From the Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du CNRS, 15 rue René Descartes, 67084 Strasbourg Cedex, France
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ABSTRACT |
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The dorsoventral regulatory gene pathway
(spätzle/Toll/cactus)
controls the expression of several antimicrobial genes during the
immune response of Drosophila. This regulatory cascade
shows striking similarities with the cytokine-induced activation
cascade of NF-B during the inflammatory response in mammals. Here,
we have studied the regulation of the I
B homologue Cactus in the fat
body during the immune response. We observe that the cactus gene is up-regulated in response to immune challenge. Interestingly, the expression of the cactus gene is controlled by the
spätzle/Toll/cactus gene
pathway, indicating that the cactus gene is autoregulated. We also show that two Cactus isoforms are expressed in the cytoplasm of
fat body cells and that they are rapidly degraded and resynthesized after immune challenge. This degradation is also dependent on the Toll
signaling pathway. Altogether, our results underline the striking
similarities between the regulation of I
B and cactus during the immune response.
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INTRODUCTION |
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Transcription factors containing the Rel homology domain have been implicated in a number of developmental and physiological processes, including dorsoventral patterning and immune response in Drosophila, mammalian acute phase response, and lymphocyte differentiation (reviewed in Refs. 1-4).
In mammals, NF-B is a generic name for a number of Rel proteins
(p50, p52, RelA, and RelB), which associate as homo- or heterodimers (reviewed in Refs. 1 and 2). This transactivator plays a pivotal role
in the regulation of immune and inflammatory response genes. NF-
B is
retained in unstimulated cells in the cytoplasm by its inhibitor I
B
and migrates into the nucleus after rapid degradation of I
B in
response to activation by cytokines such as interleukin-1 and tumor
necrosis factor
(reviewed in Refs. 1 and 2).
In Drosophila, the embryonic dorsoventral regulatory pathway
comprises 12 known maternal effect genes (reviewed in Ref. 5). The end
result of the activation of this pathway is the nuclear translocation
of the Rel transcription factor Dorsal. Four components of this
pathway, Toll (TL), Pelle (PLL), Cactus (CACT), and DORSAL (DL) are
homologous to members of the interleukin-1 receptor/NF-B pathway.
The cytoplasmic domain of TL, a transmembrane receptor protein (6), is
homologous to the cytoplasmic domain of the interleukin-1 receptor (7,
8). PLL (9) shares sequence homology with the interleukin-receptor
associated kinase (10). DL (11) and CACT (12, 13) are homologous to
NF-
B and I
B, respectively. Localized activation of the TL
receptor in the ventral region of the embryo by its ligand, the
spätzle (SPZ) protein, causes disruption of the DL-CACT complex
and the subsequent nuclear translocation of DL (14, 15). Genetic and
molecular analyses indicate that CACT, like I
B, is rapidly degraded
in response to signaling (16-18). The striking structural and
functional similarities between NF-
B and DL signaling pathways have
led to the proposal that they share a common ancestry (reviewed in
Refs. 3 and 19).
Rel proteins have recently been shown to be involved in the immune
response of Drosophila (reviewed in Ref. 4). In particular, it has been suggested that they control the induction of genes encoding
antibacterial and antifungal peptides in the fat body and in blood
cells. The upstream regions of these genes contain sequence motifs
similar to NF-B binding motifs of mammalian immune responsive genes
(reviewed in Ref. 20). Experiments with transgenic flies have shown
that these motifs are mandatory for immune inducibility of the insect
antibacterial peptide genes (21, 22). Several Rel proteins were
reported to be present in the fat body: DL (23), initially identified
as the dorsoventral morphogen, DIF (for dorsal-related immunity factor;
Ref. 24), and Relish, a NF-
B1 (p105)-like protein containing both
Rel and ankyrin domains (25). The precise roles of these Rel proteins
in the control of these immune genes has not yet been clarified
in vivo (26, 27). Recently, we have shown by genetic
analysis that the intracellular components of the dorsoventral pathway
(except for DL) and the extracellular TL ligand SPZ, collectively
referred to as the TL pathway, control the expression of the antifungal
peptide gene drosomycin in Drosophila adults
(27). In flies carrying loss-of-function mutations in the
pll, tub, Tl, and spz
genes, the immune inducibility of the drosomycin gene is
dramatically decreased. In contrast, in Tl gain-of-function
mutants, in which the TL pathway is signal-independently activated, and
in cact-deficient mutants, the gene encoding drosomycin is
constitutively expressed. Altogether, these data demonstrated that the
TL/interleukin-1 receptor pathway is indeed an ancient regulatory
cascade involved in the host defense of both mammals and insects
(27).
