INTRODUCTION

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Members of the Rel/NF-B family of transcription factors play a central role in the regulation of inflammatory and immune responses (for recent reviews, please see (1, 3, 4, 25, 46, 54). In vertebrates, NF-B consists of homo- or heterodimers of Rel (c-Rel), p65 (RelA), RelB, p50 (NFKB1), and p52 (NFKB2), all of which contain a conserved N-terminal Rel homology domain that contains the DNA-binding and dimerization domains and the nuclear localization signal. In most unstimulated cells, a large portion of NF-B is retained in the cytoplasm as inactive complexes by a family of inhibitory proteins called IB that bind to the Rel homology domain and mask the nuclear localization signal. There are at least five distinct IB proteins, IB, IB, IB, IB, and bcl-3; both the p105 precursor of p50 and the p100 precursor of p52 possess domains that function as IBs as well. Upon cell stimulation by a wide variety of stimuli, signal-responsive IB kinases (IKK)  and  are activated and phosphorylate two serine residues in the IB proteins (14, 19, 36, 42, 43, 61, 63, 64). For IB and IB, the inducible phosphorylation sites are serines 32 and 36 and serines 19 and 23, respectively (9, 10, 18, 51). The phosphorylated IBs are subsequently ubiquitinated and targeted for degradation by the 26S proteosome, releasing the NF-B dimers to translocate to the nucleus to activate the transcription of genes containing the so-called B-binding site (2, 26, 48). Among the NF-B-inducible genes are IB members, and the newly synthesized IBs quickly interact with and inactivate NF-B, leading to an autoregulation of the NF-B system (11, 45, 50). Both IB and IB function not only in the cytoplasm but also in the nucleus to inhibit NF-B activity; however, only IB is required for postinduction repression of NF-B (5, 52).

RelB shares many common features of the NF-B family, and is a strong transcriptional activator (7, 8, 21, 44). Unlike other NF-B members, however, RelB cannot form homodimers and only associates efficiently with p50 and p52 (22). The RelB heterodimers have a much lower affinity for IB than other NF-B complexes do and are less susceptible to inhibition by IB (22, 32). As a result, it is predicted that RelB will be located in the nucleus and represent the constitutive NF-B activity. Indeed, RelB heterodimers represent the major constitutive NF-B activity in lymphoid tissues and are expressed at high levels in the nuclei of interdigitating dendritic cells, suggesting an important role for RelB in the constitutive expression of B-regulated genes in lymphoid tissues (13, 32, 33, 40, 56).

The implicated in vivo function of RelB in lymphoid tissues is supported by studies of relb^/ mice (12, 17, 34, 57-60, 62). Mice deficient in RelB have a dramatic reduction in constitutive B-binding activity and specific defects in lymphoid tissues, including the absence of mature lymphoid dendritic cells, myeloid hyperplasia, and splenomegaly (12, 57). relb^/ mice also have multifocal defects in immune responses and fail to mount inflammatory reactions against a number of pathogens (17, 60). Surprisingly, relb^/ mice spontaneously develop a persistent noninfectious multiorgan inflammatory syndrome (12, 34, 57). This apparent discrepancy suggests additional defects in nonlymphoid tissues in relb^/ mice. In this regard, we showed in our previous report that the multifocal inflammation is due to non-bone-marrow-derived cells, that lipopolysaccharide (LPS)-stimulated relb^/ fibroblasts overexpress chemokines and induce leukocyte recruitment into tissues, and that RelB, while being a transcriptional activator for B-regulated genes in macrophages, acts as a transcription suppressor in fibroblasts (62). Our findings may provide hints about the role that fibroblasts might play in the initial leukocyte infiltration and how RelB might be involved in the regulation of this process (49, 62). Additional pathological changes, however, must be involved in the development of multiorgan inflammation in relb^/ mice. Cytokines which promote inflammatory effector functions, including tumor necrosis factor alpha (TNF-), interleukin-1 (IL-1), IL-1, and gamma interferon, are expressed at increased levels in the nonlymphoid organs of relb^/ mice but have either normal or reduced expression in lymphoid tissues; in particular, isolated relb^/ macrophages are impaired in the production of TNF- (60, 62). Since macrophages are normally the major source of TNF- production, one wonders what the probable cellular source of TNF- and other cytokines in the nonlymphoid organs of the relb^/ mice might be, what molecular mechanisms are involved, and, especially, how RelB may function in this process. In this report, we show that RelB is a key transcriptional suppressor of cytokine expression in fibroblasts and that its absence leads to the dysregulation of IL-1, IL-1, and TNF- expression in relb^/ fibroblasts. We further demonstrate that RelB exerts its transcriptional suppressor function through the stabilization of IB protein. These data suggest new physiological roles for the NF-B/Rel factors in the regulation of inflammatory and immune responses and may provide insights to uncover novel intra- and interfamily interactions of the NF-B/Rel and IB regulatory molecules.

