Modulation of p53 and MDM2 activity by novel interaction with Ras-GAP binding proteins (G3BP).

Inactivation of the p53 tumor suppressor pathway is a critical step in human tumorigenesis. In addition to mutations, p53 can be functionally silenced through its increased degradation, inhibition of its transcriptional activity and/or its inappropriate subcellular localization. Using a proteomic approach, we have found that members of the Ras network of proteins, Ras-GTPase activating protein-SH3-domain-binding proteins 1 and 2 (G3BP1 and 2), bind to p53 in vitro and in vivo. Our data show that expression of G3BPs leads to the redistribution of p53 from the nucleus to the cytoplasm. The G3BP2 isoform additionally associated with murine double minute 2 (MDM2), a negative regulator of p53. G3BP2 expression resulted in significant reduction in MDM2-mediated p53 ubiquitylation and degradation. Interestingly, MDM2 was also stabilized in G3BP2-expressing cells and its ability to ubiquitylate itself was compromised. Accordingly, short hairpin RNA (shRNA)-mediated knockdown of G3BP2 caused a reduction in MDM2 protein levels. Furthermore, expression of shRNA targeting either G3BP1 or G3BP2 in human cancer cell lines resulted in marked upregulation of p53 levels and activity. Our results suggest that both G3BP isoforms may act as negative regulators of p53.

Identification of p53 sequence elements that are required for MDM2-mediated nuclear export.

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.

Dual role of p300 in the regulation of p53 stability.

While the function of p300 as a transcriptional co-activator of p53 is well documented, its role in the regulation of p53 stability remains ill-defined since opposite effects of p300 on p53 levels have been reported. We show here that p300 stabilizes both p53 and its negative regulator MDM2, thereby enhancing the p53/MDM2 negative regulatory loop. Binding of p300 is associated with the retention of p53 in the nucleus, which results in the accumulation of p53 in an acetylase-independent manner. Stabilization of MDM2, on the other hand, requires the acetylase activity of p300. Importantly, MDM2, once expressed, is able to reverse the stabilizing effect of p300 on p53. A temperature-permissive p53-expressing cell line enabled us to demonstrate the completely opposite roles of p300 in the regulation of p53 stability, depending on the expression of MDM2. Prior to p53 activation, when MDM2 levels are low, p300 acts as a positive regulator to increase p53 levels. Upon shifting to permissive temperature, however, when MDM2 expression is induced, p300 becomes a negative regulator of p53 by stabilizing MDM2 and thereby augmenting MDM2's ability to target p53 for degradation.

Evidence for a distinct inhibitory factor in the regulation of p53 functional activity.

Under normal conditions, tumor suppressor protein p53 exists in the cell in its latent form and is unable to function as a transcription factor. The allosteric model of p53 regulation postulates that the extreme portion of p53 carboxyl terminus (aa 364-393) binds to the core domain of the protein, thereby abrogating specific DNA binding in that region. In this study we propose an alternative mechanism of p53 functional regulation, which involves a separate molecule acting in trans to inhibit p53 transcriptional activity. Through the use of chimeric proteins of p53, p63gamma and p73beta, we show that the extreme COOH-terminal domain of p53 exerts a powerful and specific inhibitory effect on the p73- and p63-driven expression of a reporter gene. Moreover, fusion of p53 extreme COOH terminus to a completely unrelated transcriptional activator Gal4-VP16 also results in significant inhibition of transactivation activity. Since p73, p63, or Gal4-VP16 cannot associate with any part of the p53 molecule, we conclude that p53(aa 364-393) represses transcriptional activity of chimeric proteins and p53 itself through the binding of external negative modulator(s) in that region and not by the allosteric mechanism of regulation. In accordance with the "distinct inhibitor" hypothesis, the activity of wild type p53 is substantially increased by overexpression of chimeric proteins bearing p53(aa 364-393), which might be due to the competitive removal of transcriptional inhibitor(s). Our findings provide the basis for the identification of such negative modulators of p53 transcriptional activity.

