Molecular cloning and characterization of murine caspase-12 gene promoter

Abstract

The activation of caspase-12 is involved in endoplasmic reticulum-mediated apoptosis. To investigate how caspase-12 is transcriptionally and translationally regulated, we isolated and sequenced the 5′-flanking region of mouse caspase-12 gene by a PCR-mediated chromosome-walking technique, using mouse genomic DNA as a template. Two DNA fragments of 3,221 and 800 bp were isolated and cloned into pGL3 promoterless vector upstream of the luciferase gene. The small DNA fragment contains the first intron sequence located downstream of the first exon and 27 bp from the second exon, whereas the large fragment contains the small fragment and the 5′-flanking region. Reporter constructs generated from these DNA fragments showed a substantial promoter activity in mouse NIH 3T3 or human embryonic kidney 293 cells grown in the presence of 10% serum. In the absence of serum, the luciferase activity was drastically reduced. However, the luciferase mRNA was higher in serum-starved cells than in control cells, suggesting that translation of luciferase mRNA was drastically inhibited. However, Western blot analysis revealed that the quantity of procaspase-12 is actually higher in serum-starved cells relative to that cultured in the presence of 10% serum. Progressive deletion analysis of the 3,221-bp sequence revealed that the highest luciferase activity was observed with the construct containing 700 bp upstream of ATG. The transcriptional initiation site was identified by 5′ RACE techniques using total RNA from NIH 3T3 cells. Our results should facilitate studies on the mechanism regulating the expression of this important gene.

Caspases, a family of cysteine proteases, play an important role in many forms of cell death by apoptosis and in proinflammatory cytokine maturation. To date, 14 caspases have been identified in mammalian cells, of which 12 human enzymes are known. Caspases are synthesized as zymogens with the general organization consisting of an N-terminal prodomain, followed by sequences encoding a large subunit, followed by a small subunit. Conversion of each caspase inactive precursor to the mature active enzyme requires a minimum of two cleavages; one separating the prodomain from the large subunit, and another separating the large subunit and the small subunit. Upon activation, caspases cleave a variety of cellular protein substrates. This process disrupts the survival pathways and disassembles important architectural components of a cell, contributing to the morphological and biochemical changes that characterize apoptotic cell death. Given the potentially devastating effect of caspase activation, it is not surprising that caspases production, processing, and activity are tightly regulated. In fact, active caspases appear to be regulated by posttranslational modifications, including phosphorylation (1, 2), ubiquitinylation-mediated degradation (3, 4), and nitrosylation (5–9). In addition, caspase activation and activity can also be regulated by interactions with inhibitor-of-apoptosis proteins and the broad-spectrum caspase inhibitor p35 (10, 11). Although apoptosis can occur in many cell types in the absence of mRNA or protein synthesis (12), several observations suggest that transcriptional regulation of procaspase gene expression might be important under certain circumstances. Up-regulation of caspases expression was observed during kidney ischemia (13, 14). In addition, it has been shown that in certain childhood neuroblastomas and neuroectodermal brain tumors, and in some type of lung cancers, caspase-8 level is low or even abolished. This observation was explained by both somatic gene mutations and deletions and silencing due to the hypermethylation of genomic caspase-8 sequence (15–18).

The murine caspase-12, cloned in 1997 from a murine L929r2 fibrosarcoma cDNA library (19), is localized on the cytoplasmic side of the endoplasmic reticulum, revealed with immunofluorescence and subcellular fractionation techniques (20, 22). It was described to play a role in endoplasmic reticulum-induced apoptosis (20, 23, 24). The caspase-12 gene consists of 11 exons, and is localized to the murine chromosome 9 downstream of the gene encoding caspase-1 and -11. The amino acid sequence of caspase-12 shares high homology with murine caspase-1 (39% identity) and caspase-11 (38% identity), and with human caspase-4 (48% identity) and caspase-5 (45% identity). Gene regulation of caspase-12 is largely unknown, in part because of the lack of information on the gene promoter. However, it has been shown that caspase-12 is regulated by IFN-γ at both protein and mRNA levels in L929r and B16/B16 cells (25).