The fat body of Drosophila provides a unique experimental system to dissect in vivo the TL/interleukin-1 receptor signaling pathway in the context of the immune response. In this study, we have focused our interest on the regulation of cact, the last element of the genetically characterized cascade. We have first observed that the cact gene is up-regulated in response to immune challenge and that the expression of cact is controlled by the spz/Tl/cact gene regulatory cascade. We have also noted that two CACT isoforms are expressed in the cytoplasm of fat body cells and that they are rapidly degraded and resynthesized after immune challenge. This degradation is dependent on the TL signaling pathway.
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EXPERIMENTAL PROCEDURES |
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Drosophila Stocks-- The cact255 strain contains an FZ enhancer trap (28) in the first intron of the cact gene. The cact255 FZ line exhibits an embryonic pattern of lacZ expression similar to that of the resident cact gene as detected by in situ hybridization of its transcripts (12). This insertion causes a strong CACT phenotype (13, 29). Tl10b and Tl9Q are two dominant gain-of-function ventralizing alleles of Toll (TlD) caused by a single amino acid change (30). Other dorsoventral mutant stocks used in this study have been described elsewhere (26, 31). All experiments were performed at 25 °C except when otherwise stated.
Infection Experiments--
Bacterial challenges were performed
by pricking third instar larvae or adults with a needle dipped into a
concentrated culture pellet of Escherichia coli and
Micrococcus luteus (OD of the pellet 100). Natural
infection with entomopathogenic fungi was performed by shaking
anesthetized flies for a few minutes in a Petri dish containing a
sporulating culture of Beauveria bassiana (strain 80.2).
Flies covered with spores were then placed onto fresh
Drosophila medium and incubated at 29 °C. Natural
infection with entomopathogenic fungi induces a strong and sustained
expression of the antifungal peptide gene drosomycin,
through the selective activation of the TL signaling pathway (32).
-Galactosidase and Immunolocalization Stainings--
The
-galactosidase activity measurement and staining method were as
described in Ref. 33. Immunolocalization experiments were performed as
in Ref. 26. A monoclonal anti-CACT mouse antibody (2C2-50; Ref. 34)
was applied to the fat bodies at a 1:100 dilution. The second antibody
was an alkaline phosphatase-linked sheep anti-mouse-IgG (Boehringer
Mannheim) diluted 1:500.
RNA Preparation and Analysis-- Crosses were performed at 25 °C, and third instar larvae or 2-4-day-old adult flies were collected. Total RNA was extracted from dissected larval or adult fat body with the RNA Trizol (Life Technologies, Inc.) method. Total RNA extraction and Northern blotting experiments were performed as in Ref. 35. The following probes were used: cecropin A1 cDNA (36), diptericin cDNA (37), drosomycin cDNA (38), a CACT cDNA (a polymerase chain reaction product of approximately 1.5 kb1 corresponding to the N-terminal part of cact), and rp49 cDNA (a polymerase chain reaction fragment of approximately 400 base pairs generated between two oligonucleotides designed after the rp49 coding sequence; Ref. 39). The cecropin A1 probe cross-reacts with cecropin A2 transcripts (36).
