extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Effect of tumour cell-conditioned medium on endothelial macromolecular permeability and its correlation with collagen.

Conditioned medium prepared from mouse melanoma B16 cells (B16-CM) increases the macromolecular permeability of bovine aortic, venous and human umbilical vein endothelial monolayer. Collagen, which is synthesised by endothelial cells, has an important function in regulating the permeability of endothelial monolayer. Briefly, low collagen content leads to hyperpermeable structure of the endothelial monolayer. In the present studies, we examined the relationship between the increase of endothelial permeability and content of synthesised collagen of endothelial cells cultured with B16-CM. The B16-CM reduced endothelial collagen content but did not digest collagen directly. Matrix metalloproteinase inhibitor, 1,10-phenanthroline, inhibited the increase in permeability due to addition of B16-CM. These data suggest that B16-CM acts on endothelial cells, stimulating the digestion of endothelial collagen, and that the reduced content of collagen leads to the hyperpermeability of the endothelial monolayer.

It is now well established that there is great heterogeneity among microvessel endothelial cells from different organ sites. Tumour blood vessels also differ from those of normal tissues in some important structural and functional aspects. Many investigators have reported that tumour vessels are hyperpermeable compared with normal vessels (Song and Levitt, 1971;Dvorak et al., 1984;O'Connor and Bale, 1984;Gerlowski and Jain, 1986). However, the cause and mechanism of the hyerpermeability of tumour vessels are unclear. In previous studies, we found that conditioned medium prepared from mouse melanoma B16 (B16-CM) increases the macromolecular permeability of bovine aortic, venous and human umbilical vein endothelial cell monolayers (Utoguchi et al., 1995a). These data suggest that the properties of normal tissue-derived endothelial cells can be changed, resembling those expressed in tumour vessels, by culture with tumour cell-conditioned medium. We have also reported that the collagen, which is synthesised by endothelial cells themselves, has an important role in the regulation of the endothelial cell permeability in the steady state (Utoguchi et al., 1995b). The mechanism of hyperpermeability of the endothelial monolayer cultured with B16-CM is unclear. In the present studies, we examined the relationship between the collagen content and hyperpermeability of the endothelial monolayer when cultured with B16-CM.
Preparation of continued medium Bovine smooth muscle cells were isolated from calf aorta by mechanical scraping and cultured with DMEM containing 10% FCS. When the B16 melanoma cells or bovine smooth muscle cells were at subconfluence in the culture dishes, the cells were added to ASF104 medium. After 24 h, the medium was collected and centrifuged at 1600 g for 5 min. In the preparations subjected to measurement of synthesised collagen content, the supernatant of the conditioned medium was dialysed against DMEM to remove proline. Before the conditioned medium was used it was mixed with the same volume of DMEM supplemented with 20% FCS.

Endothelial cell culture
Bovine aortic endothelial cells (BAECs) were isolated from calf aorta by mechanical scraping and cultured with DMEM containing 10% FCS. An Intercell permeation chamber was used for measurement of endothelial permeability. Before plating of endothelial cells, the Teflon membrane (pore size, 0.45 psm) of Intercell was coated with collagen type I (100 ILg ml-') for 2 h and washed three times with phosphatebuffered saline (PBS). BAECs (seventh to tenth passages) were seeded on the permeation chamber (2.0 x 105 cells cm-2) and during culture, the luminal (upper) and the abluminal (lower) compartment contained 200 ll and 700 tll of culture medium respectively. Four hours after seeding, the cells were cultured with the B16-CM, smooth muscle cell-conditioned medium or 3,4-dihydroxybenzoic acid ethyl ester (DHB). The medium was changed every 2 days. In the cultures for collagen content measurement, the cells were cultured with each medium containing 10 tCi ml-' 2,3-[3H]proline. After 5 days of cultivation, the cells were confluent and the permeability assay and measurement of collagen content were performed.
Permeability of endothelial monolayer FITC-dextran (average molecular weight, 70000) was used for an index of the macromolecular permeability of the endothelial monolayer; FITC-dextran is transported through the intercellular spaces of the endothelial junctions (Hashida et al., 1986). The FITC-dextran permeability assay was performed by the method described previously (Mizuguchi et al., 1994). Briefly, FITC-dextran was dissolved to 50 J.M in MEM containing 10% FCS without phenol red. To the luminal compartment was added 200 pl of this solution, and to the abluminal compartment was added 700 itl of the same medium without FITC-dextran. The experiments were performed at 37°C in a 95% air/5% carbon dioxide-humidified atmosphere with gentle shaking. After 1 h, the chamber was removed and the permeated FITC-dextran was quantified using a fluorescence spectrophotometer (excitation 495 nm, emission 550 nm). The permeability coefficient of the endothelial monolayers alone was calculated from the following relationship (Siflinger-Birnboim et al., 1987): Measurement of collagen content Collagen synthesis by the BAECs was determined by a slight modification of the method previously described (Geesin et al., 1991). BAECs were seeded on permeation chambers, and after 4 h of cultivation, the cells were cultured with control medium or B 16-CM containing 2,3-[3H]proline (10 ytCi ml ').
After appropriate times of incubation, the cells were washed with PBS and were solubilised with 0.5 N sodium hydroxide. After 12 h of incubation at 37°C, 0.5 N hydrochloric acid was added. This lysate was digested by non-specific protease-free collagenase. After 3 h of incubation at 37°C the samples were precipitated with 25% trichloroacetic acid and centrifuged at 13 000 g for 10 min. The radioactivity of the collagenasesoluble and -insoluble fractions was determined by a liquid scintillation counter. The relative rate of collagen synthesis was calculated based on the principle that collagen has an imino acid content 5.4 times higher than that of other proteins (Peterkofsky and Diegelmann, 1971). The ratio of collagen to total protein (R) was calculated from the following relationship: R = C/(N x 5.4 + C) x 100 (%) where C and N are the radioactivity in collagenase-soluble (collagenous) and -insoluble (non-collagenous) fractions respectively.
Preparation of extracellular matrix The BAECs, which were cultured on the permeation chamber with the medium containing [3H]proline for 3 days, were treated with 0.02 N ammonium hydroxide. After 10 min of incubation, the Teflon membrane was washed three times with PBS. The extracellular matrix that was produced by the BAECs was labelled with [3H]proline and left on the membrane (Laug et al., 1985;Partridge et al., 1992). This extracellular matrix-coated membrane was incubated with B16-CM at 37°C for 2 days and collagen content of the extracellular matrix on the membrane was measured.
Tumour-conditioned medium and endothelial permeability N Utoguchi et al x 25

Statistical analysis
All results were expressed as the mean value ± standard deviation. Statistical analysis between two groups were made by Student's t-test and one-way analysis of variance (ANOVA) was used for single and multiple comparisons. P-values of 0.05 or less were considered to be significant.

