This study identifies a member of the FHL subfamily of LIM-only proteins, DRAL, as a novel target gene for the tumor suppressor protein p53. Intriguingly, DRAL protein was capable of efficiently triggering apoptosis in a wide range of cell types upon ectopic expression.
We provide three different experimental criteria suggesting that DRAL might be a direct transcriptional target of p53. First, induction of wild-type p53 in RMS cells through a temperature-sensitive p53 allele specifically increased transcription of endogenous DRAL, in a manner comparable to known p53 target genes like p21WAF1 and thrombospondin-1. Second, exposure of primary myoblasts and fibroblasts to γ-irradiation induced an increase in DRAL mRNA that paralleled the increase in p21WAF1 mRNA only in cells with wild-type p53. Because of different probes and exposure times, the mRNA levels of p21 and DRAL are not directly comparable. However, cells with mutated or no p53 consistently showed a striking reduction in the expression from both genes whereby basal levels of DRAL were higher in wild-type p53 cells than p21WAF1. This could be a possible explanation for the lower induction of DRAL mRNA after irradiation. Also, the basal expression level of DRAL was higher in fibroblasts than in myoblasts, possibly accounting for the more moderate induction observed in this cell type. Finally, analysis of the genomic structure of DRAL in the promoter region revealed five potential consensus p53-binding sites. Interestingly, the DRAL gene could be identified on recently published draft sequences of the human genome project (GenBank/EMBL/DDBJ accession numbers AC069576 and AC012360) indicating that the entire gene consists of seven exons.
In Northern blot experiments, DRAL expression was predominantly found in fetal and adult heart. The five putative NKX2.5-binding sites identified in the promoter region are in concurrence with this expression pattern. In fact the homeobox gene Nkx2.5 represents the earliest known marker of the cardiac lineage in vertebrates and its expression is maintained throughout the developing and adult heart (Schwartz and Olson 1999
). Additionally, a MEF-2–binding site was found in the DRAL
promoter. MEF-2 factors are known coregulators for myogenic basic helix-loop-helix proteins and play a pivotal role in determination and differentiation of skeletal and cardiac muscle cells (Black and Olson 1998
). Finally, five E-boxes, which can be found in the control regions of many genes specifically expressed in skeletal muscle, are likely to contribute to expression of DRAL in skeletal muscle.
Since transcription of DRAL can be induced by wild-type p53, the next question was if DRAL would participate in any of the known functions of p53-like growth arrest or apoptosis. To test this notion, ectopic expression of DRAL was achieved in a range of cell types including human RMS cells. Interestingly, this resulted in efficient induction of apoptosis in all cell types analyzed, which was confirmed on the molecular level by annexin V staining and caspase-3 activity measurements. Indeed, we were unable to generate stable cell lines expressing DRAL. Even RMS cells ectopically expressing Bcl-2 (to protect cells from apoptosis) failed to produce DRAL expressing clones (results not shown). Therefore, DRAL might participate in the apoptotic response following activation of p53. Very recently, another LIM-only protein was described to induce apoptosis in myoblasts upon ectopic expression, namely the paxillin homologue Hic-5 (Hu et al. 1999
), which is an LIM-only protein containing four LIM domains. Hic-5 is also known to bind to cell adhesion kinase β, which can induce apoptosis in fibroblasts (Xiong and Parsons 1997
) and probably other cell types. Remarkably, like DRAL, Hic-5 levels are downregulated in transformed cells compared with normal cells (Shibanuma et al. 1994
). Since a number of other LIM proteins are downregulated in transformed cells, such as Ril (Kiess et al. 1995
) and Zyxin (Schenker and Trueb 1998
), DRAL might not be the only LIM domain protein to play a suppressive role in tumor development.
To begin to investigate the mechanisms by which ectopic expression of DRAL can induce apoptosis, its intracellular localization was determined. Staining of RD-tsp53 cells with the polyclonal DRAL antibody at the permissive temperature provided the first evidence for localization of DRAL at focal contacts (data not shown). Later, DRAL was found in focal contacts, the cytoplasm, nucleus as well as the Z-discs and the M-band of cardiac myofibrils. These localizations were observed both by staining of the endogenous DRAL protein or staining of an ectopically expressed tagged version of the protein. As a further confirmation, a recently carried out yeast two-hybrid screen using DRAL as a bait identified several transcription factors, myofibrilar proteins and focal contact proteins as potential interaction partners (data not shown). These potential interactions are currently being analyzed in more detail.
Focal contacts are known to establish a transmembrane linkage between the actin cytoskeleton and the extracellular matrix and are implicated in a number of signaling pathways. Some of the proteins at these specialized sites (e.g., focal adhesion kinase, paxillin) serve as scaffolding molecules and can act as signaling centers by providing docking sites for other proteins (Burridge and Chrzanowska-Wodnicka 1996
). Intriguingly, several of these scaffolding molecules are characterized LIM domain proteins, such as Hic-5, paxillin, PINCH, and zyxin (Salgia et al. 1995
; Beckerle 1997
; Hagmann et al. 1998
; Tu et al. 1999
). Hence, DRAL might fulfil a similar function at this site.
On the other hand, nuclear localization of DRAL might also be responsible or contribute to its pro-apoptotic function. Interestingly, dual localization in focal contacts and the nucleus is known for a number of other protein, e.g., zyxin, Hic-5, and β-catenin, a multi-functional protein activated by the Wnt signaling pathway (Miller et al. 1999
). Whether DRAL localization could be influenced by external signals in a manner analogous to β-catenin is currently not known.
The Z-disc localization of DRAL is shared with a second LIM domain protein, MLP (Arber et al. 1997
), whose expression seems to be very important for the structural organization of myofibrils, since mice deficient in this protein show a disrupted cytoarchitecture leading to dilated cardiomyopathy and finally heart failure. A possible function of DRAL in this respect will have to be investigated. MLP also shares nuclear expression with DRAL, but only MLP is associated with the actin filaments. Hence, the two proteins might have some overlapping, but not completely redundant functions.
Although the knowledge of the function of FHL proteins is still very limited, they may act either as scaffolding molecules to link several proteins and thus activating distinct signaling pathways or as modulators of transcription by complexing transcription factors (Chan et al. 2000
; Muller et al. 2000
) or both. The possible role of DRAL as a p53-dependent class II tumor suppressor molecule might involve one of these mechanisms.