Humoral factor activating the Sarcophaga lectin gene in cultured fat body

Insect Biochem. Vol. 19, No. 3, pp. 261-267, 1989

0020-1790/89 $3.00+ 0.00 Pergamon Press pie

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H U M O R A L F A C T O R ACTIVATING THE S A R C O P H A G A LECTIN GENE IN C U L T U R E D FAT BODY AKIHIRO SHIRAISHIand SHUNJI NATORI Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

(Received 15 August 1988; revised and accepted 21 November 1988) Abstract--On culture of the fat body of Sarcophaga peregrina (flesh fly) larvae in modified Grace's medium, the Sarcophaga lectin gene was activated only when the medium was supplemented with larval hemolymph. The expressions of the genes for the storage protein and the sarcocystatin A were not affected by supplementing the medium with the hemolymph. Thus the hemolymph contained a factor that specifically activated the Sarcophaga lectin gene. This factor seemed to be a heat-stable, low molecular weight compound that was probably not a peptide, and to activate genes in the fat body for defense proteins that are known to be expressed in response to injury of Sarcophaga larvae.

Key Word Index: Sarcophaga peregrina, Sarcophaga lectin gene, fat body, humoral factor

INTRODUCTION

MATERIALS

When the body wall of Sarcophaga peregrina (flesh fly) larvae is pricked with a hypodermic needle, a galactose-binding lectin (Sarcophaga lectin) and several antibacterial proteins (sareotoxins) are known to be induced in the hemolymph (Komano et al., 1980; Okada and Natori, 1983). These proteins are defense proteins, and their role is to prevent the invasion of bacteria or other foreign substances into the hemolymph through the wound. In fact, Sarcophaga lectin was shown to participate in the elimination of sheep red blood cells introduced into the abdominal cavity of Sarcophaga larvae (Komano and Natori, 1985). Northern blot hybridization experiments with cDNAs for these proteins demonstrated that these proteins are synthesized in the fat body in response to body injury and are eventually secreted into the hemolymph (Takahashi et al., 1985; Matsumoto et al., 1986b; Ando and Natori, 1988). Among these proteins, Sarcophaga lectin and sarcotoxin IA are especially interesting, because their genes were also shown to be expressed in the early embryonic stage and pupal stage, indicating that these proteins have functions in both defense and development (Takahashi et al., 1986; Nanbu et al., 1988). These findings raise the important question of how the same genes are expressed under different physiological conditions. Previously we found that the stimulus of body injury is first transmitted to a certain tissue in the anterior part of the body, and from there a mediator molecule that interacts directly with fat body cells is secreted into the hemolymph, resulting in activation of the Sarcophaga lectin gene in the fat body (Shiraishi and Natori, 1988). This paper describes a system for/n vitro culture of Sarcophaga fat body that mimicks the situation /n vivo. Namely, in this system addition of hemolymph induces expression of the Sarcophaga lcctin gene, but not of other genes. This finding shows that the hemolymph contains a specific factor that is essential for activation of the Sarcophaga lectin gene in vitro.

AND

METHODS

Animals Flesh flies, Sarcophagaperegrina, were kept at 27°C with dry milk, sugar cubes and fresh water. Larvae were reared on pork liver, and when they crawled upward at the third instal they were collected, washed well and kept in a plastic container with a small amount of water. Before injury of the body wall, larvae were anesthetized by keeping them on a glass plate in ice for a few minutes. Then the posterior half of their body wall was pricked with a hypodermic needle.

Isolation of hemolymph Hemolymph was collected by cutting off the anterior tip of larvae with fine scissors and collecting the drop of hemolymph that exuded in a Petri dish on ice. Usually, I mi of hemolymph was collected from about 150 larvae. The resulting hemolymph was centrifuged for 5 rain at 130g to remove hemocytes, and the clear supernatant was added to cultures of the fat body.

Culture of fat body The fat body was excised from third instar larvae under a binocular microscope and rinsed well in ice-cold insect saline 030 nM NaCI, 5 mM KCI and I mM CaCI2). The fat bodies from two larvae were placed in 300/~1 of modified Grace's insect medium (GIBCO) or in 300/tl of the same medium containing various amounts of hemolymph. After incubation for 2 h at 25°C, the fat body was removed from the medium, promptly frozen on dry ice and stored at - 80oc.

