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 May 2007, revised 15 June 2007, accepted 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 interleukin1b, 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 protein 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-ainduced 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 hairpin 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 mediated mostly through tumor necrosis factor receptor-1 (TNF-R1), a cell-surface receptor TNF-R1 is a type transmembrane protein that contains four cysteine-rich repeats in the extracellular domain The distal cysteinerich domain mediates homophilic interaction of the receptor molecules, thereby keeping the receptors in a silent, homomultimerized state [1] Binding of the trimeric 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 4396 FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS N Jain et al factor and the serine threonine kinase receptor interacting protein 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 regulatory factor-1 (IRF-1) [4,5] Caspase-1 is a cysteine protease that catalyses the proteolytic processing of the pro-inflammatory cytokine, interleukin-1b Caspase-1 plays a pivotal role in inflammation and apoptosis Caspase-1 knockout mice are resistant to bacterial lipopolysaccharideinduced septic shock and are also defective in the production of the active cytokines interleukin-1b, interleukin-18 and interleukin-33 [6–9] Involvement of caspase-1 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 doxorubicin, cisplatin and UV radiation also induces caspase-1 mRNA [18–20] However, the mechanism of activation of caspase-1 gene expression by TNF-a is unknown, although signaling by TNF-a has been studied extensively The p73 protein belongs to the p53 family of transcription 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 cisplatin, camptothecin and doxorubicin, causes the stabilization and activation of the p73 protein [27–29] When overexpressed, p73 binds to p53 DNA target sites, transactivates p53-responsive genes and is capable of inducing cell cycle arrest and apoptosis in a p53-like manner Clues to the physiological roles of p53 and p73 came from the respective knockout 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] Previous 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] Activation of caspase-1 gene expression can be mediated by IRF-1, signal transducer and activator of TNF-a-induced caspase-1 expression requires p73 transcription 1, p53, p73 and E26 transformation-specific sequence (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 promoter [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 exogenous, 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 activation 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 In addition, our results revealed that TNF-a induces p73 gene expression Results TNF-a activates caspase-1 promoter The human lung carcinoma cell line A549 was treated with TNF-a and RNA was isolated from TNF-a-treated and -untreated cells at the time-points indicated The level of caspase-1 mRNA was determined by semiquantitative 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 h of treatment with TNF-a and remained high up to 24 h The caspase-1 protein level also increased upon treatment of cells with TNF-a, as shown by western blot analysis (Fig 1B) The level of IRF-1 mRNA and protein 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 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 caspase-1 gene expression in cells treated with TNF-a A caspase-1 promoter reporter plasmid was transfected into A549 cells and, 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 FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS 4397 TNF-a-induced caspase-1 expression requires p73 N Jain et al Fig Induction of caspase-1 gene expression and promoter activation by TNF-a (A) A549 cells were treated with 10 ngỈmL)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 (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 h, were treated with the indicated concentrations of TNF-a for 24 h Chloramphenicol acetyltransferase (CAT) activities relative to the untreated control are shown promoter activity in a dose-dependent manner (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 activity from 4.7-fold to 2.3-fold (Fig 2B,D); however, the basal activity was not affected, as reported previously [19] These results suggested that, in addition to IRF-1, a p53 family member is also required for optimal 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-ainduced caspase-1 promoter activity Previously, it has been shown that p73DD, a deletion mutant of p73a, 4398 inhibits p73 function without affecting p53-dependent transcriptional activation [39,40] We observed that TNF-a-induced caspase-1 promoter activity was inhibited by p73DD (60% inhibition, P < 0.05) but not by the p53-specific inhibitor, p53DD (Fig 3A) To provide further evidence for the requirement of p73 in TNF-a-induced activation of the caspase-1 promoter, 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-directed 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 FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS N Jain et