Transforming growth factor-b (TGF-b) inhibits osteoblast differentiation through inhibition of the function of Runx2 (Cbfa1) by Smad3. The mechanism through which TGF-b/ Smad3 inhibits Runx2 function has not been characterized. We show that TGF-b induces histone deacetylation, primarily of histone H4, at the osteocalcin promoter, which is repressed by TGF-b, and that histone deacetylation is required for repression of Runx2 by TGF-b. This repression occurs through the action of the class IIa histone deacetylases (HDAC)4 and 5, which are recruited through interaction with Smad3 to the Smad3/Runx2 complex at the Runx2-binding DNA sequence. Accordingly, HDAC4 or 5 is required for efficient TGF-b-mediated inhibition of Runx2 function and is involved in osteoblast differentiation. Our results indicate that class IIa HDACs act as corepressors for TGF-b/Smad3-mediated transcriptional repression of Runx2 function in differentiating osteoblasts and are cell-intrinsic regulators of osteoblast differentiation.
IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions as a molecular switch for antiviral activity. IRF-3 uses an autoinhibitory mechanism to suppress its transactivation potential in uninfected cells, and virus infection induces phosphorylation and activation of IRF-3 to initiate the antiviral responses. The crystal structure of the IRF-3 transactivation domain reveals a unique autoinhibitory mechanism, whereby the IRF association domain and the flanking autoinhibitory elements condense to form a hydrophobic core. The structure suggests that phosphorylation reorganizes the autoinhibitory elements, leading to unmasking of a hydrophobic active site and realignment of the DNA binding domain for transcriptional activation. IRF-3 exhibits marked structural and surface electrostatic potential similarity to the MH2 domain of the Smad protein family and the FHA domain, suggesting a common molecular mechanism of action among this superfamily of signaling mediators.
Cell cycle exit is required for proper differentiation in most cells and is critical for normal development, tissue homeostasis, and tumor suppression. However, the mechanisms that link cell cycle exit with differentiation remain poorly understood. Here, we show that the master melanocyte differentiation factor, microphthalmia transcription factor (MITF), regulates cell cycle exit by activating the cell cycle inhibitor INK4A, a tumor suppressor that frequently is mutated in melanomas. MITF binds the INK4A promoter, activates p16Ink4a mRNA and protein expression, and induces retinoblastoma protein hypophosphorylation, thereby triggering cell cycle arrest. This activation of INK4A was required for efficient melanocyte differentiation. Interestingly, MITF was also required for maintaining INK4A expression in mature melanocytes, creating a selective pressure to escape growth inhibition by inactivating INK4A. These findings demonstrate that INK4A can be regulated by a differentiation factor, establish a mechanistic link between melanocyte differentiation and cell cycle exit, and potentially explain the tissue-specific tendency for INK4A mutations to occur in melanoma.
An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-AspTyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.Vibrio tubiashii is a marine organism that causes bacillary necrosis in larval and juvenile bivalve mollusks (24,25). The disease is characterized by a rapid onset of symptoms, such as a generalized reduction in larval motility, an increase in larval quiescence, and extensive soft tissue necrosis. The pathogen has been isolated from hard clam larvae, juvenile hard clams, and Eastern oyster spat and larvae (7,12,25). V. tubiashii has been isolated from diseased mollusks in the United States and United Kingdom and has been associated with red tides caused by Mesodinum rubrum along the northwest coast of Spain (12,23,24,25). In spite of the economic importance of V. tubiashii in the cultivation of bivalve mollusks, nothing is known about the virulence mechanisms of this pathogen. Romalde et al. (23) reported that culture supernatants of the pathogen exhibited cytotoxicity towards fathead minnow peduncle cells and mouse lung fibroblasts in tissue culture. We describe the purification and properties of a cytolysin that lyses various types of erythrocytes and Chinese hamster ovary (CHO) cells and is cytotoxic to human intestinal (Caco-2) cells and fish (Atlantic menhaden liver [AML]) cells in tissue culture.Cytolysin production and purification. Two V. tubiashii strains (ATCC 19105 and ATCC 19109) were obtained from the American Type Culture Collection (Manassas, Va). Both strains were confirmed to be V. tubiashii using biochemical tests and were stored at Ϫ70°C. The ATCC 19105 frozen culture was rapidly thawed and inoculated onto two plates containing Trypticase soy agar (BBL, Cockeysville, Md.) supplemented with 1% NaCl. The plates were incubated at 30°C for 16 to 18 h, and the bacteria were harvested in 5 ml of Casamino Acids-yeast extract broth (3% Casamino Acids, 0.4% yeast extract, 0.05% K 2 HPO 4 [pH 7.4] supplemented with 1% NaCl). A 2-liter flask containing 500 ml of Casamino Acids-yeast extract broth was inoculated with the seed culture suspension (25 optical density units at 650 nm; ca. 10 10 CFU), and the culture was incubated for 7 h at 37°C on a rotary shaker at 100 rpm. Culture supernatant fluids (stage 1) were recovered by centrifugation at 16,000 ϫ g (20 min). Disodium hydrogen phosphate and sodium chloride were dissolved in the sta...
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