Mechanism of functional inactivation of a Li-Fraumeni syndrome p53 that has a mutation outside of the DNA-binding domain.

The majority of p53 mutations are located in the DNA-binding domain of the protein. However, recently a family suffering from Li-Fraumeni syndrome (LFS) has been discovered, some of whom harbor a p53 mutation in exon 4, outside of the core domain. How this mutation affects p53 function and subsequently leads to malignant transformation is not yet clear. Interestingly, the p53 mutation found in this LFS family is localized to the p53 region that we have recently identified as necessary for Mdm2-mediated p53 degradation. We therefore endeavored to study further the LFS-associated p53 mutation at the molecular level by creating an equivalent lesion in a p53 expression construct and functionally characterizing it. Here we demonstrate that a mutation in this region is associated not only with resistance of the mutant p53 to Mdm2-mediated degradation, but also with an impaired response of mutant protein to DNA damage. In addition, the p53(LFS) mutant was found to be defective in its transactivation function, which correlated with its inability to suppress cell growth and to induce apoptosis. The molecular basis for p53(LFS) functional impairment appears to be its predominantly cytoplasmic localization caused by faulty nuclear import mechanism, which, at least in part, resulted from the mutant's decreased affinity to importin.

Dynamics of extracellular matrix production and turnover in tissue engineered cardiovascular structures.

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial requirements for their long-term patency and function. This complex process requires the deposition and accumulation of extracellular matrix molecules as well as the remodeling of this extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). In this study, we have investigated the dynamics of ECM production and the activity of MMPs and TIMPs in long-term tissue-engineered vascular conduits using quantitative ECM analysis, substrate gel electrophoresis, radiometric enzyme assays and Western blot analyses. Over a time period of 169 days in vivo, levels of elastin and proteoglycans/glycosaminoglycans in tissue-engineered constructs came to approximate those of their native tissue counter parts. The kinetics of collagen deposition and remodeling, however, apparently require a much longer time period. Through the use of substrate gel electrophoresis, proteolytic bands whose molecular weight was consistent with their identification as the active form of MMP-2 (approximately 64--66 kDa) were detected in all native and tissue-engineered samples. Additional proteolytic bands migrating at approximately 72 kDa representing the latent form of MMP-2 were detected in tissue-engineered samples at time points from 5 throughout 55 days. Radiometric assays of MMP-1 activity demonstrated no significant differences between the native and tissue-engineered samples. This study determines the dynamics of ECM production and turnover in a long-term tissue-engineered vascular tissue and highlights the importance of ECM remodeling in the development of successful tissue-engineered vascular structures.

Inhibition of MAP kinase kinase causes morphological reversion and dissociation between soft agar growth and in vivo tumorigenesis in angiosarcoma cells.

Activated ras causes increased activity of several signal transduction systems, including the mitogen-activated protein kinase kinase (MAPKK) pathway and the phosphoinositol-3-kinase (PI-3-K) pathway. We have previously shown that the PI-3-K pathway plays a major role in regulation of ras-mediated tumor angiogenesis in angiosarcoma cells. However, the contribution of the MAPKK pathway to tumorigenesis and angiogenesis is not fully understood. Overexpression of constitutively active forms of MAPKK has previously been shown to transform nonmalignant NIH3T3 fibroblasts, but the effect of down-regulation of MAPKK on tumorigenesis and angiogenesis in a well established tumor has not been fully explored. We introduced a dominant negative MAPKK gene into SVR murine angiosarcoma cells. Introduction of a dominant negative MAPKK causes a significant decrease in proliferation rate in vitro and morphological reversion. Cells expressing the dominant negative MAPKK have a greatly decreased ability to form colonies in soft agar compared with wild-type cells. Despite the decreased cell growth in vitro and inability to grow in soft agar, the cells were equally tumorigenic in nude mice. Our results suggest that the MAPKK pathway is required for soft agar growth of angiosarcoma cells, and separates the phenotypes of soft agar growth versus in vivo tumorigenicity. These findings have implications in the development of signal transduction modulators as potential antineoplastic agents.

