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Departments of
1 Plant Sciences and
5 Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv, Israel
2 Zoology Department, Stockholm University, Stockholm, Sweden
3 Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay
4 Laboratory of Immunoregulation, Institut Pasteur, Paris, France
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Abstract |
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B activity in CSN5 down-regulated mammalian cells can be induced following activation of the Toll/IL-1 pathway. csn5null larvae contained more hemocytes than wild-type (wt) larvae. A large portion of these cells have differentiated to lamellocytes (LM), a hemocyte cell type rarely seen in normal larvae. The results presented here indicate that CSN5 is a negative regulator of Dorsal subcellular localization, and of hemocyte proliferation and differentiation. These results further indicate that nuclear localization of Dorsal can be uncoupled from its activation. Surprisingly, CSN5 is not necessary for immune-induced degradation of Cactus. |
Introduction |
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The humoral response to infection in Drosophila, characterized primarily by synthesis of AMPs in the fat body, the Drosophila equivalent to a liver, is mediated by the Toll/IL-1 receptor (Wasserman 2000; Hultmark 2003; Brennan & Anderson 2004). The accepted model for Toll signaling in the fat body is as follows: Cactus, the homolog of mammalian IB, forms a cytoplasmic complex with either Rel transcription factor Dorsal or Dif, the homologs of mammalian NF-B. Following fungal or gram-positive bacterial infection, a proteolytic event activates a ligand of the Toll receptor, which in complex with Toll activates an intracellular signaling cascade resulting in the degradation of Cactus (Nicolas et al. 1998; Wu & Anderson 1998). The Rel protein relocates to the nucleus, where it can activate target AMP-encoding genes such as Drosomycin (Drs) (Wu & Anderson 1998; Manfruelli et al. 1999). During embryogenesis, signal-dependent degradation of Cactus requires its phosphorylation (Reach et al. 1996), which together with analogous IB studies (Alkalay et al. 1995), suggest that once phosphorylated, Cactus is degraded in a proteasome-dependent fashion.
The cellular response to infection in Drosophila is mediated through different classes of hemocytes. The Drosophila blood, also called the hemolymph, circulates in an open circulatory system. Drosophila hematopoiesis during larval development occurs in the lymph glands, which serve as a hemocyte reservoir (Rizki 1978; Shrestha & Gateff 1982; Lanot et al. 2001; Sorrentino et al. 2004). Hematopoiesis gives rise to three independent cell lineages: plasmatocytes (PL), phagocytes that resemble the monocyte/macrophage lineage, crystal cells, which play a critical role in defense-related melanization, and lamellocytes (LM) that encapsulate large invaders (Meister et al. 2000; Minakhina & Steward 2006). The regulation of hematopoietic cell proliferation and lineage specification in Drosophila involves several signaling pathways, including the Toll pathway.
Both dominant gain-of-function mutations in Toll, and loss-of-function mutations in Cactus, lead to constitutive activation of immune signaling with nuclear accumulation of Dorsal and induction of AMPs in the fat body, hemocyte proliferation and formation of melanotic bodies (Lemaitre et al. 1996; Qiu et al. 1998; Minakhina & Steward 2006). Down-regulation of the Toll-pathway caused by mutations in Toll, Tube or Pelle, on the contrary, lead to reduced hemocyte numbers (Qiu et al. 1998). However, mutations in Dorsal or Dif, two other key members of the Toll pathway, apparently have little or no effect on hemocyte numbers (Sorrentino et al. 2004).
The COP9 signalosome (CSN) is a conserved eight-subunit protein complex that functions in the ubiquitin-proteasome (Ub) system and thus has multiple roles in plants and animals. Within the Ub system, CSN regulates substrate phosphorylation and subcellular localization, and Cullin E3-ligase activity (Deng & Serino 2003). However, evidence from several systems indicates that there is an intricate equilibrium between the complex and specific individual subunits, which are also detected in forms independent of the CSN complex and may have independent activity. For example, CSN5, the subunit that contains deneddylase activity towards Cullin proteins, performs this enzymatic function only within the complex (Lyapina et al. 2001). However, CSN5 is also detected in smaller complexes and as a monomeric form, which may have independent signaling activities (Oron et al. 2002; Fukumoto et al. 2005). In Drosophila, null mutations in Csn5 leave an intact CSN, indicating that CSN5 is not essential for complex formation (Oron et al. 2002).
