Insulin-like growth factor (IGF) I down-regulates type 1 IGF receptor (IGF 1R) and reduces the IGF I response in A549 non-small-cell lung cancer and Saos-2/B-10 osteoblastic osteosarcoma cells.

The insulin-like growth factor type 1 receptor (IGF 1R) mediates the acute metabolic effects of IGF I as well as IGF I-stimulated cell proliferation and protection from apoptosis. IGF binding proteins (IGFBPs) can modulate these responses. We, therefore, investigated whether intrinsic IGFBPs interfere with IGF I-induced regulation of IGF 1R expression and with the biological response to IGF I in two human tumor cell lines, the non-small-cell lung cancer cell line A549 and the osteoblastic osteosarcoma cell line Saos-2/B-10. We compared the growth rates, IGFBP production, IGF I binding characteristics, IGF 1R protein and mRNA levels, and the acute IGF I response (stimulation of glycogen synthesis) after pretreatment of the cells in serum-free medium with or without added IGF I or medium supplemented with 5% fetal calf serum (FCS). In contrast to A549 cells, which produce IGF I and significant amounts of IGFBPs, survival and proliferation of Saos-2/B-10 cells, which do not produce IGF I or significant amounts of IGFBPs, depended on the addition of exogenous IGF I. IGF I increased the concentration of IGFBP-2 and -3 and decreased the concentration of IGFBP-4 in the medium of A549 cells. As compared to FCS, IGF I pretreatment in both cell lines decreased the number of specific IGF I binding sites, down-regulated total and membrane IGF 1R protein, and largely reduced or abolished the acute IGF I response without affecting IGF 1R mRNA levels. The data suggest that the IGF 1R protein of the two cell lines is translationally and/or posttranslationally down-regulated by its ligand in the presence and in the absence of locally produced IGFBPs and that the cell lines have retained this negative feedback to counteract IGF I stimulation.

Localization of organic anion transporting polypeptide 4 (Oatp4) in rat liver and comparison of its substrate specificity with Oatp1, Oatp2 and Oatp3.

Organic anion transporting polypeptides (rodents: Oatps; human: OATPs) are involved in the absorption and elimination of a wide variety of structurally unrelated amphipathic organic compounds. Several members of this protein family mediate the uptake of substrates across the basolateral membrane of hepatocytes as the first step in hepatic elimination. In contrast to the well-characterized Oatp1 and Oatp2, the localization and substrate specificity of the recently cloned Oatp4 have not been investigated in detail. Therefore, we raised an antibody against the C-terminal end of Oatp4 and localized this 85-kDa protein to the basolateral membrane of rat hepatocytes. Similar to Oatp1 and Oatp2, Oatp4 is a multispecific transporter with high affinities for bromosulfophthalein, dehydroepiandrosterone sulfate, leukotriene C4, and anionic peptides. In addition, we compared the substrate specificity of Oatp4 to that of Oatp3, which so far has mainly been shown to mediate intestinal bile acid transport. Oatp3 had a similar broad substrate specificity, but in general much lower affinities than Oatp4. Thus, while Oatp4 seems to work in concert with Oatp1 and Oatp2 in the basolateral membrane of rat hepatocytes, Oatp3 is a multispecific transport system in the small intestine.

Type I collagen induces expression of bone morphogenetic protein receptor type II.

The extracellular matrix regulates many fundamental cellular processes such as proliferation, migration, and differentiation. Among the ECM components, type I collagen induces endothelial tube formation in vitro. By analysing genes participating in this event, the bone morphogenetic protein receptor-II (BMPR-II) was detected to be upregulated in cells cultured on or within fibrillar type I collagen. Furthermore, the basement membrane type IV collagen or amorphous type I collagen did not show an induction of BMPR-II. Addition of the BMPR-II specific ligands, BMP2 and BMP4, in the culture medium of the endothelial cells seeded on type I collagen increased [(3)H]-thymidine incorporation into cellular DNA, indicating that endothelial cells were able to form a functional receptor. In addition, in the chick chorioallantoic membrane (CAM), an in vivo angiogenesis model, BMPR-II and BMPR-I were upregulated in the growing phase and ceased in the mature CAM.

