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

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

Role of p73

Nishant Jain, Ch Sudhakar and Ghanshyam Swarup

Centre for Cellular and Molecular Biology, Hyderabad, India

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

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

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.

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]

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 to bacterial

lipopolysaccharide-induced 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

doxorubi-cin, cisplatin and UV radiation also induces caspase-1

mRNA [18–20] However, the mechanism of

activa-tion 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

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 cisplatin, 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]

Activation of caspase-1 gene expression can be

mediated by IRF-1, signal transducer and activator of

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 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-trea-ted and -untreaTNF-a-trea-ted cells at the time-points indicaTNF-a-trea-ted 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

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

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

activ-ity 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

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,

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)

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

Fig 1 Induction of caspase-1 gene expression and promoter activation by TNF-a (A) A549 cells were treated with 10 ngÆmL)1of 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.

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

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

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 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; 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 caspase-1 mRNA levels in the Adp73shRNA-infected cells as compared with the con-trol adenovirus-infected cells (Fig 4B)

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

C B A

D

Fig 2 Effect of mutation of the

p73-respon-sive and IRF-1-responp73-respon-sive 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).

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

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

B A

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

(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

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).

B

C A

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.

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)

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

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

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

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 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

A

C

B

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.

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-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-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 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

A

B

C

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 cascas-pase-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.

A

B

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.

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

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

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

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

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 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 regula-tion 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-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

Experimental procedures

Cell culture and transfections

A549, HeLa and 293T cells were maintained at 37C in a

CO2incubator in Dulbecco’s modified Eagle’s medium sup-plemented with 10% fetal bovine serum The transfections were carried out using Lipofectamine PlusTMreagent (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

wherever indicated at a final concentration of 10–

20 ngÆmL)1

RT-PCR

Total RNA was isolated using the TRIzol reagent

(Invi-trogen) 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

TGAGGATGAGGAAGGG-3¢) were used for the

amplifi-cation of human IRF-1 mRNA Primers C5F2 (5¢-CCT

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 95C; 37 cycles of 1 min at 95 C, 1 min at 60 C and

1 min at 72C; and 1 cycle of 7 min 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

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

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-lows: the p73a or -b cDNA was isolated from the

pcDNA3.1-p73 plasmid by KpnI⁄ XhoI digestion and cloned

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

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 WT (or 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 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

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 BankTMaccession num-ber: NM_005427) A mutant of this shRNA was made by substituting two bases in the middle of the target sequence

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 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

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

Med-ical Research to GS

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