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Regulation of the expression and processing of caspase-12

Regulation of the expression and processing of caspase-12 JCB Article Regulation of the expression and processing of caspase-12 Michael Kalai, Mohamed Lamkanfi, Geertrui Denecker, Michael Boogmans, Saskia Lippens, Ann Meeus, Wim Declercq, and Peter Vandenabeele Department of Molecular Biomedical Research, Unit of Molecular Signalling and Cell Death, VIB and Ghent University, B-9000 Ghent, Belgium hylogenetic analysis clusters caspase-12 with the in- These stimuli also induce caspase-1 and -11 but inhibit the flammatory caspases 1 and 11. We analyzed the expres- expression of caspase-3 and -9. In contrast to caspase-1 P sion of caspase-12 in mouse embryos, adult organs, and -11, no caspase-12 protein was detected in macro- and different cell types and tested the effect of interferons phages in any of these treatments. Transient overexpression (IFNs) and other proinflammatory stimuli. Constitutive expres- of full-length caspase-12 leads to proteolytic processing of sion of the caspase-12 protein was restricted to certain cell the enzyme and apoptosis. Similar processing occurs in types, such as epithelial cells, primary fibroblasts, and L929 TNF-, LPS-, Fas ligand–, and thapsigargin (Tg)-induced fibrosarcoma cells. In fibroblasts and B16/B16 melanoma apoptosis. However, B16/B16 melanoma cells die when cells, caspase-12 expression is stimulated by IFN- but not treated with the ER stress–inducing agent Tg whether they by IFN- or -. The effect is increased further when IFN- express caspase-12 or not. is combined with TNF, lipopolysaccharide (LPS), or dsRNA. Introduction Caspases are a family of cysteinyl aspartate proteinases central and Yang, 2000). The final outcome of these cascades is the in mediating cellular signals in apoptosis and inflammation specific proteolysis of a wide variety of substrates, leading either (Lamkanfi et al., 2003). The enzymes are produced as zy- to the loss of a life-saving function or the activation of mogens consisting of a NH -terminal prodomain of variable a proapoptotic or proinflammatory function (Chang and length followed by a large and a small catalytic subunit. The Yang, 2000; Lamkanfi et al., 2003). large prodomains of certain members of this protein family, So far, 11 human and 10 murine caspases were identified referred to as initiator caspases (e.g., caspase-8, -9, and -10), (Chang and Yang, 2000; Lamkanfi et al., 2002). Phylogenetic contain interaction motifs consisting of six or seven antiparallel analysis reveals that caspases segregate into three major amphipathic  helices. These prodomains were shown to allow groups with different functions and substrate specificities. the recruitment and proximity-induced activation of the The three groups are often referred to as inflammatory caspases, enzymes in protein complexes (Stennicke and Salvesen, apoptotic executioner caspases, and apoptotic initiator caspases 2000). Caspases are usually activated by proteolysis that (Lamkanfi et al., 2002). Caspase-12 segregates with the inflam- leads to conformational changes of their catalytic parts matory caspases including caspase-1, -4, -5, and -11 (Chang (Riedl et al., 2001). The mature caspase is a heterotetramer and Yang, 2000; Lamkanfi et al., 2002). The amino acid consisting of two large and two small catalytic subunits sequence identity of murine caspase-12 to the other members aligned in a head-to-tail configuration and contains an active of this group is as follows: murine caspase-1 (42%), human site in each of the opposite ends. Caspases typically signal caspase-1 (40%), murine caspase-11 (41%), human caspase-4 through proteolytic cascades in which initiator caspases (48%), and caspase-5 (45%). These caspases seem to be cleave and activate executioner caspases that lack a large pro- involved more in the maturation and secretion of proin- domain (Slee et al., 1999; Van de Craen et al., 1999; Chang flammatory cytokines such as interleukin (IL)-1 and IL-18 than in the induction of death receptor–mediated apoptosis Address correspondence to Peter Vandenabeele, Department of Molecular (Martinon et al., 2002). All of the caspases included in the Biomedical Research, Unit of Molecular Signalling and Cell Death, Ghent group have a large prodomain containing a typical caspase University, Ledeganckstraat 35, B-9000 Ghent, Belgium. Tel.: 32-9-264-51- 31. Fax: 32-9-264-53-48. email: [email protected] Abbreviations used in this paper: FasL, Fas ligand; IFN, interferon; IL, Key words: apoptosis; caspase-12; inflammation; interferon; ER stress interleukin; LPS, lipopolysaccharide; Tg, thapsigargin.  The Rockefeller University Press, 0021-9525/2003/08/457/11 $8.00 The Journal of Cell Biology, Volume 162, Number 3, August 4, 2003 457–467 http://www.jcb.org/cgi/doi/10.1083/jcb.200303157 457 The Journal of Cell Biology 458 The Journal of Cell Biology | Volume 162, Number 3, 2003 recruitment domain (CARD). The genes encoding the mu- rine members of the group are located on chromosome 9 in the following order: caspase-1, caspase-11, and caspase-12. The genes encoding their human counterparts reside on chromosome 11q22, a region with synteny to murine chro- mosome 9, and are arranged as follows from telomere to cen- tromere: caspase-1, caspase-5, caspase-4, and finally a gene encoding a pseudo caspase-12 (Fischer et al., 2002). The predicted product of the latter gene is reported to be a cas- pase-12–like protein with a premature stop and several point mutations preventing the translated protein from having any caspase-like enzymatic activity (Fischer et al., 2002). Studies in caspase-12–deficient mice suggested that the protein plays a major role in ER stress–induced apoptosis and in the devel- opment of Alzheimer’s disease (Nakagawa et al., 2000). Since then, several other reports have linked processing of caspase-12 to ER stress–induced apoptosis (Rao et al., 2001, 2002; Diaz-Horta et al., 2002; Morishima et al., 2002). In addition, caspase-12 seems to be involved in apoptosis in- duced by viral infection (Bitko and Barik, 2001; Jordan et al., 2002) or by serum starvation (Kilic et al., 2002). Several reports have shown that the expression of certain caspases is induced during inflammation. Treatment with interferon (IFN)- increases the expression of caspase-8 in many types of cells, thus sensitizing the cells to death do- main receptor–mediated apoptosis (Dai and Krantz, 1999; Ruiz-Ruiz et al., 2000; Fulda and Debatin, 2002). Both IFN- and lipopolysaccharide (LPS) induce the expression of human caspase-1, human caspase-5, and murine caspase- 11 but not of human caspase-4 (Tamura et al., 1996; Dai Figure 1. Differential gene expression patterns of the mouse inflammatory caspase subfamily. Single-stranded cDNAs from and Krantz, 1999; Lin et al., 2000; Hur et al., 2001; Schauv- multiple mouse tissues (panel I and II) were amplified by PCR using liege et al., 2002). Although phylogenetic analysis suggests primers specific for caspase-1, -11, or -12. PCR for glyceraldehyde-3- that caspase-12 resembles other inflammatory caspases, it is phosphate dehydrogenase (G3DPH) was performed to ensure that still unclear whether the regulation and processing of this an equal quality and quantity of cDNA was used. Negative () and caspase is modulated in inflammation. Therefore, we ana- positive () PCR controls (CTRL) lacking or containing the specific cDNAs, respectively. (A) Expression of caspase-1, -11, and -12 lyzed the constitutive expression pattern of caspase-1, -11, mRNA during mouse embryonic development. (B) Expression of and -12 in mouse organs and during embryonic develop- caspase-1, -11, and -12 mRNA in different mouse organs. ment. We also tested the effect of IFNs and other proinflam- matory stimuli on the expression of caspase-12 in several cell lines both at the mRNA and protein levels. We compared caspase-12 by RT-PCR during embryonic development and these effects with those observed with other caspases. In ad- in different adult mouse organs and by Western blotting in dition we tested the effect of TNF-, Fas ligand (FasL)–, different cell lines (Figs. 1 and 2). As caspase-12 is most sim- LPS-, and thapsigargin (Tg)-induced apoptosis on the pro- ilar to caspase-1 and -11, we compared the constitutive ex- cessing state of caspase-12. pression pattern of these three caspases. The mRNAs of the three proteins were highly expressed at day 7 of embryonic development. All three caspases showed a drop in expression Results at day 11, but whereas the mRNA of caspase-1 disappeared Analysis of the expression of caspase-12 during mouse by day 17, the expression levels of caspase-11 and -12 embryonic development and in mouse organs mRNAs increased gradually and reached the initial levels and cell lines again by that day (Fig. 1 A). The highest level of caspase-12 A previous analysis of the expression pattern of caspase-12 mRNA expression in adult mouse organs was observed in in newborn mouse organs demonstrated that the enzyme is lymph node, lung, and spleen (Fig. 1 B), confirming our found in many tissues and suggested that the protein is previous results (Van de Craen et al., 1997). The lowest cas- widely expressed in a variety of cells (Nakagawa and Yuan, pase-12 mRNA expression levels were detected in brain, 2000). However, the Northern blot analysis presented in bone marrow, eye, and liver (Fig. 1 B). Interestingly, a dou- our previous report demonstrated that the mRNA of cas- ble band appeared, for example, in the testis, suggesting the pase-12 is mainly expressed in the lungs and skeletal muscle existence of a splice variant. Indeed, splice variants of mu- (Van de Craen et al., 1997). In an attempt to resolve this ap- rine caspase-12 and human pseudo caspase-12 were re- parent discrepancy, we analyzed the expression pattern of ported before (Van de Craen et al., 1997; Fischer et al., The Journal of Cell Biology Analysis of caspase-12 expression and processing | Kalai et al. 459 dominant in skeletal muscle followed by the heart and the brain (Fig. 2 A). Next, we analyzed, by Western blotting, extracts from seven murine cell lines representing different cell types (B cell hybridoma, T cells, preB cells, fibrosarcoma, melanoma, and promyelocytes). A control lysate of HEK293T cells transiently transfected with a plasmid leading to overexpres- sion of caspase-12 revealed the 49-kD band expected for full-length pro–caspase-12 and two additional bands run- ning at 38- and 28-kD, respectively (Fig. 2 B). Caspase-12 protein was detected only in cells derived from the murine fibrosarcoma cell line L929 (Fig. 2 B). In agreement with the apparent absence of caspase-12 in lymphoid organs, no caspase-12 protein was detected in cell lines of hematopoi- etic origin. The strongest expression was detected in L929r2, the cells from which the cDNA of caspase-12 was originally cloned (Van de Craen et al., 1997). Analysis of 13.5-d-post- coitum mouse embryos by immunohistochemistry revealed that the most positively stained organ is the heart (Fig. 3, A and B). In several other organs, including the lung and the nose, the expression of the protein was confined to certain cell types or layers, such as epithelium of the trachea and the nose trail (Fig. 3, C and D). These results suggest that the constitutive expression of caspase-12 is probably