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J. Biol. Chem., Vol. 275, Issue 51, 39920-39926, December 22, 2000

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Expression Analysis of the Human Caspase-1 Subfamily Reveals Specific Regulation of the CASP5 Gene by Lipopolysaccharide and Interferon-^*

Xiao Yu Lin, Michael Shui Kuen Choi, and Alan G. Porter^§

From the Institute of Molecular and Cell Biology, Singapore 117609, Republic of Singapore

Received for publication, August 10, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Based on high sequence homology, there are six members in the caspase-1 subfamily: caspases 1, 4, 5, and 13 in humans and caspases 1, 11, and 12 in mice. Only caspase-1 is known to activate interleukin-1 and interleukin-18, and caspase-11 activates pro-caspase-1 in vivo. Almost nothing is known about caspases 4, 5, and 13. Here we report a sensitive and specific polymerase chain reaction system to analyze closely related genes. We employed this system to analyze the gene expression and regulation of human caspases 1, 4, 5, and 13, demonstrating that they have different expression patterns in normal tissues and cell lines. Interferon- strongly induced CASP1 and CASP5 but not CASP4 or CASP13 gene expression in HT-29 colon carcinoma cells. In contrast to the mRNA, interferon- up-regulated caspase-1 but not caspase-5 protein. In the monocytic cell line THP-1, CASP1 mRNA and caspase-1 protein are expressed constitutively, and their levels were not increased by lipopolysaccharide, whereas both CASP5 mRNA and caspase-5 protein were induced by lipopolysaccharide. Caspase-1 subfamily members displayed different in vitro activities toward pro-caspases 1 and 3 and pro-interleukin-1. Our results demonstrate that caspase-1 and caspase-5 levels are modulated by interferon- and lipopolysaccharide, respectively, and suggest that caspase-1 subfamily members are differentially regulated and may have distinct functions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Caspases are a family of aspartic acid-specific proteases that fulfill varied and often critical roles in mammalian apoptosis or proteolytic activation of cytokines (1-3). Currently, 14 caspases have been discovered in mice and humans that represent at least 11 different enzymes (2-4). Whereas caspases 1-3, 6-9, and 14 have homologues in mice and humans, caspases 4, 5, 10, and 13 have no known counterparts in mice; and conversely, caspases 11 and 12 have no known counterparts in humans (2-4).

Based on amino acid sequence homology and to a lesser extent the presence of a long N-terminal prodomain, caspases 1, 4, and 5 and caspases 11-13 constitute one distinct group: the caspase-1 subfamily (4). The in vitro substrate preferences of caspases 1, 4, and 5 have been determined using small peptides, and they are very similar (5). Despite the fact that the caspase-1 subfamily is large and constitutes almost half the total number of known caspases, an appreciation of their regulation, functions, and activities is still inadequate. Caspase-1 is the best characterized and appears to be involved in some pathophysiological cell deaths (6). Importantly, it fulfills a major physiological role as an essential mediator of inflammation and immune regulation through the proteolytic processing and activation of IL-1¹ in macrophages and IL-18 (also called interferon--inducing factor) in T cells (3, 6, 7).

Murine caspase-11 is the only other partially characterized member of the caspase-1 subfamily. Mice deficient in caspase-11 have a very similar phenotype to Casp-1^/ mice; for example, Casp-11^/ mice are resistant to endotoxic shock induced by bacterial LPS and fail to produce mature interleukin-1 and IL-1 after LPS stimulation (8). Moreover, Casp-11^/ embryonic fibroblasts are resistant to apoptosis induced by ectopic expression of caspase-1, suggesting that caspase-11 is an upstream activator of caspase-1 (3, 8). Unlike caspase-1, the expression of caspase-11 is LPS-inducible (8, 9), and it is reasonable to anticipate that other members of the family are regulated at the transcriptional or translational level by extracellular stimuli. The human homologue of caspase-11 is unknown because of the dearth of information about the expression, induction, and in vivo substrates of human proteases related to caspase-1, but it could be caspase-4, -5, or -13 based on sequence homology (4).

Here we report the development of a highly specific PCR approach to elucidate and compare the gene expression and regulation of very closely related members of the caspase-1 subfamily. Our PCR approach is generally applicable, enabling very highly homologous genes to be clearly distinguished, which is frequently uncertain using Northern blotting. We find that IFN- strongly induces expression of the human CASP1 and CASP5 but not the CASP4 or CASP13 genes. Whereas IFN- up-regulates the CASP5 gene but not the caspase-5 protein, bacterial LPS specifically induces both CASP5 mRNA and caspase-5 protein. Thus, the human CASP5 gene resembles the murine Casp-11 gene in its LPS inducibility. We also show that the caspase-1 family members show different in vitro activities toward selected substrates.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Biological Reagents-- Human cell lines 293T, HeLa (S3, D98, H21), HT-29, Jurkat T, MCF-7, SW480, THP-1, and U937 were maintained in RPMI 1640 supplemented with 10% (v/v) fetal calf serum (10). Human Hs68, MRC-5, and MRHF fibroblast cell lines and the mouse macrophage cell line RAW 264.7 were maintained in Dulbecco's modified Eagle's medium supplemented with 4.5 mg/ml glucose and 10% (v/v) fetal calf serum.