Extraction of RNA and Northern blot hybridization RNA was extracted from the frozen fat body as described before, and subjected to electrophoresis in a horizontal slab of 1.2% (w/v) agarose gel containing 2.2 M formaldehyde in 20 mM morpholinopropanesulphonic acid (MOPS), 5 mM sodium acetate, I raM EDTA (pH 7.0) (Takaha~ et ai., 1986). Before application to the gel, the RNA was heated at 60°C for 7 rain in the same MOPS-sodium acetate buffer containing 50%(v/v) formamide and 2.2 M formaldehyde. After electrophoresis, the RNA was transferred from the gel to nitrocellulose filter paper as described by Thomas (1980), and hybridized with nick-translated DNA probes. T h e hybridization mixture consisted of 500 (v/v) formamide/ 5 x standard sodium citrate (SSC) (I x SSC is 0.15M

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AKIHIROSHIRAISHIand SHUNnNATORI

sodium chloride, and 0.05 M trisodium citrate)/5 x Denhardt's solution [1 x Denhardt's solution is 0.02% (w/v) each of bovine serum albumin, polyvinylpyrrolidone-40and Ficoll-400]/20mM sodium phosphate (pH 6.5)/50/~g/ml of denatured and sonicated salmon sperm DNA. Hybridization was carried out with a radiolabeled probe at 42°C for 12-16 h. Before hybridization, the filter was preincuhated in the absence of probe DNA under the same conditions except that I x Denhardt's solution was used instead of 5 x Denhardt's solution and 1% (w/v) giycine was added. The filters were washed successively with 2 x SSC containing 0.1% (w/v) sodium dodecyl sulfate (SDS) for 15 min at room temperature and 0.5 x SSC containing 0.1% SDS for 15 min at 42°C and than autoradiographed at -80°C. A cDNA clone for Sarcophaga lectin, pLE10 (Takahashi et al., 1985)was used throughout to detect Sarcophaga lectin mRNA. As controls of other fat body protein genes, the BalI-AsuII fragment of pSCl (Saito et al., 1989), a cDNA clone for sarcocystatin A (Suzuki and Natori, 1985), a genomic clone for storage protein (arylphorin), pSP2 (Matsumoto et al., 1986a) and a cDNA clone for sarcotoxin IIA, pTII20 (Ando and Natori, 1988), were used.

Measurement of total RNA synthesis in the fat body in vitro The fat body was cultured as described above. For labeling RNA, 1#Ci of [3H]uridine (40.3Ci/mmol) was added to 300#1 of medium. When necessary, 3/~g/ml of actinomycin D was added to the medium. After incubation for 2 h at 25°C, the fat body was rinsed well in 2 ml of phosphate buffered saline [PBS: 130 mM NaCl, 3 mM KCI in l0 mM phosphate buffer (pH 7.4)], and homogenized in 0.5 ml PBS containing 0.1% SDS. The resulting homogehate was extracted successively with equal volumes of phenol:chloroform:isoamyl alcohol (25:25: I, by vol) and chloroform:isoamyl alcohol (50: 1). Then, 200#1 of the aqueous phase was mixed with 200gl of 10% (w/v) and 800 #f of~5% trichloroacetic acid solution containing 0.5% (w/v) sodium pyrophosphate, and the acid-precipitabl¢ radioactivity was collected on a glass-fiber filter and counted. RESULTS

Expression of the Sarcophaga lectin gene in cultured

fat body Previously, we demonstrated that the larval hemolymph contains a factor that activates expression of the Sarcophaga lectin gene in the fat body (Shiraishi and Natori, 1988). Namely, when a third instar larva was cut in half, and hemolymph was injected into the posterior half, which contains the posterior half of fat body, significant expression of the Sarcophaga lectin gene was detected in the fat body, whereas the gene was not expressed in the absence of the hemolymph. Hemolymph from injured larvae induced much greater expression than that from uninjured larvae. To characterize this humeral factor, we established a system for in vitro culture of fat body in which expression of the Sarcophaga lectin gene is controlled by hemolymph added to the medium. Fat body was isolated from third instar larvae and cultured in modified Grace's insect medium at 25°C. Under these conditions, the activity of the fat body in terms of the syntheses of RNA and protein was maintained for several hours. On replacement of part of the medium by hemolymph, expression of the Sarcopimga lectin gene in the fat body was found to depend on the amount of bemolymph added to the medium. As shown in Fig. l, on incubation for 2 h, no Sarcophaga lectin mRNA was synthesized when fat body was