	MATERIALS AND METHODS

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Animals. relb^/ mice were generated and characterized as previously described (12). The mice were generated on an inbred C57BL/6J background and bred onto a B10.D2 background. The control mice were of B10.D2 origin. All experimental procedures were carried out according to the guidelines listed in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Cell culture and in vitro LPS stimulation. Fibroblasts were isolated from the kidneys of normal control and relb^/ mice as previously described (62) and were cultured in Dulbecco modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS). Stable transfection of fibroblasts with a RelB cDNA construct was performed as described previously (62). For LPS stimulation, cells were serum starved for 24 h and then treated with 1 µg of LPS (List Biological Laboratories, Campbell, Calif.) per ml in DMEM plus 0.5% FBS. The cells were harvested at different time points for RNA and protein extraction.

RNA extraction and RNase protection assay. Total RNA was isolated from fibroblasts by a single-step method (16). cDNA fragments of mouse IL-1 (bp 172 to 362; accession no. X01450), IL-1 (bp 500 to 672; accession no. M15131), TNF- (bp 429 to 556; accession no. M11731), RelB (bp 1350 to 1603; accession no. M83380), and IB (bp 372 to 632; accession no. U36277) were generated by reverse transcription-PCR or by PCR amplification of cDNA templates. The PCR products were cloned into pGEM4Z (Promega Corp., Madison, Wis.) for the generation of -³²P-incorporated riboprobes. RNase protection assays were performed as previously described (62).

Luciferase assay. The luciferase reporter plasmids containing the wild-type or mutant TNF promoter were constructed as described by others (23, 27) except that the chloramphenicol acetyltransferase reporter vector was replaced by luciferase reporter vector pGL2-Basic (Promega). The plasmids were transfected into fibroblasts by electroporation with the setting of 280 V, 600 µF and 48  (BTX Electro Cell Manipulator 600; Genetronics, San Diego, Calif.). At 48 h after the transfection, the cells were stimulated with LPS for 12 h and were harvested for the luciferase assay. This assay was performed with a luciferase assay kit (Promega), and the light production was measured with a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, Calif.).

Nuclear extract and EMSA. Nuclear extracts were prepared by a published procedure (20). Electrophoretic mobility shift assay (EMSA) was performed as described previously (62). Briefly, 2 µg of nuclear extract was incubated for 15 min at 25°C with a ³²P-labeled oligonucleotide containing the B site from the murine intronic  chain (5'-AGTTGAGGGGACTTTCCCAGG-3' [the NF-B consensus DNA binding motif is underlined]; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), and the reaction mixtures were electrophoresed in a 6% polyacrylamide sequencing gel. For supershift assays, the nuclear extract was preincubated with 1 µg of rabbit anti-RelA serum for 20 min at 25°C before the addition of oligonucleotides.

