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Coffin-Lowry Syndrome (CLS) is an X-linked mental retardation condition associated with skeletal abnormalities. The gene mutated in CLS, RSK2, encodes a growth factor-regulated kinase. However, the cellular and molecular bases of the skeletal abnormalities associated with CLS remain unknown. Here, we show that RSK2 is required for osteoblast differentiation and function. We identify the transcription factor ATF4 as a critical substrate of RSK2 that is required for the timely onset of osteoblast differentiation, for terminal differentiation of osteoblasts, and for osteoblast-specific gene expression. Additionally, RSK2 and ATF4 posttranscriptionally regulate the synthesis of Type I collagen, the main constituent of the bone matrix. Accordingly, Atf4-deficiency results in delayed bone formation during embryonic development and low bone mass throughout postnatal life. These findings identify ATF4 as a critical regulator of osteoblast differentiation and function, and indicate that lack of ATF4 phosphorylation by RSK2 may contribute to the skeletal phenotype of CLS.

Runx2 is necessary and sufficient for osteoblast differentiation, yet its expression precedes the appearance of osteoblasts by 4 days. Here we show that Twist proteins transiently inhibit Runx2 function during skeletogenesis. Twist-1 and -2 are expressed in Runx2-expressing cells throughout the skeleton early during development, and osteoblast-specific gene expression occurs only after their expression decreases. Double heterozygotes for Twist-1 and Runx2 deletion have none of the skull abnormalities observed in Runx2(+/-) mice, a Twist-2 null background rescues the clavicle phenotype of Runx2(+/-) mice, and Twist-1 or -2 deficiency leads to premature osteoblast differentiation. Furthermore, Twist-1 overexpression inhibits osteoblast differentiation without affecting Runx2 expression. Twist proteins' antiosteogenic function is mediated by a novel domain, the Twist box, which interacts with the Runx2 DNA binding domain to inhibit its function. In vivo mutagenesis confirms the antiosteogenic function of the Twist box. Thus, relief of inhibition by Twist proteins is a mandatory event precluding osteoblast differentiation.

The perichondrium, a structure made of undifferentiated mesenchymal cells surrounding growth plate cartilage, regulates chondrocyte maturation through poorly understood mechanisms. Analyses of loss-and gain-of-function models show that Twist-1, whose expression in cartilage is restricted to perichondrium, favors chondrocyte maturation in a Runx2-dependent manner. Runx2, in turn, enhances perichondrial expression of Fgf18, a regulator of chondrocyte maturation. Accordingly, compound heterozygous embryos for Runx2 and Fgf18 deletion display the same chondrocyte maturation phenotype as Fgf18-null embryos. This study identifies a transcriptional basis for the inhibition of chondrocyte maturation by perichondrium and reveals that Runx2 fulfills antagonistic functions during chondrogenesis.Supplemental material is available at http://www.genesdev.org.Received August 16, 2006; revised version accepted September 11, 2006. Endochondral bone formation is a complex process that involves several cell types with distinct patterns of gene expression. The initial step in this process is characterized by the aggregation of undifferentiated mesenchymal cells expressing ␣1(I) and ␣1(III) Collagen into condensations at the location and with the overall shape of future skeletal elements. Subsequently, cells within these condensations differentiate into chondrocytes that do not express any more ␣1(I) Collagen but instead, and among other genes, ␣1(II) b Collagen. Cartilage anlagen then enlarge through proliferation of chondrocytes that elongate to form prehypertrophic chondrocytes that will eventually exit the cell cycle to become bona fide hypertrophic chondrocytes. Genetically, hypertrophic chondrocytes differ from proliferating chondrocytes as they express ␣1(X) Collagen but not ␣1(II) Collagen (Kronenberg 2003). Throughout skeletogenesis, layers of ␣1(I) Collagen-expressing undifferentiated mesenchymal cells persist and surround cartilage anlagen to form a structure called the perichondrium (Kronenberg 2003).The transcriptional control of cell differentiation during skeletogenesis has been a topic of intense studies in the last 10 yr, leading to the identification of several key genes. For instance, Sox9 along with Sox5 and Sox6 are seen as the main transcription factors triggering mesenchymal condensations and initiating chondrocyte differentiation (Lefebvre et al. 1998;Bi et al. 1999;Smits et al. 2001). On the other hand, Runx2 appears to be the earliest transcriptional determinant of osteoblast differentiation (Ducy et al. 1997;Komori et al. 1997;Otto et al. 1997). Runx2 has broader functions during skeletogenesis since it is, along with Runx3, an inducer of chondrocyte hypertrophy (Takeda et al. 2001;Ueta et al. 2001;Yoshida et al. 2004). This latter role of Runx2 is explained by its transient expression in prehypertrophic chondrocytes.Runx2 is also expressed at high levels and throughout skeletogenesis in cells of the perichondrium, suggesting that it may have additional roles during chondrogenesis (Ducy et al. 1997). T...

Myostatin is a member of the bone morphogenetic protein/transforming growth factor-β (BMP/TGFβ) 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.

