Analysis of the heteromeric DNA binding protein GABP has revealed the interaction of two distinct peptide sequence motifs normally associated with proteins located in different cellular compartments. The alpha subunit of GABP contains an 85-amino acid segment related to the Ets family of DNA binding proteins. The ETS domain of GABP alpha facilitates weak binding to DNA and, together with an adjacent segment of 37 amino acids, mediates stable interaction with GABP beta. The beta subunit of GABP contains four imperfect repeats of a sequence present in several transmembrane proteins including the product of the Notch gene of Drosophila melanogaster. These amino-terminal repeats of GABP beta mediate stable interaction with GABP alpha and, when complexed with GABP alpha, directly contact DNA. These observations provide evidence for a distinct biochemical role for the 33-amino acid repeats, and suggest that they may serve as a module for the generation of specific dimerization interfaces.

Members of the transforming growth factor-␤ (TGF-␤)superfamily of growth and differentiation factors have been identified in a wide variety of organisms, ranging from invertebrates to mammals. Bone morphogenetic proteins (BMPs) constitute a subgroup of proteins belonging to the TGF-␤ superfamily. BMPs were initially identified by their ability to induce endochondral bone formation at ectopic sites, suggesting a critical role for this family in development and regeneration of the skeleton. They are also expressed at a variety of nonskeletal sites during development, suggesting possible extraskeletal roles for these proteins. We cloned a novel member of the BMP family that is expressed at high levels in the placenta and the prostate and that we have designated as prostate-derived factor (PDF). Based on cDNA sequence analysis, the predicted PDF protein contains two cysteines in addition to the seven conserved cysteines that are the hallmark of the members of the TGF-␤ superfamily. In addition, Northern blot hybridization to poly(A) ؉ RNA showed low levels of expression in the kidney and pancreas. We further characterized the expression of this member of the BMP family by in situ hybridization and immunohistochemistry. These results show high expression in the terminal villae of the placenta. The expression of the protein as visualized by immunohistochemistry shows an expression pattern identical to that of the message in the terminal villae of the placenta. In day 18 rat embryos, protein expression was also seen in the skin and in the cartilaginous tissue of developing skeleton. Orchidectomy and dihydrotestosterone treatment of rats revealed that PDF expression is regulated by androgens in the prostate. In addition, subcutaneous implantation of recombinant PDF induced cartilage formation and the early stages of endochondral bone formation. These data indicate that PDF has a functional relationship to the BMPs.

We have previously described osteoblast/osteocyte factor 45 (OF45), a novel bone-specific extracellular matrix protein, and demonstrated that its expression is tightly linked to mineralization and bone formation. In this report, we have cloned and characterized the mouse OF45 cDNA and genomic region. Mouse OF45 (also called MEPE) was similar to its rat orthologue in that its expression was increased during mineralization in osteoblast cultures and the protein was highly expressed within the osteocytes that are imbedded within bone. To further determine the role of OF45 in bone metabolism, we generated a targeted mouse line deficient in this protein. Ablation of OF45 resulted in increased bone mass. In fact, disruption of only a single allele of OF45 caused significantly increased bone mass. In addition, knockout mice were resistant to aging-associated trabecular bone loss. Cancellous bone histomorphometry revealed that the increased bone mass was the result of increased osteoblast number and osteoblast activity with unaltered osteoclast number and osteoclast surface in knockout animals. Consistent with the bone histomorphometric results, we also determined that OF45 knockout osteoblasts produced significantly more mineralized nodules in ex vivo cell cultures than did wild type osteoblasts. Osteoclastogenesis and bone resorption in ex vivo cultures was unaffected by OF45 mutation. We conclude that OF45 plays an inhibitory role in bone formation in mouse.The constant modulation of the balance between skeletal strength and mineral availability in bone is effected by competing cell types in response to physiological needs. Osteoblasts produce, organize, and mineralize bone matrix in forming bone. Osteoclasts break down matrix by forming a lytic pocket in which bone is degraded and calcium is released. The generation and activity of these cell types is tightly regulated to provide equilibrium between formation and resorption and, thereby, an appropriate balance of strength and mineral release. Under certain conditions, such as aging, postmenopausal estrogen deficiency, or some pathophysiological states, there can exist an imbalance between bone resorption and bone formation. As a result, skeletal mass and strength are compromised and osteoporotic fractures can occur in the afflicted individuals.Bone is produced by the organization and mineralization of the extracellular matrix produced by osteoblasts. The major component of the extracellular matrix of these cells is Type I collagen, which functions as a scaffold for new bone. In addition, non-collagenous matrix proteins have been identified that influence the operations of bone turnover, formation, and repair. These proteins are generally acidic and highly posttranslationally modified by phosphorylation, glycosylation, or sulfation (1).Targeted deletion of extracellular matrix genes in mice has been a useful method to determine the in vivo functions of several matrix proteins. For example, osteocalcin is an abundant gamma carboxyl glutamic acid-containing bone matrix...

