Targeted Disruption of the ζPKC Gene Results in the Impairment of the NF-κB Pathway

Molecular Cell, Vol. 8, 771–780, October, 2001, Copyright 2001 by Cell Press

Targeted Disruption of the ␨PKC Gene Results in the Impairment of the NF-␬B Pathway Michael Leitges,1 Laura Sanz,3 Pilar Martin,3 Angeles Duran,3 Uschi Braun,1 Juan F. Garcı´a,2 Fanny Camacho,2 Marı´a T. Diaz-Meco,3 Paul D. Rennert,4 and Jorge Moscat3,5 1 Max-Planck-Institut fu¨r experimentelle Endokrinologie Feodor-Lynen-Str. 7 30625 Hannover Germany 2 Centro Nacional de Investigaciones Oncolo´gicas Majadahonda, 28220 Madrid 3 Centro de Biologı´a Molecular Severo Ochoa Canto Blanco, 28049 Madrid Spain 4 Biogen, Inc. 14 Cambridge Center Cambridge, Massachusetts 02142

Summary Here we have addressed the role that ␨PKC plays in NF-␬B activation using mice in which this kinase was inactivated by homologous recombination. These mice, although grossly normal, showed phenotypic alterations in secondary lymphoid organs reminiscent of those of the TNF receptor-1 and of the lymphotoxin-␤ receptor gene-deficient mice. The lack of ␨PKC in embryonic fibroblasts (EFs) severely impairs ␬B-dependent transcriptional activity as well as cytokine-induced phosphorylation of p65. Also, a cytokine-inducible interaction of ␨PKC with p65 was detected which requires the previous degradation of I␬B. Although in ␨PKC⫺/⫺ EFs this kinase is not necessary for IKK activation, in lung, which abundantly expresses ␨PKC, IKK activation is inhibited.

1997). The IKK complex can be activated by direct phosphorylation of the ␤ subunit by a putative IKK kinase (Karin, 1999; Lallena et al., 1999; Lee et al., 1997, 1998). Several candidates have been proposed to be the physiologically relevant IKK activator, including the atypical PKC (aPKC) isoforms ␨PKC and ␭/␫PKC (Lallena et al., 1999). Overexpression experiments show that ectopically expressed wild-type or activated ␨PKC is sufficient to trigger both the NF-␬B nuclear activity and ␬B-dependent transcriptional activation, as well as to stimulate IKK␤ (Diaz-Meco et al., 1999; Lallena et al., 1999). In contrast, the expression of dominant negative mutants of either ␭/␫PKC or ␨PKC inhibit ␬B-dependent transcription as well as IKK activation (Lallena et al., 1999). This suggests that the aPKCs could be considered critical players in the activation of the NF-␬B pathway. Consistent with this notion, the expression of Par-4 (Diaz-Meco et al., 1996), a protein that selectively binds the aPKCs, inhibiting their enzymatic activity, severely abrogates the activation of ␬B-dependent promoters and also IKK activation (Diaz-Meco et al., 1999). Recent evidence indicates that the aPKCs interact through their specific adaptor protein p62 with RIP and TRAF6, two critical components of TNF␣ and IL-1 signaling (Sanz et al., 1999, 2000). Collectively, these observations suggest that both aPKCs may be critically involved in the activation of NF␬B. To definitively assess the specific role of ␨PKC in this signaling mechanism, we have generated ␨PKCdeficient mice by homologous recombination and have investigated the activation of the NF-␬B pathway in embryonic fibroblasts (EFs) and lung, a tissue in which ␨PKC is abundantly expressed. Our results demonstrate a role for ␨PKC in NF-␬B signaling. Results

Introduction The transcription factor NF-␬B is critical in a number of cell functions, including growth, survival, and key inflammatory and immune responses (Baldwin, 1996). The most classical form of NF-␬B is a heterodimer of p50 and p65 (RelA) (Thanos and Maniatis, 1995), which is sequestered in the cytosol by I␬B, preventing its nuclear translocation (Thanos and Maniatis, 1995; Verma et al., 1995). Upon cell activation by TNF␣ or IL-1, I␬B␣ is phosphorylated at residues 32 and 36, which triggers the ubiquitination and subsequent degradation of I␬B through the proteosome (Karin and Ben-Neriah, 2000). These events serve to release NF-␬B, which translocates to the nucleus, activating a number of important genes. The kinase responsible for the signal-induced phosphorylation of I␬B is a heterodimer of three subunits: two catalytic (IKK␣ and IKK␤) and another regulatory, termed IKK␥ or Nemo (Israel, 2000; Karin and BenNeriah, 2000; Mercurio et al., 1997; Woronicz et al., 5

Correspondence: [email protected]

Generation and Initial Characterization of ␨PKC⫺/⫺ Mice To elucidate the role of ␨PKC in NF-␬B signaling, we disrupted this gene in mice by homologous recombination. Chimeric germline transmitting males were crossed to 129/SV females and gave rise to F1 heterozygous offspring on a pure 129 background. Intercrosses of such were used to establish a homozygote ␨PKC-deficient mouse line. Wild-type and mutant alleles were identified by Southern blot analysis of an EcoRI RFLP employing the probe indicated in Figure 1A. Targeted disruption of the ␨PKC gene was confirmed by RFLP analysis of genomic DNA (Figure 1A). We first determined the levels of ␨PKC, ␭PKC, ␣PKC, and ePKC in immunoblots of extracts from different tissues of wild-type and ␨PKC-deficient mice using selective antibodies for each PKC isotype. These analyses demonstrated that ␨PKC was expressed very abundantly in lung and at moderate levels in kidney, whereas ␭PKC was expressed in all tested tissues (Figure 1B). The lack of ␨PKC protein in extracts from homozygous ␨PKC⫺/⫺ tissues was also confirmed, as was the fact