In mammals stanniocalcin (STC) is widely expressed, and in the kidney and gut it regulates serum calcium levels by promoting phosphate reabsorption. To shed further light on its functional significance in mammals we have created several lines of mice that express a human STC (hSTC) transgene. Three lines expressed the hSTC transgene, but only two lines exhibited high expression and contained circulating hSTC, and in these animals there was a reduction in postnatal growth (30-50%) that persisted after weaning. Moreover, even wild-type pups exhibited a growth retardation phenotype when nursed by a transgenic foster mother, and this implies that hSTC overexpression deleteriously affects maternal behavior and/or lactation. The reproductive potential of female transgenic mice was also compromised, as evidenced by significantly smaller litter sizes, but transgenic male fertility was unchanged even though the transgene was most highly expressed in testes. Interestingly, transgene-derived serum hSTC increased significantly after puberty and was severalfold higher in females than in males, suggesting a gender-specific mechanism for maintaining elevated circulating levels of STC. Blood analysis revealed that both transgenic lines had elevated phosphate and decreased alkaline phosphatase levels, indicative of altered kidney and bone metabolism. These studies provide the first evidence that STC is involved in growth and reproduction and reaffirm its role in mineral homeostasis.

BackgroundThe treatments currently approved for Duchenne muscular dystrophy (DMD), a progressive skeletal muscle wasting disease, address the needs of only a small proportion of patients resulting in an urgent need for therapies that benefit all patients regardless of the underlying mutation. Myostatin is a member of the transforming growth factor-β (TGF-β) family of ligands and is a negative regulator of skeletal muscle mass. Loss of myostatin has been shown to increase muscle mass and improve muscle function in both normal and dystrophic mice. Therefore, myostatin blockade via a specific antibody could ameliorate the muscle weakness in DMD patients by increasing skeletal muscle mass and function, thereby reducing patients’ functional decline.MethodsA murine anti-myostatin antibody, mRK35, and its humanized analog, domagrozumab, were developed and their ability to inhibit several TGB-β ligands was measured using a cell-based Smad-activity reporter system. Normal and mdx mice were treated with mRK35 to examine the antibody’s effect on body weight, lean mass, muscle weights, grip strength, ex vivo force production, and fiber size. The humanized analog (domagrozumab) was tested in non-human primates (NHPs) for changes in skeletal muscle mass and volume as well as target engagement via modulation of circulating myostatin.ResultsBoth the murine and human antibodies are specific and potent inhibitors of myostatin and GDF11. mRK35 is able to increase body weight, lean mass, and muscle weights in normal mice. In mdx mice, mRK35 significantly increased body weight, muscle weights, grip strength, and ex vivo force production in the extensor digitorum longus (EDL) muscle. Further, tibialis anterior (TA) fiber size was significantly increased. NHPs treated with domagrozumab demonstrated a dose-dependent increase in lean mass and muscle volume and exhibited increased circulating levels of myostatin demonstrating target engagement.ConclusionsWe demonstrated that the potent anti-myostatin antibody mRK35 and its clinical analog, domagrozumab, were able to induce muscle anabolic activity in both rodents, including the mdx mouse model of DMD, and non-human primates. A Phase 2, potentially registrational, clinical study with domagrozumab in DMD patients is currently underway.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.spinal muscular atrophy | lncRNA | PRC2 | SMN S pinal muscular atrophy is the leading genetic cause of infant mortality and is caused by deletions or mutation of Survival Motor Neuron 1 (SMN1) (1). Unique to humans, SMN1 is duplicated in the genome as SMN2, which is nearly identical in sequence. However, a C-to-T point mutation in exon 7 of SMN2 results in preferential skipping of this exon during pre-mRNA splicing and production of a truncated and unstable protein. A small fraction (10-20%) of pre-mRNA transcribed from SMN2 is spliced correctly to include exon 7 and produces a full-length SMN (SMN-FL, inclusive of exon 7) that is identical to the SMN1 gene product (2-4).Spinal motor neurons are highly sensitive to SMN1 deficiency, and their premature death causes motor function deficit in SMA patients (5, 6). The SMN2-derived SMN protein can extend spinal motor neuron survival, yet insufficient levels of SMN eventually lead to cell death. Overall, SMA patients with higher SMN2 genomic copy number have a less severe disease phenotype (7, 8). Type 0 or I patients, carrying one or two copies of SMN2, show onset of SMA within a few months of life with a life expectancy of less than 2. In contrast, type III and IV patients, carrying three or more copies, respectively, show juvenile or adult onset and slower disease progression (9). As further genetic evidence, SMA mouse models have been produced in which smn1 −/− mice, which would otherwise be embryonic lethal (10), can be rescued in the presence of high copy numbers of the human SMN transgene (11-13). Similar to the human disease spectrum, increased copy number of a human SMN transgene is inversely associated with decreased disease severity and mortality. We reasoned that increasing SMN2 transcription could phenocopy the beneficiary effect of SMN2 gene amplification and compensate for SMN1 deficiency. In addition, SMN1 heterozygotes are asymptomatic, whereas affected homozygotes have 10-20% of normal SMN levels. Therefore, we predict that modest SMN2 up-regulation will provide significant therapeutic benefit. H...