incubated in modified Grace's insect medium in the absence of hemolymph, but when 30-150 #l of the culture medium was replaced by hemolymph, expression of the Sarcophaga lectin gene became detectable by Northern blot hybridization. Moreover, heinelymph from injured larvae seemed to have more effect than the same volume of hemolymph from uninjured larvae. The same results were obtained with hemolymph collected by incising the body wall of the larvae with a razor blade to avoid contamination of the hemolymph with gut contents. Therefore, these results indicate that a humeral factor that interacts directly with the fat body and activates the Sarcophaga lectin gene is present in the hemolymph. This factor was probably the same as that reported in our previous paper, and was secreted from a certain tissue present in the anterior part of the larval body in response to body injury (Shiraishi and Natori, 1988). The activity in the hemolymph from uninjured larvae may have been due to this factor secreted during preparation of the hemolymph, since it was impossible to collect hemolymph without injuring the body wall of larvae.

Gene specificity of the humeral factor It is possible that gene activation in the fat body in the presence of hemolymph is a general event and not restricted to the Sarcophaga lectin gene. To examine this possibility, we investigated the effect of hemolymph on overall RNA synthesis in cultured fat body. As shown in Fig. 2, significant RNA synthesis was detected in cultured fat body, and this RNA synthesis was completely repressed in the presence of 3 #g/ml of actinomycin D. On addition of increasing amounts of hemolymph to this culture medium, the level of RNA synthesis increased slightly in the presence of lower concentrations of bemolymph, but was more or less constant, indicating that the effect of bemolymph on overall RNA synthesis was not significant. We then examined the effect of hemolymph on the expressions of specific genes. Two genes are known to be expressed in the fat body of third instar larvae. One is the storage protein (arylphorin) gene (Mat-

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Fig. 2. Effect of hemolymph on overall RNA synthesis in cultured fat body. Increasing amounts of hemolymph from injured larvae were added to the culture medium without changing the total volume, and incorporation of [3H]uridine into the acid-insoluble RNA fraction during incubation for 2 h was measured. @ @, RNA synthesis; m - - - I , RNA synthesis in the presence of 3 #g/nil of actinomycin D. Bars, SD.

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Fig. 1. Effect of hemolymph on transcription of the Sarcophaga lectin gene in cultured fat body. Fat bodies from two third instar larvae were combined and cultured in 300 #1 modified Grace's insect medium for 2 h at 25°C (standard conditions). The indicated amounts of culture medium were replaced by hemolymph without change in the total volume. Hemolymph was prepared from uninjured larvae (A) or injured larvae (B). RNA was extracted from the fat body, and analyzed by Northern blot hybridization with pLEI0 as a probe. Arrows indicate the bands of Sarcophaga leetin mRNA.

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Fig. 3. Northern blot hybridization analysis of fat body RNA. (A) Third instar larvae were pricked with a hypodermic needle (injury +). After 6h, the fat body was excised and its RNA was analyzed by Northern blot hybridization with various probes. (B) Fat body was cultured for 2 h in the presence of increasing amounts of hemolymph from injured larvae. Then RNA was extracted and analyzed by Northern blot hybridization. The probes used were pLEI0 (Sarcophaga ieetin eDNA) (a), pSP2 (storage protein genomie clone) (b) and the BalI-AsulI fragment of pSCI (sarcocystatin A eDNA) (c).

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Fig. 4. Comparison of expressions of the Sarcophaga lectin gene and the sarcotoxin IIA gene. Fat body was cultured in the presence ( + ) or absence ( - ) of 150/~1 of hemolymph from injured larvae. After 2 h, RNA was extracted and analyzed by Northern blot hybridization. The probes used were pLE10 (A) and pTII20 (sarcotoxin IIA cDNA) (B).