THP-1 cells were cultured for 24 h before LPS (Escherichia coli serotype 055:B5, Sigma) treatment and, together with LPS, were plated onto 100-mm Petri dishes to facilitate harvesting of the cells at each time point. CHX (Sigma), when used, was added 30 min before LPS treatment. HT-29 cells were plated under serum starvation conditions overnight before treatment with IFN- (Pharmingen).

Plasmid DNA Construction-- The cDNAs encoding full-length caspases were obtained by PCR with Pfu DNA polymerase (Promega) from cDNAs made from THP-1 cells (for human caspase-1, -4, and -5) and NIH 3T3 cells (for mouse caspase-11). The resulting PCR products were cloned into the mammalian expression vector pcDNA3 (Invitrogen). The CASP13 full-length cDNA in pcDNA3 was obtained from Dr. V. Dixit (Genentech, Inc.).

Recombinant caspases were expressed from DNA encoding the p30 portion of the caspase (with prodomain deleted) using the pGEX-4T-3 bacterial expression plasmid (Amersham Pharmacia Biotech). First, CASP p30 DNAs were generated by PCR using the full-length CASP clones as template. The forward primers used were: 5'-AAA GAT ATC TGA ACC CAG CTA TGC CCA CAT CCT CA-3' (CASP1); 5'-AAA GAT ATC TGC AAA TAT CCC CCA ATA AAA AAG CT-3' (CASP4); 5'-AAA GAT ATC TGC AAA AGA TCA CCA GTG TAA AAC CT-3' (CASP5); 5'-AAA GAT ATC TGC CAG GCA GCC ACC ATG GTG AAG CT-3' (Casp-11); and 5'-AAA GAT ATC TGA AAA ATT CCA CCA GTA TAA AAG CT-3' (CASP13). The reverse primers for CASP p30 were derived from the 3' sequence of each CASP cDNA with the addition of a XhoI restriction site. The resultant PCR products were digested with EcoRV and XhoI and were cloned into pGEX-4T-3 digested with SmaI and XhoI to generate DNA encoding GST-CASP p30.

RT-PCR Analysis-- Human multiple tissue cDNA (MTC^TM) panels I and II were purchased from CLONTECH. All other first strand cDNAs were synthesized from total RNA using the SuperScript II preamplification system (Life Technologies, Inc.) according to the manufacturer's protocol. Total RNA from various cell lines was extracted using the RNeasy mini kit from Qiagen. PCR was performed in a total volume of 50 µl of PCR buffer containing 1.5 mM MgCl₂, 0.2 mM deoxynucleoside triphosphates, 0.4 µM each primer, 2.5 units of HotStarTaq DNA polymerase (Qiagen), and 2 µl of first strand cDNA from cell lines or 5 µl of tissue cDNA from CLONTECH. The PCR cycle started at 95 °C for 15 min followed by a 3-step cycling: denaturation at 94 °C for 45 s, annealing at either 60 or 68 °C (depending on the primers used) for 1 min, and extension at 72 °C for 1 min. This was followed by a final extension step at 72 °C for 10 min. In each experiment, PCR for GAPDH was performed to ensure that an equal quality and quantity of cDNA was used. The primers for human CASP1, -4, -5, and -13 are shown in Fig. 1A. The primers for mouse Casp-11, human IL-1, and human IL-18 were derived from the 5' and 3' ends of each full-length cDNA. The primers for GAPDH were 5'-TGA AGG TCG GTG TGA ACG GAT TTG-3' and 5'-GCC TAA ATG GCC TCC AAG GAG TAA-3'. The annealing temperature for CASP1, Casp-11, and CASP13 and IL-1, IL-18, and GAPDH was 60 °C, whereas for CASP4 and -5 the annealing temperature was 68 °C (see "Results"). To avoid PCR products reaching saturation, the cycling number was adjusted depending on the endogenous expression level of each gene in various cell lines. For CASP1, a cycling number of 30 was used for THP-1 cell cDNA and 35 for cDNA from the remaining cell lines. For CASP4 and -5, the cycling number was 35 for all the cell lines used unless otherwise indicated. For Casp-11, the cycling number was 30 for cDNA from RAW 264.7 cells, whereas for CASP13, the cycling number was always 40. For IL-1 and IL-18, the cycling number was 30 for THP-1 cell cDNA. For GAPDH, the cycling number was only 25 for all the cell lines.