Identification of a novel neuroligin in humans which binds to PSD-95 and has a widespread expression.

Neuroligins, first discovered in rat brain, form a family of three synaptically enriched membrane proteins. Using reverse transcription-PCR of human brain polyadenylated RNA and extensive database searches, we identified the human homologues of the three rat neuroligins and a cDNA encoding a fourth member, which we named neuroligin 4. Neuroligin 4 has 63-73% amino acid identity with the other members of the human neuroligin family, and the same predicted domain structure. DNA database analyses, furthermore, indicated that a possible fifth neuroligin gene may be present in the human genome. Northern-blot analysis revealed expression of neuroligin 4 in heart, liver, skeletal muscle and pancreas, but barely at all in brain. Overexpression of neuroligin 4 cDNA in COS-7 cells led to the production of a 110 kDa protein. Immunofluorescence analysis demonstrated that the protein was integrated into the plasma membrane. Overexpression of cDNAs encoding neuroligin 4 and the PDZ-domain protein, PSD-95, in COS-7 cells resulted in the formation of detergent-resistant complexes. Neuroligin 4 did not bind to ZO-1, another PDZ-domain protein. Together, our data show that the human neuroligin family is composed of at least one additional member, and suggest that neuroligin 4 may also be produced outside the central nervous system.

Brain derived versican V2 is a potent inhibitor of axonal growth.

In this paper, we identify the chondroitin sulfate proteoglycan versican V2 as a major inhibitor of axonal growth in the extracellular matrix of the mature central nervous system. In immunohistochemical and in situ hybridization experiments we show that this tissue-specific splice variant of versican is predominantly present in myelinated fiber tracts of the brain and in the optic nerve, most likely being expressed by oligodendrocytes. We demonstrate that isolated versican V2 strongly inhibits neurite outgrowth of central and peripheral neurons in stripe-choice assays using laminin-1 as permissive substrate. The inhibitory character of versican V2 is maintained after removal of chondroitin sulfate and N- and O-linked oligosaccharide side chains, but it is abolished after core protein digestion with proteinase-K. Our data support the notion, that intact versican V2 prevents excessive axonal growth during late phases of development and hereby participates in the structural stabilization of the mature central nervous system.

Crystallization and initial X-ray analysis of rabbit mature sterol carrier protein 2.

Sterol carrier protein 2 (SCP2) is a basic intracellular protein which facilitates the in vitro intermembrane transfer of cholesterol, phospholipids and glycolipids. SCP2 was expressed in Escherichia coli, purified to apparent electrophoretic homogeneity and crystallized. Single crystals were obtained by hanging-drop vapour diffusion using ammonium sulfate as precipitant. These crystals belong to space group P4(1)2(1)2 or its enantiomorph, with unit-cell parameters a = b = 57.5, c = 86.5 A, and have one molecule in the crystallographic asymmetric unit. Intensity data to 1.8 A resolution were collected from native SCP2 crystals using synchrotron radiation, were processed and scaled with an R(linear) = 4.9%.

Purification, crystallization and preliminary crystallographic studies of a two fibronectin type-III domain segment from chicken tenascin encompassing the heparin- and contactin-binding regions.

A fragment of chicken tenascin consisting of fibronectin type-III domains 5 and 6 has been expressed in Escherichia coli. After modifying a previously reported purification protocol, an electrophoretically homogeneous recombinant protein was obtained from which various crystal forms could be grown under identical conditions. Only one form was suitable for structure determination. These crystals belong to space group P21, with unit-cell parameters a = 45.2, b = 57.9, c = 72.2 A, beta = 91.4 degrees, and diffract to at least 2.6 A resolution using synchrotron radiation. From density measurements of the crystals, it was found that there are two molecules in the asymmetric unit. Diffraction data of native, two platinum-derivative and one palladium-derivative crystals were collected.