Western blot analysis and ELISA. The cytoplasm and nuclear proteins from LPS-stimulated fibroblasts (5 µg per sample) were electrophoresed in a NuPAGE gel (Novex, San Diego, Calif.) and electroblotted onto a nitrocellulose membrane. The protein blots were probed with rabbit antibodies against mouse RelA, RelB, IB, IB (Santa Cruz), actin (Sigma, St. Louis, Mo.), or NF-B-inducing kinase (NIK) (Torrey Pines Biolabs, San Marcos, Calif.). The bound antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G antibody (Pierce, Rockford, Ill.) and the SuperSignal Kit (Pierce). TNF- protein was measured with a mouse enzyme-linked immunosorbent assay (ELISA) kit (Biosource International, Inc., Camarillo, Calif.).

Metabolic labeling and immunoprecipitation. Prior to the metabolic labeling, 2 × 10⁶ fibroblasts were treated with 1 µg of LPS per ml for 1 h. The cells were transferred to 1 ml of methionine- and cysteine-free DMEM plus 5% dialyzed FCS for 1 h, and then 0.2 mCi of Tran[³⁵S] (ICN, Costa Mesa, Calif.) was added to the medium. After 30 min, the cells were washed with phosphate-buffered saline and incubated with the regular medium of DMEM plus 10% FBS. The cells were harvested at different time points and were lysed in 20 mM Tris-HCl (pH 7.4)-100 mM NaCl-1 mM EDTA-0.2% Nonidet P-40-0.1% Triton X-100. The cell lysates were incubated with 1 µg of normal rabbit immunoglobulin G (Sigma) at 4°C for 1 h and with 10 µl of protein A-Sepharose (Pharmacia, Piscataway, N.J.) for 30 min and then centrifuged for 10 min. The precleared lysates were reacted with 1 µl of rabbit anti-IB antibody (Santa Cruz) at 4°C for 1 h and then with 10 µl of protein A-Sepharose for 8 h. The immune complexes were centrifuged, washed twice in phosphate-buffered saline, electrophoresed in a Nu-PAGE gel (Novex), and visualized by autoradiography.

Production of adenovirus vectors and infection of fibroblasts. cDNA fragments containing the coding region of the murine IB or a dominant negative mutant IB (53) were subcloned into the shuttle plasmid pAdv/CMV (55). The resulting plasmids, pAdv/CMV-IBwt and pAdv/CMV-IBmut, were each cotransfected with a helper plasmid, pJM17, into 293T cells to generate recombinant adenoviruses by a previously described method (55). Recombinant adenoviruses confirmed by PCR analysis were plaque purified and amplified in 293T cells. Concentrated adenoviruses were prepared by CsCl gradient ultracentrifugation. The titer of adenovirus was determined from DNA content of the viral solution, with 1.0 optical density at 260 nm unit being equivalent to 1.0 × 10¹² viral particles/ml. The adenovirus construct Adv/-gal, containing the -galactosidase (-Gal) cDNA, was generated by a similar strategy. Infection of fibroblasts was done by adding adenoviruses to the culture medium to a titer of 5,000 viral particles/cell.

IB kinase assay. The activities of IB kinases of fibroblasts were determined by an immunokinase assay (19, 39). After 24 h of serum starvation, the fibroblasts were stimulated with LPS at 1 µg/ml of medium and collected at 0, 15, and 30 min and 1 and 4 h. The cells were disrupted in lysis buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM -glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 µM Na₃VO₄, 1 mM benzamidine, 1 mM dithiothreitol, proteinase inhibitors) by repeated aspiration through a 21-gauge needle. The supernatant was incubated with 1.0 µg of anti-mouse IKK polyclonal antibody (M280; Santa Cruz) at 4°C for 1 h and then with 20 µl of protein A-Sepharose (Pierce) for 8 h. The immunocomplexes were then collected, washed, and suspended in 30 µl of kinase buffer (20 mM HEPES [pH 7.6], 100 mM NaCl, 20 mM -glycerophosphate, 10 mM MgCl₂, 10 mM P-nitrophenyl phosphate, 100 µM Na₃VO₄, 10 µg of aprotinin per ml, 2 mM dithiothreitol, 20 µM ATP, 5 µCi of [-³²P]ATP [ICN]) with 5 µg of glutathione S-transferase-IB-(1-54) as a substrate (19, 39). The reaction was stopped by adding 10 µl of 4× sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer after a 30-min incubation at 30°C. The samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% polyacrylamide) (Novex) and exposed to a film.

