William A. Horton

Expression of the human chondrocyte phenotype in vitro

SummaryWe report a culture scheme in which human epiphyseal chondrocytes lose their differentiated phenotype in monolayer and subsequently reexpress the phenotype in an agarose gel. The scheme is based on a method using rabbit chondrocytes. Culture in monolayer allowed small quantities of cells to be amplified and provided a starting point to study expression of the differentiated human chondrocyte phenotype. The cells cultured in monolayer produced type I procollagen, fibronectin, and small noncartilaginous proteoglycans. Subsequent culture in agarose was associated with the acquisition of typical chondrocyte ultrastructural features and the synthesis of type II collagen and cartilage-specific proteoglycans. The switch from the nonchondrocyte to the differented chondrocyte phenotype occurred under these conditions between 1 and 2 wk of agarose culture and was not necessarily homogeneous throughout a culture. This culture technique will facilitate direct investigation of human disorders of cartilage that have been addressed in the past by alternative approaches.

Chondrocyte Differentiation in a Rat Mesenchymal Cell Line

We used a combination of morphologic and histochemical methods to demonstrate that rat calvaria-derived mesenchymal cells, RCJ 3.1C5.18, in culture progress through the differentiation pathway exhibited by chondrocytes in the endochondral growth plate. The cells were grown either as monolayer or suspension cultures. Subconfluent monolayer cultures did not express markers typical of chondrocyte phenotypes. However, after reaching confluency the cells formed nodules of chondrocytic cells separated by cartilage-appearing matrix and encapsulated by fibroblast-like cells. Suspension culture produced cell aggregates with similar characteristics. Matrix in both the nodules and aggregates stained for collagen Types II and XI and aggrecan, and some cells displayed a distinctive pericellular matrix that stained for Type X collagen. Mineralization was evident in older cultures. By electron microscopy, most cells in the aggregates appeared as typical chondrocytes. However, some larger cells were surrounded by a “mat” of matrix comprised of hexagonal arrays of dense nodules interconnected by a filamentous network. Immunogold localization confirmed the presence of collagen Type X in this matrix. Analysis of markers of chondrocyte differentiation and terminal differentiation over time showed that these markers were acquired sequentially over 2 weeks of culture. This model system will be useful to study the regulation of various steps in the chondrocyte differentiation pathway.

Skeletal dysplasia and defective chondrocyte differentiation by targeted overexpression of fibroblast growth factor 9 in transgenic mice

Mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human chondrodysplasias, including achondroplasia, the most common form of dwarfism in humans. From in vitro studies, the skeletal defects observed in these disorders have been attributed to constitutive activation of FGFR3. Here we show that FGF9 and FGFR3, a high‐affinity receptor for this ligand, have similar developmental expression patterns, particularly in areas of active chondrogenesis. Targeted overexpression of FGF9 to cartilage of transgenic mice disturbs postnatal skeletal development and linear bone growth. The growth plate of these mice exhibits reduced proliferation and terminal differentiation of chondrocytes similar to that observed in the human disorders. The observations provide evidence that targeted, in vivo activation of endogenous FGFR3 inhibits bone growth and demonstrate that signals derived from FGF9–FGFR3 interactions can physiologically block endochondral ossification to produce a phenotype characteristic of the achondroplasia group of human chondrodysplasias.

Fibroblast Growth Factor Receptor 3 (FGFR3) Is a Strong Heat Shock Protein 90 (Hsp90) Client IMPLICATIONS FOR THERAPEUTIC MANIPULATION

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of growth and differentiation, whose aberrant activation causes a number of genetic diseases including achondroplasia and cancer. Hsp90 is a specialized molecular chaperone involved in stabilizing a select set of proteins termed clients. Here, we delineate the relationship of Hsp90 and co-chaperone Cdc37 with FGFR3 and the FGFR family. FGFR3 strongly associates with these chaperone complexes and depends on them for stability and function. Inhibition of Hsp90 function using the geldanamycin analog 17-AAG induces the ubiquitination and degradation of FGFR3 and reduces the signaling capacity of FGFR3. Other FGFRs weakly interact with these chaperones and are differentially influenced by Hsp90 inhibition. The Hsp90-related ubiquitin ligase CHIP is able to interact and destabilize FGFR3. Our results establish FGFR3 as a strong Hsp90 client and suggest that modulating Hsp90 chaperone complexes may beneficially influence the stability and function of FGFR3 in disease.

Abnormal ossification in thanatophoric dysplasia.

Thanatophoric dysplasia (TD) is a lethal human bone dysplasia characterized by severe dwarfism. It pathogenesis is thought to involve an abnormal ossifying fibrous tissue that disrupts the skeletal growth plate. We employed a combined morphologic, immunohistochemical and biochemical approach to better define the nature of this tissue in growth plate cartilage from 15 TD fetuses and infants in whom the abnormality was observed. The tissue was organized into tufts comprised of a cap of interstitial connective tissue, a transition region containing preosteoblastic cells near the cap and osteoblastic cells near the base, and a mineralized base which formed the subchondral bone trabeculae. The morphology of the cells and the presence of type I collagen in all regions of the tufts suggested that they were foci of membraneous ossification. However, elements of cartilage matrix (type II collagen, proteoglycan, link protein) were identified in the pericellular matrix of the osteoblastic cells and adjacent osteocytic cells. These observations suggest that a peculiar form of ossification occurs in the TD growth plate and may be involved in the pathogenesis of the disorder.

Bovine achondrogenesis: Evidence for defective chondrocyte differentiation

A survey study of growth cartilage abnormalities in bovine bone dysplasias revealed that a disorder in Holstein cattle called bulldog calf closely resembles human achondrogenesis Type II. Substantial amounts of Type I collagen and other non Type II collagens were detected in the bulldog cartilage which was comprised primarily of extensive vascular canals and cells having the characteristics of hypertrophic and degenerative chondrocytes normally found in the growth plate. It is proposed that chondrocytes throughout the bulldog growth cartilage prematurely differentiate into hypertrophic cells that degenerate and predispose the cartilage to vascular invasion and the formation of cartilage canals. The presence of these canals probably accounts for most of the observed collagen abnormalities.

In-vitro Proteoglykansynthese in redifferenzierten Chondrozyten

Human chondrocytes growing in monolayer cultures de-differentiate and produce type I collagen. They re-differentiate and resume their in-vivo characteristics (including the production of type II collagen) when cultured in an agarose-gel. To characterize the modulated cells in more detail, biochemical studies were performed in chondrocytes suspended in agarose for 1 to 3 weeks.