Matrix metalloproteinase-2 is required for the switch to the angiogenic phenotype in a tumor model.

Among the earliest and most important stages during tumorigenesis is the activation of the angiogenic process, an event that is termed the "switch to the angiogenic phenotype." We have developed an in vivo system that can reliably recapitulate the stages in tumor development that represent this transition. Using this model, we have harvested and studied tumor nodules that can be distinguished from each other on the basis of their degree of vascularization. Angiogenic tumor nodules were characterized by the presence of capillary vessels as determined by factor VIII immunohistochemistry, and both angiogenic and proteolytic activities in vitro. In contrast, preangiogenic nodules were devoid of microvessels and showed little angiogenic or proteolytic activity in vitro. Addition of a specific metalloproteinase inhibitor resulted in the abrogation of both angiogenic and proteolytic activities of the angiogenic nodules in vitro. Comparative substrate gel electrophoresis detected the presence of a prominent matrix metalloproteinase (MMP-2) in the angiogenic nodules when compared with the preangiogenic ones. Suppression of MMP-2 activity by antisense oligonucleotides in the vascular nodules resulted in the loss of angiogenic potential both in vitro and in vivo in the chick chorioallantoic membrane assay. Moreover, this suppression of MMP-2 activity in angiogenic nodules inhibited tumor growth in vivo by approximately 70%. These results strongly implicate the activity of MMP-2 as a requirement for the switch to the angiogenic phenotype and validate this model as a reliable and reproducible tool by which to study other cellular and biochemical factors involved in the acquisition of the angiogenic phenotype.

Regulation of angiostatin production by matrix metalloproteinase-2 in a model of concomitant resistance.

We have previously reported the identification of the endogenous angiogenesis inhibitor angiostatin, a specific inhibitor of endothelial cell proliferation in vitro and angiogenesis in vivo. In our original studies, we demonstrated that a Lewis lung carcinoma (LLC-LM) primary tumor could suppress the growth of its metastases by generating angiostatin. Angiostatin, a 38-kDa internal fragment of plasminogen, was purified from the serum and urine of mice bearing LLC-LM, and its discovery provides the first proven mechanism for concomitant resistance (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M. A., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315-328). Subsequently, we have shown that systemic administration of angiostatin can regress a wide variety of malignant tumors in vivo. However, at the time of our initial discovery of angiostatin, the source of the protein was unclear. We hypothesized that the tumor or stromal cells might produce an enzyme that could cleave plasminogen sequestered by the primary tumor into angiostatin. Alternatively, we speculated that the tumor cells might express angiostatin. By Northern analysis, however, we have found no evidence that the tumor cells express angiostatin or other fragments of plasminogen (data not shown). We now report that gelatinase A (matrix metalloproteinase-2), produced directly by the LLC-LM cells, is responsible for the production of angiostatin, which suppresses the growth of metastases in our original model.

The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro.

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.

Troponin I is present in human cartilage and inhibits angiogenesis.

Cartilage is an avascular and relatively tumor-resistant tissue. Work from a number of laboratories, including our own, has demonstrated that cartilage is an enriched source of endogenous inhibitors of angiogenesis. In the course of a study designed to identify novel cartilage-derived inhibitors of new capillary growth, we have purified an inhibitory protein that was identified by peptide microsequencing and protein database analysis as troponin I (TnI). TnI is a subunit of the troponin complex (troponin-C and troponin-T being the other two), which, along with tropomyosin, is responsible for the calcium-dependent regulation of striated muscle contraction; independently, TnI is capable of inhibiting actomyosin ATPase. Because troponin has never previously been reported to be present in cartilage, we have cloned and expressed the cDNA of human cartilage TnI, purified this protein to apparent homogeneity, and demonstrated that it is a potent and specific inhibitor of angiogenesis in vivo and in vitro, as well as of tumor metastasis in vivo.

