Rondom 6-midden

Afscheidscollege van Prof.Dr. A. Staal uitgesproken op 1 februari 199

Modulation of vitamin D action by local factors in bone

Bone provides rigid mechanical support, protects vital organs and serves as a reservoir of ions, such as calcium and phosphate, that can be mobilized from bone. There are two types of bone: cortical bone, which is composed of densely packed, mineralized collagen laid down in layers and provides rigidity, and trabecular bone which is spongy in appearance, and also consists of mineralized collagen and provides strength and elasticity. There are also two types of bone formation: intramembranous ossification, which primarily takes place in the calvarium and starts within the connective tissue, and endochondral ossification, which takes place in long bones and starts with the formation of cartilage followed by bone formation and mineralization. The mineralized matrix is composed of collagenous and noncollagenous proteins and contains a variety of locally produced growth factors

Antagonistic effects of transforming growth factor-beta on vitamin D3 enhancement of osteocalcin and osteopontin transcription: reduced interactions of vitamin D receptor/retinoid X receptor complexes with vitamin E response elements

Osteocalcin and osteopontin are noncollagenous proteins secreted by osteoblasts and regulated by a complex interplay of systemic and locally produced factors, including growth factors and steroid hormones. We investigated the mechanism by which transforming growth factor-beta (TGF beta) inhibits 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-enhanced expression of the osteocalcin (OC) and osteopontin (OP) genes. ROS 17/2.8 cells, in which both genes are expressed, were transfected with reporter constructs driven by native (i.e. wild-type) rat OC and mouse OP promoters. TGF beta abrogated the 1,25-(OH)2D3 enhanced transcription of both the OC and OP genes. The inhibitory TGF beta response for each requires vitamin D response element (VDRE) sequences, although there are additional contributions from proximal basal regulatory elements. These transcriptional effects were further investigated for contribution of the trans-activating factors, which interact with OC and OP VDREs, involving the vitamin D receptor (VDR) and retinoid X receptor (RXR). Gel mobility shift assays show that TGF beta significantly reduces induction of the heterodimers VDR/RXR complexes in 1,25-(OH)2D3-treated ROS 17/2.8 cells. However, Western blot and ligand binding analysis reveal that TGF beta does not affect nuclear availability of the VDR. We also show that activator protein-1 activity is up-regulated by TGF beta; thus, activator protein-1 binding sites in the OC promoter may potentially contribute to inhibitory effects of TGF beta on basal transcription. Our studies demonstrate that the inhibitory action of TGF beta on the 1,25-(OH)2D3 enhancement of OC and OP transcription in osteoblastic cells results from modulations of protein-DNA interactions at the OC and OP VDRE, which cannot be accounted for by changes in VDR protein levels. As OC and OP participate in bone turnover, our results provide insight into the contributions of TGF beta and 1,25-(OH)2D3 to VDR-mediated gene regulatory mechanism operative in bone formation and/or resorption events

Molecular characterization of celtix-1, a bromodomain protein interacting with the transcription factor interferon regulatory factor 2

Transcriptional control at the G1/S-phase transition of the cell cycle requires functional interactions of multimeric promoter regulatory complexes that contain DNA binding proteins, transcriptional cofactors, and/or chromatin modifying enzymes. Transcriptional regulation of the human histone H4/n gene (FO108) is mediated by Interferon Regulatory Factor-2 (IRF-2), as well as other histone gene promoter factors. To identify proteins that interact with cell-cycle regulatory factors, we performed yeast two-hybrid analysis with IRF-2 and identified a novel human protein termed Celtix-1 which binds to IRF-2. Celtix-1 contains several phylogenetically conserved domains, including a bromodomain, which is found in a number of transcriptional cofactors. Using a panel of IRF-2 deletion mutants in yeast two-hybrid assays, we established that Celtix-1 contacts the C-terminus of IRF-2. Celtix-1 directly interacts with IRF-2 based on binding studies with glutathione S-transferase (GST)/IRF-2 fusion proteins, and immunofluorescence studies suggest that Celtix-1 and IRF-2 associate in situ. Celtix-1 is distributed throughout the nucleus in a heterodisperse pattern. A subset of Celtix-1 colocalizes with the hyperacetylated forms of histones H3 and H4, as well as with the hyperphosphorylated, transcriptionally active form of RNA polymerase II. We conclude that the bromodomain protein Celtix-1 is a novel IRF-2 interacting protein that associates with transcriptionally active chromatin in situ