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Receptor Control IL-12 p40 Synthesis and NF-
B Activation1,2
* Immunobiology Center and
Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, NY 10029;
Division of Gastroenterology, Hepatology, and Nutrition, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
Department of Urology, New York University School of Medicine, New York, NY 10016; and
¶ Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112
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Abstract |
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, IL-6, IL-1, IL-8, IL-12, and CD80. Macrophage LPS-induced synthesis of IL-12 is inhibited following Fc
R ligation; TNF- secretion is unchanged. We report that microtubule-associated serine/threonine kinase-205 kDa (MAST205) is required for LPS-induced IL-12 synthesis. RNA interference-mediated suppression of MAST205 results in the inhibition of LPS-stimulated IL-12 promoter activity and IL-12 secretion, from both J774 cells and bone marrow-derived macrophages. Similarly, dominant-negative MAST205 mutants inhibit LPS-stimulated IL-12 synthesis and NF-
B activation, but do not affect IL-1 or TNF- signaling. Finally, macrophage FcR ligation regulates MAST205 by inducing the rapid ubiquitination and proteasomal degradation of the protein. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophage FcR ligation inhibits the expression of IL-12 following LPS stimulation without affecting the synthesis of TNF- (5, 6). This property is shared by many other receptors, including histamine (7), complement (8), chemokine (9), and 
-adrenergic (10) receptors. In vivo, Ag presented in immune complexes results in polarization toward a Th2 response reflected in increased macrophage IL-10 and decreased IL-12 synthesis, and preferential production of murine IgG1 (11). Regulation of IL-12 transcription is complex, but the murine IL-12 p40 promoter appears to be controlled primarily by NF-B and C/EBP sites (12, 13).
Microtubule-associated serine/threonine kinase-205 kDa (MAST205)6 was initially isolated by screening a testes library with a microtubule-associated protein antiserum (14). MAST205 is highly expressed in developing spermatids, but is also found ubiquitously at much lower levels (15). The expression of MAST205 mRNA is controlled by the class II MHC transactivator in B cells, and is induced by IFN- in the A431 epithelial carcinoma cell line (16). The goal of this study was to examine the role of MAST205 in the FcR-mediated regulation of the immune response. A MAST205 ATP-binding domain mutant and another N-terminal deletion mutant act as dominant negatives (DNs) to block macrophage synthesis of IL-12 p40 following LPS stimulation. We show that LPS-stimulated NF-B activity is inhibited both by FcR ligation, and by DN-MAST205 mutants. Furthermore, macrophage FcR ligation triggers rapid proteasome-mediated degradation of MAST205. RNA interference (RNAi)-targeted inhibition of MAST205 synthesis results in inhibition of LPS-stimulated IL-12 p40 promoter activation and IL-12 release. These data indicate that MAST205 is part of the LPS signal transduction pathway leading to activation of NF-B, and suggest that MAST205 may provide a novel target for immunotherapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peritoneal macrophages were isolated by lavage from C57BL/6 mice 4 days after i.p. injection of 0.5 ml of thioglycolate medium. Cells were cultured in DMEM containing 10% FCS and antibiotics. P388D1 cells transfected with FcRIIA (17) were maintained in the same medium supplemented with 100 µg/ml G418. Bone marrow-derived macrophages were prepared from C57BL/6 mice driven to proliferate with GM-CSF (20 ng/ml) for 1 wk.
Synthesis of meso-P-2-O-(6-biotinoylaminohexyl)-1,2,3,4,5,6-inositol hexakisphosphate (biotin-IP6) and screening
Biotin-IP6 was synthesized from the primary amino derivative (18) by reaction with N-hydroxysuccinimidyl-biotin (Pierce, Rockford, IL). A 
-ZAP (Stratagene, La Jolla, CA) P388D1 cDNA expression library was screened with a labeled inositol hexakisphosphate (IP6) complex analogous to the biotinylated phosphatidylinositol phosphates previously used to screen expression libraries (19). Streptavidin (200 ng) was preincubated with three molar equivalents of biotin-IP6. To the resulting complex, 125I-labeled biotin-BSA (50 ng) was added, which resulted in large complexes that failed to enter a 3% polyacrylamide stacking gel under non-denaturing PAGE. The cDNA library, after isopropyl -D-thiogalactoside induction, was screened with the large multimeric IP6 complexes using standard immunoblotting protocols.
