BBA - Molecular Cell Research (v.1643, #1-3)

Measuring the cell death induced by tumor necrosis factor (TNF-α) in L929 cells, we discovered for the first time that l-cycloserine, an established inhibitor of serine palmitoyltransferase, as well as dl-threo-dihydrosphingosine (threo-DHS, threo-sphinganine) significantly protected against TNF-α-induced cytotoxicity. Under the same conditions sphingosine and dl-erythro-dihydrosphingosine (erythro-DHS) did not change TNF-α-induced cytotoxicity, thus underlining the specificity of threo-DHS. In serine-labeled cells, newly (de novo) synthetized labeled ceramide was significantly diminished by threo-DHS alone or together with TNF-α, which makes the (dihydro) ceramide synthase the likely target of threo-DHS. These results suggest the decisive role of ceramide de novo synthesis in TNF signaling.
Keywords: Ceramide de novo synthesis; Safingol; Sphinganine; Sphingosine; Threo-dihydrosphingosine; Tumor necrosis factor-α (TNF-α);

Roc, a Ras/GTPase domain in complex proteins by Leonard Bosgraaf; Peter J.M. Van Haastert (5-10).
We identified a novel group of the Ras/GTPase superfamily, termed Roc, that is present as domain in complex proteins together with other domains, including leucine-rich repeats (LRRs), ankyrin repeats, WD40 repeats, kinase domains, RasGEF and RhoGAP domains. Roc is always succeeded by a novel 300–400-amino-acid-long domain, termed COR. Proteins with Roc/COR are present in prokaryotes, Dictyostelium, plants and metazoa.
Keywords: Roc; Ras/GTPase; Protein;

Cytokinins are important purine derivatives that act as hormones to control many processes in plants. Cytokinins such as isopentenyladenine (IPA), kinetin and benzyladenine were very effective at inducing the granulocytic differentiation of human myeloid leukemia HL-60 cells. The metabolism of cytokinins to their nucleotides was closely associated with cytokinin-induced differentiation and growth inhibition. When the cells were incubated with [14C]-benzyladenine, radioactivity was significantly incorporated into RNA and DNA. However, the radioactive nucleotides in RNA or DNA were adenine nucleotides, not benzyladenine nucleotides, suggesting that cytokinins were not incorporated into RNA and DNA. The benzyladenine nucleotides were not stably released into the medium in intact form. Cytokinins effectively induced a phosphorylated (active) form of mitogen-activated protein kinase (MAPK). MAPK activation was necessary for cytokinin-induced differentiation, because PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by cytokinins. These results suggest that cytokinin nucleotides themselves play an important role in inducing the differentiation of HL-60 cells.
Keywords: Cytokinin; Plant hormone; Nucleotide; Metabolism; Granulocytic differentiation; Mitogen-activated protein kinase; Leukemia cell;

The major component of the epidermis, keratinocytes, must continuously proliferate and differentiate to form the mechanical and water permeability barrier of the skin. Our previous data have suggested a potential role in these processes for phospholipase D (PLD), an enzyme that hydrolyzes phospholipids to generate phosphatidic acid. In the presence of primary alcohols, PLD also catalyzes a transphosphatidylation reaction to produce phosphatidylalcohols, and this characteristic has been exploited to monitor the activity of PLD in intact cells. In this report, PLD was demonstrated to utilize the physiological alcohol glycerol to form phosphatidylglycerol (PG) in vitro. In intact primary murine epidermal keratinocytes treated for 24 h with elevated extracellular Ca2+ levels, but not 1,25-dihydroxyvitamin D3, incubation with radioactive glycerol resulted in an increase in PLD-mediated radiolabeled PG production. This effect was dose-dependent and biphasic, with maximal PG formation detected after exposure to an intermediate (125 μM) Ca2+ concentration. Furthermore, the biphasic nature of the response was due, in part, to a corresponding biphasic change in glycerol uptake. Finally, short-term treatment of keratinocytes with phorbol 12-myristate 13-acetate (PMA) failed to increase PG synthesis and inhibited glycerol uptake. Since (1) PMA is reported to activate PLD-1 to a greater extent than PLD-2, (2) 1,25-dihydroxyvitamin D3 increases the expression/activity of PLD-1 in keratinocytes, and (3) PLD-2 is co-localized with a glycerol channel in keratinocyte membrane microdomains, we speculate that radiolabeled PG production from radioactive glycerol is a measure of PLD-2 activation in these cells. Our results also suggest that PLD-mediated PG synthesis may be regulated at the level of both PLD activity and alcohol substrate availability via changes in glycerol uptake.
Keywords: Aquaporin-3; Glycerol; Phorbol 12-myristate 13-acetate (PMA); Phospholipase D-2 (PLD-2); Transphosphatidylation;

