The protein p73 is a structural and functional homologue of the p53 tumour-suppressor protein but, unlike p53, it is not induced in response to DNA damage. The tyrosine kinase c-Abl is activated by certain DNA-damaging agents and contributes to the induction of programmed cell death (apoptosis) by p53-dependent and p53-independent mechanisms. Here we show that c-Abl binds to p73 in cells, interacting through its SH3 domain with the carboxy-terminal homo-oligomerization domain of p73. c-Abl phosphorylates p73 on a tyrosine residue at position 99 both in vitro and in cells that have been exposed to ionizing radiation. Our results show that c-Abl stimulates p73-mediated transactivation and apoptosis. This regulation of p73 by c-Abl in response to DNA damage is also demonstrated by a failure of ionizing-radiation-induced apoptosis after disruption of the c-Abl-p73 interaction. These findings show that p73 is regulated by a c-Abl-dependent mechanism and that p73 participates in the apoptotic response to DNA damage.

MDMX, an MDM2-related protein, has emerged as yet another essential negative regulator of p53 tumor suppressor, since loss of MDMX expression results in p53-dependent embryonic lethality in mice. However, it remains unknown why neither homologue can compensate for the loss of the other. In addition, results of biochemical studies have suggested that MDMX inhibits MDM2-mediated p53 degradation, thus contradicting its role as defined in gene knockout experiments. Using cells deficient in either MDM2 or MDMX, we demonstrated that these two p53 inhibitors are in fact functionally dependent on each other. In the absence of MDMX, MDM2 is largely ineffective in down-regulating p53 because of its extremely short half-life. MDMX renders MDM2 protein sufficiently stable to function at its full potential for p53 degradation. On the other hand, MDMX, which is a cytoplasmic protein, depends on MDM2 to redistribute into the nucleus and be able to inactivate p53. We also showed that MDMX, when exceedingly overexpressed, inhibits MDM2-mediated p53 degradation by competing with MDM2 for p53 binding. Our findings therefore provide a molecular basis for the nonoverlapping activities of these two p53 inhibitors previously revealed in genetic studies.The tumor suppressor gene p53 encodes a transcription factor that is activated in response to various forms of stress, leading to the induction of a number of genes whose products mediate either cell cycle arrest or apoptosis (1). Under most physiological conditions, p53 activity is tightly controlled, primarily through the ability of MDM2 to target p53 for degradation, which ensures cell survival. Current model of p53 activation suggests that diverse stress signals converge on a single regulatory node, namely the p53-MDM2 module, and interfere with the ability of MDM2 to target p53 for degradation (2). Analogous to MDM2, MDMX ablation is also associated with p53-dependent embryonic death in mice, placing MDMX in the category of essential p53 negative regulators (3). In contrast to MDM2, however, MDMX lacks ubiquitin E3 ligase activity and is unable to target p53 for ubiquitin-proteasome-dependent proteolysis (4). Moreover, MDMX was reported to inhibit MDM2-mediated p53 degradation (4 -6), contradicting the role of MDMX as defined by the genetic study. To resolve these conflicting results and gain better understanding of why neither gene product can compensate for the loss of the other, we generated MDMX-deficient cells using small interference RNA (siRNA) 1 and carried out biochemical analysis of MDM2 in these cells. In conjunction with the use of MEFs derived from either single or double knock-out mice, our loss-of-function approach allowed us to obtain compelling evidence at the molecular level to highlight mutual dependence of MDM2 and MDMX in their functional inhibition of p53 and provide support for the findings obtained in genetic studies. /MDM2Ϫ/Ϫ MEFs (Dr. Carl Maki, Harvard School of Public Health), were maintained in minimal essential medium supplemented with 10% fetal bovin...

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Attractin is a normal human serum glycoprotein of 175 kDa that is rapidly expressed on activated T cells and released extracellularly after 48-72 hr. We have cloned attractin and find that, as in its natural serum form, it mediates the spreading of monocytes that become the focus for the clustering of nonproliferating T lymphocytes. There are two mRNA species with hematopoietic tissue-specific expression that code for a 134-kDa protein with a putative serine protease catalytic serine, four EGF-like motifs, a CUB domain, a C type lectin domain, and a domain homologous with the ligand-binding region of the common ␥ cytokine chain. Except for the latter two domains, the overall structure shares high homology with the Caenorhabditis elegans F33C8.1 protein, suggesting that attractin has evolved new domains and functions in parallel with the development of cell-mediated immunity.