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Sarcophaga lectin gene activation by humoral factor sumoto et al., 1986a) and the other is the sarcocystatin A gene (Saito et al., 1989). First, we tested whether the expressions of these two genes were enhanced like that of the Sarcophaga lectin gene when the body wall of larvae was injured. For this, R N A was extracted from the fat body, and the expressions of these genes were investigated by Northern blot hybridization with appropriate probes. As shown in Fig. 3 (A), in contrast to the Sarcophaga lectin gene, the expressions of the storage protein gene and the sarcocystatin A gene in situ were not changed by injury of the larvae. Then we tested the effect of hemolymph prepared from injured larvae on the expressions of the storage protein gene and the sarcocystatin A gene in cultured fat body. As is evident from Fig. 3(B), on culture for 2 h, increasing amounts of hemolymph had less effect on the expressions of these two genes than on that of the Sarcophaga lectin gene. These results strongly suggest that the activation of gene expression in the fat body by hemolymph in vitro is restricted to the Sarcophaga lectin gene, which is expressed selectively in response to body injury. Recently, we cloned a eDNA for sarcotoxin IIA, which is an antibacterial protein of Sarcophaga larvae produced in the fat body in response to body injury (Ando and Natori, 1988). Therefore, we compared the effect of hemolymph on the expression of the Sarcophaga lectin gene and the sarcotoxin IIA gene by cultured fat body. As shown in Fig. 4, expression of the sarcotoxin IIA gene was detected in the presence of hemolymph, but the expression of this gene was not so strict as that of the Sarcophaga lectin, and a faint band was detected even in the absence of hemolymph. Therefore, the factor in the hemolymph probably activates specific fat body genes of larvae such as the Sarcophaga lectin gene and the sarcotoxin IIA gene, whose expressions are triggered by body injury.

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heated hemolymph contained about twice as much activity as the original hemolymph, indicating that the factor was a heat-stable substance and was apparently activated in some way during heat treatment. We tried to concentrate this factor in the heated hemolymph by ultrafiltration on a membrane filter (Amicon, YM-2) that traps materials of > 1000 Da. Then we measured the activities in the concentrated solution and the filtrate simultaneously. The doseresponse curves for these two samples were almost the same, as shown in Fig. 5(C) and (D), indicating that this factor passed through this filter and was not concentrated by this procedure. These results suggest that this factor is a relatively small, heat-stable, organic compound. To obtain some information about the molecular nature of this factor, we treated this filtrate with 1 mg/ml of subtilisin or proteinase K for 12 h at 37°C. By this treatment, most peptides should be digested, but the activity in the filtrate was not changed appreciably (data not shown), suggesting that this factor is not a peptide. DISCUSSION

Previously, we demonstrated that hemolymph from injured Sarcophaga larvae contains a factor which is essential for activation of the Sarcophaga lectin gene in the fat body (Shiraishi and Natori, 1988). As a first step in identifying this factor, we tried to establish an in vitro system for culture of the fat body that mimicks the situation in vivo. The Sarcophaga lectin gene is not expressed when the fat body was cultured in modified Grace's insect medium only, but this gene was found to be activated by addition of hemolymph from injured larvae to the culture medium. Under these conditions, the expressions of the genes for the storage protein and sarcocystatin A, fat body proteins that are constitutively synthesized in the fat body of third instar larvae, were not appreciably affected, but expression of the sarcoCharacterization of hemolymph factor toxin IIA gene, an antibacterial protein synthesized in Since with our system for culture of the fat body, the fat body in response to body injury, was enwe could detect activation of the Sarcophaga lectin hanced. Thus this factor probably participates in the gene in the presence of hemolymph, we tried to expressions of genes for specific defense proteins. characterize the hemolymph factor participating in This factor was found to be a heat-stable, low activation of the Sarcophaga lectin gene. molecular weight compound and was probably not a When the hemolymph was heated in boiling water, peptide, but at present we have no information about the activity of the factor for inducing expression of its molecular nature. We found that this factor is the Sarcophaga lectin gene apparently increased. This secreted somewhere in the anterior part of the larvae was shown as follows. Hemolymph from injured in response to body injury (Shiraishi and Natori, larvae was heated at 60°C for 1 5 imn , denatured 1988). For isolation and identification of this factor, protein was removed by centrifugation and the super- a large amount of hemolymph must be collected. natant was heated in boiling water for 15 rain and This factor seems to be a small substance of centrifuged. By these procedures, most of the hemo- < 1000 Da. The molecular mechanism of its activalymph protein was removed and a clear supernatant tion of the Sarcophaga lectin gene in the fat body cells was obtained. We then compared the activity of the must be studied. At least two mechanisms are possiresultant supernatant with that of the original hemo- ble. One is similar to that of gene activation by lymph. For this, R N A was extracted from fat body steroid hormones. This factor may be taken up by fat cultured in the presence of various hemolymph sam- body cells and carried to the nuclei where it interacts ples and subjected to Northern blot hybridization directly with the Sarcophaga lectin gene. The other and the resulting bands corresponding to Sarcophaga possible mechanism of gene activation is like that by lectin m R N A in autoradiograms were scanned by peptide hormone although this factor does not apdensitometry to compare the relative amounts of pear to be a peptide. This factor may bind to a mRNAs. As shown in Fig. 5(A) and (B), the amount specific receptor on the surface of fat body cells, and of Sarcophaga lectin m R N A increased with increase some signal to activate the Sarcophaga lectin gene in the amount of added hemolymph. But clearly may be transmitted via this receptor.