Results
Effect of tumour-conditioned medium on the endothelial permeability The endothelial permeability coefficient was significantly (P<0.01) increased with B16-CM to 65.2 ± 5.6 (x I0cm h'), compared with the control value of 15.2± 1.0 (x 10cm h-') ( Figure 1). The B16-CM had no effect on the cell number at confluence. The BAECs formed monolayers and no morphological differences between the BAECs cultured in the presence of control medium or B 16-CM culture were observed by phase-contrast microscopy as previously reported (Utoguchi et al., 1995a). The BAECs came in contact with each other in both culture groups.
Therefore, the increase of the permeability of BAECs cultured with Bl 6-CM did not depend on the decrease of the total shared area of the BAECs on the Teflon membrane of the permeation chamber. When the BAECs were cultured with the conditioned medium of smooth muscle cells which were derived from normal tissue, the permeability and cell number of the BAECs had not been changed. These data suggested that the hyperpermeability of the BAEC monolayer was specific for tumour cell supernatants.
Effect of B16-CM on the BAECs collagen content Table I shows the collagen content of the BAECs cultured with each medium. 3,4-Dihydroxy benzoic acid ethyl ester (DHB), a collagen synthesis inhibitor (Sasaki et al., 1987), decreased not only the collagen content but also the collagen to total protein per cent ratio and markedly increased and cell number ( ) of BAECs. BAECs were cultured on permeation chambers with B16-CM or bovine aortic smooth muscle cell-conditioned medium (SMC-CM). When the BAECs were at confluence, permeability assay was performed and the cell number was measured. The results are expressed as the mean and standard deviation of four determinations. *P<0.01 compared with control.  Similarly, the B 16-CM decreased not only the collagen count, from 67 ± 10 (x 102 d.p.m.) in the control to 22 + 3, but also the collagen to total protein per cent ratio, from 1.30 ± 0.26% to 0.36 ± 0.07%. These observations indicated that the B16-CM specifically decreased the amount of collagen rather than non-specifically decreasing the total protein content. The magnitude of decrease was similar when the BAECs were cultured with DHB, and the increase of the permeability of BAECs cultured with B 16-CM was also similar to that of BAECs cultured with DHB. These findings suggested that the low collagen content observed when BAECs were cultured with B16-CM caused the increase of the endothelial permeability.
Digestion of collagen with B16-CM The two possible causes of the decreased collagen content by B16-CM are inhibition of the collagen synthesis of BAECs by B16-CM and stimulation of the digestion of collagen by B16-CM. Tumour cells and endothelial cells secrete many types of matrix metalloproteinases (MMPs) which degrade extracellular matrix components (McCroskery et al., 1975;Moscatelli et al., 1980;Kalebic et al., 1983). We examined whether the B16-CM digested endothelial collagen by using a pulse labelling technique. After 3 days of cultivation with the control medium containing [3H]proline, the medium was removed and the BAECs were cultured with the control medium or B16-CM without [3H]proline. After 2 additional days of cultivation, the collagen content was measured. The B16-CM digested endothelial collagen, and not only was the collagen content decreased to about 40% of the control value but the collagen to total protein per cent ratio was also decreased ( Figure 2). These data suggested that the B16-CM digested collagen specifically. The analysis of the effect of B16-CM on BAEC collagen conent had revealed reduction to 33% of the control value, a similar per cent decrease also seen in this experiment. These observations suggested that the decrease of collagen content elicited by B16-CM was almost exclusively dependent on the digestion of collagen rather than the inhibition of collagen synthesis.
Digestion activity of B16-CM against the extracellular matrix collagen To examine whether B 16-CM digests collagen directly or whether B16-CM stimulates the collagen digestion activity of endothelial cells we further examined direct digestion activity of B16-CM against the extracellular matrix which contained the collagen produced by the BAECs. The Teflon membrane was coated with extracellular matrix, which was produced by the BAECs and labelled with [3H]proline, and incubated with normal medium or B16-CM. After 2 days of incubation, the collagen content was determined. The B16-CM did not digest the collagen in the extracellular matrix on the Teflon membrane of the permeation chamber ( Figure 3). These data suggested that the B 16-CM acts on BAECs, stimulating the collagen digestion activity of the BAECs.
Effect of B16-CM on permeability and collagen content of precultured BAECs BAECs were precultured with B16-CM for 5 days, detached from dishes and plated onto the permeation chamber. After 5 days of cultivation with the normal medium containing [3H]proline, the permeability and collagen content were measured (Table II). The BAECs precultured with B16-CM presented significant hyperpermeability and significantly reduced collagen content (BI6-CM/control group). Under  Figure 2 Effect of B16-CM on the collagen content ( ) and collagen-total protein per cent ratio ( 11 ) of BAECs. BAECs were cultured on a permeation chamber with the control medium containing [3H]proline. After 3 days of cultivation, the cells were washed and cultured with [3H]proline-free control medium (control) or B16-CM. After 2 additional days, the collagen content was quantified by collagenase digestion method. The results are expressed as the mean and standard deviation of four determinations. *P<0.01 compared with control. 50.3 3.5* 1.56 ± 0.14* 41.97 ± 5.15* BAECs were cultured on the tissue culture dish with the control medium or B16-CM. After 5 days of cultivation the cells were detached from dishes and were cultured on the permeation chamber with the control or B 16-CM containing [3H]proline. After 5 additional days of cultivation the cells reached confluence and the permeability assay was performed. The amount of collagen was quantified with the collagenase digestion method. *P<0.01 compared with group control-control. this condition, the precultured BAECs were further cultured with control medium on a permeation chamber and therefore the B16-CM could not directly digest collagen.
Effect of 1,10-phenanthroline on the increase of the permeability with B16-CM We further examined the effect of the MMP inhibitor 1,10phenanthroline on the increase of the permeability with B16-CM. At the dose of 1.0 gM 1, 10-phenanthroline alone had no effect on the BAEC permeability ( Figure 4). Combination of B16-CM with 1,1O-phenanthroline (1.0 LM) completely inhibited the increase of the permeability of BAECs elicited by B16-CM. This observation suggests that MMPs, which are secreted by endothelial cells, degrade endothelial collagen, and that the presence of a low level of collagen induces the observed hyperpermeability of the endothelial monolayer when cultured with B16-CM.

Discussion
To understand the permeability of endothelial monolayer we have subdivided it into two different types, one of which is the permeability that is affected by vasoactive agents such as, histamine, bradykinin, serotonin and norepinephrine. Changes in permeability of this type can generally be observed within a short-term and are reversible. Permeability of the other type varies greatly depending on the type of endothelial cell and is dependent on the location of the tissue. For example, tumour vessels are more permeable than normal tissue vessels. Many agents that alter endothelial permeability reversibly in a short-term manner, such as vasoactive agerfts, have been reported (Svensjo et al., 1979;Rotrosen and Gallin, 1986;Langer and Van Hinsebergh, 1991). However, no agents capable of altering the permeability irreversibly over a long period other than ascorbic acid have been reported (Utoguchi et al., 1995b). In our previous studies, we found that the decrease of the collagen content putatively synthesised by endothelial cells led to increased permeability of the endothelial monolayer. The change in endothelial permeability elicited by B16-CM is long term. In our study of the mechanism by which B16-CM increases endothelial permeability, we demonstrated the hyperpermeability was associated with reduction of the level of collagen.
Tumour-conditioned medium and endothelial permeability N Utoguchi et al 27 Vascular permeability factor (VPF), which is secreted by many types of tumour cells, reversibly increases vascular permeability in vivo for a short time (Senger et al., 1983;Brock et al., 1991). VPF is thought to induce tumour vessel hyperpermeability in vivo Senger et al., 1993). In our study, BAECs that were precultured with the B 16-CM presented hyperpermeability. Although there were no data indicating that B 16-CM contains VPF, it is possible that if B 16-CM contains VPF, the BAECs might be affected by VPF in the precultured condition; however, the precultured cells were cultured in a permeation chamber with medium containing no VPF. Despite the lack of VPF in culture medium the BAECs that were precultured with B16-CM still showed hyperpermeability. If VPF caused the hyperpermeability in tumour vessels in vivo, our results suggested that the continuous presence of VPF is not required to maintain the increased permeability.
Tumour-conditioned medium contains many types of MMP (Halaka et al., 1983;Morodomi et al., 1992). However, our data show that B16-CM did not directly digest the collagen of the extracellular matrix (Figure 3). In our experiment, the B 16-CM contained FCS, which is abundant in proteins, and these proteins may have inhibited the digestive activity of MMP in B16-CM. On the other hand, the MMPs secreted from BAECs have the capacity to digest the collagen of the extracellular matrix. Collagen, which is the substrate of MMP, is present in the extracellular matrix, located just under BAECs, and MMP may act locally at highconcentration, thus, the collagen was digested even though the B16-CM contained serum. Fibronectin, which is one of the components of the extracellular matrix, regulates endothelial permeability (Partridge et al., 1992;Wheatley et al., 1993). A 96 kDa gelatinase induced by TNF-ox contributes to increased microvascular endothelial permeability in culture (Partridge et al., 1993). Heparan sulfate proteoglycan also has an important role in regulating endothelial permeability (Guretzki et al., 1994). We have no data as to whether the tumour-conditioned medium decreased the content of these extracellular matrix components of the endothelial cells. The results of the 1,10-phenanthroline experiment indicate that the digestion of collagen by endothelial cells should depend on MMPs that were secreted by endothelial cells. Degradation of the matrix by endothelial cells depends on the balance between MMP and their inhibitors, which are secreted by endothelial cells themselves (Herron et al., 1986;Matrisian, 1990;Unemori et al., 1990). We are presently examining whether decrease of collagen content by B16-CM is related to stimulation of MMP secretion or inhibition of secretion of MMP inhibitors, and we plan to report the results at a later time.
Some factors which are secreted from tumour cells induce angiogenesis and the in vivo MMP activities of endothelial cells are increased in angiogenesis (Roberts et al., 1986;Klagsbrun et al., 1986). Basic fibroblast growth factor induced the MMP production of the endothelial cells (Tsuboi et al., 1990), and vascular endothelial growth factor induces interstitial collagenase expression in human endothelial cells (Unemoru et al., 1992). It is well known that these factors are secreted by tumour cells. Based on these findings, we hypothesise that, in the induction of angiogenesis, certain factors that are secreted from tumour cells affect the activities of the MMP of endothelial cells. Our data obtained in the present studies support this hypothesis.
In electron microscopic studies, the basement membrane, which consists mainly of collagen, of a tumour vessel is visualised as fragmentary and discontinuous (Ward et al., 1974). Our culture system data are consistent with this in vivo observation. The properties of aorta-derived endothelial cells may change to those of tumour tissue-derived endothelial cells in the presence of tumour-conditioned medium. Endothelial cells cultured with tumour cell-conditioned medium may serve as a model in which to study the physiological characteristics of tumor endothelial cells. If _ :


