Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy.

Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including Gsα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of Gsα in the osteoblast lineage.

Loss of Gsα early in the osteoblast lineage favors adipogenic differentiation of mesenchymal progenitors and committed osteoblast precursors.

In humans, aging and glucocorticoid treatment are associated with reduced bone mass and increased marrow adiposity, suggesting that the differentiation of osteoblasts and adipocytes may be coordinately regulated. Within the bone marrow, both osteoblasts and adipocytes are derived from mesenchymal progenitor cells, but the mechanisms guiding the commitment of mesenchymal progenitors into osteoblast versus adipocyte lineages are not fully defined. The heterotrimeric G protein subunit Gs α activates protein kinase A signaling downstream of several G protein-coupled receptors including the parathyroid hormone receptor, and plays a crucial role in regulating bone mass.

Gsα enhances commitment of mesenchymal progenitors to the osteoblast lineage but restrains osteoblast differentiation in mice.

The heterotrimeric G protein subunit Gsα stimulates cAMP-dependent signaling downstream of G protein-coupled receptors. In this study, we set out to determine the role of Gsα signaling in cells of the early osteoblast lineage in vivo by conditionally deleting Gsα from osterix-expressing cells. This led to severe osteoporosis with fractures at birth, a phenotype that was found to be the consequence of impaired bone formation rather than increased resorption.

Cross-talk between NR4A orphan nuclear receptors and β-catenin signaling pathway in osteoblasts.

The canonical Wnt signaling pathway and its key mediator β-catenin are important regulators of osteoblast function. NR4A orphan nuclear receptors (Nurr1, NGFI-B, and Nor1) are expressed in osteoblasts and have been shown to regulate the expression of osteoblastic genes and osteoblastic differentiation. Recently, interplay between Nurr1 and the canonical Wnt signaling pathway was reported in 293F cells.

Transcriptional activity of estrogen-related receptor γ (ERRγ) is stimulated by the phytoestrogen equol.

Estrogen-related receptor γ (ERRγ) is an orphan nuclear receptor lacking identified natural ligands. The synthetic estrogen receptor ligands 4-hydroxytamoxifen and diethylstilbestrol have, however, been shown to bind to and abolish the constitutive transcriptional activity of ERRγ. Certain phytoestrogens were recently reported to act as agonists of the related ERRα.

ERRalpha regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells.

The orphan nuclear receptor estrogen-related receptor-alpha (ERRalpha) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERRalpha in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERRalpha deficient mice and their differentiation capacities were compared to that of the wild-type cells.

FGF-8 stimulates the expression of NR4A orphan nuclear receptors in osteoblasts.

Nurr1, NGFI-B, and Nor1 form the NR4A subfamily of orphan nuclear receptors. The NR4A receptors are immediate early genes that can be rapidly induced in response to a variety of stimuli in many cell types, for example, in osteoblasts. Nurr1 regulates the differentiation of osteoblasts and the expression of several osteoblastic genes.

Corepressor interaction differentiates the permissive and non-permissive retinoid X receptor heterodimers.

Nurr1 is an orphan nuclear receptor regulating transcription both as a monomer and as a heterodimer with retinoid X receptor (RXR). RXR-Nurr1 heterodimers are permissive RXR heterodimers as they activate transcription in response to RXR ligands. In contrast, heterodimers formed by RXR and retinoic acid receptor (RAR) are non-permissive as they activate transcription only upon RAR ligand binding.

Cross-talk between the NR3B and NR4A families of orphan nuclear receptors.

Estrogen-related receptors (NR3B family) and Nurr1, NGFI-B, and Nor1 (NR4A family) are orphan nuclear receptors lacking identified natural ligands. The mechanisms regulating their transcriptional activities have remained elusive. We have previously observed that the members of NR3B and NR4A families are coexpressed in certain cell types such as osteoblasts and that the ability of Nurr1 to transactivate the osteopontin promoter is repressed by ERRs.

Anti-clarin-1 AAV-delivered ribozyme induced apoptosis in the mouse cochlea.

Usher syndrome type 3 is caused by mutations in the USH3A gene, which encodes the protein clarin-1. Clarin-1 is a member of the tetraspanin superfamily (TM4SF) of transmembrane proteins, expressed in the organ of Corti and spiral ganglion cells of the mouse ear. We have examined whether the AAV-mediated anti-clarin ribozyme delivery causes apoptotic cell death in vivo in the organ of Corti.

Efficacy of gene transfer through the round window membrane: an in vitro model.

The viral vector-transgene soaked gelatin-sponge method has been shown to be successful in mediating transgene expression across an intact round window membrane (RWM) in mouse in vivo. However, there are many confounding factors which make it difficult to evaluate the role of the RWM in gene transfer. We have created an in vitro model to test the feasibility of gene delivery through an intact RWM.

Requirements for transcriptional regulation by the orphan nuclear receptor ERRgamma.

Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor lacking identified natural ligands. We have addressed the requirements for ERRgamma-mediated gene regulation. ERRgamma transactivates constitutively reporter genes driven by ERR response elements (ERREs) or estrogen response elements (EREs).