In the current study, we isolated the 5′-flanking region of murine caspase-12 by a genome-walking technique and determined its translation start site by using the 5′ RACE technique. Truncation analysis showed that the maximum reporter gene activity was obtained when 700 bp upstream of ATG start codon was cloned into a reporter gene. Our results revealed several unique properties of this murine caspase-12 gene promoter: we found two genomic fragments, one of which is an intron, and both exhibit significant promoter activity in murine and human cells as monitored by transient transfection using reporter-gene vectors. The promoter activity of all constructs exhibit dependency on growth factors in the media. In addition, a 44-bp site corresponding to the 5′ UTR is involved in either the transcription or the translation of the luciferase transcript. The main transcription start site, identified by 5′ RACE techniques using mouse NIH 3T3 cells total RNA, was found at the 44 nt upstream of ATG. The current identification and characterization of the promoter sequence of caspase-12 provides the basis for further studies on the gene regulation of the caspase-12-mediated apoptotic pathway.

Experimental Procedures

Plasmids, Bacteria, and Reagents. Promoterless pGL3 basic vector and luciferase substrate were purchased from Promega. Super-Script II reverse transcriptase, TOP10 competent cells, and T-A cloning vector pCPII were from Invitrogen. Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs. The genome-walker kit, SMART RACE kit, and pEGFP-1 vector were from Clontech. FuGENE 6 and primary monoclonal anti-GFP antibody (used at 1:1,000 dilution) were purchased from Roche Molecular Biochemicals. Anti-caspase-12 polyclonal antibody was from R & D Systems (Indianapolis; used at 1:1,000 dilution). Bradford reagent and secondary goat anti-mouse antibody (used at 1:5,000 dilution) were from Bio-Rad.

Cell Culture. NIH 3T3 cells were grown in a humidified atmosphere containing 5% CO₂ at 37°C in DMEM containing high glucose (4.5 g/liter at 25 mM) and supplemented with 50 units/ml penicillin, 50 mg/ml streptomycin, and 10% (vol/vol) FBS. Just before transfection, the medium was removed and replaced with fresh medium with or without serum.

Cloning of the Mouse Caspase-12 Gene Promoter Region. To clone the 5′-flanking region of the murine caspase-12 gene, we used a mouse genome-walker kit (Clontech). The kit contains four sets of murine genomic libraries generated from mouse genomic DNA that had been cut with different restriction enzymes (EcoRV, DraI, PvuII, and SspI) and ligated to short adaptor sequences. These ligated DNA pools were then used as templates for two rounds of PCR. Primary and nested PCRs were performed by using forward primers complementary to the adaptor sequence [adaptor primer (AP)1 and AP2 provided with the kit] and capase-12 gene-specific antisense primers (GSPs) (GSP11 and GSP12, corresponding to residues 67–90 and 10–33, respectively) that were designed based on the published 5′ end of the mouse caspase-12 cDNA sequence. The GSP11 and GSP12 primers are located at 67–90 bp and 10–33 bp downstream of ATG, respectively. Each PCR mixture contained 2.5 mM Mg(OAc)₂, 2.5 μM of each dNTPs, 1× PCR buffer, 1 μM of each primer, and 1.25 units of Advantage genomic polymerase (Clontech). The PCRs were performed in a reaction volume of 50 μl by using a PerkinElmer 2400 thermocycler. For the primary PCRs, we used 1 μl of each library as template. For the nested PCRs, the primary PCR product was diluted 50 times and 1 μl was used in a 50-μl PCR mixture (7 cycles at 94°C for 2 s at 70°C for 3 min and 33 cycles at 94°C for 2 s at 65°C for 3 min). The PCR products were separated by agarose gel electrophoresis, and DNA fragments from two PCR-positive libraries were isolated and cloned in a MluI- and BglII-digested pGL3 basic plasmid vector (Promega) using T4 DNA ligase (New England Biolabs) to generate pGL3-3 and pGL3-0.8 reporter constructs. Upon sequencing, cloned fragments were found to contain 3,221 and 800 bp, respectively.

Progressive deletion mutants of the pGL3-0.8 and pGL3-3 were made by PCR. All forward primers were preceded by a sequence, including a MluI restriction site, whereas the reverse primers were designed to include a BglII restriction site. PCR was performed under the same conditions as described above by using Vent polymerase (New England Biolabs) and pGL3–3 as template. The amplified DNA fragments were unidirectionally cloned between the MluI and BglII sites of the pGL3 basic plasmid by using standard procedures. Conditions for PCR were 9 cycles at 94°C for 30 s at 66°C for 1 min and 20 cycles at 94°C for 2 s at 63°C for 1 min. Nucleotide sequences of the cloned DNA fragments were confirmed in each case by sequencing.