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Unusual Amino Acid Determinants of Host Range in the Mtx2 Family of Mosquitocidal Toxins*

Five different mosquitocidal toxin ( mtx2 ) gene ho- mologs have been cloned from eight Bacillus sphaericus strains. Pairwise comparisons of the predicted amino acid sequences show between four and eight substitutions compared with the prototype Mtx2 from B. spha- ericus strain SSII-1. Mtx2 from strain SSII-1 was (cid:59) 7-fold more toxic to Culex mosquito larvae than the Mtx2 hom- olog from B. sphaericus strain 31-2. Conversely, Mtx2 from strain 31-2 was (cid:59) 100-fold more toxic to Aedes mosquito larvae than Mtx2 from strain SSII-1. Lys 224 in Mtx2 was found to be the most important amino acid for tox- icity to Culex larvae, and substitution of Lys 224 with threonine abolished the toxicity of Mtx2 from strain SSII-1 to these larvae. In complete contrast, Thr 224 was found to be crucial for the toxicity of Mtx2 from strain 31-2 to Aedes larvae, and substitution of Thr 224 with lysine caused a (cid:59) 100-fold drop in toxicity to these larvae. Thus, amino acid 224 in the Mtx2 family of mosqui- tocidal toxins is an unusual and important determinant of mosquito larvicidal activity and host range. Gif sur Yvette, and all other B. sphaericus strains were obtained from H. de Barjac, Pasteur Institute, Paris. All B. sphaericus strains were grown either in L-broth or NYSM medium (5). Cloning, Mutagenesis, and Sequence Analysis— The protein coding regions of mtx2 from eight different strains of B. sphaericus (31-2, IAB59, Kellen Q, 2297, 2362, 1691, 1593M, and 2317.3) was amplified by PCR from genomic DNA (9) using synthetic oligonucleotides based on the sequence of B. sphaericus SSII-1 mtx2 (4). The sequences of these oligonucleotides were: 5 (cid:57) -CCCCCCATGGATCCAATGAAAAGGAC- CAAATTACTTTTTTATATT-3 (cid:57) (TT119) for the 5 (cid:57) (upstream) primer, and 5 (cid:57) -CCCGACGTCATCGATGCATGCCTTAAGTTATTTAAAA- GAAATTTCTTTAACATCTATTA-3 (cid:57) (TT120) for the 3 (cid:57) (downstream) primer. Artificial Bam HI, Nco I, Cla I, Sph I, and Afl II restriction en- zyme sites were incorporated into these sequences for cloning purposes. 2297, 31-2, 2362,

Five different mosquitocidal toxin (mtx2) gene homologs have been cloned from eight Bacillus sphaericus strains. Pairwise comparisons of the predicted amino acid sequences show between four and eight substitutions compared with the prototype Mtx2 from B. sphaericus strain SSII-1. Mtx2 from strain SSII-1 was ϳ7-fold more toxic to Culex mosquito larvae than the Mtx2 homolog from B. sphaericus strain 31-2. Conversely, Mtx2 from strain 31-2 was ϳ100-fold more toxic to Aedes mosquito larvae than Mtx2 from strain SSII-1. Lys 224 in Mtx2 was found to be the most important amino acid for toxicity to Culex larvae, and substitution of Lys 224 with threonine abolished the toxicity of Mtx2 from strain SSII-1 to these larvae. In complete contrast, Thr 224 was found to be crucial for the toxicity of Mtx2 from strain 31-2 to Aedes larvae, and substitution of Thr 224 with lysine caused a ϳ100-fold drop in toxicity to these larvae. Thus, amino acid 224 in the Mtx2 family of mosquitocidal toxins is an unusual and important determinant of mosquito larvicidal activity and host range.
Bacillus sphaericus is an aerobic, Gram-positive, spore-forming bacterium which is widespread in soil and aquatic environments. Some strains of B. sphaericus produce protein toxins which are lethal to mosquito larvae (1,2). The best studied mosquitocidal strains of B. sphaericus are divided into a high toxicity group (e.g.. 2362, 2297, and IAB59) and a low toxicity group (e.g.. SSII-1, 31-2 and Kellen Q) (1). The high toxicity but not the low toxicity strains encode 51.4-and 41.9-kDa proteins, which together form a binary toxin expressed at high levels during sporulation. Most mosquito pathogenic B. sphaericus tested also harbor a 100-kDa toxin gene (mtx) and a 31.8-kDa toxin gene (mtx2) 1 (3,4).
Toxin production in the low toxicity strain B. sphaericus SSII-1 begins in the vegetative phase of growth before the onset of sporulation (5,6). The mtx2 gene of this strain encodes a polypeptide of 292 aa (Mtx2) with a molecular mass of 31.8 kDa, which is detected in the vegetative phase of growth (4). Mtx2 is unrelated to the binary and 100-kDa toxins but has regions of significant homology with the 33-kDa ⑀ toxin of Clostridium perfringens and the 31.68-kDa cytotoxin of Pseudomonas aeruginosa (4). In this study, we demonstrate that the Mtx2 toxins from six strains of B. sphaericus have few sequence differences, but some of these toxins exhibit major differences in larvicidal activities against two species of mosquitoes. The results permitted the design and assay of hybrid toxins and the identification of aa residues in Mtx2 that are unusual determinants of larvicidal activity and mosquito host range.
Bacterial Strains and Media-B. sphaericus SSII-1 was a gift from E. W. Davidson, Arizona State University, Tempe. Strains Kellen Q and 31 were obtained from A. A. Yousten, Virginia Polytechnic Institute and State University, Blacksburg. Strain 1593M was a gift from J. Szulmajster, C.N.R.S., Gif sur Yvette, and all other B. sphaericus strains were obtained from H. de Barjac, Pasteur Institute, Paris. All B. sphaericus strains were grown either in L-broth or NYSM medium (5).
Cloning, Mutagenesis, and Sequence Analysis-The protein coding regions of mtx2 from eight different strains of B. sphaericus (31-2, IAB59, Kellen Q, 2297, 2362, 1691, 1593M, and 2317.3) was amplified by PCR from genomic DNA (9) using synthetic oligonucleotides based on the sequence of B. sphaericus SSII-1 mtx2 (4). The sequences of these oligonucleotides were: 5Ј-CCCCCCATGGATCCAATGAAAAGGAC-CAAATTACTTTTTTATATT-3Ј (TT119) for the 5Ј (upstream) primer, and 5Ј-CCCGACGTCATCGATGCATGCCTTAAGTTATTTAAAA-GAAATTTCTTTAACATCTATTA-3Ј (TT120) for the 3Ј (downstream) primer. Artificial BamHI, NcoI, ClaI, SphI, and AflII restriction enzyme sites were incorporated into these sequences for cloning purposes. * This work was funded by the Institute of Molecular and Cell Biology, Singapore. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM 1 The abbreviations used are: mtx2, mosquitocidal toxin type 2 gene; Mtx2, mosquitocidal toxin type 2 protein; aa, amino acid(s); GST, glutathione S-transferase; LC 50 , lethal concentration of protein or cells theoretically required to kill 50% of mosquito larvae; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s). 2 Protein mutations are abbreviated using the following convention; the residue number is preceded by the symbol (in the one-letter code) for the wild-type amino acid and followed by the symbol for the mutant amino acid. Thus D31H denotes a mutation from Asp to His at position 31.
The 0.8-kb PCR products were digested with NcoI and ClaI, and cloned into the NcoI and ClaI sites of the large (vector) fragment of plasmid pTH26 (7), which was propagated in Escherichia coli DH5␣. Another set of recombinants were generated in the same way, and both sets were completely sequenced to ensure mutations were not artefactually introduced into the mtx2 genes during the PCR. Cloning and sequencing were carried out in the standard manner (10). Protein sequence analysis and alignments were performed using DNASTAR software.
Expression and Purification of Mtx2 Proteins-Synthesis and purification of Mtx2␦ fusion proteins was carried out essentially by a published procedure (8), except that E. coli DH5␣ were grown at 30°C for 2 h after induction of Mtx2␦ synthesis.
Antibodies and Western Blotting-Polyclonal antibodies against recombinant Mtx2 were raised in a previous study (4) and used to perform Western blotting as described (4) to detect recombinant Mtx2 homologs expressed in recombinant E. coli.
Mosquito Larvicidal (Toxicity) Assays-All plasmids expressing Mtx2 fusion proteins were transformed into E. coli DH5␣ for larvicidal assay. The toxic activities of different E. coli clones synthesizing the Mtx2 fusion proteins (or purified Mtx2␦ fusion proteins) (4) were measured on laboratory-reared 1st instar larvae of Culex quinquefasciatus and Aedes aegypti mosquitoes. Recombinant E. coli cultures were harvested, washed once with water, resuspended in 0.1 the original volume with water, and the A 600 was measured. The cultures were adjusted to the desired range of concentrations with water and added to 10 1st instar larvae in 1 ml of water with 50 l of a 100 mg/ml suspension of yeast. Assays using the desired range of concentrations of Mtx2␦ fusion proteins were performed in a similar manner, except that 50% PBS was used throughout. All assays were performed at least twice, and each concentration of bacteria (or purified Mtx2␦ fusion protein) was assayed in triplicate. Surviving larvae were counted at 24 h. The LC 50 values were calculated from the average mortality observed at 24 h by using the CA Cricket Graph III program. Mortality of control larvae fed with yeast suspension alone, with 50% PBS, or with E. coli transformed with plasmid vector alone was less than 10% in all assays.