Expression of Recombinant GST-CASP p30 and in Vitro Cleavage Assay-- Bacterial cells harboring either GST or GST-CASP p30 cDNA were grown to an A₆₀₀ of 0.6, and proteins were induced with 0.2 mM isopropyl -D-thiogalactopyranoside for 3 h at 37 °C. Cells were harvested, and the cell pellet was resuspended in double strength cleavage buffer (50 mM Hepes, pH 7.4, 2 mM EDTA, 0.2% CHAPS, 20% glycerol) and sonicated twice for 10 s. The supernatant was used as the source of GST or active GST-caspase. For the in vitro cleavage assay, 10 µl of GST or active GST-caspase in double strength cleavage buffer was incubated with 1 µl of [³⁵S]methionine-labeled substrate using the T7 Quick TnT coupled transcription/translation systems (Promega), 10 mM dithiothreitol, 200 mM NaCl where indicated, and water to a final volume of 20 µl. The reaction was carried out for 2 h at 37 °C. The resulting cleavage products were analyzed by 15% SDS-polyacrylamide gel electrophoresis and subjected to autoradiography.

Protein Extraction and Western Blot Analysis-- Cellular protein preparation and Western blot analysis were done as described previously (10). The antibodies for human caspase-1 (catalog number sc-622) and human IL-1 (catalog number sc-7884) were from Santa Cruz Biotechnology. The antibody for caspase-4 (catalog number M029-3) was from MBL (Nagoya, Japan). The anti-Flag M2 antibody (catalog number F3165) was from Sigma. The rat monoclonal antibody for caspase-11 was supplied by Dr. J. Yuan (Harvard Medical School, Boston, MA).

To produce the anti-caspase-5 antibody, DNAs encoding peptide sequences from two separate unique regions (DVLHGVFNYLAKHDVLTLK and MDQKITSVKPLLQIE) of caspase-5 were joined (named A2) and multiplied 10 times (named A20) through molecular cloning before being fused to the pGEX-4T-3 bacterial expression plasmid to generate DNA encoding a GST-A20 fusion protein. The purified GST-A20 was used to immunize laboratory rabbits. The anti-caspase-5 antibody was affinity purified using a SulfoLink coupling gel column (Pierce) immobilized with the two above peptides, except that a cysteine was added at the C terminus of each peptide.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Design of Specific PCR Primers for Amplifying Human CASP1 Subfamily cDNAs-- The PCR primers used to amplify and detect human CASP1, -4, -5, and -13 cDNAs are shown in Fig. 1A. The specificity of the primers was determined by ensuring that the overall degree of mismatch of the forward primers with heterologous CASP1 subfamily cDNAs exceeded 24% (Fig. 1A). The CASP1 forward primer was the most distinct (average 61% mismatch) compared with the other CASP forward primers. Specificity was further guaranteed by ensuring that at least three of the four 3'-terminal nucleotides of each forward primer are unique when aligned with the corresponding region of other members of the CASP1 subfamily (Fig. 1A). This primer design prevented the occurrence of nonspecific PCR products from other CASP1 subfamily members when used in PCR with a DNA polymerase that lacks 3'-5' exonuclease activity, like Taq. The reverse primer was the same for each caspase, except for CASP1 whose DNA sequence is more distinct.

View larger version (53K): [in this window] [in a new window]   Fig. 1.   Specificity of PCR primers for the CASP1 subfamily. A, primer sequences used in PCR. Forward and reverse primers (underlined) for CASP1, -4, -5, and -13 are aligned. Flanking sequences around each forward primer are also shown. Mismatched sequences are in white boxes. A common reverse primer was used for CASP4, -5, and -13. B, PCR primers are specific to each caspase. Plasmid DNAs (50 ng for each reaction) encoding full-length caspases were used as templates for PCR. The names of the templates and primers used in each PCR are indicated.

To test the specificities of the PCR primers, plasmid DNAs harboring the full-length cDNAs of the CASP1 subfamily were used as templates, and the annealing temperature was optimized. It was found that at the annealing temperature of 60 °C, all four primer pairs amplified only their cognate CASP cDNAs, and the annealing temperature could be elevated to 68 °C for CASP4 and -5 primers without causing any significant reduction of the desired PCR product (Fig. 1B). These optimized PCR conditions (see also "Experimental Procedures") were subsequently used in all experiments with cDNAs from various tissues and cell lines, but the number of PCR cycles for each CASP cDNA was adjusted depending on the endogenous mRNA expression level to avoid PCR products reaching saturation.

