TRE17/USP6 oncogene translocated in aneurysmal bone cyst induces matrix metalloproteinase production via activation of NF-kappaB.

Aneurysmal bone cyst (ABC) is an aggressive, pediatric bone tumor characterized by extensive destruction of the surrounding bone. Although first described over 60 years ago, its molecular etiology remains poorly understood. Recent work revealed that ABCs harbor translocation of TRE17/USP6, leading to its transcriptional upregulation. TRE17 encodes a ubiquitin-specific protease (USP), and a TBC domain that mediates binding to the Arf6 GTPase. However, the mechanisms by which TRE17 overexpression contributes to tumor pathogenesis, and the role of its USP and TBC domains, are unknown. ABCs are characterized by osteolysis, inflammatory recruitment and extensive vascularization, the processes in which matrix proteases have a prominent role. This led us to explore whether TRE17 regulates the production of matrix metalloproteinases (MMPs). In this study we show that TRE17 is sufficient to induce expression of MMP-9 and MMP-10, in a manner requiring its USP activity, but not its ability to bind Arf6. TRE17 induces transcription of MMP-9 through activation of nuclear factor-kappaB (NF-kappaB), mediated in part by the GTPase RhoA and its effector kinase, ROCK. Furthermore, xenograft studies show that TRE17 induces formation of tumors that reproduce multiple features of ABC, including a high degree of vascularization, with an essential role for the USP domain. In sum, these studies reveal that TRE17 is sufficient to initiate tumorigenesis, identify MMPs as novel TRE17 effectors that likely contribute to ABC pathogenesis and define the underlying signaling mechanism of their induction.

Stimulation of CREB binding protein nucleosomal histone acetyltransferase activity by a class of transcriptional activators.

The transcriptional coactivator CREB binding protein (CBP) possesses intrinsic histone acetyltransferase (HAT) activity that is important for gene regulation. CBP binds to and cooperates with numerous nuclear factors to stimulate transcription, but it is unclear if these factors modulate CBP HAT activity. Our previous work showed that CBP interacts with the Epstein-Barr virus-encoded basic region zipper (b-zip) protein, Zta, and augments its transcriptional activity. Here we report that Zta strongly enhances CBP-mediated acetylation of nucleosomal histones. Zta stimulated the HAT activity of CBP that had been partially purified or immunoprecipitated from mammalian cells as well as from affinity-purified, baculovirus expressed CBP. Stimulation of nucleosome acetylation required the CBP HAT domain, the Zta DNA binding and transcription activation domain, and nucleosomal DNA. In addition to Zta, we found that two other b-zip proteins, NF-E2 and C/EBPalpha, strongly stimulated nucleosomal HAT activity. In contrast, several CBP-binding proteins, including phospho-CREB, JUN/FOS, GATA-1, Pit-1, and EKLF, failed to stimulate HAT activity. These results demonstrate that a subset of transcriptional activators enhance the nucleosome-directed HAT activity of CBP and suggest that nuclear factors may regulate transcription by altering substrate recognition and/or the enzymatic activity of chromatin modifying coactivators.

Stimulation of NF-E2 DNA binding by CREB-binding protein (CBP)-mediated acetylation.

The hematopoietic transcription factor NF-E2 is an important regulator of erythroid and megakaryocytic gene expression. The transcription cofactor cAMP-response element-binding protein (CREB)-binding protein (CBP) has previously been implicated in mediating NF-E2 function. In this report, we examined the role of CBP, a coactivator with intrinsic acetyltransferase activity, in the regulation of NF-E2. We found that both the hematopoietic-specific subunit of NF-E2, p45, and the widely expressed small subunit, MafG, interact with CBP in vitro and in vivo. CBP acetylates MafG, but not p45, predominantly in the basic region of MafG. Immunoprecipitation experiments with anti-acetyl lysine antibodies demonstrate that MafG is acetylated in vivo in erythroid cells. Transfection experiments further show that CBP stimulates MafG acetylation in intact cells in an E1A-sensitive manner. Acetylation of MafG augments DNA binding activity of NF-E2, and mutations at the major acetylation sites markedly reduce DNA binding and transcriptional activation by NF-E2. Together, these results suggest that recruitment of CBP by NF-E2 to specific erythroid/megakaryocytic promoters might regulate transcription by at least two mechanisms involving both modification of chromatin structure and modulation of transcription factor activity.