There are several indications that the CSN may be involved in immune signaling: in Arabidopsis, csn mutants have defects in innate immunity (Liu et al. 2002); in human cells, the CSN co-purified with an IB
kinase activity (Seeger et al. 1998); and CSN3 interacts with IKK
(Hong et al. 2001). To further explore the role of CSN5 in regulating immune signaling, we have analyzed Drosophila csn5null mutants.
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Results |
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Early developmental progression of Drosophila csn5null mutants parallels that of wild-type (wt) strains and of their heterozygotic siblings, displaying no changes in body size, locomotor activity or in timing of the larval molts (Freilich et al. 1999). However, csn5null larvae never pupariate and die as 10- to 13-day-old larvae (Freilich et al. 1999). These mutants developed massive melanotic capsules that first appear floating in the hemolymph during the third larval instar, (Fig. 1A) and progressively increase in size (Fig. 1B).
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In larvae with mutations in other genes, the presence of melanotic bodies similar to those seen in csn5null larvae has been correlated with nuclear localization and activity of Dorsal in fat body (Lemaitre et al. 1995) or in hemocytes (Huang et al. 2005). To first assess if CSN components are present in wt fat body, we examined the subcellular localization of CSN5 and CSN7. Both subunits were enriched in fat body nuclei, accumulating against the nuclear envelope, excluded from the nucleolus (Fig. 2A). A fraction of CSN5 was also present in the cytoplasm. This localization pattern was not affected by immune challenge (not shown).
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Dorsal activation is immune signal-dependent in csn5null larvae
We next monitored Dorsal activity in csn5null larvae by analyzing the transcript levels of one of its target gene, Drosomycin (Drs). We also studied transcript levels of Diptericin (Dpt), another AMP gene, which is primarily under the control of the IMD signal pathway (Georgel et al. 2001). Any changes detected in transcription of both genes could be indicative of a general defect in immune signaling or in transcriptional repression. As expected, in wt larvae, Drs and Dpt transcript levels were undetectable in unchallenged conditions, and present in high amounts following bacterial challenge (Fig. 3). Surprisingly, despite the constitutive nuclear accumulation of Dorsal, transcript levels of Drs in csn5null larvae mirrored those found in wt, as did Dpt. These results were confirmed by employing Drosophila strains carrying a GFP-reporter gene under the Drs or Dpt promoters (Fig. 3B). An independent transcript profiling experiment on csn5null larvae (Oron et al. 2005; Tuller et al. 2005) also did not identify any induction of other Dorsal targets such as cactus or attacin in csn5null larvae 96 h AED (not shown). Furthermore, promoter analysis of the genes misregulated in csn5null did not point to enrichment of Dorsal binding sites in the region extending from –400 to +200 bp relative to their annotated transcription start sites (not shown). These results indicate that Dorsal, although constitutively nuclear in csn5null fat body, is inactive in the absence of immune signal and that CSN5 is not essential for Dorsal activation.
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B activation in human cell lines
To determine if CSN5 has a role in mediating Toll-related signaling in mammals, we monitored the activity of the Dorsal homolog, NF-B, in human cell lines with reduced levels of CSN5. The levels of CSN5 were reduced at least 75% by transfection of a siRNA vector targeting CSN5 (iCSN5) in 293T and HeLa cells (Fig. 4A and not shown). NF-B activity in response to IL-1 or TNF stimulation was monitored by measuring expression of a luciferase reporter gene under the control of a NF-B dependent promoter (IgGK promoter). IL-1 or TNF treatment activated the NF-B promoter both in control and in CSN5 knockdown cells (Fig. 4B, C). This indicates that in human cells, as for the Drosophila fat body, a reduction in CSN5 levels does not affect NF-B activation.
