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Regulation of the expression and processing of caspase-12.

Kalai M, Lamkanfi M, Denecker G, Boogmans M, Lippens S, Meeus A, Declercq W, Vandenabeele P - J. Cell Biol. (2003)

Bottom Line: The effect is increased further when IFN-gamma is combined with TNF, lipopolysaccharide (LPS), or dsRNA.Transient overexpression of full-length caspase-12 leads to proteolytic processing of the enzyme and apoptosis.Similar processing occurs in TNF-, LPS-, Fas ligand-, and thapsigargin (Tg)-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biomedical Research, Unit of Molecular Signalling and Cell Death, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium.

ABSTRACT
Phylogenetic analysis clusters caspase-12 with the inflammatory caspases 1 and 11. We analyzed the expression of caspase-12 in mouse embryos, adult organs, and different cell types and tested the effect of interferons (IFNs) and other proinflammatory stimuli. Constitutive expression of the caspase-12 protein was restricted to certain cell types, such as epithelial cells, primary fibroblasts, and L929 fibrosarcoma cells. In fibroblasts and B16/B16 melanoma cells, caspase-12 expression is stimulated by IFN-gamma but not by IFN-alpha or -beta. The effect is increased further when IFN-gamma is combined with TNF, lipopolysaccharide (LPS), or dsRNA. These stimuli also induce caspase-1 and -11 but inhibit the expression of caspase-3 and -9. In contrast to caspase-1 and -11, no caspase-12 protein was detected in macrophages in any of these treatments. Transient overexpression of full-length caspase-12 leads to proteolytic processing of the enzyme and apoptosis. Similar processing occurs in TNF-, LPS-, Fas ligand-, and thapsigargin (Tg)-induced apoptosis. However, B16/B16 melanoma cells die when treated with the ER stress-inducing agent Tg whether they express caspase-12 or not.

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Analysis of the expression of caspase-12 in macrophages and primary fibroblasts. (A) Western blot analysis of Mf4/4 macrophages treated for 24 h with IFN-α, -β or, -γ (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml), LPS (1 μg/ml), or dsRNA (50 μg/ml). C-12 indicates a lysate of HEK293T cells overexpressing caspase-12, used as a positive control. *, nonspecific bands. (B) Western blot analysis of primary fibroblasts and peritoneal macrophages treated for 24 h with IFN-γ (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml) or LPS (1 μg/ml). B16/B16 cells treated or not with IFN-γ (1,000 IU/ml) were used as a positive control. An anti–β-actin antibody was used to verify that equal amounts of protein were loaded.
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fig7: Analysis of the expression of caspase-12 in macrophages and primary fibroblasts. (A) Western blot analysis of Mf4/4 macrophages treated for 24 h with IFN-α, -β or, -γ (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml), LPS (1 μg/ml), or dsRNA (50 μg/ml). C-12 indicates a lysate of HEK293T cells overexpressing caspase-12, used as a positive control. *, nonspecific bands. (B) Western blot analysis of primary fibroblasts and peritoneal macrophages treated for 24 h with IFN-γ (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml) or LPS (1 μg/ml). B16/B16 cells treated or not with IFN-γ (1,000 IU/ml) were used as a positive control. An anti–β-actin antibody was used to verify that equal amounts of protein were loaded.

Mentions: Macrophages play a major role in the response to both viral and bacterial infection and thus present a cellular model that is highly relevant for inflammation (Stoy, 2001). Moreover, macrophages express caspase-1 and -11 and secrete activated IL-1β in response to infection and proinflammatory stimuli (Lin et al., 2000; Schauvliege et al., 2002). Therefore, we determined the effects of the three IFNs alone and of IFN-γ in combination with LPS, TNF, or dsRNA on the protein expression levels of caspase-1, -11, and -12 in the Mf4/4 murine macrophage cell line. IFN-γ, and to a lesser extent also LPS and IFN-β, clearly induced the expression of caspase-11. The expression of caspase-1 remained high and stable (Fig. 7 A). However, no caspase-12–specific bands were detected in Mf4/4 cells in any of the tested conditions (Fig. 7 A). Similar results were obtained with J774 and pU518, two other murine macrophage cell lines (unpublished data) and primary peritoneal macrophages (Fig. 7 B). The nonspecific bands detected in Fig. 7 A appeared after a long exposure of the blot to the film. Similar bands were detected by the G149 antiserum in HEK293T cells that do not express murine caspase-12. Moreover, these bands were not detected using two other distinct anti–caspase-12 antisera (unpublished data).


Regulation of the expression and processing of caspase-12.