GATA-1 and erythropoietin cooperate to promote erythroid cell survival by regulating bcl-xL expression.

The transcription factor GATA-1 is essential for normal erythropoiesis. By examining in vitro-differentiated embryonic stem cells, we showed previously that in the absence of GATA-1, committed erythroid precursors fail to complete maturation and instead undergo apoptosis. The mechanisms by which GATA-1 controls cell survival are unknown. Here we report that in erythroid cells, GATA-1 strongly induces the expression of the anti-apoptotic protein bcl-xL, but not the related proteins bcl-2 and mcl-1. Consistent with a role for bcl-xL in mediating GATA-1-induced erythroid cell survival, in vitro-differentiated bcl-xL-/- embryonic stem cells fail to generate viable mature definitive erythroid cells, a phenotype resembling that of GATA-1 gene disruption. In addition, we show that erythropoietin, which is also required for erythroid cell survival, cooperates with GATA-1 to stimulate bcl-xL gene expression and to maintain erythroid cell viability during terminal maturation. Together, our data show that bcl-xL is essential for normal erythroid development and suggest a regulatory hierarchy in which bcl-xL is a critical downstream effector of GATA-1 and erythropoietin-mediated signals.

CREB-Binding protein acetylates hematopoietic transcription factor GATA-1 at functionally important sites.

The transcription factor GATA-1 is a key regulator of erythroid-cell differentiation and survival. We have previously shown that the transcriptional cofactor CREB-binding protein (CBP) binds to the zinc finger domain of GATA-1, markedly stimulates the transcriptional activity of GATA-1, and is required for erythroid differentiation. Here we report that CBP, but not p/CAF, acetylates GATA-1 at two highly conserved lysine-rich motifs present at the C-terminal tails of both zinc fingers. Using [3H]acetate labelling experiments and anti-acetyl lysine immunoprecipitations, we show that GATA-1 is acetylated in vivo at the same sites acetylated by CBP in vitro. In addition, we show that CBP stimulates GATA-1 acetylation in vivo in an E1A-sensitive manner, thus establishing a correlation between acetylation and transcriptional activity of GATA-1. Acetylation in vitro did not alter the ability of GATA-1 to bind DNA, and mutations in either motif did not affect DNA binding of GATA-1 expressed in mammalian cells. Since certain functions of GATA-1 are revealed only in an erythroid environment, GATA-1 constructs were examined for their ability to trigger terminal differentiation when introduced into a GATA-1-deficient erythroid cell line. We found that mutations in either acetylation motif partially impaired the ability of GATA-1 to induce differentiation while mutations in both motifs abrogated it completely. Taken together, these data indicate that CBP is an important cofactor for GATA-1 and suggest a novel mechanism in which acetylation by CBP regulates GATA-1 activity in erythroid cells.

CREB-binding protein cooperates with transcription factor GATA-1 and is required for erythroid differentiation.

The transcription factor GATA-1 coordinates multiple events during terminal erythroid cell maturation. GATA-1 participates in the transcription of virtually all erythroid-specific genes, blocks apoptosis of precursor cells, and controls the balance between proliferation and cell cycle arrest. Prior studies suggest that the function of GATA-1 is mediated in part through association with transcriptional cofactors. CREB-binding protein (CBP) and its close relative p300 serve as coactivators for a variety of transcription factors involved in growth control and differentiation. We report here that CBP markedly stimulates GATA-1's transcriptional activity in transient transfection experiments in nonhematopoietic cells. GATA-1 and CBP also coimmunoprecipitate from nuclear extracts of erythroid cells. Interaction mapping pinpoints contact sites to the zinc finger region of GATA-1 and to the E1A-binding region of CBP. Expression of a conditional form of adenovirus E1A in murine erythroleukemia cells blocks differentiation and expression of endogenous GATA-1 target genes, whereas mutant forms of E1A unable to bind CBP/p300 have no effect. Our findings add GATA-1, and very likely other members of the GATA family, to the growing list of molecules implicated in the complex regulatory network surrounding CBP/p300.

