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Cited by 133Inflammasome and immune disordersProtease and Inhibitor MechanismsCell death mechanisms and regulation

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The Serpin Proteinase Inhibitor 9 Is an Endogenous Inhibitor of Interleukin 1β–Converting Enzyme (Caspase-1) Activity in Human Vascular Smooth Muscle Cells

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Young, James L., Sukhova, Galina K., Foster, Don, Kisiel, Walter, Libby, Peter, Schönbeck, Uwe·2000·The Journal of Experimental Medicine
DOI 10.1084/jem.191.9.1535PMC PMC2213432
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Abstract

Interleukin-1beta-converting enzyme (ICE, caspase-1) regulates key steps in inflammation and immunity, by activating the proinflammatory cytokines interleukin (IL-)1beta and IL-18, or mediating apoptotic processes. We recently provided evidence for the regulation of caspase-1 activity via an endogenous inhibitor expressed by human vascular smooth muscle cells (SMCs) (Schönbeck, U., M. Herzberg, A. Petersen, C. Wohlenberg, J. Gerdes, H.-D. Flad, and H. Loppnow. 1997. J. Exp. Med. 185:1287-1294). However, the molecular identity of this endogenous inhibitor remained undefined. We report here that the serine proteinase inhibitor (serpin) PI-9 accounts for the endogenous caspase-1 inhibitory activity in human SMCs and prevents processing of the enzyme's natural substrates, IL-1beta and IL-18 precursor. Treatment of SMC lysates with anti-PI-9 antibody abrogated the caspase-1 inhibitory activity and coprecipitated the enzyme, demonstrating protein-protein interaction. Furthermore, PI-9 antisense oligonucleotides coordinately reduced PI-9 expression and promoted IL-1beta release. Since SMCs comprise the majority of cells in the vascular wall, and because IL-1 is implicated in atherogenesis, we tested the biological validity of our in vitro findings within human atheroma in situ. The unaffected arterial wall contains abundant and homogeneously distributed PI-9. In human atherosclerotic lesions, however, PI-9 expression correlated inversely with immunoreactive IL-1beta, supporting a potential role of the endogenous caspase-1 inhibitor in this chronic inflammatory disease. Thus, our results provide new insights into the regulation of this enzyme involved in immune and inflammatory processes of chronic inflammatory diseases, and point to an endogenous antiinflammatory action of PI-9, dysregulated in a prevalent human disease.

