Apoptosis induced by cAMP requires Smac/DIABLO transcriptional upregulation

doi:10.1016/j.cellsig.2007.01.001

Cellular Signalling

Volume 19, Issue 6, June 2007, Pages 1212-1220

https://doi.org/10.1016/j.cellsig.2007.01.001 Get rights and content

Abstract

Smac/DIABLO is a mitochondrial protein that participates in apoptotic cell death by means of sequestering several members of the inhibitor of apoptosis protein family. This action allows caspase activation, cleavage of key cellular substrates and death. Release from mitochondria is considered the main regulatory step of Smac/DIABLO activity. Nevertheless, the fact that at least one isoform, Smac-beta, does not reside in this organelle implies that transcriptional regulation could also be important. cAMP is a well known second messenger with important apoptotic effects. To analyze if cAMP could be involved in Smac/DIABLO gene regulation, we analyzed 2903 base pairs upstream of the coding sequence and characterized the minimal promoter, which contains a consensus CRE site. We found that cAMP/PKA/CREB pathway is indeed an important regulator of Smac/DIABLO transcription, since exposure to the cAMP analog 8-CPT-cAMP, the adenylyl cyclase activator forskolin, the inhibitor of phosphodiesterase isobutylmethylxanthine or by hindering PKA activation with H89, regulated the promoter activity, as shown by gene reporter and RT-PCR assays. Additionally, the results of site-directed mutagenesis revealed that the consensus CRE site was biologically functional and required for cAMP-induced promoter activity in human HeLa cells. Supporting these results, a negative dominant version of the protein kinase A responsive factor, KCREB, reduced basal Smac/DIABLO expression and rendered the promoter unresponsive to cAMP. Reducing Smac expression using an antisense approach blocked the apoptosis effects of cAMP in cervical cancer cells. These results show that cAMP is an important modulator of the apoptotic threshold in cancer cell by means of regulating Smac/DIABLO expression.

Introduction

Smac (Second mitochondria derived activator of caspase) and its murine ortholog DIABLO (Direct IAP binding with Low pI) are mitochondrial proteins encoded by nuclear DNA which are released into the cytosol in response to apoptotic stimuli that disrupt the integrity of the mitochondria. Smac/DIABLO participates in the two main apoptotic pathways, the intrinsic or mitochondrial pathway [1], [2] and the extrinsic or death receptor pathway [3], [4]. After apoptotic stimuli, released Smac acts as a dimer in the cytosol, activating caspases by means of sequestering and neutralizing members of the inhibitor of apoptosis proteins family (IAPs) [5], [6]. Although the exact mechanism that regulates Smac mitochondrial release is unknown, there is evidence that some cellular transduction signal pathways are able to regulate it [7], [8], [9], [10], [11]. In addition, as with cytochrome c, Smac release is modulated by members of the Bcl-2 family (Bcl-2, BID and Bcl-w) [12]. Interestingly, mitochondrial release of Smac is blocked by a broad-spectrum caspase inhibitor [13], [14], [15], showing the presence of a positive cellular feedback loop.

Aside from the mitochondrial release of Smac/DIABLO, additional regulation mechanisms have been far less studied. Modulation of Smac mRNA and protein has been found in some systems. For example, folic acid and tumor necrosis factor increases Smac/DIABLO mRNA in kidney tubular cells [16]. This increase correlates with apoptosis in vivo. It has also been found that Smac is deregulated in cervical cancer [17], sarcomas [18], lung cancer carcinomas [19] and renal tumors [20]. The reason for the differences in the expression of this molecule in normal cells versus cancer cells is unknown. Although the possible implications of these differences have not been thoroughly studied, alterations in Smac/DIABLO expression are able to influence the apoptotic threshold. For example, ectopic overexpression of Smac/DIABLO sensitizes hepatic cancer cells to apoptosis induced by anti-neoplasic drugs [21], [22]. For these reasons, Smac/DIABLO is an interesting target for therapy. Indeed, it has been shown that peptides corresponding to the IAP recognition motif of Smac sensitize cancer cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL), and chemotherapeutic agents [23], [24], [25].

The second messenger cAMP is able to induce growth arrest and apoptosis in diverse cancer cell lines [26]. cAMP can also potentate chemotherapeutic agents such as paclitaxel, an important antineoplasic agent used in a variety of tumors [27]. The higher susceptibility to cAMP-mediated apoptosis of certain tumor cells compared to their normal counterpart has spurred interest in developing analogs as anti-cancer drugs [28]. The mechanisms of these effects are poorly understood. In the present paper we have investigated the role of cAMP in the transcriptional regulation of Smac/DIABLO as a potential mechanism for the second messenger apoptosis effects.

Section snippets

Cloning of the Smac/DIABLO genomic 5′ untranslated region

Genomic DNA was prepared with DNAzol (Invitrogen, MD USA) from a pool of human leukocytes isolated by Ficoll density gradient centrifugation. In silico analysis was used to identify the 5′untranslated region from the chromosome 12 clone RP11-512M8 (GenBank accession no. AF62240 and GenBank accession no. AC048338). Two fragments of 2903 and 1398 bp of the 5′untranslated flanking region of the DIABLO gene were amplified by nested PCR using the proofreading Pfu polymerase (Stratagene, CA USA). The

Cloning and analysis of the human DIABLO promoter region

In order to investigate if Smac/DIABLO transcription could be regulated by cAMP and to characterize the mechanism of this regulation, we first isolated and analyzed the human promoter for this gene. Based on similarity between the Smac/DIABLO sequence (GenBank accession no. AF262240) and the chromosome 12 clone RP11-512M8 (GenBank accession no. AC048338), the human DIABLO gene has been mapped to chromosome 12, region 12q24.31 [34]. Using high fidelity long range PCR and information derived from

Discussion

The mechanism of cAMP signal transduction in mammalian cells is one of the best understood biochemical pathways. Phosphorylation of CREB mediated by the cAMP signaling pathway can be initiated by a plethora of physiological stimuli and is critically involved in the regulation of metabolism, cell growth and differentiation, apoptosis, and gene expression [43]. Transcriptional regulation by cAMP is mediated by a group of nuclear factors that bind to and regulate the expression of genes containing

Acknowledgements

This work was supported by grants from the Consejo Nacional de Ciencia y Tecnologia (CONACyT) A2039/A1 to VM and SEP-2004-C01-45728 to JMZ. Moises Martinez-Velazquez was also supported by a graduate fellowship from CONACyT. We thank Dr. Brent Zanke for his kind gift of SEK-AL plasmid and Dr. Richard Goodman for the kind gift of KCREB plasmid.

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Apoptosis induced by cAMP requires Smac/DIABLO transcriptional upregulation

Abstract

Introduction

Section snippets

Cloning of the Smac/DIABLO genomic 5′ untranslated region

Cloning and analysis of the human DIABLO promoter region

Discussion

Acknowledgements

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Kidney Inter., Suppl.

J. Biol. Chem.

Neuron

J. Biol. Chem.

Int. J. Cancer

Trends Neurosci.

J. Biol. Chem.

J. Biol. Chem.

Blood

Exp. Cell Res.

Free Radic. Biol. Med.

J. Cell Biol.

Genes Dev.

Biochem. J.

Apoptosis

Cell Death Differ.

Eur. J. Neurosci.

EMBO J.

BMC Cancer

Apmis

Oncol. Rep.