Genome-wide analyses of gene expression during mouse endochondral ossification.

BACKGROUND: Endochondral ossification is a complex process involving a series of events that are initiated by the establishment of a chondrogenic template and culminate in its replacement through the coordinated activity of osteoblasts, osteoclasts and endothelial cells. Comprehensive analyses of in vivo gene expression profiles during these processes are essential to obtain a complete understanding of the regulatory mechanisms involved. METHODOLOGY/PRINCIPAL FINDINGS: To address these issues, we completed a microarray screen of three zones derived from manually segmented embryonic mouse tibiae. Classification of genes differentially expressed between each respective zone, functional categorization as well as characterization of gene expression patterns, cytogenetic loci, signaling pathways and functional motifs both confirmed reported data and provided novel insights into endochondral ossification. Parallel comparisons of the microdissected tibiae data set with our previously completed micromass culture screen further corroborated the suitability of micromass cultures for modeling gene expression in chondrocyte development. The micromass culture system demonstrated striking similarities to the in vivo microdissected tibiae screen; however, the micromass system was unable to accurately distinguish gene expression differences in the hypertrophic and mineralized zones of the tibia. CONCLUSIONS/SIGNIFICANCE: These studies allow us to better understand gene expression patterns in the growth plate and endochondral bones and provide an important technical resource for comparison of gene expression in diseased or experimentally-manipulated cartilages. Ultimately, this work will help to define the genomic context in which genes are expressed in long bones and to understand physiological and pathological ossification.

The role of Akt1 in terminal stages of endochondral bone formation: angiogenesis and ossification.

Longitudinal bone growth is the result of endochondral bone formation which takes place in the growth plate. The rate of chondrocyte proliferation and hypertrophy, vascular invasion with the formation of primary ossification centers and cartilage replacement by bone tissue are all important processes required for normal growth. We have shown a role for the PI3K signaling pathway in chondrocyte hypertrophy and bone growth in tibia explant cultures. In this current study, we aimed to investigate the role of Akt1, an important target of PI3K, in endochondral ossification. Akt1 KO mice showed reduced size compared to their littermates throughout life, but the largest difference in body size was observed around 1 week of age. Focusing on this specific developmental stage, we discovered delayed secondary ossification in the long bones of Akt1 KO mice. A delay in formation of a structure resembling a secondary ossification center was also seen in tibia organ cultures treated with the PI3K inhibitor LY294002. The expression of matrix metalloproteinase-14 (MMP-14), the main protease responsible for development of secondary ossification centers, was decreased in the epiphysis of Akt1 KO mice, possibly explaining the delay in secondary ossification centers seen in the Akt1 KO mice. Bone mineral density (BMD) and bone mineral content (BMC) measured in the proximal tibia of 1-year-old mice were decreased in Akt1 KO mice, suggesting that the original delay in ossification might affect bone quality in older animals.

Nitric oxide, C-type natriuretic peptide and cGMP as regulators of endochondral ossification.

Coordinated proliferation and differentiation of growth plate chondrocytes is required for endochondral bone growth, but the mechanisms and pathways that control these processes are not completely understood. Recent data demonstrate important roles for nitric oxide (NO) and C-type natriuretic peptide (CNP) in the regulation of cartilage development. Both NO and CNP stimulate the synthesis of cGMP and thus the activation of common downstream pathways. One of these downstream mediators, cGMP-dependent kinase II (cGKII), has itself been shown to be essential for normal endochondral bone formation. This review summarizes our knowledge of the roles and mechanisms of NO, CNP and cGKII signaling in cartilage and endochondral bone development.

Genetic ablation of Rac1 in cartilage results in chondrodysplasia.

