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.

Novel type II collagen reporter mice: New tool for assessing collagen 2α1 expression in vivo and in vitro.

We report the generation of a new mouse strain harboring a Col2-pd2EGFP reporter transgene; pd2EGFP has a much shorter half-life than EGFP, making it a near real-time reporter for Col2α1 expression in vivo and in vitro. In the post-natal growth plate, pd2EGFP fluorescence was expressed in almost all proliferative chondrocytes and in some hypertrophic chondrocytes based on localization with type X collagen. In articular cartilage, pd2EGFP fluorescence diminished over time, nicely illustrating the decrease of type II collagen synthesis in articular chondrocytes during growth. Monolayers of FACS-sorted chondrocytes from P1-2 mice showed faster loss of pd2EGFP compared to EGFP, reflecting rapid chondrocyte de-differentiation. High-density culture of FACS-pd2EGFP- growth plate chondrocytes revealed the typical temporal expression pattern in which type II collagen preceded type X collagen matrix deposition. The Col2-pd2EGFP reporter mouse will be a valuable tool for studies of growth plate chondrocyte biology.

Sprouty 2 disturbs FGFR3 degradation in thanatophoric dysplasia type II: a severe form of human achondroplasia.

Thanatophoric dysplasia is a member of the achondroplasia family of human skeletal dysplasias, which result from FGFR3 mutations that exaggerate this receptor's inhibitory influence on chondrocyte proliferation and differentiation in the skeletal growth plate. We have previously reported that defective lysosomal degradation of activated receptor contributes to the gain-of-function of the mutant FGFR3. We now provide evidence that this disturbance is mediated by the receptor's kinase activity and involves constitutive induction and activation of Spry2. Our findings suggest that activated Spry2 may interfere with c-Cbl-mediated ubiquitination of FGFR3 by sequestering c-Cbl. They provide novel insight into the pathogenesis of this group of human skeletal dysplasias and identify a mechanism that potentially could be targeted therapeutically.

Transient dimerization and interaction with ERGIC-53 occur in the fibroblast growth factor receptor 3 early secretory pathway.

The fibroblast growth factor receptor 3 (FGFR3) secretory pathway includes N-linked glycosylation in the endoplasmic reticulum where a stringent quality control system ensures that only correctly folded receptor reaches the cell surface from where mature-functional FGFR3 signals upon ligand-mediated dimerization. We have previously shown that the increased kinase activity associated with FGFR3 bearing the thanatophoric dysplasia type II (TDII) mutation hampers its maturation, enabling the receptor to signal from the endoplasmic reticulum. Here we investigate if this biosynthetic disturbance could be explained by premature dimerization of the receptor. Our observations show that a limited fraction of the immature high-mannose, mutant receptor dimerizes in the early secretory pathway, as does the immature wild type FGFR3. In contrast, the mature fully glycosylated wild type receptor reaches the cell surface as monomer suggesting that dimerization is a transient event. The kinase activity of mutant FGFR3 is not required for dimerization to occur, although it increases dimerization efficiency. Furthermore, mutant FGFR3 trans-phosphorylates the immature wild type receptor indicating that dimerization occurs in the endoplasmic reticulum. Visualization of protein interaction inside the secretory pathway confirms receptor dimerization. In addition, it shows that both wild type and TDII FGFR3 interact with the mannose-specific lectin ERGIC-53. We conclude that transient dimerization is an obligatory step in FGFR3 biosynthesis acting as a pre-assembly quality control mechanism. Furthermore, the TDII/ERGIC-53 complex formation may function as a checkpoint for FGFR3 sorting downstream the endoplasmic reticulum. These findings have implications for understanding the pathogenesis of FGFR3-related disorders.

A degradation fragment of type X collagen is a real-time marker for bone growth velocity.

Despite its importance as a key parameter of child health and development, growth velocity is difficult to determine in real time because skeletal growth is slow and clinical tools to accurately detect very small increments of growth do not exist. We report discovery of a marker for skeletal growth in infants and children. The intact trimeric noncollagenous 1 (NC1) domain of type X collagen, the marker we designated as CXM for Collagen X Marker, is a degradation by-product of endochondral ossification that is released into the circulation in proportion to overall growth plate activity. This marker corresponds to the rate of linear bone growth at time of measurement. Serum concentrations of CXM plotted against age show a pattern similar to well-established height growth velocity curves and correlate with height growth velocity calculated from incremental height measurements in this study. The CXM marker is stable once collected and can be accurately assayed in serum, plasma, and dried blood spots. CXM testing may be useful for monitoring growth in the pediatric population, especially responses of infants and children with genetic and acquired growth disorders to interventions that target the underlying growth disturbances. The utility of CXM may potentially extend to managing other conditions such as fracture healing, scoliosis, arthritis, or cancer.

