Báo cáo khoa học: Tumor necrosis factor-a-induced caspase-1 gene expression

Tumor necrosis factor-a-induced caspase-1 gene expression Role of p73

Nishant Jain, Ch Sudhakar and Ghanshyam Swarup

Centre for Cellular and Molecular Biology, Hyderabad, India

Keywords caspase-1; caspase-5; IRF-1; p73; TNF-a

Correspondence G. Swarup, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad)500 007, India Fax: +91 40 27160591 ⁄ +91 40 27160311 Tel: +91 40 27192616 ⁄ +91 40 27160222 E-mail: gshyam@ccmb.res.in

(Received 2 May 2007, revised 15 June 2007, accepted 2 July 2007)

doi:10.1111/j.1742-4658.2007.05969.x

Tumour necrosis factor-a (TNF-a) is a cytokine that is involved in many functions, including the inflammatory response, immunity and apoptosis. Some of the responses of TNF-a are mediated by caspase-1, which is involved in the production of the pro-inflammatory cytokines interleukin- 1b, interleukin-18 and interleukin-33. The molecular mechanisms involved in TNF-a-induced caspase-1 gene expression remain poorly defined, despite the fact that signaling by TNF-a has been well studied. The present study was undertaken to investigate the mechanisms involved in the induction of caspase-1 gene expression by TNF-a. Treatment of A549 cells with TNF-a resulted in an increase in caspase-1 mRNA and protein expression, which was preceded by an increase in interferon regulatory factor-1 and p73 pro- tein levels. Caspase-1 promoter reporter was activated by the treatment of cells with TNF-a. Mutation of the interferon regulatory factor-1 binding site resulted in the almost complete loss of basal as well as of TNF-a- induced caspase-1 promoter activity. Mutation of the p53 ⁄ p73 responsive site resulted in reduced TNF-a-induced promoter activity. Blocking of p73 function by a dominant negative mutant or by a p73-directed small hairpin RNA reduced basal as well as TNF-a-induced caspase-1 promoter activity. TNF-a-induced caspase-1 mRNA and protein levels were reduced when p73 mRNA was down-regulated by small hairpin RNA. Caspase-5 gene expression was induced by TNF-a, which was inhibited by the small hair- pin RNA-mediated down-regulation of p73. Our results show that TNF-a induces p73 gene expression, which, together with interferon regulatory factor-1, plays an important role in mediating caspase-1 promoter activation by TNF-a.

Tumor necrosis factor-a (TNF-a) is a multifunctional cytokine that plays an important role in the immune response, inflammation, control of cell death and cell proliferation. The biological effects of TNF-a are med- iated mostly through tumor necrosis factor receptor-1 (TNF-R1), a cell-surface receptor. TNF-R1 is a type 1 transmembrane protein that contains four cysteine-rich repeats in the extracellular domain. The distal cysteine- rich domain mediates homophilic interaction of the

receptor molecules, thereby keeping the receptors in a silent, homomultimerized state [1]. Binding of the tri- meric TNF-a ligand results in the re-organization of pre-assembled TNF-R1 complexes. These events signal the recruitment of tumor necrosis factor-a receptor associated death domain to the intracellular death domain of TNF-R1. TNF-R1-bound tumor necrosis factor-a receptor associated death domain serves as platform for the binding of TNF receptor-associated

Abbreviations CAT, chloramphenicol acetyltransferase; Cdk-2, cyclin dependent kinase 2; CMV, cytomegalovirus; Ets-1, E26 transformation-specific sequence 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN, interferon; IRF-1, interferon regulatory factor-1; NF-jB, nuclear factor-jB; shRNA, short hairpin RNA; TNF-a, tumor necrosis factor-a; TNF-R1, tumor necrosis factor receptor-1.

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factor and the serine threonine kinase receptor inter- acting protein 1. These proteins recruit key enzymes to TNF-R1 that orchestrate the inducible expression of genes for diverse biological processes, including cell death, cell growth, stress response and inflammation [2,3]. One of the major signaling pathways induced by TNF-a leads to the activation of transcription factor nuclear factor-jB (NF-jB), which directly mediates the induction of several genes, including interferon regula- tory factor-1 (IRF-1) [4,5].