Western Blot Analysis--
The monoclonal anti-DL antibody
(7A4-25, 34) used in this study is directed against the C-terminal
domain of the DL protein. The monoclonal anti-CACT antibody 2C2-50 was
described by Whalen and Steward (34). A monoclonal anti--tubulin
antibody (Boehringer Mannheim) was used as a loading control. Larval or
adult fat bodies from 30-40 insects were collected and frozen at
80 °C. Fat bodies were lysed in 2× Laemmli solution. 15 µg of
fat body extract were loaded on a 7.5% SDS-polyacrylamide gel.
Following SDS-polyacrylamide gel electrophoresis, proteins were blotted
to Hybond ECL nitrocellulose membranes (Amersham Life Science). The
blots were developed using the ECL system (Amersham) and x-ray film to
detect the signal. Cycloheximide treatment was performed by
injecting ~20 µl of a mixture of cycloheximide (10 µg/ml) and
bacterial suspension into the thorax of Drosophila adults
using a Nanoject apparatus (DrumondTM).
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RESULTS |
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The results reported in this study were obtained with fat body extracted from either larvae or adults. The fat body, a functional analog of the mammalian liver, is the major site of antimicrobial peptide production in Drosophila. In larvae, it consists of a mass of large polyploid cells that can easily be dissected out. In contrast, adult fat body is a thin and loose tissue difficult to excise. Our analysis was performed with extracts of fat body cells and occasionally, when indicated, of adult abdominal carcass, which allows the extraction predominantly of fat body with minor contaminations from epidermal and muscle cells.
Expression of the cact Gene Is Induced in the Fat Body by Immune Challenge-- In a previous study, we had observed that 3 h after a bacterial challenge, cact gene expression was markedly up-regulated in adults (27). We have now extended this study by analyzing the time course of cact gene expression both in excised larval fat body and in male adult carcass tissues. The Northern blot analysis, presented in Fig. 1 (A and B) shows a faint signal for cact transcripts in unchallenged fat body and adult carcass and a remarkably rapid and strong up-regulation following bacterial challenge. In both larvae and adults, peak values were observed after 2 or 3 h, after which the signals of cact transcripts leveled off. These kinetics of induction/up-regulation, frequently referred to as acute phase kinetics, were similar to those of the cecropin A gene in these experiments. In contrast, the drosomycin and the diptericin genes reached their highest level of expression only 6-16 h postchallenge (Fig. 1, A and B).
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cact Expression Is Autoregulated--
We have further analyzed the
expression of the cact gene in Drosophila
carrying mutations that affect the dorsoventral signaling pathway. We
have first examined the expression of the
cact255 FZ reporter gene in dominant
gain-of-function Tl (TlD) and
cact-deficient mutant larvae in which the TL pathway is signal-independently activated and the drosomycin gene is
constitutively turned on (27). A first striking result, shown in Fig.
2, C and D, was that in both mutant contexts, the
reporter gene was expressed in the absence of immune challenge in
larvae. The level of -galactosidase activity was higher than that
induced by bacterial challenge in wild-type insects. Similar results
were obtained in adult fat body (data not shown).
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Two CACT Isoforms Are Present in Cytoplasm in the Fat Body Cells-- With appropriate antibodies, we examined the subcellular localization of the CACT protein in excised fat body. We restricted our analysis to the large polyploid cells of larval fat body from control and bacteria-challenged Drosophila. Staining with an anti-CACT monoclonal antibody revealed only a faint cytoplasmic reaction in control larval fat body. The staining was however more conspicuous with fat body from challenged insects (data not shown). These data indicating that CACT proteins have similar subcellular localizations in the fat body and in embryos are consistent with their putative function as a cytoplasmic inhibitor.