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According to the accepted model, Dorsal activity is inhibited mainly by its cytoplasmic sequestering via its interaction with Cactus. The constitutive nuclear accumulation of Dorsal would thus suggest that Cactus is constitutively degraded in csn5null larval fat body. On the other hand, the lack of Dorsal activity in unchallenged csn5null larvae suggests that Dorsal is inhibited, possibly by still being complexed to Cactus. We therefore monitored the levels and subcellular localization of Cactus. Total protein extracts from csn5null third instar larvae, as well as from fat body isolated from these larvae, displayed elevated levels of Cactus in comparison with wt larvae or their heterozygous siblings (Fig. 5A). In agreement with this finding, we observed higher immunofluorescence of anti-Cactus in the fat body of csn5 mutants (see below, Fig. 5C) However, Cactus was apparently degraded in csn5null following bacterial infection, as the Cactus band was not detected following infection (Fig. 5A), consistent with the immune inducibility of Drs in csn5null larvae. Additional high molecular weight species that cross-react with the anti-Cactus antibodies were also detected. These appear specific for Cactus as they over-accumulated in csn5null, and were not detected following immune challenge (Fig. 5A, right panel). The increase in Cactus in csn5null larvae was not due to increased Cactus transcript levels (Fig. 5B).
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csn5null larvae show hematopoietic phenotypes
Having ascertained that immune defenses are defective in csn5null mutants, though the humoral output is apparently normal, we then examined the cellular arm of the immune response. The formation of melanotic bodies similar to those seen in csn5null larvae has been correlated with hematopoietic defects, specifically arising from defects in Toll signaling (Lemaitre et al. 1995). To first assess if CSN components are present in wt hemocytes, we examined the subcellular localization of CSN5 and CSN7 in these cells. Both subunits were enriched in PL nuclei. This localization pattern was not affected by immune challenge (not shown).
Observation of living mutant larvae under the microscope indicated that they contain abnormally large numbers of hemocytes, many of which are intimately associated with melanotic cells (Fig. 6A,B). To confirm this, circulating hemocytes were counted in blood samples extracted from mutant and wt larvae. As seen in Fig. 6, csn5null larvae contain more hemocytes than the wt (2.14-fold increase, P < 0.05). A similar ratio of 2.16 was obtained by a second counting method based on sampling representative microscope frames of fixed cells from different larvae. Hemocyte numbers from heterozygotic siblings mirrored those of the wt (not shown).
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10% of the hemocytes in csn5null larvae (899 LM out of 8307 cells counted from 11 larvae). Mutant larvae contained normal numbers of crystal cells, although these cells were larger compared with wt cells (Fig. 7C).
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Discussion |
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CSN5 appears also to be necessary for proper hemocyte differentiation. In wt larvae, LM differentiation is normally repressed but rapidly induced as an immune response to parasites (Lanot et al. 2001; Sorrentino et al. 2004). LM differentiation is also induced during metamorphosis, when LM have been proposed to have a role in remodeling larval tissues (Rizki & Rizki 1978). Consequently, very few LM are observed in normal larvae prior to metamorphosis (Luo et al. 2002; Evans et al. 2003). The abnormally large numbers of LM in mutant larvae indicates that CSN5 normally represses their differentiation during larval life. Although it is generally unclear what seeds formation of melanotic capsules, it is clear that in our larval cultures the melanotic capsules generated within csn5 mutant larvae are not a response to parasitism or penetration of other foreign bodies into their body cavity; hence, the encapsulation reaction in csn5 mutant larvae may represent an immune response to self tissues. Furthermore, the physical association of LM with melanotic bodies from the earliest stage of melanization suggests a direct relationship between the abnormally high number of LM and the melanotic phenotype. At this stage, however, we cannot discern whether the increased numbers of LM are induced by the melanotic bodies or if melanization itself is initiated by the misregulation of LM differentiation.