RESULTS
Cloning and Comparison of mtx2 Genes-Using PCR primers based on the sequences at the extremities of the region coding for the mtx2 gene from B. sphaericus SSII-1 (4), 0.8-kb PCR products were generated from the genomic DNA of eight strains of B. sphaericus, namely 31-2, IAB59, Kellen Q, 2297, 2362, 1691, 1593M, and 2317.3. The amplified DNAs were cloned in a plasmid vector, pTH26 (7), and two independent clones of each mtx2 gene were completely sequenced to exclude the possibility of PCR-derived mutations.
The predicted aa sequences of the eight Mtx2 homologs were compared with each other and with the known sequence of B. sphaericus SSII-1 Mtx2, and aa variations were found at 10 positions between aa 31 and aa 279 (Fig. 1). The Mtx2 homologs from B. sphaericus 2362, 1691, 1593M, and 2317.3 were identical to each other (Table I). Overall, there were between four and eight aa substitutions when the Mtx2 sequences from strains 31-2, IAB59, Kellen Q, 2297, and 2362 were compared in a pairwise fashion with the prototype Mtx2 sequence from SSII-1 (Table I). Mtx2 from SSII-1 was unique in having Ser 37 , Ser 67 , Lys 224 , and Thr 279 , while Phe 239 was only found in Mtx2 from Kellen Q, and Thr 171 was unique to Mtx2 from strain 2297 (Table I).
Synthesis and Mosquito Larvicidal Activity of Mtx2 Homologs in E. coli-In order to compare the mosquitocidal activities of the six Mtx2 homologs, the mtx2 coding regions were fused in frame to GST (see "Experimental Procedures"). Recombinant E. coli cells were incubated with isopropyl-␤-D-thiogalactopyranoside to induce the synthesis of GST-Mtx2, and cell extracts were examined by Western blotting using a polyclonal antiserum to Mtx2 from B. sphaericus SSII-1. All recombinant cells produced a protein of ϳ58 kDa, the expected size of GST-Mtx2 (Fig. 2). The synthesis in E. coli of Mtx2 from B. sphaericus strains 2362 and Kellen Q was lower than that of Mtx2 from SSII-1, 31-2, IAB59, and 2297, but expression of all fusion proteins did not vary by more than 2.7-fold (Fig. 2).
Intact E. coli cells were fed to larvae of the mosquito C. quinquefasciatus, and larvicidal activities of the various recombinant E. coli clones harboring Mtx2 fusion proteins were quantitated (Table II), taking into account the different levels of Mtx2 fusion proteins (Fig. 2 legend). All clones exhibited significant toxicity to C. quinquefasciatus, and E. coli synthesizing Mtx2 from strains SSII-1 and 2362 were the most toxic. E. coli synthesizing Mtx2 from SSII-1 were ϳ7 to ϳ14 times more toxic than E. coli synthesizing Mtx2 from strains 31-2, IAB59, 2297, and Kellen Q (Table II). E. coli synthesizing Mtx2 from SSII-1 were ϳ7 times more toxic to C. quinquefasciatus larvae than clones synthesizing Mtx2 from strain 31-2 (Table  II). Surprisingly, the opposite result was obtained when E. coli synthesizing Mtx2 from SSII-1 and 31-2 were assayed against larvae of the mosquito, A. aegypti. Mtx2 from strain 31-2 was about 100-fold more toxic to A. aegypti than Mtx2 from SSII-1 (Table III). SSII-1 and 31-2 Mtx2 differ in four aa positions, namely 37, 67, 224, and 279 (Table I), and any or all of these aa could contribute to the significant differences in the toxicity and mosquito host range of these toxins.
Single or double amino acid substitutions were introduced by  site-directed mutagenesis into selected Mtx2 homologs to test the contribution of individual aa to toxicity and host range. Changes were made to render the Mtx2 proteins more or less like Mtx2 from strain SSII-1 ( Table I). None of the single substitutions in Mtx2, including IAB59 (D31H or R50K), 31-2 (P67S), 2297 (T171K), 2362 (K40Q), SSII-1 (S37I), and SSII-1 (S67P) ( Table I) had any detectable effect on toxicity toward C. quinquefasciatus larvae (data not shown).
Completely opposite results were obtained when the aa 224 and 279 mutants were assayed against A. aegypti larvae. SSII-1 (T279A) was about as toxic to these larvae as the weakly toxic SSII-1 Mtx2 protein, but SSII-1 (K224T) and the double mutant SSII-1 (K224T,T279A) were ϳ50 -150-fold more toxic than the parental SSII-1 Mtx2. The excellent A. aegypti toxicity of 31-2 Mtx2 was not significantly affected by the A279T mutation, but the double mutant 31-2 (T224K,A279T) was only weakly toxic to A. aegypti due to a drop in larvicidal activity of ϳ100-fold compared with 31-2 (A279T) ( Table III).
Assays against C. quinquefasciatus and A. aegypti larvae were performed with purified Mtx2␦ fusion proteins in which the N-terminal 15 amino acids containing the putative signal sequence of Mtx2 were deleted from GST-Mtx2 (Fig. 3) to exclude the possibility that the different larvicidal activities of E. coli expressing SSII-1 and 31-2 Mtx2 were due to differences in toxin solubility or stability in the bacteria. Table IV shows that SSII-1 Mtx2␦ was over 4-fold more toxic than 31-2 Mtx2␦, and that the 31-2 (T224K,A279T) mutant was about 17-fold more toxic than parental 31-2 Mtx2␦ to C. quinquefasciatus larvae. Both SSII-1 mutants with the K224T substitution were nontoxic to C. quinquefasciatus (Table IV). Conversely, purified toxins with T224 were the most toxic to A. aegypti, and toxins with K224 (SSII-1 and 31-2 (T224K,A279T)) were much less toxic to these larvae (Table IV). These results mirror the larvicidal activities of these toxins in live E. coli (Table III), suggesting that the different toxicities of the recombinant bacteria are due to variations in the aa sequences of the Mtx2 homologs, particularly in residue 224.