Gene Expression of the CASP1 Subfamily in Various Human Tissues and Cultured Cell Lines-- Using the optimized PCR conditions, we examined the endogenous mRNA levels of CASP1, -4, -5, and -13 in various human tissues (Fig. 2A). Consistent with other reports (11-14), the CASP1, -4, and -5 genes are expressed in many human tissues, although brain, muscle, and testis have very low levels of all three mRNAs. The overall levels of CASP4 expression are higher than those of CASP1 and CASP5. Notable differences in expression levels were found in colon and pancreas. In the colon, CASP5 levels are very high, and CASP1 and -4 levels are low. However, in the pancreas, CASP4 levels are very high compared with the levels of CASP1 and -5 (Fig. 2A). With the exception of placenta, lung, spleen, small intestine, colon, and peripheral blood lymphocytes, the expression levels of CASP5 are extremely low (Fig. 2A). Except in heart and colon, the tissue distributions of CASP1 and -5 mRNAs are quite similar. Surprisingly, we were consistently unable to detect CASP13 gene expression in any of the human tissues examined, even in peripheral blood lymphocytes, spleen, and placenta (Fig. 2A) in which CASP13 mRNA was reported to be abundant by Northern blot analysis (15). This was not due to failure of the primers, because they correctly amplified cloned CASP13 cDNA (Fig. 1B).

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Differential gene expression patterns of the human CASP1 subfamily. A, tissue distribution of the human CASP1 subfamily. Human multiple tissue cDNA panels I and II (CLONTECH) were used in PCR with indicated CASP-specific primers. 5 µl of cDNA was used in each PCR. B, expression pattern of the human CASP1 subfamily in different cell lines. RT-PCR was performed with total RNA from the indicated cell lines. PCR results with specific CASP and GAPDH primers are shown. The representative data from at least five independent experiments are shown. C, no CASP13 was detected in HeLa cells. RT-PCR was performed for CASP13 with the total RNA from three different HeLa sublines. A prolonged PCR cycling number (40) was used in B and C. PBL, peripheral blood lymphocytes.

The varied patterns of CASP1, -4, and -5 gene expression and the more widespread occurrence of CASP4 mRNA were also observed in diverse human tumor and fibroblast cell lines (Fig. 2B). Taking into account the relatively low expression of CASP5 mRNA (especially in Hs68 cells), the expression patterns of the CASP1 and -5 mRNAs were qualitatively similar (as in tissues) (Fig. 2B). Only CASP4 mRNA was present in SW480 and MCF-7 cells (Fig. 2B). Again, CASP13 mRNA was undetectable in all cell lines examined, except in RNA prepared from one batch of MRC-5, MRHF, and Hs68 cells in one of five independent experiments (Fig. 2B and data not shown). The amount of CASP13 mRNA in this batch of cells was very low, because it was only detected with a prolonged PCR cycling number of 40 and after a long exposure of the agarose gel (Fig. 2B). Nevertheless, the PCR product represents genuine CASP13 mRNA based on nucleotide sequence analysis. It is worth noting that MRHF and Hs68 are human foreskin fibroblast lines, and CASP13 cDNA was originally isolated from a skin fibroblast cDNA library (15). CASP13 has previously been shown to be highly expressed in HeLa S3 cells by Northern blot analysis (15), but in support of our data, CASP13 mRNA was not detected in three different HeLa sublines (Fig. 2C). In addition, the published CASP13 mRNA is around 2 kilobases, but the size of CASP13 mRNA observed by Northern blot analysis is 1.5 kilobases (15), which is similar to CASP4 and CASP5 mRNAs (11).

IFN- Up-regulates CASP1 and CASP5 mRNAs-- IFN- is known to up-regulate CASP1 mRNA and protein in several different cell lines including HeLa, U937, and HT-29 (16-18), and it has been suggested that CASP4 mRNA is elevated by IFN- in HT-29 cells (17). Using our PCR approach, we confirmed that CASP1 mRNA was strongly induced by IFN- in HT-29 cells and found that CASP5 mRNA was also induced in IFN--stimulated HT-29 cells (Fig. 3). In contrast to the previous finding (17), the CASP4 mRNA level was not changed by IFN- stimulation in these cells. Because the basal level of CASP4 mRNA is quite high in HT-29 cells, the failure to observe up-regulation of CASP4 mRNA following IFN- treatment might be due to the PCR reaching saturation, even in the untreated samples. However, when the PCR cycling number was reduced from 35 to 30, although the PCR product for CASP4 reduced significantly, there was still no up-regulation, confirming that CASP4 is not regulated by IFN- in HT-29 cells (Fig. 3). When the yield of amplified cDNA for CASP5 was raised to a level comparable with that of CASP4 by increasing the PCR cycling number from 35 to 40, the CASP5 mRNA induction was still clearly evident (Fig. 3). CASP13 mRNA expression was undetectable in both IFN--treated and untreated HT-29 cells (data not shown).

View larger version (41K): [in this window] [in a new window]   Fig. 3.   IFN- up-regulates CASP1 and CASP5 mRNAs in HT-29 cells. RT-PCR was performed with total RNA from HT-29 cells either untreated () or treated (+) for 24 h with 10 ng/ml IFN-. Experiments were carried out in duplicate. PCR results with specific CASP and GAPDH primers are shown. Different PCR cycling numbers (30, 35, or 40) for CASP4 and -5 are indicated.

