Promoter haplotype combinations of the platelet-derived growth factor alpha-receptor gene predispose to human neural tube defects.

Neural tube defects (NTDs), including anencephaly and spina bifida, are multifactorial diseases that occur with an incidence of 1 in 300 births in the United Kingdom. Mouse models have indicated that deregulated expression of the gene encoding the platelet-derived growth factor alpha-receptor (Pdgfra) causes congenital NTDs (refs. 2-4), whereas mutant forms of Pax-1 that have been associated with NTDs cause deregulated activation of the human PDGFRA promoter. There is an increasing awareness that genetic polymorphisms may have an important role in the susceptibility for NTDs (ref. 6). Here we identify five different haplotypes in the human PDGFRA promoter, of which the two most abundant ones, designated H1 and H2 alpha, differ in at least six polymorphic sites. In a transient transfection assay in human bone cells, the five haplotypes differ strongly in their ability to enhance reporter gene activity. In a group of patients with sporadic spina bifida, haplotypes with low transcriptional activity, including H1, were under-represented, whereas those with high transcriptional activity, including H2 alpha, were over-represented. When testing for haplotype combinations, H1 homozygotes were fully absent from the group of sporadic patients, whereas H1/H2 alpha heterozygotes were over-represented in the groups of both sporadic and familial spina bifida patients, but strongly under-represented in unrelated controls. Our data indicate that specific combinations of naturally occurring PDGFRA promoter haplotypes strongly affect NTD genesis.

Role of the prostanoid FP receptor in action potential generation and phenotypic transformation of NRK fibroblasts.

By using an shRNA approach to knockdown the expression of the prostaglandin (PG)-F(2alpha) receptor (FP-R), the role of PGF(2alpha) in the process of phenotypic transformation of normal rat kidney (NRK) fibroblasts has been studied. Our data show that PGF(2alpha) up-regulates Cox-2 expression both at the mRNA and protein level, indicating that activation of FP-R in NRK fibroblasts induces a positive feedback loop in the production PGF(2alpha). Knockdown of FP-R expression fully impaired the ability of PGF(2alpha) to induce a calcium response and subsequent depolarization in NRK cells. However, these cells could still undergo phenotypic transformation when treated with a combination of EGF and retinoic acid, but in contrast to the wild-type cells, this process was not accompanied by a membrane depolarization to -20 mV. Knockdown of FP-R expression also impaired the spontaneous firing of calcium action potentials by density-arrested NRK cells. These data show that a membrane depolarization is not a prerequisite for the acquisition of a transformed phenotype. Furthermore, our data provide the first direct evidence that activity of PGF(2alpha) by putative pacemaker cells underlies the generation of calcium action potentials in NRK monolayers.

Local induction of pacemaking activity in a monolayer of electrically coupled quiescent NRK fibroblasts.

Cultures of normal rat kidney (NRK) fibroblasts may display spontaneous calcium action potentials which propagate throughout the cellular monolayer. Pacemaking activity of NRK cells was studied by patch clamp electrophysiology and vital calcium imaging, using a new experimental approach in which a ring was placed on the monolayer in order to physically separate pacemakers within or under the ring and follower cells outside the ring. Stimulation of cells inside the ring with IP(3)-generating hormones such as prostaglandin F(2alpha) (PGF(2alpha)) resulted in the induction of periodic action potentials outside the ring, which were abolished when the L-type calcium channel blocker nifedipine was added outside the ring, but not inside the ring. PGF(2alpha)-treated cells displayed asynchronous IP(3)-mediated calcium oscillations of variable frequency, while follower cells outside the ring showed synchronous calcium transients which coincided with the propagating action potential. Mathematical modelling indicated that addition of PGF(2alpha) inside the ring induced both a membrane potential gradient and an intracellular IP(3) gradient, both of which are essential for the induction of pacemaking activity under the ring. These data show that intercellular coupling between PGF(2alpha)-treated and non-treated cells is essential for the generation of a functional pacemaker area whereby synchronization of calcium oscillations occurs by activation of L-type calcium channels.

Neuroblastoma cells express c-sis and produce a transforming growth factor antigenically related to the platelet-derived growth factor.