In addition, our

results

caspase-1

transcription 1, p53, p73 and E26 transformation-spe- cific sequence 1 (Ets-1) [13,18,19,35–37]. Analysis of the human caspase-1 promoter has shown functional binding sites for IRF-1 and p53 in the minimal pro- moter [18,38]. An Ets-1-binding site has also been identified in the caspase-1 promoter upstream of the minimal promoter [36]. Endogenous, as well as exoge- nous, p73 activates caspase-1 promoter primarily through the p53 ⁄ p73-binding site. Optimal activation of the caspase-1 promoter by IFN-c requires p73 [19]. However, the transcription factors involved in the acti- vation of the caspase-1 promoter by TNF-a are not known. In the present study we analyzed the role of p73 and IRF-1 in mediating TNF-a-induced caspase-1 promoter activation. Our results showed that p73 plays an important role in TNF-a-induced caspase-1 gene expression from endogenous, as well as exogenous, promoters. revealed that TNF-a induces p73 gene expression.

Results

TNF-a activates caspase-1 promoter

caspase-1 gene

Caspase-1 is a cysteine protease that catalyses the proteolytic processing of the pro-inflammatory cyto- kine, interleukin-1b. Caspase-1 plays a pivotal role in inflammation and apoptosis. Caspase-1 knockout mice are resistant lipopolysaccharide- to bacterial induced septic shock and are also defective in the interleukin-1b, the active cytokines production of interleukin-18 and interleukin-33 [6–9]. Involvement of in TNF-a-induced cytotoxicity has been determined by employing inhibitors of caspase-1 [10–12]. Caspase-1 gene expression is induced by interferon (IFN)-a, IFN-c and TNF-a [13–17]. In addition, treatment of tumor cell lines with doxorubi- cin, cisplatin and UV radiation also induces caspase-1 mRNA [18–20]. However, the mechanism of activa- expression by TNF-a is tion of unknown, although signaling by TNF-a has been studied extensively.

cisplatin,

The human lung carcinoma cell line A549 was treated with TNF-a and RNA was isolated from TNF-a-trea- ted and -untreated cells at the time-points indicated. The level of caspase-1 mRNA was determined by semi- quantitative RT-PCR. There was a time-dependent increase in caspase-1 mRNA levels upon treatment of the cells with TNF-a (Fig. 1A). There was no change in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, which was used as a control. Caspase-1 mRNA levels reached maximum levels after 9 h of treatment with TNF-a and remained high up to 24 h. The caspase-1 protein level also increased upon treat- ment of cells with TNF-a, as shown by western blot analysis (Fig. 1B). The level of IRF-1 mRNA and pro- tein also increased upon TNF-a treatment of these cells and this increase was transient (Fig. 1A,C). There was a decrease in IRF-1 mRNA as well as in protein levels when cells were treated for longer than 3 h with TNF-a (Fig. 1A,C). The levels of p73 mRNA and protein increased upon treatment of cells with TNF-a (Fig. 1A,C). By employing specific primers, we detected that the alpha-isoform of p73 was induced in A549 cells. These results raised the possibility that IRF-1 and p73 may be involved in regulating or maintaining cas- pase-1 gene expression in cells treated with TNF-a.

The p73 protein belongs to the p53 family of tran- scription factors. Unlike the p53 gene, which shows only little alternative splicing, the p73 gene encodes multiple protein isoforms, which arise as a result of alternative promoter usage and differential mRNA splicing [21–26]. Exposure to chemotherapeutic agents, such as camptothecin and doxorubicin, causes the stabilization and activation of the p73 pro- tein [27–29]. When overexpressed, p73 binds to p53 DNA target sites, transactivates p53-responsive genes and is capable of inducing cell cycle arrest and apopto- sis in a p53-like manner. Clues to the physiological roles of p53 and p73 came from the respective knock- out mice. The main phenotype of the p53-deficient mouse is the high incidence of spontaneous tumours [30]. In contrast, p73-deficient mice exhibit chronic infections, inflammation and neural defects [31]. Pre- vious reports have shown that p73 contributes to TNF-a-induced apoptosis in mouse thymocytes and vascular smooth muscle cells [32,33]. These findings are consistent with a recent study in a human B-cell lymphoblastoid cell line (Ramos cells) in which TNF-a increased p73 protein levels [34].