Our microarray analysis indicates that hemocyte over-proliferation and differentiation phenotypes of csn5null mutants are also reflected at the molecular level (Oron et al. 2005; Tuller et al. 2005). For example, Pendulin, a blood cell tumor suppressor (Kussel & Frasch 1995), is strongly down-regulated in csn5null. A loss-of-function mutation of pendulin causes lethality, melanotic bodies formation, abnormal growth of larval tissues, enlarged lymph glands and over-proliferation of abnormal hemocytes (Kussel & Frasch 1995). These phenotypes resemble the phenotypes of csn5null mutants suggesting that some of the hematopoietic phenotypes of csn5null larvae are the result of the pendulin suppression.
On the other hand, the up-regulation of several genes involved in hematopoiesis may simply represent higher mRNA levels due to increased hemocyte numbers. For example, Cg25C, expressed in all hemocytes, is 3.5-fold increased in csn5null. Similarly, transcript levels of Dox-A3, which is expressed specifically in LMs (Irving et al. 2005) are increased -fold in csn5null mutants, reflecting the increased numbers of LMs in the mutant.
In humoral signaling, CSN5 appears to be essential for the steady-state regulation of Cactus and Dorsal in Drosophila larval fat body, but dispensable for Toll/IL-1 activation in fat body. Our data indicate that the same is probably true for the homologous pathway in mammalian cells. We propose a model in which CSN5 is involved in Cactus and Dorsal regulation at two levels (Fig. 8B).
First, CSN5 is a negative regulator of Cactus and Dorsal nuclear accumulation in the fat body in unchallenged conditions. The Cactus–Dorsal complex is mainly cytoplasmic in wt fat body cells under naive conditions but, in the absence of Csn5, both Cactus and Dorsal are found mainly in the nucleus. Nuclear localization of Cactus in wt larvae has been previously reported in larval brain cells in response to dark–light cycle (Cantera et al. 1999b) and also in somatic muscles of dorsal mutant larvae (Cantera et al. 1999a). However, while the subcellular distribution of Cactus has been extensively studied during embryogenesis and in relation to the immune response during larval and adult stages, nuclear accumulation of Cactus in fat body cells has not been reported so far. For lack of immunoprecipitating antibodies we could not directly confirm that Cactus and Dorsal remain bound to each other in the csn5null fat body nuclei. However, our data support this hypothesis as Dorsal transcriptional activity on the drosomycin gene in unchallenged larvae remains repressed, and Dorsal targets are not preferentially induced in csn5null larvae, as shown by transcript profiling experiments (Oron et al. 2005; Tuller et al. 2005), despite nuclear localization of Dorsal. In mammals, IB is also found in the nucleus, where it not only serves to transport NFB back to the cytoplasm, but also inhibits DNA binding of NFB (Arenzana-Seisdedos et al. 1995; Turpin et al. 1999). Furthermore, a recent study shows that nuclear IB can also directly repress transcription (Aguilera et al. 2004). Our results add to the accumulating evidence which points to a repressive role for Cactus in the nucleus of larval fat body.
The mechanistic basis of the CSN5-dependent repression on Cactus–Dorsal nuclear localization is unclear, and indeed the nuclear localization of CSN5 might suggest that this regulation is indirect. However, a direct role involving either a cytoplasmic fraction of CSN5, or regulation of nuclear import or export, must also be considered. Consistently, CSN5 is essential for proper subcellular localization of signaling molecules such as p27kip1 and COP1 (Chamovitz et al. 1996; Bianchi et al. 2000; Tomoda et al. 2002).
Second, CSN5 is a negative regulator of Cactus steady state levels. Neither RT-PCR analysis, nor microarray analysis, detected significant changes in Cactus transcript levels, indicating that the increase in Cactus protein observed in csn5null larvae is due to post-transcriptional mechanisms. Previous studies on Drosophila embryos (Belvin et al. 1995) and human cells (Van Antwerp & Verma 1996) demonstrated an equilibrium between Dorsal/NFB-bound and unbound Cactus/IB. Unbound Cactus/IB is unstable and marked for protease-dependent degradation. This steady-state degradation is promoted by CK2 (Schwarz et al. 1996; Liu et al. 1997; Packman et al. 1997). As CK2 activity co-purifies with CSN (Uhle et al. 2003), we propose that CSN5 negatively regulates steady-state Cactus levels by promoting the immune signal-independent degradation of unbound Cactus (Fig. 8B).