RACE. The transcription start site of the murine caspase-12 gene was also mapped by using the SMART RACE kit (Clontech) following the manufacturer's protocol. This technique involved the incorporation of a “smart oligo” onto the 5′ end of the reverse-transcribed cDNA for the 5′ RACE analysis. The 5′ end of the mouse caspase-12 was then amplified by using a standard PCR protocol. Primers were designed so that a relatively large section of the coding region (244–385 bp) was amplified during the nested PCR, along with the 5′ UTR. Reaction products were analyzed by 1.5% agarose gel electrophoresis, purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA). A purified 5′ RACE fragment was cloned into the T-A cloning vector pCPII and sequenced.

Transfection and Luciferase Assay. Murine NIH 3T3 or human embryonic kidney (HEK) 293 cells were seeded to six-well plates 24 h before transfection at a density of 4 × 10⁵ cells per well. Cells were transfected by using FuGENE 6 according to the manufacturer's suggestion. Measurement of luciferase activity was performed 24 h after transfection by using the luciferase assay kit (Promega) according to the manufacturer's protocol. Briefly, cells were washed twice with PBS solution, solubilized with 150 μl of lysis buffer supplemented with 1 mM DTT, and detached with a cell scraper. The lysates were centrifuged at 12,000 × g for 2 min to remove cell debris. The supernatant protein concentration was estimated by a standard Bradford protein assay (Pierce). Luciferase assay was determined in duplicate by using 10-μl aliquots of supernatants using a TD-20/20 Turner Designs luminometer. Each lysate was measured twice. Promoter activities were expressed as relative light units normalized for the protein content in each extract. The luciferase activity of each construct was compared with that of the promoterless pGL3 basic vector.

RT-PCR. RT-PCR was carried out by using total RNA extracted from the NIH 3T3 cells that had been transfected with the caspase-12 reporter gene in the presence or absence of serum, using TRIzol reagent according to the manufacturer's instructions. cDNA was transcribed from 1 μg of total RNA by using superscript RNase H^- reverse transcriptase and polydT primers as recommended by the manufacturer. RT-PCR primer were designed to amplify a portion of enhanced GFP (EGFP) coding transcript, which was under the control of caspase-12 promoter region in transfected cells.

Western Blot Analysis. Transfected NIH 3T3 cell extracts (20 μgof protein) were fractionated in SDS/4–20% PAGE, and the fractionated proteins were electrophoretically transferred to a nitrocellulose membrane. Immunoblotting was performed by using monoclonal anti-GFP antibody (Roche Molecular Biochemicals) or anti-caspase-12 polyclonal antibody as the primary antibody, and horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG as the secondary antibody. Bands were detected by using a chemiluminescence detection kit (Amersham Pharmacia Life Science, Rockville, MD).

Results

Identification of the Transcription Start Sites for Mouse Caspase-12. To identify the transcription start sites for mouse caspase-12, a SMART RACE analysis was performed by using three reverse oligonucleotides derived from the coding region (549, 385, and 244 bp downstream of start codon) and two adapter primers provided with the kit for the primary and nested PCR. A weak smear on the agarose gel was observed in the first PCR with outer primer AP1 and reverse primers GSP1 or GSP2 by using the total RNA from mouse NIH 3T3 cells (Fig. 1B, lane 1 and 2, respectively). The primary PCR products were used as template for three different nested PCRs by using AP2 and GSP2, or GSP3. Only one single product was obtained from all three nested PCRs (Fig. 1B, lanes 3–5). This result indicates that caspase-12 transcript may have one single translational site, although it does not rule out the possibility that another transcription site lies so close to the first one that PCR products were inseparable on the agarose gel electrophoresis. The 5′ RACE product was cloned into T-A cloning vector pCPII. The sequencing of 10 different clones, randomly selected, shows that they have the same sequence (Fig. 1C). The first base of this product was therefore assigned position -44, and it is located 44 bp upstream of caspase-12 start codon.

Fig. 1.

RACE analysis of the transcription start site of the caspase-12 gene. (A) Schematic presentation of the primers used in RACE. Three RACE assays were conducted by using two adapter primers (AP1 and AP2) and three caspase-12 GSPs (GSP1, GSP2, and GSP3, corresponding to nucleotide residues 524–549, 361–385, and 220–244, respectively). In the first RACE analysis, AP1-GSP1 and AP1-GSP2 primer pairs were used in the primary (lanes 1 and 2 in B) and secondary PCRs (lanes 3–5 in B), respectively. (B) Analysis of the primary and nested PCR products on agarose gel. Total RNA was extracted from NIH 3T3 cells by using TRIzol reagent. RACE was conducted as described in Experimental Procedures following the manufacturer's instructions. Nested PCR product was cloned into the T-A cloning vector pCRII, and then clones were sequenced. (C) Nucleotides of the murine caspase-12 5′ UTR. The first methionine codon is numbered +1 and upstream sequences are indicated by negative numbers.