al TNF-a-induced caspase-1 expression requires p73 A B C D Fig Effect of mutation of the p73-responsive 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 h, were treated with TNF-a (10 ngỈmL)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 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 control virus expresses the mutated shRNA These adenoviruses 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, 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 western blot analysis The p73 protein level was knocked down by Adp73shRNA virus but not by control virus (Fig 4A) C3G protein levels or endogenous Cdk-2 levels were not 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; subsequently, the cells were treated with TNF-a for or h RNA was then isolated and subjected to semiquantitative 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 shRNAinfected cells There was a significant decrease of TNF-a-induced caspase-1 mRNA levels in the Adp73shRNA-infected cells as compared with the control adenovirus-infected cells (Fig 4B) We also investigated whether knockdown of p73 would affect caspase-1 protein expression A549 cells FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS 4399 TNF-a-induced caspase-1 expression requires p73 A N Jain et al A B B C Fig 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 h the cells were treated with TNF-a (10 ngỈmL)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 transfected 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 (control) After 28 h of transfection, cell lysates were made for reporter assays CAT activities relative to the control without p73 are shown (C) Effect of p73-directed shRNA on caspase-1 promoter 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 h of transfection, 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 Adp73shRNAinfected 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 suggest that p73 plays an important role in TNF-ainduced caspase-1 gene and protein expression p73 induces caspase-1 gene and protein expression 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 4400 C Fig 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 expression plasmids; after 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 analysis using specific antibodies for p73 (anti-HA), C3G and tubulin C3G served as a transfection control and tubulin as a loading control (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 timeperiods Total RNA was isolated and semiquantitative RT-PCR analysis 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 FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS N Jain et al TNF-a-induced caspase-1 expression requires p73 A B C Fig Adenovirus-mediated expression of p73 induces caspase-1 mRNA and protein (A) A549 cells were infected with adenoviruses Ad Con, Ad p73a or Ad p73b After 24 or 48 h of infection, cell lysates were prepared for western blotting with antibodies for caspase-1, p73 and Cdk-2 (B) A549 cells were infected with the indicated adenoviruses 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 Expression of p73a and p73b in A549 cells resulted in the induction of caspase-1 protein expression, as determined by western blotting (Fig 5A) Infection with control 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) TNF-a-induced caspase-5 gene expression: role of p73 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 levels increased in TNF-a-treated A549 cells, reaching maximum levels after 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 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 Caspase-5 gene expression was induced by the overexpression 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 Effect of TNF-a on p73 promoter 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 demonstrated 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 activation requires p73 and that p73 protein accumulates in FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS 4401 TNF-a-induced caspase-1 expression requires p73 N Jain et al A A B C B Fig TNF-a enhances caspase-5 mRNA levels (A) Total RNA was isolated from A549 cells treated with TNF-a at the indicated timepoints and subjected to semiquantitative RT-PCR analysis for caspase-5 and GAPDH (B) TNF-a-induced caspase-5 gene expression is inhibited by p73 shRNA 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 Total RNA was isolated and semiquantitative RT-PCR analysis for caspase-5 and GAPDH was performed Numbers at the top indicate the relative amount of caspase-5 PCR product (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 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), increase in p73 promoter 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-ainduced increase in p73 mRNA level may not be a result of promoter activation but may involve a posttranscriptional mechanism Alternatively, it is possible that the DNA elements which mediate the TNF-a4402 Fig 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 ngỈmL)1) and interferon-c (IFN-c) (100 ngỈmL)1) for 24 h Luciferase activities relative to the untreated control are shown (n ¼ 3) after normalizing with b-galactosidase 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 