	RESULTS

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IL-1, IL-1, and TNF- overexpression and TNF- promoter activation in LPS-stimulated relb^/ fibroblasts. Normal fibroblasts do not express IL-1, IL-1, and TNF-, even when treated with LPS (Fig. 1A) (31). However, when relb^/ fibroblasts were stimulated with LPS, the mRNA expression of all three cytokines was dramatically induced (Fig. 1A). The induction was readily detectable at 1 h after the LPS stimulation, peaked at 4 to 8 h, and persisted through 24 h. To examine the expression of cytokine proteins, the relb^/ fibroblasts culture medium was analyzed by ELISA. We chose to analyze the TNF- protein level because its synthesis is regulated at both the transcription and translation levels (6). The expression of TNF- protein in LPS-stimulated fibroblasts correlated well with its mRNA expression (Fig. 1B).

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Expression of proinflammatory cytokines in normal and relb/ fibroblasts. (A) IL-1, IL-1, and TNF- mRNA expression in normal (N) and mutant (M) fibroblasts. Total RNA from fibroblasts treated with LPS for the indicated times was analyzed by the RNase protection assay. Riboprobes contain polylinker sequences and are longer than the protected bands. The mouse L32 gene was used as a housekeeping gene. (B) TNF- protein expression in normal and mutant fibroblasts. Fibroblasts were treated with LPS, and the culture medium was collected at the indicated time points for ELISA analysis. (C) TNF- promoter activity in fibroblasts as determined by the luciferase assay. Normal and relb/ fibroblasts were transfected with TNF- promoter-luciferase plasmids, and the transfected cells were treated with LPS for 12 h and then assayed for luciferase activities (arbitrary units), as described in Materials and Methods. The results are from three independent experiments (means ± standard deviations). Columns 1 and 2, normal and relb/ fibroblasts transfected with TNF promoter-luciferase plasmid; column 3, relb/ fibroblasts transfected with a mutant TNF- promoter-luciferase plasmid. (D) RelB cDNA-transfection of relb/ fibroblasts. The expression vector pcDNA, containing a RelB cDNA, was used to transfect relb/ fibroblasts. Total RNA from positive clones was analyzed for RelB mRNA expression (a). relb/ fibroblasts transfected with pcDNA vector only (b) and normal fibroblasts (c) were used as controls. (E) Reversal of LPS-induced cytokine overexpression in relb/ fibroblasts by RelB cDNA transfection. Normal fibroblasts (a), relb/ fibroblasts (b), relb/ fibroblasts transfected with pcDNA plasmid (c), and relb/ fibroblasts transfected with pcDNA vector containing a mouse RelB cDNA fragment (d) were treated with LPS for the indicated times and then analyzed for cytokine expression by the RNase protection assay.

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Reverse of cytokine overexpression in relb^/ fibroblasts by RelB cDNA transfection. Since the relb^/ fibroblasts were isolated from the kidneys of relb^/ mice, the dysregulation of proinflammatory cytokine expression in these cells could be the result of developmental changes that were secondary to the RelB deficiency. To verify the casual effect of RelB on the suppression of cytokine expression in fibroblasts, RelB cDNA was transfected into relb^/ fibroblasts. The expression of the transfected RelB in relb^/ fibroblasts (Fig. 1D) completely abolished the overexpression of IL-1, IL-1, and TNF- mRNA induced by LPS (Fig. 1E). This result strongly suggests an indispensable role of RelB in the transcription suppression of proinflammatory cytokines in fibroblasts.