Earlier Western blot analyses of CACT protein expression had revealed three polypeptides, which are differentially expressed during development (Refs. 12 and 34; see also Fig. 6). In male extracts, two major proteins of 69 and 71 kDa cross-react with an anti-CACT monoclonal antibody (Refs. 12 and 34; Fig. 6). These proteins are also detected in female ovaries, where a third form of 72 kDa is present. The latter species is the major form of CACT in late stage oocytes and early embryos. Phosphatase treatment revealed that the 72-kDa protein is a phosphorylated form of the 71-kDa protein and that both are encoded by the 2.2-kb maternal/zygotic mRNA (12, 34, 40).
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Bacterial Challenge Induces Degradation of CACT in Wild-type Larvae
and Adults--
By Western blot analysis, we next studied the level of
CACT proteins in the fat body during the immune response. Fat body from
larvae and adults were collected at different time intervals after
bacterial challenge. Fig. 7 (A
and B) shows that in response to this challenge, both the
69- and 71-kDa forms were degraded. The signals corresponding to both
protein bands decreased 30-90 min postchallenge but afterward began to
increase until they reached the initial or an even higher level. It
should be noted that the 71-kDa form was more sensitive to immune
induced degradation than was the 69-kDa form, since the latter never
totally disappeared. The kinetics of degradation were essentially
similar to those observed for IB
in cell culture (41).
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Tl Controls the Immune Induced Degradation of CACT--
We have
also examined the immune induced degradation of CACT in larvae and
adults carrying mutations that alter the dorsoventral signaling
pathway. No immune induced degradation of CACT was observed in fat body
extracts derived from Tl-deficient mutants (Fig. 7, E and F), indicating that the immune induced
degradation of CACT requires the TL signaling cascade. It should be
noted that, in contrast to adults, only the 69-kDa zygotic CACT form
was detected in Tl larvae (Fig.
7E).
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DISCUSSION |
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Transcriptional Regulation-- In a previous study, we had shown that the genes encoding the components of the embryonic dorsoventral pathway are expressed at a low but detectable level in control adults. They are significantly up-regulated upon septic injury (27). The high transcriptional level of these genes in challenged insects obviously allows for amplification of antimicrobial peptide gene expression, by increasing the amount of SPZ/TL/CACT components able to respond to the signal.
Here, we have analyzed in detail the kinetics of expression of the cact gene during the immune response. We have found that cact expression is rapidly and markedly induced and, after a peak value at 3 h, gradually levels off, this profile of expression being evocative of that of mammalian acute phase response genes. Interestingly, we have also observed that cact gene expression is controlled by the SPZ/TL/CACT signaling pathway. Indeed, the activation of the TL signaling pathway in TlD gain-of-function and cact-deficient mutants is sufficient for a strong induction of the cact gene, whereas loss of function in any of the genes extending in the dorsoventral regulatory cascade from spz to pll results in a markedly impaired induction of the cact gene by bacterial challenge. In contrast, the cact gene remains fully inducible in imd mutants. In essence, the transcriptional profile of cact in dorsoventral mutants parallels that earlier observed for the drosomycin gene (27). We hypothesize that both genes are induced via a Rel protein (possibly DIF or an as yet unidentified Rel protein, but not DL alone), which is retained in the cytoplasm of the fat body by binding to the CACT protein. Our results indicate that the dissociation of this CACT-Rel complex is mediated by the TL signaling pathway. This autoregulatory loop allows for the rapid resynthesis of inhibitors, which can in turn shut down the response when the extracellular signal levels off (Fig. 8). In agreement with this hypothesis, several putative Rel binding sites are observed in the genomic region flanking the cact255 FZ insertion site. Indeed, the observation that the expression of the cact255 FZ enhancer trap insertion is inducible after microbial challenge strongly suggests that this element is inserted in the vicinity of immune responsive regulatory sequences.