DISCUSSION
The Mtx2 homologs from six strains of B. sphaericus differ in only a few aa positions, and some of the homologs were found to be more toxic to mosquito larvae than others. These observations allowed us to design mutagenesis experiments which pinpointed Lys 224 as an important aa in the toxicity of Mtx2 to C. quinquefasciatus larvae. The SSII-1 (T279A) mutant was 5-6-fold more toxic to C. quinquefasciatus larvae than SSII-1 Mtx2, but the double mutant SSII-1 (K224T,T279A) was inactive, showing that, although Ala 279 is more favorable than Thr 279 in the context of SSII-1 Mtx2, Lys 224 is an overriding determinant of larvicidal activity. Why did 31-2 Mtx2 exhibit significant toxicity to C. quinquefasciatus despite the absence of Lys 224 ? The highly active 31-2 Mtx2 toxin differs from the inactive SSII-1 (K224T,T279A) mutant in having Ile 37 and Pro 67 , suggesting that one or both of these aa contributes to the toxicity of 31-2 Mtx2 and compensates for the absence of Lys 224 . This assumption is strengthened by the observation that the 31-2 (T224K,A279T) mutant, which is identical to SSII-1 Mtx2 except for the presence of Ile 37 and Pro 67 , was over 7-fold more toxic to C. quinquefasciatus larvae than SSII-1 Mtx2. Therefore, it was surprising that the single substitutions 31-2 (P67S), SSII-1 (S37I), and SSII-1 (S67P) had no effect on toxicity. Further reciprocal constructs (e.g. 31-2 (I37S), SSII-1 (S37I,K224T), and SSII-1 (S67P,K224T)) are needed to resolve the issue of the relative importance of Ile 37 and Pro 67 in the context of 31-2 Mtx2.
When E. coli synthesizing Mtx2 from SSII-1, 31-2, and their respective mutants were assayed against A. aegypti larvae, even greater differences in toxicity were observed. However, in complete contrast to the results of larvicidal assays against C. quinquefasciatus, SSII-1 Mtx2 was found to be ϳ100-fold less toxic to A. aegypti than was 31-2 Mtx2; and surprisingly, the difference was also due to aa position 224. Parental or mutant Mtx2 proteins with Thr 224 were always ϳ50 -150-fold more toxic to A. aegypti than their counterparts with Lys 224 (Table III).
How can a single aa substitution in a toxin substantially increase toxicity to one species of mosquito and virtually abolish toxicity to another species of mosquito? Although this is a new phenomenon, there are earlier studies on lepidopteran and dipteran toxins which are instructive and allow us to speculate on the possible mechanism of action of aa 224 (11)(12)(13)(14)(15)(16). Important genetic determinants of mosquito host range in the binary toxin from B. sphaericus have been localized to aa positions 99 and 104 in the 41.9-kDa subunit, but other aa in the 51.4-kDa subunit also contribute to toxicity (16). This is analogous to aa 224 in Mtx2 playing a major role in mosquito host range and aa 37 and/or 67 playing an accessory role. Several other studies have also concluded that one or a very few aa are major determinants of larvicidal activity and host range in a variety of toxins active against Lepidoptera and/or Diptera (11)(12)(13)(14)(15). However, in many cases insect specificity could not easily be attributed to particular aa, as different toxin segments were found to determine specificity by interacting in an undefined manner (11)(12)(13). Nevertheless, it is evident that different and sometimes overlapping segments of many toxins carry determinants of specificity for different insects (11)(12)(13).
In one interesting study, Ile 545 of a dual specificity larvicidal protein from Bacillus thuringiensis aizawai ICI was found to be essential for toxicity to A. aegypti larvae, but not to larvae of the caterpillar Pieris brassicae; conversely, the single substitution I568T abolished toxicity to P. brassicae larvae but not to A. aegypti larvae (12). Together, the results suggested that the I568T mutation destroyed proteolytic cleavage activation of the 130 ϫ 10 3 M r protoxin to a known ϳ55 ϫ 10 3 M r Lepidopteraactive toxin, while the I545P mutation inhibited proteolytic activation at a different site of the protoxin and prevented the formation of a known ϳ53 ϫ 10 3 M r Diptera-active toxin (12).
Although proteolytic cleavage by larval gut proteases activates many insecticidal toxins (1,2,12), it is unclear whether this also occurs in Mtx2 as the mechanism of action of the recently discovered Mtx2 family of mosquitocidal toxins is unknown (4). However, it is worth speculating that K224 in SSII-1 Mtx2 may be a major site of proteolytic cleavage activation by C. quinquefasciatus gut trypsin-like proteases (1,2), and that other as yet unknown neighboring site(s) in 31-2 Mtx2 (which has Thr 224 ) are exposed for cleavage activation by the compensating aa, Ile 37 and Pro 67 . By analogy with the dual specificity larvicidal toxin from B. thuringiensis aizawai ICI, we would have to postulate that in the A. aegypti larval gut a different protease is responsible for cleavage activation of Mtx2 at a distinct site from the one cleaved in the C. quinquefasciatus gut, and that the T224K mutation in 31-2 Mtx2 adversely affects substrate specificity or denies access to the putative A. aegypti protease.
An alternative and perhaps more plausible hypothesis is simply that there are subtle (but vital) aa differences in the C. quinquefasciatus and A. aegypti gut receptors in the domains which interact with position 224 on the surface of Mtx2. For example, a crucial electrostatic interaction might form only with Thr 224 in A. aegypti, while in C. quinquefasciatus a similar crucial interaction might form only with Lys 224 . Position 224 is predicted to lie at the peak of 1 of 11 predicted hydrophilic regions in Mtx2, which is consistent with a surface location. Regardless of the mechanism, it is clear that aa position 224 in the Mtx2 family of mosquitocidal toxins is an important and unusual determinant of toxicity and mosquito host range.
Finally, it is worth noting that some of the mutant Mtx2 toxins were significantly more toxic to C. quinquefasciatus  a Variation between replicates within one assay Ϯ 10%. Values are means of two or more assays using equivalent amounts of GST-Mtx2␦ fusion proteins based on SDS-polyacrylamide gel electrophoresis analysis (Fig. 3). b Ͼ20 g/ml is nontoxic.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy:
Biology
Computer Science

text:
Activation of phospholipase D and phosphatidylinositol 4-phosphate 5-kinase in HL60 membranes is mediated by endogenous Arf but not Rho.

Membrane-associated phospholipase D (PLD) in HL60 cells can be activated by the small GTP-binding proteins Arf and RhoA, but polyphosphorylated inositol lipids were required for maximum activity. The intact lipid was required because neither inositol 1,4,5-trisphosphate nor stearoyl-arachidonyl glycerol could substitute for phosphatidylinositol 4,5-bisphosphate (PIP2). Arf-stimulated but not Rho-stimulated PLD activity was increased by the inclusion of Mg2+ and ATP. ATP-dependent PLD activation occurred when phosphatidylinositol 4-phosphate (PIP), PIP2, or phosphatidylinositol 3,4,5-trisphosphate (PIP3) were included, but PIP2 formation was only detected with PIP; no PIP3 production was detected under any conditions. Therefore, the ATP-dependent increase in PLD activity cannot be explained by PIP2 or PIP3 formation. Association of endogenous Arf and RhoA with membranes was increased by incubation with GTPγS. This treatment increased membrane PLD and PIP kinase activities in the absence of exogenous p21 proteins. Reduction of Arf translocation suppressed the increase in PLD and PIP kinase activities, whereas complete removal of Rho but not Arf from membranes with RhoGDI was without effect on PLD activity but increased PIP kinase activity. Therefore, although recombinant Arf and Rho can activate PLD and PIP kinase in HL60 cells, it is the endogenous Arf but not Rho that regulates PLD, and thus a role for Rho in the physiological regulation of PLD in HL60 cells is unlikely.