restricted to certain cell types. Figure 2. Western blot analysis of the expression of caspase-12 protein in lysates of different mouse organs and cell lines. IFN- induces the expression of caspase-12 in mouse (A) Expression of caspase-1, -11, and -12 in adult mouse organs fibrosarcoma and melanoma cells: synergy with other using anti–caspase-12 antiserum (Ab-1). *, antiserum G149 failed proinflammatory stimuli to detect caspase-12 expression in the brain (not depicted). (B) Analysis of the expression of caspase-12 protein in mouse cell Several reports have demonstrated that the expression of lines representing different cell lineages. The cell lines used were caspase-1 and -11 can be induced by proinflammatory stim- 7TD1 (mouse/rat B cell hybridoma), D10(N1) T cell lymphoma, uli such as IFN-, TNF, or LPS (Lin et al., 2000; Schauv- FDCP1 (promyelocytoma), L929r2 and L929sA (fibrosarcoma), liege et al., 2002). The L929r2 murine fibrosarcoma and B16/B16 (melanoma), CTLrIL-2 (cytotoxic T cells), and WEHI-3 (myelomonocytoma). Lysates of HEK293T cells transfected () or B16/B16 murine melanoma cells respond to TNF only not () with a cDNA encoding for the corresponding caspase were when cotreated with IFN- (Mareel et al., 1988; Vanhaese- used as positive and negative controls (CTRL), respectively. Arrows broeck et al., 1991). Therefore, we compared the effect of indicate bands corresponding to full-length (49 kD) or processed IFN- with and without TNF on the expression levels of (38 and 28 kD) caspase-12. caspase-1, -11, and -12 in these two cell lines. Northern blot analysis demonstrated that the expression of caspase-12 2002). Caspase-11 mRNA was observed in all analyzed or- mRNA is further enhanced in L929r2 and induced in Bl6/ gans, though the expression levels in the lung, spleen, B16 cells by IFN- (Fig. 4). Synergism between TNF and uterus, and smooth muscle were clearly the highest (Fig. 1 IFN- was observed with caspase-12 mRNA in B16/B16 B). Caspase-1 mRNA expression was detected in all of the cells and with caspase-11 mRNA in both L929r2 and B16/ organs, with the lowest level in the liver and the kidney and B16 cells. Such an effect was not observed for caspase-1 and the highest in the spleen. -12 in L929r2 cells. Treatment of L929r2 cells for 24 h Using Western blot analysis, we checked the expression of with IFN- and TNF leads to cell death, about half of the caspase-1, -11, and -12 at the protein level in lysates of sev- cells are propidium iodide positive at 24 h, whereas B16/ eral organs from 5-wk-old mice (Fig. 2 A). The expression B16 cells still survive at that time point. This may explain pattern of caspase-1 at the protein level corresponded with the apparent difference observed in the response of these the RT-PCR results in Fig. 1 B. However, for caspase-12 cell lines. An increase in caspase-1 mRNA was observed and caspase-11, this correlation between protein and mRNA only in L929r2 treated with IFN-, whereas no caspase-1 expression levels was not obvious (Fig. 2). Expression of cas- mRNA expression was observed in B16/B16 cells in any of pase-12 protein was highest in skeletal muscle, heart, brain, the treatments. testis, and eye (Fig. 2 A). However, no, or hardly any, cas- Using Western blot analysis, we determined if the induc- pase-12 protein was detected in the other organs tested. Al- tion of caspase-12 mRNA expression in B16/B16 cells was though high caspase-12 mRNA expression levels were de- also reflected at the protein level and compared the effect tected in the lymph node and the spleen, only very low with that observed with other caspases (Fig. 5). Treatment amounts of the protein could be detected in any of the he- with IFN- increased the expression of caspase-1, -11, and matopoietic organs tested. Caspase-11 expression was pre- -12. When the treatment with IFN- was combined with The Journal of Cell Biology 460 The Journal of Cell Biology | Volume 162, Number 3, 2003 Figure 3. Immunohistochemical analysis of the expression of caspase-12 in the mouse embryo. Caspase-12 expression was detected in sections using either anti–caspase-12 rabbit antiserum Ab-1 or Ab-2. Results obtained with both antisera are similar (not depicted). Only those obtained with Ab-2 are presented. Negative controls of adjacent sections were stained by the anti–cas- pase-12 antisera depleted with recombinant caspase-12. Sections stained by secondary antibody alone were completely negative (not depicted). (A) Sections of a 13.5-d-postcoitum embryo. Note the strong staining of heart, lung, and nose. White box shows the embryo at its relative real size (6 mm). (B) A close-up of the heart (1) in A. (C) A close-up of the lung (2) in A. Note the positive staining of the epithelial layer of the trachea. (D) A close-up of the nose (3) in A. Note the positive staining of the epithelium in the entrance of the nose. Bars: (A) 455 m; (B) 80 m; (C) 30 m; (D) 45 m. either TNF or LPS, the effect on the expression of caspase-1, expression of the different caspases intensified in time, sug- -11, and -12 was intensified (Fig. 5; Fig. 6 A). In contrast to gesting that the expression of caspase-1, -11, and -12 was the caspases mentioned above, IFN- treatment led to a de- induced, whereas that of the proapoptotic caspases 3 and 9 crease in the expression levels of caspase-3 and -9. The dis- was suppressed (Fig. 5). appearance of the full-length form of these caspases (Fig. 5) The expression of caspase-12 is not induced by in B16/B16 cells was not due to proteolytic processing be- type I IFNs cause no proteolytic fragments of caspase-3 and -9 were de- tected. The antisera used reveal specific proteolytic process- Next, we tested whether the effect on caspase-12 expression ing of caspase-3 and -9 during apoptosis (Fig. 8). As is specific for IFN- or whether it also occurs with IFN- or expected, IFN- also increased the expression of the IFN- -. Both IFN- and IFN- failed to induce the expression inducible dsRNA-activated protein kinase (PKR), which we of caspase-12. In contrast with the results obtained with used as a positive control (Baier et al., 1993). No effect was IFN-, only a very faint caspase-12 signal could be detected observed on the level of -actin used as internal negative in Western blots of lysates of B16/B16 cells treated with the control. Treatment with either TNF or LPS alone did not combinations of type I IFNs with either TNF or LPS (Fig. 6 affect the expression levels of any of the proteins. We ob- A). However, in cells treated with any one of the IFNs, the served a similar effect of IFN- with and without TNF or expression level of caspase-3 was reduced (Fig. 6 A), con- LPS on caspase-12 expression in another murine melanoma firming the results presented in Fig. 5. All three IFNs in- cell line (PG19; unpublished data). These results suggest duced the expression of PKR, demonstrating that the failure that the activation of the gene coding for caspase-12 is of type I IFNs to induce the expression of caspase-12 did not tightly regulated. Interestingly, the distinct effects on the result from an incapacity of the cells to respond to these cy- The Journal of Cell Biology Analysis of caspase-12 expression and processing | Kalai et al. 461 Figure 5. Analysis of the expression of caspase-12 and other caspases at the protein level in B16/B16 cells treated with IFN-, TNF, and LPS. Western blot analysis of lysates of B16/B16 cells treated for 9 or 24 h with IFN- (1,000 U/ml), TNF (5,000 U/ml), Figure 4. Effect of IFN- and TNF on the expression of the mRNAs or LPS (1 g/ml) alone or in combinations, using antisera specific of the mouse inflammatory caspase subfamily in L929r2 fibrosarcoma for the indicated murine caspases. Blots were revealed also with and B16/B16 melanoma cells. Cells were treated or not with IFN- an anti–-actin antibody to verify that equal amounts of protein (1,000 U/ml) or TNF (5,000 U/ml) alone or in combination. The next were loaded. The IFN-inducible kinase PKR was used as a positive day, mRNA was extracted and analyzed by Northern blotting using control, demonstrating responsiveness to IFN. CTRL indicates specific probes. The size of the hybridizing bands is indicated in kb. untreated controls. UV imaging of ethidium bromide intercalated in the 18S and 28S rRNAs was used to monitor loading of the gels (bottom). tected by the G149 antiserum in HEK293T cells that do not express murine caspase-12. Moreover, these bands were not tokines. IFNs are known inducers of antiviral response. Be- detected using two other distinct anti–caspase-12 antisera cause caspase-12 was implicated in the response to a viral in- (unpublished data). fection (Bitko and Barik, 2001; Jordan et al., 2002), we Untreated primary fibroblasts, like the L929 fibrosarcoma wondered if dsRNA, a typical virus by-product, would fur- cells, constitutively expressed caspase-12 (Fig. 7 B). Treat- ther modulate caspase-12 expression in the presence of IFN. ment of primary fibroblasts with IFN- strongly enhanced Interestingly, IFN-, but not IFN-, which is the antiviral the expression of the protein, and addition of either LPS or IFN per se, increased the expression of caspase-12 in synergy TNF even intensified the effect further (Fig. 7 B). These re- with dsRNA (Fig. 6 B). sults demonstrate that the expression of caspase-12, like that Macrophages play a major role in the response to both vi- of the proinflammatory caspases 1 and 11, can be induced in ral and bacterial infection and thus present a cellular model cells exposed to IFN-. However, the response might be re- that is highly relevant for inflammation (Stoy, 2001). More- stricted to certain cell types, for example fibroblasts and me- over, macrophages express caspase-1 and -11 and secrete ac- lanocytes, because macrophages that constitutively express tivated IL-1 in response to infection and proinflammatory caspase-1 and can be induced to produce caspase-11 appar- stimuli (Lin et al., 2000; Schauvliege et al., 2002). There- ently fail to express caspase-12. fore, we determined the effects of the three IFNs alone and of IFN- in combination with LPS, TNF, or dsRNA on the Caspase-12 is processed in apoptotic cell death protein expression levels of caspase-1, -11, and -12 in the induced by FasL, TNF, or LPS Mf4/4 murine macrophage cell line. IFN-, and to a lesser extent also LPS and IFN-, clearly induced the expression of Transient overexpression of full-length wild-type caspase-12, caspase-11. The expression of caspase-1 remained high and but not of its inactive C298A mutant, in HEK293T cells led stable (Fig. 7 A). However, no caspase-12–specific bands to processing of the protein and to apoptosis (Fig. 2 B; Fig. were detected in Mf4/4 cells in any of the tested conditions 8 A; Fig. 9 B). Processing of caspase-12 in these conditions (Fig. 7 A). Similar results were obtained with J774 and resulted in fragments of 38 and 28 kD. These results sug- pU518, two other murine macrophage cell lines (unpub- gest that a link exists between the processing of caspase-12 lished data) and primary peritoneal macrophages (Fig. 7 B). and the induction of an apoptotic cell death. In rapid apop- The nonspecific bands detected in Fig. 7 A appeared after a tosis occurring in L929sAhFas cells treated with FasL, cas- long exposure of the blot to the film. Similar bands were de- pase-12 was fully processed, and only the 28-kD fragment The Journal of Cell Biology 462 The Journal of Cell Biology | Volume 162, Number 3, 2003 Figure 7. 