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Figure 1. ␨PKC⫺/⫺ Mice Phenotypic Analysis (A) Restriction map of the ␨PKC wild-type locus (wt). The targeting vector was integrated into the endogenous locus by homologous recombination and gave rise to the mutant LacZ allele (mt). E, EcoRI; X, XhoI; LacZneo, ␤-Galactosidase/neomycin selections cassette (upper panel). Southern blot analysis of a litter derived from a heterozygote intercross. Genomic EcoRI fragments detected with a 3⬘ probe (shown as box in [A]) indicating wild-type and mutant alleles. Homozygote animals are represented in lanes 1, 3, and 8, and heterozygote animals are represented in lanes 6 and 7; lanes 2, 4, and 5 are representative for wild-type animals (lower panel). (B) Immunoblot analysis using different anti-PKC antibodies of tissues and EFs extracts from wild-type (⫹/⫹) and ␨PKC-deficient (⫺/⫺) mice. (C) Photomicrographs of representative hematoxilin-eosin stainings of PP (C, upper panels) and spleen (C, lower panels) of 2-week-old mutant and wild-type mice. Original magnification: upper panels (⫻50); lower panels (⫻400). (D and E) Immunohistochemistry of PP (D) and spleen (E) from the above mice with different antibodies. Original magnification ⫻100 (B) and ⫻200 (C). This is representative of another three mice with similar results.

that ␨PKC deficiency does not alter the levels of the other PKC isotypes (Figure 1B). When the blots were overexposed, it became apparent that ␨PKC was also detectably expressed in thymus and spleen (not shown). The ␨PKC-deficient mice were born in Mendelian proportions and were grossly normal. However, at 2 weeks of age, they showed a severely reduced number of Peyer’s patches (PP; 1 ⫾ 1 in the knock out mice versus 6 ⫾ 1 in the wild-type controls; n ⫽ 6 mice for each group). Histological examination of these mice also re-

vealed significant alterations in PP and spleen. Thus, the structural features of individual PP were altered, showing a reduction in the number and size of follicles in each PP of the mutant mice (Figure 1C, upper panels). The overall structure of the spleens from the ␨PKCdeficient mice was preserved, but a defect in the marginal zone was detected together with smaller B cell follicles in the white pulp (Figure 1C, lower panels). Immunohistochemical studies using anti-B220, anti-CD3, and anti-CD21 antibodies indicate an impaired segrega-

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Table 1. Analysis of Lymphocyte Subpopulations % CD4⫹ Peripheral LNs Mesenteric LNs

WT KO WT KO

33.1 42.8 26.3 28.3 Cellularity a (⫻10⫺6)

Peyer’s patches 4 weeks Peyer’s patches 7–8 weeks

WT KO WT KO

3.6 1.5 3.1 2.1

⫾ ⫾ ⫾ ⫾

0.3 0.2 0.1 0.4

⫾ ⫾ ⫾ ⫾

% CD8⫹

0.2 2.3 3.1 2.9

15.3 16.8 11.4 11.4

%CD4⫹

%CD8⫹

9.4 ⫾ 0.4 9.5 ⫾ 0.7 16.6 ⫾ 0.6 13.6 ⫾ 1.1

5.1 3.1 4.8 4.7

⫾ ⫾ ⫾ ⫾

0.7 0.2 0.8 0.2

⫾ ⫾ ⫾ ⫾

% B220⫹

0.4 1.6 0.6 2.6

35.9 23.5 40.1 30.3

%B220⫹ 80.6 81.2 75.9 76.9

⫾ ⫾ ⫾ ⫾

2.5 0.7 2.3 3.3

⫾ ⫾ ⫾ ⫾

1.1 4.2 2.1 1.3

b %B220high MHC IIhigh

c

%B220low MHC IIlow

54 38 80 57

46 62 20 43

Cells from peripheral and mesenteric lymph nodes (LNs) and Peyer’s patches of wild-type (WT) and ␨PKC⫺/⫺ (KO) mice were stained with different antibodies and analyzed by flow cytometry. Numbers indicate mean percentages ⫾ SD of positive cells per total cells (n ⫽ 4). a Numbers indicate total cell number of a single PP from each mouse (⫾ SD, n ⫽ 7). b Relative percentage of mature B cells (B220high MHC IIhigh IgMlow IgDhigh) within the total B220⫹ cells. c Relative percentage of immature B cells (B220low MHC IIlow IgMhigh IgDlow) within the total B220⫹ cells.

