Isolation of Chinese hamster ovary cell lines expressing human acyl-coenzyme A/cholesterol acyltransferase activity.

We have previously reported the isolation of Chinese hamster ovary cell mutants deficient in acylcoenzyme A/cholesterol acyltransferase (ACAT) activity (Cadigan, K. M., J. G. Heider, and T. Y. Chang. 1988, J. Biol. Chem. 263:274-282). We now describe a procedure for isolating cells from these mutants that have regained the ability to synthesize cholesterol esters. The protocol uses the fluorescent stain Nile red, which is specific for neutral lipids such as cholesterol ester. After ACAT mutant populations were subjected to chemical mutagenesis or transfected with human fibroblast whole genomic DNA, two revertants and one primary transformant were isolated by virtue of their higher fluorescent intensities using flow cytofluorimetry. Both the revertants and transformant have regained large amounts of intracellular cholesterol ester and ACAT activity. However, heat inactivation experiments revealed that the enzyme activity of the transformant had heat stability properties identical to that of human fibroblasts, while the ACAT activities of the revertants were similar to that of other Chinese hamster ovary cell lines. These results suggest that the molecular lesion in the ACAT mutants resides in the structural gene for the enzyme, and the transformant has corrected this defect by acquiring and stably expressing a human gene encoding the ACAT polypeptide. Secondary transformants were isolated by transfection of ACAT mutant cells with primary transformant genomic DNA. Genomic Southern analysis of the secondary transformants using a probe specific for human DNA revealed several distinct restriction fragments common to all the transformants which most likely comprise part or all of the human ACAT gene. The cell lines described here should facilitate the cloning of the gene encoding the human ACAT enzyme.

Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)-cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.

The structure of acyl coenzyme A-cholesterol acyltransferase and its potential relevance to atherosclerosis.

Acyl coenzyme A-cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters. It is present in a variety of tissues and is believed to play significant roles in cholesterol homeostasis. Under pathologic conditions, accumulation of the ACAT reaction product as cytoplasmic cholesterol ester lipid droplets within macrophages and smooth muscle cells is a characteristic feature of early lesions of human atherosclerotic plaques. ACAT is a membrane protein located in the endoplasmic reticulum. Its activity is susceptible to inactivation by detergents, and it has never been purified to homogeneity; no antibodies directed against it have been reported. Through a somatic cell and molecular genetic approach, we have recently succeeded in molecular cloning and functional expression of a human macrophage ACAT cDNA. This cDNA contains an open reading frame of 1650 base pairs encoding an integral membrane protein of 550 amino acids. Protein homology analysis shows that the predicted protein sequence shares short regions of homology with other enzymes involved in the catalysis of acyl adenylate formation with subsequent acyl thioester formation and acyl transfer. The ACAT cDNA will enable the investigation of ACAT biochemistry and molecular biology. It will speed up the design of specific ACAT inhibitors as drugs that may provide more effective therapeutic treatment or prevention of atherosclerosis. In addition, studies on the physiologic roles of ACAT in various tissues can now be undertaken through transgenic animal research.

How do they do Wnt they do?: regulation of transcription by the Wnt/ß-catenin pathway.

Wnt/ß-catenin signalling is known to play many roles in metazoan development and tissue homeostasis. Misregulation of the pathway has also been linked to many human diseases. In this review, specific aspects of the pathway's involvement in these processes are discussed, with an emphasis on how Wnt/ß-catenin signalling regulates gene expression in a cell and temporally specific manner. The T-cell factor (TCF) family of transcription factors, which mediate a large portion of Wnt/ß-catenin signalling, will be discussed in detail. Invertebrates contain a single TCF gene that contains two DNA-binding domains, the high mobility group (HMG) domain and the C-clamp, which increases the specificity of DNA binding. In vertebrates, the situation is more complex, with four TCF genes producing many isoforms that contain the HMG domain, but only some of which possess a C-clamp. Vertebrate TCFs have been reported to act in concert with many other transcription factors, which may explain how they obtain sufficient specificity for specific DNA sequences, as well as how they achieve a wide diversity of transcriptional outputs in different cells.

A simple method for reconstitution of CHO cell and human fibroblast acyl coenzyme A: cholesterol acyltransferase activity into liposomes.

