Gamma-secretase limits the inflammatory response through the processing of LRP1 - PubMed (original) (raw)

Gamma-secretase limits the inflammatory response through the processing of LRP1

Kai Zurhove et al. Sci Signal. 2008.

Abstract

Inflammation is a potentially self-destructive process that needs tight control. We have identified a nuclear signaling mechanism through which the low-density lipoprotein receptor-related protein 1 (LRP1) limits transcription of lipopolysaccharide (LPS)-inducible genes. LPS increases the proteolytic processing of the ectodomain of LRP1, which results in the gamma-secretase-dependent release of the LRP1 intracellular domain (ICD) from the plasma membrane and its translocation to the nucleus, where it binds to and represses the interferon-gamma promoter. Basal transcription of LPS target genes and LPS-induced secretion of proinflammatory cytokines are increased in the absence of LRP1. The interaction between LRP1-ICD and interferon regulatory factor 3 (IRF-3) promotes the nuclear export and proteasomal degradation of IRF-3. Feedback inhibition of the inflammatory response through intramembranous processing of LRP1 thus defines a physiological role for gamma-secretase.

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Figures

Figure 1

Figure 1. Nuclear localization of the free LRP1 ICD

A) Wildtype (a and d), LRP1-deficient (b and e), and murine embryonic fibroblasts stably expressing the free LRP1 ICD (LRP1-105, c and f) were analyzed by immunocytochemistry with an antibody directed against the LRP1 C-terminus after treatment with 1 µM epoxomicin for 9h. Nuclear counterstaining was done by DAPI (d–f). A representative experiment of n=5 is shown. B) Nuclear and cytosolic extracts were prepared from wildtype (WT) and LRP1-deficient (k.o.) MEF after treatment with 1 µM epoxomicin or no treatment for 12h and were subsequently analyzed by Western Blotting with the C-terminal LRP1-antibody, a nuclear marker (α-Histone 2b), and a cytosolic marker (α-Hsp90). A representative experiment of n=5 is shown.

Figure 2

Figure 2. LPS enhances proteolytical processing of LRP1

A) Wildtype primary macrophages were pretreated with the γ-secretase inhibitor DAPT at a final concentration of 10 µM for 2 h. Then cells were treated with 1 µg/ml LPS or left untreated for 9 h and cell membranes were prepared and analyzed by Western Blotting with the C-terminal LRP1 antibody. A representative experiment of n=4 is shown. B) Schematic representation of LPS-induced signaling pathways. Binding of LPS to its receptor complex leads to the activation of MyD88 (and Mal)-dependent signaling that results in the early activation of NFκB, and of MyD88-independent (TRIF and TRAM-dependent) pathways that lead to the activation of IRF-3 and late NFκB activation. TRIF – TIR-domain containing adaptor molecule, TRAM – TRIF-related adapter molecule, MyD88 – myeloid differentiation marker 88, MAL – MyD88-like, TRAF6 – tumor necrosis factor receptor-associated factor, TBK1 – TANK-binding protein kinase, NEMO – NFκB essential modulator, IKK – IκB kinase.

Figure 3

Figure 3. LRP1 modulates LPS-activated signaling through direct interaction with IRF-3