Identification and Cloning of the Mouse Caspase-12 Gene 5′-Flanking Region. The 5′-flanking region of the mouse caspase-12 gene was cloned by using the mouse genome-walker kit (Clontech). Primers (GSP11 and GSP12) were designed based on the 5′ end of the published caspase-12 cDNA. We isolated two caspase-12 5′-flanking regions fragments from four genomic DNA pools, with lengths ranging from 0.8 to 3.2 kbp (Fig. 2A). The fragments were cloned into the promoterless pGL3 basic reporter plasmid (Promega) to generate pGL3-3 and pGL3-0.8. The complete nucleotide sequence of these fragments was determined (Fig. 2B). blast analysis confirms that the genomic sequences isolated by genomic walking correspond to the mouse caspase-12 gene. blast analysis of the isolated caspase-12 genomic DNA against the mouse or human genome database did not reveal any homology with any gene. By using computer analysis, we identify several putative transcription factor-binding sites in the caspase-12 promoter region, including binding site for AP1, Oct-1, specificity protein 1 (Sp1), and NF-κB.

Fig. 2.

Nucleotide sequence of the 5′-flanking region of the mouse caspase-12 gene. (A) Two genomic DNA fragments, corresponding to the murine caspase-12 5′-flanking region, were amplified by a PCR-mediated chromosome-walking technique using four mouse genomic libraries from Clontech, previously digested with four different restriction enzymes: EcoRV, lane 1; DraI, lane 2; PvuII, lane 3; and SspI, lane 4; M, DNA marker. (B) Both DNA fragments were cloned in pGL3 basic vector and sequenced. The coding region, including the ATG start codon, are red, and the 5′ UTR, obtained by RACE analysis, is blue.

Deletion Analysis of the Mouse Caspase-12 Promoter. Fig. 3A depicts the schematic representation of the two DNA fragments, comprised of 3,221 and 800 bp, isolated and cloned onto the promoter-less vector upstream of the luciferase gene, denoted as pGL3-3 and pGL3-0.8, respectively. When these constructs were transfected into murine NIH 3T3 or human HEK 293 cells for 24 h, the resulting luciferase activity revealed that both DNA fragments process promoter activity in murine and human cells. This result suggests that the cell-specific elements may not be present in those sequences. As shown in Fig. 3B, the promoter activity in the intron-only fragment (pGL3-08) is more pronounced than that in the 3,221-bp fragment, particularly when transfected in NIH 3T3 cells.

Fig. 3.

Analysis of murine caspase-12 promoter activity. (A) Schematic illustration of the two genomic fragments, obtained by a genome-walking technique, as described in Experimental Procedures, cloned into the pGL3 promoterless vector upstream of luciferase coding region. (B) Constructs were transfected in murine NIH 3T3 or human HEK 293 cells by using a ratio of 6:1 FuGENE 6 transfection reagent to 1 μg of DNA reporter gene. The luciferase activity, normalized to the protein concentration in the sample, was determined 24 h after transfection and expressed as relative light units per microgram of protein. The control used was pGL3 basic vector in the absence of the inserted DNA fragments.

To localize the active promoter regions and to determine the important DNA element regulating mouse caspase-12 gene expression, a series of six truncated promoter fragments (pGL3-1/2 to pGL3-9/8), starting from nucleotide -2235 to nucleotide -311 of the upstream sequence, and ending before ATG, were prepared by PCR (Fig. 4A, upper scheme). They were cloned unidirectionally into a promoterless luciferase reporter vector, pGL3 basic, and the fusion promoter-luciferase constructs were transiently transfected into murine NIH 3T3 cells. The luciferase activity was normalized to the protein concentration. All six PCR-generated fragments have substantial luciferase activities. However, the highest promoter activity was shown by the construct containing 700 bp upstream of the ATG. The deletion of the sequence corresponding to the caspase-12 5′ UTR has a significant negative effect on the reporter gene (Fig. 4B Left). This region may contain sequences necessary either for the translation or transcription of the luciferase reporter gene.

Fig. 4.