Mouse neuroblastoma Neuro-2A cells produce transforming growth factors during exponential growth in a defined hormone-free medium, which, on Bio-Gel columns in 1 M HAc, elute at a molecular size of 15 to 20 kilodaltons (kDa). These neuroblastoma-derived transforming growth factors have strong mitogenic activity, but they do not compete with epidermal growth factor for receptor binding (E. J. J. van Zoelen, D. R. Twardzik, T. M. J. van Oostwaard, P. T. van der Saag, S. W. de Laat, and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 81:4085-4089, 1984). In this study approximately 80% of the mitogenic activity was immunoprecipitated by antibodies raised against platelet-derived growth factor (PDGF). Immunoblotting indicated a true molecular size of 32 kDa for this PDGF-like growth factor. Analysis of poly(A)+ RNA from Neuro-2A cells demonstrated the expression of the c-sis oncogene in this cell line, whereas in vitro translation of the RNA yielded a 20-kDa protein recognized by anti-PDGF antibodies. Separation by reverse-phase high-pressure liquid chromatography demonstrated the presence of two distinct mitogenic activities in neuroblastoma-derived transforming growth factor preparations, one of which is antigenically related to PDGF. Both activities had the ability to induce anchorage-independent growth in normal rat kidney cells, both in the presence and in the absence of epidermal growth factor. It is concluded that Neuro-2A cells express c-sis with concomitant production and secretion of a PDGF-like growth factor, which plays a role in the induction of phenotypic transformation on normal rat kidney cells.

Isolation of autonomously growing dog mammary tumor cell lines cultured in medium supplemented with serum treated to inactivate growth factors.

Conventional methods for isolation of cell lines from carcinomas suffer inherently from a lack of advantage for proliferation of transformed cells as opposed to contaminating fibroblasts and normal epithelial cells. To isolate cell lines from metastases of estrogen receptor-negative mammary carcinomas in dogs, we applied a novel method using medium supplemented with serum treated to inactivate growth factors. Under these conditions, autonomously growing tumor cells are selectively allowed to proliferate. In this way, four autonomously growing tumor cell lines were obtained from metastases of two dogs. Tumors formed from cells implanted in C3H nude mice closely resembled the original dog tumors, indicating that the main result of this selective procedure was suppression of normal cell proliferation. Serum treated to inactivate growth factors seems to be an important medium supplement for isolation of autonomously growing tumor cell lines, which may be valuable tools for future studies on regulation of cell proliferation in advanced hormone-independent mammary tumors.

Developmentally regulated expression of two novel platelet-derived growth factor alpha-receptor transcripts in human teratocarcinoma cells.

Two novel platelet-derived growth factor (PDGF) alpha-receptor transcripts of 1.5 kilobases and 5.0 kilobases are expressed in the human teratocarcinoma cell line Tera-2 only while the cells are in an undifferentiated state. After retinoic acid-induced differentiation, expression of these mRNAs is completely shut off and instead, the cells express a single 6.4-kilobase mRNA species which is also expressed in many other cell types. The 1.5-kilobase mRNA initiates within intron 12, contains the correctly spliced exons 13, 14, 15, and 16, and contains a cryptic exon, designated teratocarcinoma cryptic exon, at the 3' end. Teratocarcinoma cryptic exon contains a functional polyadenylation signal. Exons 13 to 16 correspond to the first tyrosine kinase domain and to part of the interkinase domain of the PDGF alpha-receptor. Recently, a splice variant lacking exon 14 was identified. These results show that a combination of alternative promoter usage and alternative splicing of the human PDGF alpha-receptor gene occur in a developmentally regulated fashion. In vitro translation of the 1.5-kilobase mRNA generates protein products which can be specifically immunoprecipitated with a PDGF alpha-receptor-specific antibody. The significance of the expression of this transcript for the growth factor-independent proliferation of undifferentiated Tera-2 cells is unclear. Expression of PDGF alpha-receptor transcripts containing the cryptic exon may be useful as a marker for undifferentiated stem cells in human teratocarcinomas.

Production of transforming growth factors by simian sarcoma virus-transformed cells.

Cellular transformation of normal rat kidney (NRK) cells by simian sarcoma virus (SSV) results in a complete loss of the cellular requirement of externally added polypeptide growth factors for proliferation. Moreover, SSV-transformed NRK cells have a strongly reduced ability to bind both external platelet-derived growth factor and epidermal growth factor, when compared to nontransformed NRK cells. Analysis of serum-free medium conditioned by SSV-transformed NRK cells shows that this cell line secretes both types alpha and beta transforming growth factor (TGF). The level of TGF alpha production (300 ng/liter conditioned medium) by SSV-transformed NRK is among the highest described to date. Since addition of TGF alpha and beta in combination is sufficient to induce phenotypic transformation of NRK cells, it is concluded that although expression of the sis oncogene is essential for transformation, expression of additional genes may be required for the phenotypic alterations accompanying complete cellular transformation.

