The Roles And Mechanisms Of Cdc42 In Long Bone Development

Elucidating the mechanisms that control endochondral bone development is critical for understanding human skeletal diseases, injury response, and aging.The osseous tissues in mammals are formed via two distinct processes during embryogenesis. Intramembranous bone formation produces many of the craniofacial bones directly from mesenchymal condensations. In contrast, endochondral ossification, the principal process responsible for forming much of the mammalian skeleton, generates bone via a cartilage intermediate. The transition from cartilage to bone is tightly coupled with several key processes of endochondral ossification, including formation of the mesenchymal condensation, chondrocyte differentiation and maturation, and osteoblast development.The Rho family of small GTPases including Racl, Cdc42 and RhoA are molecular switches that control a wide variety of signal transduction pathways in all eukaryotic cells, such as regulation of the cytoskeleton, cell movement, cell cycle control, and regulation of transcription factor activity. Have bene demonstrated that Racl regultate osteogenic differentiation via Wnt signaling. Racl and Cdc42 have been shown to promote chondrogenesis. But the molecular mechanisms controlling differentiation of mesenchymal precursor cells into chondrocytes are not completely understood.Here we utilized tissue- and cell-type specific gene targeting of Cdc42 in mice demonstrate that Cdc42 play a critical role in endochondral ossification. The molecular mechanisms have been shown in this article. It is well understood the functions of small GTPases in limb bud development.The apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) are major morphogen sources in the limb bud formation of vertebrates. At first, we generated the AER inactivated Cdc42 mice (Msx2Cre;Cdc42f/f). But they are almost the same as compared to their Cdc42f/f littermates. Then, we generated limb bud mesenchyme-specific inactivated Cdc42 mice (PrxCre;Cdc42f/f). They had thickening and massive accumulation of hypertrophic chondrocytes in long bone growth plate cartilage, resulting in delayed endochondral bone formation associated with reduced bone growth. These indicated that Cdc42 is involved in the endochondral ossification.H&E staining results suggested that Cdc42 may regulate the osteogenic differentiation. In situ hybridization analyses of PrxCre;Cdc42f/f femur obtained at E18.5 showed that the expression levels of Col10 and IHH which were hypertrophic chondrocytes and prehypertrophic chondrocytes maker genes were increased as compared to Cdc42f/f control. The expression pattern of the marker genes of blood vessel invade, osteogenesis and chondrocytes mature are not different as control. Then we tested the effects of Cdc42 in osteogenic differentiation by knocking down Cdc42 in vitro. These studies revealed nomal mRNA levels of marker genes of osteogenic differentiation and transcript levels of the transcription factor of osteogenesis-Runx2. We also generated the osteoblast inactivated Cdc42 mice (Co11Cre;Cdc42f/f). Whole-mount skeletal staining with alcian blue and alizarin red revealed that they are almost the same as compared to their Cdc42f/f littermates. These results indicated that Cdc42 is not involved in the osteogenic differentiation.Endochondral bone development begins with the condensation of mesenchymal cells. In situ hybridization analyses suggested that Sox9 was ectopically expressed in the PrxCre;Cdc42f/f limbs at E12.5. To study the role of Cdc42 as a potential mediator of mesenchymal stem cell condensation further, an in vitro model was established using micromass cultures of C3H10T1/2 cells. Micromass cultures of C3H10T1/2 cells treated with BMP-2 (100 ng/ml) remarkably resulted in nodule formation. While this effect was blocked in cultures infected by Cdc42-shRNA-expressing viruses compared to the control. The expression of N-cadherin, an important molecular marker of condensation, was up-regulated stimulating by BMP-2 for 3 days and redueed in C3H10T1/2 infected by Cdc42-shRNA-expressing viruses, following BMP-2 treatment. These data suggested that Cdc42 plays a positive role in condensation of mesenchymal stem cell.Western blot results showed that knockdown Cdc42 or Pakl greatly blocked BMP-2 induced phosphorylation of Smal/5 and p38. In those cells pretreated with SB203580 followed by BMP-2 induction, both the intensity and nuclear translocation of Smad were substantially suppressed. Co-immunoprecipitation results indicated that p38 did interact with Smad5 forming a complex. Then we performed the micromass cultures, the results showed that the inhibition of p38 blocked the condensation of C3H10T1/2 induced by BMP-2 and the anisomycin rescued the condensation blocked by Cdc42-shRNA-expressing viruses. To confirm our results, we detected the expression of p-Smadl/5 and p-p38 in PrxCre;Cdc42 limbs at E11.5 by IHC and western blot. The expression were faintly reduced in PrxCre;Cdc42f/f limbs compared with control mice. Together these data indicated Cdc42 plays a positive role in condensation of mesenchymal stem cell via BMP2/Pakl/p38/Smad signaling.Following mesenchymal condensation, cells in the core of the condensations differentiate into chondrocytes. In order to know the role of Cdc42 in chondrogenic differentiation, we then tested the expression of a variety of cartilage-related genes using qPCR. Knockdown of Cdc42 or Pakl in C3H10T1/2 cells resulted in reduced the expression of Col2a1 and the luciferase activity of Sox9, stimulated by TGF-β. Knockdown of Cdc42 or Pakl almost completely abolished TGF-β-induced activation of AKT1. Sox9 is a critical transcription factor responsible for the differentiation of all chondroprogenitors except for the hypertrophy, and the DNA binding activity and cellular localization of Sox9 is controlled through post-translational modifications including phosphorylation. TGF-β increased the phosphorylation of Sox9 at Ser181 (p-Sox9), whereas knockdown of Cdc42 reduced TGF-β-induced p-Sox9 levels. Moreover, constitutive activation of AKT1 by dominant-active form of AKT1 (da-AKT1) increased the p-Sox9 activities in both the absence and presence of TGF-P, whereas da-AKT almost completely reversed Cdc42-shRNA-negated Sox9 phosphorylation, suggesting that Cdc42 lies on the upstream of AKT1 to activate Sox9 in response to TGF-p.We also examined the expression of Col10 and Runx2 in ATDC5 cells, which were hypertrophic chondrocytes and mineralization maker genes, while they were not remarkable different as control. To confirm our results, we got Co110Cre;Cdc42f/f mice. Whole-mount skeletal staining and H&E staining at P1 revealed that they are almost the same as wild type mice. These results indicated that Cdc42 is not involved in hypertrophy of chondrocytes.Together these data indicated Cdc42 plays a positive role in condensation of mesenchymal stem cell via BMP2/Pakl/p38/Smad signaling and mediate TGF-β/Pak1/AKT1/Sox9 signaling to promote the transactivity of Sox9 and subsequent chondrogenic differentiation but not hypertrophy of chondrocytes.