Hepatocyte growth factor activates several transduction pathways in rat pancreatic acini by I.M Aparicio; L.J Garcia-Marin; A.G Andreolotti; G Bodega; R.T Jensen; M.J Bragado (37-46).
The receptor of hepatocyte growth factor (HGF), c-met induces different physiological responses in several cell types. Little is known about the role of HGF in exocrine pancreas. However, abnormal HGF signaling has been strongly implicated in pancreatic tumorigenesis and association of HGF with pancreatitis has been demonstrated. We have studied the presence of c-met and activation of their intracellular pathways associated in rat pancreatic acini in comparison with cholecystokinin (CCK) and epidermal growth factor (EGF). C-met expression in rat exocrine pancreas was identified by immunohistochemistry and immunoprecipitation followed by Western analysis. Tyrosine phosphorylation of c-met is strongly stimulated as well as kinase pathways leading to ERK1/2 cascade. HGF, but not CCK or EGF, selectively caused a consistent increase in the amount of p85 regulatory subunit of PI3-K present in anti-phosphotyrosine immunoprecipitates. Downstream of PI3-K, HGF increased Ser473 phosphorylation of Akt selectively, as CCK or EGF did not affect it. HGF selectively stimulated tyrosine phosphorylation of phosphatase PTP1D. HGF failed to promote the well-known CCK effects in pancreatic acini such as amylase secretion and intracellular calcium mobilization. Although HGF shares activation of ERK1/2 with CCK, we demonstrate that it promotes the selective activation of intracellular pathways not regulated by CCK or EGF. Our results suggest that HGF is an in vivo stimulus of pancreatic acini and provide novel insight into the transduction pathways and effects of c-met/HGF in normal pancreatic acinar cells.
Keywords: Pancreatic acini; Hepatocyte growth factor; PI3-Kinase; ERK1/2; PTP1D; Amylase secretion;

Recovery from heat shock injury by activation of Na+-glucose cotransporter in renal epithelial cells by Akira Ikari; Mika Nakano; Mihoko Ishibashi; Kazuya Kawano; Yasunobu Suketa; Hitoshi Harada; Kuniaki Takagi (47-53).
Exposure of cells or organs to sublethal physical or chemical stresses induces disruption of cellular structures and functions. Here, we examined whether Na+-glucose cotransporter (SGLT1) is involved in the recovery from heat shock (HS) injury in porcine renal epithelial LLC-PK1 cells. Recovery from HS (42 °C for 3 h, then 37 °C for 12 h) increased SGLT1 activity, assessed by [14C]α-methyl glucopyranoside uptake, and a maximal transport rate (V max) from 2.4 to 5.9 nmol/mg protein/30 min, but did not alter an apparent affinity constant (K m). Protein distribution of SGLT1 in apical membrane fraction was also increased after recovery from HS without changing in total membrane fraction. Membrane integrity assessed by calcein accumulation was decreased by HS, and then returned to basal level. This recovery was inhibited by phloridzin, a potent SGLT1 inhibitor, and nonmetabolizable glucose analogues. Anti-transforming growth factor-β1 (TGF-β1) antibody inhibited both elevation of SGLT1 distribution in apical membrane and recovery of calcein accumulation induced by HS. Taken together, HS increases in the number of SGLT1 protein in apical membrane mediated via TGF-β1 signaling pathway. The increase of glucose uptake is necessary to repair plasma membrane integrity.
Keywords: Heat shock; Kidney; Membrane integrity; SGLT1;

Nuclear localization domains in human thyroid transcription factor 2 by Maria Grazia Romanelli; Luciano Tato'; Pamela Lorenzi; Carlo Morandi (55-64).
Thyroid transcription factor-2 (TTF2) is a nuclear protein involved in morphogenesis and gene expression in thyroid gland, belonging to the family of the forkhead/winged-helix transcription factors. In the present study we have investigated the sequence determinants for transport and accumulation into the nucleus of the TTF2 protein. By transient expression of fusion proteins constructed by joining different parts of TTF2 to the reporter gene of the jellyfish green fluorescent protein (GFP) and, in a separate set of deleted constructs, the glutathione S-transferase (GST) coding sequence, we have demonstrated that a basic amino acid stretch present at both ends of the DNA-binding domain is a bona fide nuclear localization signal (NLS). We have analyzed the subcellular localization of deleted GFP-GST-TTF2 fusion proteins and have shown that residues inside the forkhead domain (FHD) contributed to the complete nuclear TTF2 protein accumulation. Furthermore, by means of GST binding assays we have shown that distinct TTF2 fragments, containing the NLS, were able to bind the nuclear import receptor importin α. Taken together, our results provide the first documentation about nuclear targeting of a forkhead protein containing two identical NLS signal flanking the DNA-binding domain.
Keywords: Nuclear localization signal; TTF2; Forkhead; FOXE1; FKHL15; Importin α;

