-cadherin. We observed an increased association between E-cadherin and β-catenin in the lysyl-oxidase down-regulated cells during serum starvation. Moreover, we found that β-catenin cytoplasmic and nuclear levels were increased, suggesting a failure of its down-regulation by the APC-GSK-3β system, in particular the GSK-3β phosphorylation of ser-33/37 and thr-41 of β-catenin. Finally, we investigated the mechanisms leading to the observed cyclin D1 up-regulation. We showed that in the antisense lysyl oxidase cells the cyclin D1 promoter was activated through the LEF and the ATF/CRE sites in the proximal promoter. While the promoter activation through LEF is compatible with β-catenin signaling, we investigated the possibility that the CRE-dependent activation might be linked to the down-regulation of lysyl oxidase. In fact, up-regulation of lysyl oxidase in a COS-7 cell model showed a significant diminution of the CREB protein binding to the cyclin D1 promoter, leading to a dramatic inhibition of its activity and a significant down-regulation of cyclin D1 protein level in vivo. Finally, our study describes some major anomalies occurring in lysyl oxidase down-regulated fibroblasts, related to β-catenin signaling and cyclin D1 expression.
Keywords: Lysyl oxidase; Cross-link; Gene promoter regulation; ras; β-catenin; Cyclin D1; TCF; CREB;

The localization of estrogen receptors alpha (ERα) and beta (ERβ) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In both cell types, ERα was localized mainly in the nucleus, particularly concentrated on nuclear structures, which on the basis of their staining with pyronin and with antibodies against the nucleoli-specific Ki67 antigen and C23-nucleolin, were characterized as nucleoli. A faint, diffuse ERα staining was also observed in the cytoplasm. ERβ was specifically enriched at the site of the mitochondria, visualized by labelling with the vital dye CMX and antibody against the mitochondrial-specific cytochrome oxidase subunit I. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of ERα and ERβ. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of ERα in nucleolar-related processes.
Keywords: Estrogen receptor α; Estrogen receptor β; Mitochondrion; SaOS-2; HepG2;

Cyclooxygenase (COX)-2 expression in intestinal epithelial cells is associated with colorectal carcinogenesis. COX-2 expression is induced by numerous growth factors and gastrointestinal hormones through multiple protein kinase cascades. Here, the role of mitogen activated protein kinases (MAPKs) and small GTPases in COX-2 expression was investigated. Anisomycin and sorbitol induced COX-2 expression in non-transformed, intestinal epithelial IEC-18 cells. Both anisomycin and sorbitol activated p38MAPK followed by phosphorylation of CREB. SB202190 and PD169316 but neither PD98059 nor U0126 blocked COX-2 expression and CREB phosphorylation by anisomycin or sorbitol. Clostridium difficile toxin B inhibition of small GTPases did not affect anisomycin-induced COX-2 mRNA expression or phosphorylation of p38MAPK and CREB but did inhibit sorbitol-dependent COX-2 expression and phosphorylation of p38MAPK and CREB. Angiotensin (Ang) II-dependent induction of COX-2 mRNA and induced phosphorylation of p38MAPK and CREB were inhibited by toxin B. Reduction of CREB protein in cells transfected with CREB siRNAs inhibited anisomycin-induced COX-2 expression. These results indicate that activation of p38MAPK signaling is sufficient for COX-2 expression in IEC-18 cells. Ang II and sorbitol require small GTPase activity for COX-2 expression via p38MAPK while anisomycin-induced COX-2 expression by p38MAPK does not require small GTPases. This places small GTPase activity down-stream of the AT1 receptor and hyperosmotic stress and up-stream of p38MAPK and CREB.
Keywords: Cyclooxygenase-2; p38MAPK; CREB; anisomycin; Gene expression;

Rapid inhibition of MAPK signaling and anti-proliferation effect via JAK/STAT signaling by interferon-α in hepatocellular carcinoma cell lines by Kentaro Inamura; Yasushi Matsuzaki; Naoya Uematsu; Akira Honda; Naomi Tanaka; Kazuhiko Uchida (401-410).
The potential anti-proliferation effect of interferon-alpha (IFN-α) against hepatocellular carcinoma (HCC) and its growth inhibitory mechanisms remain unclear. We examined four human HCC cell lines and every cell line had the anti-proliferative effect of IFN-α. The PLC/PRF/5 cell line, which expressed the IFN receptor most abundantly, responded most effectively to IFN-α stimulation. Here, we delineate the anti-proliferative effect of IFN-α via the MAPK pathway in human HCC cell lines. IFN-α retarded G1/S transition with no evidence of apoptosis and inhibited cell proliferation. IFN-α diminished the phosphorylation of both extracellular signal-regulated kinase (ERK) and mitogen-activated ERK-regulating kinase (MEK), but not Raf, within 5 min. Knockdown of signal transducers of activation and transcription1 (STAT1) or Janus kinase1 (JAK1) suppressed the reduction of phosphorylation both of ERK and MEK and diminished the growth inhibition by IFN-α. These results suggest that IFN-α induces anti-proliferative signaling via the JAK/STAT pathway downstream of IFN-α receptors and may reduce the growth stimulation signaling by cross-talk with the MEK/ERK pathway without IFN-α-induced transcription.
Keywords: Interferon (IFN); Extracellular signal-regulated kinase (ERK); Mitogen-activated ERK-regulating kinase (MEK); Signal transducers of activation and transcription1 (STAT1); Janus kinase1 (JAK1); Small interfering RNA (siRNA);