Estrogen-induced apoptosis by inhibition of the erythroid transcription factor GATA-1.

Steroid hormones regulate diverse biological functions, including programmed cell death (apoptosis). Although steroid receptors have been studied extensively, relatively little is known regarding the cellular targets through which apoptosis is triggered. We show here that the ligand-activated estrogen receptor (ER) induces apoptosis in an erythroid cell line by binding to, and consequently inhibiting the activity of, GATA-1, an erythroid transcription factor essential for the survival and maturation of erythroid precursor cells. GATA-1 inhibition is reflected in the downregulation of presumptive GATA-1 target genes. Constitutive overexpression of a GATA-binding protein resistant to the effects of the ER partially rescues ER-induced apoptosis. Induction of apoptosis by a mutant ER defective in binding to the estrogen response element but active in GATA-1 inhibition suggests that ER-mediated inhibition of GATA-1 is direct and does not require estrogen response element-dependent transcriptional activation. Thus, a lineage-restricted transcription factor, such as GATA-1, constitutes one cellular target through which steroid hormones may control apoptosis. As GATA-binding proteins are evolutionarily conserved, we speculate that members of the steroid receptor family may exert some of their diverse biological functions in different cellular contexts through interference with the function of GATA-binding proteins.

Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

Rescue of GATA-1-deficient embryonic stem cells by heterologous GATA-binding proteins.

Totipotent murine embryonic stem (ES) cells can be differentiated in vitro to form embryoid bodies (EBs) containing hematopoietic cells of multiple lineages, including erythroid cells. In vitro erythroid development parallels that which is observed in vivo. ES cells in which the gene for the erythroid transcription factor GATA-1 has been disrupted fail to produce mature erythroid cells either in vivo or in vitro. With the EB in vitro differentiation assay, constructs expressing heterologous GATA-binding proteins were tested for their abilities to correct the developmental defect of GATA-1-deficient ES cells. The results presented here show that the highly divergent chicken GATA-1 can rescue GATA-1 deficiency to an extent similar to that of murine GATA-1 (mGATA-1), as determined by size and morphology of EBs, presence of red cells, and globin gene expression. Furthermore, GATA-3 and GATA-4, which are normally expressed in different tissues, and a protein consisting of the zinc fingers of GATA-1 fused to the herpes simplex virus VP16 transcription activation domain were able to compensate for the GATA-1 defect. Chimeric molecules in which both zinc fingers of mGATA-1 were replaced with the zinc fingers of human GATA-3 or with the single finger of the fungal GATA factor areA, as well as a construct bearing the zinc finger region alone, displayed rescue activity. These results suggest that neither the transcription activation domains of mGATA-1 nor its zinc fingers impart erythroid cell specificity for its action in vivo. Rather, it appears that specificity is mediated through the cis-acting control regions which determine spatial and temporal expression of the GATA-1 gene. Furthermore, our results demonstrate that the zinc finger region may have a biological function in addition to mediating DNA binding.

The v-src inducible gene 9E3/pCEF4 is regulated by both its promoter upstream sequence and its 3' untranslated region.

The 9E3/pCEF4 mRNA is strongly induced in Rous sarcoma virus-transformed chicken embryo fibroblasts when compared to untransformed cells. To identify cis-acting transcriptional elements that confer inducibility by v-src, we isolated the 9E3 promoter upstream region. We found that 1.53 kilobases upstream of the transcriptional start site, when placed in front of a reporter gene, conferred a small degree of inducibility by v-src, in both transient and stable transfections. Two potential AP-1 sites were identified in the 9E3 promoter. AP-1 elements have been implicated previously in mediating a transcriptional response to v-src in fibroblast cell lines. These elements alone do not confer a significant inducibility by v-src in primary chicken embryo fibroblasts. Since the 9E3 mRNA is stabilized in transformed cells, we replaced the 3' untranslated region of the reporter gene with the 9E3 3' untranslated region and found this construct to be strongly responsive to stimulation by v-src. In addition, the 9E3 3' untranslated region increased the response to serum and the tumor promoter phorbol 12-myristate 13-acetate. This suggests that a posttranscriptional mechanism plays a major role in the induction of 9E3 expression.

