p300 is a multifunctional transcriptional coactivator that serves as an adapter for several transcription factors including nuclear steroid hormone receptors. p300 possesses an intrinsic histone acetyltransferase (HAT) activity that may be critical for promoting steroid-dependent transcriptional activation. In osteoblastic cells, transcription of the bone-specific osteocalcin (OC) gene is principally regulated by the Runx2/Cbfa1 transcription factor and is stimulated in response to vitamin D 3 via the vitamin D 3 receptor complex. Therefore, we addressed p300 control of basal and vitamin D 3 -enhanced activity of the OC promoter. We find that transient overexpression of p300 results in a significant dose-dependent increase of both basal and vitamin D 3 -stimulated OC gene activity. This stimulatory effect requires intact Runx2/Cbfa1 binding sites and the vitamin D-responsive element. In addition, by coimmunoprecipitation, we show that the endogenous Runx2/ Cbfa1 and p300 proteins are components of the same complexes within osteoblastic cells under physiological concentrations. We also demonstrate by chromatin immunoprecipitation assays that p300, Runx2/Cbfa1, and 1␣,25-dihydroxyvitamin D 3 receptor interact with the OC promoter in intact osteoblastic cells expressing this gene. The effect of p300 on the OC promoter is independent of its intrinsic HAT activity, as a HAT-deficient p300 mutant protein up-regulates expression and cooperates with P/CAF to the same extent as the wild-type p300. On the basis of these results, we propose that p300 interacts with key transcriptional regulators of the OC gene and bridges distal and proximal OC promoter sequences to facilitate responsiveness to vitamin D 3 .The rat osteocalcin (OC) gene encodes a 10-kDa bonespecific protein that is induced in osteoblasts with the onset of mineralization at late stages of differentiation (26). Transcription of the OC gene is controlled by modularly distributed basal and hormone-responsive elements, located within two DNase I-hypersensitive sites (distal site, positions Ϫ600 to Ϫ400; proximal site, positions Ϫ170 to Ϫ70) that are present only in bone-derived cells expressing this gene (23,24). Thus, chromatin remodeling of the OC gene promoter accompanies the onset in OC gene expression during osteoblast differentiation (Fig. 1). A key regulatory element that controls OC gene expression is recognized by the 1␣,25-dihydroxyvitamin D 3 receptor (VDR) complex upon ligand activation. This vitamin D 3 -responsive element (VDRE) is located in the distal region (Fig. 1) of the OC promoter (positions Ϫ465 to Ϫ437) and functions as an enhancer to increase OC gene transcription by three-to fivefold (20). Binding of the ligand 1␣,25-dihydroxyvitamin D 3 (vitamin D 3 ) induces conformational changes in the receptor that enable it to interact with several coactivators, such as NCoA-1/SRC-1 (nuclear receptor coactivator 1/steroid receptor coactivator 1), NCoA-2/GRIP/TIF2 (nuclear receptor coactivator 2/glucocorticoid receptor-interacting protein/transcrip...