Augmented IB mRNA expression in relb^/ fibroblasts. The mutual regulations of NF-B and IB activities play a primary role in the control of NF-B-activated genes. Since NF-B molecules interact with IB molecules differently, this could lead to a hierarchy or mutual regulation among different NF-B members (22). In particular, RelB is relatively resistant to IB inhibition and can strongly induce the expression of IB. In our previous study, we showed that LPS treatment of relb^/ fibroblasts led to an exaggerated and persistent activation of NF-B activity that was mainly attributed to RelA/p50 (62). Since IB is the major inhibitor of RelA/p50 activity, RelB may exert its transcriptional suppressor function in fibroblasts through the regulation of IB activities. The mRNA expression of IB in normal and relb^/ fibroblasts was therefore analyzed. While IB mRNA was induced in both type of cells by LPS, the induction was significantly augmented in mutants (Fig. 2A), probably the result of enhanced RelA/p50 activity. We then examined the stability of IB mRNA but found no significant difference in the half-life of IB mRNAs in normal and mutant fibroblasts (Fig. 2B and C). These results indicate that IB mRNA expression is not down-regulated by RelB deficiency in relb^/ fibroblasts.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Analysis of IB mRNA expression in fibroblasts. (A) IB mRNA expression in normal (N) and relb/ (M) fibroblasts treated with LPS for different periods. (B) Analysis of IB mRNA stability in LPS-treated fibroblasts. Normal and mutant fibroblasts were treated with LPS for 1 h before actinomycin D was added to stop mRNA synthesis. Cells were harvested at 5, 10, 20, 30, and 60 min after the addition of actinomycin D and analyzed for IB mRNA levels by an RNase protection assay. (C) Graphic representation of the IB mRNA half-life. The IB and L32 bands in panel B were quantitated by phosphorimager scanning. The count of each IB band was factored by that of the corresponding L32 band. The final value of each time point is expressed as a percentage of that at time zero.

Rapid degradation of IB protein in relb^/ fibroblasts. Analysis of IB protein levels, however, revealed a dramatic difference between normal and relb^/ fibroblasts (Fig. 3A). The down-regulation of IB protein in relb^/ fibroblasts in the presence of higher mRNA levels could be due to either translation suppression or a decrease in protein stability. A metabolic labeling experiment was carried out with LPS-stimulated normal and mutant cells to determine the actual mechanism. The level of metabolically labeled IB protein in relbsup/ fibroblasts at the initial time point after a pulse-labeling was comparable to that in normal fibroblasts (Fig. 3B), suggesting that the IB mRNA in relb^/ fibroblasts can be translated and that translational suppression is not the primary cause of IB protein down-regulation in these cells. However, as early as 30 min after the pulse-labeling, most of the newly synthesized IB protein disappeared in the relb^/ fibroblasts (Fig. 3B), indicating a very rapid degradation. In contrast, the observed half-life of IB in normal fibroblasts was on the order of 2 to 3 h, significantly longer than that in the mutant cells. This indicated that the accelerated degradation of IB protein was responsible for the decreased IB protein in relb^/ fibroblasts.

View larger version (47K): [in this window] [in a new window]   FIG. 3.   Analysis of IB protein level in fibroblasts. (A) IB protein level in normal (N) and mutant (M) fibroblasts. Fibroblasts were treated with LPS for the indicated times. The cell lysates were analyzed for IB protein by Western blotting. A total of 5 µg of protein was used for each sample. The same blot was probed for actin to ensure even loading. (B) Pulse-chase experiment to determine the stability of IB in normal and relb/ fibroblasts. Cells were treated with LPS for 1 h before being pulse-labeled for 1 h with [35S]Met-[35S]Cys. The cells were then chased with cold medium for the indicated times. The cell lysates were immunoprecipitated with anti-IB antibody and analyzed as described in Materials and Methods. (C) Western blot analysis of different IB family members in normal (N) and relb/ (M) fibroblasts. Cells were treated with LPS for the times shown. The cell lysates were analyzed for IB, IB, and IB protein levels by Western blotting. The same blots were reprobed with anti-actin antibody to show comparable amounts of proteins in different lanes.