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 Discussion 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 protein The increase in caspase-1 gene expression is probably caused by activation of the promoter because the caspase-1 promoter is activated in response to TNF-a FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS N Jain et al Mutation of the IRF-1-binding site abolished TNF-ainduced caspase-1 promoter activity Optimal activation of the caspase-1 promoter by TNF-a required the p73 ⁄ p53 responsive site Moreover, blocking the function of p73 by employing specific inhibitors significantly compromised the activation of the caspase-1 promoter However, blocking the function of p53 had no significant effect on TNF-a-induced promoter activity 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-ainduced p73 and IRF-1 contribute to caspase-1 promoter activation and gene expression Various lines of evidence have established a requirement of p73 for TNF-a-induced signaling to caspase-1, namely (i) mutation of the p73-responsive site compromises TNF-a-induced caspase-1 promoter activity, (ii) knockdown of p73 by shRNA (or a dominant negative mutant) reduces the activation of the caspase-1 promoter 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 expression 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 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 promoter by TNF-a requires p73 but not p53 These results are consistent with previous reports that TNFa-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 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 TNF-a-induced caspase-1 expression requires p73 In murine cells, caspase-1 activation requires caspase-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 caspase-5 by TNF-a provides further support to the suggestion that in human cells caspase-5 serves a function similar to that of caspase-11 in murine cells TNF-a-induced caspase-5 gene expression, like that 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 regulation of other genes 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 caspase-1 and -5 gene expression Although the primary role of caspase-1 and -5 is believed to be in the production of cytokines, we speculate that they may also contribute, to some extent, to TNF-ainduced 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 respective 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 provide support to the suggestion that p73 is an important component of the TNF-a-induced signaling pathway leading to gene expression Experimental procedures Cell culture and transfections A549, HeLa and 293T cells were maintained at 37 °C in a CO2 incubator in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum The transfections were carried out using Lipofectamine PlusTM reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’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 FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS 4403 TNF-a-induced caspase-1 expression requires p73 N Jain et al wherever indicated at a final concentration of 10– 20 ngỈmL)1 RT-PCR Total RNA was isolated using the TRIzol reagent (Invitrogen) Semiquantitative RT-PCR was carried 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] Primers IRF-2 (5¢-CGGAATTCTACGGTGCA CAGGGAATGGCC-3¢) and IRF-3 (5¢-TACAACAGA TGAGGATGAGGAAGGG-3¢) were used for the amplification of human IRF-1 mRNA Primers C5F2 (5¢-CCT GCAAGGAATGGGGCTCACTAT-3¢) and RCASP (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: cycle of at 95 °C; 37 cycles of at 95 °C, at 60 °C and at 72 °C; and cycle of at 72 °C The PCR reaction mixture for p73 contained 10% dimethylsulfoxide Expression vectors and antibodies 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 medicine 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-hemagglutinin (HA) was from Roche Molecular Biochemicals (Indianapolis, IN, USA); p73 monoclonal antibody (IMG 259) was from Imgenex (San Diego, CA, USA) and Cy-3-conjugated anti-mouse immunoglobulin was from Amersham Pharmacia Biotech (Piscataway, NJ, USA) Construction of adenoviral vectors All adenoviral vectors were generated using the AdEasy System [46] kindly provided by B Vogelstein (Howard Hughes Medical Institute and The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, Baltimore, MD, USA) Adp73a or Adp73b, expressing the p73a or -b isoform, was constructed as follows: the p73a or -b cDNA was isolated from the pcDNA3.1-p73 plasmid by KpnI ⁄ XhoI digestion and cloned 4404 into the pAdtrack-cytomegalovirus (CMV) plasmid under the control of the CMV promoter terminated by the simian virus 40 (SV40) polyadenylation signal, resulting in pAdtrack-CMV-p73a or -p73b The pAdtrack-CMV plasmid was utilized as a control vector The adenovirus-based shRNA vector was generated by subcloning the transcriptional 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 CMVgreen 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 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-1responsive 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 pCMT-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-transfection 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 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 promoter