A) Whole cell lysates from wildtype and LRP1-deficient fibroblasts were prepared after treatment with 10 µg/ml LPS for the times indicated and from untreated controls. Lysates were analyzed by Western Blotting and staining with an anti-IκBα antibody. Anti-β-actin was used as a loading control. For quantification of Western Blotting results see supplementary Figure 1A. B) Nuclear extracts were prepared from wildtype and LRP1-deficient fibroblasts as well as from LRP1-deficient cells stably transfected with the LRP1-cDNA (k.o.-LRP1), the empty plasmid vector (k.o.-ctrl.), or the LRP1-βchain (k.o.-LRP1-βchain). The extracts were analyzed by Western Blotting and staining with an anti-phospho-IRF-3 antibody. Staining for β-actin served as loading control. Inset: Analysis of nuclear extracts from LRP1-deficient cells stably transfected with the LRP1-ICD-cDNA (k.o.-LRP1-105) and the empty plasmid vector (k.o.-ctrl.) A representative experiment of n=5 is shown. C) Total IRF-3 levels in the cell lines described under B) were identical as judged by analysis of whole cell lysates by Western Blotting and staining with an IRF-3 antibody. Staining with a β-actin antibody served as loading control. D) Schematic representation of LRP1 and the LRP1 mutants retransfected into the LRP1-deficient fibroblasts. E)/F) Wildtype (wt) and LRP1-deficient (k.o.) MEF and k.o. cells retransfected with LRP1, the LRP1-β-chain, or the empty plasmid vector were analyzed for LRP1 expression by Western Blotting with the C-terminal LRP1 antibody. G) Schematic representation of the GST-LRP1 fusion proteins used in a pull-down assay to detect direct interaction of IRF-3 with LRP1. The LRP ICD was N-terminally fused to a GST tag. In addition a C-terminal LRP1 ICD deletion mutant and constructs with mutations in the first, second, or both NPxY motifs were employed. The black box indicates a putative casein kinase II phosphorylation site in the distal LRP1 ICD. H) GST-LRP1 ICD fusion proteins (see G) were used to pull down IRF-3 from whole cell lysates of MEF. Equal input of fusion proteins was made visible by Ponceau staining of the transfer membrane (see supplementary figure 7). A representative experiment of n=3 is shown.

Figure 4

Figure 4. The LRP1 ICD regulates the expression of a subset of LPS-induced genes by direct nuclear signaling and limiting nuclear pIRF-3

A) Whole cell lysates were prepared from wildtype and LRP1-deficient macrophages after treatment with 1 µg/ml LPS for the times indicated and from untreated controls. The lysates were examined as described under 3A). Pooled macrophages from 4 k.o. and 4 wildtype mice were used for each repetition of the experiment (n=3). For quantification of Western Blotting results see supplementary Figure 1B. B) Whole cell lysates were prepared from wildtype (WT) and LRP1-deficient macrophages (k.o.) treated with 1 µg/ml LPS for the times indicated. After Western Blotting antibodies directed against LRP1, pIRF-3, IRF-3 and β-actin were used for staining. Pooled macrophages from 3 k.o. and 3 wildtype mice were used for each repetition of the experiment (n=3). For quantification of Western Blotting results see supplementary Figure 1C. C) Total RNA was prepared from wildtype and LRP1-deficient macrophages either treated with 1 µg/ml LPS or left untreated. Quantitative real-time PCR was used to assess expression levels of interferon γ. Bars represent the mean of 10 independent experiments, error bars depict SEM. Statistical analysis was done using the Student’s t-test. * p<0.05. For each of the 10 experiments one conditional k.o. and one control mouse were used. D) Total RNA was prepared from wildtype and LRP1-deficient macrophages either treated with 1 µg/ml LPS or left untreated. Quantitative real-time PCR was used to assess expression levels of interferon β. Bars represent the mean of 6 independent experiments, error bars depict SEM. Statistical analysis was done using the Student’s t-test. * p<0.01. For each of the 6 experiments one conditional k.o. and one control mouse were used. E) Wildtype and LRP1-deficient macrophages were treated with 1 µg/ml LPS for 15 min. or left untreated. Then DNA-binding proteins were cross-linked to the chromatin and cell lysates were prepared. After shearing of the DNA by sonification an immunoprecipitation with the C-terminal LRP1 antibody was carried out. The precipitate was used in a PCR reaction with primers binding to the interferon γ promoter. A representative experiment of n=3 is shown. For each of the 3 experiments one conditional k.o. and one control mouse were used. F) Wildtype and LRP1-deficient macrophages were treated with 1 µg/ml LPS for the times indicated or left untreated. Chromatin immunoprecipitation was carried out as described above. Primers specific for the interferon β promoter were used in the PCR reaction. A representative experiment of n=3 is shown. For each of the 3 experiments one conditional k.o. and one control mouse were used. G) Wildtype and LRP1-deficient murine embryonic fibroblasts were treated with 4 ng/ml leptomycin B for 19h or were left untreated. Nuclear extracts were prepared and analyzed by immunoblotting with a pIRF-3 antibody. β-actin served as a loading control. A representative experiment of n=3 is shown. H) Wildtype and LRP1-deficient murine embryonic fibroblasts were treated with 1 µM epoxomicin or 10 µM lactacystin for 19h or were left untreated. Nuclear extracts were prepared and analyzed by immunoblotting with a pIRF-3 antibody. β-actin served as a loading control. I) Wildtype and LRP1-deficient macrophages were treated with 10 ng/ml LPS for 4h or left untreated. Supernatants were then collected and TNF-α concentrations were determined by ELISA. Bars represent the mean of 6 independent experiments, error bars depict SEM. Statistical analysis was done using the Student’s t-test. * p<0.005. For each of the 6 experiments one conditional k.o. and one control mouse were used. J) Wildtype and LRP1-deficient macrophages were treated with 1 µg/ml LPS for 24h or left untreated. Supernatants were then collected and IL-6 concentrations were determined by ELISA. Bars represent the mean of 6 independent experiments, error bars depict SEM. Statistical analysis was done using the Student’s t-test. * p<0.05. For each of the 6 experiments one conditional k.o. and one control mouse were used.