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-, - or, - (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml), LPS (1 g/ml), or dsRNA (50 g/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- (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml) or LPS (1 g/ml). B16/B16 cells treated or not with IFN- (1,000 IU/ml) were used as a positive control. Figure 6. Comparison of the effect of type I and type II IFNs on An anti–-actin antibody was used to verify that equal amounts of the expression of caspase-12 and -3 by B16/B16 cells. (A) Western protein were loaded. blot analysis of cells treated for 24 h with IFN-, -, or - (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml) or LPS (1 g/ml). (B) Western blot analysis of cells treated for 24 h with IFN- or - (1,000 IU/ml) in the presence or absence of dsRNA (100 g/ml). Caspase-12 is dispensable for Tg-induced The IFN-inducible kinase PKR was used as a positive control for ER stress–mediated apoptosis responsiveness to the different IFNs. An anti–-actin antibody was used to verify that equal amounts of protein were loaded. 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/ was observed (Fig. 8 B). The processing of caspase-12 in the B16 cells express caspase-12 only when they are treated with latter cells coincided with the activation of caspase-3, -7, and IFN-, we used these cells to test if caspase-12 is required for -9 (Fig. 8 B). Treatment of B16/B16 cells with TNF alone ER stress–mediated apoptosis induced by Tg. B16/B16 cells had no effect on their growth, whereas treatment with either died in response to Tg in the presence or absence IFN- IFN- or LPS alone led to a decrease in the proliferation rate (Fig. 10 A). Light microscopy analysis revealed that in both without any apparent signs of cell death (Fig. 9 A). How- cases, cells dying in response to Tg were blebbing and had ever, light microscopy analysis demonstrated that cells the typical apoptotic morphology similar to that seen with treated with a combination of IFN- with LPS or TNF HEK293T overexpressing caspase-12 (Fig. 8 A). Although rounded up and started floating and eventually died and dis- IFN- treatment clearly sensitized the cells to TNF-induced integrated (Fig. 9 A; unpublished data). We tested whether cytotoxicity (Fig. 10 A), the effect of IFN- on Tg-induced caspase-12 is also processed in this kind of cell death. As seen cell death was minor. To analyze the expression and process- before (Figs. 5 and 6), the protein was highly expressed in all ing pattern of caspase-12 in response to Tg and compare it the conditions that included IFN-. However, processing of with that observed in IFN-  TNF–treated cells, we used caspase-12 was detected only when the cells were treated two different anti–caspase-12 antisera. The first antiserum, with the combination of IFN- with either LPS or TNF Ab-2, was raised against a peptide spanning residues 2–17 in (Fig. 9 B). These results show that caspase-12 is processed in the amino acid sequence of caspase-12 and thus recognized different proapoptotic conditions, including those induced fragments containing the prodomain of the protein, and the by TNF, LPS, and FasL, and suggest that it may actively second antiserum, G149, was raised against the catalytic contribute to the cell death process. parts of the enzyme (Fig. 10 B). Both antisera detected the The Journal of Cell Biology Analysis of caspase-12 expression and processing | Kalai et al. 463 Figure 9. Caspase-12 is processed in B16/B16 cells dying from the combination of IFN- with either LPS or TNF. (A) Analysis of the cytotoxic potential of IFN-, TNF, or LPS. Cells were treated with IFN- (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 g/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- (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 g/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 Figure 8. Processing of caspase-12 in apoptosis induced by fragments of the processed caspase. *, nonspecific bands. 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 thus, probably corresponds to the mature large catalytic sub- localization signal containing GFP and a plasmid encoding for unit (LCSU) (Fig. 10, B and C). Taken together, these re- either the wild type (C-12 wt) or an inactive caspase-12 mutant sults demonstrate that caspase-12 is not required for Tg- (C-12 C298A). Fluorescent microscopy photographs and Western blot analysis demonstrate that although both of the caspase-12 induced cell death, although treatment with Tg can induce variants are expressed in equal levels, only the wild-type protein the proteolytic cleavage of the protease. is processed and leads to apoptotic cell death. Bar, 5 m. Arrows indicate fragments corresponding to caspase-12 and their size in kD. (B) L929sAhFas cells were treated with recombinant soluble Discussion FasL for the indicated time. Cytotoxicity was measured by trypan blue (TB) exclusion. CTRL indicates untreated controls. Processing In the current report, we investigated the expression of cas- of caspase-3, -7, -9, and -12 after a 2-h treatment with FasL was pase-12 during mouse embryonic development and in adult determined by Western blotting. Arrows indicate the proteolytically mouse organs. We compared the regulation of the expres- processed caspase fragments and their size in kD. sion of caspase-12 by proinflammatory stimuli with that of other caspases. Although the mRNAs of caspase-1, -11, and full-length 49-kD protein in cells treated with IFN- but -12 are expressed in most of the organs tested, the level of failed to detect the expression of caspase-12 in cells treated expression varies substantially. For example, all three cas- with Tg alone (Fig. 10 C). The level of caspase-12 ex- pases were highly expressed in the spleen, whereas only cas- pression seen in cells cotreated with IFN- and Tg was pase-11 and -12 were highly expressed in the lungs. Similar lower than in cells treated with IFN- in the absence of Tg. results were obtained before for murine caspase-1, -11, and This may be explained by a decrease in translation due to -12 using Northern blots (Van de Craen et al., 1997) and Tg treatment (Wong et al., 1993). Caspase-12 processing for human caspase-1, -4, and -5 by RT-PCR (Lin et al., clearly occurred in cells treated with the combination of 2000). By analyzing a battery of mouse cell lines of various IFN- and TNF or Tg (Fig. 10 C). Ab-2 detected the 38- lineages, we show that the expression of caspase-12 at the kD fragment of caspase-12 in cells treated with IFN-  protein level is more restricted than expected. Indeed, the TNF but failed to detect this fragment in cells treated with only cells we observed to constitutively express the protein IFN-  Tg, whereas G149 detected the 28-kD fragment are primary fibroblasts and L929 fibrosarcoma cells. This in both types of treatments (Fig. 10 C). These results suggest suggests that although caspase-12 can be detected in most that the 38-kD fragment lacks only the COOH-terminal organs, only some of the cells in these organs are expressing small catalytic subunit (SCSU), and that the 28-kD frag- the protease. This suggestion was clearly supported by the ment is missing both the SCSU and the prodomain and, immunohistochemical analysis that demonstrated that in The Journal of Cell Biology 464 The Journal of Cell Biology | Volume 162, Number 3, 2003 1998; Burgess et al., 1998). Caspase-11–deficient mice present a similar phenotype in addition to the lack of the ability to respond to LPS by caspase-1 processing, a function that apparently requires caspase-11 (Wang et al., 1998). In humans, caspase-1 and IL-1 activation was shown to in- volve the formation of a complex including caspase-1 and caspase-5 (Martinon et al., 2002). Murine caspase-11 can also form a complex with, and is required for the activation of, caspase-1 (Wang et al., 1998) and therefore may have a function similar to that of human caspase-5. The phylogenic clustering of caspase-1, -4, -5, -11, and -12 suggests a role for the latter caspase in inflammatory responses. As seen with caspase-1 and -11, the expression of caspase-12 is stimulated by IFN- in fibrosarcoma and melanoma cells. In contrast to IFN- and -, which are produced by most cell types in re- sponse to viruses or dsRNA, production of IFN- is re- stricted to cells of the immune system (Katze et al., 2002). These include natural killer (NK) cells, CD4 T helper 1 (T 1) cells, and CD8 cytotoxic T cells stimulated by IL-12 and IL-18 secreted from activated macrophage or dendritic cells (Murphy and Reiner, 2002). IFN- and - are mainly involved in the response to viral infection (Katze et al., 2002; Taniguchi and Takaoka, 2002). Although IFN- has an an- tiviral function, the cytokine acts mainly as an effector cyto- kine required for cell-mediated immunity (Murphy and Reiner, 2002; Gordon, 2003). Moreover, IFN- is a strong activator of proinflammatory and microbicidal functions of macrophages (Gordon, 2003). Remarkably, expression of caspase-12 is induced exclusively by IFN- and not by IFN- Figure 10. Caspase-12 is dispensable for Tg-induced ER stress– or -. Does this suggest that caspase-12 plays a role in anti- mediated apoptosis in B16/B16 cells. (A) Analysis of the cytotoxic bacterial or proinflammatory activities? The answer is far effect of Tg (2 M) alone or in combination with IFN- (1,000 IU/ml) from clear, as the cellular substrates of the protease are still compared with TNF (5,000 IU/ml). Cell viability was measured at unknown. In contrast to caspase-1 and -11, caspase-12 is not the indicated time using MTT. CTRL is untreated control. Error bars, expressed in macrophages, a typical example of inflammatory standard deviation of six replicates of a representative experiment. cells. Moreover, overexpression of caspase-12 in several cell (B) Schematic representation of pro–caspase-12 indicating the relative location of the peptides used to produce the anti–caspase-12 lines together with pro–IL-1 does not lead to secretion of antisera Ab-2 and G149. Ab-2 was raised against a peptide spanning active IL-1 (Van de Craen et al., 1997; unpublished data), amino acids 2–17 of caspase-12 and is specific for the NH -terminal excluding a direct role for the protease in IL-1 maturation. prodomain. G149 was raised against a recombinant protein spanning In addition, although caspase-12–deficient mice were made amino acids 116–419 of caspase-12 and recognizes the catalytic (Nakagawa et al., 2000), nothing has been published yet on part of the protein. LCSU and SCSU indicate the large and the small catalytic subunits of the caspase, respectively. (C) Western blot the response of these deficient mice to proinflammatory or analysis of cells treated for 72 h with IFN- (1,000 IU/ml), TNF endotoxic stimuli or to bacterial infection. (5,000 IU/ml), or Tg (2 M) alone or in combinations. C-12 indicates Activation of caspase-1 or -11 due to exposure to LPS or lysate of HEK293T cells overexpressing caspase-12, used as a positive bacterial infection can also lead to apoptosis (Chen et al., control. Arrows indicate bands corresponding to the 49-kD full-length 1996; Hilbi et al., 1998; Kang et al., 2000, 2002; Hisahara pro–caspase-12 and to the 38- and 28-kD fragments of the processed et al., 2001). Caspase-12 may exert