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Fig. 5. Dose-responses to the hemolymph factor of transcription of Sarcophaga lectin mRNA in cultured fat body. Hemolymph from injured larvae was heated, and the resulting supernatant was concentrated by ultrafiltration through a YM-2 membrane filter as described in the text. The fat body was incubated in medium containing increasing amounts of each sample for 2 h. The RNA was extracted from the fat body and analyzed by Northern blot hybridization. The intensities of the bands of Sarcophaga lectin mRNA in the autoradiograms were scanned by densitometry, and relative amounts of Sarcophaga lectin mRNA are plotted against the volume of sample added to the culture medium. Bars, SD. (A) Original hemolymph; (B) h~nolymph after heat treatment; (C) fraction concentrated by YM-2; (D) filtrate through YM-2.

The present results show that this hemolymph factor is needed for activation of the Sarcophaga lectin gone in the fat body. This gone is known to be transiently expressed in the embryonic and pupal stages (Takahashi et al., 1986), so if this factor is a prerequisite for activation of the Sarcophaga lectin gone, it must be produced at these stages. This factor may be secreted by the same anterior tissue in pupae as in injured larvae, but the trigger for secretion in pupae is unknown. Moreover, nothing is known about the mechanism of activation of the Sarcophaga lectin geno in the embryonic stage. As an embryonic cell line of Sarcophaga peregrina, NIH-Sape-4, produces Sarcophaga lcctin constitutively when cultured in vitro (Komano et al., 1987), the Sarcophaga le~tin gone may be activated in embryos without mediation of the hemolymph factor. We assume that this factor causes simultaneous activations of the genes for various defense proteins expressed in the fat body in response to body injury (Natori, 1987). When this factor has been identified, it will be possible to investigate the expressions of these genes at the molecular level.

Acknowledgements--Tl~s work was supported by a Grantin-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a grant from the Tokyo Biochemical Research Foundation. REFERENCES Ando K. and Natori S. (1988) Molecular cloning, sequencing, and characterization of eDNA for sarcotoxin IIA, and inducible antibacterial protein of Sarcophaga peregrina (flesh fly). Biochemistry 27, 1715-1721. Komano H. and Natori S. 0985) Participation of Sarcophaga peregrina humeral lectin in the lysis of sheep red blood cells injected into the abdominal cavity of larvae. Dee. comp. Immun. 9, 31-40. Komano H., Mizuno D. and Natori S. 0980) Purification of lectin induced in the hemolymph of Sarcophaga peregrina larvae on injury. J. biol. Chem. 255, 2919-2924. Komano H., Kasama E., Nagasawa Y., Nakanishi Y., Matsuyama K., Ando K. and Natori S. (1987) Purification of Sarcophaga (flashily) lectin and detection of sarcotoxins in the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina. Bioehem. J. 248, 217-222. Matsumoto N., Nakanishi Y. and Natori S. (1986a) Homologies of nucleotide sequences in the Y-end regions of two

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analysis of the expression of the sarcocystatin A gene during development of Sareophaga peregrina. Biochemistry. 28, 1749-1755. Shiraishi A. and Natori S. (1988) Humoral mediatordependent activation of the Sarcophaga lectin gone. FEBS Lett. 232, 163-166. Suzuki T. and Natori S. (1985) Purification and characterization of an inhibitor of the cysteine proteas¢ from the hemolymph of Sarcophagaperegrina larvae. J. biol. Chem. 260, 5115-5120. Takahashi H., Komano H. and Natori S. 0986) Expression of the lectin gene in Sarcophaga peregrina during normal development and under conditions where the defence mechanism is activated. J. Insect Physiol. 32, 771-779. Takahashi H , Komano H., Kawaguchi N., Kitamura N., Nakanishi S. and Natori S. (1985) Cloning and sequencing of eDNA of Sarcophaga peregrina humoral lectin induced on injury of the body wall. J. biol. Chem. 260, 12228-12233. Thomas P. S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. nam. Acad. Sci. U.S.A. 77, 5201-5205.