tion between B and T cell zones in the PP (Figure 1D), but not in the spleen (Figure 1E), of the ␨PKC⫺/⫺ mice as compared with wild-type animals. In addition, a decrease was detected in the follicular dendritic cell network in B cell follicles of both lymphoid tissues in the ␨PKC-deficient mice, as demonstrated by CD21 staining (Figures 1D and 1E). The alterations in the spleen were less apparent in 4-week-old ␨PKC⫺/⫺ mice and become normal at 7–8 weeks of age (not shown). However, although the differences in the number and size of PP between the ␨PKC⫺/⫺ and wild-type mice are still detectable in 4-week-old mice, older knockout animals showed a normal number of PP, but these were still smaller, with fewer follicles. Next, we analyzed the lymphocyte populations of spleen, thymus, lymph nodes, and PP from wild-type and ␨PKC-deficient mice. Results shown in Table 1 show that in 2-week-old ␨PKC⫺/⫺ mice, there is a reproducible diminution in the relative percentage of B cells in peripheral and mesenteric lymph nodes. In spleen, these alterations were also detected, although they were less apparent (not shown). The lymph node B cell population of the ␨PKC⫺/⫺ mice was significantly enriched in a precursor B220lowIgM⫺ cell population (not shown). These defects become less dramatic in older animals, particularly in those of 7–8 weeks of age (not shown). No alterations in the thymi of the mutant mice were detected at any age (not shown). The PP from the ␨PKC⫺/⫺ mice, although displaying a normal relative percentage of B and T cells, have a significant increase in the relative percentage of immature B cells (Table 1). This alteration was observed irrespective of the age of the mutant mice. Although less penetrant, this phenotype is reminiscent of that of the TNF receptor-1 (TNF-R1) and Lymphotoxin-␤ receptor (LT-␤R) knockout mice (Fu and Chaplin, 1999; Neumann et al., 1996; Pasparakis et al., 1997). This would be at least in part consistent with previous observations linking ␨PKC to TNF␣ signaling (Muller et al., 1995; Sanz et al., 1999). The observation that ␨PKCdeficient mice had defects in the development and organization of spleen and PP led us to investigate in detail aspects of splenic architecture known to be dependent on signals from TNF-R1 and/or LT-␤R for development and maintenance. Immunohistochemical staining with

antibodies to B220 (Figure 1) and Cr1 (not shown) indicated that, in young mice, proper B cell follicles had not formed and that B cells were present throughout the PALS, including within the T cell zone. Furthermore, T cells were not tightly packed within the PALS as expected, but appeared diffuse, with staining present in immediately adjacent follicular areas (data not shown). In addition, the population of B cell normally present in the marginal zone of the spleen appeared absent, or perhaps displaced. Normally, absence of marginal zone B cell staining correlates with the dissolution of the marginal zone structure, as shown by the loss of staining for markers of macrophage and sinus-lining cells. However, in ␨PKC⫺/⫺ mice, marginal zone populations remain intact, as shown by staining for MAdCAM-1 on sinuslining cells, sialoadhesin, and ER-TR9 on marginal zone macrophages, and MOMA-1 on metallophilic macrophages (not shown). Furthermore, stromal and dendritic cell populations in the B cell zone remained undisturbed, as demonstrated by the intact CD11b and CD11c staining and normal appearance of ER-TR7⫹ cells (data not shown). Immunohistochemical staining had indicated a decrease in the FDC network (Figure 1), and staining with anti-FDCM2 confirmed that, while present, FDC networks were less extensive and not as strongly stained in the ␨PKC⫺/⫺ mice (data not shown). Inhibition of NF-␬B-Dependent Transcription in ␨PKC⫺/⫺ EFs In order to determine whether the lack of ␨PKC impairs NF-␬B activation, we initially tested in primary cultures of EFs the transcriptional activation of I␬B␣, a wellestablished NF-␬B-dependent gene, in response to TNF␣ and an agonistic anti-LT-␤R antibody (ACH6). The data shown in Figure 2A clearly demonstrate that both TNF␣ and the agonistic ACH6 antibody provoked a robust increase in I␬B␣ transcription in the wild-type EFs but that this response is completely ablated in the ␨PKC⫺/⫺ EFs. Therefore, it seems that, at least in fibroblasts, ␨PKC appears to be essential for the ␬B-dependent transcription. To study the signaling pathways in more detail, we used immortalized embryonic fibroblasts from either wild-type or ␨PKC⫺/⫺ mice. In the next experiments, the activity of a ␬B-dependent luciferase reporter plasmid

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Figure 2. ␨PKC Is Required for NF-␬B-Dependent Transcription in EFs (A) Primary EFs, wild-type or ␨PKC-deficient, were incubated or not with TNF␣ (10 ng/ml) or the agonistic anti-LT-␤R antibody ACH6 (2 and 5 ␮g/ml) for 1 hr, after which RNA was extracted and the levels of I␬B␣ or GAPDH (as a negative control) mRNA were determined. EFs, either wild-type or ␨PKC⫺/⫺, were transfected with 250 ng of the ␬B-luciferase reporter gene plasmid and 2.5 ng of the Renilla control reporter pRL-CMV. In parallel cultures, ␨PKC⫺/⫺ EFs were also transfected with 5 ␮g of a ␨PKC expression vector (gray bars). After 24 hr, cells were stimulated or not for 6 hr with different concentrations of TNF␣ (B) or IL-1 (C), extracts were prepared, and the levels of luciferase activity were determined. (D) EFs, either wild-type or ␨PKC⫺/⫺, were treated or not with 20 ng/ml of TNF␣, either in the absence or in the presence of increasing concentrations of cycloheximide for 12 hr, after which the percentage of apoptotic cells (with subG1 DNA content) was determined by flow cytometry analysis. (E) EFs, wild-type or ␨PKC-deficient, were incubated or not with IL-1 (10 ng/ml) for 5 hr, after which RNA was extracted and the levels of IL-6 or GAPDH (as a negative control) mRNA were determined. Experiments shown in (A) and (E) are representative of another two with very similar results. Experiments shown in (B), (C), and (D) are the mean ⫾ SD of three independent experiments with incubations in duplicate.