a similar function. Our caspase. *, nonspecific bands. results demonstrate that overexpression of caspase-12 in HEK293T cells leads to processing of the enzyme and to most positive organs, only certain cell layers, such as epithe- apoptosis. We observed similar processing of caspase-12 in lium of the trachea in the lung and the nose trail, express B16/B16 cells dying in response to IFN- combined with the caspase. either TNF or LPS and in L929sAhFas cells dying by Fas- Protein sequence comparison shows that caspase-12 most mediated apoptosis. Interestingly, in B16/B16 cells treated resembles caspase-1 and -11. Caspase-1 and its cytokine sub- with IFN-, expression of the typical apoptotic caspases 3 strates IL-1 and IL-18 play an important role in immune and 9 decreased, whereas that of caspases 1, 11, and 12 went and inflammatory responses to endotoxins and bacterial up. This down-regulation of the apoptotic caspases versus infection such as Shigella flexneri and Escherichia coli the up-regulation of the inflammatory caspases may suggest (Dinarello, 1998; Sansonetti et al., 2000; Joshi et al., 2002). that IFN- prepares the cells for an alternative caspase cas- Mice deficient in caspase-1 are resistant to endotoxic shock cade, avoiding the intrinsic apoptotic pathway. and are defective in LPS-induced secretion of IL-1, IL-1, The inducible expression of caspase-12 in B16/B16 cells IL-18, and IFN- (Li et al., 1995; Fantuzzi et al., 1997, allowed us to study the involvement of the protein in Tg- The Journal of Cell Biology Analysis of caspase-12 expression and processing | Kalai et al. 465 Preparation of protein extracts from mouse organs induced ER stress–mediated apoptosis. Our results clearly Mouse organs were isolated, and samples from three to six mice were show that the cells die in response to Tg whether caspase-12 pooled and frozen in liquid nitrogen. Tissues were thawed and homoge- is present or not. They also demonstrate that in cells express- nized on ice with an RZR homogenizer (Heidolph-Instruments) in PBS-A ing caspase-12, the enzyme is processed and probably acti- supplemented with 1 mM leupeptin, 1 mM aprotinin, 0.1 mM PMSF, and 1 mM oxidized glutathione. The homogenates were diluted in an equal vated in a variety of apoptotic conditions. In view of the re- volume of caspase lysis buffer containing 0.05% Nonidet-P40, 220 mM stricted expression pattern of caspase-12 to certain cell types PO , 10 mM Hepes, mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH 2 4 and the fact that its closest homologue encoded in the hu- pH 7.4, 1 mM leupeptin, 1 mM aprotinin, 0.1 mM PMSF, and 1 mM oxi- man genome is not a functional protease, we suggest that al- dized glutathione and incubated on ice for 10 min. Cell debris was re- moved by centrifugation, protein concentrations were measured using a though the protein may have a function in apoptotic cell Bio-Rad Laboratories protein assay, and 50 g from each sample was death, it is not likely to be the initiator of ER stress–medi- taken for Western blot analysis. ated apoptosis. Despite the absence of caspase-12 in stimu- lated macrophages, the induction of the expression and pro- Immunohistochemistry BALB/c mouse embryos, aged 13.5 d postcoitum, were fixed in 4% para- cessing of the protease by proinflammatory stimuli in formaldehyde, embedded in paraffin, and sliced (4–6 m). Endogenous fibroblasts may still suggest a role in the immune or inflam- peroxidase was blocked with peroxidase blocking reagent (DakoCytoma- matory response in the mouse. tion). Caspase-12 expression was detected in sections using either anti– caspase-12 rabbit antiserum, Ab-1 or Ab-2. Binding of primary antibody was revealed by 45 min incubation with a rat anti–rabbit antibody conju- gated to peroxidase (DakoCytomation) followed by incubation with the Materials and methods 3,3 -diaminobenzidine chromogen (DakoCytomation). Negative controls Antibodies, cytokines, and reagents were stained by the secondary antibody alone or by the anti–caspase-12 Recombinant murine TNF (specific biological activity of 2.2  10 IU/ml) rabbit antiserum depleted with a His -tagged recombinant caspase-12 pro- was produced in E. coli and purified in our laboratory. Recombinant mu- duced in E. coli and captured on Ni-NTA (QIAGEN). The specificity of the rine IFN- was from BioSource International. Recombinant murine IFN- depletion procedure was controlled by passing anti–caspase-12 antiserum and IFN- were produced in E. coli and purified in our laboratory. Flag- over a Ni-NTA column. Furthermore, the specificity of the antisera to cas- tagged human FasL was expressed in HEK293T cells and purified in house. pase-12 was verified by immunostaining of HEK293T transfected or not Poly(I)-poly(C) (synthetic dsRNA) was dissolved at 3.5 mg/ml in water (Am- with pCAGGS-caspase-12 C298A using a similar staining technique. ersham Biosciences). LPS (Salmonella abortus equi) was from Sigma- Aldrich. Anti–murine caspase-9 antibody was from New England Biolabs, Northern blot analysis Inc. Rabbit polyclonal antiserum against recombinant murine caspase-1, 3 2. B16/B16 or L929r2 cells were seeded at 1.2  10 cells per cm The next -3, -7, and -11 and caspase-12 (G149) were prepared at the Centre d’Econ- day, cells were left untreated or treated with either IFN- (1,000 U/ml) or omie Rurale (Laboratoire d’Hormonologie Animale). Purified anti–cas- TNF (5,000 U/ml) alone or in combination. Cells were harvested 20 h pase-12 antisera, Ab-1 and Ab-2, were from Oncogene Research Products. 6 cells using RNAzol™ later, and total RNA was extracted from 4  10 The anti–caspase-12 antiserum used in all Western blot analyses, unless (WAK-Chemie Medical GmbH). After electrophoretic separation on a 1% otherwise indicated, is G149. Antiserum specific for mPKR was from Santa mem- formaldehyde agarose gel, RNA was transferred to a Hybond-N Cruz Biotechnology, Inc., and antibody to -actin was from ICN Biomedi- brane and fixed by UV light. Membranes were incubated in prehybridiza- cals. Determination of protein concentration in cell lysates was performed tion solution (50 mM Tris-HCl, pH 7.4, 40% formamide, 4 SSC, 10 using a Bio-Rad Laboratories protein assay. P O , 1% SDS, and 20 g/ml herring sperm Denhardt’s solution, 0.1% Na 4 2 7 DNA) for 2 h at 42 C. DNA probes for caspase-1, -11, and -12 were la- RT-PCR analysis P]dCTP using random primering (Boehringer) and incubated beled with [ TM Mouse multiple tissue cDNA (MTC ) panels I and II were from CLON- overnight at 42 C with the prehybridized membrane. Filters were washed l PCR TECH Laboratories, Inc. PCR was performed in a total volume of 50 with 2 SSC, 0.1% SDS at room temperature and then exposed to an X-ray , 0.4 mM deoxynucleoside triphosphates, buffer containing 2 mM MgSO 4 film at 80 C. 0.5 M each primer, 2.5 U of Pfx Platinum DNA polymerase (Invitrogen), and 5 l of first strand cDNA of tissue or embryo cDNA. The PCR cycle Cell culture and treatment started at 94 C for 2 min, followed by a three-step cycling: denaturation at Murine Mf4/4 macrophages (Desmedt et al., 1998) and B16/B16 cells 94 C for 15 s, annealing at either 58 C or 62 C (depending on the primers were maintained in LPS-free RPMI 1640 medium (GIBCO BRL) supple- used) for 30 s, and extension at 72 C for 90 s. This was followed by a final mented with 10% FCS (GIBCO BRL), 0.03% L-Gln (Merck), 100 U/ml pen- extension step at 72 C for 5 min. In each experiment, PCR for glyceralde- icillin, 100 g/ml streptomycin, and 50 M -mercaptoethanol at 37 C in hyde-3-phosphate dehydrogenase (G3DPH) was performed according to a humidified 5% CO atmosphere. Before analysis, cells were seeded at the manufacturer’s instructions using primers provided by CLONTECH 3  10 cells/2 ml/well in six-well tissue culture plates (Falcon; Becton Laboratories, Inc., to ensure that an equal quality and quantity of cDNA Dickinson) in fresh growth medium. The next day, cells were left untreated was used. To avoid PCR products reaching saturation, 30 PCR cycles were or were treated as indicated. used for caspase-1, -11, and -12 and 22 cycles for G3DPH. The primers for Mouse embryonic fibroblasts were isolated from BALB/c mouse embryos mouse caspase-12 were derived from the 5 end of the full-length cDNA by collagenase treatment and incubated in DMEM.NUT.MIX.F-12 medium and from the 3 end of the predicted large catalytic subunit (Van de Craen supplemented with GLUTAMAX-I, pyridoxine, and 15% FCS. For analysis, et al., 1997). The primers for caspase-1 and -11 were derived from the 5 cells were seeded at 3  10 cells/2 ml/well in six-well tissue culture plates and 3 ends of the respective open reading frames. in fresh growth medium. The next day, cells were treated as indicated. Peritoneal macrophages were obtained by washing the peritoneal cavity Plasmids and transfection of HEK293T cells of three adult C57/BL6 mice with PBS-A. May-Grünwald/Giemsa (Sigma- The cloning of the cDNA encoding for full-length caspase-12 from a Aldrich) staining performed after the removal of erythrocytes by a hypo- cDNA library derived from L929r2 cells and its transfer to the pCAGGS eu- tonic treatment demonstrated that 90% of the recovered cells had a karyotic expression vector were described previously (Van de Craen et al., monocyte/macrophage-like morphology. The remaining cells were seeded 1997). In pCAGGS-caspase-12 C298A, the codon for the catalytic cysteine at 10 cells/1 ml/well in six-well tissue culture plates in LPS-free RPMI 1640 was substituted by that coding for alanine, leading to the expression of an medium supplemented with 10% FCS, 0.03% L-Gln, 100 U/ml penicillin, inactive form of the enzyme. pNLS-EGFP is a modified pEGFP-N1 plasmid 100 g/ml streptomycin, and 50 M -mercaptoethanol and allowed to ad- (CLONTECH Laboratories, Inc.) encoding GFP with a nuclear localization here for 1.5 h. After the removal of suspension cells and refreshment of the signal. HEK293T cells were transiently cotransfected using calcium phos- medium (2 ml/well), adherent cells were treated as indicated. phate precipitation in six-well culture dishes with 50 ng of pNLS-EGFP and The human Fas-expressing L929sA cells (L929sAhFas) were described 600 ng of either pCAGGS-caspase-12 or pCAGGS-caspase-12 C298A. The previously (Vercammen et al., 1998). L929sAhFas cells were seeded in next day, cells were examined and photographed using a fluorescent mi- 9-cm plates at 3.10 /plate. The next day, cells were treated with medium croscope. Cells were harvested and lysed for Western blot analysis as de- or recombinant Flag-tagged FasL (1/500 dilution), and cell death was de- scribed below. termined by trypan blue staining in 2-h intervals. 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Acta. 1477:299–306. thapsigargin in cultured cells. Biochem. J. 289(Pt. 1):71–79. The Journal of Cell Biology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Pubmed Central

Regulation of the expression and processing of caspase-12

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Pubmed Central
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Copyright © 2003, The Rockefeller University Press
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Abstract