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Post-translational Regulation-- We have detected two CACT isoforms of 69 and 71 kDa in the fat body but did not observe the 72-kDa phosphorylated CACT species, which is the predominant form in late ovaries and early embryos. We have no idea whether the two CACT isoforms have distinct regulatory properties in the control of antimicrobial peptide gene expression. However, in agreement with previous studies (12, 16), our data indicate that the 69-kDa protein, encoded by the 2.6-kb maternal/zygotic transcript, is more stable than the maternal form; although the maternal 2.2-kb mRNA is more abundant than the 2.6-kb zygotic transcript, the 69-kDa protein is predominant in the fat body.
The Western blot analysis of the fluctuations of CACT protein in the fat body following bacterial challenge points to several successive phases. In control insects, both CACT isoforms are expressed at a low level, the 69-kDa protein being predominant. In response to immune challenge, a rapid depletion of both CACT isoforms is observed with the maternal/zygotic 71-kDa species disappearing completely. This CACT degradation is mediated by the TL signaling pathway as demonstrated by the fact that it does not occur in Tl-deficient mutants. This short depletion phase (30-90 min) is rapidly followed by the regeneration of both isoforms by de novo synthesis, as illustrated by our cycloheximide studies. During this phase, the CACT levels reach an equilibrium between signal-induced degradation and de novo synthesis of CACT following intense expression of its gene. A similar situation is observed in TlD mutants, where the TL pathway is constitutively activated and where a high level of CACT protein (particularly of the 71-kDa form) is detected. The observation that bacterial challenge failed to induce the depletion of CACT in TlD mutants also suggests that a state of equilibrium has been reached under constitutive signaling. We may anticipate that in wild-type challenged animals, at a later stage, the decrease of signaling is correlated with a return to the normal situation. The findings that TlD mutants or persistently infected adults express high titers of CACT are at first sight paradoxical, since in these backgrounds the Rel proteins DIF and DL are predominantly nuclear (24, 26) and the drosomycin gene is constitutively turned on (27). Several explanations can account for the activation of Rel proteins in the presence of a high level of inhibitor. One possibility is that the levels of Rel proteins (the dl and dif genes are themselves up-regulated upon bacterial challenge; Refs. 23 and 46) are in excess of that of the inhibitor CACT. Alternatively, we propose that the nuclear translocation of the Rel proteins is not strictly correlated to the level of CACT proteins but rather to the intensity of CACT degradation. This implies that once dissociated from the Rel-CACT complexes, the Rel proteins cannot be inhibited by free CACT (e.g. because of structural modifications). Such a model would ensure a strict correlation between the level of signaling and the level of Rel nuclear translocation. However, it excludes the possibility of an active inhibitory mechanism by CACT of the cognate Rel proteins, in contrast to IConclusions-- In Drosophila, as in other organisms, signal transduction pathways are involved in various developmental and physiological processes. These cascades exhibit subtle differences to account for their respective functions in these tissues. The TL signaling pathway, which is involved in embryonic dorsoventral patterning, in the antimicrobial response, and probably in several other processes (reviewed in Ref. 3) is a good example. It is interesting in this context to note that in contrast to embryonic development, the regulation of CACT in the fat body involves an autoregulatory loop.
Finally, the data in this paper reveal striking functional similarities between transcriptional and post-translational regulation of I ![]() |
ACKNOWLEDGEMENTS |
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We are indebted to Ruth Steward (Rutgers) for the gift of anti-DL and anti-CACT antibodies and to Marie Meister, Philippe Georgel, Jean Luc Imler, and Isabelle Gross for stimulating discussions. The technical assistance of Reine Klock and Raymonde Syllas is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by CNRS, the University Louis Pasteur of Strasbourg, and Rhone Poulenc-Agro.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Institut de Biologie
Moléculaire et Cellulaire, UPR 9022 du CNRS, 15 rue René Descartes, 67084 Strasbourg Cedex, France. Tel.: 33 03 88 41 70 77;
Fax: 33 03 88 60 69 22; E-mail: lemaitre{at}ibmc.u-strasbg.fr.
1 The abbreviation used is: kb, kilobase pair(s).
2 Berkeley Drosophila Genome Project, unpublished results.
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REFERENCES |
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