Agonist-activated phospholipase D (PLD) 1 activity is an important step in signal transduction. The product of PLD activity, phosphatidate, can be degraded to diacylglycerol by phosphatidate phosphohydrolase or can be metabolized by phospholipases of the A class to lysophosphatidate and fatty acid. These lipids have all been proposed to function as signaling molecules. For example, diacylglycerol and unsaturated fatty acids are activators of most forms of the protein kinase C isoenzymes, and arachidonate is the precursor of leukotrienes and thromboxanes, mediators of inflammatory responses. The actions of phosphatidate are more complex because it has been suggested to function as an agonist and can regulate the activity of several intracellular proteins (for a detailed list see Ref. 1). Lysophosphatidate functions as an extracellular agonist rather than an intracellular agent.
The regulation of stimulated PLD activity is multifactorial, with protein kinase Cs, tyrosine kinases, and G-proteins all being suggested as regulators in various systems (2,3). PLD does not appear to be directly receptor-coupled, and recently a role for small G-proteins in its regulation has been proposed (4,5). Arf, first identified as the protein responsible for enhancing the cholera toxin catalyzed ADP-ribosylation of the Gs␣ subunit, has a key role in vesicular transport and has been shown to activate membrane bound PLD in a GTP-dependent manner in HL60 membranes.
The small G-protein RhoA has also been proposed to be a regulator of PLD activity (6). This protein is found predominantly in the cytosol of unstimulated cells in its GDP bound state associated with RhoGDI. The exact sequence of events remains ill defined, but RhoA binds to membranes in its GTPbound state following disassociation from GDP dissociation inhibitor (7). RhoA can be inactivated by incubation with the Clostridium bottulinum C3 toxin, which ADP-ribosylates RhoA in the putative effector domain on Asn 41 (8). This inhibition has been used to identify RhoA-mediated events, such as stress fiber formation, focal adhesion complex formation, and recently PIP kinase activation. However, it has been reported that C3 treatment of liver plasma membranes did not inhibit a RhoAregulated PLD activity (6).
The activation of PLD by Arf or RhoA is strongly dependent upon the presence of polyphosphoinositides, and it has been considered possible that the availability of PIP 2 (4) or PIP 3 (9) is an important factor in PLD activation. Because RhoA has been reported to activate PIP kinase (10), thereby increasing PIP 2 biosynthesis, it is possible that this stimulation is the means of PLD activation. However, a recent report has shown that Rac but not RhoA interacts with and activates the type II isoform of PIP kinase, questioning the physiological role of RhoA (11).
We have investigated the activation of PLD by both Arf and RhoA in the presence of acidic lipids with or without ATP in the incubations to identify the potential role of PIP kinase or other kinases in PLD activation. Furthermore, by manipulating the localization of Arf and RhoA under various conditions and relating the redistribution of these proteins to PLD and PIP kinase activation in the same membranes, we have assessed the relative physiological roles of the GTP-binding proteins in PLD and PIP kinase activation.
GST-RhoGDI expressed in Escherichia coli was generous gift from Dr. Alan Hall, University College London, UK. PIP 3 was a generous gift from Dr. Roy Gigg, NIMR, Mil Hill, London, UK.
Antibodies against synthetic peptide sequences from Arf1 and 3 (amino acids 98 -112) and RhoA (amino acids 87-101) were raised in sheep. The whole immunoglobulin fraction was prepared and used.
Preparation of Recombinant Proteins-Glu-Glu epitope tagged Arf was prepared from Sf9 cells using the method given previously for the cloning and purification of such proteins (12). GST-RhoA, expressed in Sf9 cells and GST-RhoGDI expressed in E. coli were prepared as given previously (13).
Cell Culture and Fractionation-HL60 cells were grown in RPMI ϩ Glutamax medium supplemented with 15% (v/v) heat-inactivated fetal calf serum to a density of 1.0 -1.2 ϫ 10 6 /ml. Cells were harvested by centrifugation (800 ϫ g for 3 min) and washed three times using 137 mM NaCl, 8.1 mM Na 2 HPO4, pH 7.5, 2.7 mM KCl, 2.5 mM EDTA, 1 mM dithiothreitol, and 0.1 mM phenylmethanesulfonyl fluoride (harvesting solution). The washed cells were sonicated in harvesting solution on ice for three 10-s bursts using a probe sonicator. Membrane and cytosolic fractions were prepared by centrifugation in a Beckman TLA 120.2 rotor at 435,000 ϫ g for 20 min at 4°C.
Translocation of G-proteins-Endogenous monomeric G-proteins from HL60 cytosol were translocated to membranes in the presence of 100 M GTP␥S according to the method of Bokoch et al. (7). Where indicated 10 mM EDTA or 2 M recombinant RhoGDI were included in the incubation. Re-isolated membranes were washed in harvesting solution containing the same additions as the initial translocation and stored as aliquots at Ϫ70°C until used.

SDS-Polyacrylamide Gel Electrophoresis and Western
Blotting-SDS-polyacrylamide gel electrophoresis was performed using 12.5% gels. Following transfer, membranes were probed with antibodies to RhoA and a common sequence from Arf1 and Arf3. Rho and Arf antibodies were used at 1:1000 and 1:3000, respectively. The proteins were visualized using the Amersham Enhanced Chemiluminescence kit.
Enzyme Assays-PLD assays were performed using the method of Brown et al. (4), with slight modifications. The reaction was carried out in a final volume of 50 l. All assays contained 10 g of membrane protein in harvesting buffer, 3 mM Mg 2ϩ , 2 mM Ca 2ϩ , guanine nucleotide (30 M GTP␥S or 2 mM GDP␤S) and 5 l of 4 M NaCl, which increased the PLD activity (results not shown). Where indicated, 0.5 g of Arf and or RhoA, 2 mM Mg/ATP (in 20 mM HEPES, pH 7.5), were included. The membranes and the above constituents in a volume of 30 l were pre-incubated for 10 min at 37°C, and the reaction was started by the addition of 20 l of lipid substrate. The final concentration of lipids in the assay were 137 M phosphatidylethanolamine, 8.6 M phosphatidylcholine (specific activity, 58 Ci/mol, 25 nCi/assay) and where indicated 6 M SAG or 50 M PIP and unless indicated 12 M PIP 2 , or 12 M PIP 3 . When PIP 2 or PIP 3 were used at less than 12 M, sufficient PS was added such that the combined acidic lipid concentration was maintained at 12 M. The dried lipids were sonicated using a probe sonicator in a buffer such that the final concentrations in the assay were 50 mM HEPES, pH 7.5, 3 mM EGTA, 80 mM KCl, and 1 mM dithiothreitol. The incubation was performed for 1 h at 37°C and stopped by the addition 750 l of methanol/chloroform (2:1 v/v). Phases were split with 250 l of chloroform and 400 l of water. After vortexing and centrifugation (2000 ϫ g for 10 min), 600 l of the methanol/ aqueous phase was taken, and the choline was separated using Dowex columns, as given previously (14).
PIP kinase was assayed under essentially identical conditions to PLD except that 20 g of membrane protein was employed and 2 mM [␥-32 P]ATP (specific activity, 10 Ci/mol, 1 Ci/assay) was used in all assays. The reaction was initiated by the addition of lipid substrate and continued for 7 min at 37°C. The reaction was stopped with 750 l of methanol/chloroform/HCl (150:75:1, v/v/v), and the phases were split with 250 l of chloroform and 400 l of water. The upper phase containing the unreacted [ 32 P]ATP was removed, and the chloroform-rich phase was washed once with 1 ml of water. The organic phase was then dried under vacuum, and the lipids were spotted onto a Merck silica gel 60 aluminium 10-cm plate that had previously been soaked in 1% (w/v) potassium oxalate (in methanol/water, 3:2) and dried at 100°C for 20 min. Authentic PIP, PIP 2 , or PIP 3 was spotted on each plate, and the lipids were separated using chloroform/acetone/methanol/acetic acid/ water, (40:15:13:12:5 v/v/v/v/v) or when PIP 3 was separated propan-1ol/2 M acetic acid (65/35). The lipid standards were located using iodine vapor, and the [ 32 P]lipids were identified and quantified using a Molecular Dynamics PhosphorImager.
Protein was assayed using the Bio-Rad protein assay. For each experiment means and ranges or standard deviations as appropriate are given. The number of separate experiments performed for each figure are given in the legends.