Oxidative stress induced by a glucose/glucose oxidase (G/GO) generator system dose-dependently decreased the viability of cultured vascular smooth muscle cells (VSMC) as estimated by MTT assay. Cell death was induced in 40% of cells exposed to 0.2 IU/ml of the free radical generating mixture. Annexin-V labeling, Hoechst staining together with DNA laddering demonstrated that apoptosis was responsible for this cell loss. Pretreatment of the cells with 10−8 M calcitonin gene-related peptide (CGRP) significantly attenuated the damaging effect of the oxidative stress. Indeed, cell viability was estimated to be 80% in CGRP-treated group, instead of 60% in absence of CGRP treatment. This protective effect of CGRP was antagonized by 8-37 CGRP, an antagonist of CGRP-1 receptors, whereas it was not reproduced by amylin, a CGRP analogue. As indicated by the reduction in Hoechst staining and in DNA laddering, CGRP prevented the onset of apoptosis. We also demonstrated that the peptide significantly up-regulated the activation of ERK1/2 and P38 kinases. Inhibitors of the kinases prevented the protective effect of CGRP. We conclude that CGRP antagonizes oxidative stress-induced apoptosis by up-regulating MAP kinase activation and that activation of these kinases was necessary to protection.
Keywords: Vascular smooth muscle cell; Apoptosis; Hydrogen peroxide; CGRP; MAPK; SAPK; Neuropeptide;

Substance P induces TNF-α and IL-6 production through NFκB in peritoneal mast cells by Antonina Azzolina; Antonella Bongiovanni; Nadia Lampiasi (75-83).
The neuropeptide Substance P (SP) is an important mediator of neuroimmunomodulatory activity. The aim of this study is to elucidate the mechanism used by SP to promote increased production of pro-inflammatory cytokines in fresh isolated rat peritoneal mast cells (rPMC). We have demonstrated that SP induces production of interleukin-6 (IL-6) in rPMC through the PI-3K, p42/44 and p38 MAP kinase pathways. SP-stimulated rPMC also exhibited an enhanced nuclear translocation of the nuclear factor κ B (NFκB). The tumour necrosis factor-α (TNF-α) and IL-6 production was completely inhibited by using (E)-4-hydroxynonenal (HNE) as an inhibitor of IκB-α and -β phosphorylation. Further, TNF-α and IL-6 expression was significantly inhibited by the oligonucleotides (ODNs) containing the NFκB element (NFκB decoy ODNs) but not by the scrambled control ODNs. These findings indicate that the NFκB pathway is involved in the transcriptional regulation of the TNF-α and IL-6 overexpression in primary SP-stimulated mast cells.
Keywords: Mast cell; TNF-α; IL-6; SP; MAPK; NFκB; ODN decoy;

Cell-to-cell variability in the differentiation program of human megakaryocytes by Els den Dekker; Monique van Abel; Hans van der Vuurst; Guillaume J.J.M. van Eys; Jan-Willem N. Akkerman; Johan W.M. Heemskerk (85-94).
Differentiation of CD34+ stem/progenitor cells into megakaryocytes is thought to be a uniform, unidirectional process, in which cells transform step by step from less differentiated precursor stages to more differentiated megakaryocytes. Here we propose the concept and present evidence based on single-cell analysis that differentiation occurs along multiple, partially asynchronous routes. In all CD34+ cells cultured with thrombopoietin, surface appearance of glycoprotein IIIa (GPIIIa) preceded that of GPIb, indicating that the expression of these glycoproteins occurs in a timely ordered manner. Cellular F-actin content increased in parallel with GPIb expression. Only cells that expressed GPIb were polyploid, pointing to co-regulation of GPIb expression, actin cytoskeleton formation and polyploidization during megakaryocytopoiesis. On the other hand, most progenitor cells responded to thrombin but not to thromboxane A2 analogue by rises in cytosolic [Ca2+]i. The appearance of thromboxane-induced responses during megakaryocytopoiesis was not strictly linked to glycoprotein expression, because cells showed responsiveness either before or after GPIb expression. The same non-strictly sequential pattern was observed for disappearance of the Ca2+ response by prostacyclin mimetic; in some megakaryocytes it occurred before and in others after GPIb expression. Thus, megakaryocytic differentiation follows along independent routes that are either strictly sequential (GPIIIa and GPIb expression) or proceed at different velocities (Ca2+ signal regulation).
Keywords: Differentiation; Glycoprotein; Megakaryocyte; Thromboxane; Prostacyclin;