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Impaired postinduction repression of NF-B activity by IB in relb^/1 fibroblasts. One irreplaceable function of IB is the postinduction repression of NF-B activation, whereas its cytoplasmic retention of NF-B can be compensated for by other IB proteins (5, 52). Newly synthesized IB not only stops cytoplasmic NF-B from entering the nucleus but also enters the nucleus to dissociate NF-B binding to DNA and exports the bound NF-B to the cytoplasm. The accelerated degradation may preempt the IB in LPS-stimulated relb^/ fibroblasts from carrying out its function in the postinduction repression, and this may be the underlying cause of the prolonged activation of NF-B activity and persistent expression of cytokine mRNAs in LPS-stimulated relb^/ fibroblasts. Since LPS-induced NF-B activity in fibroblasts consists mainly of p50 and RelA, we assessed the pattern of RelA translocation in LPS-stimulated normal and relb^/ fibroblasts by Western blot analysis. In resting normal and mutant fibroblasts, RelA was located in the cytoplasm. LPS stimulation induced a rapid RelA translocation from cytoplasm to nucleus in both types of cells, peaking 30 min after LPS stimulation. At 1 h after LPS stimulation, nuclear RelA in normal fibroblasts was significantly reduced, and 3 h later it was much lower than that in the cytoplasm (Fig. 4A, top). In mutant cell, however, the nuclear localization of RelA in relb^/ fibroblasts was significantly prolonged. A large portion of RelA persisted in the nucleus even at 3 h after the LPS stimulation (Fig. 4A, bottom), indicating an impaired postinduction termination of RelA nuclear localization in the mutant cells.

View larger version (34K): [in this window] [in a new window]   FIG. 4.   LPS induction and postinduction repression of NF-B nuclear localization. Normal and relb/ fibroblasts were treated with LPS for the times shown. Cytoplasmic (cy) and nuclear (nu) extracts were prepared for Western blot analysis of RelA protein (A) or RelB protein (B).

Presence of IB in the nucleus of normal fibroblasts. A similar level of nuclear translocation of RelA during the first 30 min of LPS stimulation of normal and relb^/ fibroblasts suggests a difference in the RelA activity in these cells: the B-binding of RelA is much lower in the normal cells than in the mutants (62). In the presence of proteosome inhibitor, IB has been detected in the nucleus in unstimulated endothelial cells (41). To investigate whether IB localizes in the nuclei of unstimulated fibroblasts, we performed Western blot analysis of subcellular fractions of normal fibroblasts. As shown in Fig. 5, IB was clearly detectable in the nucleus, albeit at a lower level than in the cytoplasm. To rule out the possibility of contamination of the nuclear fraction by cytoplasm, the same blot was reprobed with antiserum to NIK, a cytoplasmic protein (35). The exclusive localization of NIK in the cytoplasm indicated that our preparation of the nuclear fraction was free of cytoplasmic input. The preexistence of IB in the nuclei of normal fibroblasts may account for the low B-binding activity of RelA in these cells.

View larger version (37K): [in this window] [in a new window]   FIG. 5.   Analysis subcellular distribution of IB protein in fibroblasts. Cytoplasmic (cy) and nuclear (nu) extracts were prepared from resting normal fibroblasts and analyzed by Western blotting. IB protein can be detected in both the cytoplasmic and nuclear extracts. The same blot was reprobed with anti-NIK antibody to ensure that the nuclear extract was free of cytosolic proteins.

