A myostatin and activin decoy receptor enhances bone formation in mice.

Myostatin is a member of the bone morphogenetic protein/transforming growth factor-ß (BMP/TGFß) super-family of secreted differentiation factors. Myostatin is a negative regulator of muscle mass as shown by increased muscle mass in myostatin deficient mice. Interestingly, these mice also exhibit increased bone mass suggesting that myostatin may also play a role in regulating bone mass. To investigate the role of myostatin in bone, young adult mice were administered with either a myostatin neutralizing antibody (Mstn-mAb), a soluble myostatin decoy receptor (ActRIIB-Fc) or vehicle. While both myostatin inhibitors increased muscle mass, only ActRIIB-Fc increased bone mass. Bone volume fraction (BV/TV), as determined by microCT, was increased by 132% and 27% in the distal femur and lumbar vertebrae, respectively. Histological evaluation demonstrated that increased BV/TV in both locations was attributed to increased trabecular thickness, trabecular number and bone formation rate. Increased BV/TV resulted in enhanced vertebral maximum compressive force compared to untreated animals. The fact that ActRIIB-Fc, but not Mstn-mAb, increased bone volume suggested that this soluble decoy receptor may be binding a ligand other than myostatin, that plays a role in regulating bone mass. This was confirmed by the significant increase in BV/TV in myostatin deficient mice treated with ActRIIB-Fc. Of the other known ActRIIB-Fc ligands, BMP3 has been identified as a negative regulator of bone mass. However, BMP3 deficient mice treated with ActRIIB-Fc showed similar increases in BV/TV as wild type (WT) littermates treated with ActRIIB-Fc. This result suggests that BMP3 neutralization is not the mechanism responsible for increased bone mass. The results of this study demonstrate that ActRIIB-Fc increases both muscle and bone mass in mice. Therefore, a therapeutic that has this dual activity represents a potential approach for the treatment of frailty.

Bone biomechanical properties in LRP5 mutant mice.

The mutation responsible for the high bone mass (HBM) phenotype has been postulated to act through the adaptive response of bone to mechanical load resulting in denser and stronger skeletons in humans and animals. The bone phenotype of members of a HBM family is characterized by normally shaped bones that are exceptionally dense, particularly at load bearing sites [Cancer Res. 59 (1999) 1572]. The high bone mass (HBM) mutation was identified as a glycine to valine substitution at amino acid residue 171 in the gene coding for low-density lipoprotein receptor-related protein 5 (LRP5) [Bone Miner. Res. 16(4) (2001) 758]. Thus, efforts have focused on the examination of the role of LRP5 and the G171V mutation in bone mechanotransduction responses [J. Bone Miner. Res 18 (2002) 960]. Transgenic mice expressing the human G171V mutation have been shown to have skeletal phenotypes remarkably similar to those seen in affected individuals. In this study, we have identified differences in biomechanical (structural and apparent material) properties, bone mass/ash, and bone stiffness of cortical and cancellous bone driven by the G171V mutation in LRP5. As in humans, the LRP5 G171V plays an important role in regulating bone structural phenotypes in mice. These bone phenotypes include greater structural and apparent material properties in HBM HET as compared to non-transgenic littermates (NTG) mice. Body size and weight in HBM HET were similar to that in NTG control mice. However, the LRP5 G171V mutation in HET mice results in a skeleton that has greater structural (femoral shaft, femoral neck, tibiae, vertebral body) and apparent material (vertebral body) strength, percent bone ash weight (ulnae), and tibial stiffness. Despite similar body weight to NTG mice, the denser and stiffer bones in G171V mice may represent greater bone formation sensitivity to normal mechanical stimuli resulting in an overadaptation of skeleton to weight-related forces.

Herpes simplex virus transcriptional activator VP16 is detrimental to preimplantation development in mice.

