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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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Comparison of the effect of type I and type II IFNs on the expression of caspase-12 and -3 by B16/B16 cells. (A) Western blot analysis of cells 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) or LPS (1 μg/ml). (B) Western blot analysis of cells treated for 24 h with IFN-β or -γ (1,000 IU/ml) in the presence or absence of dsRNA (100 μg/ml). The IFN-inducible kinase PKR was used as a positive control for responsiveness to the different IFNs. An anti–β-actin antibody was used to verify that equal amounts of protein were loaded.
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fig6: Comparison of the effect of type I and type II IFNs on the expression of caspase-12 and -3 by B16/B16 cells. (A) Western blot analysis of cells 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) or LPS (1 μg/ml). (B) Western blot analysis of cells treated for 24 h with IFN-β or -γ (1,000 IU/ml) in the presence or absence of dsRNA (100 μg/ml). The IFN-inducible kinase PKR was used as a positive control for responsiveness to the different IFNs. An anti–β-actin antibody was used to verify that equal amounts of protein were loaded.

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)

Comparison of the effect of type I and type II IFNs on the expression of caspase-12 and -3 by B16/B16 cells. (A) Western blot analysis of cells 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) or LPS (1 μg/ml). (B) Western blot analysis of cells treated for 24 h with IFN-β or -γ (1,000 IU/ml) in the presence or absence of dsRNA (100 μg/ml). The IFN-inducible kinase PKR was used as a positive control for responsiveness to the different IFNs. An anti–β-actin antibody was used to verify that equal amounts of protein were loaded.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Comparison of the effect of type I and type II IFNs on the expression of caspase-12 and -3 by B16/B16 cells. (A) Western blot analysis of cells 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) or LPS (1 μg/ml). (B) Western blot analysis of cells treated for 24 h with IFN-β or -γ (1,000 IU/ml) in the presence or absence of dsRNA (100 μg/ml). The IFN-inducible kinase PKR was used as a positive control for responsiveness to the different IFNs. An anti–β-actin antibody was used to verify that equal amounts of protein were loaded.
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