A new method for reconstituting acyl coenzyme A: cholesterol acyltransferase (ACAT) activity from either Chinese hamster ovary (CHO) or human fibroblast cell extracts into cholesterol-phosphatidylcholine liposomes is described. The method is rapid (less than 60 min) and easy to perform. The procedure involves solubilizing the cell extracts with deoxycholate followed by dilution into preformed liposomes. Ficoll gradient analysis demonstrated that, after reconstitution, almost all of the detectable ACAT activity co-migrated with the liposomes. Exogenous cholesterol in the liposomes was absolutely necessary for providing ACAT activity, but not for incorporation of the ACAT enzyme into the vesicle bilayer. Human fibroblast cell extracts prepared from cells grown in medium containing 10% fetal calf serum were found to contain a 10-fold higher microsomal ACAT activity compared to extracts from cells grown in 10% delipidated fetal calf serum. In contrast, when the ACAT activity from these extracts was measured using the reconstitution assay, there was no difference in the specific activities. These results support our previous work (Doolittle, G. M., and T. Y. Chang. 1982. Biochim. Biophys. Acta. 713: 529-537; and Chang, C. C. Y., et al. 1986. Biochemistry. 25: 1693-1699), and suggest that cholesterol regulates ACAT activity in CHO cells and human fibroblasts by mechanism(s) other than modulation of the amount of enzyme.

wingless signaling in the Drosophila eye and embryonic epidermis.

After the onset of pupation, sensory organ precursors, the progenitors of the interommatidial bristles, are selected in the developing Drosophila eye. We have found that wingless, when expressed ectopically in the eye via the sevenless promoter, blocks this process. Transgenic eyes have reduced expression of acheate, suggesting that wingless acts at the level of the proneural genes to block bristle development. This is in contrast to the wing, where wingless positively regulates acheate to promote bristle formation. The sevenless promoter is not active in the acheate-positive cells, indicating that the wingless is acting in a paracrine manner. Clonal analysis revealed a requirement for the genes porcupine, dishevelled and armadillo in mediating the wingless effect. Overexpression of zeste white-3 partially blocks the ability of wingless to inhibit bristle formation, consistent with the notion that wingless acts in opposition to zeste white-3. Thus the wingless signaling pathway in the eye appears to be very similar to that described in the embryo and wing. The Notch gene product has also been suggested to play a role in wingless signaling (J. P. Couso and A. M. Martinez Arias (1994) Cell 79, 259-72). Because Notch has many functions during eye development, including its role in inhibiting bristle formation through the neurogenic pathway, it is difficult to assess the relationship of Notch to wingless in the eye. However, we present evidence that wingless signaling still occurs normally in the complete absence of Notch protein in the embryonic epidermis. Thus, in the simplest model for wingless signalling, a direct role for Notch is unlikely.

Three maternal coordinate systems cooperate in the patterning of the Drosophila head.

In contrast to the segmentation of the embryonic trunk region which has been extensively studied, relatively little is known about the development and segmentation of the Drosophila head. Proper development of the cephalic region requires the informational input of three of the four maternal coordinate systems. Head-specific gene expression is set up in response to a complex interaction between the maternally provided gene products and zygotically expressed genes. Several zygotic genes involved in head development have recently been characterized. A genetic analysis suggests that the segmentation of the head may use a mechanism different from the one acting in the trunk. The two genes of the sloppy paired locus (slp1 and slp2) are also expressed in the embryonic head. slp1 plays a predominant role in head formation while slp2 is largely dispensible. A detailed analysis of the slp head phenotype suggests that slp is important for the development of the mandibular segment as well as two adjacent pregnathal segments (antennal and ocular). Our analysis of regulatory interactions of slp with maternal and zygotic genes suggests that it behaves like a gap gene. Thus, phenotype and regulation of slp support the view that slp acts as a head-specific gap gene in addition to its function as a pair-rule and segment polarity gene in the trunk. We show that all three maternal systems active in the cephalic region are required for proper slp expression and that the different systems cooperate in the patterning of the head. The terminal and anterior patterning system appear to be closely linked. This cooperation is likely to involve a direct interaction between the bcd morphogen and the terminal system. Low levels of terminal system activity seem to potentiate bcd as an activator of slp, whereas high levels down-regulate bcd rendering it inactive. Our analysis suggests that dorsal, the morphogen of the dorsoventral system, and the head-specific gap gene empty spiracles act as repressor and corepressor in the regulation of slp. We discuss how positional information established independently along two axes can act in concert to control gene regulation in two dimensions.