was determined in EFs activated with TNF␣, IL-1, or ACH6 antibody. Interestingly, the activation of the ␬Bdependent transcription by TNF␣ and IL-1 (Figures 2B and 2C), as well as by ACH6 (not shown), was dramatically reduced in the ␨PKC⫺/⫺ EFs as compared to the wild-type controls. Of note, the transfection of a ␨PKC expression plasmid along with the reporter construct restored to near normal levels the activation of the ␬Bdependent transcription in the ␨PKC-deficient cells (Figures 2B and 2C), indicating that the defect in this pathway can only be ascribed to the lack of ␨PKC in the knockout EFs. Numerous studies have demonstrated that fibroblasts stimulated with TNF␣ can undergo apoptosis if the NF-␬B pathway is impaired. In order to address if this is the case in the ␨PKC⫺/⫺ EFs, wild-type and ␨PKC-deficient cells were treated or not with 20 ng/ ml of TNF␣, either with or without different concentrations of cycloheximide. As shown in Figure 2D, the presence of TNF␣ dramatically induces apoptosis in the ␨PKC⫺/⫺ EFs, but not in the wild-type controls, at all the cycloheximide concentrations tested. On the other hand, IL-1 is known to induce the production of inflammatory cytokines such as IL-6 through a transcriptional mechanism dependent on NF-␬B (Tanaka et al., 1999; Thomas et al., 1999). Results shown in Figure 2E demonstrate that this parameter is significantly reduced in ␨PKC-deficient cells, confirming that ␨PKC is important for NF-␬B-dependent gene transcription in response to IL-1. Control experiments using a ␬B mutant reporter gene and cotransfection experiments with I␬B demonstrate that the reporter assays actually correspond to NF-␬B-dependent transcription (not shown). Collectively, these results indicate that ␨PKC is an essential component in the activation of NF-␬B-dependent transcription at a step that is common to at least three different signaling pathways.

Lack of Effect of ␨PKC Deficiency on IKK Activation and p65 Nuclear Translocation in EFs The inhibition of ␬B-dependent transcription observed in the ␨PKC-deficient EFs could be explained by an impairment in the activation of the IKK complex, the subsequent degradation of I␬B, and the nuclear translocation of the NF-␬B heterodimer. However, the activation of IKK was intact in ␨PKC-deficient EFs when activated with TNF␣ and IL-1 (Figure 3A) or ACH6 (not shown). Consistent with these observations, the nuclear accumulation of NF-␬B was not impaired in the ␨PKCdeficient EFs activated either with TNF␣ or IL-1 (Figure 3B). Therefore, it seems that, at least in EFs, ␨PKC, although important for the control of NF-␬B transcriptional activity, does not seem to be essential for IKK activation. Interestingly, although TNF␣ and IL-1 activated NF-␬B DNA binding in ␨PKC⫺/⫺ EFs, this response was substantially reduced when compared with that of the wild-type cells (Figure 3C). Therefore, even though IKK activation and NF-␬B translocation is intact in the ␨PKC-deficient cells, the ability of the NF-␬B complex to bind its DNA enhancer element, as well as its transcriptional activity, are seriously inhibited. TNF␣ and IL-1 are potent activators of the stress MAPK pathway. Of note, the lack of ␨PKC has no effect in the activation of p38 or JNK (not shown) in response to IL-1 or TNF␣ in EFs. In addition, stimulation of ERK by EGF in EFs is not affected by the ␨PKC deficiency (not shown). Critical Role of ␨PKC in p65 Phosphorylation In order to begin to address the possible mechanism whereby ␨PKC may regulate this function in EFs, we next determined the phosphorylation state of p65 in immortalized wild-type and ␨PKC⫺/⫺ EFs. Cells labeled with 32P were stimulated with TNF␣ or IL-1 for 10 min,

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lated or not with TNF␣ or IL-1 either in the absence or in the presence of the proteosome inhibitor lactacystin, after which cell extracts were immunoprecipitated with the anti-␨PKC antibody and the immunoprecipitates were analyzed by immunoblotting with the anti-p65 antibody. Interestingly, the stimulation with TNF␣ and IL-1 promotes a rapid association of ␨PKC with NF-␬B, which is severely impaired when the degradation of I␬B is blocked with lactacystin (Figure 4C). This suggests that, for ␨PKC to interact with NF-␬B, I␬B has first to be degraded.

Figure 3. ␨PKC Is Not Required for IKK Activation in EFs (A) EFs, wild-type or ␨PKC⫺/⫺, were stimulated either with TNF␣ or IL-1, extracts were immunoprecipitated with an anti-IKK␥ antibody, and the IKK enzymatic activity was determined. Nuclear extracts from EFs, either wild-type or ␨PKC⫺/⫺, stimulated or not for 30 min with different concentrations of TNF␣ or IL-1, were analyzed by EMSA (C) for NF-␬B binding activity and by immunobloting with an anti-p65 antibody (B). These are representative experiments of another three with similar results.

after which the phosphorylation of p65 was analyzed. The stimulation with either IL-1 (Figure 4A) or TNF␣ (not shown) leads to a detectable increase in p65 phosphorylation which is dramatically inhibited in ␨PKC⫺/⫺ cells (Figure 4A). Therefore, these results indicate that ␨PKC plays an essential role in directly or indirectly phosphorylating p65. Previous studies have shown in cotransfection experiments that ␨PKC could induce the phosphorylation of the p65 Rel-homology domain (RHD). In order to address if p65 is a direct substrate of ␨PKC, a recombinant bacterially expressed RHD p65 construct was incubated with recombinant pure ␨PKC and the phosphorylation of p65 was determined. The results shown in Figure 4B demonstrate that p65 is efficiently phosphorylated by ␨PKC in vitro. Kinases that have been implicated in the control of the phosphorylation state in NF-␬B have been reported to interact with this transcription factor (Wang et al., 2000; Zhong et al., 1997). To determine if this is the case for ␨PKC, EFs were stimu-