Regulation of Arf-and RhoA-stimulated PLD Activity by
Acidic Lipids and ATP-Initial experiments confirmed that HL60 cell membranes contained a PLD activity that was stimulated by recombinant Arf in the presence of GTP␥S (30 M) but not GDP␤S (2 mM). In agreement with previous reports this activity was also enhanced by the inclusion in the assay of a cytosolic fraction prepared from these cells (4). This cytosolic preparation contained negligible PLD activity (results not shown). The activity was dependent on the presence of the polyphosphoinositides PIP 2 or PIP 3 . Fig. 1A shows that PS (in the absence of phosphoinositide) did not support PLD activity and that over the range of 1-12 M phospholipid PIP 3 was a slightly more potent activator than PIP 2 , whereas PIP was a poor activator (Fig. 1B). The polyphosphoinositide-dependent PLD activity was increased in the presence of Mg 2ϩ and ATP compared with Mg 2ϩ alone (Fig. 1B). In the absence of GTP␥S, ATP did not support PLD activity (data not shown).
Recombinant RhoA also activated the PLD in HL60 membranes in a GTP␥S-dependent manner. The activation by RhoA was less potent than by Arf (Fig. 2), whereas in combination the activation was approximately additive. The inclusion of Mg 2ϩ and ATP did not increase the RhoA-stimulated PLD activity when either PIP or PIP 2 ( Fig. 2) were employed.
Stimulated PIP 2 hydrolysis precedes agonist-activated PLD activity in many cells, thus the ability of the inositol trisphosphate head group and the diacylglycerol moieties of PIP 2 to stimulate Arf-dependent PLD were examined. 6 M SAG was unable to activate PLD. IP 3 at 50 M or 100 M did not activate PLD either in the absence or the presence of SAG. IP 3 also failed to function an inhibitor of PIP 2 -stimulated PLD (results not shown).
PIP Kinase Activity and Its Relationship to PLD Activity-The results given above demonstrated that Arf-and RhoAstimulated PLD activity was dependent upon the presence of PIP 2 or PIP 3 . The possibility that the increased PLD activity seen the presence of ATP (Fig. 1B) was the result of the GTPbinding proteins stimulating the production of polyphosphoinositides was investigated by measuring the incorporation of [ 32 P] from [␥-32 P]ATP into phosphoinositides. These experiments utilized conditions similar to the measurement of PLD activity, so that the PLD and PIP kinase assays were comparable. When PIP (50 M) was employed, PIP kinase activity was increased by Arf (control activity of 85.5 Ϯ 28. 9 Fig. 5C). When the PIP was replaced by 12 M PIP 2 or 12 M PIP 3 , the 32 P incorporation into PIP 2 was lower than that seen with PIP and not stimulated by Arf (Fig. 3A). Furthermore, the incorporation of phosphate into PIP 3 was considerably lower than that into PIP 2 in the presence or the absence of Arf using PIP, PIP 2 , or PIP 3 (Fig. 3B).
The Effect of Membrane Translocation of Endogenous Arf and RhoA upon on PLD and PIP Kinase Activities-The experiments described above utilized exogenous recombinant GTPbinding proteins; it was therefore considered important to ex-amine regulation of PLD activity by the endogenous RhoA and Arf proteins. The intracellular location of RhoA and Arf was modulated as described by Bokoch et al. (7). Translocation of endogenous Arf and RhoA from the cytosol to membranes was assessed using specific antibodies against each of the G-proteins. Figs. 4A and 5A show that the inclusion of 100 M GTP␥S in incubations of membranes and cytosol induced a translocation of both endogenous Arf and RhoA to the membranes. However, when 10 mM EDTA was also included in the incubations, the translocation of Arf was decreased, whereas that of RhoA was clearly increased (Fig. 4A). The membranes re-isolated from these incubations were used in assays for PLD and PIP kinase. Control incubations were performed in the absence of GTP␥S or EDTA, and the membranes were isolated.
When assayed in the absence of exogenous Arf, the PLD activity in the membranes that had been pre-incubated with GTP␥S (80 Ϯ 15.9 pmol choline liberated/mg protein/min; mean Ϯ S.D. of six separate experiments) was increased by an average of 2.5-fold compared with control (32.8 Ϯ 12.8 pmol choline liberated/mg protein/min; mean Ϯ S.D. of six separate experiments) membranes that had been pre-incubated in the absence of GTP␥S. When EDTA was included in the pre-incubation, the increase was 1.8-fold (54.6 Ϯ 19.4 pmol choline liberated/mg protein/min; mean Ϯ S.D. of six separate experiments). This lower PLD activity was not due to loss of PLD protein from the membranes because the inclusion of recombinant Arf in assays of the membranes from the GTP␥S/EDTA and control pre-incubations produced equivalent choline generation (Fig. 4B). In the same samples a small but consistent increase in PIP kinase activity of 1.2-fold was observed in the membranes pre-incubated with GTP␥S (135.6 Ϯ 12.1 pmol PIP 2 formed/mg protein/min; mean Ϯ S.D. of three separate experiments compared with control incubations (112.5 Ϯ 9.4 pmol PIP 2 formed/mg protein/min; mean Ϯ S.D. of three separate experiments), whereas a decrease to 0.76-fold (84.0 Ϯ 10.3 pmol PIP 2 formed/mg protein/min; mean Ϯ S.D. of three separate experiments) was observed in membranes where EDTA had been included in the pre-incubation. This decrease could be due to loss of PIP kinase protein from the membranes because Arf failed to increase the PIP 2 production to levels observed in membranes prepared in the absence of EDTA (Fig. 4C).
Membranes re-isolated from incubations containing control, GTP␥S, or GTP␥S plus RhoGDI were probed for Arf and RhoA.
The inclusion of RhoGDI completely extracted RhoA from the membranes without affecting the membrane-association of Arf (Fig. 5A). PLD and PIP kinase assays performed on these membranes are shown in Fig. 5 (B and C). The extraction of the RhoA had very little effect on the PLD activity in these samples The results given in A show the pmol of phosphate incorporated into PIP 2 , and those in B show the pmol of phosphate incorporated into PIP 3 . It was possible that more than 1 [ 32 P]P i was incorporated into lipids in some conditions (for example PIP 3 when PIP was used in the assays), meaning that the specific radioactivity of the product could vary. Therefore, the results are expressed as pmol phosphate incorporated into lipid. The production of PIP 2 from PIP given in A is taken as an assay of PIP kinase. The data were calculated by spotting a portion of the radioactive substrate onto the TLC plate prior to phosphorimaging. The image units produced by the known quantity in this sample was used to calculate the pmol of phosphate incorporated. The results shown are the means Ϯ ranges of two independent experiments. The proteins were detected using antibodies raised against specific amino acid sequences contained in Arf (upper blot) and Rho (lower blot), respectively. Similar results were obtained in a total of six independent experiments. Recombinant Arf and Rho were also blotted to demonstrate the specificity of the antibodies. Arf could be detected in the control membranes (lane 1) when the time of exposure of the the film to the chemiluminesence was increased. The recombinant Arf runs slightly higher than the HL60 Arf because of the epitope tag, and a doublet was seen that corresponded to myrisyoylated and nonmyristoylated forms (16). B, phospholipase D activity in HL60 membranes loaded with differing amounts of endogenous Arf or Rho. Pre-isolated membranes from the translocation system described in A were assayed for PLD activity in the presence of GTP␥S (30 M) in the absence or the presence of exogenous Arf (0. PIP kinase activity increased in membranes pre-incubated with GTP␥S as reported above; however, GST-RhoGDI inclusion caused a further increase in the activity (GTP␥S-preincubated membranes 104.7 Ϯ 0.6 and GTP␥S/RhoGDI-preincubated membranes 169.0 Ϯ 20.6 pmol PIP 2 formed/mg protein/ min; means Ϯ range of two independent experiments when assayed in the absence of exogenous p21 protein). PIP kinase activity in RhoGDI-treated membranes was higher than in the control membranes in both experiments under all assay conditions employed. DISCUSSION The results presented in this paper demonstrate that although both RhoA and Arf can activate PLD in HL60 membranes in a PIP 2 -and GTP␥S-dependent manner, it is probable that only Arf is the physiologically relevant activator.
