Steven J. Berberich

MdmX Protects p53 from Mdm2-Mediated Degradation

ABSTRACT The p53 tumor suppressor protein is stabilized in response to cellular stress, resulting in activation of genes responsible for either cell cycle arrest or apoptosis. The cellular pathway for releasing normal cells from p53-dependent cell cycle arrest involves the Mdm2 protein. Recently, a p53-binding protein with homology to Mdm2 was identified and called MdmX. Like Mdm2, MdmX is able to bind p53 and inhibit p53 transactivation; however, the ability of MdmX to degrade p53 has yet to be examined. We report here that MdmX is capable of associating with p53 yet is unable to facilitate nuclear export or induce p53 degradation. In addition, expression of MdmX can reverse Mdm2-targeted degradation of p53 while maintaining suppression of p53 transactivation. Using a series of MdmX deletions, we have determined that there are two distinct domains of the MdmX protein that can stabilize p53 in the presence of Mdm2. One domain requires MdmX interaction with p53 and results in the retention of both proteins within the nucleus and repression of p53 transactivation. The second domain involves the MdmX ring finger and results in stabilization of p53 and an increase in p53 transactivation. The potential basis for stabilization and increased p53 transactivation by the MdmX ring finger domain is discussed. Based on these observations, we propose that the MdmX protein may function to maintain a nuclear pool of p53 protein in undamaged cells.

Human NM23/nucleoside diphosphate kinase regulates gene expression through DNA binding to nuclease-hypersensitive transcriptional elements.

NM23-H2/NDP kinase B has been identified as a sequence-specific DNA-binding protein withaffinity for a nuclease-hypersensitive element of the c-MYC gene promoter (Postel et al.,1993). The ability of Nm23-H2 to activate c-MYC transcription in vitro and in vivo via thesame element demonstrates the biological significance of this interaction. Mutational analyseshave identified Arg34, Asn69 and Lys135 as critical for DNA binding, but not required forthe NDP kinase reaction. However, the catalytically important His118 residue is dispensiblefor sequence-specific DNA binding, suggesting that sequence-specific DNA recognition andphosphoryl transfer are independent properties. Nm23-H2 also has an activity that cleavesDNA site-specifically, involving a covalent protein-DNA complex. In a DNAsequence-dependent manner, Nm23-H2 recognizes additional target genes for activation, includingmyeloperoxidase, CD11b, and CCR5, all involved in myeloid-specific differentiation. Moreover,both NM23-H1 and Nm23-H2 bind to nuclease hypersensitive elements in the platelet-derivedgrowth factor PDGF-A gene promoter sequence-specifically, correlating with either positiveor negative transcriptional regulation. These data support a model in which NM23/NDP kinasemodulates gene expression through DNA binding and subsequent structural transactions.

MdmX Binding to ARF Affects Mdm2 Protein Stability and p53 Transactivation

Regulation of p53 involves a complex network of protein interactions. The primary regulator of p53 protein stability is the Mdm2 protein. ARF and MdmX are two proteins that have recently been shown to inhibit Mdm2-mediated degradation of p53 via distinct associations with Mdm2. We demonstrate here that ARF is capable of interacting with MdmX and in a manner similar to its association with Mdm2, sequestering MdmX within the nucleolus. The sequestration of MdmX by ARF results in an increase in p53 transactivation. In addition, the redistribution of MdmX by ARF requires that a nucleolar localization signal be present on MdmX. Although expression of either MdmX or ARF leads to Mdm2 stabilization, coexpression of both MdmX and ARF results in a decrease in Mdm2 protein levels. Similarly, increasing ARF protein levels in the presence of constant MdmX and Mdm2 leads to a dose-dependent decrease in Mdm2 levels. Under these conditions, ARF can synergistically reverse the ability of Mdm2 and MdmX to inhibit p53-dependent transactivation. Finally, the association and redistribution of MdmX by ARF has no effect on the protein stability of either ARF or MdmX. Taken together, these results demonstrate that the interaction between MdmX and ARF represents a novel pathway for regulating Mdm2 protein levels. Additionally, both MdmX and Mdm2, either individually or together, are capable of antagonizing the effects of the ARF tumor suppressor on p53 activity.