Potential Role of ␨PKC in IKK Activation Although in EFs, ␨PKC does not appear to be essential for the stimulation of IKK, it is possible that in other tissues where ␨PKC is more abundantly expressed, this isotype may also control the stimulation of IKK. Since lung is the tissue where ␨PKC is more predominantly expressed as compared with other organs and EFs (Figure 1B), we tested if the activation of IKK in lung depends on ␨PKC. ␨PKC⫺/⫺ and control mice were intravenously injected with murine TNF␣ or control buffer. At 7 min after the challenge, lungs were extracted and NF-␬B activity was determined in nuclear extracts by electrophoretic mobility shift assays (EMSA) using an NF-␬Bspecific probe. Figure 5A shows the results of one representative experiment. Although the injection of TNF␣ provokes a robust activation of NF-␬B in the lungs of wild-type animals, this response was severely abrogated in the ␨PKC⫺/⫺ mice. This defect was specific for NF-␬B, since the DNA binding of Oct-1 was unaltered (Figure 5A). This NF-␬B complex is most likely composed of p65/p50 dimers (Figure 5A). In similar experiments, mice were inoculated intraperitoneally either with control buffer, murine IL-1, or E. coli-derived LPS, and NF-␬B activity was determined in lungs as above. It was clear that the lack of ␨PKC severely inhibits NF-␬B activation in response to both stimuli (Figure 5B). The data shown in Figure 5C show a quantification of the changes in NF-␬B from all the experiments. The lack of ␨PKC reproducibly inhibits the activation of NF-␬B in lung, although a complete blockade of this parameter was not always detected. Consistently, the nuclear accumulation of p65 in lungs in response to TNF␣ and IL-1 (Figure 5D) as well as to LPS (not shown) was severely reduced in the ␨PKC⫺/⫺ mice, as compared to the controls. We next determined if the activation of IKK in response to these three stimuli was affected in the ␨PKC⫺/⫺ mice. Figure 5E shows the results of determining IKK activity in the lungs of the experiments shown in Figure 5A. The lack of ␨PKC leads to a severe impairment of the activation of IKK in this system (Figure 5E). When all the experiments were quantified and represented as in Figure 5F, it was clear that, although not always completely inhibited, IKK activation is severely impaired by the lack of ␨PKC. Taken together, these results indicate that ␨PKC is an essential step in the control of the IKK/NF-␬B signaling cascade in lung, an organ in which this PKC isoform is particularly abundant. As a control of the specificity of the lung experiments, NF-␬B activity was determined in liver extracts from the same experiments shown in Figure 5. Consistent with the low levels of ␨PKC in that tissue, NF-␬B activation

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Figure 4. ␨PKC Phosphorylates and Interacts with p65 (A) EFs were labeled with 32P, after which they were stimulated or not with IL-1 (10 ng/ml; 10 min), and p65 was immunoprecipitated. (B) Recombinant pure RHD p65 (3 ␮g) was incubated with baculovirus-expressed pure ␨PKC (30 ng) for 30 min at 30⬚C, after which the phosphorylation of p65 was determined. (C) EFs were incubated or not with lactacystin prior to the stimulation with TNF␣ or IL-1 for different times, after which ␨PKC was immunoprecipitated and the associated p65 was determined by immunoblotting. These are representative experiments of at least another two with very similar results.

was not affected or was affected to a small degree (not shown). Discussion The results shown here demonstrate that ␨PKC is essential in the control of p65 transcriptional function in response to the activation of several signaling pathways, including that of lymphotoxin-␤ receptor. Furthermore, in a tissue in which this kinase is abundantly expressed, such as lung, the ␨PKC deficiency significantly impairs the activation of IKK and the subsequent nuclear accumulation of p65. Our observation that ␨PKC is required for the activation of NF-␬B transcription with no effects on IKK activation in EFs is reminiscent of the reported role of GSK-3␤ (Hoeflich et al., 2000), T2K (Bonnard et al., 2000), and NIK (Yin et al., 2001) in this pathway. Thus, cells from mice deficient in any of these kinases display, like the ␨PKC⫺/⫺ EFs, a significantly reduced cytokine-stimulated ␬B-dependent transcriptional activity with no defect on I␬B degradation or the nuclear translocation of NF-␬B. This suggests that ␨PKC may share parts of the pathways controlled by these kinases. Interestingly, whereas GSK-3␤ and T2K are both neces-

sary for TNF␣ and IL-1 signaling, NIK is not, but is selectively located in the LT-␤R pathway (Matsushima et al., 2001; Yin et al., 2001). In marked contrast, ␨PKC deficiency impairs NF-␬B signaling in response to not only TNF␣ and IL-1 but also to lymphotoxin-␤ in EFs, emerging as a common step in NF-␬B activation in response to these three stimuli. Another intriguing aspect of our results is the reduction of the NF-␬B DNA binding activity observed in the stimulated ␨PKC⫺/⫺ EFs that does not correlate with an inhibition of IKK activation in this system. We do not have a clear explanation for this phenomenon, but it is worth mentioning that recent data demonstrate that expression of PTEN, which antagonizes PI 3-kinase signaling, inhibits NF-␬B DNA binding and transcriptional activities without affecting IKK activation in response to IL-1 (Koul et al., 2001). Interestingly, this effect was ascribed to the phosphorylation state of NF-␬B (Koul et al., 2001). Although Akt/PKB was implicated as potentially being responsible for PTEN actions, it should be borne in mind that ␨PKC has also been shown to be an important downstream target of PI 3-kinase (Standaert et al., 1997). Of note, EFs from the GSK-3␤ knockout mice also display a reduction in IL-1 and TNF␣-activated