No abstract

Tissue-specific activation of the osteocalcin (OC) gene is associated with changes in chromatin structure at the promoter region. Two nuclease-hypersensitive sites span the key regulatory elements that control basal tissue-specific and vitamin D 3 -enhanced OC gene transcription. To gain understanding of the molecular mechanisms involved in chromatin remodeling of the OC gene, we have examined the requirement for SWI/SNF activity. We inducibly expressed an ATPase-defective BRG1 catalytic subunit that forms inactive SWI/SNF complexes that bind to the OC promoter. This interaction results in inhibition of both basal and vitamin D 3 -enhanced OC gene transcription and a marked decrease in nuclease hypersensitivity. We find that SWI/SNF is recruited to the OC promoter via the transcription factor CCAAT/enhancer-binding protein ␤, which together with Runx2 forms a stable complex to facilitate RNA polymerase II binding and activation of OC gene transcription. Together, our results indicate that the SWI/SNF complex is a key regulator of the chromatin-remodeling events that promote tissue-specific transcription in osteoblasts.Within the eukaryotic nucleus, the packaging of DNA into nucleosomes and higher order chromatin structures have been implicated in the regulation of key cellular events, such as replication and transcription. During the last decade, a large family of protein complexes that promote transcription by altering chromatin structure have been described (1-3). Among them is the SWI/SNF complex subfamily that remodels chromatin in an ATP-dependent manner (1-3). SWI/SNF complexes are composed of several subunits, which have been implicated in a wide range of cellular events, including gene regulation, cell cycle control, development, and differentiation (1, 3). The mammalian SWI/SNF complexes contain a catalytic subunit that can be either BRG1 or BRM, which includes ATPase activity. Mutations in the ATPase domain of BRG1 or BRM that abrogate the ability of these proteins to bind ATP result in the formation of inactive SWI/SNF complexes (4 -6). Furthermore, expression of mutant BRG1 or BRM proteins in NIH3T3 cells impairs the ability of these cells to activate endogenous stress response genes in the presence of arsenite (5) and to differentiate into muscle or adipocytic cells (4, 5, 7). In addition, we have recently shown that the presence of the mutant BRG1 protein in these NIH3T3 cell lines inhibits BMP2-induced differentiation into the osteoblast lineage (8).The rat osteocalcin (OC) 3 gene encodes a 10-kDa bone-specific protein that is expressed at late stages of osteoblast differentiation, concomitant with the mineralization of the extracellular matrix (9). Osteoblast-specific transcription of the OC gene is controlled by modularly organized basal and hormoneresponsive elements located within two DNase I-hypersensitive sites (distal site Ϫ605 to Ϫ400 and proximal site Ϫ170 to Ϫ70; see Fig. 1) that are present only in osteoblastic cells expressing this gene (10). Thus, chromatin remodeling of the OC gene p...

Glycine receptors (GlyRs), together with GABA(A) and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein betagamma dimer (Gbetagamma). We found that the amplitude of the glycine-activated Cl- current was enhanced after application of purified Gbetagamma or after activation of a G protein-coupled receptor. Overexpression of three distinct G protein alpha subunits (Galpha), as well as the Gbetagamma scavenger peptide ct-GRK2, significantly blunted the effect of G protein activation. Single-channel recordings from isolated membrane patches showed that Gbetagamma increased the GlyR open probability (nP(o)). Our results indicate that this interaction of Gbetagamma with GlyRs regulates both motor and sensory functions in the central nervous system.

Kinetic analysis of vitamin C uptake demonstrated that different specialized cells take up ascorbic acid through sodium-vitamin C cotransporters. Recently, two different isoforms of sodium-vitamin C cotransporters (SVCT1/SLC23A1 and SVCT2/SLC23A2) have been cloned. SVCT2 was detected mainly in choroidal plexus cells and neurons; however, there is no evidence of SVCT2 expression in glial and endothelial cells of the brain. Certain brain locations, including the hippocampus and hypothalamus, consistently show higher ascorbic acid values compared with other structures within the central nervous system. However, molecular and kinetic analysis addressing the expression of SVCT transporters in cells isolated from these specific areas of the brain had not been done. The hypothalamic glial cells, or tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid with different neurons of the region. Our hypothesis postulates that SVCT2 is expressed selectively in tanycytes, where it is involved in the uptake of the reduced form of vitamin C (ascorbic acid), thereby concentrating this vitamin in the hypothalamic area. In situ hybridization and optic and ultrastructural immunocytochemistry showed that the transporter SVCT2 is highly expressed in the apical membranes of mouse hypothalamic tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of the SVCT2 isoform in these cells. The results demonstrate that tanycytes express a high-affinity transporter for vitamin C. Thus, the vitamin C uptake mechanisms present in the hypothalamic glial cells may perform a neuroprotective role concentrating vitamin C in this specific area of the brain.