Regulation of p63 Function by Mdm2 and MdmX

p63, a p53-related protein, has been shown to activate p53-responsive genes and induce apoptosis in certain cell types. In this study, we examined the effects of Mdm2 and MdmX proteins on p63 transactivation, apoptosis, and protein levels. The isoforms of p63 most structurally similar to p53, p63gamma (p51A) and p63alpha (p51B), were chosen for study. Our results confirm earlier reports demonstrating that although both p63 isoforms can transactivate p53-responsive promoters and induce apoptosis, p63gamma has a stronger transactivation potential and is a more potent inducer of apoptosis than is p63alpha. In addition, both Mdm2 and MdmX were able to inhibit the transactivation induced by p63gamma and p63alpha. However, only Mdm2 overexpression led to a detectable decrease in p63-induced apoptosis. Although Mdm2 binding to p53 triggers ubiquitin-mediated proteosome degradation, p63 protein levels were unaltered by association with either Mdm2 or MdmX. Finally, immunofluorescence experiments showed that both p63 isoforms were localized in the nucleus and could be exported when coexpressed with Mdm2 but not with MdmX. These findings suggest that both Mdm2 and MdmX can downregulate p63 transactivation potential; however, only Mdm2 is capable of inhibiting the apoptotic function of p63 by removing it from the nucleus.

Phospholipase D (PLD) drives cell invasion, tumor growth and metastasis in a human breast cancer xenograph model

Breast cancer is one of the most common malignancies in human females in the world. One protein that has elevated enzymatic lipase activity in breast cancers in vitro is phospholipase D (PLD), which is also involved in cell migration. We demonstrate that the PLD2 isoform, which was analyzed directly in the tumors, is crucial for cell invasion that contributes critically to the growth and development of breast tumors and lung metastases in vivo. We used three complementary strategies in a SCID mouse model and also addressed the underlying molecular mechanism. First, the PLD2 gene was silenced in highly metastatic, aggressive breast cancer cells (MDA-MB-231) with lentivirus-based short hairpin RNA, which were xenotransplanted in SCID mice. The resulting mouse primary mammary tumors were reduced in size (65%, P<0.05) and their onset delayed when compared with control tumors. Second, we stably overexpressed PLD2 in low-invasive breast cancer cells (MCF-7) with a biscistronic MIEG retroviral vector and observed that these cells were converted into a highly aggressive phenotype, as primary tumors that formed following xenotransplantation were larger, grew faster and developed lung metastases more readily. Third, we implanted osmotic pumps into SCID xenotransplanted mice that delivered two different small-molecule inhibitors of PLD activity (5-fluoro-2-indolyl des-chlorohalopemide and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide). These inhibitors led to significant (>70%, P<0.05) inhibition of primary tumor growth, metastatic axillary tumors and lung metastases. In order to define the underlying mechanism, we determined that the machinery of PLD-induced cell invasion is mediated by phosphatidic acid, Wiscott–Aldrich Syndrome protein, growth receptor-bound protein 2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows for the first time that PLD2 has a central role in the development, metastasis and level of aggressiveness of breast cancer, raising the possibility that PLD2 could be used as a new therapeutic target.

MicroRNA-34a Modulates MDM4 Expression via a Target Site in the Open Reading Frame

Background MDM4, also called MDMX or HDMX in humans, is an important negative regulator of the p53 tumor suppressor. MDM4 is overexpressed in about 17% of all cancers and more frequently in some types, such as colon cancer or retinoblastoma. MDM4 is known to be post-translationally regulated by MDM2-mediated ubiquitination to decrease its protein levels in response to genotoxic stress, resulting in accumulation and activation of p53. At the transcriptional level, MDM4 gene regulation has been less clearly understood. We have reported that DNA damage triggers loss of MDM4 mRNA and a concurrent increase in p53 activity. These experiments attempt to determine a mechanism for down-regulation of MDM4 mRNA. Methodology/Principal Findings Here we report that MDM4 mRNA is a target of hsa-mir-34a (miR-34a). MDM4 mRNA contains a lengthy 3′ untranslated region; however, we find that it is a miR-34a site within the open reading frame (ORF) of exon 11 that is responsible for the repression. Overexpression of miR-34a, but not a mutant miR-34a, is sufficient to decrease MDM4 mRNA levels to an extent identical to those of known miR-34a target genes. Likewise, MDM4 protein levels are decreased by miR-34a overexpression. Inhibition of endogenous miR-34a increased expression of miR-34a target genes and MDM4. A portion of MDM4 exon 11 containing this 8mer-A1 miR-34a site fused to a luciferase reporter gene is sufficient to confer responsiveness, being inhibited by additional expression of exogenous mir-34a and activated by inhibition of miR-34a. Conclusions/Significance These data establish a mechanism for the observed DNA damage-induced negative regulation of MDM4 and potentially provide a novel means to manipulate MDM4 expression without introducing DNA damage.