A caspase-1 promoter reporter plasmid was trans- fected into A549 cells and, 6 h after transfection, the cells were treated with TNF-a for 24 h. TNF-a treatment of cells resulted in an increase in caspase-1

Activation of caspase-1 gene expression can be mediated by IRF-1, signal transducer and activator of

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Fig. 1. Induction of caspase-1 gene expression and promoter activation by TNF-a. (A) A549 cells were treated with 10 ngÆmL)1 of TNF-a for 3, 6, 9, 12 or 24 h. After total RNA isolation, caspase-1, IRF-1, p73 and GAPDH mRNA levels were analyzed by semiquantitative RT-PCR. C, untreated control cells. Numbers at the top of the lower panel indicate the relative amount of the p73 PCR product. (B,C) Immunoblotting was performed with total proteins isolated from A549 cells treated with TNF-a for the indicated time. The immunoblot was performed with antibodies against caspase-1, IRF-1, p73 and cyclin dependent kinase 2 (Cdk-2). Cdk-2 was used as a loading control. Numbers at the top of (C) indicate the relative amount of the p73 protein. (D) TNF-a activates caspase-1 promoter. A549 cells were transfected with pC-WT (100 ng), and, after 6 h, were treated with the indicated concentrations of TNF-a for 24 h. Chloramphenicol acetyltransferase (CAT) activities relative to the untreated control are shown.

in

activity

inhibits p73 function without affecting p53-dependent transcriptional activation [39,40]. We observed that TNF-a-induced caspase-1 promoter activity was inhib- ited by p73DD (60% inhibition, P < 0.05) but not by the p53-specific inhibitor, p53DD (Fig. 3A).

suggested that,

promoter dose-dependent manner a (Fig. 1D). Functional binding sites for IRF-1 and p53 ⁄ p73 have been identified in the human caspase-1 promoter [18,19,38]. Mutation of the IRF-1-binding site resulted in a near-complete loss of basal, as well as of TNF-a-induced, promoter activity (Fig. 2A,C). Mutation of the p53 ⁄ p73 responsive site resulted in a reduction of TNF-a-induced caspase-1 promoter activ- ity from 4.7-fold to 2.3-fold (Fig. 2B,D); however, the basal activity was not affected, as reported previously in addition to [19]. These results IRF-1, a p53 family member is also required for opti- mal activation of the caspase-1 promoter by TNF-a.

Role of p73 in TNF-a-induced activation of the caspase-1 promoter

We used dominant negative mutants of p53 and p73 to assess the requirement of these proteins for TNF-a- induced caspase-1 promoter activity. Previously, it has been shown that p73DD, a deletion mutant of p73a,

To provide further evidence for the requirement of p73 in TNF-a-induced activation of the caspase-1 pro- moter, we used a p73-directed short hairpin RNA (shRNA). This shRNA has been shown to reduce p73 levels and was presumed to be specific for p73 because it did not affect the level of C3G or other endogenous proteins tested [19]. The mutation of two nucleotides inactivated this shRNA, which was used as a control. The p73-directed shRNA strongly reduced p73-induced caspase-1 promoter activity (Fig. 3B). TNF-a-induced caspase-1 promoter activity was inhibited by p73-direc- ted shRNA (67% inhibition; P < 0.05) (Fig. 3C). Basal caspase-1 promoter activity was also inhibited by this shRNA. These results suggest that p73 plays an important role in the TNF-a-induced activation of caspase-1 promoter.

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A

B

D

C

Fig. 2. Effect of mutation of the p73-respon- sive and IRF-1-responsive sites on TNF-a-- induced caspase-1 promoter activity. (A,B) Schematic representations of wild-type and mutated caspase-1 promoter-reporter constructs. (C,D) pC-WT, pC-MT-IRF-1 or pC-MT-p53 (100 ng) were transfected into A549 cells, and, after 6 h, were treated with TNF-a (10 ngÆmL)1) for 24 h. CAT activities relative to the untreated control are shown (n ¼ 3).

Knockdown of endogenous p73 inhibits TNF-a-induced caspase-1 gene expression

levels were not

caspase-1 mRNA levels

in

western blot analysis. The p73 protein level was knocked down by Adp73shRNA virus but not by con- trol virus (Fig. 4A). C3G protein levels or endogenous Cdk-2 affected significantly by Adp73shRNA. To determine the effect of knockdown of endogenous p73 on caspase-1 gene expression, A549 cells were infected with adenoviruses for 24 h; subse- quently, the cells were treated with TNF-a for 6 or 9 h. RNA was then isolated and subjected to semi- quantitative RT-PCR analysis. As expected, adenoviral p73shRNA abrogated endogenous p73 mRNA levels as compared with the control shRNA-infected cells (Fig. 4B). The level of TNF-a-induced p73 mRNA was also reduced by p73shRNA. Next, we determined caspase-1 mRNA levels in the TNF-a-treated shRNA- infected cells. There was a significant decrease of TNF-a-induced the Adp73shRNA-infected cells as compared with the con- trol adenovirus-infected cells (Fig. 4B).