Macrophage stimulation and receptor ligation
FcRIIA-expressing P388D1 cells were primed with 10 ng/ml IFN- (Genzyme, Cambridge, MA) for 2 h before addition of LPS (Sigma-Aldrich, St. Louis, MO). Proteins (anti-FcRIIA mAb IV.3 or trinitrophenylated BSA (TNP-BSA)) were covalently coupled to glass as previously described (20). To trigger FcRIIA, cells were seeded on mAb IV.3-coated glass. Peritoneal macrophages seeded on TNP-BSA glass were triggered by addition of 5 µg/ml IgG2b anti-DNP mAb U12.5. Peptidoglycan was from Fluka (Buchs, Switzerland), and human IL-1 and human TNF- were from PeproTech (Rocky Hill, NJ).
Cytokine ELISAs and RT-PCR
Murine cytokines IL-12, IL-10, and TNF- were measured by ELISA (R&D Systems, Minneapolis, MN). Nitrite was assayed by the Griess reaction, as described (21). For RT-PCR assays, total RNA was extracted using TRIzol (Life Technologies, Grand Island, NY). cDNA was prepared from 1–3 µg of RNA with Superscript II RT (Life Technologies) and random hexamer primers (Promega, Madison, WI). The following primers were used for PCR amplification: IL-12 p40, 5'-TCGCAGCAAAGCAAGATGTG-3' and 5'-GAGCAGCAGATGTGAGTGGC-3'; IL-10, 5'-CCAGTTTTACCTGGTAGAAGTGATG-3' and 5'-TGTCTAGGTCCTGGAGTCCAGCAGACTCAA-3'; and TNF-, 5'-GTTCTATGGCCCAGACCCTCACA-3' and 5'-TACCAGGGTTTGAGCTCAGC-3'. PCR was conducted by a standard protocol for appropriate cycles. An equal aliquot of cDNA was amplified with -actin primers.
Transfections and retroviral transductions
SuperFect (Qiagen, Valencia, CA) was used to transfect RAW 264.7 cells with promoter constructs and a constitutively active CMV promoter--galactosidase plasmid to monitor transfection efficiency. 293T cells were transfected with Lipofectamine (Life Technologies). Cytokine promoter analyses were conducted with luciferase reporter plasmids coupled to the following: the murine IL-12 p40 promoter, residues -355 to +55 with respect to the transcription start site (13); the murine IL-12 p40 promoter with the NF-B site deleted (-101D) (22); the IL-10 promoter, residues -1538 to +64 (23); the TNF- promoter, residues -283 to +113 (24); and a multimeric NF-B (25). The constitutively active Toll-like receptor (TLR)4 expression plasmid was from Dr. R. Medzhitov (Yale University School of Medicine, New Haven, CT). The IB kinase (IKK) expression plasmid was from Dr. A. Ting (Mount Sinai Medical School).
For retroviral transductions, we used a derivative of the Moloney murine leukemia virus vector pMMP412 (26) (pRetro), courtesy of Dr. A. Ting (Mount Sinai School of Medicine). Pseudotyped virus was produced by triple transfection of human 293 EbnaT cells with plasmids encoding vesicular stomatitis virus (VSV) G protein, gag-pol, and the retroviral construct. The viral supernatants were used to transduce macrophage cell lines as described (27). The hemagglutinin (HA)-ubiquitin expression construct (28) was from Dr. Z. Ronai (Mount Sinai School of Medicine). The cDNA encoding HA-ubiquitin was excised from this plasmid with NotI and EcoR1, blunted with T4 polymerase, and ligated into the blunted cloning site of pRetro (27). HA was detected on immunoblots with mAb 12CA5 (Roche, Basel, Switzerland). The macrophage cell line transduction efficiency, based on expression of green fluorescent protein (GFP) from an internal internal ribosome entry site, was 60–80%.