Functional redundancy: the respective roles of the two sloppy paired genes in Drosophila segmentation.

The sloppy paired (slp) locus consists of two genes, slp1 and slp2, both of which encode proteins containing a forkhead domain (a DNA-binding motif). Previous work has shown that a severe segmentation phenotype is obtained only when both slp genes are deleted. Here we examine the functional redundancy of the locus in more detail. The phenotypes of embryos containing various combinations of functional slp genes suggest that for early slp function, until gastrulation, only slp1 is required. At later times, there is still a greater requirement for slp1, but in many respects the two slp genes are completely redundant. Both slp genes produce similar phenotypes when ubiquitously expressed via a heat shock promoter. We propose that the slp proteins are biochemically equivalent and that the greater requirement for slp1 in some functions can be explained in large part by its earlier expression.

Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity.

A protocol has been developed for isolating cholesterol ester-deficient cells from the Chinese hamster ovary cell clone 25-RA. This cell line previously was shown to be partially resistant to suppression of cholesterogenic enzyme activities by 25-hydroxycholesterol and to accumulate a large amount of intracellular cholesterol ester when grown in medium containing 10% fetal calf serum (Chang, T. Y., and Limanek, J. S. (1980) J. Biol. Chem. 255, 7787-7795). The higher cholesterol ester content of 25-RA is due to an increase in the rate of cholesterol biosynthesis and low density lipoprotein receptor activity compared to wild-type Chinese hamster ovary cells, and not due to an abnormal acyl-CoA:cholesterol acyltransferase enzyme. The procedure to isolate cholesterol ester-deficient mutants utilizes amphotericin B, a polyene antibiotic known to bind to cholesterol and to form pore complexes in membranes. After incubation in cholesterol-free medium plus an inhibitor of endogenous cholesterol biosynthesis, 25-RA cells were found to be 50-500 times more sensitive to amphotericin B killing than were mutant cells containing reduced amounts of cholesterol ester. Twelve amphotericin B-resistant mutants were isolated which retained the 25-hydroxycholesterol-resistant phenotype. These mutants did not exhibit the perinuclear lipid droplets characteristic of 25-RA cells, and lipid analysis revealed a large (up to 40-fold) reduction in cellular cholesterol ester. The acyl-CoA:cholesterol acyltransferase activities of these cholesterol ester-deficient mutants were markedly lower than 25-RA when assayed in intact cells or in an in vitro reconstitution assay. The tightest mutant characterized, AC29, was found to have less than 1% of the parental acyl-CoA:cholesterol acyltransferase activity. These mutants all have reduced rates of sterol synthesis and lower low density lipoprotein receptor activity compared to 25-RA, probably as a consequence of their reduced enzyme activities. Cell fusion experiments revealed that the phenotypes of all the mutants examined are not dominant and that the mutants all belong to the same complementation group. We conclude that these mutants contain a lesion in the gene encoding acyl-CoA:cholesterol acyltransferase or in a gene encoding a factor needed for enzyme production.

Localized expression of sloppy paired protein maintains the polarity of Drosophila parasegments.

During germ-band extension in the Drosophila embryo, intercellular communication is required to maintain gene expression patterns initiated at cellular blastoderm. For example, the wingless (wg) single-cell-wide stripe in each parasegment (PS) is dependent on a signal from the adjacent, posterior cells, which express engrailed (eN). This signal is thought to be the hedgehog (hh) gene product, which antagonizes the activity of patched (ptc), a repressor of wg expression. Genetic evidence indicates that the hh signal is bidirectional, but wg transcription is only derepressed on the anterior side of the en/hh stripes. To explain the asymmetric response of the wg promoter to the hh signal, current models predict that each PS is divided into cells that are competent to express either wg or en, but not both. The sloppy paired (slp) locus contains two transcription units, both encoding proteins containing a forkhead domain, a DNA-binding motif. Removal of slp gene function causes embryos to exhibit a severe pair-rule/segment polarity phenotype. We show that the en stripes expand anteriorly in slp mutant embryos and that slp activity is an absolute requirement for maintenance of wg expression at the same time that wg transcription is dependent on hh. The slp proteins are expressed in broad stripes just anterior of the en-positive cells, overlapping the narrow wg stripes. We propose that by virtue of their ability to activate wg and repress en expression, the distribution of the slp proteins define the wg-competent and en-competent groups. Consistent with this hypothesis, ubiquitous expression of slp protein throughout the PS abolishes en expression and, in ptc mutant embryos, results in a near ubiquitous distribution of wg transcripts. In addition to demonstrating the role of slp in maintaining segment polarity, our results suggest that slp works in, or parallel with, the ptc/hh signal transduction pathway to regulate wg transcription.

Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells.

Accumulation of cholesterol esters as cytoplasmic lipid droplets within macrophages and smooth muscle cells is a characteristic feature of early lesions of atherosclerotic plaque. Intracellularly, an essential element in forming cholesterol ester from cholesterol is the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT). ACAT is a membrane protein located in the endoplasmic reticulum. The ACAT protein has never been purified to homogeneity, and no antibodies directed against ACAT have been reported. The gene(s) encoding this enzyme had not been isolated. This laboratory had previously reported the isolation of Chinese hamster ovary cells expressing human ACAT activity. From DNAs of these cells, we have cloned a 1.2-kb exonic human genomic DNA. This led to the eventual cloning of a 4-kb cDNA clone (K1) from a human macrophage cDNA library. Transfection of K1 in ACAT-deficient mutant Chinese hamster ovary cells complemented the mutant defect and resulted in the expression of human ACAT activity. K1 contained an open reading frame of 1650 bp encoding an integral membrane protein of 550 amino acids. Protein homology analysis showed that the predicted K1 protein shared homologous peptide sequences with other enzymes involved in the catalysis of acyl adenylate formation followed by acyl thioester formation and acyl transfer. These results indicate that K1 encodes a structural gene for ACAT. The cDNA reported here should facilitate future molecular studies on ACAT.

Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development.

In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, we have conducted a genetic analysis of frizzled and Dfrizzled2 in the embryo. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled, Dfrizzled2 mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled, Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning.

Wingless repression of Drosophila frizzled 2 expression shapes the Wingless morphogen gradient in the wing.

In Drosophila wing imaginal discs, the Wingless (Wg) protein acts as a morphogen, emanating from the dorsal/ventral (D/V) boundary of the disc to directly define cell identities along the D/V axis at short and long range. Here, we show that high levels of a Wg receptor, Drosophila frizzled 2 (Dfz2), stabilize Wg, allowing it to reach cells far from its site of synthesis. Wg signaling represses Dfz2 expression, creating a gradient of decreasing Wg stability moving toward the D/V boundary. This repression of Dfz2 is crucial for the normal shape of Wg morphogen gradient as well as the response of cells to the Wg signal. In contrast to other ligand-receptor relationships where the receptor limits diffusion of the ligand, Dfz2 broadens the range of Wg action by protecting it from degradation.

Naked cuticle targets dishevelled to antagonize Wnt signal transduction.

In Drosophila embryos the protein Naked cuticle (Nkd) limits the effects of the Wnt signal Wingless (Wg) during early segmentation. nkd loss of function results in segment polarity defects and embryonic death, but how nkd affects Wnt signaling is unknown. Using ectopic expression, we find that Nkd affects, in a cell-autonomous manner, a transduction step between the Wnt signaling components Dishevelled (Dsh) and Zeste-white 3 kinase (Zw3). Zw3 is essential for repressing Wg target-gene transcription in the absence of a Wg signal, and the role of Wg is to relieve this inhibition. Our double-mutant analysis shows that, in contrast to Zw3, Nkd acts when the Wg pathway is active to restrain signal transduction. Yeast two hybrid and in vitro experiments indicate that Nkd directly binds to the basic-PDZ region of Dsh. Specially timed Nkd overexpression is capable of abolishing Dsh function in a distinct signaling pathway that controls planar-cell polarity. Our results suggest that Nkd acts directly through Dsh to limit Wg activity and thus determines how efficiently Wnt signals stabilize Armadillo (Arm)/beta-catenin and activate downstream genes.