We hypothesized that p73 is required for the optimal activation of caspase-1 gene expression by TNF-a, as evident from our dominant negative and shRNA-based promoter assay experiments. To test this assumption, we generated an adenovirus- based vector, which expressed shRNA, to knock down the expression of p73. We derived recombinant adenoviruses encoding control shRNA or p73shRNA under the control of the U6 promoter (Ad control shRNA or Adp73shRNA), as described in the Experimental procedures. The con- trol virus expresses the mutated shRNA. These adeno- viruses co-expressed green fluorescent protein as a reporter for infection efficiency. To determine the knockdown efficacy of this virus, HeLa cells were transfected with p73a and C3G expression plasmids and, 4 h later, the cells were infected with control or Adp73shRNA viruses. After another 24 h, the cells were harvested and the cell lysates were subjected to

We also investigated whether knockdown of p73 would affect caspase-1 protein expression. A549 cells

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A

A

B

B

C

C

Fig. 3. Role of p73 in TNF-a-induced caspase-1 promoter activity. (A) pC-WT reporter plasmid was transfected along with p53DD or p73DD (100 ng of each) or control plasmid. After 6 h the cells were treated with TNF-a (10 ngÆmL)1) for 24 h. CAT activities relative to the untreated control are shown (n ¼ 3). (B) shRNA for p73 inhibits p73-induced caspase-1 promoter activity. A549 cells were transfect- ed with pC-WT reporter plasmid (100 ng) and p73b (5 ng), along with 200 ng of p73 shRNA (shRNA) or 200 ng of a control shRNA lysates were made for (control). After 28 h of transfection, cell reporter assays. CAT activities relative to the control without p73 are shown. (C) Effect of p73-directed shRNA on caspase-1 pro- moter activity induced by TNF-a. A549 cells were cotransfected with pC-WT reporter plasmid (100 ng) along with shRNA for p73 or control shRNA-expressing plasmids (200 ng). After 6 h of transfec- tion, cells were treated with TNF-a or left untreated for 24 h. CAT activities relative to the untreated control are shown (n ¼ 3).

were infected with control or Adp73shRNA viruses and then treated with TNF-a. In the Adp73shRNA- infected cells, the TNF-a-induced caspase-1 protein level was also markedly lower than that of the control virus-infected cells (Fig. 4C). Overall, these results sug- gest that p73 plays an important role in TNF-a- induced caspase-1 gene and protein expression.

p73 induces caspase-1 gene and protein expression

Fig. 4. TNF-a-induced caspase-1 gene expression is inhibited by p73 shRNA. (A) Efficacy of adenovirus expressing p73-directed shRNA. HeLa cells were transfected with p73a and C3G expres- sion plasmids; after 4 h the cells were infected with control or p73shRNA-expressing adenovirus. After another 24 h, the cells were harvested and extracts were subjected to western blot analy- sis using specific antibodies for p73 (anti-HA), C3G and tubulin. C3G served as a transfection control and tubulin as a loading con- trol. (B) A549 cells were infected with adenoviruses expressing control shRNA (Ad con) or p73shRNA (Ad shRNA). After 24 h of infection, the cells were treated with TNF-a for the indicated time- periods. Total RNA was isolated and semiquantitative RT-PCR anal- ysis for p73, caspase-1 and GAPDH was performed. (C) A549 cells were infected with adenoviruses expressing control shRNA (Ad con) or p73shRNA (Ad shRNA) for 24 h, followed by treatment with TNF-a for 12 or 18 h. Western blot analysis for caspase-1 and Cdk-2 is shown.

To determine the effect of p73 on caspase-1 protein expression, adenoviruses were constructed that express the a and b isoforms of p73. A549 cells were infected with adenoviruses expressing p73 proteins or with

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B

A

C

Fig. 5. Adenovirus-mediated expression of p73 induces caspase-1 mRNA and protein. (A) A549 cells were infected with adeno- viruses Ad Con, Ad p73a or Ad p73b. After 24 or 48 h of infection, cell lysates were prepared for western blotting with antibod- ies for caspase-1, p73 and Cdk-2. (B) A549 cells were infected with the indicated ade- noviruses. RNA was isolated 24 and 48 h postinfection and caspase-1 mRNA levels were analyzed by RT-PCR. GAPDH was used as a control. (C) Activation of caspase-1 promoter by p73a, p73b and IRF-1. A549 cells were transfected with 100 ng of pC-WT and the indicated amounts of p73a, p73b or IRF-1 expression plasmids. CAT activities relative to the control are shown.