The intermediate filament cytoskeleton of malignant mesotheliomas and its diagnostic significance.

The intermediate filament cytoskeleton of epithelial, biphasic, and fibrous malignant pleural mesotheliomas was studied by immunohistochemistry and gel electrophoresis. The results were compared with data similarly obtained from lung adenocarcinomas. All mesotheliomas immunostained with various monoclonal and polyclonal antibodies against cytokeratins. By double immunofluorescence microscopy, coexpression of cytokeratins and vimentin was found in the fusiform cells of biphasic and fibrous mesotheliomas. As determined by two-dimensional gel electrophoresis, lung adenocarcinomas exclusively expressed Cytokeratins 7, 8, 18, and 19, and the same polypeptides were found in the fibrous mesotheliomas. These four cytokeratins were also found in the epithelial and biphasic mesotheliomas, most of which, however, also expressed, additional cytokeratins, such as the basic Polypeptide 5 and, in some cases, Cytokeratins 4, 6, 14, and 17. The results demonstrate the epithelial nature of all types of malignant mesotheliomas and thus justify their classification as carcinomas. When epithelial morphology is evident, the pattern of cytokeratin expression is usually more complex, as indicated by the synthesis, in addition to the "simple epithelial" pattern (7, 8, 18, and 19), of certain cytokeratin polypeptides which hitherto have been presumed to be typical of stratified epithelia. This cytokeratin complexity and the coexpression of vimentin and cytokeratins in certain forms of mesotheliomas indicate that these tumors are a clearly distinct and complex group of carcinomas. Their special cytoskeletal filament protein expression should prove useful in differentiating mesotheliomas from other carcinomas, particularly from adenocarcinomas growing in the lung.

[Neuroendocrine larynx cancer of the small cell (oat cell) type. Morphologic and immunohistochemical findings and their significance for therapy]. / Das neuro-endokrine Larynxkarzinom vom kleinzelligen (Oat-Cell) Typ. Morphologische und immunhistochemische Befunde und ihre Bedeutung für Therapie.

By combining immunohistochemical, biochemical, and light and electron microscopy techniques, the following original observations concerning the rare and highly malignant small cell ("oat cell") carcinoma of the larynx were made: The typical electron microscopic feature was the presence of neurosecretory granules in some cells. This indicates that this tumour was a carcinoma with endocrinal differentiation. The demonstration of cytokeratins as major cytoskeletal components, as well as the presence of desmosomes, confirmed the epithelial origin of the tumour cells. The detection of neuroendocrinal markers such as calcitonin and neuronspecific enolase (NSE) in some of the tumour cells, as well as the presence of neurosecretory granules allow us to classify this tumour as a neuroendocrinal carcinoma of the small-cell type. This is in accordance with the latest classification of neuroendocrine carcinomas of the lung. The rapid clinical evolution of the disease is typical for patients with anaplastic carcinomas. Laryngectomy followed by adjuvant chemotherapy and radiotherapy is indicated only in cases without metastases. The detection of regional metastases suggests haematogenous spread with distinct metastases, restricting treatment to chemotherapy and radiotherapy.

Coexpression of neuroendocrine markers and epithelial cytoskeletal proteins in bronchopulmonary neuroendocrine neoplasms.