MdmX Inhibits Smad Transactivation

Mdm2 overexpression confers a growth promoting activity upon cells primarily by downregulating the p53 tumor suppressor protein. Nevertheless, Mdm2 deregulation has also been implicated in inhibiting TGF-β growth repression in a p53 independent manner. Our goal in this study was to examine whether overexpression of Mdm2 or MdmX, a Mdm2-related protein, could affect Smad-induced transactivation. As downstream signaling elements of the TGF-β pathway, Smads represent one potential target for Mdm2 and MdmX. Here we show that MdmX but not Mdm2 is capable of inhibiting Smad induced transactivation. Based on deletion mutant analysis, MdmX inhibition of Smad transactivation was independent of the p53 and Mdm2 interaction domains, yet required amino acid residues 128–444. Using TGF-β sensitive HepG2 cells, MdmX overexpression was shown to inhibit TGF-β induced Smad transactivation. Additionally, mouse embryo fibroblasts (MEFs) lacking p53 and MdmX showed enhanced Smad transactivation when compared to MEFs lacking either p53 or p53 and Mdm2. Interestingly, the inhibition of Smad transactivation by MdmX could be reversed by p300, a functional co-activator of Smads and a necessary factor for Mdm2 nuclear export and did not result from altered Smad localization. In vitro studies demonstrate that MdmX binds to p300 as well as Smad3 and Smad4. Taken together, these results suggest that inhibition of Smad-induced transactivation by MdmX occurs by altering Smad interaction with its coactivator p300.

p53 binds to cisplatin-damaged DNA.

We have previously shown that bacterially expressed p53 protein or p53 protein isolated from cis-diamminedichloroplatinum II (cisplatin)-damaged cells is capable of binding to double-stranded platinated DNA molecules lacking any p53 DNA binding sites. Here we report using various p53 mutants that two separate domains of p53 protein affect p53 binding to platinated DNA. Mutations within the central core of p53, the domain responsible for sequence-specific DNA binding activity, completely eliminated p53 binding to platinated DNA. Based on competition experiments p53 preferred binding to sequence-specific DNA molecules over platinated DNA molecules. However, p53 binding to platinated DNA molecules was significantly stronger than p53 interactions with DNA molecules lacking damage and a p53 consensus site. Finally, an antibody specific to the C-terminal domain of p53 (pAb421) which activates sequence-specific DNA binding activity inhibited p53 binding to platinated DNA. Taken together, these results suggest that in addition to binding to p53 DNA binding sites, p53 also interacts with cisplatin-damaged DNA molecules.

Full-length hdmX transcripts decrease following genotoxic stress.

Previous studies have suggested that the mdmX gene is constitutively transcribed, and that MdmX protein activity is instead controlled by cellular localization and DNA damage induced Mdm2-mediated ubiquitination leading to proteasomal degradation. In these studies, we report that the human mdmX (hdmX) mRNA is reproducibly decreased in various human cell lines following treatment with various DNA-damaging agents. Repression of hdmX transcripts is observed in DNA-damaged HCT116 colon cancer cells and in isogenic p53−/− cells, suggesting that this effect is p53-independent. Reduction in the amount of hdmX transcript occurs in both human tumor cell lines and primary human diploid fibroblasts, and results in a significant reduction of HdmX protein. Examination of hdmX promoter activity suggests that damage-induced repression of hdmX mRNA is not significantly impacted by transcription initiation. In contrast, changes in hdmX mRNA splicing appear to partly explain the reduction in full-length hdmX mRNA levels in tumor cell lines with the destabilization of full-length hdmX transcripts, potentially through microRNA miR-34a regulation, also impacting transcript levels. Taken together, this study uncovers previously unrecognized cellular mechanisms by which hdmX mRNA levels are kept low following genotoxic stress.

Constitutive mdmx expression during cell growth, differentiation, and DNA damage

The mdmx gene was shown to possess high homology to the mdm-2 gene and to encode a protein that can bind p53 and block p53 transactivation. Because Mdm-2 protein blocks the growth-suppressive activity of the p53 tumor-suppressor protein through similar activities, we examined the expression patterns of mdmx to determine how MdmX expression correlates with p53 protein levels. In this study, the expression pattern and protein levels of mdmx were examined in a number of cell culture systems. Like mdm-2, mdmx gene expression was constitutive during serum deprivation/restimulation of murine fibroblasts and differentiation of either murine teratocarcinoma or preadipocyte cells. In contrast, whereas mdm-2 gene expression was induced after cisplatin damage to ovarian carcinoma cells, mdmx expression remained constitutive. Because p53 transactivation is critical following a genotoxic stress, we examined p53:MdmX complexes after in vitro DNA-PK phosphorylation, a posttranslational modification that blocks p53 association with Mdm-2. The DNA-PK phosphorylation of p53 was capable of inhibiting p53:MdmX association. Thus, whereas DNA damage does not regulate mdmx mRNA levels, posttranslational modifications induced during DNA damage may block p53:MdmX association in vivo. These results demonstrate that, in the cell lines examined, mdmx gene expression remains constitutive during cell proliferation and differentiation or following DNA damage. Taken together, the data suggest that cells retain a constant level of MdmX. Thus, in undamaged cells, there exists the potential for an MdmX:p53 reservoir.