Regulation of the osteopontin gene by the orphan nuclear receptor NURR1 in osteoblasts.

The orphan nuclear receptor Nurr1 is mainly expressed in the central nervous system but is also detected in certain peripheral tissues such as bone. To elucidate the role of Nurr1 in bone, we examined the ability of Nurr1 to regulate osteopontin (OPN) expression in osteoblastic cell lines. Transfection of Nurr1 in osteoblastic cells increased OPN mRNA expression.

Dimerization modulates the activity of the orphan nuclear receptor ERRgamma.

Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor lacking identified natural ligands. However, 4-hydroxytamoxifen and diethylstilbestrol were recently shown to bind to and inhibit ERRgamma activity. ERR activates transcription constitutively as a monomer.

Defining an N-terminal activation domain of the orphan nuclear receptor Nurr1.

Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons. Activation of Nurr1 depends on two so-called activation functions (AFs) situated in the N- and C-terminal regions, respectively. The region important for activation within the C-terminal domain has been shown to promote activation in a highly cell-type specific fashion in the absence of added exogenous ligands.

Nurr1 regulates dopamine synthesis and storage in MN9D dopamine cells.

Nurr1, a transcription factor belonging to the nuclear receptor family, is essential for the generation of midbrain dopamine (DA) cells during embryonic development. Nurr1 continues to be expressed in adult DA neurons but the role for Nurr1 in inducing and regulating basic dopaminergic functions such as dopamine synthesis and storage has remained unknown. We have previously used MN9D dopamine cells to analyze the role of Nurr1 and retinoids in DA cell maturation.

Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors.

Members of the nuclear receptor (NR) superfamily of transcription factors modulate gene transcription in response to small lipophilic molecules. Transcriptional activity is regulated by ligands binding to the carboxy-terminal ligand-binding domains (LBDs) of cognate NRs. A subgroup of NRs referred to as 'orphan receptors' lack identified ligands, however, raising issues about the function of their LBDs.

Defining requirements for heterodimerization between the retinoid X receptor and the orphan nuclear receptor Nurr1.

Nurr1, an orphan nuclear receptor mainly expressed in the central nervous system, is essential for the development of the midbrain dopaminergic neurons. Nurr1 binds DNA as a monomer and exhibits constitutive transcriptional activity. Nurr1 can also regulate transcription as a heterodimer with the retinoid X receptor (RXR) and activate transcription in response to RXR ligands.

Induction of cell cycle arrest and morphological differentiation by Nurr1 and retinoids in dopamine MN9D cells.

Dopamine cells are generated in the ventral midbrain during embryonic development. The progressive degeneration of these cells in patients with Parkinson's disease, and the potential therapeutic benefit by transplantation of in vitro generated dopamine cells, has triggered intense interest in understanding the process whereby these cells develop. Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons.

Coregulator small nuclear RING finger protein (SNURF) enhances Sp1- and steroid receptor-mediated transcription by different mechanisms.

The small nuclear RING finger protein SNURF is not only a coactivator in steroid receptor-dependent transcription but also activates transcription from steroid-independent promoters. In this work, we show that SNURF, via the RING finger domain, enhances protein binding to Sp1 elements/GC boxes and interacts and cooperates with Sp1 in transcriptional activation. The activation of androgen receptor (AR) function requires regions other than the RING finger of SNURF, and SNURF does not influence binding of AR to cognate DNA elements.

Transcription activating and repressing functions of the androgen receptor are differentially influenced by mutations in the deoxyribonucleic acid-binding domain.

Despite the wide spectrum of androgen receptor (AR) mutants described in androgen insensitivity syndromes (AIS), their influence on transactivating and, in particular, transrepressing functions of AR are poorly defined. Rat AR mutants with substitutions in the DNA-binding domain, corresponding to several mutations in AIS patients, were examined for these activities. AR variants (G551V and C562G) with mutations in the first zinc finger (ZF) exhibited reduced DNA binding activity and attenuated transactivation.

Ubc9 interacts with the androgen receptor and activates receptor-dependent transcription.

Ubc9, a homologue of the class E2 ubiquitin-conjugating enzymes, has recently been shown to catalyze conjugation of a small ubiquitin-like molecule-1 (SUMO-1) to a variety of target proteins. SUMO-1 modifications have been implicated in the targeting of proteins to the nuclear envelope and certain intranuclear structures and in converting proteins resistant to ubiquitin-mediated degradation. In the present work, we find that Ubc9 interacts with the androgen receptor (AR), a member of the steroid receptor family of ligand-activated transcription factors.

CREB-binding protein in androgen receptor-mediated signaling.

CREB-binding protein (CBP) is a transcriptional coregulator that interacts with different DNA binding proteins and components of the general transcription machinery. CBP enhanced androgen receptor (AR)-dependent transcription under transient transfection conditions in CV-1 cells. The ligand binding domain (LBD) and residues 38-296 of the N-terminal region of AR are not required because the activity of a receptor mutant devoid of these domains was augmented by coexpressed CBP.