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Figure 1 The human serpin PI-9 inhibits native and recombinant caspase-1 processing activity. (A) Human recombinant PI-9 (100 nM) was either preincubated for 30 min (−30) with native, monocyte-derived (Mφ-Lysates; equivalent to 106 Mφ/ml) or recombinant (rec.) caspase-1 (15 nM), applied simultaneously (0), or added 30 min after incubation (+30) of proIL-1β (20 nM) with the enzyme for 30 min at 37°C in a final volume of 50 μl processing buffer. (B) Human recombinant caspase-1 (15 nM, top) or human recombinant IL-1β precursor (20 nM, bottom) was preincubated for 30 min with the indicated concentrations of PI-9, before application to the processing assay. Processing was stopped by heating the samples in 10 μl SDS-PAGE sample buffer. The preparations were analyzed by 15% SDS-PAGE and subsequent Western blot analysis using anti–human IL-1β. For control purposes, recombinant mature (mIL-1β, 20 nM) and precursor (pIL-1β, 20 nM) IL-1β were applied. The positions of the molecular weight markers are indicated on the left (in kD). Similar data were obtained in three (A) or five (B) independent experiments.
Figure 1 The human serpin PI-9 inhibits native and recombinant caspase-1 processing activity. (A) Human recombinant PI-9 (100 nM) was either preincubated for 30 min (−30) with native, monocyte-derived (Mφ-Lysates; equivalent to 106 Mφ/ml) or recombinant (rec.) caspase-1 (15 nM), applied simultaneously (0), or added 30 min after incubation (+30) of proIL-1β (20 nM) with the enzyme for 30 min at 37°C in a final volume of 50 μl processing buffer. (B) Human recombinant caspase-1 (15 nM, top) or human recombinant IL-1β precursor (20 nM, bottom) was preincubated for 30 min with the indicated concentrations of PI-9, before application to the processing assay. Processing was stopped by heating the samples in 10 μl SDS-PAGE sample buffer. The preparations were analyzed by 15% SDS-PAGE and subsequent Western blot analysis using anti–human IL-1β. For control purposes, recombinant mature (mIL-1β, 20 nM) and precursor (pIL-1β, 20 nM) IL-1β were applied. The positions of the molecular weight markers are indicated on the left (in kD). Similar data were obtained in three (A) or five (B) independent experiments.CC BY-NC-SA · PubMed Central
Figure 2 PI-9 concentration-dependently inhibits recombinant caspase-1 processing activity. (A) Human recombinant caspase-1 (15 nM) was preincubated with the indicated concentrations of PI-9 for 30 min at 37°C, before being added for 30 min (37°C) to proIL-1β (20 nM) in 50 μl processing buffer. Reactions were stopped by heating the samples in 10 μl SDS-PAGE sample buffer, and the preparations were applied to 15% SDS-PAGE and subsequent Western blot analysis using anti–human IL-1β. For control purposes, recombinant mature and precursor IL-1β were applied in combination (m/pIL-1β, both at 20 nM). The positions of the molecular weight markers are indicated on the left (in kD). (B) Densitometric analysis of immunoreactive cleavage products obtained in the processing assays described in A. The IC50 was determined in reference to untreated recombinant proIL-1β (20 nM, 33-kD form) or mature IL-1β (20 nM, 17-kD form). Similar data were obtained in eight independent experiments.
Figure 2 PI-9 concentration-dependently inhibits recombinant caspase-1 processing activity. (A) Human recombinant caspase-1 (15 nM) was preincubated with the indicated concentrations of PI-9 for 30 min at 37°C, before being added for 30 min (37°C) to proIL-1β (20 nM) in 50 μl processing buffer. Reactions were stopped by heating the samples in 10 μl SDS-PAGE sample buffer, and the preparations were applied to 15% SDS-PAGE and subsequent Western blot analysis using anti–human IL-1β. For control purposes, recombinant mature and precursor IL-1β were applied in combination (m/pIL-1β, both at 20 nM). The positions of the molecular weight markers are indicated on the left (in kD). (B) Densitometric analysis of immunoreactive cleavage products obtained in the processing assays described in A. The IC50 was determined in reference to untreated recombinant proIL-1β (20 nM, 33-kD form) or mature IL-1β (20 nM, 17-kD form). Similar data were obtained in eight independent experiments.CC BY-NC-SA · PubMed Central
Figure 3 The human serpin PI-9 inhibits processing of the IL-18 precursor by caspase-1. Human recombinant caspase-1 (15 nM, top) or human recombinant IL-18 precursor (equivalent to 50,000/ml THP.1 cells) was preincubated for 30 min (37°C) with the indicated concentrations of PI-9, before either the substrate (IL-18, top) or the enzyme (bottom) was added (30 min, 37°C). Processing was stopped by heating the samples in 10 μl SDS-PAGE sample buffer. Samples were analyzed by 15% SDS-PAGE and subsequent Western blot analysis using anti–human IL-18. For control purposes, THP.1 lysate (THP.1; equivalent to 106 cells/ml) was applied. The positions of the molecular weight markers are indicated on the left (in kD). Similar data were obtained in three independent experiments.
Figure 3 The human serpin PI-9 inhibits processing of the IL-18 precursor by caspase-1. Human recombinant caspase-1 (15 nM, top) or human recombinant IL-18 precursor (equivalent to 50,000/ml THP.1 cells) was preincubated for 30 min (37°C) with the indicated concentrations of PI-9, before either the substrate (IL-18, top) or the enzyme (bottom) was added (30 min, 37°C). Processing was stopped by heating the samples in 10 μl SDS-PAGE sample buffer. Samples were analyzed by 15% SDS-PAGE and subsequent Western blot analysis using anti–human IL-18. For control purposes, THP.1 lysate (THP.1; equivalent to 106 cells/ml) was applied. The positions of the molecular weight markers are indicated on the left (in kD). Similar data were obtained in three independent experiments.CC BY-NC-SA · PubMed Central
Figure 4 Human vascular SMCs express PI-9 constitutively and in a cell-associated manner. (A) Lysates of SMCs, cultured for 24 h in IT medium in the absence (None) or presence of the respective concentrations of human recombinant mature IL-1β/TNF-α, were obtained by three freeze–thaw cycles. Lysates (Lys), equilibrated for total protein (50 μg/lane), as well as supernatants (SN, 50 μl; obtained from cultures stimulated with 30 ng/ml IL-1β/TNF-α) were analyzed by Western blotting with anti–human PI-9 antibody. (B) Similarly, lysates of peripheral blood mononuclear cells cultured for 1, 3, or 10 d (50 μg total protein/lane) were applied to Western blot analysis using anti–human PI-9 antibody. Recombinant PI-9 (recPI-9, 20 nM) was applied for control purposes. The positions of the molecular weight markers are indicated on the left (in kD). Similar data were obtained in three independent experiments.