Reverse of cytokine overexpression in relb^/ fibroblasts by a dominant negative mutant of IB. Our data presented above suggested the importance of IB stability in the regulation of NK-B activity in fibroblasts as well as a connection between IB destabilization in relb^/ fibroblasts and the overexpression of cytokines in these cells. To further demonstrate the relationship between the IB stability and the regulation of cytokine expression in fibroblasts, we used an adenovirus vector carrying a dominant negative mutant of IB (Adv/IM) (53). In this IB variant, serines 32 and 36 were substituted with alanines. IM is therefore resistant to phosphorylation-induced degradation and is a potent and specific inhibitor of NF-B. Adv/IM and a control adenovirus vector, Adv/-Gal, were used to infect normal and relb^/ fibroblasts, and the infected cells were studied for LPS stimulation. Infection of Adv/IM resulted in a high level of IB mRNA in both normal and mutant fibroblasts, indicating that the infection was successful (data not shown). Adv/IM infection of relb^/ fibroblasts recovered IB protein to a level comparable to that in the normal fibroblasts (Fig. 6A). The overexpression of cytokine mRNAs in LPS-stimulated relb^/ fibroblasts was dramatically reversed by Adv/IM infection, although a weak induction of IL-1 and IL-1 mRNAs was still detectable compared with that in the normal fibroblasts (Fig. 6B). This indicates that the transcription suppression of cytokines by RelB in fibroblasts can be partially compensated by the expression of a stable IB.

View larger version (45K): [in this window] [in a new window]   FIG. 6.   Effects of a dominant negative mutant IB (IM) on cytokine expression and NF-B activity in fibroblasts. (A) Analysis of IB and IB proteins in resting or LPS-treated normal fibroblasts, relb/ fibroblasts, and Adv/IM-infected relb/ fibroblasts. Cells were treated with LPS for 3 h or left untreated. Cytoplasmic (Cy) and nuclear (nu) extracts were prepared and analyzed by Western immunoblotting. (B) Suppression of LPS-induced cytokine overexpression in relb/ fibroblasts by Adv/IM infection. Normal and relb/ fibroblasts with no adenovirus infection (N), Adv/-Gal infection (G), or Adv/IM infection (IM) were treated with LPS for 4 h. IL-1, IL-1, and TNF- mRNA expression in these cells were analyzed by an RNase protection assay. (C) Effect of IM on RelA nuclear localization. Adv/IM- or Adv/-Gal-infected relb/ fibroblasts were treated with LPS for the indicated times. Cytoplasmic (cy) and nuclear (nu) extracts of these cells were prepared and analyzed for RelA protein by Western blotting. (D) Effect of IM on NF-B activity in fibroblasts. Normal fibroblasts (a) relb/ fibroblasts (b), relb/ fibroblasts infected with Adv/-Gal (c), or relb/ fibroblasts infected with Adv/IM (d) were treated with LPS for 2 h or left untreated. Nuclear extracts of these cells were analyzed by EMSA or supershift EMSA, as described in Materials and Methods.

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Increased IB phosphorylation by IB kinases. IKK phosphorylation of IB is the key step in LPS-induced IB degradation. To address the potential cause of accelerated degradation of IB protein in LPS-treated relb^/ fibroblasts, we compared the expression level and activity of IKK and IKK in normal and mutant cells. We did not detect a difference in IKK or IKK mRNA (data not shown) and protein levels in normal and relb^/ fibroblasts (Fig. 7). In resting cells, the basal IKK activity was low and comparable in normal and mutant cells (Fig. 7). However, after LPS stimulation, the IKK activity was significantly augmented and prolonged in relb^/ fibroblasts compared with normal fibroblasts, causing an increased and persistent IB phosphorylation (Fig. 7). This prolonged activation of IKK may explain the observed destabilization of IB, IB, and IB in relb^/ fibroblasts.