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Figure 5. Lungs from ␨PKC-Deficient Mice Show Impaired NF-␬B and IKK Activities Nuclear extracts of lungs from four wild-type (WT) and four ␨PKC⫺/⫺ (KO) mice that have been either treated with control buffer or TNF␣, IL-1, or LPS were analyzed by EMSA for NF-␬B binding activity ([A], upper panel; [B]) or for Oct-1 DNA binding as a negative control ([A], lower panel), or were analyzed by immunoblot with anti-p65 antibody (D). Incubation of nuclear lung extracts from TNF␣-treated mice with anti-p50 and anti-p65 antibodies demonstrate that the NF-␬B band is formed by p50/p65 dimers ([A], right panel). IKK activity was also determined in whole lung extracts (E). These are representative experiments of another two with similar results. Gels of all the experiments were quantified in an Instantimager, and the mean ⫾ SD of the CPMs of the NF-␬B bands and IKK activities are shown in panels (C) and (F), respectively.

NF-␬B DNA binding with no inhibition of IKK activation (Hoeflich et al., 2000). Together, these results could be consistent with a model according to which p65 phosphorylation may be important not only for its transcriptional activity but also for the interaction with its enhancer elements in the promoter. Our results provide genetic support for the proposed role of p65 phosphorylation in the activation by ␨PKC of ␬B-dependent transcription (Anrather et al., 1999). We show here that ␨PKC associates with, and directly phosphorylates, p65, and that ␨PKC is required for the cytokine-induced p65 phosphorylation in EFs. Interestingly, other kinases, such as PKA and CKII, have been reported to target p65 function by phoshorylation of the C-terminal transactivation domain, through a process that involves their interaction following I␬B degradation (Wang et al., 2000; Zhong et al., 1997, 1998). We demonstrate that for ␨PKC to interact with p65 in cytokineactivated cells, I␬B has to be first degraded by the proteosome. Therefore, a model can be proposed according to which, upon cytokine stimulation, ␨PKC—or another IKK kinase, depending on the cell type or tissue—triggers the degradation of I␬B, which is a required step for the interaction of ␨PKC with p65 and its subsequent phosphorylation. In this model, ␨PKC would phosphorylate the RHD of p65, whereas other kinases would target the C-terminal transactivation domain.

The in vivo data of this paper support the notion that, at least in some cell systems such as the lung, ␨PKC can also be involved in the activation of IKK. Recent data indicate that MEKK3 is required for the activation of IKK and that it may interact, at least in cotransfection experiments, with RIP. Our previous data demonstrated that ␨PKC interacts, through the adaptor p62 (Sanchez et al., 1998), with RIP and TRAF6 (Sanz et al., 2000, 1999). All these molecules serve to link the cytokine receptors to IKK activation (Goeddel, 1999; Kelliher et al., 1998; Lomaga et al., 1999). The inhibition of IKK activation in lungs from ␨PKC⫺/⫺ mice reported here genetically supports the involvement of this kinase in these pathways. Whether MEKK3 is also required for IKK activation in lung or not is unknown. One possibility is that ␨PKC, RIP, and MEKK3 form part of a sequential multiprotein complex that controls IKK activation. The activation of NF-␬B by TNF␣ provides a potent antiapoptotic signal that prevents TNF␣-induced cell death (Beg and Baltimore, 1996). The inhibition of NF␬B signaling in the pathways of the different knockout mice sensitizes cells to the proapoptotic actions of TNF␣. In this regard, there are intriguing differences in the phenotypes of the GSK-3␤, T2K, and ␨PKC mutant mice. Whereas both the GSK-3␤- and T2K-deficient mice die of liver apoptosis, only the EFs from the GSK3␤-deficient mice undergo apoptosis in response to