The generation of endostatin is mediated by elastase.

Endostatin, a potent inhibitor of angiogenesis and tumor growth, is a COOH-terminal fragment of collagen XVIII derived through cleavage of an Ala-His linkage by an as yet unidentified endostatin-processing enzyme. Endostatin was originally isolated from the conditioned medium of hemangioendothelioma (EOMA) cells. By investigating the processing of collagen XVIII to endostatin by EOMA cells, we show here that the generation of endostatin can be mediated by an elastase activity. We also show that several members of the elastase family can act as an endostatin-processing enzyme by specifically cleaving the Ala-His linkage and releasing endostatin from a precursor molecule. We further suggest that the generation of endostatin from collagen XVIII is at least a two-step process, involving a metal-dependent early step and an elastase activity-dependent final step.

Increased incidence of matrix metalloproteinases in urine of cancer patients.

Matrix metalloproteinases (MMPs) have been implicated in mechanisms of metastasis in experimental cancer models and in human malignancies. In this study, we used substrate gel electrophoresis (zymography) to determine the frequency of detection of MMPs in urine of patients with a variety of cancers. Three molecular weight classes of urinary MMPs, Mr 72,000, Mr 92,000, and high molecular weight (Mr > or = 150,000) species, were detected reproducibly and correlated with disease status. The Mr 72,000 and Mr 92,000 species were identified as MMP-2 and MMP-9, respectively, by Western blot analysis. The presence of biologically active MMP-2 (P < 0.001) or MMP-9 (P = 0.002) was an independent predictor of organ-confined cancer, and the high molecular weight species (P < 0.001) was an independent predictor of metastatic cancer. This is the first study to demonstrate that analysis of urinary MMPs may be useful in determining disease status in a variety of human cancers, both within and outside of the urinary tract.

Dysregulated proteolytic balance as the basis of excess extracellular matrix in fibrotic disease.

To investigate mechanisms of tissue fibrosis, we developed a model of ovine fetal bladder fibrosis due to surgically induced obstruction. Tissues were analyzed for matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Active MMP-2 was not detected in obstructed bladders, while latent and active forms were detected in normal bladders. MMP-1 (interstitial collagenase) activity was lower in obstructed bladders. MMP inhibitory activity was increased with obstruction, as were levels of TIMP mRNA and protein. These results indicate that the proteins responsible for collagen degradation are present in the developing bladder, and a shift in the proteolytic balance favoring inhibition of degradation occurs in a model of obstruction-induced fibrosis. This altered proteolytic balance favors accumulation of extracellular matrix and decreased tissue compliance characteristic of this and perhaps other fibrotic conditions.

Matrix metalloproteinases and their inhibitors in aqueous humor.

Matrix metalloproteinase activity is the rate-limiting step in extracellular matrix degradation. One mechanism by which metalloproteinases are regulated is through the activity of their endogenous inhibitors, the tissue inhibitors of metalloproteinases. Since metalloproteinase activity is a key component of the angiogenic process and many anterior segment structures are largely avascular, we became interested in examining aqueous humor for the presence of metalloproteinases and their endogenous inhibitors. Using zymography, we have identified the presence of several metalloproteinases in normal aqueous humor. Treatment with 4-aminophenylmercuric acetate, an organomercurial which activates latent metalloproteinases, revealed that all metalloproteinases were in their active state. By Western blot analysis, normal aqueous humor was also found to contain at least two tissue inhibitors of metalloproteinases. Subsequent partial purification by two successive chromatographic steps revealed the presence of inhibitory activity against collagenase, endothelial cell DNA synthesis, and angiogenesis on the chick chorioallantoic membrane. The presence of metalloproteinases and their inhibitors in normal aqueous humor, a fluid which bathes avascular ocular structures, suggests that future studies should examine whether an imbalance in this protease/inhibitor family may contribute to the anterior chamber extracellular matrix alterations associated with diseases such as ocular neovascularization and glaucoma.