Role of estrogen-induced insulin-like growth factors in the proliferation of human breast cancer cells.

Insulin-like growth factor I (IGF-I) is the most potent growth factor for estrogen (E2)-dependent breast cancer cell lines. It has been reported that such cell lines produce an immunoreactive IGF-I-related protein in an E2-dependent fashion, while autonomously growing cell lines constitutively produce these factors, indicating that they might be involved in autocrine growth stimulation of breast cancer cells. We have studied the role of IGFs in autocrine growth stimulation of the E2-dependent breast cancer cell line MCF7 by using IGF-binding proteins (BPs) that were able to neutralize completely the mitogenic effect of IGF-I on this cell line. These BPs, however, did not have any inhibitory effect on E2-induced mitogenesis, suggesting that secretion of autocrine IGFs is not involved in growth stimulation by E2. To exclude the possibility that variants of IGF are produced by the cells that are not recognized by the BPs, we also studied the production of biologically active IGF using the same MCF7 cell line in a sensitive bioassay in which the various forms of IGF and insulin can be detected. Although the conditioned medium (CM) of human fibroblasts used as a control showed IGF-like activity in this assay, no such activity was detected in CM of both untreated and E2-stimulated MCF7 cells, while the CM of the E2-independent cell lines BT20 and Hs578T had only slight mitogenic activity or was even growth inhibitory, respectively. These data show that no significant secretion of biologically active IGFs by human breast cancer cells could be detected and do not support a possible autocrine function of IGFs in the proliferation of such cells. Because E2-dependent cells strongly react to externally added IGF-like factors produced by human fibroblasts, a role for IGFs in paracrine regulation of proliferation is suggested.

Molecular cloning and functional characterization of the human platelet-derived growth factor alpha receptor gene promoter.

Expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) is strictly regulated during mammalian development and tumorigenesis. The molecular mechanisms involved in the specific regulation of PDGF alpha R expression are unknown, but transcriptional regulation of the PDGF alpha R gene is most likely to be involved. This study describes the molecular cloning of the non-coding exon 1 and approximately 2 kb of 5' flanking region of the human PDGF alpha R gene. This 5' flanking region is a functional promoter of the PDGF alpha R gene as concluded from its capacity to drive luciferase reporter gene expression in an orientation dependent way. Analysis of 5' promoter deletion mutants revealed that the region from -441 to +118, relative to the transcription initiation site, is sufficient to establish high level promoter activity. In addition, the morphogen retinoic acid, alone or in combination with dibutyryl cAMP, gives a 22-fold induction of PDGF alpha R gene promoter activity in human teratocarcinoma cells. This effect is mediated through specific transcription factor binding within the -52/+118 region of the PDGF alpha R gene.

The oncogenic ErbB-2/ErbB-3 heterodimer is a surrogate receptor of the epidermal growth factor and betacellulin.

The ErbB-1 receptor tyrosine kinase binds to six different growth factors, whose prototype is the epidermal growth factor (EGF). Two homologous epithelial receptors, ErbB-3 and ErbB-4, bind all isoforms of another family of growth factors, the Neu differentiation factors (NDFs/neuregulins). The fourth member of the ErbB family, ErbB-2, acts as the preferred heterodimeric partner of ligand-occupied complexes of the three other ErbB proteins. Here we report that at high concentrations, EGF can induce cell growth and differentiation in the absence of ErbB-1. This function is shared by betacellulin, but not by three other ligands, including the transforming growth factor alpha (TGFalpha). The functional receptor was identified as a heterodimer between ErbB-3 and ErbB-2, a previously identified oncogenic complex. When singly expressed, neither ErbB-3 nor ErbB-2 can mediate signaling by EGF. In addition, when co-expressed, blocking either receptor by using site-specific antibodies inhibited EGF and betacellulin activities, indicating strict cooperativity between ErbB-3 and ErbB-2. Through analysis of chimeras between EGF and TGFalpha, we identified the middle portion of EGF (loop B) as the site that enables activation of ErbB-2/ErbB-3. In conclusion, cooperative and promiscuous binding of stroma-derived growth factors by the epithelium-expressed ErbB-2/ErbB-3 heterodimer may be significant to cancer development. The mechanistic implications of our results for a model that attributes receptor dimerization to ligand bivalency, as well as to a recently proposed mechanism of secondary dimerization, are discussed.