Figure 4 Human vascular SMCs express PI-9 constitutively and in a cell-associated manner. (A) Lysates of SMCs, cultured for 24 h in IT medium in the absence (None) or presence of the respective concentrations of human recombinant mature IL-1β/TNF-α, were obtained by three freeze–thaw cycles. Lysates (Lys), equilibrated for total protein (50 μg/lane), as well as supernatants (SN, 50 μl; obtained from cultures stimulated with 30 ng/ml IL-1β/TNF-α) were analyzed by Western blotting with anti–human PI-9 antibody. (B) Similarly, lysates of peripheral blood mononuclear cells cultured for 1, 3, or 10 d (50 μg total protein/lane) were applied to Western blot analysis using anti–human PI-9 antibody. Recombinant PI-9 (recPI-9, 20 nM) was applied for control purposes. The positions of the molecular weight markers are indicated on the left (in kD). Similar data were obtained in three independent experiments.CC BY-NC-SA · PubMed Central
Figure 5 Human vascular SMCs depleted of endogenous PI-9 lack caspase-1 inhibitory activity. Human vascular SMCs were cultured for 24 h serum-free in IT medium, before being incubated for 24 h with fresh medium in the absence (None) or presence of human recombinant IL-1β/TNF-α (10/50 ng/ml). (A) Culture lysates of SMCs (equivalent to 107 cells/ml) were incubated with the anti–human PI-9 antibody (24 h, 4°C) and subsequently precipitated with protein A–Sepharose (500 g, 10 min). The remaining supernatants were incubated with human recombinant caspase-1 (15 nM; 1 h, 37°C) before being added to recombinant IL-1β precursor (20 nM) for 30 min at 37°C, and application to Western blot analysis using anti-human IL-1β. Combined mature (20 nM) and precursor (20 nM) IL-1β were applied as controls (m/pIL-1β). (B) Culture lysates of SMCs (equivalent to 107 cells/ml) were incubated with either affinity- or protein A–purified anti–human PI-9 or affinity-purified PI-8 antibody (24 h, 4°C), and subsequently precipitated with protein A–Sepharose (500 g, 10 min). The precipitates were applied to Western blot analysis using anti–human ICEP20. The positions of the molecular weight markers are indicated on the left (in kD). Similar data were obtained in three independent experiments, using SMC isolates of five different donors.
Figure 5 Human vascular SMCs depleted of endogenous PI-9 lack caspase-1 inhibitory activity. Human vascular SMCs were cultured for 24 h serum-free in IT medium, before being incubated for 24 h with fresh medium in the absence (None) or presence of human recombinant IL-1β/TNF-α (10/50 ng/ml). (A) Culture lysates of SMCs (equivalent to 107 cells/ml) were incubated with the anti–human PI-9 antibody (24 h, 4°C) and subsequently precipitated with protein A–Sepharose (500 g, 10 min). The remaining supernatants were incubated with human recombinant caspase-1 (15 nM; 1 h, 37°C) before being added to recombinant IL-1β precursor (20 nM) for 30 min at 37°C, and application to Western blot analysis using anti-human IL-1β. Combined mature (20 nM) and precursor (20 nM) IL-1β were applied as controls (m/pIL-1β). (B) Culture lysates of SMCs (equivalent to 107 cells/ml) were incubated with either affinity- or protein A–purified anti–human PI-9 or affinity-purified PI-8 antibody (24 h, 4°C), and subsequently precipitated with protein A–Sepharose (500 g, 10 min). The precipitates were applied to Western blot analysis using anti–human ICEP20. The positions of the molecular weight markers are indicated on the left (in kD). Similar data were obtained in three independent experiments, using SMC isolates of five different donors.CC BY-NC-SA · PubMed Central
Figure 6 PI-9 antisense treatment induces the release of IL-1β into the supernatant of vascular SMC cultures. Cultures of SMCs were treated with lipofectin (1 μg/ml; GIBCO BRL) and PI-9 antisense or scrambled (Scr) phosphorothioate oligodeoxynucleotides for 72 h in the absence (None) or presence of recombinant TNF-α (50 ng/ml) during the last 24 h. Lysates of these cultures were analyzed by Western blotting using anti–PI-9 (top) or anti–caspase-1 (bottom), whereas supernatants were assayed for IL-1β by ELISA (middle), using recombinant IL-1β (recIL-1β) as standard. The positions of the molecular weight markers are indicated on the left (in kD). Similar data were obtained in three independent experiments.
Figure 6 PI-9 antisense treatment induces the release of IL-1β into the supernatant of vascular SMC cultures. Cultures of SMCs were treated with lipofectin (1 μg/ml; GIBCO BRL) and PI-9 antisense or scrambled (Scr) phosphorothioate oligodeoxynucleotides for 72 h in the absence (None) or presence of recombinant TNF-α (50 ng/ml) during the last 24 h. Lysates of these cultures were analyzed by Western blotting using anti–PI-9 (top) or anti–caspase-1 (bottom), whereas supernatants were assayed for IL-1β by ELISA (middle), using recombinant IL-1β (recIL-1β) as standard. The positions of the molecular weight markers are indicated on the left (in kD). Similar data were obtained in three independent experiments.CC BY-NC-SA · PubMed Central
Figure 7 Differential expression of caspase-1 and PI-9 in human atherosclerotic lesions. Serial cryostat sections of frozen specimens of human nonatherosclerotic aorta (A–C) and carotid atheroma (D–G) were stained with (A and D) goat anti–human ICEP20 (1:100), (B and E) rabbit anti–human PI-9 (1:50), or (C and F) mouse anti–human IL-1β (1:100) antibody. (G) For colocalization of PI-9 (green) with caspase-1 (red), double-immunofluorescence staining was performed. The asterisk indicates the lumen of the vessels. (H) Frozen tissue from nonatherosclerotic (Normal) as well as stable or unstable atheromatous carotid plaques was analyzed by Western blotting using the anti–PI-9 (top) or anti–IL-1β (bottom) antibody. The positions of the molecular weight markers are indicated on the left (in kD). Analysis of tissue obtained from five nonatherosclerotic as well as five stable and seven vulnerable atherosclerotic surgical specimens of different donors showed similar results.
Figure 7 Differential expression of caspase-1 and PI-9 in human atherosclerotic lesions. Serial cryostat sections of frozen specimens of human nonatherosclerotic aorta (A–C) and carotid atheroma (D–G) were stained with (A and D) goat anti–human ICEP20 (1:100), (B and E) rabbit anti–human PI-9 (1:50), or (C and F) mouse anti–human IL-1β (1:100) antibody. (G) For colocalization of PI-9 (green) with caspase-1 (red), double-immunofluorescence staining was performed. The asterisk indicates the lumen of the vessels. (H) Frozen tissue from nonatherosclerotic (Normal) as well as stable or unstable atheromatous carotid plaques was analyzed by Western blotting using the anti–PI-9 (top) or anti–IL-1β (bottom) antibody. The positions of the molecular weight markers are indicated on the left (in kD). Analysis of tissue obtained from five nonatherosclerotic as well as five stable and seven vulnerable atherosclerotic surgical specimens of different donors showed similar results.CC BY-NC-SA · PubMed Central