control adenovirus, and, after 24 or 48 h of infection, cell lysates were prepared for western blotting. Expres- sion of p73a and p73b in A549 cells resulted in the induction of caspase-1 protein expression, as deter- mined by western blotting (Fig. 5A). Infection with con- trol virus did not induce caspase-1. Caspase-1 mRNA levels were also increased upon the expression of p73a or p73b (Fig. 5B). Caspase-1 promoter was strongly activated by p73a and p73b in A549 cells (Fig. 5C).

expression, A549 cells were infected with adenovirus (Adp73shRNA) and then treated with TNF-a. The induction of caspase-5 mRNA by TNF-a was reduced in cells infected with Adp73shRNA compared with control virus-infected cells (Fig. 6B), although the basal level of caspase-5 mRNA was not reduced. Cas- pase-5 gene expression was induced by the overexpres- sion of p73a and also by p73b (Fig. 6C). These results suggest that caspase-5 gene expression is induced by p73 and that TNF-a-induced caspase-5 gene expression is mediated, in part, by p73.

TNF-a-induced caspase-5 gene expression: role of p73

Effect of TNF-a on p73 promoter

The treatment of murine osteoblastic cells with TNF-a has been shown to induce caspase-11 gene expression, in addition to the induction of caspase-1 and -7 [41]. Caspase-5 is believed to be a human counterpart of murine caspase-11 [42,43]. Caspase-11 is an upstream regulator of caspase-1 activation [44]. Therefore, we explored the possibility of regulation of caspase-5 by TNF-a and p73. We found that caspase-5 mRNA lev- els increased in TNF-a-treated A549 cells, reaching maximum levels after 9 h of treatment, and remained high up to 24 h (Fig. 6A). To determine the effect of knockdown of endogenous p73 on caspase-5 gene

The treatment of cells with TNF-a has been shown to increase the p73 protein level [32,34]. The promoter of p73 has E2F1-binding sites and the TNF-a treatment of cells has been shown to recruit E2F1 to these sites in the p73 promoter that are occupied by E2F3 in unstimulated cells [34]. However, activation of p73 promoter activity by TNF-a has not been demon- strated. We found that the p73 promoter reporter was not activated by TNF-a (Fig. 7A). We have previously found that IFN-c-induced caspase-1 promoter activa- tion requires p73 and that p73 protein accumulates in

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A

A

B

C

B

Fig. 6. TNF-a enhances caspase-5 mRNA levels. (A) Total RNA was isolated from A549 cells treated with TNF-a at the indicated time- points and subjected to semiquantitative RT-PCR analysis for cas- pase-5 and GAPDH. (B) TNF-a-induced caspase-5 gene expression is inhibited by p73 shRNA. A549 cells were infected with adenovi- ruses expressing control shRNA (Ad con) or p73shRNA (Ad shRNA). After 24 h of infection, the cells were treated with TNF-a for the indicated time. Total RNA was isolated and semiquantitative RT-PCR analysis for caspase-5 and GAPDH was performed. Num- bers at the top indicate the relative amount of caspase-5 PCR prod- uct. (C) Adenovirus-mediated expression of p73 induces caspase-5 mRNA. A549 cells were infected with the adenoviruses Ad con, Ad p73a or Ad p73b. Total RNA was isolated 24 h postinfection and caspase-5 mRNA levels were analyzed by RT-PCR. GAPDH was used as a control.

Fig. 7. Effect of TNF-a on p73 promoter activity. (A) A549 cells were transfected with 100 ng of p73 promoter-reporter plasmid (p73Pr-Luc) treated with TNF-a (10 ngÆmL)1) and interferon-c (IFN-c) (100 ngÆmL)1) for 24 h. Luciferase activities relative to the untreated control are shown (n ¼ 3) after normalizing with b-galac- tosidase activities. (B) A549 cells were treated with IFN-c for the indicated periods of time; subsequently, total RNA was isolated and subjected to semiquantitative RT-PCR analysis for p73, GAPDH, caspase-1 and IRF-1. Cells treated with TNF-a for 6 h were used for comparison.

induced increase in p73 mRNA are not present in this promoter and may be present upstream or downstream of this promoter.

promoter

increase

p73

in

Discussion

response to treatment with IFN-c [19]. We explored the possibility of regulation of p73 gene expression by IFN-c. To achieve this, we treated A549 cells with IFN-c for various periods of time; the p73 mRNA level was enhanced by IFN-c treatment of cells but to a much lesser extent than that induced by TNF-a (Fig. 7B). In contrast to TNF-a, the IFN-c treatment of A549 cells resulted in a small, but significant (P < 0.01), activity (Fig. 7A), which is consistent with a small increase in the p73 mRNA level observed upon IFN-c treatment of cells. These observations indicate that the TNF-a- induced increase in p73 mRNA level may not be a result of promoter activation but may involve a post- transcriptional mechanism. Alternatively, it is possible that the DNA elements which mediate the TNF-a-

The results presented here show that stimulation of the human lung carcinoma cell line, A549, with TNF-a increases the expression of caspase-1 mRNA and pro- tein. The increase in caspase-1 gene expression is prob- ably caused by activation of the promoter because the caspase-1 promoter is activated in response to TNF-a.

