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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.