MAST205 plasmid constructs and RNAi vector
pUCMAST205 full-length cDNA, pcDNAI-MAST205 full-length cDNA, and all pcDNAI-MAST205 in-frame deletion constructs were gifts from Dr. P. Walden (New York University). To construct the C-terminal myc-tagged MAST205 pcDNAIII vector, the following PCR primers were used: 5' primer, 5'-gcatatGGCGCGCCTTGCCATGGTTACTGGACTTAGTCC-3', which has an AscI site (underlined) and the start ATG (bold); and 3' primer, 5'-gcatatGCCCGGGCTGCTGGTTTGCTTTAAGAGCTCATC-3', which mutates the termination codon and creates an SrfI site (underlined). These primers and Pfu DNA polymerase (Stratagene) were used to generate a full-length mouse MAST205 PCR fragment from pUCMAST205. The PCR fragment was inserted into the AscI/SrfI sites of a modified pSR expression vector, which has a triple myc-epitope-tag inserted at the 3' end of the multiple cloning site. The full-length cDNA of MAST205 with the C-terminal myc tag was excised from pSR-MAST205myc with XhoI and NotI. The resulting 5.4-kb fragment was cloned into the corresponding XhoI/NotI site in pcDNAIII (Invitrogen, San Diego, CA) to generate pcDNAIII-myc-MAST205. The MAST205 ATP binding domain K482R/K483A mutant was generated by excising an internal BamHI/SacI fragment from pUCMAST205, which was subcloned into pBluescript II SK+, followed by two-step PCR using overlapping internal primers containing the mutation, which was confirmed by DNA sequencing. Finally, the mutated fragment was inserted into pcDNAIII-myc-MAST205 using the same internal restriction sites to generate pcDNAIII-myc-K482R/K483A-MAST205. MAST205 constructs with C-terminal myc tags were excised from pcDNAIII vectors with AscI and NotI, and ligated into pRetro with an adapter oligo encoding a consensus Kozak sequence, and an N-terminal FLAG epitope, resulting in doubly tagged proteins.
Pseudotyped retroviruses encoding MAST205 small interfering RNA (siRNA) were generated as described above. The pSuper.retro vector (Oligoengine, Seattle, WA) was used according to manufacturer’s protocols. siRNA sequences were designed according to software from Oligoengine. Nineteen-nucleotide inverted repeats were separated by a 9-nt linker. The inverted repeats corresponded to nt 125–143 (A) and 1530–1548 (B) of the murine MAST205 coding sequence.
MAST205 antiserum
Rabbit anti-MAST205 serum was generated by immunization with a 45-kDa GST-fusion protein containing aa 241–340 of MAST205. The protein was purified from insoluble inclusion bodies in BL21cells by SDS-PAGE and electroelution from the gel slices.
EMSA
RAW 264.7 cell nuclear extracts were prepared as described (29). EMSA probes were made by annealing equal amounts of complementary single-stranded oligonucleotides with 5'-GATC overhangs (Genosys Biotechnologies, The Woodlands, TX), and labeled with 32Pi as described (13). The IL-12 NF-B probe was -146/-107 of the IL-12 p40 promoter. EMSAs were performed as described (22), using 105 cpm of labeled probe and 5 µg of nuclear extract protein per reaction.
Immunofluorescence microscopy
Cells were fixed with 4% paraformaldehyde in PBS for 20 min, washed, permeabilized with 0.1% Triton X-100 for 30 min, incubated 2 h with a 1/100 dilution of rabbit anti-MAST205, and finally stained with a 1/100 dilution of FITC-F(ab')2 goat anti-rabbit Ig (Jackson ImmunoResearch, West Grove, PA) in PBS for 1 h. Fluorescence digital images were acquired using an OMA Vision charge-coupled device camera (EG&G PARC, Princeton, NJ) and an Axiovert inverted microscope (Zeiss, Thornwood, NY) fitted with a Plan-Apochromat x63, 1.4 numerical aperture, or a Plan-Neofluar x40, 1.3 numerical aperture objective, and an FITC filter set (Omega Optical, Brattleboro, VT).
Immunoprecipitation and immunoblotting
Cells were lysed in buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Nonidet P-40, 0.1 M NaF, 10 mM PPi, and 0.2 mM Na3VO4) supplemented with protease inhibitors (10 µg/ml chymostatin, leupeptin, and aprotonin, 5 µg/ml pepstatin, and 1 mM PMSF) for 15 min on ice, lysates were cleared by centrifugation at 20,000 x g for 15 min, and aliquots normalized for protein (15 µg/lane) were subjected to SDS-PAGE. Proteins were electroeluted to nitrocellulose, and incubated with either anti-myc mAb 9E10 (5 µg/ml) or rabbit anti-MAST205 overnight, and stained with appropriate HRP-conjugated antiserum. For immunoprecipitation, lysates were incubated with 9E10 (4 µg/ml) overnight, and immunoprecipitated with protein G-Sepharose. Immunoprecipitates were washed extensively in lysis buffer and subjected to SDS-PAGE. Proteins were electoeluted as before, and visualized with anti-HA mAb 12CA5 and HRP-anti-mouse IgG.