The crystal structure of lignin peroxidase at 1.70 A resolution reveals a hydroxy group on the cbeta of tryptophan 171: a novel radical site formed during the redox cycle.

The crystal structure of lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium was refined to an R-factor of 16.2 % utilizing synchrotron data in the resolution range from 10 to 1.7 A. The final model comprises all 343 amino acid residues, 370 water molecules, the heme, four carbohydrates, and two calcium ions. Lignin peroxidase shows the typical peroxidase fold and the heme has a close environment as found in other peroxidases. During refinement of the LiP model an unprecedented modification of an amino acid was recognized. The surface residue tryptophan 171 in LiP is stereospecifically hydroxylated at the Cbeta atom due to an autocatalytic process. We propose that during the catalytic cycle of LiP a transient radical at Trp171 occurs that is different from those previously assumed for this type of peroxidase. Recently, the existence of a second substrate-binding site centered at Trp171 has been reported, by us which is different from the "classical heme edge" site found in other peroxidases. Here, we report evidence for a radical formation at Trp171 using spin trapping, which supports the concept of Trp171 being a redox active amino acid and being involved in the oxidation of veratryl alcohol. On the basis of our current model, an electron pathway from Trp171 to the heme is envisaged, relevant for the oxidation of veratryl alcohol and possibly lignin. Beside the opening leading to the heme edge, which can accommodate small aromatic substrate molecules, a smaller channel giving access to the distal heme pocket was identified that is large enough for molecules such as hydrogen peroxide. Furthermore, it was found that in LiP the bond between the heme iron and the Nepsilon2 atom of the proximal histidine residue is significantly longer than in cytochrome c peroxidase (CcP). The weaker Fe-N bond in LiP renders the heme more electron deficient and destabilizes high oxidation states, which could explain the higher redox potential of LiP as compared to CcP.

Versican V2 is a major extracellular matrix component of the mature bovine brain.

We have isolated and characterized the proteoglycan isoforms of versican from bovine brain extracts. Our approach included (i) cDNA cloning and sequencing of the entire open reading frame encoding the bovine versican splice variants; (ii) preparation of antibodies against bovine versican using recombinant core protein fragments and synthetic peptides; (iii) isolation of versican isoforms by ammonium sulfate precipitation followed by anion exchange and hyaluronan affinity chromatography; and (iv) characterization by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining or immunoblotting. Our results demonstrate that versican V2 is, together with brevican, a major component of the mature brain extracellular matrix. Versicans V0 and V1 are only present in relatively small amounts. Versican V2 migrates after chondroitinase ABC digestion with an apparent molecular mass of about 400 kDa, whereas it barely enters a 4-15% polyacrylamide gel without the enzyme treatment. The 400-kDa product is recognized by antibodies against the glycosaminoglycan-alpha domain and against synthetic NH2- and COOH-terminal peptides. Our preparations contain no major proteolytic products of versican, e.g. hyaluronectin or glial hyaluronate-binding protein. Having biochemical quantities of versican V2 available will allow us to test its putative modulatory role in neuronal cell adhesion and axonal growth.

DNA methylation accounts for the inhibition of collagen VI expression in transformed fibroblasts.

The expression of collagen VI, an adhesive glycoprotein of the extracellular matrix, is completely inhibited in virally transformed fibroblasts and in many cell lines derived from spontaneous mesenchymal tumors. Here we present evidence that DNA methylation plays an important role in this inhibition: (a) The mRNA level for DNA methyltransferase is highly increased in simian virus 40 (SV40)-transformed fibroblasts compared with normal cells and this increase correlates with the decrease of the mRNA level for collagen VI. (b) Methylation of the alpha2(VI) collagen promoter in vitro abolishes promoter activity in a transient transfection assay. (c) Genomic sequencing reveals extensive methylation of the promoter region in SV40-transformed cells, but virtually no methylation of the corresponding region in normal cells. Increased methylation is also observed in a rhabdomyosarcoma cell line. (d) Two of the cis-acting elements of the alpha2(VI) collagen promoter lose their affinity for transcription factor AP2 when methylated in vitro as demonstrated by gel retardation experiments. DNA methylation is therefore involved in the silencing of the alpha2(VI) collagen gene. It seems likely that the same mechanism is also responsible for the repression of other transformation-sensitive proteins.