It is surprising that CSN5 is not essential for immune-dependent degradation of Cactus as the most central role postulated for CSN5 is deneddylation of the Cullin subunit of the SCF ubiquitin ligase complex. This activity is necessary for controlled degradation of specific regulatory proteins (Cope & Deshaies 2003). Neddylation and the coordinated activity of SCFß-TrCP towards IB (Read et al. 2000) and p105 (Amir et al. 2002) control NFB transcriptional activity. The Drosophila homolog, SCFslimb, was suggested to be required for Cactus degradation in embryos, but may be dispensable in Toll signaling in larval fat body (Khush et al. 2002; Leulier et al. 2003).
Given the importance of SCF complexes in promoting signal-dependent degradation of IB (Read et al. 2000), and the regulation by CSN of SCF activity (Cope & Deshaies 2003), we had hypothesized that CSN5 would be required for signal-dependent degradation of Cactus. Unexpectedly, our findings show that while Cactus over-accumulates in the absence of CSN5, degradation of Cactus and activation of Dorsal/NFB in response to immune stimulation can still occur in the absence of CSN5. This suggests that SCF dependent degradation of Cactus does not require CSN5 under physiological conditions.
Signal-dependent activation of Dorsal/NFkB classically requires both Cactus/IB degradation and Dorsal/NFkB nuclear localization. These two steps however can be uncoupled (Uv et al. 2000; Bhaskar et al. 2002). In TlD naive larva, Dorsal is constitutively nuclear and active even though Cactus remains at high levels in the cytoplasm (Nicolas et al. 1998). In csn5null larvae, Cactus also accumulates to high levels, but co-localizes with Dorsal in the nucleus, where Dorsal activity remains repressed in the absence of an immune signal. Consistently, microarray analysis did not reveal preferential induction of Dorsal targets in csn5null mutants despite constitutive Dorsal nuclear localization.
Our results clearly indicate a role for CSN5 in mediating immune responses in Drosophila larvae. The phenotypes described could be the result of a loss of CSN5 as a component of the CSN complex, or the result of a loss of the CSN-independent forms of CSN5 (Oron et al. 2002). As additional available csn mutants (e.g. csn4null, csn51) die at earlier stages before immune competence, this question could not be further pursued.
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Experimental procedures |
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Growth conditions and strains are as in Oron et al. (2002). Egg depositions lasted for 4 h. All larvae were taken for analysis at 110 h after egg deposition (AED). Drs-GFP (Ferrandon et al. 1998) and Dpt-GFP (Tzou et al. 2000) flies were crossed to csn5null background to create Drs-GFP; csn5null/TM3, Ser act-GFP and csn5null Dpt-GFP/TM3, Ser act-GFP, respectively.
Immune response and statistical analysis
Third instar larvae, 72 h AED, were challenged by pricking with a needle dipped in E. coli and Micrococcus luteus cultures, and collected 30 min, 60 min, 4 h, and 24 h post-infection for analysis of Cactus degradation, Dorsal localization, RNA expression, and viability, respectively. For the survival experiments, two controls were used, Canton-S, and heterozygote Csn5+/csn5null siblings of the mutants. Relative risk value shows the ratio of the risk of death relative to exposure in the mutant versus the control group.
Immunohistochemistry
Larval fat bodies were dissected and immunostained as described (Cantera et al. 1999b). Primary antibodies: -Dorsal (Gillespie & Wasserman 1994) at 1 : 1500; -Cactus (Reach et al. 1996) at 1 : 1000; -Lamin (Harel et al. 1989) at 1 : 2, -CSN5 and -CSN7 at 1 : 1500 (Freilich et al. 1999). Secondary antibodies conjugated to Cy2 or Cy3 (Jackson Laboratories) were used at 1 : 1000.