LPS Induces CASP5 and IL-1 mRNAs in Human THP-1 Cells-- The human monocytic leukemia cell line THP-1 has been widely used to study the signaling pathways induced by bacterial LPS in the inflammatory response, but the LPS-induced activation of caspases is poorly understood. To investigate the regulation of CASP1 subfamily genes by LPS, we analyzed their mRNA levels by RT-PCR in THP-1 cells either treated with LPS at different times or left untreated (Fig. 4A). CASP1 and -4 mRNA levels showed no change in the presence or absence of LPS stimulation, even after a prolonged incubation; in contrast, CASP5 mRNA was strongly induced by LPS (Fig. 4A). The CASP5 mRNA level increased sharply at 4 h after the addition of LPS, peaked at 8 h, and remained high for at least 24 h (Fig. 4A). The induction of CASP5 mRNA was not due merely to prolonged incubation of THP-1 cells, because the control phosphate-buffered saline-treated cells did not show induction of CASP5 mRNA at any time. Once again, CASP13 mRNA was not detectable in THP-1 cells and was not induced by LPS (data not shown).

View larger version (46K): [in this window] [in a new window]   Fig. 4.   LPS induces CASP5 mRNA in THP-1 cells. A, RT-PCR was performed with total RNA from THP-1 cells either untreated () or treated (+) with 10 µg/ml LPS for the indicated times. PCR results with indicated gene-specific primers are shown. B, RT-PCR was performed with total RNA from THP-1 cells treated with 10 µg/ml LPS and 10 µg/ml CHX either individually or in combination for the indicated times. PCR results with CASP5-, IL-1-, or GAPDH-specific primers are shown.

The levels of mRNAs encoding pro-IL-1 and pro-IL-18, two physiological substrates of caspase-1 (6, 7), were also examined by RT-PCR following LPS treatment of THP-1 cells. As expected (19), pro-IL-1 mRNA was rapidly and dramatically induced by LPS, and maximal induction was achieved within 2-3 h after LPS treatment (Fig. 4A). On the other hand, the basal level of pro-IL-18 mRNA in THP-1 cells was much higher than that of pro-IL-1 mRNA and showed no increase, but rather a delayed reduction upon LPS treatment (Fig. 4A). These results indicate that, although pro-IL-1 and pro-IL-18 are both processed by caspase-1, the transcriptional regulation of their genes is different in THP-1 cells.

The protein synthesis inhibitor CHX was employed to determine whether the induction of CASP5 mRNA by LPS is a direct process or an indirect process that requires an intermediate step of new protein synthesis. CASP5 mRNA induction was greatly diminished when THP-1 cells were pre-treated with CHX (Fig. 4B, upper panels). The residual induction of CASP5 mRNA in cells treated with both LPS and CHX is likely to be due to the superinductive effect of CHX, because CHX treatment alone also displayed a steady yet weak increase of CASP5 mRNA (Fig. 4B, top right panel). In contrast, the large increase in IL-1 mRNA following LPS treatment was not inhibited by CHX in THP-1 cells (Fig. 4B, middle panel). These results indicate that in THP-1 cells the induction of CASP5 mRNA by LPS is dependent on an intermediate step(s) of protein synthesis.

A 35-kDa Form of Caspase-5 Is Induced by LPS in THP-1 Cells-- To detect caspase-5 protein, an anti-caspase-5 antibody was generated against multimerized peptide sequences from two separate unique regions of caspase-5 fused to GST (see "Experimental Procedures"). The purified anti-caspase-5 antibody showed no cross-reactivity against other members of the caspase-1 subfamily or GST (data not shown). Cell lysates from 293T cells transfected with C-terminal Flag-tagged CASP5 cDNA show specific bands at 46 kDa and at 36 kDa when probed with anti-caspase-5 antibody (Fig. 5B). The band at 46 kDa corresponds in size to the full-length caspase-5, whereas the bands at 36 kDa suggest cleavage product(s) of caspase-5 resulting from the loss of the C-terminal p10 subunit. This is supported by the observation that the anti-Flag antibody directed to the C terminus of caspase-5 detected the 46-kDa band but not the 36-kDa bands (Fig. 5B, arrow).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   LPS induces a 35-kDa form of caspase-5 protein in THP-1 cells. A, total cell extracts were prepared from THP-1 cells treated with 10 µg/ml LPS for the indicated times and analyzed by Western blotting with the indicated antibodies. Arrows indicate the specific protein bands. B, total cell extracts were prepared from 293T cells transiently transfected with either pcDNA3 or CASP5flag/pcDNA3 for 40 h and analyzed by Western blotting with the anti-caspase-5 antibody or anti-Flag antibody (Ab). C, upper two panels, RT-PCR was performed with total RNA from mouse RAW 264.7 cells treated with 10 µg/ml LPS for the indicated times. PCR results with Casp-11- and GAPDH-specific primers are shown. Lower two panels, total cell extracts were prepared from RAW 264.7 cells treated with 10 µg/ml LPS for the indicated times and analyzed by Western blotting with an anti-caspase-11 rat monoclonal antibody. The same blot was stripped and immunoblotted with an anti-actin antibody to show the equal loading of sample in each lane.

