| Cartilage, which is the principal engine for conducting growth of most mammalian skeletons, is developmentally regulated by multiple signaling pathways whcih are driven by multiple factors including cytokines and hormones such as Ihh (Indian hedgehog), PTHrP (parathyroid hormone-related peptide), members of TGF-β(transforming growth factorβ) superfamily, FGF (fibroblast growth factor), as well as transcription factors such as Sox (SRY-related high mobility group-box gene) family, Runx2 (runt-related transcription factor 2), HIF-1α(hypoxia-inducible factor-1α), and stresses such as ERSS (endoplasmic reticulum stress). They are vitally engaged in the processes of coordinating chondrocytic proliferation, differentiation, migration, apoptosis and cellular homeostasis. Disruption of these processes may impair endochondral ossification, leading to various bone defects. Dyschondroplasia is a defect of cartilage development which is characterized by multiple enchondromatosis within the bone marrow cavity. This defect mainly harms youths by means of pain, skeletal deformities and pathological fractures. However, the molecular mechanisms underlying dyschondroplasia are poorly understood.The tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) is involved in the regulation of cell proliferation, lineage determination, motility, adhesion and apoptosis, as well as in the maintenance of intracellular homeostasis through coordinating the PI3K (phosphatidylinositol-3-kinase) signaling. Loss or mutation of PTEN has been implicated in various hereditary disorders as well as several sporadic human cancers. However, the functions of PTEN in endochondral ossification and dyschondroplasia inhibition remain largely unknown. In this study, we have created chondrocyte-specific PTEN knockout mice (PTENCo/Co;Col2a1-Cre-2) using the Cre–LoxP system. The employment of this genetic model would facilitate our understanding the role of PI3K/Akt signaling in cartilage development and bone defects.Following Akt activation, PTEN mutant mice exhibited excessive body length and dyschondroplasia resembling human enchondroma. By using BrdU incorporation and in situ hybridization/BrdU labeling-chasing assay, we observed asynchronous proliferation and differentiation of the PTEN mutant chondrocytes that led to the formation of neoplastic cores within the central region of growth plates. Abnormal resting chondrocytes within these cores are pinched off from the developing growth plates, gradually forming cartilaginous nodules within the bone marrow cavity. Histological observations showed pathological similarities between these cartilaginous neoplasms and human enchondroma. Additionally, an abnormal arrangement and differentiation of growth plate cartilage coupled with joint deformation as well as depletion of articular cartilage was also observed. Therefore, these knockout mice provided us with powerful genetic models on studying dyschondroplasia.The formation of neoplastic cores which were considered to be the rudiments of enchondromas, was the most remarkable even during the oncogenesis within the PTEN mutant mice. Immunohistochemistry and electron microscopy analyses revealed aberrant properties and distribution of ColII (type II collagen) fibrils within the neoplastic cores. Chondrocytes within the cores experienced severe ERSS characterized by an up-regulation of ERSS related genes as well as engorged and fragmented ER in which extracellular matrix proteins were trapped. We further found ERSS only occurred under hypoxic conditions within the PTEN mutant chondrocytes, leading to impaired maintenance of chondrocytic differentiation. This result suggested the synergistic function of PTEN deficiency and hypoxia in triggering ERSS.An up-regulation of HIF-1αand downstream targets VEGF (vascular endothelial growth factor), PGK (phosphoglycerate-kinase), p21, p57 followed by an emergence of ERSS and neoplastic core was demonstrated via immunohistochemistry and in situ hybridization. Activated HIF-1αsignaling within PTEN mutant growth plates resulted in halted chondrocytic proliferation as well as excessive blood vessel growth and invasion. In vitro study showed that under hypoxia, PTEN mutant chondrocytes exhibited over-reacted HIF-1αsignaling in advance of ERSS, suggesting that PI3K/Akt signaling may regulate the responses of chondrocytes to hypoxia and trigger ERSS via up-regulation of HIF-1αsignaling.In this study, we provided the first genetic evidence to show that PTEN coordinates proliferation and differentiation of growth plate chondrocytes via inhibiting overactive HIF-1αsignaling and ERSS, thereby guides the uniform development of growth plate cartilage and suppresses dyschondroplasia. Our data provided new clues for better understanding molecular mechanisms of dyschondroplasia. |
