Wnt proteins are secreted signaling factors with critical roles in various types of tissue organization. The proteins bind to cell surface receptors and transmit signals by several pathways, including the beta-catenin pathway. Upon receptor activation, cytoplasmic beta-catenin transits to the nucleus and forms complexes with resident transcription factors (LEF or TCFs) that bind response sequences and modulate target gene expression. In recent studies, we have shown that the Wnt/beta-catenin pathway operates in growth plate chondrocytes during skeletal formation. We found that beta-catenin is cytoplasmic in proliferating and prehypertrophic cells, but undergoes a dramatic nuclear re-localization in hypertrophic chondrocytes. In good agreement, promoter-driven expression of constitutive-active LEF/TCF proteins boosted chondrocyte maturation, expression of genes such as MMP-13, and matrix mineralization. Dominant-negative constructs blocked these events. These and other findings lead us to our central hypothesis: activation of Wnt/beta-catenin signaling is required for chondrocyte hypertrophy and function and for progression and completion of endochondral ossification. To test this hypothesis, we will identify Wnt and LEF/TCF molecules expressed by growth plate chondrocytes and determine their activity and function during chondrocyte maturation and endochondral ossification. We will also characterize mechanisms by which beta-catenin-LEF/TCF complexes regulates gene expression in hypertrophic cells. Approaches to be used will include: cell cultures; in vivo analyses of avian and mammalian embryos; RNA interference; creation and analysis of transgenic mice; promoter reporter tests. The results will shed important light into a previously unsuspected pathway that operates in hypertrophic chondrocytes and controls their function and roles in endochondral ossification. The data will also suggest future targets of investigation to determine the possible involvement of Wnt/beta-catenin signaling in pathologies of cartilage and bone, a presumption based on well-established roles of Writ signaling in the pathology of other tissues and organs.
