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Horton Laboratory

William Horton, MD

 

Senior Investigator
Director of Research
,
Portland Shriners Research Center
Professor of Molecular & Medical Genetics,
Oregon Health & Science University

"Cellular Modulation of Growth Plate Regulatory Signals"

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 differentiatng cells synthesize matrix and facilitate its maturation into a functional template and a trailing edge where the template is degraded and replaced by bone. 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. Our experimental approach utilizes a wide variety of biochemical, molecular genetic, immunologic, molecular biology and cell biology 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.

Influence of cartilage matrix on growth plate function 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 are being 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 invesigated in real time in living cells and animals. 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.

Fates of cells in the mammalian growth plate Bone growth can ultimately be explained in terms of the fates of individual growth plate cells. After they are 'born' at the leading edge of the growth plate, they theoretically have options to enter the cell cycle, to engage in chondrogenesis, to enter the apoptotic pathway, to terminally differentiate and even to transdifferentiate into osteoblasts. The decisions that cells make en masse have profound effects on formation and maturation of the template and therefore on bone growth. However, relatively little is actually known about how these decisions are made, the extent to which they are coordinated and the functional impacts of the different fates on growth plate function. We are using conventional methods and live-cell confocal microscopy to explore the cell cycle progression, apoptosis, terminal differentiation and transdifferentiation in the growth plates from transgenic reporter mice specific for these cell fates.

FGFR3 signaling pathways in the growth plate

Activating mutations of FGFR3 (fibroblast growth factor receptor 3) are responsible for the most common human chondrodysplasias (the achondroplasias). From this observation and from analysis of FGFR3 null mice, it is clear that FGFR3 signals inhibit bone growth. We have helped to show that signals transmitted through STAT1 and the antimitotic factor, p21CIP1/WAF1, contribute to the inhibitory effects of FGFR3. Now we are searching for other pathways used by growth plate chondrocytes to transmit FGFR3 signals. Our goal is to define comprehensively the nature and local consequences of FGFR3 signals in the mammalian growth plate. It is anticipated that this will lead to identification of agents that will safely block transmission of unwanted signals and to new strategies for treatment of the human achondroplasias.

Much of our recent work has focused on defective degradation of mutant FGFR3 receptors as a mechanism to amplify FGFR3 growth inhibitory signals. We 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 where they are destroyed. Addition of ubiquitin to the activated receptors by the adaptor protein – c-Cbl – serves as a “targeting signal” to direct the receptors to lysosomes. Activated FGFR3 receptors bearing achondroplasia mutations are not ubiquitinated properly and as a result escape degradation, accumulate and continue to propagate signals.

Recent observations suggest that the activated mutant receptors reside in endosomes that reflect disturbed packaging of mutant receptors into multivesicular bodies during lysosomal targeting and that these endosomes move regularly between centrosomes and the cell periphery on microtubules. We are trying to understand the relationship between defective FGFR3 ubiquitination, disturbed lysosomal targeting and mircrotuble-mediated endosomal trafficking; and we are exploring how these disturbances might be ameliorated therapeutically, such as through the use of FGFR3-selective tyrosine kinase inhibitors.

Fig: Endosomes containing mutant FGFR3-GFP counterstained for vimentin.

 

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