The herpes simplex virus transactivator protein VP16 is frequently used to regulate gene expression in several experimental systems, including transgenic mice. It has been suggested that high levels of VP16 expression in mice may be lethal. In order to systematically address this issue, we linked the VP16 gene to promoters that are active early and in a variety of tissues throughout development, such as the human beta-actin promoter or the rat nestin gene enhancer. VP16 expression was assayed using a LacZ reporter gene linked to a VP16-responsive immediate early gene promoter. We show here that expression of VP16 at high levels is detrimental to pre-implantation development. By culturing embryos in vitro, we demonstrate that this effect is exerted at the transition from the 2-cell to the 4-cell stage, reducing survival to the blastocyst stage dramatically. On the other hand, transgenic mice expressing VP16 transgenes at postimplantation stages are viable. These results suggest a differential sensitivity to VP16 expression in different cell types and stages of development. The reduction of embryo survival by VP16 implicates herpes virus infection as a potential cause of infertility.

Heterogeneity of neural progenitor cells revealed by enhancers in the nestin gene.

Using transgenic embryos, we have identified two distinct CNS progenitor cell-specific enhancers, each requiring the cooperation of at least two independent regulatory sites, within the second intron of the rat nestin gene. One enhancer is active throughout the developing CNS, while the other is specifically active in the ventral midbrain. These experiments demonstrate that neural progenitor cells in the midbrain constitute a unique subpopulation based upon their ability to activate the midbrain regulatory element. Our finding of differential enhancer activity from a gene encoding a structural protein reveals a previously unrecognized diversity in neural progenitor cell populations.

Transgenic analyses reveal developmentally regulated neuron- and muscle-specific elements in the murine neurofilament light chain gene promoter.

We report here the developmental activity of regulatory elements that reside within 1.7 kilobases of the murine neurofilament light chain (NF-L) gene promoter. NF-L promoter activity is first detected at embryonic day 8.5 in neuroepithelial cells. Neuron-specific gene expression is maintained in the spinal cord until embryonic day 12.5 and at later developmental stages in the brain and sensory neuroepithelia. After day 14.5, the promoter becomes active in myogenic cells. Transgene expression in both neurons and muscle is consistent with the detection of endogenous NF-L transcript in both neuronal and myogenic tissues of neonates by reverse transcriptase-polymerase chain reaction. Neuron- and muscle-specific activities of the NF-L promoter decrease and are nearly undetectable after birth. Thus, the 1.7-kilobase NF-L promoter contains regulatory elements for initiation but not maintenance of transcription from the NF-L locus. Deletion analyses reveal that independent regulatory elements control the observed tissue-specific activities and implicate a potential MyoD binding site as the muscle-specific enhancer. Our results demonstrate that the NF-L promoter contains distinct regulatory elements for both neuron- and muscle-specific gene expression and that these activities are temporally separated during embryogenesis.

The asparaginyl-tRNA synthetase gene encodes one of the complementing factors for thermosensitive translation in the Escherichia coli mutant strain, N4316.

Escherichia coli strain N4316 is a mutant that exhibits temperature-sensitive growth at 43 degrees C and temperature-sensitive translation in vivo and in vitro. Extracts of the mutant produce an aberrant pattern of translation products of MS2 bacteriophage RNA. Previous work has shown that a protein, called 'rescue', isolated from the parental strain partly corrects the defective translation in vitro. Here we report the purification to homogeneity of a second factor from ribosomal eluates of the wild-type parental strain; the purified protein is a homodimer of 54 kDa. The partial sequence of the second protein was determined, and a recombinant plasmid was isolated based on its ability to complement the temperature-sensitive growth phenotype of the mutant at the non-permissive temperatures. The cloned gene was sequenced, mapped to the 20.9-min region of the E. coli chromosome and shown to code for a 466-amino-acid protein with a molecular mass of 52 kDa. Analysis of the DNA sequence and the correspondence to that of the partial protein sequence has identified the complementing factor as asparaginyl-tRNA synthetase. Marker rescue experiments indicate that the asnS mutation in N4316 resides within the motif 2 domain of the synthetase. A potential role of this synthetase in restoring normal protein synthesis with respect to ribosomal frameshifting, read-through of nonsense codons and protein copy number is discussed.