Functional expression and purification of histidine-tagged rat renal Na/Phosphate (NaPi-2) and Na/Sulfate (NaSi-1) cotransporters.

Two mammalian sodium-dependent anion-cotransporters (NaPi-2 for phosphate and NaSi-1 for sulfate) have been expressed in Sf9 insect cells using the baculovirus expression system. A histidine tag was introduced at the C-termini in order to facilitate purification by metal-affinity chromatography. Sf9 cells infected with the histidine-tagged Ni/Pi-cotransporter exhibited more than 60-fold higher sodium-dependent transport of phosphate compared to noninfected cells. Expressed Na/Pi-cotransport exhibited a Km of Pi of 0.21 mm and an apparent Km of sodium of 92 mm. Infected cells expressed a 65 kDa polypeptide as detected by Western blotting and immunoprecipitation. Sf9 cells infected with the histidine-tagged NaSi-1 or untagged NaSi-1 protein expressed sodium-dependent sulfate cotransport up to 60-fold higher compared to noninfected cells. Transport of sulfate was highly dependent on sodium exhibiting a Km of SO2-4 of about 0.3-0.4 mm and a Km of sodium of 55 mm. By Western blotting and immunoprecipitation expressed NaSi-1 proteins were detected at 55-60 kDa. These studies demonstrate that histidine tagged proximal tubular Na-dependent cotransporters for phosphate and sulfate can be expressed functionally in Sf9 cells and that the kinetic characteristics were not altered by the introduction of a histidine tag at the C-termini. Furthermore, it is demonstrated that after solubilization under denaturing conditions histidine-tagged cotransporter proteins can be purified by metal-chelate affinity chromatography.

Distribution pattern of tenascin-C in normal and neoplastic mesenchymal tissues.

Descriptions for tenascin-C distribution are largely restricted to epithelial tumours. The present study utilized newly developed and characterized monoclonal (hT191) and polyclonal antibodies to investigate the distribution pattern of tenascin-C in a panel of mesenchymal tumours, which was contrasted with normal tissue. The specific antibodies recognized the distinctive star-like hexabrachion protein isolated from transformed cell-culture medium and serum from normal individuals. In normal tissues, a strong tenascin-C expression in the extracellular matrix was largely restricted to basement-membrane regions of epithelium and tonsilar sinusoids, pericellularly within smooth-muscle bundles, associated with perimysial, -chondrial, -neurial and -tendon surfaces, and diffusely within vascular adventitia. It was found in the corresponding tumours of the neural sheath (schwannoma) and smooth muscle (leiomyosarcoma), and was abundantly present around certain blood vessels of mesenchymal tumours. Although not detected in normal muscle, or in adipose or fibrous connective tissue, neo-expression of tenascin-C was shown in more than half of the rhabdomyosarcomas, fibromas and liposarcomas, with an increased positive percentage in variably malignant myxoid liposarcomas compared with lipoma-like sarcomas. Tenascin-C was typically found in the extracellular matrix of soft-tissue tumours, but was notably absent from the epithelial-cell components of mixed epithelial/mesenchymal tumours. Its apparently enhanced expression in soft-tissue tumours differs from that of most other large extracellular-matrix proteins, suggesting possible functional involvement of the cell-adhesion molecule, tenascin-C, in the neoplastic phenotype.

t-Butylhydroperoxide and gliotoxin stimulate Ca2+ release from rat skeletal muscle mitochondria.