Neuroendocrine (NE) neoplasms of the human bronchopulmonary tract were examined by electron microscopy, immunocytochemistry, and gel electrophoresis of cytoskeletal proteins from microdissected tissue samples. All samples (carcinoids, well-differentiated NE carcinoma, NE carcinomas of intermediate type, NE carcinomas of the small cell type) contained significant numbers of cells that immunostained for one or more of the following neuroendocrine markers tested: bombesin, calcitonin, ACTH, leu-enkephalin, gastrin, serotonin, somatostatin, alpha-melanocyte-stimulating hormone, vasoactive intestinal peptide, glucagon, insulin, substance P, and neuron-specific enolase. Electron microscopy revealed typical NE cell features, including variable abundant and frequently heterogeneous neurosecretory granules. Tumor cells contained filaments specifically stained with different conventional and monoclonal antibodies to cytokeratins and displayed punctate plasma membrane staining with antibodies to desmoplakins, in agreement with the electron microscopic demonstration of tonofilament bundles and desmosomes. Immunocytochemistry for NE markers and cytoskeletal proteins on consecutive sections revealed both cytokeratins and neuroendocrine substances in single cells. Using gel electrophoresis of cytoskeletal proteins of tissue regions extracted with high salt buffer and detergent, we could detect, in the tumors tested, appreciable amounts of cytokeratin polypeptides 8, 18, and 19, i.e., major cytokeratins also found in certain other lung carcinomas such as adenocarcinomas. Tumor cells were not significantly stained with antibodies to other intermediate filament proteins such as vimentin, desmin, glial filament protein, and neurofilament protein. The results show that NE substances can be synthesized in cells containing a typical epithelial cytoskeleton, i.e., cytokeratin filaments and desmosomes. These findings support the notion of an epithelial character of these tumors and appear in contrast with recent reports that neurofilaments are the only type of intermediate filaments present in carcinoids and other pulmonary NE tumors. These observations may have important implications for the histogenesis of NE carcinomas and for diagnostic pathology.

Cytokeratins in normal lung and lung carcinomas. I. Adenocarcinomas, squamous cell carcinomas and cultured cell lines.

The various epithelial cells of the lower respiratory tract and the carcinomas derived from them differ markedly in their differentiation characteristics. Using immunofluorescence microscopy and two-dimensional gel electrophoresis of cytoskeletal proteins from microdissected tissues we have considered whether cytokeratin polypeptides can serve as markers of cell differentiation in epithelia from various parts of the human and bovine lower respiratory tract. In addition , we have compared these protein patterns with those found in the two commonest types of human lung carcinoma and in several cultured lung carcinoma cell lines. By immunofluorescence microscopy, broad spectrum antibodies to cytokeratins stain all epithelial cells of the respiratory tract, including basal, ciliated, goblet, and alveolar cells as well as all tumor cells of adenocarcinomas and squamous cell carcinomas. However, in contrast, selective cytokeratin antibodies reveal cell type-related differences. Basal cells of the bronchial epithelium react with antibodies raised against a specific epidermal keratin polypeptide but not with antibodies derived from cytokeratins characteristic of simple epithelia. When examined by two-dimensional gel electrophoresis, the alveolar cells of human lung show cytokeratin polypeptides typical of simple epithelia (nos. 7, 8, 18 and 19) whereas the bronchial epithelium expresses, in addition, basic cytokeratins (no. 5, small amounts of no. 6) as well as the acidic polypeptides nos. 15 and 17. Bovine alveolar cells also differ from cells of the tracheal epithelium by the absence of a basic cytokeratin polypeptide. All adenocarcinomas of the lung reveal a "simple-epithelium-type" cytokeratin pattern (nos. 7, 8, 18 and 19). In contrast, squamous cell carcinomas of the lung contain an unusual complexity of cytokeratins. We have consistently found polypeptides nos. 5, 6, 8, 13, 17, 18 and 19 and, in some cases, variable amounts of cytokeratins nos. 4, 14 and 15. Several established cell lines derived from human lung carcinomas (SK-LU-1, Calu -1, SK-MES-1 and A-549) show a uniform pattern of cytokeratin polypeptides (nos. 7, 8, 18 and 19), similar to that found in adenocarcinomas. In addition, vimentin filaments are produced in all the cell lines examined, except for SK-LU-1.(ABSTRACT TRUNCATED AT 400 WORDS)