fragment of 930 bp was cloned into the pMOSBlue vector (Amersham) and sequenced The p73 promoter fragment was then excised by digestion with HindIII and XhoI, subcloned into the pGL3-BASIC vector (Promega, Madison, WI, USA) and named p73Pr-Luc Vector expressing p73-directed shRNA 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 number: NM_005427) A mutant of this shRNA was made by substituting two bases in the middle of the target sequence FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS N Jain et al TNF-a-induced caspase-1 expression requires p73 and was found to be functionally inactive This mutant shRNA expression plasmid was used as a control Western blot analysis Cells were washed twice with PBS and lysed in · SDS sample buffer Proteins were separated on 10% SDS-polyacrylamide 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 : 1000 dilutions, and the blot was incubated for h at room temperature 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 immunoblotting procedure for the p73 blot has been described previously [19] 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 Medical Research to GS References Micheau O & Tschopp J (2003) Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes Cell 114, 181–190 Wajant H, Pfizenmaier K & Scheurich P (2003) Tumor necrosis factor signaling Cell Death Differ 10, 45–65 Chen C & Goeddel DV (2002) TNF-R1 signaling: a beautiful pathway Science 296, 1634–1635 Pine R (1997) Convergence of TNFa and IFNc signalling pathways through synergistic induction of IRF-1 ⁄ ISGF-2 is mediated by a composite GAS ⁄ kB promoter element Nucleic Acids Res 25, 4346–4354 Ohmori Y, Schreiber RD & Hamilton TA (1997) Synergy between interferon-c and tumor necrosis factor-a in transcriptional activation is mediated by cooperation between signal transducer and activator of transcription and nuclear factor jB J Biol Chem 272, 14899–14907 Kuida K, Lippke JA, Ku G, Harding MW, Livingston DJ, Su MS & Flavell RA (1995) Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme Science 267, 2000–2003 Li P, Allen H, Banerjee S, Franklin S, Herzog L, Johnston C, McDowell J, Paskind M, Rodman L, Salfeld J et al (1995) Mice deficient in IL-1-beta- 10 11 12 13 14 15 16 17 18 19 converting enzyme are defective in production of mature IL-1-beta and resistant to endotoxic shock Cell 80, 401–411 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 interferongamma inducing factor mediated by interleukin-1beta converting enzyme Science 5297, 206–209 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 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 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 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 Chin YE, Kitagawa M, Kuida K, Flavell RA & Fu XY (1997) Activation of the STAT signaling pathway can cause expression of caspase and apoptosis Mol Cell Biol 17, 5328–5337 Wang J & Lenardo MJ (2000) Roles of caspases in apoptosis, development, and cytokine maturation revealed by homozygous gene deficiencies J Cell Sci 113, 753–757 Dai C & Krantz SB (1999) Interferonc induces upregulation and activation of caspases 1, 3, and to produce apoptosis in human erythroid progenitor cells Blood 80, 3309–3316 Zhang HH, Kumar S, Barnett AH & Eggo MC (2001) Dexamethasone inhibits tumor necrosis factor-alphainduced apoptosis and interleukin-1 beta release in human subcutaneous adipocytes and preadipocytes J Clin Endocrinol Metab 86, 2817–2825 Tamura T, Ueda S, Yoshida M, Matsuzaki M, Mohri H & Okubo T (1996) Interferon-gamma induces Ice gene expression and enhances cellular susceptibility to apoptosis in the U937 leukemia cell line Biochem Biophys Res Commun 1, 21–26 Gupta S, Radha V, Furukawa Y & Swarup G (2001) Direct transcriptional activation of human caspase-1 by tumor suppressor p53 J Biol Chem 276, 10585–10588 Jain N, Gupta SCh, Sudhakar Radha V & Swarup G (2005) Role of p73 in regulating human caspase-1 gene transcription induced by interferon-c and cisplatin J Biol Chem 280, 36664–36673 FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS 4405 TNF-a-induced caspase-1 expression requires p73 N Jain et al 20 Takahashi H, Kinouchi M & Iizuka H (1997) Interleukin-1beta-converting enzyme and CPP32 are involved in ultraviolet B-induced apoptosis of SV40-transformed human keratinocytes Biochem Biophys Res Commun 236, 194–198 21 Yang A & McKeon F (2000) P63 and P73: P53 mimics, menaces and more Nat Rev Mol Cell Biol 1, 199–207 22 Pozniak CD, Radinovic S, Yang A, McKeon F, Kaplan DR & Miller FD (2000) An anti-apoptotic role for the p53 family member, p73, during developmental neuron death Science 289, 304–306 23 De Laurenzi V, Costanzo A, Barcaroli D, Terrinoni A, Falco M, Annicchiarico-Petruzzelli M, Levrero M & Melino G (1998) Two new p73 splice variants, gamma and delta, with different transcriptional activity J Exp Med 188, 1763–1768 24 DeLaurenzi V, Catani MV, Terrinoni A, Corazzari M, Melino G, Costanzo A, Levrero M & Knight RA (1999) Additional complexity in p73: induction by mitogens in lymphoid cells and identification of two new splicing variants epsilon and zeta Cell Death Differ 6, 389–390 25 Casciano I, Ponzoni M, Lo CC, Tonini GP & Romani M (1999) Different p73 splicing variants are