Nitric oxide (NO) plays an important role in the progression of osteoarthritis (OA), in part by influencing the physiology of chondrocytes (1, 2). NO is synthesized through L-arginine by NO synthases (NOS) in many cell types. Three different types of NOS have been identified. The neuronal NOS (nNOS or NOS1) and endothelial NOS (eNOS or NOS3) forms are constitutively expressed, and their activity is regulated by intracellular signaling and the calcium-binding protein calmodulin (3). The inducible form (iNOS or NOS2) is stimulated at the level of expression by factors including lipopolysaccharide and cytokines, such as interleukin-1, tumor necrosis factor α, and interferon-α, and leads to sustained and high levels of NO, mainly in inflammatory disease (1, 2, 4).
Changes in the articular chondrocyte phenotype, such as initiation of hypertrophic differentiation, are thought to play an important role in the pathogenesis of OA (5–7). Physiologically, chondrocyte hypertrophy occurs in the process of endochondral ossification that gives rise to most bones in the vertebrate skeleton, such as the long bones of the limbs (8, 9). Endochondral ossification involves the formation of a highly controlled and precisely shaped cartilage template that is subsequently replaced by bone tissue and bone marrow (10). Once mesenchymal cells commit to the chondrogenic lineage, the subsequent events of endochondral bone formation occur through the epiphyseal growth plate (11), which consists of zones of resting, proliferating, and hypertrophic chondrocytes organized in distinguishable columnar arrays (10). Within the growth plate, proliferation of chondrocytes occurs in a unidirectional manner, resulting in longitudinal bone growth (12, 13). After exiting the cell cycle, chondrocytes start to differentiate into prehypertrophic and eventually hypertrophic chondrocytes (14–16). Hypertrophic chondrocytes direct mineralization of the surrounding extracellular matrix, attract blood vessel invasion, and ultimately undergo apoptosis while being replaced by bone and bone marrow (13, 17). Proliferation and hypertrophy of growth plate chondrocytes and extracellular matrix production drive the longitudinal growth of endochondral bones and eventually determine body length in mammals (13, 18).
Since both NO signaling and altered chondrocyte differentiation contribute to OA, it is important to examine whether both processes are connected. Studies in the chicken suggest that NO signaling promotes chondrocyte hypertrophy (19, 20), but the roles of NO and in particular the individual NOS genes in mammalian cartilage development are not well understood. In this study, we used a genetic approach to address the role of eNOS in chondrocyte differentiation and endochondral ossification in vivo. Inactivation of the eNOS gene resulted in a number of phenotypes, including reduced proliferation and earlier cell cycle exit of chondrocytes. Overall, these data provide novel information regarding the importance of eNOS/NO signaling in chondrocyte development and identify downstream regulators of endochondral ossification.
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While NO has been shown to play an important role in controlling chondrocyte physiology in OA, the roles of the 3 NO-synthesizing enzymes in cartilage development are less clear. In this study, we provide evidence for an important function of eNOS in the regulation of chondrocyte proliferation and differentiation. Our data show that genetic ablation of the eNOS gene in vivo results in reduced chondrocyte proliferation and endochondral bone growth. Analyses of the underlying molecular mechanisms suggest that these effects are likely due to altered expression of several cell cycle proteins and the modulation of intracellular pathways with known roles in chondrocyte differentiation, including Sox9 and Hif1α.
Although there were no gross morphologic changes in the axial and appendicular skeleton of mutant mice, staining confirmed smaller rib cages, skull, and spine and shorter long bones. Some knockout mice showed more pronounced growth retardation and other defects such as kinky tails. The reasons for the variability in the phenotype are not known, but are consistent with the results of previous studies (22). Mice deficient in eNOS share some similarities with mice that lack C-type natriuretic peptide (CNP) (39), especially dwarfism and a reduction in chondrocyte proliferation. Because both NO and CNP stimulate the production of cGMP via soluble or particulate guanylyl cyclases (19), these similarities are not surprising and provide further evidence of the importance of cGMP signaling in endochondral bone formation. However, the phenotype of eNOS-null mice does not exactly resemble that of mice lacking CNP or the main effector of cGMP in cartilage, cGMP-dependent kinase II (40). For example, mice lacking CNP have strikingly narrow growth plates and shorter proliferating and hypertrophic zones (39), which we did not see in the eNOS-deficient mice used in the present study.