Maximum GTP-regulated PLD activity was dependent upon the presence of polyphosphorylated inositol lipids (Fig. 1) and (4). In agreement with previous reports, PS was not a PLD activator, and PIP was a weak stimulator, whereas PIP 2 and PIP 3 were both potent activators. These data indicate that there was not a general stimulation of PLD activity by acidic lipids. PIP 2 hydrolysis is frequently observed when PLD is stimulated in an intact cell, raising the possibility that it is actually one of the products of this reaction that is the physiological activator. Thus SAG, the lipid portion, and the water soluble head group, IP 3 , of PIP 2 were added alone or in combination to PLD assays. This protocol was also employed in an attempt to define which moiety of the PIP 2 molecule is the activator. The concentration of SAG employed (6 M) was intended to represent a concentration that could be produced in actual assays. On the other hand, the concentration of IP 3 used (50 -100 M) was higher than the PIP 2 concentration because this metabolite is susceptible to hydrolysis catalyzed by membrane-associated inositol polyphosphate 5-phosphatase under the PLD assay conditions (15). In an attempt to minimize IP 3 degradation, the concentration of Mg 2ϩ in these assays was reduced to 1 mM (this decreased the PLD activity by approximately 50%). SAG and IP 3 alone or in combination could neither replace PIP 2 nor inhibit the PLD activity detected in the presence of PIP 2 . Thus an intact PIP 2 molecule is essential for PLD activation, and it is possible that the lipid binds either to the enzyme itself or to an associated regulatory molecule. This binding is unlikely to be via a Pleckstrin Homology domain because it has been shown previously that IP 3 blocks the binding of PIP 2 to proteins containing such a domain (16).
The Arf-stimulated PLD activity was enhanced in the presence of ATP (Figs. 1B and 2). Because PIP can be phosphorylated to PIP 2 , it is possible that this increase was secondary to PIP kinase activation, as suggested by Pertile et al. (17). ATP also increased PLD activity in the presence of PIP 2 or PIP 3 , raising the possibility that re-synthesis of PIP 2 or PIP 3 under the assay conditions was responsible for the increased PLD activity. This was investigated by measuring the incorporation of 32 P from [␥-32 P]ATP into PIP 2 and PIP 3 in the presence of PIP, PIP 2 , or PIP 3 under the conditions employed in the PLD assays. When PIP was present [ 32 P]PIP 2 was produced, and Arf stimulated this activity (Fig. 3A). Therefore, the ATP-dependent increase in PLD activity seen under these conditions could be mediated by PIP kinase generated PIP 2 . However, when PIP 2 or PIP 3 were employed, little PIP 2 was made, and no activation by Arf was seen. Furthermore, very little [ 32 P]PIP 3 was generated under any conditions tested here, demonstrating that the ATP-mediated increase in Arf-stimulated PLD activity in the presence of PIP 2 or PIP 3 was not the result of phosphoinositide phosphorylation. It is also unlikely that the conversion of PIP 2 to PIP 3 would result in a significant activation of PLD in this system because PIP 3 is only slightly more potent than PIP 2 as an activator (Fig. 1A). This suggests that the ATP was being utilized by a different kinase activity. The ATP-dependent increase in PLD activity was observed in the presence of Arf but not RhoA; thus the ATP effect upon PLD activity was probably on an Arf-mediated process rather than on PLD directly. A number of potential regulatory proteins such as Arf-GTPase-activating protein, Arf-guanine nucleotide exchange factor, or even Arf itself are targets of a putative regulatory kinase. The notion that a kinase other than PIP kinase is responsible for the increased PLD activity when Mg 2ϩ /ATP and PIP 2 or PIP 3 were included in the assay also argues against the proposal that PIP 2 production was involved in Mg 2ϩ /ATP plus PIP-stimulated PLD activation. Nevertheless, the inability of RhoA to activate PIP kinase in these cells (see Fig. 5C, control incubations) could explain why ATP did not increase PLD activity when RhoA was used as the activator and provides support for the proposal that PIP 2 production from PIP indeed activates PLD, unless the Rho regulatory proteins are not controlled by phosphorylation.
In an intact cell, stimulated translocation will be a fundamental point of regulation of any pathway regulated by small GTP-binding proteins. Thus the preparation of membranes containing variable levels of endogenous Arf and RhoA was an ideal system in which to test for regulation of enzyme activities by such proteins. A small but consistent increase in the activity of PIP kinase was detected in membranes loaded with both Arf and RhoA measured in the absence of exogenous Arf. This presumably reflects stimulation by the translocated proteins. The inclusion of 10 mM EDTA during translocation decreased the Arf, increased the RhoA membrane localization (Fig. 4A), and blocked the increase in PIP kinase activity (Fig. 4C). However, the PIP kinase activity measured in the presence of exogenous Arf was lower in the EDTA-treated membranes than the control membranes, indicating that the PIP kinase protein itself or an additional regulator had been lost from the membranes (Fig. 4C). The data in Figs. 3, 4C, and 5C showed that under control conditions PIP kinase was activated by exogenous Arf, and in Fig. 5C a small inhibition by exogenous RhoA was seen. Selective, complete blockage of RhoA, but not Arf, translocation with GST-RhoGDI increased the PIP kinase activity compared with RhoA containing membranes. This was particularly surprising because it has been proposed that RhoA is an activator of PIP kinase (10). Although this has been questioned (11) and our data would also tend to argue against this proposal, inhibition has not been previously described. However, the addition of exogenous RhoA to the assays was without effect upon the PIP kinase activity in either the Arfand RhoA-loaded or the Arf-loaded RhoA-depleted membranes (Fig. 5C). Thus RhoA was not acting as a direct regulator of PIP kinase in the Arf-and RhoA-loaded membranes. It seems most likely that the GST-RhoGDI removed another factor that was limiting the activity of PIP kinase in this system. Thus the role of RhoA in PIP kinase regulation is not clear. However, in addition to the proposed function for this protein, and for Rac (11), in regulating PIP kinase, our results suggest that Arf may also play a role. It is unclear if this reflects differences in PIP kinase subtypes.
The PLD activity in membranes loaded with Arf and RhoA was activated by GTP␥S and PIP 2 in the absence of exogenous p21 protein. Reducing the translocation of Arf and increasing that of RhoA by the inclusion of EDTA in the pre-incubation, resulted in membranes in which stimulated PLD activity was decreased. However, in contrast to PIP kinase, these mem-branes did not contain less PLD because the addition of exogenous Arf stimulated PLD activity to an approximately equal extent in each group of membranes (Fig. 4B). The use of RhoGDI to prevent RhoA binding to membranes did not decrease the measurable PLD activity when compared with the Arf-and RhoA-loaded membranes (Fig. 5B). Therefore, the membrane PLD activity was dependent on the presence of Arf but not RhoA. Activation of PLD by exogenous (recombinant) RhoA was partially inhibited by Bottulinum C3 toxin (results not shown), with no effect upon Arf-activated PLD, indicating that Arf did not require the presence of RhoA to activate PLD. Furthermore, the PLD activity in the Arf and RhoA loaded membranes was not inhibited by the C3 toxin (not shown), indicating that RhoA was not involved in the PLD activation in this system, in agreement with the data obtained with RhoGDI-treated membranes. The activation of membrane PLD by exogenous Rho has been previously reported (18), indeed it has been demonstrated that recombinant Rho and Arf activated partially purified porcine brain PLD synergistically (19). Treatment of liver membranes with GST-RhoGDI was shown to reduce basal PLD activity, which was partially restored by exogenous Rho (6). It was unclear what level of Arf was present in the treated membranes, which may be relevant because in the experiments reported here Arf could be detected. We have not observed synergy, as reported by Singer et al. (19), rather we detected additivity in the effects of submaximal exogenous Arf and Rho in the stimulation of PLD activity; thus it is possible that Rho can regulate PLD activity when the Arf concentration is limiting. Nevertheless, an increase in endogenous membrane Rho concomitant with a decreased Arf association (Fig. 4A) failed to maintain PLD activity (Fig. 4B). This suggests that endogenous Rho does not regulate PLD activity in HL60 membranes. It is perhaps relevant that activation of partially purified phospholipase C by the G i and G o GTP binding proteins was demonstrated (20) prior to the identification of G q and G 11 and that nonphysiological activation of the same phospholipase was detected when p21 ras was over-expressed in NIH3T3 cells (21). Thus, under certain conditions it may be possible to activate phospholipases with inappropriate G-proteins. However in an intact cell, when the translocation of Arf is stimulated, as will occur upon receptor activation, PLD will be regulated by this G-protein. Therefore, whereas Rho and other small molecular weight G-proteins can function in an in vitro assay as PLD regulators, we believe that as originally suggested (4,5), the physiological regulator of phospholipase D, at least in HL60 membranes, is Arf.