Figure 5

Figure 5. Identification of a subset of LPS-inducible genes that are repressed by the LRP1 ICD

A) Total RNA from wildtype and LRP1-deficient MEF and from LRP1-deficient fibroblasts stably re-transfected with LRP1, the membrane-bound LRP1 ICD (LRP-β-chain), the LRP1 ECD, the free LRP1 ICD (LRP1-105) or the empty plasmid vector was analyzed by quantitative real-time PCR for expression levels of Usp18, Rsad2, Tyki, and Oas1. Bars represent the mean of 6 independent experiments, error bars depict SEM. Statistical analysis was done using the Student’s t-test. * p<0.001. B) Wildtype fibroblasts were treated with 2 µg/ml LPS for 3h or were left untreated. RNA was prepared and examined by real-time PCR for expression of Usp18, Rsad2, Tyki, and Oas1. ** p<0.05, n=5.

Figure 6

Figure 6. LRP1 is involved in multiple signaling pathways

A) RNA samples from wildtype and LRP1-deficient MEF were analyzed by Northern Blotting with cDNA probes for the genes indicated. Cyclophilin served as a loading control. A representative experiment of n=3 is shown. Dkk-3 – dickkopf-3, C3 – complement factor 3, sfrp-1 – secreted frizzled-related protein-1, AHR – aryl hydrocarbon receptor. B) RNA samples from wildtype and LRP1-deficient MEF and from LRP1-deficient fibroblasts stably re-transfected with an expression plasmid for LRP1 (control: empty plasmid vector) were examined for 25-cholesterol-hydroxylase expression. A representative experiment of n=3 is shown.

Figure 7

Figure 7. Proposed model for the negative feedback regulation of LPS-induced inflammatory signaling by γ-secretase-dependent LRP1 ICD generation

Expression of a subset of LPS-induced genes is activated by IRF-3. Activation of IRF-3 and other LPS-induced signaling pathways leads to increased expression of metalloproteases. In addition, LPS-dependent PKC-activation also occurs (55). The proteolytical processing of LRP1 is therefore augmented. Increased shedding provides more substrate for the γ-secretase-mediated cleavage step that leads to LRP1 ICD release. The LRP1 ICD interacts with IRF-3 and displaces it from its binding to CBP/p300, thereby unmasking its nuclear export signal (yellow) and facilitating its nuclear export. Subsequently, IRF-3 target gene expression is reduced.

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References

    1. Parks AL, Curtis D. Presenilin diversifies its portfolio. Trends Genet. 2007;23:140–150. - PubMed
    1. Ehebauer M, Hayward P, Martinez-Arias A. Notch signaling pathway. Sci STKE. 2006;2006:cm7. - PubMed
    1. Ni CY, Murphy MP, Golde TE, Carpenter G. gamma -Secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase. Science. 2001;294:2179–2181. - PubMed
    1. Sardi SP, Murtie J, Koirala S, Patten BA, Corfas G. Presenilin-dependent ErbB4 nuclear signaling regulates the timing of astrogenesis in the developing brain. Cell. 2006;127:185–197. - PubMed
    1. Herz J, Strickland DK. LRP: a multifunctional scavenger and signaling receptor. J Clin Invest. 2001;108:779–784. - PMC - PubMed

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