Effects of the antimicrobial peptide PGLa on live Escherichia coli by Arnaldo da Silva; Omar Teschke (95-103).
The activity of PGLa, an antimicrobial peptide isolated from hemocytes of frog skin and its secretions on living Escherichia coli, was investigated by imaging the cells with atomic force microscopy (AFM) in physiological conditions and by measuring its cellular stiffness. The treatment of bacteria with the antimicrobial peptide PGLa in the culture medium had two stages. The first was characterized by the loss of surface stiffness and consequent loss of bacteria topographic features and the formation of micelles probably originating from the disruption of the outer membrane. The formation of outer membrane originated micelles is in agreement with the carpet-like mechanism of action proposed for antimicrobial peptides of the magainin family. The peptide action also resulted in the removal of bacterial pili. In a second stage there was further damage which resulted in total cell rupture. The addition of Mg2+ ions prior to peptide treatment partially inhibited the effects of PGLa on bacteria. This result suggests that PGLa interacts with the outer membrane by displacing Mg2+ from LPS, inserting itself into the bilayer and cross-bridging the negative charges of LPS lipids as proposed in the self-promoted pathway mechanism. The peptide effect on the bacteria was compared to the activity of the chelating agent EDTA that damages the bacterial outer membrane by removing Mg2+ ions.
Keywords: Escherichia coli; Antimicrobial peptide PGLa; EDTA; Cellular stiffness; Structural cellular variation;

C-type natriuretic peptide decreases soluble guanylate cyclase levels by activating the proteasome pathway by Sergio de Frutos; Marta Saura; Francisco J. Rivero-Vilches; D. Rodriguez-Puyol; M. Rodriguez-Puyol (105-112).
Natriuretic peptides (NP) activate particulate guanylate cyclase (pGC) and nitric oxide (NO) activates soluble guanylate cyclase (sGC). Both guanylate cyclases catalyse the formation of the same second messenger, cyclic guanosine 3′,5′-monophosphate (cGMP), which activates the cGMP-dependent protein kinases (PKG). PKG then starts a signalling cascade that mediates many cardiovascular and renal effects, such as smooth muscle relaxation and diuresis. Many cell types possess both sGC and pGC. Because both GC-cGMP systems play complementary roles, an interaction between the two pathways might represent an important physiological control mechanism. In this report we demonstrate an interaction between the two pathways. C-type natriuretic peptide (CNP) decreased the β-subunit of sGC (sGC-β) steady-state protein levels and enzymatic activity in cultured human mesangial cells (HMC) in a time- and dose-dependent manner. This down-regulation was not dependent on changes in sGC-β mRNA levels. Treatment of the cells with the stable cGMP analogue 8-Br-cGMP or the phosphodiesterase type-5 inhibitor Zaprinast produced the same down-regulatory effect. Inhibition of PKG or proteasome activity prevented the CNP-induced reduction of sGC-β protein levels and activity. Taken together, these results demonstrate that pGC activation induces a post-transductional down-regulation of sGC by a mechanism involving PKG and the proteasome pathway.
Keywords: Soluble guanylate cyclase; Particulate guanylate cyclase; Cross-regulation; Protein degradation; Proteasome; cGMP;

Glycosylation profile of integrin α3β1 changes with melanoma progression by Ewa Pocheć; Anna Lityńska; Angela Amoresano; Annarita Casbarra (113-123).
Glycosylation of integrins has been implicated in the modulation of their function. Characterisation of carbohydrate moieties of α3 and β1 subunits from non-metastatic (WM35) and metastatic (A375) human melanoma cell lines was carried out on peptide-N-glycosidase F-released glycans using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). β1 integrin subunit from both cell lines displayed tri- and tetraantennary oligosaccharides complex type glycans, but only in A375 cell line was the sialylated tetraantennary complex type glycan (Hex7HexNAc6FucSia4) present. In contrast, only α3 subunit from metastatic cells possessed β1–6 branched structures. Our data indicate that the β1 and α3 subunits expressed by the metastatic A375 cell line carry β1–6 branched structures, suggesting that these cancer-associated glycan chains may modulate tumor cell adhesion by affecting the ligand binding properties of α3β1 integrin. In direct ligand binding assays, α3β1 integrin from both cell lines binds strongly to fibronectin and to much lesser degree to placental laminin. No binding to collagen IV was observed. Enzymatic removal of sialic acid residues from purified α3β1 integrin stimulates its adhesion to all examined ECM proteins. Our data suggest that the glycosylation profile of α3β1 integrin in human melanoma cells correlates with the acquisition of invasive capacity during melanoma progression.
Keywords: α3β1 integrin; Glycosylation; MALDI MS; Cell adhesion; ELISA;