JCB Article Regulation of the expression and processing of caspase-12 Michael Kalai, Mohamed Lamkanfi, Geertrui Denecker, Michael Boogmans, Saskia Lippens, Ann Meeus, Wim Declercq, and Peter Vandenabeele Department of Molecular Biomedical Research, Unit of Molecular Signalling and Cell Death, VIB and Ghent University, B-9000 Ghent, Belgium hylogenetic analysis clusters caspase-12 with the in- These stimuli also induce caspase-1 and -11 but inhibit the flammatory caspases 1 and 11. We analyzed the expres- expression of caspase-3 and -9. In contrast to caspase-1 P sion of caspase-12 in mouse embryos, adult organs, and -11, no caspase-12 protein was detected in macro- and different cell types and tested the effect of interferons phages in any of these treatments. Transient overexpression (IFNs) and other proinflammatory stimuli. Constitutive expres- of full-length caspase-12 leads to proteolytic processing of sion of the caspase-12 protein was restricted to certain cell the enzyme and apoptosis. Similar processing occurs in types, such as epithelial cells, primary fibroblasts, and L929 TNF-, LPS-, Fas ligand–, and thapsigargin (Tg)-induced fibrosarcoma cells. In fibroblasts and B16/B16 melanoma apoptosis. However, B16/B16 melanoma cells die when cells, caspase-12 expression is stimulated by IFN- but not treated with the ER stress–inducing agent Tg whether they by IFN- or -. The effect is increased further when IFN- express caspase-12 or not. is combined with TNF, lipopolysaccharide (LPS), or dsRNA. Introduction Caspases are a family of cysteinyl aspartate proteinases central and Yang, 2000). The final outcome of these cascades is the in mediating cellular signals in apoptosis and inflammation specific proteolysis of a wide variety of substrates, leading either (Lamkanfi et al., 2003). The enzymes are produced as zy- to the loss of a life-saving function or the activation of mogens consisting of a NH -terminal prodomain of variable a proapoptotic or proinflammatory function (Chang and length followed by a large and a small catalytic subunit. The Yang, 2000; Lamkanfi et al., 2003). large prodomains of certain members of this protein family, So far, 11 human and 10 murine caspases were identified referred to as initiator caspases (e.g., caspase-8, -9, and -10), (Chang and Yang, 2000; Lamkanfi et al., 2002). Phylogenetic contain interaction motifs consisting of six or seven antiparallel analysis reveals that caspases segregate into three major amphipathic  helices. These prodomains were shown to allow groups with different functions and substrate specificities. the recruitment and proximity-induced activation of the The three groups are often referred to as inflammatory caspases, enzymes in protein complexes (Stennicke and Salvesen, apoptotic executioner caspases, and apoptotic initiator caspases 2000). Caspases are usually activated by proteolysis that (Lamkanfi et al., 2002). Caspase-12 segregates with the inflam- leads to conformational changes of their catalytic parts matory caspases including caspase-1, -4, -5, and -11 (Chang (Riedl et al., 2001). The mature caspase is a heterotetramer and Yang, 2000; Lamkanfi et al., 2002). The amino acid consisting of two large and two small catalytic subunits sequence identity of murine caspase-12 to the other members aligned in a head-to-tail configuration and contains an active of this group is as follows: murine caspase-1 (42%), human site in each of the opposite ends. Caspases typically signal caspase-1 (40%), murine caspase-11 (41%), human caspase-4 through proteolytic cascades in which initiator caspases (48%), and caspase-5 (45%). These caspases seem to be cleave and activate executioner caspases that lack a large pro- involved more in the maturation and secretion of proin- domain (Slee et al., 1999; Van de Craen et al., 1999; Chang flammatory cytokines such as interleukin (IL)-1 and IL-18 than in the induction of death receptor–mediated apoptosis Address correspondence to Peter Vandenabeele, Department of Molecular (Martinon et al., 2002). All of the caspases included in the Biomedical Research, Unit of Molecular Signalling and Cell Death, Ghent group have a large prodomain containing a typical caspase University, Ledeganckstraat 35, B-9000 Ghent, Belgium. Tel.: 32-9-264-51- 31. Fax: 32-9-264-53-48. email: [email protected] Abbreviations used in this paper: FasL, Fas ligand; IFN, interferon; IL, Key words: apoptosis; caspase-12; inflammation; interferon; ER stress interleukin; LPS, lipopolysaccharide; Tg, thapsigargin.  The Rockefeller University Press, 0021-9525/2003/08/457/11 $8.00 The Journal of Cell Biology, Volume 162, Number 3, August 4, 2003 457–467 http://www.jcb.org/cgi/doi/10.1083/jcb.200303157 457 The Journal of Cell Biology 458 The Journal of Cell Biology | Volume 162, Number 3, 2003 recruitment domain (CARD). The genes encoding the mu- rine members of the group are located on chromosome 9 in the following order: caspase-1, caspase-11, and caspase-12. The genes encoding their human counterparts reside on chromosome 11q22, a region with synteny to murine chro- mosome 9, and are arranged as follows from telomere to cen- tromere: caspase-1, caspase-5, caspase-4, and finally a gene encoding a pseudo caspase-12 (Fischer et al., 2002). The predicted product of the latter gene is reported to be a cas- pase-12–like protein with a premature stop and several point mutations preventing the translated protein from having any caspase-like enzymatic activity (Fischer et al., 2002). Studies in caspase-12–deficient mice suggested that the protein plays a major role in ER stress–induced apoptosis and in the devel- opment of Alzheimer’s disease (Nakagawa et al., 2000). Since then, several other reports have linked processing of caspase-12 to ER stress–induced apoptosis (Rao et al., 2001, 2002; Diaz-Horta et al., 2002; Morishima et al., 2002). In addition, caspase-12 seems to be involved in apoptosis in- duced by viral infection (Bitko and Barik, 2001; Jordan et al., 2002) or by serum starvation (Kilic et al., 2002). Several reports have shown that the expression of certain caspases is induced during inflammation. Treatment with interferon (IFN)- increases the expression of caspase-8 in many types of cells, thus sensitizing the cells to death do- main receptor–mediated apoptosis (Dai and Krantz, 1999; Ruiz-Ruiz et al., 2000; Fulda and Debatin, 2002). Both IFN- and lipopolysaccharide (LPS) induce the expression of human caspase-1, human caspase-5, and murine caspase- 11 but not of human caspase-4 (Tamura et al., 1996; Dai Figure 1. Differential gene expression patterns of the mouse inflammatory caspase subfamily. Single-stranded cDNAs from and Krantz, 1999; Lin et al., 2000; Hur et al., 2001; Schauv- multiple mouse tissues (panel I and II) were amplified by PCR using liege et al., 2002). Although phylogenetic analysis suggests primers specific for caspase-1, -11, or -12. PCR for glyceraldehyde-3- that caspase-12 resembles other inflammatory caspases, it is phosphate dehydrogenase (G3DPH) was performed to ensure that still unclear whether the regulation and processing of this an equal quality and quantity of cDNA was used. Negative () and caspase is modulated in inflammation. Therefore, we ana- positive () PCR controls (CTRL) lacking or containing the specific cDNAs, respectively. (A) Expression of caspase-1, -11, and -12 lyzed the constitutive expression pattern of caspase-1, -11, mRNA during mouse embryonic development. (B) Expression of and -12 in mouse organs and during embryonic develop- caspase-1, -11, and -12 mRNA in different mouse organs. ment. We also tested the effect of IFNs and other proinflam- matory stimuli on the expression of caspase-12 in several cell lines both at the mRNA and protein levels. We compared caspase-12 by RT-PCR during embryonic development and these effects with those observed with other caspases. In ad- in different adult mouse organs and by Western blotting in dition we tested the effect of TNF-, Fas ligand (FasL)–, different cell lines (Figs. 1 and 2). As caspase-12 is most sim- LPS-, and thapsigargin (Tg)-induced apoptosis on the pro- ilar to caspase-1 and -11, we compared the constitutive ex- cessing state of caspase-12. pression pattern of these three caspases. The mRNAs of the three proteins were highly expressed at day 7 of embryonic development. All three caspases showed a drop in expression Results at day 11, but whereas the mRNA of caspase-1 disappeared Analysis of the expression of caspase-12 during mouse by day 17, the expression levels of caspase-11 and -12 embryonic development and in mouse organs mRNAs increased gradually and reached the initial levels and cell lines again by that day (Fig. 1 A). The highest level of caspase-12 A previous analysis of the expression pattern of caspase-12 mRNA expression in adult mouse organs was observed in in newborn mouse organs demonstrated that the enzyme is lymph node, lung, and spleen (Fig. 1 B), confirming our found in many tissues and suggested that the protein is previous results (Van de Craen et al., 1997). The lowest cas- widely expressed in a variety of cells (Nakagawa and Yuan, pase-12 mRNA expression levels were detected in brain, 2000). However, the Northern blot analysis presented in bone marrow, eye, and liver (Fig. 1 B). Interestingly, a dou- our previous report demonstrated that the mRNA of cas- ble band appeared, for example, in the testis, suggesting the pase-12 is mainly expressed in the lungs and skeletal muscle existence of a splice variant. Indeed, splice variants of mu- (Van de Craen et al., 1997). In an attempt to resolve this ap- rine caspase-12 and human pseudo caspase-12 were re- parent discrepancy, we analyzed the expression pattern of ported before (Van de Craen et al., 1997; Fischer et al., The Journal of Cell Biology Analysis of caspase-12 expression and processing | Kalai et al. 459 dominant in skeletal muscle followed by the heart and the brain (Fig. 2 A). Next, we analyzed, by Western blotting, extracts from seven murine cell lines representing different cell types (B cell hybridoma, T cells, preB cells, fibrosarcoma, melanoma, and promyelocytes). A control lysate of HEK293T cells transiently transfected with a plasmid leading to overexpres- sion of caspase-12 revealed the 49-kD band expected for full-length pro–caspase-12 and two additional bands run- ning at 38- and 28-kD, respectively (Fig. 2 B). Caspase-12 protein was detected only in cells derived from the murine fibrosarcoma cell line L929 (Fig. 2 B). In agreement with the apparent absence of caspase-12 in lymphoid organs, no caspase-12 protein was detected in cell lines of hematopoi- etic origin. The strongest expression was detected in L929r2, the cells from which the cDNA of caspase-12 was originally cloned (Van de Craen et al., 1997). Analysis of 13.5-d-post- coitum mouse embryos by immunohistochemistry revealed that the most positively stained organ is the heart (Fig. 3, A and B). In several other organs, including the lung and the nose, the expression of the protein was confined to certain cell types or layers, such as epithelium of the trachea and the nose trail (Fig. 3, C and D). These results suggest that the constitutive expression of caspase-12 is probably restricted to certain cell types. Figure 2. Western blot analysis of the expression of caspase-12 protein in lysates of different mouse organs and cell lines. IFN- induces the expression of caspase-12 in mouse (A) Expression of caspase-1, -11, and -12 in adult mouse organs fibrosarcoma and melanoma cells: synergy with other using anti–caspase-12 antiserum (Ab-1). *, antiserum G149 failed proinflammatory stimuli to detect caspase-12 expression in the brain (not depicted). (B) Analysis of the expression of caspase-12 protein in mouse cell Several reports have demonstrated that the expression of lines representing different cell lineages. The cell lines used were caspase-1 and -11 can be induced by proinflammatory stim- 7TD1 (mouse/rat B cell hybridoma), D10(N1) T cell lymphoma, uli such as IFN-, TNF, or LPS (Lin et al., 2000; Schauv- FDCP1 (promyelocytoma), L929r2 and L929sA (fibrosarcoma), liege et al., 2002). The L929r2 murine fibrosarcoma and B16/B16 (melanoma), CTLrIL-2 (cytotoxic T cells), and WEHI-3 (myelomonocytoma). Lysates of HEK293T cells transfected () or B16/B16 murine melanoma cells respond to TNF only not () with a cDNA encoding for the corresponding caspase were when cotreated with IFN- (Mareel et al., 1988; Vanhaese- used as positive and negative controls (CTRL), respectively. Arrows broeck et al., 1991). Therefore, we compared the effect of indicate bands corresponding to full-length (49 kD) or processed IFN- with and without TNF on the expression levels of (38 and 28 kD) caspase-12. caspase-1, -11, and -12 in these two cell lines. Northern blot analysis demonstrated that the expression of caspase-12 2002). Caspase-11 mRNA was observed in all analyzed or- mRNA is further enhanced in L929r2 and induced in Bl6/ gans, though the expression levels in the lung, spleen, B16 cells by IFN- (Fig. 4). Synergism between TNF and uterus, and smooth muscle were clearly the highest (Fig. 1 IFN- was observed with caspase-12 mRNA in B16/B16 B). Caspase-1 mRNA expression was detected in all of the cells and with caspase-11 mRNA in both L929r2 and B16/ organs, with the lowest level in the liver and the kidney and B16 cells. Such an effect was not observed for caspase-1 and the highest in the spleen. -12 in L929r2 cells. Treatment of L929r2 cells for 24 h Using Western blot analysis, we checked the expression of with IFN- and TNF leads to cell death, about half of the caspase-1, -11, and -12 at the protein level in lysates of sev- cells are propidium iodide positive at 24 h, whereas B16/ eral organs from 5-wk-old mice (Fig. 2 A). The expression B16 cells still survive at that time point. This may explain pattern of caspase-1 at the protein level corresponded with the apparent difference observed in the response of these the RT-PCR results in Fig. 1 B. However, for caspase-12 cell lines. An increase in caspase-1 mRNA was observed and caspase-11, this correlation between protein and mRNA only in L929r2 treated with IFN-, whereas no caspase-1 expression levels was not obvious (Fig. 2). Expression of cas- mRNA expression was observed in B16/B16 cells in any of pase-12 protein was highest in skeletal muscle, heart, brain, the treatments. testis, and eye (Fig. 2 A). However, no, or hardly any, cas- Using Western blot analysis, we determined if the induc- pase-12 protein was detected in the other organs tested. Al- tion of caspase-12 mRNA expression in B16/B16 cells was though high caspase-12 mRNA expression levels were de- also reflected at the protein level and compared the effect tected in the lymph node and the spleen, only very low with that observed with other caspases (Fig. 5). Treatment amounts of the protein could be detected in any of the he- with IFN- increased the expression of caspase-1, -11, and matopoietic organs tested. Caspase-11 expression was pre- -12. When the treatment with IFN- was combined with The Journal of Cell Biology 460 The Journal of Cell Biology | Volume 162, Number 3, 2003 Figure 3. Immunohistochemical analysis of the expression of caspase-12 in the mouse embryo. Caspase-12 expression was detected in sections using either anti–caspase-12 rabbit antiserum Ab-1 or Ab-2. Results obtained with both antisera are similar (not depicted). Only those obtained with Ab-2 are presented. Negative controls of adjacent sections were stained by the anti–cas- pase-12 antisera depleted with recombinant caspase-12. Sections stained by secondary antibody alone were completely negative (not depicted). (A) Sections of a 13.5-d-postcoitum embryo. Note the strong staining of heart, lung, and nose. White box shows the embryo at its relative real size (6 mm). (B) A close-up of the heart (1) in A. (C) A close-up of the lung (2) in A. Note the positive staining of the epithelial layer of the trachea. (D) A close-up of the nose (3) in A. Note the positive staining of the epithelium in the entrance of the nose. Bars: (A) 455 m; (B) 80 m; (C) 30 m; (D) 45 m. either TNF or LPS, the effect on the expression of caspase-1, expression of the different caspases intensified in time, sug- -11, and -12 was intensified (Fig. 5; Fig. 6 A). In contrast to gesting that the expression of caspase-1, -11, and -12 was the caspases mentioned above, IFN- treatment led to a de- induced, whereas that of the proapoptotic caspases 3 and 9 crease in the expression levels of caspase-3 and -9. The dis- was suppressed (Fig. 5). appearance of the full-length form of these caspases (Fig. 5) The expression of caspase-12 is not induced by in B16/B16 cells was not due to proteolytic processing be- type I IFNs cause no proteolytic fragments of caspase-3 and -9 were de- tected. The antisera used reveal specific proteolytic process- Next, we tested whether the effect on caspase-12 expression ing of caspase-3 and -9 during apoptosis (Fig. 8). As is specific for IFN- or whether it also occurs with IFN- or expected, IFN- also increased the expression of the IFN- -. Both IFN- and IFN- failed to induce the expression inducible dsRNA-activated protein kinase (PKR), which we of caspase-12. In contrast with the results obtained with used as a positive control (Baier et al., 1993). No effect was IFN-, only a very faint caspase-12 signal could be detected observed on the level of -actin used as internal negative in Western blots of lysates of B16/B16 cells treated with the control. Treatment with either TNF or LPS alone did not combinations of type I IFNs with either TNF or LPS (Fig. 6 affect the expression levels of any of the proteins. We ob- A). However, in cells treated with any one of the IFNs, the served a similar effect of IFN- with and without TNF or expression level of caspase-3 was reduced (Fig. 6 A), con- LPS on caspase-12 expression in another murine melanoma firming the results presented in Fig. 5. All three IFNs in- cell line (PG19; unpublished data). These results suggest duced the expression of PKR, demonstrating that the failure that the activation of the gene coding for caspase-12 is of type I IFNs to induce the expression of caspase-12 did not tightly regulated. Interestingly, the distinct effects on the result from an incapacity of the cells to respond to these cy- The Journal of Cell Biology Analysis of caspase-12 expression and processing | Kalai et al. 461 Figure 5. Analysis of the expression of caspase-12 and other caspases at the protein level in B16/B16 cells treated with IFN-, TNF, and LPS. Western blot analysis of lysates of B16/B16 cells treated for 9 or 24 h with IFN- (1,000 U/ml), TNF (5,000 U/ml), Figure 4. Effect of IFN- and TNF on the expression of the mRNAs or LPS (1 g/ml) alone or in combinations, using antisera specific of the mouse inflammatory caspase subfamily in L929r2 fibrosarcoma for the indicated murine caspases. Blots were revealed also with and B16/B16 melanoma cells. Cells were treated or not with IFN- an anti–-actin antibody to verify that equal amounts of protein (1,000 U/ml) or TNF (5,000 U/ml) alone or in combination. The next were loaded. The IFN-inducible kinase PKR was used as a positive day, mRNA was extracted and analyzed by Northern blotting using control, demonstrating responsiveness to IFN. CTRL indicates specific probes. The size of the hybridizing bands is indicated in kb. untreated controls. UV imaging of ethidium bromide intercalated in the 18S and 28S rRNAs was used to monitor loading of the gels (bottom). tected by the G149 antiserum in HEK293T cells that do not express murine caspase-12. Moreover, these bands were not tokines. IFNs are known inducers of antiviral response. Be- detected using two other distinct anti–caspase-12 antisera cause caspase-12 was implicated in the response to a viral in- (unpublished data). fection (Bitko and Barik, 2001; Jordan et al., 2002), we Untreated primary fibroblasts, like the L929 fibrosarcoma wondered if dsRNA, a typical virus by-product, would fur- cells, constitutively expressed caspase-12 (Fig. 7 B). Treat- ther modulate caspase-12 expression in the presence of IFN. ment of primary fibroblasts with IFN- strongly enhanced Interestingly, IFN-, but not IFN-, which is the antiviral the expression of the protein, and addition of either LPS or IFN per se, increased the expression of caspase-12 in synergy TNF even intensified the effect further (Fig. 7 B). These re- with dsRNA (Fig. 6 B). sults demonstrate that the expression of caspase-12, like that Macrophages play a major role in the response to both vi- of the proinflammatory caspases 1 and 11, can be induced in ral and bacterial infection and thus present a cellular model cells exposed to IFN-. However, the response might be re- that is highly relevant for inflammation (Stoy, 2001). More- stricted to certain cell types, for example fibroblasts and me- over, macrophages express caspase-1 and -11 and secrete ac- lanocytes, because macrophages that constitutively express tivated IL-1 in response to infection and proinflammatory caspase-1 and can be induced to produce caspase-11 appar- stimuli (Lin et al., 2000; Schauvliege et al., 2002). There- ently fail to express caspase-12. fore, we determined the effects of the three IFNs alone and of IFN- in combination with LPS, TNF, or dsRNA on the Caspase-12 is processed in apoptotic cell death protein expression levels of caspase-1, -11, and -12 in the induced by FasL, TNF, or LPS Mf4/4 murine macrophage cell line. IFN-, and to a lesser extent also LPS and IFN-, clearly induced the expression of Transient overexpression of full-length wild-type caspase-12, caspase-11. The expression of caspase-1 remained high and but not of its inactive C298A mutant, in HEK293T cells led stable (Fig. 7 A). However, no caspase-12–specific bands to processing of the protein and to apoptosis (Fig. 2 B; Fig. were detected in Mf4/4 cells in any of the tested conditions 8 A; Fig. 9 B). Processing of caspase-12 in these conditions (Fig. 7 A). Similar results were obtained with J774 and resulted in fragments of 38 and 28 kD. These results sug- pU518, two other murine macrophage cell lines (unpub- gest that a link exists between the processing of caspase-12 lished data) and primary peritoneal macrophages (Fig. 7 B). and the induction of an apoptotic cell death. In rapid apop- The nonspecific bands detected in Fig. 7 A appeared after a tosis occurring in L929sAhFas cells treated with FasL, cas- long exposure of the blot to the film. Similar bands were de- pase-12 was fully processed, and only the 28-kD fragment The Journal of Cell Biology 462 The Journal of Cell Biology | Volume 162, Number 3, 2003 Figure 7. 