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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 dispensable for Tg-induced ER stress–mediated apoptosis in B16/B16 cells. (A) Analysis of the cytotoxic effect of Tg (2 μM) alone or in combination with IFN-γ (1,000 IU/ml) compared with TNF (5,000 IU/ml). Cell viability was measured at the indicated time using MTT. CTRL is untreated control. Error bars, standard deviation of six replicates of a representative experiment. (B) Schematic representation of pro–caspase-12 indicating the relative location of the peptides used to produce the anti–caspase-12 antisera Ab-2 and G149. Ab-2 was raised against a peptide spanning amino acids 2–17 of caspase-12 and is specific for the NH2-terminal prodomain. G149 was raised against a recombinant protein spanning amino acids 116–419 of caspase-12 and recognizes the catalytic part of the protein. LCSU and SCSU indicate the large and the small catalytic subunits of the caspase, respectively. (C) Western blot analysis of cells treated for 72 h with IFN-γ (1,000 IU/ml), TNF (5,000 IU/ml), or Tg (2 μM) 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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fig10: Caspase-12 is dispensable for Tg-induced ER stress–mediated apoptosis in B16/B16 cells. (A) Analysis of the cytotoxic effect of Tg (2 μM) alone or in combination with IFN-γ (1,000 IU/ml) compared with TNF (5,000 IU/ml). Cell viability was measured at the indicated time using MTT. CTRL is untreated control. Error bars, standard deviation of six replicates of a representative experiment. (B) Schematic representation of pro–caspase-12 indicating the relative location of the peptides used to produce the anti–caspase-12 antisera Ab-2 and G149. Ab-2 was raised against a peptide spanning amino acids 2–17 of caspase-12 and is specific for the NH2-terminal prodomain. G149 was raised against a recombinant protein spanning amino acids 116–419 of caspase-12 and recognizes the catalytic part of the protein. LCSU and SCSU indicate the large and the small catalytic subunits of the caspase, respectively. (C) Western blot analysis of cells treated for 72 h with IFN-γ (1,000 IU/ml), TNF (5,000 IU/ml), or Tg (2 μM) 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: Several reports have proposed that caspase-12 plays a major role in ER stress–induced apoptosis (Rao et al., 2001, 2002; Diaz-Horta et al., 2002; Morishima et al., 2002). As B16/B16 cells express caspase-12 only when they are treated with IFN-γ, we used these cells to test if caspase-12 is required for ER stress–mediated apoptosis induced by Tg. B16/B16 cells died in response to Tg in the presence or absence IFN-γ (Fig. 10 A). Light microscopy analysis revealed that in both cases, cells dying in response to Tg were blebbing and had the typical apoptotic morphology similar to that seen with HEK293T overexpressing caspase-12 (Fig. 8 A). Although IFN-γ treatment clearly sensitized the cells to TNF-induced cytotoxicity (Fig. 10 A), the effect of IFN-γ on Tg-induced cell death was minor. To analyze the expression and processing pattern of caspase-12 in response to Tg and compare it with that observed in IFN-γ + TNF–treated cells, we used two different anti–caspase-12 antisera. The first antiserum, Ab-2, was raised against a peptide spanning residues 2–17 in the amino acid sequence of caspase-12 and thus recognized fragments containing the prodomain of the protein, and the second antiserum, G149, was raised against the catalytic parts of the enzyme (Fig. 10 B). Both antisera detected the full-length 49-kD protein in cells treated with IFN-γ but failed to detect the expression of caspase-12 in cells treated with Tg alone (Fig. 10 C). The level of caspase-12 expression seen in cells cotreated with IFN-γ and Tg was lower than in cells treated with IFN-γ in the absence of Tg. This may be explained by a decrease in translation due to Tg treatment (Wong et al., 1993). Caspase-12 processing clearly occurred in cells treated with the combination of IFN-γ and TNF or Tg (Fig. 10 C). Ab-2 detected the 38-kD fragment of caspase-12 in cells treated with IFN-γ + TNF but failed to detect this fragment in cells treated with IFN-γ + Tg, whereas G149 detected the 28-kD fragment in both types of treatments (Fig. 10 C). These results suggest that the 38-kD fragment lacks only the COOH-terminal small catalytic subunit (SCSU), and that the 28-kD fragment is missing both the SCSU and the prodomain and, thus, probably corresponds to the mature large catalytic subunit (LCSU) (Fig. 10, B and C). Taken together, these results demonstrate that caspase-12 is not required for Tg-induced cell death, although treatment with Tg can induce the proteolytic cleavage of the protease.