Using the anti-caspase-5 antibody, we examined caspase-5 protein in THP-1 cells stimulated with LPS (Fig. 5A). Bands at 46 and 42 kDa (Fig. 5A, upper two arrows) were detected in the unstimulated THP-1 cells. However, these two bands diminished in the LPS-stimulated cells; the larger band was lost during the first 2 h of treatment. Meanwhile, a 35-kDa band (Fig. 5A, lower arrow) was induced 8 h after the addition of LPS and increased steadily over the remaining period of treatment. All three bands were specific to caspase-5, because they were not recognized by the preimmune antibody, and were blocked by the peptides used to purify the caspase-5 antibody. Because the major decrease of the 46- and 42-kDa forms of caspase-5 was complete during the early period of LPS stimulation, the induction of the 35-kDa form of caspase-5 must reflect a protein induction of caspase-5 in the late period of LPS stimulation. Importantly, the kinetics of induction of the 35-kDa form of caspase-5 correlated with the observed up-regulation of CASP5 mRNA (Fig. 4A). Currently, we are investigating whether the 35-kDa form of caspase-5 corresponds to the active form or an intermediate form of caspase-5.

The protein levels of caspases 1 and 4 and IL-1 were also examined in LPS-stimulated THP-1 cells. Consistent with the results from RT-PCR (Fig. 4A), the protein levels of caspase-1 and caspase-4 showed no change at any time during LPS treatment (Fig. 5A). pro-IL-1 protein was not detectable in untreated THP-1 cells, but its induction by LPS was, as expected, rapid and sustained (Fig. 5A).

The induction of Casp-11 mRNA and caspase-11 protein in mice by LPS injection (9) and our finding that human CASP5 mRNA and protein are also induced by LPS in THP-1 cells raises the question of whether caspase-5 is the human homologue of caspase-11. The induction of caspase-11 protein was detected in mice 4 h after LPS injection (9). Our RT-PCR analysis shows that LPS induced Casp-11 mRNA in the mouse macrophage RAW 264.7 cell line after only 1 h; Casp-11 mRNA reached a peak at 4 h after LPS stimulation (Fig. 5C). Consistent with these results, caspase-11 protein was induced and was first observed in RAW 264.7 cells at 4 h after the addition of LPS (Fig. 5C). Whereas we demonstrated the induction of one form of caspase-5 in THP-1 cells (Fig. 5A), we detected the induction of two forms of caspase-11 in RAW 264.7 cells, as reported by others (8, 9, 20) (Fig. 5C). Together, our results show that LPS induces a strong and sustained level of the related CASP5 and Casp-11 mRNAs and proteins in human and mouse macrophage cell lines (Figs. 4 and 5C).

IFN- Induces Caspase-1 but not Caspase-5 Protein in HT-29 Cells-- The protein levels of human caspases 1, 4, and 5 were examined in HT-29 cells treated with IFN-. In accord with the data from RT-PCR (Fig. 3), caspase-1 protein was sharply induced in IFN--treated HT-29 cells, whereas caspase-4 protein showed no change (Fig. 6). Surprisingly, the anti-caspase-5 antibody only detected a weak band at 42 kDa, which showed little difference in intensity between IFN--treated and untreated cells (Fig. 6). This 42-kDa band is identical in size to one of the three bands observed in THP-1 cells (Fig. 5A). Because IFN- up-regulates CASP5 mRNA (Fig. 3), these results suggest that the synthesis of caspase-5 protein is post-transcriptionally regulated in IFN--treated HT-29 cells, unlike the up-regulation of caspase-5 that we observed in LPS-stimulated THP-1 cells.

View larger version (60K): [in this window] [in a new window]   Fig. 6.   IFN- induces caspase-1 but not caspase-5 protein in HT-29 cells. Total cell extracts were prepared from HT-29 cells either untreated () or treated (+) for 24 h with 10 ng/ml IFN- and analyzed by Western blotting with the indicated antibodies. Experiments were carried out in duplicate.