RNase protection assay
RNase protection was performed using the mCK-2b RiboQuant Multiprobe RNase protection assay system (BD PharMingen, San Diego, CA), according to the manufacturer’s protocols. RAW264.7 cells were cotransfected with BX-MAST205 and a GFP construct or GFP alone, and after 24 h, GFP-positive cells were isolated by FACS sorting. These cells were primed with 10 ng/ml IFN- for 2 h and activated with 5 µg/ml LPS for 4 h. Total RNA was isolated with TRIzol (Life Technologies).
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Results |
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R ligation regulates macrophage IL-12 p40 synthesis induced by LPS
Immune complexes binding to FcR polarize the immune response toward a Th2 phenotype (11), in part by inhibiting the synthesis of IL-12. To study the effect of FcR ligation on cytokine protein secretion after LPS activation, we seeded a P388D1 mouse macrophage cell line expressing human FcRIIA (17) on anti-FcRIIA mAb IV.3-coated glass, which results in the activation of tyrosine kinases (20) and dramatic cell spreading. In agreement with previous results (5), ligation of FcRIIA 2 h before addition of IFN- and LPS markedly inhibited the induction of IL-12 p40 protein (Fig. 1). However, although the amount of secreted TNF- was not significantly inhibited, the amount of IL-10 was increased, which was also reported by Sutterwala et al. (30).
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Intrigued by the report of increased concentration of IP6 following FcR ligation (31), we screened a P388D1 cDNA -ZAP expression library with an 125I-labeled multimeric IP6 complex, and identified a strongly positive clone, containing a 2-kB cDNA fragment consisting of the C-terminal 40% (residues 1015–1731) of MAST205, a murine microtubule-associated serine/threonine kinase (14). MAST205 cDNA encodes an open reading frame of 1734 aa (117 ATG-5318 TAG) with a 274-aa catalytic domain (residues 453–726) related to protein kinase A and protein kinase C. The microtubule-binding region of MAST205 encompasses both the kinase domain and a postsynaptic density protein-95, discs large protein, zonula occludens (PDZ) domain (aa 1050–1130) (14). The clone we isolated included the PDZ domain, but not the kinase domain. MAST205 transcripts were initially identified only in total RNA from testis, where the protein is associated with the spermatid manchette microtubular array (14). However, using a MAST205-specific probe on a poly(A)+ Northern blot, a 5.5-kb mRNA transcript was later detected in all tissues, with highest levels in heart and testis (15). In agreement with this result, we found widespread expression of a 
200-kDa (Mr) protein, using a rabbit antiserum directed against a GST fusion protein containing residues 241–340 of MAST205 (our unpublished data). MAST205 in B cells is under the control of the class II MHC transactivator, and is induced by IFN- in A431 cells (16), suggesting that MAST205 may be involved in intracellular signaling pathways in the immune system.
MAST205 DN mutants inhibit LPS-stimulated IL-12 p40 synthesis
We analyzed the role of MAST205 in LPS signaling pathways by cotransfecting MAST205 mutants (Fig. 2A) and either IL-12 p40 or IL-10 promoter-luciferase reporter constructs into the RAW264.7 macrophage cell line (13, 32). Cotransfection of the N-terminal deletion BX-MAST205 mutant or the ATP-binding domain K482R/K483A mutant resulted in inhibition of IL-12 p40 promoter activity, without inhibition of IL-10 promoter activity (Fig. 2B). To quantify mRNA levels in the transfected cells, we had to enrich the transfected population, because only a small percentage of macrophages was transfected. We cotransfected RAW264.7 cells with a 10:1 ratio of plasmid encoding the BX-MAST205 mutant:plasmid encoding GFP, sorted GFP-positive cells by FACS, and extracted RNA for RT-PCR and RNase protection assays. LPS stimulation of macrophages expressing the BX-MAST205 mutant resulted in inhibition of IL-12 p40 mRNA relative to control macrophages, with no change in the mRNAs for other cytokines (Fig. 2, C and D), results strikingly similar to those obtained after ligation of FcR (Fig. 1).