Rat liver mitochondria have a specific Ca2+ release pathway which operates when NAD+ is hydrolysed to nicotinamide and ADPribose. NAD+ hydrolysis is Ca(2+)-dependent and inhibited by cyclosporine A (CSA). Mitochondrial Ca2+ release can be activated by the prooxidant t-butylhydroperoxide (tbh) or by gliotoxin (GT), a fungal metabolite of the epipolythiodioxopiperazine group. Tbh oxidizes NADH to NAD+ through an enzyme cascade consisting of glutathione peroxidase, glutathione reductase, and the energy linked transhydrogenase, whereas GT oxidizes some vicinal thiols to the disulfide form, a prerequisite for NAD+ hydrolysis. We report now that rat skeletal muscle mitochondria also contain a specific Ca2+ release pathway activated by both tbh and GT. Ca2+ release increases with the mitochondrial Ca2+ load, is completely inhibited in the presence of CSA, and is paralleled by pyridine nucleotide oxidation. In the presence of tbh and GT, mitochondria do not lose their membrane potential and do not swell, provided continuous release and re-uptake of Ca2+ ('Ca2+ cycling') is prevented. These data support the notion that both tbh- and GT-induced Ca2+ release are not the consequence of an unspecific increase of the inner membrane permeability ('pore' formation). Tbh induces Ca2+ release from rat skeletal muscle less efficiently than from liver mitochondria indicating that the coupling between tbh and NADH oxidation is much weaker in skeletal muscle mitochondria. This conclusion is corroborated by a much lower glutathione peroxidase activity in skeletal muscle than in liver mitochondria. The prooxidant-dependent pathway promotes, under drastic conditions (high mitochondrial Ca2+ loads and high tbh concentrations), Ca2+ release to about the same extent and rate as the Na+/Ca2+ exchanger. This renders the prooxidant-dependent pathway relevant in the pathophysiology of mitochondrial myopathies where its activation by an increased generation of reactive oxygen species probably results in excessive Ca2+ cycling and damage to mitochondria.

Binding of contactin/F11 to the fibronectin type III domains 5 and 6 of tenascin is inhibited by heparin.

The structural basis for the interaction between tenascin-C and the neuronal cell adhesion molecule, contactin/F11, was investigated using plasmon surface resonance technology. The binding site on tenascin-C for contactin/F11 is shown to span the two fibronectin type III homology domains 5 and 6. Either domain alone is insufficient for binding. Heparin, heparan sulfate and dermatan sulfate inhibit this interaction through binding to a conserved heparin-binding site on domain 5. In contrast, chondroitin sulfates A and C have no such effect.

Contactin/F11 and tenascin-C co-expression in the chick retina correlates with formation of the synaptic plexiform layers.

The neural immunoglobulin-like cell adhesion molecule contactin/F11 and the extracellular matrix glycoprotein tenascin-C are prominent molecules in the developing nervous system which interact in in vitro assays (Zisch et al., J. Cell Biol. 119, 203-213). To determine their potential role in neural development, the distribution of tenascin-C and contactin/F11 was examined in the developing chick retina. The onset of both tenascin-C and contactin/F11 expression coincides with the appearance of ganglion cell dendrides and neurites from bipolar and amacrine cells in the inner layer (IPL) at E8, and the extension of bipolar and horizontal cell processes in the outer plexiform layer (OPL) at E9. Contactin/F11 expression is co-ordinately upregulated with the TN190 and TN200 tenascin-C isoforms between embryonic day 8 (E8) and E17, while little, if any, of the TN220 isoform, which does not bind contactin/F11, is detected. In situ hybridization reveals that tenascin-C and contactin/F11 mRNAs are synthesized by different neuronal types. Tenascin-C mRNA probes hybridize to amacrine and displaced amacrine neurons, and horizontal neurons. In cultured retinal cells, tenascin-C is also present on process-bearing neurofilament-positive cells. Contactin/F11 mRNA is detected in bipolar cells or their precursors from E8-9, and later in horizontal and ganglion neurons. The highest levels and greatest overlap in the synaptic IPL and OPL are reached at E17, when the stratification of the retina is nearly complete. These results are consistent with a putative role for contactin/F11-tenascin-C interactions in the establishment of synaptic layers in the retina.