expressed in distinct tumour areas of a multifocal neuroblastoma Cell Death Differ 6, 391–393 26 Fillippovich I, Sorokina N, Gatei M, Haupt Y, Hobson K, Moallem E, Spring K, Mould M, McGuckin MA, Lavin MF et al (2001) Transactivation deficient p73alpha (p73Deltaexon2) inhibits apoptosis and competes with p53 Oncogene 20, 514–522 27 Gong JG, Costanzo A, Yang HQ, Melino G, Kaelin WG Jr, Levrero M & Wang JY (1999) The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage Nature 399, 806–809 28 Truong T, Sun G, Doorly M, Wang JY & Schwartz MA (2003) Modulation of DNA damage-induced apoptosis by cell adhesion is independently mediated by p53 and c-Abl Proc Natl Acad Sci USA 100, 10281–10286 29 Strano S, Monti O, Pediconi N, Baccarini A, Fontemaggi G, Lapi E, Mantovani F, Damalas A, Citro G, Sacchi A et al (2005) The transcriptional coactivator Yes-associated protein drives p73 gene-target specificity in response to DNA damage Mol Cell 4, 447–459 30 Donehower LA, Harvey BL, Slagle BL, McArthur MJ, Montgomery CA, Butel JS & Bradley A (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours Nature 356, 215–221 31 Yang A, Walker N, Bronson R, Kaghad M, Oosterwegel M, Bonnin J, Vagner C, Bonnet H, Dikkes P, Sharpe A et al (2000) p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours Nature 404, 99–103 32 Chau BN, Chen TT, Wan YY, DeGregori J & Wang JY (2004) Tumor necrosis factor alpha-induced apoptosis 4406 33 34 35 36 37 38 39 40 41 42 43 44 45 requires p73 and c-ABL activation downstream of RB degradation Mol Cell Biol 10, 4438–4447 Tang V, Dhirapong A, Yabes AP & Weiss RH (2005) TNF-alpha-mediated apoptosis in vascular smooth muscle cells requires p73 Am J Physiol Cell Physiol 289, 199–206 Dasgupta P, Betts V, Rastogi S, Joshi B, Morris M, Brennan B, Ordonez-Ercan D & Chellappan S (2004) Direct binding of apoptosis signal-regulating kinase to retinoblastoma protein: novel links between apoptotic signaling and cell cycle machinery J Biol Chem 279, 38762–38769 Tamura T, Ishihara M, Lamphier MS, Tanaka N, Oishi I, Aizawa S, Matsuyama T, Mak TW, Taki S & Taniguchi T (1995) An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogen-activated T lymphocytes Nature 376, 596–599 Pei H, Li C, Adereth Y, Hsu T, Watson DK & Li R (2005) Caspase-1 is a direct target gene of ETS1 and plays a role in ETS1-induced apoptosis Cancer Res 65, 5–13 Kumar A, Commane M, Flickinger TW, Horvath CM & Stark GR (1997) Defective TNF-a-induced apoptosis in STAT1-null cells due to low constitutive levels of caspases Science 278, 1630–1632 Iwase S, Furukawa Y, Kikuchi J, Saito S, Nakamura M, Nakayama R, Horiguchi-Yamada J & Yamada H (1999) Defective binding of IRFs to the initiator element of interleukin-1beta-converting enzyme (ICE) promoter in an interferon-resistant Daudi subline FEBS Lett 450, 263–267 Irwin M, Marin MC, Phillips AC, Seelan RS, Smith DI, Liu W, Flores ER, Tsai KY, Jacks T, Vousden KH et al (2000) Role for the p53 homologue p73 in E2F-1induced apoptosis Nature 407, 645–648 Zaika A, Irwin M, Sansome C & Moll UM (2001) Oncogenes induce and activate endogenous p73 protein J Biol Chem 276, 11310–11316 Chua CC, Chua BH, Chen Z, Landy C & Hamdy RC (2002) TGF-beta1 inhibits multiple caspases induced by TNF-alpha in murine osteoblastic MC3T3–E1 cells Biochim Biophys Acta 1, 1–8 Lin XY, Choi MS & Porter AG (2000) Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-gamma J Biol Chem 51, 39920–39926 Lamkanfi M, Declercq W, Kalai M, Saelens X & Vandenabeele P (2002) Alice in caspase land A phylogenetic analysis of caspases from worm to man Cell Death Differ 4, 358–361 Wang S, Miura M, Jung YK, Zhu H, Li E & Yuan J (1998) Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE Cell 4, 501–509 Kamatkar S, Radha V, Nambirajan S, Reddy RS & Swarup G (1996) Two splice variants of a tyrosine FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS N Jain et al phosphatase differ in substrate specificity, DNA binding, and subcellular location J Biol Chem 271, 26755– 26761 46 He TC, Zhou S, da Costa LT, Yu J, Kinzler KW & Vogelstein B (1998) A simplified system for generating recombinant adenoviruses Proc Natl Acad Sci USA 95, 2509–2514 47 Yu JV, Deruiter SL & Turner DL (2002) RNA interference by expression of short-interfering RNAs and TNF-a-induced caspase-1 expression requires p73 hairpin RNAs in mammalian cells Proc Natl Acad Sci USA 99, 6047–6052 48 Seelan RS, Irwin M, van der Stoop P, Qian C, Kaelin WG Jr & Liu W (2002) The human p73 promoter: characterization and identification of functional E2F binding sites Neoplasia 4, 195–203 FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS 4407 ... role in TNF-ainduced caspase-1 gene and protein expression p73 induces caspase-1 gene and protein expression To determine the effect of p73 on caspase-1 protein expression, adenoviruses were constructed... of endogenous p73 inhibits TNF-a-induced caspase-1 gene expression We hypothesized that p73 is required for the optimal activation of caspase-1 gene expression by TNF-a, as evident from our dominant... TNF-a-induced caspase-1 gene expression from endogenous, as well as exogenous, promoters In addition, our results revealed that TNF-a induces p73 gene expression Results TNF-a activates caspase-1