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B activity is inhibited by DN-MAST205 mutants and FcRIIA ligation
Because NF-B is important for IL-12 promoter activation (12), comparable cotransfection experiments were performed using a multimerized NF-B luciferase reporter. The BX-MAST205 mutant strongly inhibited the LPS-induced luciferase signal from the multimerized NF-B reporter (Fig. 3A), as shown previously for the IL-12 p40 promoter (Fig. 2B). To confirm further the importance of MAST205 in NF-B activation, we examined the effect of the BX-MAST205 mutant on LPS-induced activity of an IL-12 p40 promoter truncation lacking the NF-B site. Cotransfection of the BX-MAST205 mutant with an IL-12 p40 reporter from which the NF-B site was deleted resulted in a 20% inhibition of LPS-stimulated luciferase activity (Fig. 3B), compared with 85% for wild-type (wt) IL-12 p40 promoter (A).
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B activity induced by a constitutively active TLR4 chimera and IKK. We chose IKK for transfection, because it is required for NF-B activation through the LPS/TLR4 pathway (33). BX-MAST205 inhibited NF-B activation by a constitutively active TLR4 chimera (Fig. 3C). However, activation of NF-B by overexpression of IKK was not inhibited by cotransfection of BX-MAST205 (Fig. 3D), suggesting that MAST205 is acting on elements upstream of the IKK complex.
We have examined the effect of BX-MAST205 on several pathways that result in NF-B activation. The activation of NF-B by heat-killed Listeria monocytogenes and peptidoglycan, which activate TLR2 (34, 35), was inhibited 80% by transfection of BX-MAST205 into RAW264.7 cells (Table I). Other inflammatory cytokines such as IL-1 and TNF- also induce NF-B activation. Because the macrophage cell lines respond only weakly to IL-1 and TNF-, we used 293T cells, which respond well to these cytokines, to analyze the effect of BX-MAST205 on NF-B activity. BX-MAST205 had no effect on either IL-1- or TNF--stimulated NF-B activation (Table I). However, in both RAW264.7 cells and 293T cells transfected with a constitutively active TLR4 chimera, BX-MAST205 cotransduction inhibited NF-B activation comparably (40–60%) (Table I, Fig. 3C). Thus, MAST205 may act on elements specific to the TLR2/TLR4 pathways.
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B activity, we performed EMSAs to analyze binding of cell extracts to the IL-12 p40 NF-B site and a consensus NF-B site. To perform this experiment, we constructed retroviral vectors, and used VSV pseudotyped virus to transduce macrophage cell lines. The efficiency of transduction of the macrophage cell lines with control GFP-virus was 80%, as determined by FACS (our unpublished data). The assignment of the hetero- and homodimer complexes was made on the basis of supershift data with anti-p50, -p65, and -c-Rel sera (our unpublished data and Ref. 13). Transient transduction of macrophages with wt MAST205 had little effect on the induction of binding activity for the IL-12 p40 NF-B site, but the MAST205 ATP-binding domain K482R/K483A mutant resulted in a significant decrease in binding of the p50/c-Rel complex from LPS-stimulated cells (Fig. 3E). Similar results were obtained with an NF-B consensus probe.
Because we (Fig. 1) and others (5, 6) have shown that FcR ligation blocks IL-12 synthesis stimulated by LPS, we next analyzed the effect of FcR ligation on NF-B activation. The P388D1 mouse macrophage cell line expressing human FcRIIA (17) was transfected with the NF-B reporter construct and FcRIIA ligated by seeding on mAb IV.3-coupled glass (20). Two hours after FcR ligation, the cells were stimulated with LPS, and 10 h later, cell lysates were assayed for luciferase activity. Results were normalized by activity from a cotransfected CMV promoter-driven -galactosidase reporter. FcRIIA ligation, although not altering background NF-B promoter activity, inhibited subsequent LPS activation of NF-B reporter activity by 70% (Table I). If FcR ligation alters LPS-induced NF-B promoter activity, this should also be reflected in altered abundance of the p50/p65/c-Rel complexes. We therefore performed EMSA analysis as before using an IL-12 p40-specific NF-B oligonucleotide probe. Two hours after FcR ligation, cells were incubated with or without 100 ng/ml LPS, and nuclear extracts were prepared 1 h later. FcR ligation causes a clear reduction in the amount of p50/c-Rel stimulated by LPS (Fig. 3F).