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in human cells

Mutation of the IRF-1-binding site abolished TNF-a- induced caspase-1 promoter activity. Optimal activa- tion of the caspase-1 promoter by TNF-a required the p73 ⁄ p53 responsive site. Moreover, blocking the function of p73 by employing specific inhibitors signifi- cantly compromised the activation of the caspase-1 promoter. However, blocking the function of p53 had no significant effect on TNF-a-induced promoter activ- ity. TNF-a also enhances the gene expression of the full-length isoform of p73, p73a. Taken together, these results are consistent with a pathway in which TNF-a- induced p73 and IRF-1 contribute to caspase-1 pro- moter activation and gene expression.

In murine cells, caspase-1 activation requires cas- pase-11 [44]. Caspase-5 is believed to be the human ortholog of caspase-11 because both are expressed at a low level in most tissues and are induced by IFN-c and lipopolysaccharide in responsive cells. Expression of caspase-11 mRNA is induced by TNF-a in murine osteoblastic cells [41]. We found that caspase-5 gene expression is induced by TNF-a in A549 cells and also by the overexpression of p73. Induction of cas- pase-5 by TNF-a provides further support to the sug- gestion that caspase-5 serves a function similar to that of caspase-11 in murine cells. like that TNF-a-induced caspase-5 gene expression, of caspase-1, was partly inhibited by p73-directed shRNA. Thus, it is probable that the role of p73 in TNF-a-induced gene expression is not restricted to caspase-1 and that p73 may be involved in the regula- tion of other genes.

Various lines of evidence have established a require- ment of p73 for TNF-a-induced signaling to caspase-1, namely (i) mutation of the p73-responsive site compro- mises TNF-a-induced caspase-1 promoter activity, (ii) knockdown of p73 by shRNA (or a dominant nega- tive mutant) reduces the activation of the caspase-1 pro- moter in response to TNF-a and (iii) knockdown of p73 by shRNA reduces the expression of caspase-1 mRNA and protein in response to TNF-a. Further support for a role of p73 in TNF-a-induced caspase-1 gene expres- sion is provided by the observation that p73 mRNA and protein are up-regulated by TNF-a, which precedes the maximal induction of caspase-1 mRNA.

to some extent,

Although the requirement of p73 for TNF-a-induced apoptosis has been demonstrated in various cells [32,33], the precise role of p73 in this pathway is not known. It has been speculated that p73 contributes to a mitochondria-dependent apoptotic mechanism in the TNF-a-induced pathway [32]. In the present study we have shown that p73 contributes to TNF-a-induced expression. Although the caspase-1 and -5 gene primary role of caspase-1 and -5 is believed to be in the production of cytokines, we speculate that they may also contribute, to TNF-a- induced apoptosis in some cells.

In conclusion, our results show that TNF-a-induced caspase-1 gene expression is mediated by IRF-1 and p73, which activate the promoter through their respec- tive binding sites. TNF-a induces p73 and IRF-1 gene expression, which precede caspase-1 gene expression. TNF-a induces caspase-5 gene expression, which is also mediated, in part, by p73. These observations pro- vide support to the suggestion that p73 is an important component of the TNF-a-induced signaling pathway leading to gene expression.

IRF-1, p53, Ets-1 and p73 have been reported to be direct transcriptional activators of caspase-1 [18,19,36, 38]. We evaluated their ability to affect the activation of caspase-1 promoter by TNF-a. Our experiments revealed that the optimal activation of caspase-1 pro- moter by TNF-a requires p73 but not p53. These results are consistent with previous reports that TNF- a-induced apoptosis requires p73 and not p53 [32]. An Ets-1-binding site has been identified in the upstream region of the caspase-1 promoter, which is not present in the promoter constructs used in this study. As the caspase-1 promoter-reporter construct does not have an Ets-1-binding site but is activated by TNF-a to the same extent as that with an Ets site (data not shown), a role of Ets-1 in caspase-1 promoter activation by TNF-a is very unlikely.

