Endothelial nitric oxide synthase deficiency in mice results in reduced chondrocyte proliferation and endochondral bone growth

Abstract

Objective. Nitric oxide (NO) and aberrant chondrocyte differentiation have both been implicated in the pathogenesis of osteoarthritis, but whether these processes are connected is unknown, and the role of specific NO synthase (NOS) enzymes in chondrocyte physiology is unclear. This study was undertaken to examine the effects of inactivation of endothelial cell NOS (eNOS) on cartilage development in mice.Methods. Skeletal growth and development of mice carrying a null mutation in the eNOS gene was compared with that of their control littermates. In situ analyses were complemented by experiments with primary chondrocytes and tibial explants from these mice.Results. Mice that were deficient in eNOS showed increased fatality and reduced bone growth, with hypocellular growth plates and a marked reduction in the number of proliferating chondrocytes. In vitro studies demonstrated lower chondrocyte numbers and reduced endochondral bone growth in mutant mice, suggesting that the role of eNOS signaling in chondrocyte proliferation is cell autonomous. Reduced chondrocyte numbers appear to be caused by decreased cyclin D1 and increased p57 expression in mutant mice, resulting in slower cell cycle progression and earlier cell cycle exit. In addition, expression of early chondrocyte markers such as SOX9 was reduced, and prehypertrophic markers were expressed prematurely in mutant mice.Conclusion. Our findings identify a novel and important role of eNOS in chondrocyte proliferation and endochondral bone growth and demonstrate that loss of eNOS results in premature cell cycle exit and prehypertrophic chondrocyte differentiation during cartilage development.

Inducible nitric oxide synthase–nitric oxide signaling mediates the mitogenic activity of Rac1 during endochondral bone growth

“…The similar phenotypes found in this and our earlier study (Yan et al, 2010) also suggest partial overlap of iNOS and eNOS function in chondrocytes. NO generated from either source inhibits the onset of ATF3 expression, allowing continued expression of cyclin D1 and thus delaying cell cycle exit.…”

“…Interestingly, we recently demonstrated a similar phenotype as described here (e.g. reduced proliferation and cyclin D1 expression, increased levels of ATF3 and premature cell cycle exit) in eNOS-deficient mice (Yan et al, 2010). Together, these studies suggest a dose-dependent effect of NO in the growth plate where low levels are required for proliferation, but high levels promote terminal differentiation and ultimately apoptosis.…”

“…Cell proliferation assays were performed using CyQUANT NF Cell Proliferation Assay Kit (Invitrogen) according to manufacturer's protocol, as described previously (Yan et al, 2010). Chondrocytes were isolated as described above and seeded in 96-well NUNC plates at a density of 1000 cells/100 ml/well.…”

Millimeter wave treatment promotes chondrocyte proliferation via G1/S cell cycle transition

“…Cell cycle control is a highly regulated process that involves a complex cascade of events, in which regulation can be realized through a complicated network by cyclins, cyclin-dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CDKI). G 1 /S transition, one of the two main checkpoints, is a rate-limiting step in the cell cycle and regulates cell proliferation (5,6). Cyclin D1 is a key cell cycle regulatory protein, which binds to CDK4 or CDK6 to control cell cycle progression from the G 1 to the S phase (7).…”

Function and Regulation of Pannexin 1 Channels in the Vascular Endothelium

“…For example, NO promotes bone and cartilage development by influencing osteoblast and chondrocyte differentiation, processes that are influenced by activation of Panx3 channels at the plasma membrane and ER [290,291]. In this context, targeted S-nitrosylation of Panx3 in these cells may serve as an important regulatory element promoting cell growth and differentiation.…”