Degradation of MAST205 protein after FcR ligation
The hypothesis that MAST205 plays a central role in NF-B and IL-12 p40 regulation upon FcR ligation requires that the activity of MAST205 be subject to regulation under these conditions. MAST205 mRNA level in P388D1 cells was unaffected by ligation of FcRIIA, as shown both by Northern blot hybridization 45 min after ligation, and by RT-PCR 6 h after ligation (our unpublished data). However, ligation of FcRIIA led, within 1 h, to a precipitous decrease in MAST205 protein, detected by immunofluorescence (Fig. 4A) and immunoblotting (B) with a polyvalent rabbit antiserum directed against the N-terminal domain of MAST205. The same effect was also evident in thioglycolate-elicited peritoneal macrophages plated on TNP-BSA-coated glass. Within 3 h after addition of IgG2b anti-DNP mAb 12.5, MAST205 was nearly undetectable (Fig. 4A). The disappearance of MAST205 is due to proteasome-mediated proteolysis. Addition of 25 µM MG132, an aldehyde inhibitor of proteasomes (36), to P388D1 cells blocked the degradation of MAST205 completely (Fig. 4A). Similar results were obtained with lactacystin (36) (our unpublished data). Incubation of macrophages with LPS alone had no effect on the level of expression of MAST205 (our unpublished data). The degradation of MAST205 following FcR ligation provides an effective way to regulate the activity of this enzyme.
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RIIA were transduced with retrovirus encoding myc-tagged MAST205. After SDS-PAGE, immunoblotting for myc revealed the transduced protein at a Mr of 200 kDa (Fig. 5A). A 2-h incubation with MG132 resulted in increased expression of the transduced wt MAST205, suggesting that the enzyme turns over rapidly even in the absence of FcR ligation. Furthermore, there was a shift of MAST205, after incubation of cells with MG132, to a slightly higher Mr, which is consistent with ubiquitin conjugation to the protein. FcRIIA ligation, as shown before, resulted in degradation of the transduced protein, and the degradation was blocked completely by MG132, as shown previously (Fig. 4A).
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R ligation did not alter the expression of BX-MAST205 protein, whereas under the same conditions, the expression of wt MAST205 was significantly inhibited. If the N-terminal domain, which is strikingly conserved in Drosophila and Caenorhabditis elegans MAST205 orthologues, is necessary for function, the dramatically higher level of expression of BX-MAST205 relative to the ATP binding domain mutation may explain why BX-MAST205 is a more efficient DN.
To analyze ubiquitination, the same P388D1 cells were cotransduced with myc-tagged MAST205 and HA-ubiquitin. After 2 days, cells were plated on mAb IV.3-coated glass for 30 min to ligate FcRIIA, and detergent lysates were prepared. The lysates were immunoprecipitated to isolate myc-tagged proteins, and after resolution on SDS-PAGE, were immunoblotted for HA reactivity. The results (Fig. 5B) show clearly that there is a background level of ubiquitination that is not greatly increased by preincubation with MG132 for 2 h. However, within 30 min of FcRIIA ligation, essentially all HA reactivity is gone. In this case, addition of MG132 resulted in the accumulation of ubiquitinated MAST205 in the high-molecular-mass portion of the gel relative to controls, showing an induction of ubiquitination stimulated by FcR ligation.
RNAi of MAST205 synthesis
FcR ligation results not only in prompt degradation of MAST205 and down-regulation of LPS-stimulated IL-12 synthesis, but also in the activation of protein tyrosine kinase pathways. To examine the phenotype resulting from lack of MAST205, without the complications of FcR-associated signaling events, we constructed retroviral RNAi vectors targeting MAST205. For this experiment, we used a mixture of viruses encoding two siRNA sequences. Transduction of J774 cells with VSV G protein pseudotyped pSuper.retroMAST205 A and B virus resulted in clear inhibition of MAST205 expression (Fig. 6A). Transfection of J774 cells with the retroviral vectors and an IL-12 p40 promoter luciferase construct resulted in 55% inhibition of promoter activity (Table II). Finally, J774 cells transduced with control or pSuper.retro MAST205 A and B pseudotyped viruses were stimulated with LPS, and analyzed for secretion of IL-12. In agreement with the inhibition of the IL-12 promoter shown in Table II, there was a dramatic inhibition of the LPS-stimulated synthesis and release of IL-12 (Fig. 6B). The production of NO, and secretion of TNF-, analyzed from the same cells, were largely unaffected by the RNAi targeting of MAST205. The transduction of the cells by MAST205 retroviral vectors did not impair viability, nor the capability of the cells to respond to LPS, but resulted in the specific blockade of the synthesis of IL-12.
