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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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Processing of caspase-12 in apoptosis induced by overexpression of the full-length protein in HEK293T cells and in L929sAhFas cells treated with FasL. (A) HEK293T cells were transiently cotransfected with a plasmid encoding for a nuclear localization signal containing GFP and a plasmid encoding for either the wild type (C-12 wt) or an inactive caspase-12 mutant (C-12 C298A). Fluorescent microscopy photographs and Western blot analysis demonstrate that although both of the caspase-12 variants are expressed in equal levels, only the wild-type protein is processed and leads to apoptotic cell death. Bar, 5 μm. Arrows indicate fragments corresponding to caspase-12 and their size in kD. (B) L929sAhFas cells were treated with recombinant soluble FasL for the indicated time. Cytotoxicity was measured by trypan blue (TB) exclusion. CTRL indicates untreated controls. Processing of caspase-3, -7, -9, and -12 after a 2-h treatment with FasL was determined by Western blotting. Arrows indicate the proteolytically processed caspase fragments and their size in kD.
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fig8: Processing of caspase-12 in apoptosis induced by overexpression of the full-length protein in HEK293T cells and in L929sAhFas cells treated with FasL. (A) HEK293T cells were transiently cotransfected with a plasmid encoding for a nuclear localization signal containing GFP and a plasmid encoding for either the wild type (C-12 wt) or an inactive caspase-12 mutant (C-12 C298A). Fluorescent microscopy photographs and Western blot analysis demonstrate that although both of the caspase-12 variants are expressed in equal levels, only the wild-type protein is processed and leads to apoptotic cell death. Bar, 5 μm. Arrows indicate fragments corresponding to caspase-12 and their size in kD. (B) L929sAhFas cells were treated with recombinant soluble FasL for the indicated time. Cytotoxicity was measured by trypan blue (TB) exclusion. CTRL indicates untreated controls. Processing of caspase-3, -7, -9, and -12 after a 2-h treatment with FasL was determined by Western blotting. Arrows indicate the proteolytically processed caspase fragments and their size in kD.

Mentions: Using Western blot analysis, we determined if the induction of caspase-12 mRNA expression in B16/B16 cells was also reflected at the protein level and compared the effect with that observed with other caspases (Fig. 5). Treatment with IFN-γ increased the expression of caspase-1, -11, and -12. When the treatment with IFN-γ was combined with either TNF or LPS, the effect on the expression of caspase-1, -11, and -12 was intensified (Fig. 5; Fig. 6 A). In contrast to the caspases mentioned above, IFN-γ treatment led to a decrease in the expression levels of caspase-3 and -9. The disappearance of the full-length form of these caspases (Fig. 5) in B16/B16 cells was not due to proteolytic processing because no proteolytic fragments of caspase-3 and -9 were detected. The antisera used reveal specific proteolytic processing of caspase-3 and -9 during apoptosis (Fig. 8). As expected, IFN-γ also increased the expression of the IFN-inducible dsRNA-activated protein kinase (PKR), which we used as a positive control (Baier et al., 1993). No effect was observed on the level of β-actin used as internal negative control. Treatment with either TNF or LPS alone did not affect the expression levels of any of the proteins. We observed a similar effect of IFN-γ with and without TNF or LPS on caspase-12 expression in another murine melanoma cell line (PG19; unpublished data). These results suggest that the activation of the gene coding for caspase-12 is tightly regulated. Interestingly, the distinct effects on the expression of the different caspases intensified in time, suggesting that the expression of caspase-1, -11, and -12 was induced, whereas that of the proapoptotic caspases 3 and 9 was suppressed (Fig. 5).


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)

Processing of caspase-12 in apoptosis induced by overexpression of the full-length protein in HEK293T cells and in L929sAhFas cells treated with FasL. (A) HEK293T cells were transiently cotransfected with a plasmid encoding for a nuclear localization signal containing GFP and a plasmid encoding for either the wild type (C-12 wt) or an inactive caspase-12 mutant (C-12 C298A). Fluorescent microscopy photographs and Western blot analysis demonstrate that although both of the caspase-12 variants are expressed in equal levels, only the wild-type protein is processed and leads to apoptotic cell death. Bar, 5 μm. Arrows indicate fragments corresponding to caspase-12 and their size in kD. (B) L929sAhFas cells were treated with recombinant soluble FasL for the indicated time. Cytotoxicity was measured by trypan blue (TB) exclusion. CTRL indicates untreated controls. Processing of caspase-3, -7, -9, and -12 after a 2-h treatment with FasL was determined by Western blotting. Arrows indicate the proteolytically processed caspase fragments and their size in kD.
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fig8: Processing of caspase-12 in apoptosis induced by overexpression of the full-length protein in HEK293T cells and in L929sAhFas cells treated with FasL. (A) HEK293T cells were transiently cotransfected with a plasmid encoding for a nuclear localization signal containing GFP and a plasmid encoding for either the wild type (C-12 wt) or an inactive caspase-12 mutant (C-12 C298A). Fluorescent microscopy photographs and Western blot analysis demonstrate that although both of the caspase-12 variants are expressed in equal levels, only the wild-type protein is processed and leads to apoptotic cell death. Bar, 5 μm. Arrows indicate fragments corresponding to caspase-12 and their size in kD. (B) L929sAhFas cells were treated with recombinant soluble FasL for the indicated time. Cytotoxicity was measured by trypan blue (TB) exclusion. CTRL indicates untreated controls. Processing of caspase-3, -7, -9, and -12 after a 2-h treatment with FasL was determined by Western blotting. Arrows indicate the proteolytically processed caspase fragments and their size in kD.
Mentions: Using Western blot analysis, we determined if the induction of caspase-12 mRNA expression in B16/B16 cells was also reflected at the protein level and compared the effect with that observed with other caspases (Fig. 5). Treatment with IFN-γ increased the expression of caspase-1, -11, and -12. When the treatment with IFN-γ was combined with either TNF or LPS, the effect on the expression of caspase-1, -11, and -12 was intensified (Fig. 5; Fig. 6 A). In contrast to the caspases mentioned above, IFN-γ treatment led to a decrease in the expression levels of caspase-3 and -9. The disappearance of the full-length form of these caspases (Fig. 5) in B16/B16 cells was not due to proteolytic processing because no proteolytic fragments of caspase-3 and -9 were detected. The antisera used reveal specific proteolytic processing of caspase-3 and -9 during apoptosis (Fig. 8). As expected, IFN-γ also increased the expression of the IFN-inducible dsRNA-activated protein kinase (PKR), which we used as a positive control (Baier et al., 1993). No effect was observed on the level of β-actin used as internal negative control. Treatment with either TNF or LPS alone did not affect the expression levels of any of the proteins. We observed a similar effect of IFN-γ with and without TNF or LPS on caspase-12 expression in another murine melanoma cell line (PG19; unpublished data). These results suggest that the activation of the gene coding for caspase-12 is tightly regulated. Interestingly, the distinct effects on the expression of the different caspases intensified in time, suggesting that the expression of caspase-1, -11, and -12 was induced, whereas that of the proapoptotic caspases 3 and 9 was suppressed (Fig. 5).

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