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-, - or, - (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml), LPS (1 g/ml), or dsRNA (50 g/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- (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml) or LPS (1 g/ml). B16/B16 cells treated or not with IFN- (1,000 IU/ml) were used as a positive control. Figure 6. Comparison of the effect of type I and type II IFNs on An anti–-actin antibody was used to verify that equal amounts of the expression of caspase-12 and -3 by B16/B16 cells. (A) Western protein were loaded. blot analysis of cells treated for 24 h with IFN-, -, or - (1,000 IU/ml) in the presence or absence of TNF (5,000 IU/ml) or LPS (1 g/ml). (B) Western blot analysis of cells treated for 24 h with IFN- or - (1,000 IU/ml) in the presence or absence of dsRNA (100 g/ml). Caspase-12 is dispensable for Tg-induced The IFN-inducible kinase PKR was used as a positive control for ER stress–mediated apoptosis responsiveness to the different IFNs. An anti–-actin antibody was used to verify that equal amounts of protein were loaded. 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/ was observed (Fig. 8 B). The processing of caspase-12 in the B16 cells express caspase-12 only when they are treated with latter cells coincided with the activation of caspase-3, -7, and IFN-, we used these cells to test if caspase-12 is required for -9 (Fig. 8 B). Treatment of B16/B16 cells with TNF alone ER stress–mediated apoptosis induced by Tg. B16/B16 cells had no effect on their growth, whereas treatment with either died in response to Tg in the presence or absence IFN- IFN- or LPS alone led to a decrease in the proliferation rate (Fig. 10 A). Light microscopy analysis revealed that in both without any apparent signs of cell death (Fig. 9 A). How- cases, cells dying in response to Tg were blebbing and had ever, light microscopy analysis demonstrated that cells the typical apoptotic morphology similar to that seen with treated with a combination of IFN- with LPS or TNF HEK293T overexpressing caspase-12 (Fig. 8 A). Although rounded up and started floating and eventually died and dis- IFN- treatment clearly sensitized the cells to TNF-induced integrated (Fig. 9 A; unpublished data). We tested whether cytotoxicity (Fig. 10 A), the effect of IFN- on Tg-induced caspase-12 is also processed in this kind of cell death. As seen cell death was minor. To analyze the expression and process- before (Figs. 5 and 6), the protein was highly expressed in all ing pattern of caspase-12 in response to Tg and compare it the conditions that included IFN-. However, processing of with that observed in IFN-  TNF–treated cells, we used caspase-12 was detected only when the cells were treated two different anti–caspase-12 antisera. The first antiserum, with the combination of IFN- with either LPS or TNF Ab-2, was raised against a peptide spanning residues 2–17 in (Fig. 9 B). These results show that caspase-12 is processed in the amino acid sequence of caspase-12 and thus recognized different proapoptotic conditions, including those induced fragments containing the prodomain of the protein, and the by TNF, LPS, and FasL, and suggest that it may actively second antiserum, G149, was raised against the catalytic contribute to the cell death process. parts of the enzyme (Fig. 10 B). Both antisera detected the The Journal of Cell Biology Analysis of caspase-12 expression and processing | Kalai et al. 463 Figure 9. Caspase-12 is processed in B16/B16 cells dying from the combination of IFN- with either LPS or TNF. (A) Analysis of the cytotoxic potential of IFN-, TNF, or LPS. Cells were treated with IFN- (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 g/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- (1,000 U/ml), TNF (5,000 U/ml), or LPS (1 g/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 Figure 8. Processing of caspase-12 in apoptosis induced by fragments of the processed caspase. *, nonspecific bands. 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 thus, probably corresponds to the mature large catalytic sub- localization signal containing GFP and a plasmid encoding for unit (LCSU) (Fig. 10, B and C). Taken together, these re- either the wild type (C-12 wt) or an inactive caspase-12 mutant sults demonstrate that caspase-12 is not required for Tg- (C-12 C298A). Fluorescent microscopy photographs and Western blot analysis demonstrate that although both of the caspase-12 induced cell death, although treatment with Tg can induce variants are expressed in equal levels, only the wild-type protein the proteolytic cleavage of the protease. is processed and leads to apoptotic cell death. Bar, 5 m. Arrows indicate fragments corresponding to caspase-12 and their size in kD. (B) L929sAhFas cells were treated with recombinant soluble Discussion FasL for the indicated time. Cytotoxicity was measured by trypan blue (TB) exclusion. CTRL indicates untreated controls. Processing In the current report, we investigated the expression of cas- of caspase-3, -7, -9, and -12 after a 2-h treatment with FasL was pase-12 during mouse embryonic development and in adult determined by Western blotting. Arrows indicate the proteolytically mouse organs. We compared the regulation of the expres- processed caspase fragments and their size in kD. sion of caspase-12 by proinflammatory stimuli with that of other caspases. Although the mRNAs of caspase-1, -11, and full-length 49-kD protein in cells treated with IFN- but -12 are expressed in most of the organs tested, the level of failed to detect the expression of caspase-12 in cells treated expression varies substantially. For example, all three cas- with Tg alone (Fig. 10 C). The level of caspase-12 ex- pases were highly expressed in the spleen, whereas only cas- pression seen in cells cotreated with IFN- and Tg was pase-11 and -12 were highly expressed in the lungs. Similar lower than in cells treated with IFN- in the absence of Tg. results were obtained before for murine caspase-1, -11, and This may be explained by a decrease in translation due to -12 using Northern blots (Van de Craen et al., 1997) and Tg treatment (Wong et al., 1993). Caspase-12 processing for human caspase-1, -4, and -5 by RT-PCR (Lin et al., clearly occurred in cells treated with the combination of 2000). By analyzing a battery of mouse cell lines of various IFN- and TNF or Tg (Fig. 10 C). Ab-2 detected the 38- lineages, we show that the expression of caspase-12 at the kD fragment of caspase-12 in cells treated with IFN-  protein level is more restricted than expected. Indeed, the TNF but failed to detect this fragment in cells treated with only cells we observed to constitutively express the protein IFN-  Tg, whereas G149 detected the 28-kD fragment are primary fibroblasts and L929 fibrosarcoma cells. This in both types of treatments (Fig. 10 C). These results suggest suggests that although caspase-12 can be detected in most that the 38-kD fragment lacks only the COOH-terminal organs, only some of the cells in these organs are expressing small catalytic subunit (SCSU), and that the 28-kD frag- the protease. This suggestion was clearly supported by the ment is missing both the SCSU and the prodomain and, immunohistochemical analysis that demonstrated that in The Journal of Cell Biology 464 The Journal of Cell Biology | Volume 162, Number 3, 2003 1998; Burgess et al., 1998). Caspase-11–deficient mice present a similar phenotype in addition to the lack of the ability to respond to LPS by caspase-1 processing, a function that apparently requires caspase-11 (Wang et al., 1998). In humans, caspase-1 and IL-1 activation was shown to in- volve the formation of a complex including caspase-1 and caspase-5 (Martinon et al., 2002). Murine caspase-11 can also form a complex with, and is required for the activation of, caspase-1 (Wang et al., 1998) and therefore may have a function similar to that of human caspase-5. The phylogenic clustering of caspase-1, -4, -5, -11, and -12 suggests a role for the latter caspase in inflammatory responses. As seen with caspase-1 and -11, the expression of caspase-12 is stimulated by IFN- in fibrosarcoma and melanoma cells. In contrast to IFN- and -, which are produced by most cell types in re- sponse to viruses or dsRNA, production of IFN- is re- stricted to cells of the immune system (Katze et al., 2002). These include natural killer (NK) cells, CD4 T helper 1 (T 1) cells, and CD8 cytotoxic T cells stimulated by IL-12 and IL-18 secreted from activated macrophage or dendritic cells (Murphy and Reiner, 2002). IFN- and - are mainly involved in the response to viral infection (Katze et al., 2002; Taniguchi and Takaoka, 2002). Although IFN- has an an- tiviral function, the cytokine acts mainly as an effector cyto- kine required for cell-mediated immunity (Murphy and Reiner, 2002; Gordon, 2003). Moreover, IFN- is a strong activator of proinflammatory and microbicidal functions of macrophages (Gordon, 2003). Remarkably, expression of caspase-12 is induced exclusively by IFN- and not by IFN- Figure 10. Caspase-12 is dispensable for Tg-induced ER stress– or -. Does this suggest that caspase-12 plays a role in anti- mediated apoptosis in B16/B16 cells. (A) Analysis of the cytotoxic bacterial or proinflammatory activities? The answer is far effect of Tg (2 M) alone or in combination with IFN- (1,000 IU/ml) from clear, as the cellular substrates of the protease are still compared with TNF (5,000 IU/ml). Cell viability was measured at unknown. In contrast to caspase-1 and -11, caspase-12 is not the indicated time using MTT. CTRL is untreated control. Error bars, expressed in macrophages, a typical example of inflammatory standard deviation of six replicates of a representative experiment. cells. Moreover, overexpression of caspase-12 in several cell (B) Schematic representation of pro–caspase-12 indicating the relative location of the peptides used to produce the anti–caspase-12 lines together with pro–IL-1 does not lead to secretion of antisera Ab-2 and G149. Ab-2 was raised against a peptide spanning active IL-1 (Van de Craen et al., 1997; unpublished data), amino acids 2–17 of caspase-12 and is specific for the NH -terminal excluding a direct role for the protease in IL-1 maturation. prodomain. G149 was raised against a recombinant protein spanning In addition, although caspase-12–deficient mice were made amino acids 116–419 of caspase-12 and recognizes the catalytic (Nakagawa et al., 2000), nothing has been published yet on part of the protein. LCSU and SCSU indicate the large and the small catalytic subunits of the caspase, respectively. (C) Western blot the response of these deficient mice to proinflammatory or analysis of cells treated for 72 h with IFN- (1,000 IU/ml), TNF endotoxic stimuli or to bacterial infection. (5,000 IU/ml), or Tg (2 M) alone or in combinations. C-12 indicates Activation of caspase-1 or -11 due to exposure to LPS or lysate of HEK293T cells overexpressing caspase-12, used as a positive bacterial infection can also lead to apoptosis (Chen et al., control. Arrows indicate bands corresponding to the 49-kD full-length 1996; Hilbi et al., 1998; Kang et al., 2000, 2002; Hisahara pro–caspase-12 and to the 38- and 28-kD fragments of the processed et al., 2001). Caspase-12 may exert a similar function. Our caspase. *, nonspecific bands. results demonstrate that overexpression of caspase-12 in HEK293T cells leads to processing of the enzyme and to most positive organs, only certain cell layers, such as epithe- apoptosis. We observed similar processing of caspase-12 in lium of the trachea in the lung and the nose trail, express B16/B16 cells dying in