The ets-related protein GABPoL interacts with the four ankyrin-type (ANK) repeats of GABP[3 to form a high-affinity DNA-binding complex that recognizes a site important for herpes simplex virus type I immediate early gene activation. To investigate the selectivity and specificity of the GABP complex, we have isolated two new ETS family members, termed ER81 and ER71. ER81 and GABP~ were present in most tissues of adult mice, whereas ER71 was restricted to testis. We have compared the DNA-binding specificities of these proteins by binding site selection. GABPc~, ER71, and ER81 recognized the common pentanucleotide DNA sequence 5'-CGGAA/T-3'. Although subtle differences were observed for nucleotide preferences flanking this pentanucleotide core, the overall similarity of the selected sequences was most striking. Given the observation that GABPa interaction with GABPB requires its intact ETS domain, we further compared the ability of GABP[3 to interact with other ETS proteins. GABP[3 did not augment the DNA-binding activity of the highly similar ETS domains of ER81, ER71, or Ets-1. Moreover, probing of total tissue extracts with radiolabeled GABP[3 demonstrated its exceedingly stringent specificity for GABP~. Given that the DNA-binding specificities of these ETS proteins are similar and that the protein-protein interactions between GABPB and GABPa are highly specific, we conclude that the protein interactions determine the target site selection by GABPot.

Streptococcus mutans is a biofilm-forming bacterium that is adapted to tolerate rapid and dramatic fluctuations in nutrient availability, carbohydrate source, and pH in its natural environment, the human oral cavity. Dissecting the pathways used to form stable biofilms and to tolerate environmental stress is central to understanding the virulence of this organism. Here, we investigated the role of the S. mutans relA gene, which codes for a guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase/hydrolase, in biofilm formation and acid tolerance. Two mutants in which relA was insertionally inactivated or replaced by an antibiotic resistance determinant were constructed. Under normal growth and stress conditions, the mutants grew slower than the wild-type strain, although the final yields were similar. The mutants, which were still able to accumulate (p)ppGpp after the induction of a stringent response, showed significant reductions in biofilm formation on microtiter plates or hydroxylapatite disks. There was no difference in the sensitivities to acid killing of the parent and relA strains grown in planktonic cultures. However, when cells were grown in biofilms, the mutants became more acid resistant and could lower the pH through glycolysis faster and to a greater extent than the wild-type strain. Differences in acid resistance were not correlated with increases in F-ATPase activity, although bacterial sugar:phosphotransferase activity was elevated in the mutants. Expression of the luxS gene was increased as much as fivefold in the relA mutants, suggesting a link between AI-2 quorum sensing and the stringent response.

The objective of this study was to examine the effects of short-term exercise training, myostatin inhibition (PF-354), and exercise+PF-354, all relative to a vehicle control, on performance and metabolic measures in 24-month-old mice. At study termination, PF-354-treated mice exhibited significantly greater muscle weights. Performance measures revealed that exercise+PF-354 increased treadmill running time and distance to exhaustion (more than twofold) and increased habitual activity. Measures of strength were not different; however, all treatment groups demonstrated more than 30% reductions in muscle fatigue. Metabolic measures showed that basal metabolic rates were higher in PF-354- and exercise+PF-354-treated mice, and exercise and exercise+PF-354 groups exhibited significantly greater insulin sensitivity. PF-354 was associated with decreased Smad3 phosphorylation and increased peroxisome proliferator-activated receptor gamma coactivator-1alpha expression and, similar to exercise, decreased MuRF-1. The data suggest that the combination of exercise training and myostatin blockade may significantly improve physical function and whole-body metabolism in older individuals.

Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2؊/؊ mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2؊/؊ mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.human mesenchymal stem cell ͉ osteoclast ͉ osteoblast P roline-rich tyrosine kinase 2 (PYK2) and focal adhesion kinase (FAK) are nonreceptor tyrosine kinases, and together they constitute the focal adhesion kinase subfamily (1). Unlike FAK, PYK2 expression is relatively restricted, with highest levels in the brain and the hematopoietic system. PYK2Ϫ/Ϫ mice have been described previously and appear normally developed (2, 3). Characterization of the immune system of PYK2Ϫ/Ϫ animals revealed the absence of marginal zone B cells along and abnormal T cell-independent type II responses (2), as well as altered macrophage morphology, adhesion, and migration (3).Although PYK2 is expressed in both bone-forming osteoblasts and bone-resorbing osteoclasts, the skeletal phenotype of PYK2Ϫ/Ϫ mice has not been described. In vitro studies pointed to a positive role for PYK2 in osteoclast maturation and bone resorption. PYK2 localizes to the podosomes of osteoclasts (4), and, upon integrin binding, cell attachment, and actin ring formation, PYK2 associates with a variety of proteins including p130 CAS (5), Src (4), Cbl (6), integrins (4), gelsolin (7), and paxillin (8). Antisense depletion of PYK2 (9), but not the expression of a kinase inactive dominant negative mutant (10), blocked osteoclast spreading and bone resorption, indicating that PYK2 catalytic activity may be dispensable. The in vitro effects of bone anabolic stimuli suggested that PYK2 might have a positive role in osteoblasts as well. Treatment of osteoblast cells with fluoroaluminate led to increased PYK2 autophosphorylation, Src association, and kinase activity (11) and was associated with increased cell attachment and spreading (12). Likewise, in an anabolic model of mechanical loading, PYK2 autophosphorylation and kinase activity were stimulated in o...

Surface plasmon resonance biosensor technology was used to directly measure the binding interactions of small molecules to the ligand-binding domain of human estrogen receptor. In a screening mode, specific ligands of the receptor were easily discerned from nonligands. In a high-resolution mode, the association and dissociation phase binding responses were shown to be reproducible and could be fit globally to a simple interaction model to extract reaction rate constants. On average, antagonist ligands (such as tamoxifen and nafoxidine) were observed to bind to the receptor with association rates that were 500-fold slower than agonists (such as estriol and ␤-estradiol). This finding is consistent with these antagonists binding to an altered conformation of the receptor. The biosensor assay also could identify subtle differences in how the same ligand interacted with two different isoforms of the receptor (␣ and ␤). The biosensor's ability to determine kinetic rate constants for small molecule͞protein interactions provides unique opportunities to understand the mechanisms associated with complex formation as well as new information to drive the optimization of drug candidates.S urface plasmon resonance (SPR) biosensor technology has advanced to the point where it is possible to measure directly small molecules interacting with immobilized macromolecular targets (1, 2). This development suggests that biosensor analysis will become an important secondary screening tool in drug discovery, confirming hits from primary screens and providing detailed kinetics for lead optimization (3, 4). To illustrate the utility of current SPR technology, the binding properties of small compounds (200-500 Da) interacting with human estrogen receptor (ER) were analyzed.Ligand binding to ER is responsible for controlling the basic biology of estrogen-sensitive tissues. Using selective agonists or antagonists to modulate this biology is the focus of significant activity in the pharmaceutical industry (5-9). To date, a ligand's binding properties for ER have mainly been determined by equilibrium binding assays that often employ radiolabeled compounds and require overnight incubations. Here, we demonstrate how optical biosensors may be used to determine both kinetic and equilibrium binding constants for compounds interacting with ER in real time without labeling either binding partner.SPR biosensor experiments require immobilizing one reactant on a surface and monitoring its binding to a second reactant in solution. An antibody-capturing method was used to study the dynamics of ER͞ligand interactions. This assay format created a chemically homogenous receptor surface and allowed us to determine rapidly the binding properties of a variety of compounds. We examined the binding of 12 compounds (shown in Fig. 1) having differing receptor activities: both estrogen and non-estrogen agonists, SERMs (selective ER modulators, which for this discussion are referred to as antagonists), and nonbinding control compounds that possess core structures sim...