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R ligation of both macrophage cell lines and peritoneal macrophages results in degradation of MAST205. LPS-stimulated IL-12 p40 mRNA synthesis from J774 cells is inhibited by BX-MAST205, and LPS-stimulated IL-12 p40 protein synthesis in the J774 macrophage cell line is inhibited RNAi targeting MAST205. However, macrophage cell lines may not reflect the behavior of primary cells, which secrete far higher levels of IL-12 following LPS stimulation. Therefore, we repeated the RNAi targeting experiment using bone marrow-derived macrophages driven to proliferate by GM-CSF. The retroviral transduction efficiency in this setting was 50%, as judged by the GFP transduction control, instead of >80% for J774 cells. Under these conditions, we observed a 35% inhibition of LPS-stimulated IL-12 p40 secretion from the MAST205 RNAi-transduced cells, which is significant, because presumably half of the cells are responding normally (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation in response to LPS without altering TNF- synthesis. Furthermore, RNAi suppression of MAST205 leads to both inhibition of LPS-stimulated IL-12 promoter activity and IL-12 p40 secretion, without significantly altering NO or TNF- secretion. These effects are also observed after macrophage FcR ligation (5, 6, 37), which drives the immune response toward a Th2-dominated phenotype characterized by the secretion of murine IgG1 (11). We do not find that overexpression of MAST205 alters LPS-induced NF-B or IL-12 promoter activation, possibly due to the high level of endogenous MAST205 expression.
The dichotomy between effects of inhibition of MAST205 or FcR ligation on IL-12 and TNF- is noteworthy. Mandatory participation of NF-B in regulation of the TNF- promoter is quite controversial. Liu et al. (38) report that TNF- secretion induced by LPS in macrophages is regulated independently by c-Jun or C/EBP and NF-B (binding to the B3 site), without synergy between the sites. There may also be differences between cell lineages in TNF- regulation, as shown by Preischl et al. (39), who find that the same B3 element in the TNF- promoter is controlled by NF-B in a dendritic cell line, but by NF-AT and AP-1 factors in a mast cell line. Finally, the phenotype of a RelA (p65) knockout, which does not express NF-B is an embryonic lethal, due to massive apoptosis of the liver. This is due to unopposed action of TNF-, because in a TNF-/RelA double deletion, the liver develops normally and the mouse is viable (40). The inhibition of LPS-induced IL-12 synthesis by immune complexes supports the hypothesis of cross talk between the FcR and TLR2/4 signaling pathways. The control of MAST205 by FcR-activated proteolysis provides a bridge between the TLR2/4 inflammatory pathways and the signal pathway induced by FcR cross-linking.
Many inflammatory stimuli, acting through different pathways, induce the activation of NF-B. DN-MAST205 mutants inhibit the TLR4 and TLR2 pathways, but have no effect on IL-1 or TNF- activation of NF-B. TLR4 and IL-1R are closely related molecules, and share many signaling components proximal to NF-B activation, including myeloid differentiation factor 88, IL-1R-associated kinase, and TNFR-associated factor 6 (41). DN-MAST205 mutants do not alter NF-B activation induced by overexpression of IKK, which is an integral member of the IKK complex leading to degradation of IB and translocation of NF-B to the nucleus. This result indicates that MAST205 is positioned between TLR4 and IKK. Presumably, signaling molecules that are shared between TLR4 and IL-1R pathways are not candidates for molecules interacting with MAST205. The identification of MAST205-interacting proteins in the TLR4 signaling pathway is of great interest.
The enzymatic activity of MAST205, a serine/threonine kinase, is likely to be regulated in macrophages, as was previously shown for the kinase activity of MAST205 during spermatid maturation (42). The kinase activity of MAST205 is crucial for its biological role; the ATP-binding domain mutant of MAST205 acts as a DN. Although in vitro kinase assays (our unpublished results) show that IP6 inhibits MAST205 autophosphorylation, we do not know enough about the cellular concentrations or cellular compartmentalization of IP6, or other inositol polyphosphates, to make any conclusions about the role of these metabolites in regulating kinase activity.