Crystal structure of Asian elephant (Elephas maximus) cyano-metmyoglobin at 1.78-A resolution. Phe29(B10) accounts for its unusual ligand binding properties.

The crystal structure of Asian elephant cyano-metmyoglobin which has a glutamine instead of the usual distal site histidine has been determined to high resolution. In addition to this replacement, the substitution of a conserved leucine residue in position 29(B10) at the distal side by a phenylalanine was unambiguously identified based on the available electron density. The suspicion, that there were errors in the original sequence which has caused some confusion, is thus confirmed. Comparison with other myoglobin structures in various ligated forms reveals an essentially unchanged tertiary structure in elephant myoglobin despite the two amino acid substitutions in the heme pocket. Our current structural model shows that the N epsilon 2 atom of Gln64(E7) has moved with respect to the corresponding nitrogen position of His64(E7) in the CO complex of sperm whale myoglobin. The newly assigned residue Phe29(B10) penetrates into the distal side of the heme pocket approaching the ligand within van der Waals distance and causing a much more crowded heme pocket compared to other myoglobins. Kinetic properties of Asian elephant myoglobin, wild type, and recombinant sperm whale myoglobins are discussed in relation to the structural consequences of the two amino acid substitutions H64Q and L29F.

Versican is selectively expressed in embryonic tissues that act as barriers to neural crest cell migration and axon outgrowth.

Chondroitin sulfate proteoglycans have been implicated in the regulation of cell migration and pattern formation in the developing peripheral nervous system. To identify whether the large aggregating proteoglycan versican might be mediating these processes, we prepared monospecific antibodies against a recombinant core protein fragment of chick versican. The purified antibodies recognize the predominant versican splice-variants V0 and V1. Using these antibodies, we revealed a close correlation between the spacio-temporal expression of versican and the formation of molecular boundaries flanking or transiently blocking the migration pathways of neural crest cells or motor and sensory axons. Versican is present in the caudal sclerotome, the early dorsolateral tissue underneath the ectoderm, the pelvic girdle precursor and to a certain extent in the perinotochordal mesenchyme. Versican is completely absent from tissues invaded by neural crest cells and extending axons. Upon completion of neural crest cell migration and axon outgrowth, versican expression is shifted to pre-chondrogenic areas. Since versican inhibits cellular interactions with fibronectin, laminin and collagen I in vitro, the selective expression of versican within barrier tissues may be linked to a functional role of versican in the guidance of migratory neural crest cells and outgrowing axons.

The glypiated neuronal cell adhesion molecule contactin/F11 complexes with src-family protein tyrosine kinase Fyn.

Glycosyl phosphatidylinositol-anchored glycoproteins of the immunoglobulin superfamily play an important role in the formation of neuronal networks during development. The mechanism whereby neuronal GPI-linked molecules transduce recognition signals remains to be established. Analysis of detergent-resistant immune-complexes reveals that the glypiated neuronal cell adhesion molecule contactin/F11 specifically complexes with the cytoplasmic, nonreceptor type src-family tyrosine kinase Fyn. Antibody-mediated cross-linking of contactin/F11 on embryonic chick neuronal cells leads to an increase of the Fyn-activity coprecipitated with contactin/F11, and elevates phosphorylation of an additional 75/80 K component within the contactin/F11-immune-complex. Additionally, binding of ligands, i.e., contactin/F11-specific antibody or tenascin-R, a natural ligand of contactin/F11, to the surface of HeLa transfectants expressing contactin/F11, causes capping of contactin/F11 and a concomitant change in the distribution of the intracellular kinase Fyn, thus confirming their physical association. This indicates that contactin/F11-mediated signaling requires Fyn.