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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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Caspase-12 is processed in B16/B16 cells dying from the combination of IFN-γ with either LPS or TNF. (A) Analysis of the cytotoxic potential of IFN-γ, TNF, or LPS. Cells were treated with IFN-γ (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 μg/ml) alone or in combinations. Cell viability was measured at the indicated time using MTT. CTRL indicates untreated controls. Error bars, standard deviation of six replicates of a representative experiment. (B) Western blot analysis of cells treated for 48 h with IFN-γ (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 μg/ml) alone or in combinations. C-12 indicates lysate of HEK293T cells overexpressing caspase-12, used as a positive control. Arrows indicate bands corresponding to the 49-kD full-length pro–caspase-12 and to the 38- and 28-kD fragments of the processed caspase. *, nonspecific bands.
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fig9: Caspase-12 is processed in B16/B16 cells dying from the combination of IFN-γ with either LPS or TNF. (A) Analysis of the cytotoxic potential of IFN-γ, TNF, or LPS. Cells were treated with IFN-γ (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 μg/ml) alone or in combinations. Cell viability was measured at the indicated time using MTT. CTRL indicates untreated controls. Error bars, standard deviation of six replicates of a representative experiment. (B) Western blot analysis of cells treated for 48 h with IFN-γ (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 μg/ml) alone or in combinations. C-12 indicates lysate of HEK293T cells overexpressing caspase-12, used as a positive control. Arrows indicate bands corresponding to the 49-kD full-length pro–caspase-12 and to the 38- and 28-kD fragments of the processed caspase. *, nonspecific bands.

Mentions: Transient overexpression of full-length wild-type caspase-12, but not of its inactive C298A mutant, in HEK293T cells led to processing of the protein and to apoptosis (Fig. 2 B; Fig. 8 A; Fig. 9 B). Processing of caspase-12 in these conditions resulted in fragments of ∼38 and 28 kD. These results suggest that a link exists between the processing of caspase-12 and the induction of an apoptotic cell death. In rapid apoptosis occurring in L929sAhFas cells treated with FasL, caspase-12 was fully processed, and only the 28-kD fragment was observed (Fig. 8 B). The processing of caspase-12 in the latter cells coincided with the activation of caspase-3, -7, and -9 (Fig. 8 B). Treatment of B16/B16 cells with TNF alone had no effect on their growth, whereas treatment with either IFN-γ or LPS alone led to a decrease in the proliferation rate without any apparent signs of cell death (Fig. 9 A). However, light microscopy analysis demonstrated that cells treated with a combination of IFN-γ with LPS or TNF rounded up and started floating and eventually died and disintegrated (Fig. 9 A; unpublished data). We tested whether caspase-12 is also processed in this kind of cell death. As seen before (Figs. 5 and 6), the protein was highly expressed in all the conditions that included IFN-γ. However, processing of caspase-12 was detected only when the cells were treated with the combination of IFN-γ with either LPS or TNF (Fig. 9 B). These results show that caspase-12 is processed in different proapoptotic conditions, including those induced by TNF, LPS, and FasL, and suggest that it may actively contribute to the cell death process.


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)

Caspase-12 is processed in B16/B16 cells dying from the combination of IFN-γ with either LPS or TNF. (A) Analysis of the cytotoxic potential of IFN-γ, TNF, or LPS. Cells were treated with IFN-γ (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 μg/ml) alone or in combinations. Cell viability was measured at the indicated time using MTT. CTRL indicates untreated controls. Error bars, standard deviation of six replicates of a representative experiment. (B) Western blot analysis of cells treated for 48 h with IFN-γ (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 μg/ml) alone or in combinations. C-12 indicates lysate of HEK293T cells overexpressing caspase-12, used as a positive control. Arrows indicate bands corresponding to the 49-kD full-length pro–caspase-12 and to the 38- and 28-kD fragments of the processed caspase. *, nonspecific bands.
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Related In: Results  -  Collection

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fig9: Caspase-12 is processed in B16/B16 cells dying from the combination of IFN-γ with either LPS or TNF. (A) Analysis of the cytotoxic potential of IFN-γ, TNF, or LPS. Cells were treated with IFN-γ (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 μg/ml) alone or in combinations. Cell viability was measured at the indicated time using MTT. CTRL indicates untreated controls. Error bars, standard deviation of six replicates of a representative experiment. (B) Western blot analysis of cells treated for 48 h with IFN-γ (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 μg/ml) alone or in combinations. C-12 indicates lysate of HEK293T cells overexpressing caspase-12, used as a positive control. Arrows indicate bands corresponding to the 49-kD full-length pro–caspase-12 and to the 38- and 28-kD fragments of the processed caspase. *, nonspecific bands.
Mentions: Transient overexpression of full-length wild-type caspase-12, but not of its inactive C298A mutant, in HEK293T cells led to processing of the protein and to apoptosis (Fig. 2 B; Fig. 8 A; Fig. 9 B). Processing of caspase-12 in these conditions resulted in fragments of ∼38 and 28 kD. These results suggest that a link exists between the processing of caspase-12 and the induction of an apoptotic cell death. In rapid apoptosis occurring in L929sAhFas cells treated with FasL, caspase-12 was fully processed, and only the 28-kD fragment was observed (Fig. 8 B). The processing of caspase-12 in the latter cells coincided with the activation of caspase-3, -7, and -9 (Fig. 8 B). Treatment of B16/B16 cells with TNF alone had no effect on their growth, whereas treatment with either IFN-γ or LPS alone led to a decrease in the proliferation rate without any apparent signs of cell death (Fig. 9 A). However, light microscopy analysis demonstrated that cells treated with a combination of IFN-γ with LPS or TNF rounded up and started floating and eventually died and disintegrated (Fig. 9 A; unpublished data). We tested whether caspase-12 is also processed in this kind of cell death. As seen before (Figs. 5 and 6), the protein was highly expressed in all the conditions that included IFN-γ. However, processing of caspase-12 was detected only when the cells were treated with the combination of IFN-γ with either LPS or TNF (Fig. 9 B). These results show that caspase-12 is processed in different proapoptotic conditions, including those induced by TNF, LPS, and FasL, and suggest that it may actively contribute to the cell death process.

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