Cdc42 Is Critical for Cartilage Development During Endochondral Ossification

Abstract

Cdc42 is a widely expressed protein that belongs to the family of Rho GTPases and controls a broad variety of signal transduction pathways in a variety of cell types. To investigate the physiological functions of Cdc42 during cartilage development, we generated chondrocyte-specific inactivated Cdc42 mutant mice (Cdc42(fl/fl); Col2-Cre). The gross morphology of mutant neonates showed shorter limbs and body as compared with the control mice (Cdc42(fl/fl)). Skeletal preparations stained with alcian blue and alizarin red also revealed that the body and the long bone length of the mutants were shorter than those of the control mice. Furthermore, severe defects were found in growth plate chondrocytes in the femur sections of mutant mice, characterized by a reduced proliferating zone height, wider hypertrophic zone, and loss of columnar organization in proliferating chondrocytes. The expression levels of chondrocyte marker genes, such as Col2, Col10, and Mmp13, in mutant mice were decreased as compared with the control mice. Mineralization of trabecular bones in the femur sections was also decreased in the mutants as compared with control mice, whereas osteoid volume was increased. Together these results suggested that chondrocyte proliferation and differentiation in growth plates in the present mutant mice were not normally organized, which contributed to abnormal bone formation. We concluded that Cdc42 is essential for cartilage development during endochondral bone formation.

Signaling Cascades Governing Cdc42-Mediated Chondrogenic Differentiation and Mensenchymal Condensation

“…At E18.5, Cdc42 expression was exclusively restricted to hypertrophic chondrocytes and trabecular bone areas ( Figure 1a). Severe cartilage phenotypes have been reported by several previous studies (Aizawa et al 2012;Suzuki et al 2015); however, whether Cdc42 directly regulates osteogenesis still lacks solid evidence. To assess the physiological role of Cdc42 in bone formation, we genetically removed Cdc42 from the skeletal progenitors by using the Cre-loxP technique and harvested the embryos at E18.5 and E15.5.…”

“…Chondrocyte-specific inactivation of Cdc42 (Col2Cre;Cdc42 fl/fl ) has demonstrated that Cdc42 is essential for cartilage development during endochondral ossification (Suzuki et al 2015). To explore the underlying mechanism governing Cdc42-mediated chondrogenic differentiation, we stimulated C3H10T1/2 cells with transforming growth factor-b (TGF-b), a critical regulator in the process of chondrocyte differentiation and hypertrophy shRNA-or scramble-shRNA-expressing lentiviruses, alternatively, after cells were infected withGFP-or da-Cdc42-expressing retroviruses, cells were subjected to micromass cultures in the presence or absence of rhBMP2 at 10 ng/ml for 7 days followed by Alcian Blue staining.…”

“…chondrocytes and apoptosis in the chondro-osseous junction in Prx1Cre;Cdc42 fl/fl and Col2Cre;Cdc42 fl/fl long bones (Aizawa et al 2012;Suzuki et al 2015). Though a positive correlation between the decreased number of osteoclasts adjacent to chondrocytes and delayed mineralization formation has been found in Col2Cre;Cdc42 fl/fl long bones and Cdc42 indeed regulates osteoclast formation and function (Ito et al 2010;Suzuki et al 2015), whether osteoclasts are directly or indirectly involved in the malformation of cartilage and bone in Cdc42 mutants needs to be further studied.…”

“…Among the variety of phenotypes in Prx1Cre;Cdc42 fl/fl mice, severe defects in long bone growth plate cartilage are notable and related to loss of columnar organization of chondrocytes, as well as thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth (Aizawa et al 2012). Moreover, chondrocyte-specific inactivation of Cdc42 (Col2Cre;Cdc42 fl/fl ) shows shorter limbs and body, and severe defects were found in growth plate chondrocytes of long bone, characterized by a reduced proliferating zone height, wider hypertrophic zone, and loss of columnar organization in proliferating chondrocytes (Suzuki et al 2015).Despite data demonstrating the importance of Cdc42 in cartilage development, little is known about the molecular mechanisms underlying Cdc42-mediated chondrogenesis. On the other hand, though activation of FYVE, RhoGEF, and PH domain-containing protein 1 (FGD1), a guanine nucleotide exchange factor (GEF) of Cdc42, promotes the osteogenic differentiation in human mesenchymal stem cells (Gao et al 2011), whether Cdc42 directly regulates bone formation still lacks genetic evidence.…”

“…Among the variety of phenotypes in Prx1Cre;Cdc42 fl/fl mice, severe defects in long bone growth plate cartilage are notable and related to loss of columnar organization of chondrocytes, as well as thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth (Aizawa et al 2012). Moreover, chondrocyte-specific inactivation of Cdc42 (Col2Cre;Cdc42 fl/fl ) shows shorter limbs and body, and severe defects were found in growth plate chondrocytes of long bone, characterized by a reduced proliferating zone height, wider hypertrophic zone, and loss of columnar organization in proliferating chondrocytes (Suzuki et al 2015).…”