Effect of the phase transition on the transbilayer movement of dimyristoyl phosphatidylcholine in unilamellar vesicles.

Dimyristoyl phosphatidylcholine rapidly exchanges between vesicles at 37 degrees C without vesicle fusion. The rate of the transbilayer movement of dimyristoyl phosphatidylcholine in sonicated vesicles has been measured employing 13C NMR using N-13CH3-labeled lipids which are introduced into the outer monolayer of non-labeled vesicles by a phosphatidylcholine exchange protein. The rate of transbilayer movement of dimyristoyl phosphatidylcholine shows a distinct maximum (half-time 4 h) in the temperature range at which the hydrocarbon phase transition occurs. The activation energy of the flip-flop rate above the phase transition is 23.7 +/- 2.0 kcal/mol.

Glycophorin facilitates the transbilayer movement of phosphatidylcholine in vesicles.

The rate of transbilayer movement of dioleoylphosphatidylcholine in sonicated lipid vesicles is enhanced by at least two orders of magnitude upon incorporation of glycophorin in the bilayer.

Protein-mediated transbilayer movement of lysophosphatidylcholine in glycophorin-containing vesicles.

1. Sonicated glycophorin-containing vesicles of dioleoyl phosphatidylcholine have been made. The outside-inside distributions of the lipid molecules in these vesicles was measured with NMR and was found to be comparable with that of protein-free vesicles. 2. The transbilayer distribution of palmitoyl lysophosphatidylcholine in these vesicles is such that they have a significantly higher content of the lyso-compound in the inner monolayer when compared with vesicles without glycophorin. 3. Lysophosphatidylcholine, added to pre-existing glycophorin-containing vesicles, is incorporated in the outer monolayer of these vesicles. Subsequently it is able to move to the inner monolayer with an estimated half time of about 1.5 h at 4 degrees C. This was measured with 13C-NMR using [N-13CH3]lysophosphatidylcholine. 4. Treatment of co-sonicated vesicles of phosphatidylcholine and lysophosphatidylcholine containing glycophorin with the enzyme lysophospholipase results in a complete degradation of the lyso-compound. A half time of transbilayer movement of lysophosphatidylcholine during this experiment was estimated to be about 1 h at 37 degrees C.

A 13C NMR method for determination of the transbilayer distribution of phosphatidylcholine in large, unilamellar, protein-free and protein-containing vesicles.

(1) Large unilamellar vesicles have been prepared from N-[Ne3-13C]-18 : 1c/18 : 1c-phosphatidylcholine, both with and without the major intrinsic proteins from the human erythrocyte membrane incorporated in the bilayer. (2) It is shown that the inside-outside distribution of the lipid molecules in these large unilamellar structures can be determined using 13C NMR. (3) Large vesicles of 18 : 1c/18 : 1c-phosphatidylcholine containing glycophorin show an enhanced permeability to Dy3+. It is shown that the permeability barrier of these vesicles can be restored by addition of 10 mol% 18 : 1c/18 : 1c-phosphatidylethanolamine or 1-18 : 1c-lysophosphatidylcholine.

Evidence for the preferential interaction of glycophorin with negatively charged phospholipids.

Glycophorin, extracted from the erythrocyte membrane after treatment with lithium-diiodo-salicylate, still contains a significant amount of phospholipid, consisting predominantly of phosphatidylserine. Methods are described which lead to a full delipidation of the protein. After treatment with neuraminidase, delipidated glycophorin shows a preferential interaction with monolayers of negatively-charged phospholipids. This lipid-protein interaction is decreased by the presence of cholesterol in the lipid film.

Expression of human liver fatty acid-binding protein in Escherichia coli and comparative analysis of its binding characteristics with muscle fatty acid-binding protein.

