Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway.

Achondroplasia is the most common genetic form of human dwarfism, for which there is presently no effective therapy. C-type natriuretic peptide (CNP) is a newly identified molecule that regulates endochondral bone growth through GC-B, a subtype of particulate guanylyl cyclase. Here we show that targeted overexpression of CNP in chondrocytes counteracts dwarfism in a mouse model of achondroplasia with activated fibroblast growth factor receptor 3 (FGFR-3) in the cartilage. CNP prevented the shortening of achondroplastic bones by correcting the decreased extracellular matrix synthesis in the growth plate through inhibition of the MAPK pathway of FGF signaling. CNP had no effect on the STAT-1 pathway of FGF signaling that mediates the decreased proliferation and the delayed differentiation of achondroplastic chondrocytes. These results demonstrate that activation of the CNP-GC-B system in endochondral bone formation constitutes a new therapeutic strategy for human achondroplasia.

Complementary antagonistic actions between C-type natriuretic peptide and the MAPK pathway through FGFR-3 in ATDC5 cells.

We previously reported that C-type natriuretic peptide (CNP) stimulates endochondral ossification and corrects the reduction in body length of achondroplasia model mouse with constitutive active fibroblast growth factor receptor 3 (FGFR-3). In order to examine the interaction between CNP and FGFR-3, we studied intracellular signaling by using ATDC5 cells, a mouse chondrogenic cell line, and found that FGF2 and FGF18 markedly reduced CNP-dependent intracellular cGMP production, and that these effects were attenuated by MAPK inhibitors. Western blot analysis demonstrated that the level of GC-B, a particulate guanylyl cyclase specific for CNP, was not changed by treatment with FGFs. Conversely, CNP and 8-bromo-cGMP strongly and dose-dependently inhibited the induction of ERK phosphorylation by FGF2 and FGF18 without changing the level of FGFR-3, although they did not affect the phosphorylation of STAT-1. In the organ-cultured fetal mouse tibias, CNP and FGF18 counteracted on the longitudinal bone growth, and both the size and number of hypertrophic chondrocytes. The FGF/FGFR-3 pathway is known as the negative regulator of endochondral ossification. We found that FGFs inhibited CNP-stimulated cGMP production by disrupting the signaling pathway through GC-B while CNP antagonized the activation of the MAPK cascade by FGFs. These results suggest that the CNP/GC-B pathway plays an important role in growth plate chondrocytes and constitutes the negative cross talk between FGFs and the activity of MAPK. Our results may explain one of the molecular mechanisms of the growth stimulating action of CNP and suggest that activation of the CNP/GC-B pathway may be effective as a novel therapeutic strategy for achondroplasia.

Thyroid hormones promote chondrocyte differentiation in mouse ATDC5 cells and stimulate endochondral ossification in fetal mouse tibias through iodothyronine deiodinases in the growth plate.

Thyroid hormones (THs), 3,3',5-triiodo-L-thyronine (T3) and L-thyroxine (T4), are important for the normal development of the growth plate (GP); congenital TH deficiency leads to severe dwarfism. In mouse chondrogenic cell line, ATDC5, T3 enhanced differentiation and increased Alizarin red staining, but did not affect Alcian blue staining. In organ-cultured mouse tibias, THs stimulated the cartilage growth, especially in the hypertrophic zone. Interestingly, T4 was as equally potent as T3 in organ-cultured tibias, which suggests that T4 is metabolized locally to T3, because T4 is a prohormone and must be converted to T3 for its activity. Two enzymes catalyze the conversion; type I deiodinase (D1) and type II deiodinase (D2). D1 has a ubiquitous distribution and D2, with a high affinity for T4, is present where the maintenance of intracellular T3 concentration is critical. Messenger RNAs (mRNAs) for D1 and D2 were detected in neonatal mouse tibias and ATDC5 cells. The enzyme activity was unaffected by the D1 inhibitor 6-propyl-2-thiouracil, suggesting that D2 mainly catalyzes the reaction. D2 mRNA was detected in differentiated ATDC5 cells. In organ-cultured mouse tibias, D2 activity was greater at later stages. In contrast, thyroid hormone receptors (TRs) were expressed in neonatal mouse tibias and ATDC5 cells, but their expression levels in ATDC5 cells were stable throughout the culture periods. Therefore, increased T3 production at later stages by D2 is likely to contribute to the preferential effects of THs in the terminal differentiation of GP. This article is the first to show that T4 is activated locally in GP and enhances the understanding of TH effects in GP.

High-dose glucocorticoid treatment induces rapid loss of trabecular bone mineral density and lean body mass.

A recent large-scale study revealed that glucocorticoid treatment increased fracture risk, which occurred at a far smaller dose and by a shorter duration than previously thought. To study the underlying mechanism for the increased risk of fracture, we studied the early changes in bone mineral density (BMD) and body composition by dual energy X-ray absorptiometry (DXA) after initiating high-dose glucocorticoid treatment. High-dose glucocorticoid treatment was arbitrarily defined as daily doses of >or=40 mg of a predonisolone equivalent. The 33 patients enrolled in this study had not received glucocorticoid treatment before. Only 2 months of treatment resulted in substantial BMD loss, most markedly in the lumbar spine, followed by the femoral neck and total body, which suggests the preferential trabecular bone loss. Body composition was also greatly affected. Thus, 2-month treatment with glucocorticoid significantly reduced bone mineral content (BMC), lean body mass (LBM) and increased fat mass (FAT). Our results are likely to have some clinical relevance. First, BMD loss occurs quite rapidly after starting glucocorticoid treatment, and patients receiving glucocorticoid treatment should be more carefully monitored for their BMD. Second, LBM, which mainly represents muscle volume, decreases rapidly after initiating glucocorticoid treatment. Decreased LBM might be also responsible for the increased risk of fracture, since falling is a well-known risk factor for fracture, and patients receiving glucocorticoid treatment should also be evaluated for their body composition.

A novel interaction between thyroid hormones and 1,25(OH)(2)D(3) in osteoclast formation.

Thyroid hormones enhance osteoclast formation and their excess is an important cause of secondary osteoporosis. 3,5,3' -Triiodo-L-thyronine (T3) induced the mRNA expression of receptor activator of nuclear factor-kappa B ligand (RANKL), which is a key molecule in osteoclast formation, in primary osteoblastic cells (POB). This effect was amplified in the copresence of 1 alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Although T3 alone did not induce octeoclasts in coculture of bone marrow cells with POB, T3 enhanced 1,25(OH)(2)D(3)-induced osteoclast formation. Thyroxine (T4) also enhanced 1,25(OH)(2)D(3)-induced osteoclast formation. These data suggested that T4 was locally metabolized to T3 for its action, since T4 is a prohormone with little hormonal activity. The mRNA expression of type-2 iodothyronine deiodinase (D2), which is responsible for maintaining local T3 concentration, was induced by 1,25(OH)(2)D(3) dose- and time-dependently. Our data would facilitate our understanding of the mechanism of osteoclast formation by thyroid hormones and suggest a novel interaction between thyroid hormones and 1,25(OH)(2)D(3).