Differential in Vitro Cleavage Preference of the Caspase-1 Subfamily-- Caspases exhibit different in vitro substrate preferences, and caspases 1, 4, and 5 have been classified in the caspase-1 subfamily, in part due to their preference for the peptide sequence WEHD (5). Recently, the optimal cleavage site for murine caspase-11 was determined to be (I/L/V/P)EHD (20), which still shows some degree of similarity to that of caspases 4 and 5. However, a comparison of the substrate preference of the murine and human caspase-1 subfamily has not been made. Therefore, we expressed recombinant active caspases 1, 4, 5, 11, and 13 in bacteria and assayed their ability to cleave selected in vitro translated ³⁵S-labeled substrates (pro-caspase-1 and -3 and pro-IL-1).

They all cleaved pro-caspase-3 into fully mature small (p12) subunits, but only caspase-13 gave the fully mature large (p17) subunit of caspase-3 (the others gave an ~20-kDa (p20) peptide) (Fig. 7, middle panel). Moreover, caspase-13 was the only tested protease that completely cleaved pro-caspase-3 into the fully mature subunits. Interestingly, caspase-5 activity on pro-caspase-3 depended largely on the presence of 200 mM NaCl (Fig. 7, middle panel) (5). However, in the presence of 200 mM NaCl, caspases 1, 4, 11, and 13 all displayed reduced in vitro cleavage activity toward all the substrates tested (Fig. 7 for caspase-11; and data not shown).

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Differential in vitro cleavage preference of the caspase-1 subfamily. In vitro cleavage assays were performed as described under "Experimental Procedures". 35S-labeled in vitro translated pro-caspase-1 (A), pro-caspase-3 (B), or pro-IL-1 (C) was incubated with the indicated crude bacterial sonicates expressing GST or various active GST-caspase p30 proteins. The presence of 200 mM NaCl in some reaction mixtures is also indicated. Arrows indicate the full-length or cleaved products of each substrate. prop20, prodomain plus p20 domain; mIL-1, mouse interleukin-1.

Caspases 1, 4, and 13 processed pro-IL-1 correctly into 17.5- and 10.5-kDa species but with very different efficiencies, whereas caspase-5 (in the presence of 200 mM NaCl) and caspase-11 only partially cleaved pro-IL-1 to generate the 28-kDa intermediate form and failed to produce the 17.5-kDa mature IL-1 (Fig. 7, lower panel) (21). Caspase-1 weakly processed itself between the p20 and p10 subunits, as judged by the generation of 35-kDa (prop20) and 11-kDa (p10) bands, whereas none of the other members within the family could process pro-caspase-1 in our assay system (Fig. 7, upper panel).

These results indicate that although the caspase-1 subfamily members share extremely high sequence homology, especially in the p30 region corresponding to the active subunits, they show different cleavage patterns toward certain substrates in vitro. This implies that these caspases may have distinct functions in vivo.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Compared with conventional Northern blot analysis, RT-PCR is more sensitive, straightforward, and less time-consuming but does depend on a careful verification of the specificity of the PCR primers. In this study, we took great care that the PCR primers only amplified their cognate mRNAs (Fig. 1). Moreover, the elevation of CASP5 mRNA by IFN- and LPS that we demonstrated by RT-PCR is real, because we also verified the IFN--mediated induction of CASP1 mRNA (16-18) and the LPS-mediated induction of Casp-11 and IL-1 mRNAs (7-9) using this technique. Caspase-4, -5, and -13 proteins have 53, 51, and 47% sequence identity with caspase-1, respectively (11, 14, 15). Caspases 4, 5, and 13 are more related to each other, with 61-75% sequence identity. Even more striking, the sequence identity among caspases 4, 5, and 13 is over 80% when compared at the DNA level (11, 14, 15). When Northern blot analysis is used to assess CASP4, -5, and -13 mRNA levels, the possibility cannot be excluded that cross-hybridization may arise even under very highly stringent hybridization conditions. On the other hand, we designed short PCR primers for RT-PCR chosen from the few divergent regions of each mRNA and ensured that four 3' nucleotides from each primer are 75-100% mismatched compared with the corresponding positions in the other CASP sequences (Fig. 1, A and B). This is probably the crucial factor that ensures the total absence of cross-priming and forms the basis of our PCR approach for measuring the expression of very closely related genes. Using this approach, we have carried out for the first time a comprehensive assessment of gene expression for the human caspase-1 subfamily in normal tissues and in unstimulated or stimulated cell lines.