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Research works printed but not linked: 2

  1. Ross, 1980, Morphogenesis of vascular smooth muscle in atherosclerosis and cell structure, 66
  2. Howard, 1991, IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha, J. Immunol., 147, 2964, 10.4049/jimmunol.147.9.2964 · DOI

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Metadata

Authors
Young, J. L., Sukhova, G. K., Foster, D., Kisiel, W., Libby, P., & Schönbeck, U.
Affiliations
  • Brigham and Women's Hospital (US)
  • bZymoGenetics, Incorporated, Seattle, Washington 93102
  • University of New Mexico (US)
ORCID
Venue
The Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
2000-05-01
Volume
191
Issue
9
Pages
1535-1544
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.191.9.1535
PMC
PMC2213432
OpenAlex
W2171074690
Cited by
133
References
58
Citations by year
2026: 1 · 2024: 1 · 2023: 1 · 2022: 1 · 2021: 4 · 2020: 6 · 2019: 6 · 2018: 1 · 2017: 5 · 2016: 2 · 2015: 3 · 2014: 1 · 2013: 10 · 2012: 7
FWCI
3.62
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (5r01hl034636-23)
  • National Heart, Lung, and Blood Institute (R37HL034636, R01-HL034636, HL-34636)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Inflammasome and immune disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Protease and Inhibitor Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
Keywords
caspase-1, IL-1β, IL-18, vascular smooth muscle cells, protein-protein interaction, atherosclerosis, inflammation, anti-inflammatory regulation, atherosclerotic plaque
MeSH terms
Arteries, Arteriosclerosis, Gene Expression Regulation, Humans, Interleukin-1, Interleukins, Muscle, Smooth, Vascular, Protein Precursors, Protein Processing, Post-Translational, Serpins, Interleukin-18, Caspase Inhibitors

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