Kalai M, Lamkanfi M, Denecker G, Boogmans M, Lippens S, Meeus A, Declercq W, Vandenabeele P - J. Cell Biol. (2003)

Analysis of the expression of caspase-12 in macrophages and primary fibroblasts. (A) Western blot analysis of Mf4/4 macrophages treated for 24 h with IFN-α, -β or, -γ (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml), LPS (1 μg/ml), or dsRNA (50 μg/ml). C-12 indicates a lysate of HEK293T cells overexpressing caspase-12, used as a positive control. *, nonspecific bands. (B) Western blot analysis of primary fibroblasts and peritoneal macrophages treated for 24 h with IFN-γ (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml) or LPS (1 μg/ml). B16/B16 cells treated or not with IFN-γ (1,000 IU/ml) were used as a positive control. An anti–β-actin antibody was used to verify that equal amounts of protein were loaded.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172698&req=5

fig7: Analysis of the expression of caspase-12 in macrophages and primary fibroblasts. (A) Western blot analysis of Mf4/4 macrophages treated for 24 h with IFN-α, -β or, -γ (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml), LPS (1 μg/ml), or dsRNA (50 μg/ml). C-12 indicates a lysate of HEK293T cells overexpressing caspase-12, used as a positive control. *, nonspecific bands. (B) Western blot analysis of primary fibroblasts and peritoneal macrophages treated for 24 h with IFN-γ (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml) or LPS (1 μg/ml). B16/B16 cells treated or not with IFN-γ (1,000 IU/ml) were used as a positive control. An anti–β-actin antibody was used to verify that equal amounts of protein were loaded.
Mentions: Macrophages play a major role in the response to both viral and bacterial infection and thus present a cellular model that is highly relevant for inflammation (Stoy, 2001). Moreover, macrophages express caspase-1 and -11 and secrete activated IL-1β in response to infection and proinflammatory stimuli (Lin et al., 2000; Schauvliege et al., 2002). Therefore, we determined the effects of the three IFNs alone and of IFN-γ in combination with LPS, TNF, or dsRNA on the protein expression levels of caspase-1, -11, and -12 in the Mf4/4 murine macrophage cell line. IFN-γ, and to a lesser extent also LPS and IFN-β, clearly induced the expression of caspase-11. The expression of caspase-1 remained high and stable (Fig. 7 A). However, no caspase-12–specific bands were detected in Mf4/4 cells in any of the tested conditions (Fig. 7 A). Similar results were obtained with J774 and pU518, two other murine macrophage cell lines (unpublished data) and primary peritoneal macrophages (Fig. 7 B). The nonspecific bands detected in Fig. 7 A appeared after a long exposure of the blot to the film. Similar bands were detected by the G149 antiserum in HEK293T cells that do not express murine caspase-12. Moreover, these bands were not detected using two other distinct anti–caspase-12 antisera (unpublished data).

Bottom Line: The effect is increased further when IFN-gamma is combined with TNF, lipopolysaccharide (LPS), or dsRNA.Transient overexpression of full-length caspase-12 leads to proteolytic processing of the enzyme and apoptosis.Similar processing occurs in TNF-, LPS-, Fas ligand-, and thapsigargin (Tg)-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biomedical Research, Unit of Molecular Signalling and Cell Death, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium.

ABSTRACT
Phylogenetic analysis clusters caspase-12 with the inflammatory caspases 1 and 11. We analyzed the expression of caspase-12 in mouse embryos, adult organs, and different cell types and tested the effect of interferons (IFNs) and other proinflammatory stimuli. Constitutive expression of the caspase-12 protein was restricted to certain cell types, such as epithelial cells, primary fibroblasts, and L929 fibrosarcoma cells. In fibroblasts and B16/B16 melanoma cells, caspase-12 expression is stimulated by IFN-gamma but not by IFN-alpha or -beta. The effect is increased further when IFN-gamma is combined with TNF, lipopolysaccharide (LPS), or dsRNA. These stimuli also induce caspase-1 and -11 but inhibit the expression of caspase-3 and -9. In contrast to caspase-1 and -11, no caspase-12 protein was detected in macrophages in any of these treatments. Transient overexpression of full-length caspase-12 leads to proteolytic processing of the enzyme and apoptosis. Similar processing occurs in TNF-, LPS-, Fas ligand-, and thapsigargin (Tg)-induced apoptosis. However, B16/B16 melanoma cells die when treated with the ER stress-inducing agent Tg whether they express caspase-12 or not.

Show MeSH
Related in: MedlinePlus