Local regulation of growth plate chondrocytes : molecular mechanisms and implications for longutidinal growth

Abstract: The regulation of linear growth is a complex and intriguing process that has the growth plate cartilage as its final target. Growth plate is a specialized cartilage localized in the end of the long bones between the epiphyseal and metaphyseal bone. It consists of 3 layers (resting, proliferative and hypertrophic zones) with distinct cellular morphology and function, where the continuous process of endochondral ossification takes place. This process is tightly regulated and relies on the interaction between systemic and local action of several hormones and growth factors. Although height is a true multigenetic trait and much has been discovered on the control of skeletal growth; there is still a long way to go to understand the intrinsic control of growth plate chondrogenesis that regulates postnatal bone growth and determines individual height. In order to explore the molecular mechanisms implicated in the spatial and temporal control of growth plate, we first used microdissection of postnatal rat growth plate combined with microarray and real-time PCR analyses. Bioinformatic analysis of global gene expression implicated novel biological functions, molecular pathways, transcription factors and potential markers for spatially-associated chondrocyte differentiation and temporally-associated growth plate senescence (Paper I). We next explored some of these molecular pathways implicated in the control of postnatal chondrogenesis. The Wnt/?-catenin signaling was one of the most strongly implicated pathways in the developmental program of growth plate senescence revealed in our microarray analysis. We found that six Wnts were expressed in growth plate chondrocytes, of which Wnts-2b, -4 and -10b act through the canonical ?-catenin pathway and Wnts -5a, -5b and -11 acts through the non-canonical Calcium pathway. We observed that all the expressed Wnts exhibited a similar pattern of expression in the growth plate, showing low expression in resting zone, increasing expression as the chondrocytes differentiated into the proliferative zone, and then decreasing expression as the chondrocytes underwent hypertrophic differentiation. In addition, all identified Wnts persisted at similar levels with age. Altogether, our findings suggest that Wnts modulate growth plate senescence and chondrocyte hypertrophy through the canonical ?-catenin and non-canonical calcium pathways (Paper II). We then, characterized the expression profile of a growth-regulated network of imprinted genes implicated in embryonic growth of soft tissues. We found that the expression pattern of the network is modified in growth plate cartilage compared both to soft tissues and to bone. In particular, developmental changes in the expression of growth-promoting genes (Mest, Dlk1, Gtl2), and growth-inhibitory genes (Cdnk1c and Grb10) may contribute to the decline in longitudinal bone growth that occurs with age (Paper III). In addition, by using distinct growth inhibiting conditions, we found functional and structural delay in growth plate senescence markers that indicate that growth plate senescence is not simply a function of time per se but rather of growth, and that delayed senescence may be a general consequence of growth inhibition (Paper IV). We also identified unique microRNAs (miRNAs) that are preferentially expressed and age-regulated in growth plate chondrocytes. The role of these miRNAs in growth plate chondrogenesis were tested using in vitro system for culture and transfection of murine resting zone chondrocytes, which enabled us to study the role of miRNAs in primary chondrocytes phenotype, simulating in vivo conditions more closely. These findings suggest that miRNAs contribute to the developmentally regulated decline in longitudinal bone growth through regulation of chondrocyte proliferation and apoptosis. Altogether, our findings contribute to understand the molecular regulation of growth plate chondrocytes and its implications to postnatal linear growth (Paper V).