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TNF␣, whereas the T2K⫺/⫺ EFs do not (Matsushima et al., 2001; Yin et al., 2001). The ␨PKC⫺/⫺ EFs are sensitive to the proapoptotic actions of TNF␣, like the GSK-3␤ EFs, but the livers of the ␨PKC mutant mice do not show signs of apoptosis. It is worth mentioning that not all the mice with a knockout of genes shown to be essential for NF-␬B activation display liver cell death. Thus, for example, whereas mice deficient in RelA (p65), IKK␤, and Nemo/IKK␥, in addition to the already discussed T2K, or GSK-3␤, die around day 14 of gestation due to severe liver degeneration and apoptosis (Beg et al., 1995; Hoeflich et al., 2000; Li et al., 1999a, 1999b; Rudolph et al., 2000; Takeda et al., 1999; Tanaka et al., 1999; Bonnard et al., 2000), other mutant mice, such as the TNF-R1, TRAF2, TRAF6, RIP, MyD88, and IRAK, do not (Adachi et al., 1998; Kelliher et al., 1998; Lomaga et al., 1999; Pfeffer et al., 1993; Thomas et al., 1999; Yeh et al., 1997). It may be that the former proteins control the expression of NF-␬B-dependent antiapoptotic genes that are essential for survival of liver cells, whereas the latter do not. In the case of the ␨PKC⫺/⫺ mice, a possible explanation for the lack of liver cell death may rely on the fact that this tissue expresses little or no ␨PKC and, therefore, other kinases may have substituted for ␨PKC for this function. Upon injection of LPS, the activation of NF-␬B in lungs is severely inhibited in the ␨PKC⫺/⫺ mice but not in the livers of these animals. The phenotype of the ␨PKC⫺/⫺ mice is complex and consistent with the notion that this kinase participates in distinct NF-␬B signaling pathways. Not surprisingly, the ␨PKC-deficient mice have at least some of the features characteristic of mutant mice for the TNF-R1 and LT-␤R. For example, the ␨PKC⫺/⫺ mice have a significantly reduced number of PP, which is characteristic of the mutant mice for the TNF-R1 (Locksley et al., 2001). However, the PP of the ␨PKC-deficient mice display an impaired segregation of B and T cells, which has not been reported in the PP of the TNF-R1 animals (Pasparakis et al., 1997). However, the PP of both mutant mice are similar in that they have decreased follicular dendritic cell networks. The overall structure of the spleen is normal in the ␨PKC⫺/⫺ mice, similar to what has been reported for the TNF-R1 mutant mice (Locksley et al., 2001). In addition, the ␨PKC⫺/⫺ mice have detectable lymph nodes, like the TNF-R1 mutant mice and unlike the LT-␤R-deficient mice, which display a complete lack of lymph nodes (Locksley et al., 2001). However, the ␨PKC⫺/⫺ lymph nodes are smaller and have a lower content of B cells. Therefore, overall, the ␨PKC⫺/⫺ phenotype, although less penetrant, has characteristics of both the TNF-R1 and LT-␤R mutant phenotypes, but is more similar to the former. There is an additional important observation in the ␨PKC⫺/⫺ mice, which is the apparent delay in the maturation of B cells. A similar phenotype has been described for mutants in the BCR signaling cascade (Kurosaki, 1999). Studies are in progress to determine if ␨PKC could play a nonredundant role in this pathway that could explain, at least in part, this phenotype. Taken together, the data suggest that, rather than a fundamental defect in TNF-R1 or LT-␤R signaling in the spleen, the ␨PKC deficiency causes a developmental delay in the delivery of these signals during the first few weeks of life. Thus, it may be that the ␨PKC requirement to transduce these signals in lymph-

oid tissues is developmentally regulated, and as the animal matures, other, perhaps redundant, signaling pathways can compensate. This study highlights the role that ␨PKC plays in the transduction of various receptor signals to NF-␬B, illustrates the complexity of ␨PKC regulation in multiple tissue and cell types, and demonstrates that this kinase plays an essential role in NF-␬B activation at two different levels: IKK stimulation and NF-␬B transcriptional activity. Experimental Procedures Generation of ␨PKC⫺/⫺ Mice To clone the mouse ␨PKC locus, a 129/O1a genomic phage library (obtained from Stratagene) was screened using the full-length rat cDNA as a probe. One phage was isolated containing a single exon corresponding to nucleotides 358–444 of the rat cDNA. To generate a targeting construct, a 6.5 kb XhoI fragment containing the exon was subcloned. Subsequently a ␤-galactosidase/neomycin cassette (LacZneo) was inserted into the coding sequence, thereby disrupting the transcriptional organization of the ␨PKC locus after homologous recombination. The targeting vector was linearized at a unique Sal I site in the polylinker and introduced into E14 ES cells. We screened 1920 G418-resistant colonies by Southern blot analysis. Two ES cell clones were correctly targeted, resulting in a targeting frequency of 1 in 960. Both were used for further experiments and were microinjected into NMRI albino mouse blastocysts to generate chimeras. Five chimeras gave germline transmission, which further on were used to establish the homozygous ␨PKC-deficient mouse line. The genotyping of recombinant ES cells and adult mice was done by Southern blot analysis with a probe corresponding to a 3⬘ prime 0.6 kb XhoI fragment recognizing a 7.0 kb band in the wild-type and a 4.0 kb fragment in all targeted alleles. Immunohistopathological Analysis Freshly dissected tissue samples were formalin fixed, paraffin embedded, and stained with hematoxilin-eosin according to standard procedures. Peyer’s patches were counted, both in fresh and HEstained paraffin sections. Immunohistochemical staining was performed in PP and spleen following standard procedures. B cell immunostaining was performed with CD45R/B220 antibody (RA36B2, Pharmingen, USA). T cells were identified using CD3 polyclonal antibody (Dako, Denmark). Follicular dendritic cell staining was done using anti-CD21 (M-19, Santa Cruz, California). Sections were counterstained with hematoxylin. Immunofluorescent Analysis Immunofluorescent staining was performed using 8 ␮m frozen sections that were air dried and fixed in ice-cold acetone. Antibodies used were MOMA-1, anti-sialoadhesin, ER-TR7 (all from Serotec, Oxford, UK), ER-TR9 (Bachem, King of Prussia, PA USA), anti-CR1 (CD35), anti-CD11c, anti-CD11b (all from Pharmingen, San Diego, CA USA), and anti-FDCM2 (Immunokontact, Oxon, UK). Flow Cytometric Analysis Thymus, spleen, Peyer’s patches, and peripheral and mesenteric lymph nodes were removed from 2-, 4-, and 7-week-old mice. Single cell suspensions were made and washed in phosphate-buffered saline (PBS) supplemented with 5% FCS and 5 mM EDTA. Afterward, cells were analyzed after staining with the following monoclonal antibodies: FITC-conjugated anti-B220 (clone RA3-6B2, Pharmingen, San Diego CA), FITC-conjugated anti-IgD (Pharmingen), PE-conjugated anti-IgM (clone R4-22, Pharmingen), PE-conjugated anti-CD4 (clone GK1.5, Pharmingen), PE-conjugated anti-MHC class II (clone M5/114.15.2, Pharmingen), and TC-conjugated anti-CD8 (clone 53-6.7, Caltag). Analysis was performed on a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA) with CELLQuestTM software. Dead cells were excluded from the analysis on the basis of low forward-light scatter. Apoptosis was determined by flow cytometry according to an standard protocol using an EPICS XL flow cytometer (Coulter Electronics Inc., Hialeah, FL) by recording the propidium iodide staining