The minimal promoter region of the murine caspase-12 identified with progressive deletions of the 5′-flanking region in luciferase reporter constructs. (A) A series of heterologous luciferase reporter constructs were generated by PCR amplification and contained progressive deletions of the 5′- or 3′-flanking region of caspase-12 as illustrated. (B) These constructs were transfected into mouse NIH 3T3 cells. Cells were lysed 24 h after transfection and luciferase activities were measured. The data were presented as relative light unit per microgram of protein for five independent experiments.

It is interesting to note that four truncated fragments (pGL3-0.6 to pGL3-0.1 see Fig. 4A, lower scheme) obtained from the 800-bp fragment that contains only the sequence downstream of caspase-12 ATG, corresponding to the first intron, and all posses substantial luciferase activity (Fig. 4B Right).

Inhibition of Caspase-12 Promoter by Growth Factor Deprivation. To examine whether the activity of the caspase-12 promoter is affected by growth factor deprivation, NIH 3T3 cells were transiently transfected with pGL3-3 or pGL3-0.8 luciferase reporter plasmid, and luciferase activity was assayed after 24 h incubation with or without 10% serum. Results demonstrated an ≈80% decrease in luciferase activity in the absence of serum compared with that in the presence of serum (Fig. 5A).

Fig. 5.

Regulation of caspase-12 promoter activity in NIH 3T3 cells by trophic factor deprivation. (A) pGL3-3 and pGL3-0.8 were transfected into 3T3 cells and grown in media with or without 10% serum for 24 h. Luciferase activity was measured and expressed as relative light units per microgram of protein. (B) The caspase-12 promoter sequence was inserted upstream from the EGFP reporter in the pEGFP-1 vector and transfected into NIH 3T3 cells for 24 h. EGFP protein was monitored by using a fluorescent microscope and Western blot analysis. Intense green fluorescence was observed in many transfected cells in the presence of 10% serum, but not in the cells transfected in the absence of serum. (C), Twenty-microgram aliquots of cytosolic extracts were isolated from control cultured in the presence of 10% serum, and growth factor-deprived cells were subjected to SDS/4–20% PAGE and transferred to a nitrocellulose filters. The filters were probed with a monoclonal anti-GFP antibody. (D) EGFP mRNA expression in NIH 3T3 cells transfected in the absence or the presence of 10% serum. Levels of mRNA were measured by semiquantitative RT-PCR.

A DNA fragment comprising the promoter sequence from nucleotide 2235 was then inserted upstream from the EGFP reporter gene in the promoterless vector pEGFP-1, and the obtained construct was transiently transfected into NIH 3T3 cells. Fluorescent microscopic examination revealed low levels of green fluorescence in transfected cells grown in the absence of 10% serum. In contrast, intensive fluorescence was observed in cells cultured in the presence of 10% serum. (Fig. 5B). Significantly, cells expressing EGFP did not show any sign of apoptotic cell death.

To confirm that inhibition of the caspase-12 promoter leads to a decrease in protein levels, we examined EGFP protein expression in transiently transfected NIH 3T3 cells as a function of time after serum deprivation. By using Western blot analysis, we found that 24–48 h of serum deprivation leads to a substantial decrease in the level of EGFP protein (Fig. 5C). However, RT-PCR analysis of EGFP transcripts show that EGFP mRNA content is higher in transfected cells grown in the absence of serum than in the cells cultured in the presence of serum (Fig. 5D). This result suggests that the EGFP transcript was not efficiently translated to EGFP protein under serum starvation conditions.

Effect of Growth Factor Deprivation on Caspase-12 Protein Level. To examine the effect of growth factor deprivation on caspase-12 protein level, NIH 3T3 cells were cultured in the presence or absence of 10% serum for 24 h, and the caspase-12 protein was monitored by Western blot analysis using specific polyclonal antibody. Fig. 6 shows that the caspase-12 protein content is much higher in starved cells than in the cells grown in media containing 10% serum. However, semiquantitative RT-PCR analysis, using mRNA from starved or control cells, did not show any difference in the level of caspase-12 transcript. This result could be explained by a decrease of caspase-12 protein degradation, leading to its accumulation in starved cells relative to cells grown under 10% serum, or by an increase in the rate of the caspase-12 transcript translation under serum starvation.

Fig. 6.

Effect of growth factor deprivation on caspase-12 protein level. Confluent NIH 3T3 cells were grown for 24 h in the presence or absence of 10% serum as indicated. Equal amount of protein (20 μg of protein) were separated on SDS/4–20% PAGE and transferred to nitrocellulose membrane. Caspase-12 protein was monitored by Western blot analysis using anti-caspase-12 polyclonal antibody.