Human liver fatty acid-binding protein (L-FABP) has been efficiently expressed in Escherichia coli. The cDNA encoding human liver FABP was under the control of T7 RNA polymerase promoter in the expression vector pET-3b. Expression required overnight induction with isopropyl beta-D-thiogalactopyranoside in the presence of the bacterial RNA polymerase inhibitor, rifampicin. The protein could be purified by (NH4)2SO4 fractionation, anion-exchange and gel filtration chromatography, and was recognized by anti-(human L-FABP) antiserum. The binding characteristics of delipidated recombinant human L-FABP and muscle FABP (M-FABP) for fatty acids of different chain length and saturation grade, and for various hydrophobic ligands, were determined by radiochemical analysis and also by fluorescence for L-FABP. The apparent binding affinity of the ligands was calculated by using displacement curves of oleic acid and dansylamino-undecanoic acid (DAUDA). L-FABP showed a preference for the binding of long-chain saturated and unsaturated fatty acids up to C24:1, whereas the M-FABP has a preference for unsaturated fatty acids, especially those with 18 C atoms. L-FABP also binds palmitoyl derivatives and many other hydrophobic ligands--however, generally with a lower affinity than fatty acids. M-FABP binds--besides with fatty acids--only with oestradiol and testosterone with high affinity. Fatty acids with fluorescent reporter groups are also more tightly bound by L-FABP. A direct assay and displacement study of oleic acid gave the same Kd value of DAUDA for L-FABP. Fluorescence enhancement and displacement studies indicate that the binding of fluorescent fatty acids is determined by both the fluorescent reporter group and the acyl carbon chain.

Phenotypic transformation of normal rat kidney fibroblasts by endothelin-1. Different mode of action from lysophosphatidic acid, bradykinin, and prostaglandin f2alpha.

In the present study, we compared the effects of endothelin (ET)-1 on cell proliferation and second messenger induction in normal rat kidney (NRK) fibroblasts, with those of other activators of G-protein-coupled receptors such as prostaglandin (PG)-F2alpha, bradykinin (BK), and lysophosphatidic acid (LPA). LPA is mitogenic by itself, while the other factors require the presence of EGF. In density-arrested NRK cells, ET-1 and LPA induce phenotypic transformation rapidly, with similar kinetics as retinoic acid (RA) and transforming growth factor (TGF)-beta, while BK and PGF2alpha only do so with delayed kinetics. ET-1 and PGF2alpha are strong inducers of anchorage-independent growth, with a similar level of induction as TGFbeta, in contrast to LPA and BK. When investigating the second messenger generation, we found that ET-1 is the strongest activator of arachidonic acid release and phosphatidylinositol diphosphate hydrolysis. Only in the case of ET-1 the cell depolarization is not reversible upon removal of the factor. Similarly, only the ET-1-induced transient enhancement of intracellular calcium concentration is paralleled by both homologous and heterologous desensitization. In conclusion, these data show that ET-1 is a potent inducer of second messengers and phenotypic transformation in NRK cells, with characteristics that clearly differ from those of other activators of G-protein-coupled receptors, most likely as a result of prolonged receptor activation.

Osmotic behaviour of Acholeplasma laidlawii B cells with membrane lipids in liquid-crystalline and gel state.

The osmotic behaviour of Acholeplasma laidlawii B cells was investigated with combined spectrophotometric and enzymatic measurements. The conclusion could be drawn that this osmotic behaviour depends largely on the physical state of the membrane lipids. When part of the membrane lipids is in the liquid-crystalline phase the cell is able to swell and behaves as a good osmometer. However, when the membrane lipid is in the gel phase, the cell is unable to swell and the change in absorbance of the cell suspension is then completely due to lysis.

A molecular basis for an irreversible thermodynamic description on non-electrolyte permeation through lipid bilayers.

The non-electrolyte permeability of liposomal membranes has been investigated according to the concepts of irreversible thermodynamics. A strong interaction between the permeation of solute and water was observed. This solute-solvent interaction can be fully described by assuming that a number of water molecules will copermeate with each molecule of solute. This number of copermeating water molecules is independent of the nature of the permeant and of temperature, but depends on the osmotic concentration of impermeants inside the liposomes.

Non-electrolyte permeability as a tool for studying membrane fluidity.

1. The reflection coefficient for the permeation of thiourea through bilayers of phosphatidylcholine is a function of the fatty-acid composition of the lipid molecules. By means of these reflection coefficients an index for membrane fluidity has been given to each of those lipids, relative to that of egg phosphatidylcholine. 2. The maximum number of water molecules that can copermeate with each molecule of solute by means of solute-solvent interaction is a function of the packing of the lipid molecules in the bilayer. This parameter has been used in this paper for characterizing the fluidity of cholesterol-containing membranes and for membranes with their lipids in the gel state.