Human caspase-13 has 47, 75, and 61% sequence identity to caspase-1, -4, and -5, respectively (11, 14, 15). Caspase-13 is processed by caspase-8, and overexpressed caspase-13 induces apoptosis, suggesting a potential role of caspase-13 in a caspase-8-mediated signaling pathway (15). Recombinant caspase-13 completely processed pro-caspase-3 (Fig. 7) and poly(ADP-ribose) polymerase,² whereas other caspases within this family gave only partial cleavage (Fig. 7 and data not shown). In addition, caspase-13 completely cleaved Pro-IL-1 into the 28-kDa intermediate product, whereas caspase-1 cleaved pro-IL-1 into mature IL-1, with hardly a trace of the 28-kDa intermediate. Thus, caspase-13 may be more distinct from other members of the caspase-1 subfamily both on the basis of its substrate cleavage preference and the fact that the expression of its mRNA in many human tissues and cell lines is undetectable by RT-RCR. Nevertheless, CASP13 cDNA encodes an authentic protein, based on the sequence analysis of the RT-PCR product obtained from the skin fibroblast cells. The extremely restrictive and/or transient expression pattern of CASP13 mRNA and the strong potency of recombinant caspase-13 protein against some in vitro substrates tested suggest that caspase-13 could play an important role during embryonic development or that its expression may be specifically induced by a currently unknown stimulus.

The functions of human caspases 4 and 5 are largely unknown. We found that caspases 5 (in high salt), 4, and 1 cleaved pro-caspase-3 with equivalent efficiencies. This was not the case with pro-IL-1 as substrate, because caspase-1 cleaved pro-IL-1 efficiently, caspase-4 cleaved pro-IL-1 weakly, and caspase-5 had virtually no activity on this substrate. Our results with pro-IL-1 as substrate are very similar to previous findings using a different system (21). Although caspases 4 and 5 induced apoptosis when overexpressed in cells (11-14), there is no evidence that they play crucial roles in apoptosis, as do, for example, caspases 3, 8, and 9 (2). However, it has been shown that caspase-4 is activated during Fas-induced apoptosis in HeLa cells (22). In addition, overexpression of an inactive mutant of caspase-4 or microinjection of an anti-caspase-4 antibody delays Fas-induced apoptosis, suggesting that caspase-4 is involved (22). The protein sequence of caspase-4 is most similar to murine caspase-11 (60% identity) (9, 11). But caspase-4 is unlikely to be the human homologue of murine caspase-11, because the basal level of CASP4 mRNA is quite high in tissues and cells, which is in contrast to the very low unstimulated levels of Casp-11 in mice (9). More importantly, Casp-11 mRNA is LPS-inducible, but CASP4 mRNA is not.

Like murine Casp-11, human CASP5 expression is very low in many normal tissues, and CASP5 expression can be induced by bacterial LPS, a property so far shared only with Casp-11. We did not detect pro-caspase-5 zymogen induction by LPS, although we observed an induction of a 35-kDa form of caspase-5, which very likely represents an intermediate or active form of caspase-5, a result of the processing of caspase-5 after induction by LPS. Because very similar size bands were detected in 293T cells overexpressing caspase-5, this would suggest that increased expression of caspase-5 results in its processing. Also, despite the fact that the apparent cleavage preferences of caspases 5 and 11 toward selected substrates are similar, the activity of caspase-5 relies on a high salt concentration, unlike caspase-11. Finally, caspase-5 contains a unique region (amino acids 1-44) in its prodomain that is present neither in caspase-11 nor in any other caspase-1 subfamily members (11-15). Interestingly, frameshift mutations were detected around this unique region in CASP5 in tumors of the endometrium, colon, and stomach (23). Based on our study, caspase-5 is more likely than caspase-4 to be the human homologue of mouse caspase-11, which activates pro-caspase-1 and is required for the generation of functional IL-1. In this study, we also demonstrated that CASP5 mRNA is strongly induced by IFN-, a pleiotropic cytokine that plays important roles in both antiviral defense and immune cell activation. However, caspase-5 protein is not induced by IFN-, suggesting that caspase-5 protein synthesis is post-transcriptionally regulated. The significance of these results will be the subject of our future investigations.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Junying Yuan (Harvard Medical School) for caspase-11 antibody, Dr. V. Dixit (Genentech, Inc.) for CASP13 full-length cDNA, Dr. Jean-Paul Klein (Institut National de la Santé et de la Recherche Médicale U932, France) for THP-1 cells, Dr. Graeme Guy for MRC-5 fibroblasts, and Dr. Anthony Ting for Hs68 fibroblasts. We thank Dr. Li Peng and Dr. B. Venkatesh for reviewing the manuscript.

	FOOTNOTES

^* This work was supported by the Institute of Molecular and Cell Biology, Singapore.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors share equal senior authorship.

^§ To whom correspondence should be addressed: Inst. of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Republic of Singapore. Tel.: 65-874-3761 or 65-874-3777; Fax: 65-779-1117; E-mail: mcbagp@imcb.nus.edu.sg.

Published, JBC Papers in Press, September 13, 2000, DOI 10.1074/jbc.M007255200

² X. Y. Lin, M. S. K. Choi, and A. G. Porter, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: IL, interleukin; LPS, lipopolysaccharide; PCR, polymerase chain reaction; IFN-, interferon-; CHX, cycloheximide; GST, glutathione S-transferase; RT-PCR, reverse transcriptase-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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