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 dispensable for Tg-induced ER stress–mediated apoptosis in B16/B16 cells. (A) Analysis of the cytotoxic effect of Tg (2 μM) alone or in combination with IFN-γ (1,000 IU/ml) compared with TNF (5,000 IU/ml). Cell viability was measured at the indicated time using MTT. CTRL is untreated control. Error bars, standard deviation of six replicates of a representative experiment. (B) Schematic representation of pro–caspase-12 indicating the relative location of the peptides used to produce the anti–caspase-12 antisera Ab-2 and G149. Ab-2 was raised against a peptide spanning amino acids 2–17 of caspase-12 and is specific for the NH2-terminal prodomain. G149 was raised against a recombinant protein spanning amino acids 116–419 of caspase-12 and recognizes the catalytic part of the protein. LCSU and SCSU indicate the large and the small catalytic subunits of the caspase, respectively. (C) Western blot analysis of cells treated for 72 h with IFN-γ (1,000 IU/ml), TNF (5,000 IU/ml), or Tg (2 μM) 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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fig10: Caspase-12 is dispensable for Tg-induced ER stress–mediated apoptosis in B16/B16 cells. (A) Analysis of the cytotoxic effect of Tg (2 μM) alone or in combination with IFN-γ (1,000 IU/ml) compared with TNF (5,000 IU/ml). Cell viability was measured at the indicated time using MTT. CTRL is untreated control. Error bars, standard deviation of six replicates of a representative experiment. (B) Schematic representation of pro–caspase-12 indicating the relative location of the peptides used to produce the anti–caspase-12 antisera Ab-2 and G149. Ab-2 was raised against a peptide spanning amino acids 2–17 of caspase-12 and is specific for the NH2-terminal prodomain. G149 was raised against a recombinant protein spanning amino acids 116–419 of caspase-12 and recognizes the catalytic part of the protein. LCSU and SCSU indicate the large and the small catalytic subunits of the caspase, respectively. (C) Western blot analysis of cells treated for 72 h with IFN-γ (1,000 IU/ml), TNF (5,000 IU/ml), or Tg (2 μM) 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: Several reports have proposed that caspase-12 plays a major role in ER stress–induced apoptosis (Rao et al., 2001, 2002; Diaz-Horta et al., 2002; Morishima et al., 2002). As B16/B16 cells express caspase-12 only when they are treated with IFN-γ, we used these cells to test if caspase-12 is required for ER stress–mediated apoptosis induced by Tg. B16/B16 cells died in response to Tg in the presence or absence IFN-γ (Fig. 10 A). Light microscopy analysis revealed that in both cases, cells dying in response to Tg were blebbing and had the typical apoptotic morphology similar to that seen with HEK293T overexpressing caspase-12 (Fig. 8 A). Although IFN-γ treatment clearly sensitized the cells to TNF-induced cytotoxicity (Fig. 10 A), the effect of IFN-γ on Tg-induced cell death was minor. To analyze the expression and processing pattern of caspase-12 in response to Tg and compare it with that observed in IFN-γ + TNF–treated cells, we used two different anti–caspase-12 antisera. The first antiserum, Ab-2, was raised against a peptide spanning residues 2–17 in the amino acid sequence of caspase-12 and thus recognized fragments containing the prodomain of the protein, and the second antiserum, G149, was raised against the catalytic parts of the enzyme (Fig. 10 B). Both antisera detected the full-length 49-kD protein in cells treated with IFN-γ but failed to detect the expression of caspase-12 in cells treated with Tg alone (Fig. 10 C). The level of caspase-12 expression seen in cells cotreated with IFN-γ and Tg was lower than in cells treated with IFN-γ in the absence of Tg. This may be explained by a decrease in translation due to Tg treatment (Wong et al., 1993). Caspase-12 processing clearly occurred in cells treated with the combination of IFN-γ and TNF or Tg (Fig. 10 C). Ab-2 detected the 38-kD fragment of caspase-12 in cells treated with IFN-γ + TNF but failed to detect this fragment in cells treated with IFN-γ + Tg, whereas G149 detected the 28-kD fragment in both types of treatments (Fig. 10 C). These results suggest that the 38-kD fragment lacks only the COOH-terminal small catalytic subunit (SCSU), and that the 28-kD fragment is missing both the SCSU and the prodomain and, thus, probably corresponds to the mature large catalytic subunit (LCSU) (Fig. 10, B and C). Taken together, these results demonstrate that caspase-12 is not required for Tg-induced cell death, although treatment with Tg can induce the proteolytic cleavage of the protease.

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