Because NO is produced through different NOS isoforms, deletion of eNOS alone might be compensated for by the other 2 NOS enzymes. Indeed, we found a 2-fold up-regulation of nNOS transcript levels in eNOS-deficient mice, suggesting that these 2 constitutive forms have partially redundant roles in cartilage. Additionally, cGMP is not the only downstream target of NO; for example, NO can alter the activity of several proteins through tyrosine nitrosylation (41, 42), which could account for some of the observed differences between eNOS-deficient mice and CNP- and cGMP-dependent kinase II–deficient mice.
A requirement of eNOS for proliferation has been described for a number of cells, including endothelial cells and osteoblasts (43, 44). However, Teixeira and colleagues (20) demonstrated that NO signaling stimulates chondrocyte hypertrophy in chicken chondrocytes, raising the question of whether loss of eNOS promotes proliferation at the expense of differentiation or results in the opposite phenotype, e.g., reduced proliferation and earlier onset of maturation. Our results clearly support the latter model. While species-specific effects or differences in approaches (e.g., in vivo versus in vitro) could explain some of the differences between the results of our study and the results of the study by Teixeira (20), the more likely explanation is that NO effects in chondrocytes are differentiation stage and concentration dependent. For example, it is plausible to speculate that loss of only 1 NO-synthesizing protein has different effects from the pharmacologic inhibition of all 3 proteins.
Inactivation of eNOS results in reduced chondrocyte proliferation in vivo and in vitro. Considering that eNOS was deleted in all of the cells of the body in the mice used in this study, it was unclear whether the reduced chondrocyte proliferation was due to loss of eNOS in cartilage or was secondary to defects in other tissues (e.g., in endocrine tissues or endothelial cells). Therefore, we conducted organ culture and primary cell culture in vitro, thus removing systemic and endocrine influences. Our organ culture data demonstrated reduced tibia growth upon loss of eNOS, and chondrocytes from mutant mice showed reduced multiplication of cells in culture. While these culture systems contain perichondral and periosteal cells that might be responsible for some of the observed changes, these results are consistent with the in vivo phenotype of eNOS-knockout mice and a cell-autonomous role of eNOS in chondrocyte proliferation.
The progression of the eukaryotic cell cycle is controlled by cyclins and cyclin-dependent kinases (32). For example, cyclin D1 has been shown to regulate the G1 phase of the cell cycle, is expressed specifically in the proliferating zone of growth plates, and is required for maximal chondrocyte proliferation in vivo and in vitro (32, 33). Our data showed a decline in cyclin D1 and an increase in p57 expression, presumably resulting in slower cell cycle progression and early cell cycle exit upon loss of eNOS. The earlier cell cycle exit and resulting acceleration in the onset of prehypertrophic differentiation is also supported by increased expression of prehypertrophic markers such as RORα and HIF-1α, and by reduced expression of markers for proliferating chondrocytes, such as type II collagen and SOX9. Of note, growth plate zone measurements and analyses of type X collagen expression suggest that eNOS deficiency accelerates prehypertrophic differentiation without accompanying acceleration of terminal hypertrophic differentiation.
In summary, our data demonstrate a role of eNOS in chondrocyte proliferation and endochondral bone growth. Loss of eNOS affects numerous aspects of cartilage physiology, including chondrocyte proliferation and gene expression. Further investigations into the specific mechanisms involved will result in a better understanding of physiologic and pathologic ossification and the role of eNOS in arthritic diseases.
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- MATERIALS AND METHODS
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Beier had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Yan, Feng, Beier.
Acquisition of data. Yan.
Analysis and interpretation of data. Yan, Beier.