response to IFN- combined with the caspase. either TNF or LPS and in L929sAhFas cells dying by Fas- Protein sequence comparison shows that caspase-12 most mediated apoptosis. Interestingly, in B16/B16 cells treated resembles caspase-1 and -11. Caspase-1 and its cytokine sub- with IFN-, expression of the typical apoptotic caspases 3 strates IL-1 and IL-18 play an important role in immune and 9 decreased, whereas that of caspases 1, 11, and 12 went and inflammatory responses to endotoxins and bacterial up. This down-regulation of the apoptotic caspases versus infection such as Shigella flexneri and Escherichia coli the up-regulation of the inflammatory caspases may suggest (Dinarello, 1998; Sansonetti et al., 2000; Joshi et al., 2002). that IFN- prepares the cells for an alternative caspase cas- Mice deficient in caspase-1 are resistant to endotoxic shock cade, avoiding the intrinsic apoptotic pathway. and are defective in LPS-induced secretion of IL-1, IL-1, The inducible expression of caspase-12 in B16/B16 cells IL-18, and IFN- (Li et al., 1995; Fantuzzi et al., 1997, allowed us to study the involvement of the protein in Tg- The Journal of Cell Biology Analysis of caspase-12 expression and processing | Kalai et al. 465 Preparation of protein extracts from mouse organs induced ER stress–mediated apoptosis. Our results clearly Mouse organs were isolated, and samples from three to six mice were show that the cells die in response to Tg whether caspase-12 pooled and frozen in liquid nitrogen. Tissues were thawed and homoge- is present or not. They also demonstrate that in cells express- nized on ice with an RZR homogenizer (Heidolph-Instruments) in PBS-A ing caspase-12, the enzyme is processed and probably acti- supplemented with 1 mM leupeptin, 1 mM aprotinin, 0.1 mM PMSF, and 1 mM oxidized glutathione. The homogenates were diluted in an equal vated in a variety of apoptotic conditions. In view of the re- volume of caspase lysis buffer containing 0.05% Nonidet-P40, 220 mM stricted expression pattern of caspase-12 to certain cell types PO , 10 mM Hepes, mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH 2 4 and the fact that its closest homologue encoded in the hu- pH 7.4, 1 mM leupeptin, 1 mM aprotinin, 0.1 mM PMSF, and 1 mM oxi- man genome is not a functional protease, we suggest that al- dized glutathione and incubated on ice for 10 min. Cell debris was re- moved by centrifugation, protein concentrations were measured using a though the protein may have a function in apoptotic cell Bio-Rad Laboratories protein assay, and 50 g from each sample was death, it is not likely to be the initiator of ER stress–medi- taken for Western blot analysis. ated apoptosis. Despite the absence of caspase-12 in stimu- lated macrophages, the induction of the expression and pro- Immunohistochemistry BALB/c mouse embryos, aged 13.5 d postcoitum, were fixed in 4% para- cessing of the protease by proinflammatory stimuli in formaldehyde, embedded in paraffin, and sliced (4–6 m). Endogenous fibroblasts may still suggest a role in the immune or inflam- peroxidase was blocked with peroxidase blocking reagent (DakoCytoma- matory response in the mouse. tion). Caspase-12 expression was detected in sections using either anti– caspase-12 rabbit antiserum, Ab-1 or Ab-2. Binding of primary antibody was revealed by 45 min incubation with a rat anti–rabbit antibody conju- gated to peroxidase (DakoCytomation) followed by incubation with the Materials and methods 3,3 -diaminobenzidine chromogen (DakoCytomation). Negative controls Antibodies, cytokines, and reagents were stained by the secondary antibody alone or by the anti–caspase-12 Recombinant murine TNF (specific biological activity of 2.2  10 IU/ml) rabbit antiserum depleted with a His -tagged recombinant caspase-12 pro- was produced in E. coli and purified in our laboratory. Recombinant mu- duced in E. coli and captured on Ni-NTA (QIAGEN). The specificity of the rine IFN- was from BioSource International. Recombinant murine IFN- depletion procedure was controlled by passing anti–caspase-12 antiserum and IFN- were produced in E. coli and purified in our laboratory. Flag- over a Ni-NTA column. Furthermore, the specificity of the antisera to cas- tagged human FasL was expressed in HEK293T cells and purified in house. pase-12 was verified by immunostaining of HEK293T transfected or not Poly(I)-poly(C) (synthetic dsRNA) was dissolved at 3.5 mg/ml in water (Am- with pCAGGS-caspase-12 C298A using a similar staining technique. ersham Biosciences). LPS (Salmonella abortus equi) was from Sigma- Aldrich. Anti–murine caspase-9 antibody was from New England Biolabs, Northern blot analysis Inc. Rabbit polyclonal antiserum against recombinant murine caspase-1, 3 2. B16/B16 or L929r2 cells were seeded at 1.2  10 cells per cm The next -3, -7, and -11 and caspase-12 (G149) were prepared at the Centre d’Econ- day, cells were left untreated or treated with either IFN- (1,000 U/ml) or omie Rurale (Laboratoire d’Hormonologie Animale). Purified anti–cas- TNF (5,000 U/ml) alone or in combination. Cells were harvested 20 h pase-12 antisera, Ab-1 and Ab-2, were from Oncogene Research Products. 6 cells using RNAzol™ later, and total RNA was extracted from 4  10 The anti–caspase-12 antiserum used in all Western blot analyses, unless (WAK-Chemie Medical GmbH). After electrophoretic separation on a 1% otherwise indicated, is G149. Antiserum specific for mPKR was from Santa mem- formaldehyde agarose gel, RNA was transferred to a Hybond-N Cruz Biotechnology, Inc., and antibody to -actin was from ICN Biomedi- brane and fixed by UV light. Membranes were incubated in prehybridiza- cals. Determination of protein concentration in cell lysates was performed tion solution (50 mM Tris-HCl, pH 7.4, 40% formamide, 4 SSC, 10 using a Bio-Rad Laboratories protein assay. P O , 1% SDS, and 20 g/ml herring sperm Denhardt’s solution, 0.1% Na 4 2 7 DNA) for 2 h at 42 C. DNA probes for caspase-1, -11, and -12 were la- RT-PCR analysis P]dCTP using random primering (Boehringer) and incubated beled with [ TM Mouse multiple tissue cDNA (MTC ) panels I and II were from CLON- overnight at 42 C with the prehybridized membrane. Filters were washed l PCR TECH Laboratories, Inc. PCR was performed in a total volume of 50 with 2 SSC, 0.1% SDS at room temperature and then exposed to an X-ray , 0.4 mM deoxynucleoside triphosphates, buffer containing 2 mM MgSO 4 film at 80 C. 0.5 M each primer, 2.5 U of Pfx Platinum DNA polymerase (Invitrogen), and 5 l of first strand cDNA of tissue or embryo cDNA. The PCR cycle Cell culture and treatment started at 94 C for 2 min, followed by a three-step cycling: denaturation at Murine Mf4/4 macrophages (Desmedt et al., 1998) and B16/B16 cells 94 C for 15 s, annealing at either 58 C or 62 C (depending on the primers were maintained in LPS-free RPMI 1640 medium (GIBCO BRL) supple- used) for 30 s, and extension at 72 C for 90 s. This was followed by a final mented with 10% FCS (GIBCO BRL), 0.03% L-Gln (Merck), 100 U/ml pen- extension step at 72 C for 5 min. In each experiment, PCR for glyceralde- icillin, 100 g/ml streptomycin, and 50 M -mercaptoethanol at 37 C in hyde-3-phosphate dehydrogenase (G3DPH) was performed according to a humidified 5% CO atmosphere. Before analysis, cells were seeded at the manufacturer’s instructions using primers provided by CLONTECH 3  10 cells/2 ml/well in six-well tissue culture plates (Falcon; Becton Laboratories, Inc., to ensure that an equal quality and quantity of cDNA Dickinson) in fresh growth medium. The next day, cells were left untreated was used. To avoid PCR products reaching saturation, 30 PCR cycles were or were treated as indicated. used for caspase-1, -11, and -12 and 22 cycles for G3DPH. The primers for Mouse embryonic fibroblasts were isolated from BALB/c mouse embryos mouse caspase-12 were derived from the 5 end of the full-length cDNA by collagenase treatment and incubated in DMEM.NUT.MIX.F-12 medium and from the 3 end of the predicted large catalytic subunit (Van de Craen supplemented with GLUTAMAX-I, pyridoxine, and 15% FCS. For analysis, et al., 1997). The primers for caspase-1 and -11 were derived from the 5 cells were seeded at 3  10 cells/2 ml/well in six-well tissue culture plates and 3 ends of the respective open reading frames. in fresh growth medium. The next day, cells were treated as indicated. Peritoneal macrophages were obtained by washing the peritoneal cavity Plasmids and transfection of HEK293T cells of three adult C57/BL6 mice with PBS-A. May-Grünwald/Giemsa (Sigma- The cloning of the cDNA encoding for full-length caspase-12 from a Aldrich) staining performed after the removal of erythrocytes by a hypo- cDNA library derived from L929r2 cells and its transfer to the pCAGGS eu- tonic treatment demonstrated that 90% of the recovered cells had a karyotic expression vector were described previously (Van de Craen et al., monocyte/macrophage-like morphology. The remaining cells were seeded 1997). In pCAGGS-caspase-12 C298A, the codon for the catalytic cysteine at 10 cells/1 ml/well in six-well tissue culture plates in LPS-free RPMI 1640 was substituted by that coding for alanine, leading to the expression of an medium supplemented with 10% FCS, 0.03% L-Gln, 100 U/ml penicillin, inactive form of the enzyme. pNLS-EGFP is a modified pEGFP-N1 plasmid 100 g/ml streptomycin, and 50 M -mercaptoethanol and allowed to ad- (CLONTECH Laboratories, Inc.) encoding GFP with a nuclear localization here for 1.5 h. After the removal of suspension cells and refreshment of the signal. HEK293T cells were transiently cotransfected using calcium phos- medium (2 ml/well), adherent cells were treated as indicated. phate precipitation in six-well culture dishes with 50 ng of pNLS-EGFP and The human Fas-expressing L929sA cells (L929sAhFas) were described 600 ng of either pCAGGS-caspase-12 or pCAGGS-caspase-12 C298A. The previously (Vercammen et al., 1998). L929sAhFas cells were seeded in next day, cells were examined and photographed using a fluorescent mi- 9-cm plates at 3.10 /plate. The next day, cells were treated with medium croscope. Cells were harvested and lysed for Western blot analysis as de- or recombinant Flag-tagged FasL (1/500 dilution), and cell death was de- scribed below. termined by trypan blue staining in 2-h intervals. 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Repli- sessed at the indicated time using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- cation of a cytopathic strain of bovine viral diarrhea virus activates PERK nyltetrazolium bromide (MTT; Sigma-Aldrich) (Tada et al., 1986). The and induces endoplasmic reticulum stress-mediated apoptosis of MDBK percentage of cell death was calculated using the following equation: cells. J. Virol. 76:9588–9599. -treated cells  A medium)/(A -untreated 100%  (1  (A 595/655 595/655 595/655 Joshi, V.D., D.V. Kalvakolanu, J.R. Hebel, J.D. Hasday, and A.S. Cross. 2002. medium). cells  A 595/655 Role of caspase 1 in murine antibacterial host defenses and lethal endotox- emia. Infect. Immun. 70:6896–6903. The authors thank W. Burm and E. Van Damme for technical assistance and Kang, S.J., S. Wang, H. Hara, E.P. Peterson, S. Namura, S. Amin-Hanjani, Z. A. Raeymaekers and F. Duerinck for preparation of TNF, IFN-, and IFN-. Huang, A. Srinivasan, K.J. Tomaselli, N.A. Thornberry, et al. 2000. 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