View larger version (31K): [in this window] [in a new window]   FIG. 7.   LPS-induced IKK activity in normal and relb/ (mutant) fibroblasts. Fibroblasts were stimulated with 1 µg of LPS per ml for the indicated times, and cell lysate was prepared. IB kinase complex was immunoprecipitated from 400 µg of lysate, and kinase activity was determined by using GST-IB (top). The same samples (10-µg portions) were used for Western blot analysis by anti-mouse IKK polyclonal antibody (bottom).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have shown that RelB plays an indispensable role in the suppression of cytokine expression in fibroblasts. IL-1, IL-1, and TNF- expression are dysregulated in LPS-stimulated relb^/ fibroblasts, and this dysregulation can be reversed by RelB cDNA transfection. This result is consistent with our previous observation of overexpression of chemokines in activated relb^/ fibroblasts. Together, our findings provide a plausible explanation for the multiorgan inflammation of relb^/ mice. We have also provided evidence that the overexpression of NF-B-activated genes in relb^/ fibroblasts is a result of IB destabilization in these cells, suggesting that RelB exerts its effect in normal fibroblasts by affecting the inhibitory function of IB on RelA or other NF-B molecules. These results ascribe a transcription suppression function to the NF-B/Rel family of transcription factors previously thought of exclusively as activators, and they suggest a new mode of intra- and interfamily interaction among the NF-B/Rel and its coevolved IB families of regulators.

Multiorgan inflammation in relb^/ mice as the result of RelB deficiency-associated defects in lymphoid and nonlymphoid tissues. By virtue of its low affinity with IB (22), RelB is unique among the NF-B/Rel family of transcription factors in that it is located in the nucleus and has constitutive NF-B activity in lymphoid tissues (13, 32, 33, 56). As a result, RelB is believed to be responsible for the constitutive expression of NF-B-regulated genes in the lymphoid organs and to play important housekeeping roles in the immune system. Indeed, when RelB genes are disrupted in transgenic mice, multifocal defects develop in the lymphoid organs of relb^/ mice, including the disappearance of thymic medulla and dendritic cells, dramatically enlarged spleen, and loss of lymph nodes and Peyer's patches (12, 34, 57). Inflammatory and immune functions are also affected in these mice (17, 58, 60). Interestingly, relb^/ mice develop overwhelming inflammation in multiple nonlymphoid organs (12, 34, 57). The multiorgan inflammation is T-cell dependent and may be caused by autoreactive T-cell clones in these mice (17, 58). However, adoptive transfer and bone marrow chimera studies reveal that the inflammation is not dependent on the defects in relb^/ T cells (17, 62). We then, after careful examination of the lymphoid system, looked into possible defects in nonlymphoid tissues and investigated whether the multiorgan inflammation might be rooted in nonimmune cells.

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RelB modulation of IB stability in fibroblasts. Our studies further suggest that the suppressive effect of RelB on cytokine expression in fibroblasts is mediated, at least in part, by modulating the stability of IB protein. When stimulated by LPS, normal and relb^/ fibroblasts have a similar pattern of RelA nuclear translocation (Fig. 4A). The B-binding activity of RelA in normal fibroblasts, however, is much lower than that in mutant cells (Fig. 6D) (62), perhaps due to preexisting IB in the nucleus (Fig. 5). IB is the primary inhibitor of NF-B activity. It has been shown previously that while not efficiently inhibited by IB, RelB can strongly induce the expression of IB and may inhibit RelA or c-Rel activities by driving the expression of IB (22, 24). In normal fibroblasts, a large portion of RelB is located in the nucleus (Fig. 4B). Intuitively, one would suspect the overexpression of proinflammatory mediators in relb^/ fibroblasts to be the result of insufficient IB expression in these cells. However, we have found no reduction in the IB mRNA level in the mutant cells; in fact, IB mRNA expression in LPS-stimulated relb^/ fibroblasts was increased compared with that in similarly treated normal fibroblasts.

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