Collagen type XII and versican are present in the early stages of cartilage tissue formation by both redifferentating passaged and primary chondrocytes.

Current approaches to cartilage tissue engineering require a large number of chondrocytes. Although chondrocyte numbers can be expanded in monolayer culture, the cells dedifferentiate and unless they can be redifferentiated are not optimal to use for cartilage repair. We took advantage of the differential effect of culture conditions on the ability of passaged and primary chondrocytes to form cartilage tissue to dissect out the extracellular matrix (ECM) molecules produced and accumulated in the early stages of passaged cell cartilage tissue formation as we hypothesized that passaged bovine cells that form cartilage accumulate a pericellular matrix that differs from cells that do not form cartilage. Twice passaged bovine chondrocytes (P2) (cartilage forming), or as a control primary chondrocytes (P0) (which do not generate cartilage), were cultured on three-dimensional membrane inserts in serum-free media. P2 redifferentiation was occurring during the first 8 days as indicated by increased expression of the chondrogenic genes Sox9, collagen type II, aggrecan, and COMP, suggesting that this is an appropriate time period to examine the ECM. Mass spectrometry showed that the P2 secretome (molecules released into the media) at 1 week had higher levels of collagen types I, III, and XII, and versican while type II collagen and COMP were found at higher levels in the P0 secretome. There was increased collagen synthesis and retention by P2 cells compared to P0 cells as early as 3 days of culture. Confocal microscopy showed that types XII, III, and II collagen, aggrecan, versican, and decorin were present in the ECM of P2 cells. In contrast, collagen types I, II, and III, aggrecan, and decorin were present in the ECM of P0 cells. As primary chondrocytes grown in serum-containing media, a condition that allows for the generation of cartilage tissue in vitro, also accumulate versican and collagen XII, this study suggests that these molecules may be necessary to provide a microenvironment that supports hyaline cartilage formation. Further study is required to determine if these molecules are also accumulated by passaged human chondrocytes and their role in promoting hyaline cartilage formation.

Confocal/TEM overlay microscopy: a simple method for correlating confocal and electron microscopy of cells expressing GFP/YFP fusion proteins.

Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-microm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.

Secretion of cartilage oligomeric matrix protein is affected by the signal peptide.

Cartilage oligomeric matrix protein (COMP) is a secreted glycoprotein found in the extracellular matrices of skeletal tissues. Mutations associated with two human skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia, disturb COMP secretion leading to intracellular accumulation of mutant COMP, especially in chondrocytes. Here we show that the manifestation of this secretory defect is dramatically influenced by the signal peptide that targets COMP for secretion. The comparison of wild type and mutant COMP secretion directed by the COMP or BM40 signal peptide in HEK-293 cells and rat chondrosarcoma cells revealed that the BM40 signal peptide substantially enhances secretion of mutant COMP that accumulates in endoplasmic reticulum-like structures when targeted by its own signal peptide. Additionally, we demonstrate that mutant COMP forms mixed pentamers with wild type COMP. Our findings suggest that the secretory defect in pseudoachondroplasia and multiple epiphyseal dysplasia is not specific for chondrocytes, nor does it require interaction of mutant COMP with other matrix proteins prior to transport from the cell. They also imply a previously unappreciated role for the signal peptide in the regulation of protein secretion beyond targeting to the endoplasmic reticulum.

Defective lysosomal targeting of activated fibroblast growth factor receptor 3 in achondroplasia.

Mutations of fibroblast growth factor receptor 3 (FGFR3) are responsible for achondroplasia (ACH) and related dwarfing conditions in humans. The pathogenesis involves constitutive activation of FGFR3, which inhibits proliferation and differentiation of growth plate chondrocytes. Here we report that activating mutations in FGFR3 increase the stability of the receptor. Our results suggest that the mutations disrupt c-Cbl-mediated ubiquitination that serves as a targeting signal for lysosomal degradation and termination of receptor signaling. The defect allows diversion of actively signaling receptors from lysosomes to a recycling pathway where their survival is prolonged, and, as a result, their signaling capacity is increased. The lysosomal targeting defect is additive to other mechanisms proposed to explain the pathogenesis of ACH.