Cadmium (Cd) is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium ([Ca2+]i) homeostasis, leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium signaling in Cd-induced apoptosis in neuronal cells. Thus we investigated the role of calcium signaling in Cd-induced apoptosis in primary rat cerebral cortical neurons. Consistent with known toxic properties of Cd, exposure of cerebral cortical neurons to Cd caused morphological changes indicative of apoptosis and cell death. It also induced elevation of [Ca2+]i and inhibition of Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities. This Cd-induced elevation of [Ca2+]i was suppressed by an IP3R inhibitor, 2-APB, suggesting that ER-regulated Ca2+ is involved. In addition, we observed elevation of reactive oxygen species (ROS) levels, dysfunction of cytochrome oxidase subunits (COX-I/II/III), depletion of mitochondrial membrane potential (ΔΨm), and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) during Cd exposure. Z-VAD-fmk, a pan caspase inhibitor, partially prevented Cd-induced apoptosis and cell death. Interestingly, apoptosis, cell death and these cellular events induced by Cd were blocked by BAPTA-AM, a specific intracellular Ca2+ chelator. Furthermore, western blot analysis revealed an up-regulated expression of Bcl-2 and down-regulated expression of Bax. However, these were not blocked by BAPTA-AM. Thus Cd toxicity is in part due to its disruption of intracellular Ca2+ homeostasis, by compromising ATPases activities and ER-regulated Ca2+, and this elevation in Ca2+ triggers the activation of the Ca2+-mitochondria apoptotic signaling pathway. This study clarifies the signaling events underlying Cd neurotoxicity, and suggests that regulation of Cd-disrupted [Ca2+]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

Runx2 may play an important role in development of osteoarthritis (OA). However, the specific role of Runx2 in articular chondrocyte function and in OA development in adult mice has not been fully defined. In this study, we performed the destabilization of the medial meniscus (DMM) surgery at 12-week-old mice to induce OA in adult Runx2 Agc1CreER mice, in which Runx2 was specifically deleted in Aggrecan-expressing chondrocytes by administering tamoxifen at 8-weeks of age. Knee joint samples were collected 8- and 12-weeks post-surgery and analyzed through histology, histomorphometry and micro-computed tomography (μCT). Our results showed that severe OA-like defects were observed after DMM surgery in Cre-negative control mice, including articular cartilage degradation and subchondral sclerosis, while the defects were significantly ameliorated in Runx2 Agc1CreER KO mice. Immunohistochemical (IHC) results showed significantly reduced expression of MMP13 in Runx2 Agc1CreER KO mice compared to that in Cre-negative control mice. Results of quantitative reverse-transcription PCR (qRT-PCR) demonstrated that expression of the genes encoding for matrix degradation enzymes was significantly decreased in Runx2 Agc1CreER KO mice. Thus, our findings suggest that inhibition of Runx2 in chondrocytes could at least partially rescue DMM-induced OA-like defects in adult mice.