Small GTPases of the Rho family have been implicated in the regulation of many intracellular processes. However, their tissue-specific roles in mammalian growth and development in vivo remain largely unknown. Here we describe the effects of cartilage-specific inactivation of the Rac1 gene in mice. Mice carrying this mutation show increased lethality, skeletal deformities, severe kyphosis and dwarfism. Rac1-deficient growth plates are disorganized and hypocellular, with chondrocytes of abnormal shape and size. Rac1-deficient chondrocytes also display reduced adhesion and spreading on collagen II and fibronectin as well as altered organization of the actin cytoskeleton, suggesting that Rac1 is required for normal cell-extracellular matrix interactions in cartilage. This phenotype is accompanied by reduced proliferation, increased apoptosis and deregulated expression of the cell cycle genes cyclin D1 and p57 in vivo. Moreover, phosphorylation of p38 MAP kinases is greatly reduced and expression of a key regulator of cartilage development, Indian hedgehog, is increased in mutant mice. In summary, these data identify a novel, essential and tissue-specific role of Rac1 in skeletal development and demonstrate that Rac1 deficiency affects numerous regulatory pathways in cartilage.

C-type natriuretic peptide regulates endochondral bone growth through p38 MAP kinase-dependent and -independent pathways.

BACKGROUND: C-type natriuretic peptide (CNP) has recently been identified as an important anabolic regulator of endochondral bone growth, but the molecular mechanisms mediating its effects are not completely understood. RESULTS: We demonstrate in a tibia organ culture system that pharmacological inhibition of p38 blocks the anabolic effects of CNP. We further show that CNP stimulates endochondral bone growth largely through expansion of the hypertrophic zone of the growth plate, while delaying mineralization. Both effects are reversed by p38 inhibition. We also performed Affymetrix microarray analyses on micro-dissected tibiae to identify CNP target genes. These studies confirmed that hypertrophic chondrocytes are the main targets of CNP signaling in the growth plate, since many more genes were regulated by CNP in this zone than in the others. While CNP receptors are expressed at similar levels in all three zones, cGMP-dependent kinases I and II, important transducers of CNP signaling, are expressed at much higher levels in hypertrophic cells than in other areas of the tibia, providing a potential explanation for the spatial distribution of CNP effects. In addition, our data show that CNP induces the expression of NPR3, a decoy receptor for natriuretic peptides, suggesting the existence of a feedback loop to limit CNP signaling. Finally, detailed analyses of our microarray data showed that CNP regulates numerous genes involved in BMP signaling and cell adhesion. CONCLUSION: Our data identify novel target genes of CNP and demonstrate that the p38 pathway is a novel, essential mediator of CNP effects on endochondral bone growth, with potential implications for understanding and treatment of numerous skeletal diseases.

Dexamethasone stimulates expression of C-type Natriuretic Peptide in chondrocytes.

BACKGROUND: Growth of endochondral bones is regulated through the activity of cartilaginous growth plates. Disruption of the physiological patterns of chondrocyte proliferation and differentiation--such as in endocrine disorders or in many different genetic diseases (e.g. chondrodysplasias)--generally results in dwarfism and skeletal defects. For example, glucocorticoid administration in children inhibits endochondral bone growth, but the molecular targets of these hormones in chondrocytes remain largely unknown. In contrast, recent studies have shown that C-type Natriuretic Peptide (CNP) is an important anabolic regulator of cartilage growth, and loss-of-function mutations in the human CNP receptor gene cause dwarfism. We asked whether glucocorticoids could exert their activities by interfering with the expression of CNP or its downstream signaling components. METHODS: Primary mouse chondrocytes in monolayer where incubated with the synthetic glucocorticoid Dexamethasone (DEX) for 12 to 72 hours. Cell numbers were determined by counting, and real-time PCR was performed to examine regulation of genes in the CNP signaling pathway by DEX. RESULTS: We show that DEX does influence expression of key genes in the CNP pathway. Most importantly, DEX significantly increases RNA expression of the gene encoding CNP itself (Nppc). In addition, DEX stimulates expression of Prkg2 (encoding cGMP-dependent protein kinase II) and Npr3 (natriuretic peptide decoy receptor) genes. Conversely, DEX was found to down-regulate the expression of the gene encoding its receptor, Nr3c1 (glucocorticoid receptor), as well as the Npr2 gene (encoding the CNP receptor). CONCLUSION: Our data suggest that the growth-suppressive activities of DEX are not due to blockade of CNP signaling. This study reveals a novel, unanticipated relationship between glucocorticoid and CNP signaling and provides the first evidence that CNP expression in chondrocytes is regulated by endocrine factors.