PU.1 is regulated by NF-kappaB through a novel binding site in a 17 kb upstream enhancer element

The majority of patients with acute myeloid leukemia (AML) still die of their disease, and novel therapeutic concepts are needed. Timely expression of the hematopoietic master regulator PU.1 is crucial for normal development of myeloid and lymphoid cells. Targeted disruption of an upstream regulatory element (URE) located several kb upstream in the PU.1 promoter decreases PU.1 expression thereby inducing AML in mice. In addition, suppression of PU.1 has been observed in specific subtypes of human AML. Here, we identified nuclear factor-kappaB (NF-kappaB) to activate PU.1 expression through a novel site within the URE. We found sequence variations of this particular NF-kappaB site in 4 of 120 AML patients. These variant NF-kappaB sequences failed to mediate activation of PU.1. Moreover, the synergistic activation of PU.1 together with CEBPB through these variant sequences was also lost. Finally, AML patients with such variant sequences had suppressed PU.1 mRNA expression. This study suggests that changes of a single base pair in a distal element critically affect the regulation of the tumor suppressor gene PU.1 thereby contributing to the development of AML.

Regulation of wingless-type MMTV integration site family (WNT) signalling in pancreatic islets from wild-type and obese mice

TCF7L2 is a type 2 diabetes susceptibility gene and downstream effector of canonical wingless-type MMTV integration site family (WNT) signalling. However, it is unknown whether this pathway is active in adult pancreatic islets in vivo, and whether it is regulated in obesity.

Macular pigment density at the site of altered fundus autofluorescence

The purpose of our study was to determine whether abnormalities of increased or decreased fundus autofluorescence (FAF) are associated with local changes in macular pigment (MP) optical density in patients with age-related maculopathy (ARM) and macular degeneration (ARMD).

Effect of site of lactate infusion on regional lactate exchange in pigs

The rate of extra-hepatic lactate production and the route of influx of lactate to the liver may influence both hepatic and extra-hepatic lactate exchange. We assessed the dose-response of hepatic and extra-hepatic lactate exchange during portal and central venous lactate infusion.

Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin

Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion. We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors. The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell adhesion on the implant surface, thereby accelerating osseointegration of the implant.

An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein

The phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are modulated by a balance of kinase and phosphatase activities. Here we characterize the association of Rb with the catalytic subunit of protein phosphatase 1 (PP1c). A crystal structure identifies an enzyme docking site in the Rb C-terminal domain that is required for efficient PP1c activity toward Rb. The phosphatase docking site overlaps with the known docking site for cyclin-dependent kinase (Cdk), and PP1 competition with Cdk-cyclins for Rb binding is sufficient to retain Rb activity and block cell-cycle advancement. These results provide the first detailed molecular insights into Rb activation and establish a novel mechanism for Rb regulation in which kinase and phosphatase compete for substrate docking.