It has been demonstrated that MDM2 can differentially regulate subcellular distribution of p53 and its close structural homologue p73. In contrast to MDM2-mediated p53 nuclear export, p73 accumulates in the nucleus as aggregates that colocalize with MDM2. Distinct distribution patterns of p53 and p73 suggest the existence of unique structural elements in the two homologues that determine their MDM2-mediated relocalization in the cell. Using a series of p53/p73 chimeric proteins, we demonstrate that three regions of p53 are involved in the regulation of MDM2-mediated nuclear export. The DNA binding domain (DBD) is involved in the maintenance of a proper conformation that is required for functional activity of the nuclear export sequence (NES) of p53. The extreme C terminus of p53 harbors several lysine residues whose ubiquitination by MDM2 appears to be the initial event in p53 nuclear export, as evidenced by the impaired nucleocytoplasmic shuttling of p53 mutants bearing simultaneous substitutions of lysines 370, 372, 373, 381, 382, and 386 to arginines (6KR) or alanines (6KA). Finally, the region between the DBD and the oligomerization domain of p53, specifically lysine 305, also plays a critical role in fully revealing p53NES. We conclude that MDM2-mediated nuclear export of p53 depends on a series of ubiquitination-induced conformational changes in the p53 molecule that lead to the activation of p53NES. In addition, we demonstrate that the p53NES may be activated without necessarily disrupting the p53 tetramer.As a result of its high turnover rate, the p53 protein has a half-life of approximately 30 to 60 min and is maintained at low levels in normal proliferating cells (2). In response to genotoxic stress or oncogenic signaling, p53 levels rapidly increase, mainly through protein stabilization (2). Recent findings suggest that p53 stabilization and accumulation can be accomplished through the inhibition of nuclear export, implicating nuclear import-export as a potential mechanism of controlling p53 stability (12). An important regulator of p53 protein level is MDM2, which possesses intrinsic E3 ligase activity and thus promotes p53 ubiquitination and subsequent degradation via proteasome-mediated proteolysis (2). In addition, MDM2 functions as a mediator of p53 nuclear export (2), and MDM2 Rev-like nuclear export sequence (NES) has been shown to be essential for this function (5). p53 has its own leucine-rich, Rev-like NES in the C terminus that has been reported to be fully capable of mediating nuclear export independently of MDM2 (21). However, the p53NES lies within the oligomerization domain (OD) of the protein. Analysis of three-dimensional structure of p53OD indicates that the NES is buried at the interface of the two dimers that form active p53 tetramer (10, 13), thus rendering it inaccessible to transport proteins. It was therefore suggested that in order to reveal the buried NES, tetrameric conformation of p53 has to be disrupted (20). Results from two recent studies indicate that the ring domain of...

DNA fragmentation factor (DFF) functions downstream of caspase-3 and directly triggers DNA fragmentation during apoptosis. Here we described the identification and characterization of DFF35, an isoform of DFF45 comprised of 268 amino acids. Functional assays have shown that only DFF45, not DFF35, can assist in the synthesis of highly active DFF40. Using the deletion mutants, we mapped the function domains of DFF35/45 and demonstrated that the intact structure/conformation of DFF45 is essential for it to function as a chaperone and assist in the synthesis of active DFF40. Whereas the amino acid residues 101-180 of DFF35/45 mediate its binding to DFF40, the amino acid residues 23-100, which is homologous between DFF35/45 and DFF40, may function to inhibit the activity of DFF40. In contrast to DFF45, DFF35 cannot work as a chaperone, but it can bind to DFF40 more strongly than DFF45 and can inhibit its nuclease activity. These findings suggest that DFF35 may function in vivo as an important alternative mechanism to inhibit the activity of DFF40 and further, that the inhibitory effects of both DFF35 and DFF45 on DFF40 can put the death machinery under strict control.Apoptosis is fundamentally important in a variety of physiological and pathological processes. Apoptotic cells undergo an orchestrated cascade of events characterized by distinct morphological changes including membrane blebbing, cytoplasmic and nuclear condensation, chromatin aggregation, and formation of apoptotic bodies (1, 2). Activation of the caspase cascade is a key molecular event in the process of apoptosis (3, 4). Apoptotic signals, including growth factor and interleukin deprivation, activation of Fas, ionizing radiation, and a series of chemicals acting as upstream signals, can convert the precursors of caspases into the active enzymes (2). Several important downstream substrates of caspase, such as gelsolin (5), p21-activated kinase-2 (PAK-2) (6), and DNA fragmentation factor 45 (DFF45) 1 (7), whose cleavages clearly induce specific well characterized steps in apoptosis, have been recently identified. The cleavage of chromatin into the nucleosomal fragments, which distinguishes apoptosis from oncosis and necrosis, is a key element in the cell death process and is believed to be mediated by Mg 2ϩ /Ca 2ϩ required and Zn 2ϩ -sensitive nuclease (8 -12).We have previously identified a triplet of nuclease proteins named NP 42-50 that causes DNA degradation in vitro when cells undergo apoptosis (13). The similarity in molecular weight and biochemical characteristics between NP 42-50 and the recently identified DFF40 led us to further investigate these molecules. DFF is a heterodimeric protein composed of DFF45 and DFF40 subunits. DFF45 has been found to be the substrate of caspase-3, and DFF40 has also been cloned and found to be a DNA fragmentation nuclease (7,14,15). Cleavage of the DFF45 by caspase-3 during apoptosis releases DFF40 that degrades chromosomal DNA into nucleosomal fragments. Similar findings have also been described recently in the...