Director of Research, Portland Shriners Research Center
Professor of Molecular & Medical Genetics, Oregon Health & Science University
Biology of Linear Bone Growth
We are interested in how bones grow. More precisely, we want to understand the molecular and cellular mechanisms that control mammalian skeletal development, especially those involved in linear bone growth. Skeletal growth is primarily responsible for the final form of adult mammals. This is achieved for most bones through the generation of cartilage models that serve as a templates for bone growth, a process known as endochondral ossification. Once the embryonic bone is formed, endochondral ossification occurs near the ends of bones in so called growth plates.
The growth plate is a dynamic structure with a leading edge where new cells arise through mitosis, intermediate zones where terminally differentiating cells synthesize matrix and facilitate its maturation into a functional template and a trailing edge where the template is degraded and replaced by bone (See Fig 3, left). The synthesis of template, chondrogenesis, drives this process to a large extent.
A large number of genes must be involved in regulating these events judging from the many inherited human disorders (the chondrodysplasias) manifesting defective bone growth, as well as, the many naturally occurring skeletal mutants in mice and other species. However, there must also be much redundancy considering the many man-made misexpression and knockout mouse mutants that exhibit no abnormalities of skeletal development despite disrupting expression of genes that influence basic cell functions such as mitosis and differentiation. Our goal is to understand what the critical genes are and how they work to control the proliferation, survival and terminal differentiation of growth plate chondrocytes and the complex growth plate machinery . Our experimental approach utilizes a wide variety of biochemical, molecular genetic, immunologic, molecular biology, cell biology and mouse genetics methods. It is hoped our results will provide insight into the fundamental biologic process of growth and also establish a rational basis for new therapies for patients with bone growth disorders.
Cartilage matrix is the primary constituent of the template generated during endochondral ossification. It provides structural integrity for the template and serves as an interface to the external environment for cells participating in the process. Chondrodysplasia-causing mutations have been identified in genes encoding several cartilage matrix components including types II, IX, X and XI collagen chains and cartilage oligomeric matrix protein (COMP). To determine how such mutations adversely affect skeletal development and growth, transgenic mice have been generated in which expression of relevant mutations is targeted to cartilage. One mouse strain, Col2-GFP, carries a Green Fluorescent Protein “reporter” transgene that allows chondrogenesis to be investigated in real time in living cells and animals (Fig 1). Analysis of the mice, cells, tissues and extracts from the mice is providing considerable information about the functional consequences of the mutations to bone growth and factors that influence chondrogenesis.
FGFR3 Signaling in the Growth Plate
Gain-of-function mutations of the receptor tyrosine kinase -FGFR3 (Fibroblast Growth Factor Receptor 3) – are responsible for the most common human chondrodysplasias (the achondroplasia and related conditions). It is clear from a variety of studies that FGFR3 signals inhibit linear bone growth and that this inhibition is exaggerated in achondroplasia, but the cellular and molecular mechanisms through which the receptors act and the means through which mutations enhance theses actions are not well understood. We are interested in unraveling these mechanisms as a strategy to identify new therapeutic targets for achondroplasia. We expect that this will lead to identification of agents that will safely block transmission of unwanted signals and to new treatment modalities the human achondroplasia.
Much of our attention is focused on the intracellular fate of activated FGFR3 receptors because a receptor’s signal intensity is directly related to its signaling lifetime, i.e., the longer it survives, the greater the aggregate signal. Our studies have shown that FGFR3 receptors are normally degraded relatively quickly after they are activated through a process that involves endocytosis and transport through endosomal pathways to lysosomes or proteasomes where they are destroyed (Fig 2). They suggest that both pathways are less efficient in degrading FGFR3 receptors bearing the achondroplasia mutation compared to normal FGFR3 receptors. It is not known how the receptors are directed into these two pathways, however we discovered that the proteasomal pathway is modulated by the molecular chaperone Hsp90, which binds to and protects the receptor, especially the mutant receptor, from degradation. We are exploring strategies to interfere with the Hsp90 chaperone function as a means to selectively promote degradation of mutant FGFR3 in cell and mouse models of achondroplasia.
As a transmembrane receptor protein FGFR3 is anchored in the cell membrane with its extracellular domain extending away from the cell surface to interact with FGF ligands and an intracellular domain that contains its tyrosine kinase domain, which initiates signaling inside the cell.
We have recently discovered that upon activation, FGFR3 undergoes two sequential proteolytic cleavages that free its intracellular domain from its cell membrane anchorage into the cytoplasm from where it may enter the nucleus and directly influence nuclear events. We refer to the would-be nuclear signaling as non-cannonical FGFR3 signaling. We are currently investigating what functions the intracelluular fragment may have compared to the intact FGFR3 receptor, how it is transported to the nucleus, what molecules it interacts with in the nucleus and the role it may play in the pathogenesis of achondroplasia. A combination of mouse genetics, genome editing (CRISPR), biochemical and cell culture methods are being employed for these studies.
Growth Velocity Biomarkers
A major challenge to caring for growing children, especially those with bone growth disorders, is accurately measuring growth velocity. It generally involves waiting many months, typically 6 -12 months, for a child to grow and then calculating velocity from measured interim growth. We are developing a new test that measures growth plate “activity” as a surrogate for skeletal growth velocity.
As a dynamic structure, the growth plate degrades cartilage template constituents at its trailing edge as bone lengthens. We reasoned that the degradation products would reach the circulation and their relative abundance should be proportionate to the overall rate of bone growth. We have developed assays for these products, which we consider biomarkers for bone growth; and our preliminary results show a good correlation between growth rate and biomarker levels. Studies are underway to validate our initial results and determine how well the biomarkers predict growth responses of children whose growth is slowed by disease or environmental factors to corrective measures.