Experimental procedures

Cell culture and transfections

A composite GAS ⁄ jB promoter element present in the IRF-1 promoter mediates the induction of IRF-1 transcription in response to TNF-a. The jB motif has been demonstrated to be occupied by the p50 ⁄ p65 subunits of NF-jB [4,5]. Blocking of NF-jB by super repressor inhibitor of NF-jB (I-jB) strongly inhibited activation of the caspase-1 promoter by TNF-a but not by overexpressed IRF-1 (data not shown). Taken together, our results are consistent with the suggestion that NF-jB-mediated IRF-1 expression is required for TNF-a-induced caspase-1 promoter activation.

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A549, HeLa and 293T cells were maintained at 37 (cid:2)C in a CO2 incubator in Dulbecco’s modified Eagle’s medium sup- plemented with 10% fetal bovine serum. The transfections were carried out using Lipofectamine PlusTM reagent (Invi- trogen, San Diego, CA, USA) according to the manufac- turer’s instructions. All the plasmids for transfection were prepared by using Qiagen columns (Hilden, Germany). Human TNF-a (Sigma, St Louis, MO, USA) was added

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RT-PCR

indicated at a final concentration of 10– wherever 20 ngÆmL)1.

carried Semiquantitative RT-PCR was

IRF-2 Primers

into the pAdtrack-cytomegalovirus (CMV) plasmid under the control of the CMV promoter terminated by the simian virus 40 (SV40) polyadenylation signal, resulting in pAd- track-CMV-p73a or -p73b. The pAdtrack-CMV plasmid was utilized as a control vector. The adenovirus-based shRNA vector was generated by subcloning the transcrip- tional unit of p73 shRNA (0.4 kb) from the pmu6 vector described previously [19,47]. The U6-SH cassette was cloned into the pAdTrack plasmid upstream of the CMV- green fluorescent protein cassette (1.6 kb). Recombinant plasmids were generated by homologous recombination in AdEasier cells. The 293T cells were transfected with the recombinant adenoviral plasmids using Lipofectamine 2000 (Invitrogen), and adenoviruses were collected.

Reporter plasmids and reporter assays

and

Expression vectors and antibodies

Total RNA was isolated using the TRIzol reagent (Invi- trogen). out essentially as described previously [18,45]. RNA was reverse transcribed using reagents from the first-strand cDNA synthesis kit (Invitrogen). Primers for amplification of caspase-1 and GAPDH have been described previously [18]. (5¢-CGGAATTCTACGGTGCA CAGGGAATGGCC-3¢) and IRF-3 (5¢-TACAACAGA TGAGGATGAGGAAGGG-3¢) were used for the amplifi- cation of human IRF-1 mRNA. Primers C5F2 (5¢-CCT RCASP GCAAGGAATGGGGCTCACTAT-3¢) (5¢-CTCTGCAGGCCTGGACAATGATGAC-3¢) were used for the amplification of human caspase-5 mRNA. The primers used for p73 amplification – p73P1 (5¢-ACT TTGAGATCCTGATGAAGCTG-3¢) and p73P2 (5¢-CA GATGGTCATGCGGTACTG-3¢) – were designed in a region common to various TA isoforms (a, b, c and d) of p73. The PCR conditions for p73 were: 1 cycle of 3 min at 95 (cid:2)C; 37 cycles of 1 min at 95 (cid:2)C, 1 min at 60 (cid:2)C and 1 min at 72 (cid:2)C; and 1 cycle of 7 min at 72 (cid:2)C. The PCR reaction mixture for p73 contained 10% dimethylsulfoxide.

The reporter plasmid pC-WT, which contains the human caspase-1 promoter from positions )182 to +42, relative to the transcriptional start site, cloned upstream of the CAT reporter gene, has been described previously [38]. The reporter plasmid pC-MT-p53 and pC-MT-IRF-1, were derived from pC-WT by mutating the p53 and the IRF-1- responsive sites, respectively, and have been described previously [18,19]. Cells grown in 24-well plates were transfected with 100 ng of pC-WT (or pC-MT-p53 or pC- MT-IRF-1), 50 ng of pCMV.SPORT-b-gal (Invitrogen) and with the required amount of the other plasmids. The total amount of plasmid in each transfection was kept constant (400 ng for each well of a 24-well plate) by adding control plasmid. Lysates were generally made 30 h post-transfec- tion. Preparation of lysates and CAT assays were carried out as described previously [18]. Relative CAT activities were calculated after normalizing with b-galactosidase enzyme activities.