Hemocytes immunostaining
Pre-wandering stage larvae were washed in water, dried and the integument was disrupted in the latero-posterior region into a cold drop of PBS on a microscope glass slide. The cells were allowed to settle and adhere for 10 min and fixed for 10 min by addition of an equal volume of cold 4% paraformaldehyde, followed by four washes in PBS. Two hours blocking and over-night primary antibodies incubations were performed at 4 °C in buffer containing PBS, Triton-X, and 5% normal goat serum. The following primary antibodies were used: -CSN7 and -CSN5 diluted 1 : 1500 (Freilich et al. 1999), monoclonal -crystal cell, HC12F6 (Johansson et al. 2005) diluted 1 : 1, monoclonal -plasmatocytes, P1 diluted 1 : 10, and monoclonal anti-lamellocytes, L1 (Kurucz et al. 2003) diluted 1 : 5. Secondary antibodies as above or FITC (Molecular Probe) at 1 : 250 in PBS for 2 h. The cells were then washed in PBS and mounted in 50% glycerol in PBS for microscopy.
Hemocytes counting
Circulating hemocytes were extracted from eight mutant larvae and eight wt larvae, of same chronological age, with wt at early wandering, in phosphate-buffered saline, fixed and total hemocyte numbers counted using a Zeiss Axioplan-2 microscope. Alternatively, circulating hemocytes were fixed as above, and random microscope frames of fixed cells from different larvae were counted.
Immunoblotting
A 100 µg total protein was analyzed on 7.5% SDS-PAGE, transferred to PVDF membrane (Immobilon-P) and incubated with -Cactus at 1 : 1000 dilution and -CSN7 at 1 : 10 000.
Fluorescence microscopy and laser confocal microscopy
Epi-fluorescence imaging was performed using a Zeiss Axioplan-2 imaging fluorescence microscope equipped with an AxioCam cooled charge-coupled device camera, or with an Olympus SZX 12 fluorescent stereoscope equipped with an eGFP filter, equipped with a DVC-1310 color digital camera (DVC, Austin, TX, USA). Cy2/Cy3 double staining was viewed with a Zeiss LSM510 confocal microscope, imaging in multitracking mode using 488 and 543 nm laser lines with 545 nm dichroic filter, a 505–530 nm band pass and 560 nm long-pass filters. Image analysis was performed with Zeiss AxioVision, Zeiss LSM-5 image browser, and Adobe Photoshop 6.0.
RNA analysis
Total RNA was isolated using TRI REAGENT (MRC, Cincinnati, OH, USA). For Northern analysis, 10 µg RNA were blotted on Hybond nylon filters (Amersham Pharmacia Biotech). Random-primed Drs and rp49 probes were amplified with specific primers. For RT-PCR, SuperScript II Reverse transcriptase (Invitrogen) was used for first strand cDNA synthesis using oligo(dT) on 5 µg RNA. A 1 : 200 dilution of cDNA was amplified for 25 (for Cact) or 20 (for rp49) cycles. These conditions were within the linear PCR phase. PCR primers surrounded introns.
Small interference RNA
The human CSN5 target sequence was cloned in the mammalian expression vector pSUPER. A scrambled CSN5 oligo was cloned in the same vector as a control.
Luciferase reporter assay
The 293T cells were transfected in triplicate samples with Ig-ConA-luciferase reporter gene CMV-ß-galactosidase, and either pSUPER-scrambled iCSN5 or pSUPER-iCSN5 constructs using FuGENE6 reagent (Roche Molecular Biochemicals). TNF stimulation (10 ng/mL) was performed on day 4. Luciferase activity was analyzed after 20 h in total cell lysates and normalized to ß-galactosidase activity.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: dannyc{at}tauex.tau.ac.il
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Received: 21 September 2006
Accepted: 1 November 2006
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