Targeted protein degradation plays a central role in regulating inflammatory processes, as exemplified by the IB degradation paradigm. MAST205 is rapidly and completely degraded following FcR ligation, and the proteasome inhibitors MG132 and lactacystin blocked degradation and increased the amount of ubiquitination. This is true for ligation of transfected human FcRIIA with mAb IV.3 and for the endogenous murine receptors on peritoneal macrophages ligated with mAb anti-DNP-DNP-BSA complexes (Fig. 4). The lack of effect of MG132 on BX-MAST205 expression and degradation implicates the N-terminal domain as important for E3 ligase recognition. However, we do not know the identity of the E2 and E3 enzymes that mediate the ubiquitination of MAST205. The role of MAST205 as a bridge between the TLR4 and FcR pathways implies that it interacts with components of these signaling pathways. MAST205 has a PDZ domain, a protein/protein interaction module. Preliminary results, from pull-down experiments with GST-chimeric proteins, show that the PDZ domain associates with a complex set of proteins, and that the N-terminal domain also interacts with specific cellular components. In neuromuscular junctions, MAST205 and -2 syntrophin associate via a PDZ-PDZ domain interaction, suggesting that MAST205 links the dystrophin/utrophin network with microtubule filaments via syntrophin (15). MAST205 from spermatids is found, by gel filtration, to be associated with a large protein complex (42). We are pursuing the identity of the proteins that interact with MAST205.
IL-12 plays an essential role in the protective immunity against intracellular pathogens by directing the development of Th1 vs Th2 responses. IL-12 synthesis is induced by intracellular pathogens and bacterial products, including LPS. The synthesis of this cytokine is also regulated negatively by many effectors, including histamine (7), chemoattractants such as C5a and monocyte chemoattractant protein (MCP) (9), and -adrenergic agonists (10). MCP-1 has no significant effect on MAST205 expression in the J774 cell line (our preliminary results), suggesting that the inhibition of IL-12 by MCP, which proceeds through a pertussis toxin-inhibited pathway, is different from the FcR-mediated pathway. The inhibition of IL-12 synthesis after FcR ligation has been attributed to a Ca2+ flux (5), and to inhibition of a PU.1 complex binding to the Ets site of the human IL-12 p40 promoter (6). It is possible that these diverse inhibitory agents may also interface with MAST205.
Unregulated expression of IL-12 has been shown to be involved in the pathophysiology of several diseases, including inflammatory bowel disease (43), insulin-dependent diabetes mellitus (44), and rheumatoid arthritis (45). MAST205 may offer an attractive target for small molecule inhibitors in these settings. In contrast, immune complexes that occur in intracellular bacterial infections, such as tuberculosis and leprosy, may block an effective Th1 response. A clearer understanding of the signaling networks responsible for IL-12 regulation as well as the effectors that modulate MAST205 activity and degradation may provide novel therapeutic approaches for infectious and autoimmune diseases.
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Footnotes |
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2 We dedicate this work to the memory of Massimo Sassaroli, a stimulating colleague and good friend, who succumbed to cancer on July 6, 2003. We will always appreciate his humor, integrity, and scientific acumen.
3 Current address: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, NY 10021.
4 H.Z. and H.X. contributed equally to this work.
5 Address correspondence and reprint requests to Dr. Jay Unkeless, Immunobiology Center, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029. E-mail address: jay.unkeless{at}mssm.edu
6 Abbreviations used in this paper: MAST205, microtubule-associated serine/threonine kinase-205 kDa; DN, dominant negative; RNAi, RNA interference; biotin-IP6, meso-P-2-O-(6-biotinoylaminohexyl)-1,2,3,4,5,6-inositol hexakisphosphate; IP6, inositol hexakisphosphate; TNP-BSA, trinitrophenylated BSA; TLR, Toll-like receptor; IKK, IB kinase; VSV, vesicular stomatitis virus; HA, hemagglutinin; GFP, green fluorescent protein; siRNA, small interfering RNA; PDZ, postsynaptic density protein-95, discs large protein, zonula occludens; wt, wild type; MCP, monocyte chemoattractant protein.
Received for publication July 17, 2003. Accepted for publication December 5, 2003.
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