The expression vectors of p73a and p73b, cloned in-frame with the hemagglutinin tag into pcDNA3-HA, were a kind gift from Gerry Melino (Department of experimental medi- cine and biochemical sciences, University of Rome, Italy) [23]. pcDNA3-p73DD and pcDNA3-p53DD were gifts of William Kaelin (DFCI, Harvard Medical School, Boston, MA, USA) [39]. Cdk-2, IRF-1, C3G, tubulin and caspase-1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal anti-hemagglu- tinin (HA) was from Roche Molecular Biochemicals (India- napolis, IN, USA); p73 monoclonal antibody (IMG 259) was from Imgenex (San Diego, CA, USA) and Cy-3-conju- gated anti-mouse immunoglobulin was from Amersham Pharmacia Biotech (Piscataway, NJ, USA).

Construction of adenoviral vectors

Vector expressing p73-directed shRNA

The p73 promoter was cloned from human genomic DNA by utilizing the PCR as described previously [48]. The primers used were: forward, 5¢-CGCTCGAGGATCC AGAGCCCGAGCCCACA-3¢ and reverse, 5¢-CGAAGCT TCCGTCGCAGCCCCGGGCA-3¢ [48]. The amplified pro- moter fragment of 930 bp was cloned into the pMOSBlue vector (Amersham) and sequenced. The p73 promoter frag- ment was then excised by digestion with HindIII and XhoI, subcloned into the pGL3-BASIC vector (Promega, Madison, WI, USA) and named p73Pr-Luc.

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the p73a or The shRNA expression vector targeting p73 was constructed using the U6 promotor-based vector and has been described previously [19,47]. The p73 sequence targeted by this shRNA was from nucleotides 638–656 (Gene BankTM accession num- ber: NM_005427). A mutant of this shRNA was made by substituting two bases in the middle of the target sequence All adenoviral vectors were generated using the AdEasy System [46] kindly provided by B. Vogelstein (Howard Hughes Medical Institute and The Sidney Kimmel Compre- hensive Cancer Center, The Johns Hopkins Medical Institu- tions, Baltimore, MD, USA). Adp73a or Adp73b, expressing the p73a or -b isoform, was constructed as fol- isolated from the -b cDNA was lows: pcDNA3.1-p73 plasmid by KpnI ⁄ XhoI digestion and cloned

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TNF-a-induced caspase-1 expression requires p73

Western blot analysis

and was found to be functionally inactive. This mutant shRNA expression plasmid was used as a control. converting enzyme are defective in production of mature IL-1-beta and resistant to endotoxic shock. Cell 80, 401–411. 8 Gu Y, Kuida K, Tsutsui H, Ku G, Hsiao K, Fleming

MA, Hayashi N, Higashino K, Okamura H, Nakanishi K et al. (1997) Activation of interferon- gamma inducing factor mediated by interleukin-1beta converting enzyme. Science 5297, 206–209.

9 Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, Zurawski G, Moshrefi M, Qin J, Li X et al. (2005) IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 5, 479–490.

10 Tiwari M & Dixit VM (1995) Fas- and tumor necrosis factor-induced apoptosis is inhibited by the poxvirus gene product. J Biol Chem 270, 3255–3260.

Cells were washed twice with PBS and lysed in 1 · SDS sample buffer. Proteins were separated on 10% SDS-poly- acrylamide gels and blotted onto nitrocellulose membranes. The blot was washed twice with Tween-Tris-buffered saline before blocking nonspecific binding with 5% nonfat dry milk (BLOTTO; Santa Cruz Biotechnology). The caspase-1, C3G, Cdk-2 and other antibodies were used at 1 : 1000 dilutions, and the blot was incubated for 1 h at room tem- perature. The blots were washed three times, and detection was performed by using horseradish peroxidase-conjugated secondary antibody or alkaline phosphatase-conjugated secondary antibody, as described previously [19]. The the p73 blot has been immunoblotting procedure for described previously [19].

11 Miura M, Friedlander RM & Yuan J (1995) Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway. Proc Natl Acad Sci USA 92, 8318–8322.

Acknowledgements

We thank Drs Gerry Melino, William Kaelin and Bert Vogelstein for providing reagents, and Dr V. Radha for critical reading of the manuscript. This work was supported by a grant from the Indian Council of Med- ical Research to GS.

12 Rouquet N, Pages JC, Molina T, Briand P & Joulin V (1996) ICE inhibitor YVADcmk is a potent therapeutic agent against in vivo liver apoptosis. Curr Biol 6, 1192–1195.

13 Chin YE, Kitagawa M, Kuida K, Flavell RA & Fu XY (1997) Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis. Mol Cell Biol 17, 5328–5337.

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