Targeted Disruption of ␨PKC 779

in the red channel. The percentage of apoptotic cells was determined by calculating the fraction of cells with sub-G1 DNA content.

ported by the DFG (Sta314/2-1 and KE 246/7-2). We thank Miguel Angel Piris for comments and advice.

Animal Treatments Six-week-old sex-matched ␨PKC⫺/⫺ and control mice were studied. Mice were inoculated intraperitoneally with 1.5 ␮g/g body weight lipopolysaccharide (LPS) derived from E. coli Serotype 0111:B4 (Sigma) in PBS solution, or with 3 ng/g body weight IL-1␤ (R&D systems). For TNF␣ studies, mice were inoculated intravenously with 15 ng/g body weight TNF␣ (R&D systems). After different times, mice were sacrificed and organs were obtained. Two animals were sampled per time point per treatment. Animal handling protocols conform to the NIH guidelines.

Received October 30, 2000; revised August 15, 2001.

Electrophoretic Mobility Shift Assay and In Vitro Kinase Assay EMSA experiments were performed as previously described (DiazMeco et al., 1993). IKK activity was determined in anti-IKK␥ immunoprecipitates as described (Lallena et al., 1999). Luciferase Reporter Assay Subconfluent cultures of EFs either wild-type or ␨PKC⫺/⫺ were transfected by Lipofectamine Plus (Life Technologies) with 250 ng of the ␬B-luciferase reporter gene plasmid and 2.5 ng of the Renilla control reporter pRL-CMV. In parallel cultures, ␨PKC⫺/⫺ EFs were also transfected with 5 ␮g of a HA-␨PKC expression vector. After 24 hr, cells were stimulated or not for 6 hr with different concentrations of IL1␤, and extracts were prepared and the levels of luciferase activity were determined as described previously (Sanz et al., 2000). Measurement of I␬B␣ or IL-6 mRNA Levels EFs were stimulated or not with IL-1 (10 ng/ml) for 5 hr to measure IL-6 mRNA, or with TNF␣ (10 ng/ml) or anti-LT␤-R Mab AC.H6 (2 ␮g/ml) for 1 hr to determine I␬B␣ mRNA levels. Then, total RNA was prepared as previously described (Barradas et al., 1999) and an aliquot of 10 ng was used as template in a 20 ␮l reaction using the SuperScript One-Step RT-PCR kit (Gibco-BRL). The RT-PCR conditions and primers to measure IL-6 and the control GAPDH were as described (Goh et al., 2000). Primers for I␬B␣ were: 5⬘-GCCTTCCT CAACTTCCAGAACAAC-3⬘ and 5⬘-CAGACGCTGGCCTCCAAACA CACAG-3⬘ In Vivo Phosphorylation of p65 Metabolically 32P-labeled EFs, either wild-type or ␨PKC⫺/⫺, were stimulated with TNF␣ (20 ng/ml) or IL-1␤ (10 ng/ml) for 10 min and harvested in cold lysis buffer (25 mM Tris [pH 7.6], 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) supplemented with phosphatase and protease inhibitors. Whole cell lysates were subjected to immunoprecipitation with anti-p65 antibody. Immunoprecipitates were separated on SDS-PAGE, and the phosphorylated proteins were detected and quantified in an InstantImager (Packard). In Vitro Phosphorylation of p65 Three ␮g of recombinant p65-RHD (amino acids 12–317) were incubated at 30⬚C for 30 min in 25 ␮l of assay buffer containing 35 mM Tris (pH 7.5), 10 mM MgCl2, 0.1 mM CaCl2, 0.5 mM EGTA, and 2.5 ␮M ATP in the presence of baculovirus-expressed ␨PKC (30 ng). Association of p65 EFs were stimulated or not with TNF␣ (20 ng/ml) or IL-1␤ (10 ng/ml) either in the absence or in the presence of 30 ␮M of the proteosome inhibitor lactacystin for 4 hr prior to stimulation, after which cells were lysed in buffer PD (Sanz et al., 2000) and immunoprecipitated with anti-␨PKC antibody. The immunoprecipitates were analyzed by immunoblotting with anti-p65 antibody. Acknowledgments This work was supported by grants SAF1999-0053 (to J.M.), 2FD971429 (to J.M.), and SAF2000-0175 (to M.T.D.M.) from MCYT, and 08.1/0060/2000 from CAM and has benefited from an institutional grant from Fundacio´n Ramo´n Areces to the CBM. M.L. was sup-

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