Discussion

To date, only a few reports are available on the transcriptional regulation of caspase expression. It has recently been demonstrated that the basal caspase-3 and -8 promoter depends on an Sp1 element (18, 26). Caspase-9 promoter activation in response to sever hypoxia has been reported (27). It has also been shown that polysaccharide activates caspase-11 gene expression through NF-κB and signal transducer and activator of transcription 1 (28). To study the regulation of caspase-12 expression, we isolated and cloned the mouse caspase-12 5′-flanking promoter region, using the genome-walking technique. Our data reveal several unique properties of the murine caspase-12 gene promoter. They include: (i)the isolated two DNA fragments, the 3,221-bp fragment containing the first exon, the first intron, and the 5′-flanking region, and the 800-bp fragment containing the first intron. Both fragments exhibited significant promoter activity in murine and human cells as monitored by luciferase reporter gene (see Fig. 3). These results suggest that the major tissue-specific transcriptional activators of caspase-12 expression are either not present in the cell lines used, or could not transactivate the promoter fragments used in this analysis. It's not unusual for tissue-specific regulatory sequences to reside at a long distance from transcription start site. Moreover, tissue-specific expression of caspase-12 in vivo may require proper chromatin structure or DNA methylation, thus the function of transgenes in transfected cells does not always mimic exactly the expression in the complex milieu (29, 30). A more definitive test of promoter specificity is to express a reporter gene in vivo by using transgenic methods, which will be an important area for our future studies. (ii) Progressive deletion analysis shows that there is a lack of segment specificity for the promoter activity of the 800-bp fragment, whereas the highest promoter activity of the large fragment is located with the construct containing 700 bp upstream of ATG (see Fig. 4). (iii) The promoter activity of the caspase-12 gene is highly sensitive to the growth factors in the culture media and it also appears to depend on the nature of the protein it regulates (see below).

The growth factor responsible for the activation of caspase-12 promoter is not known. However, caspase-12 promoter can be activated by growth factors present in the serum. When the caspase-12 promoter region was inserted upstream from either the luciferase or the EGFP reporter gene in pGL3 or pEGFP-1 vector, respectively, and transfected into NIH 3T3 cells, we found that the mRNA of the reporter gene was elevated, whereas the reporter protein was dropped dramatically when cells were grown in the absence of serum (see Fig. 5). On the contrary, when the transfected cells were grown in the presence of 10% serum, the level of the reporter gene mRNA was found significantly reduced, whereas the reporter protein increased drastically (Fig. 5). Interestingly, when the caspase-12 promoter region is regulating the expression of caspase-12, serum deprivation had no effect on the level of caspase-12 mRNA (data not shown), whereas caspase-12 protein was greatly elevated in the absence of serum (Fig. 6). Together, these results indicate, under serum deprivation, the translation mechanism for the luciferase and the EGFP transcript is down-regulated, whereas the translation of the caspase-12 mRNA is up-regulated or turnover of caspase-12 protein is suppressed in serum-starved cells. Nevertheless, it should be pointed out that under our experimental conditions, we did not observe any caspase-12 cleavage product, using Western blot analysis after NIH 3T3 cells were deprived of serum for 24 h. This result did not support other findings showing the serum starvation for only 6 h induced caspase-12 activation in AKR-2B mouse fibroblasts (31). Our data support the view that when cells approach apoptotic stress conditions, the transcription and the translation are coordinated to reduce proteins not required for program cell death but elevate proteins such as caspases needed to induce apoptosis. Although logically sound, an alternative notion is suggested by another report, where Liu et al. (26) found that growth factor deprivation led to an increase in caspase-3 mRNA and in luciferase controlled by caspase-3 gene promoter transfected in PC 12 cells. In addition, their observation occurred at a time when cells are undergoing apoptosis.

No TATA-box, CAAT-box, or GC-box were found in the vicinity of the transcription start site. Despite the lack of these important elements, we could identify several putative transcription factor-binding sites in the caspase-12 promoter region, including binding sites for AP1, Oct-1, Sp1, and NF-κB. In addition, the deletion of the caspase-12 5′ UTR from the reporter gene led to a significant decrease of the promoter activity. This result suggest that the caspase-12 5′UTR contains a sequence that is necessary for either transcription or translation. In addition, we determined the murine caspase-12 transcription start site by using the RACE technique. It is localized 44 bp upstream of ATG start codon.