Session V Abstracts

Session V: Bone Development,
Repair and Related Disorders II

T. Michael Underhill, B.Sc., PhD, Assistant Professor, University of Western Ontario, School of Dentistry, Division of Oral Biology

“Importance of RAR-Mediated Gene Repression in Skeletal Development”

T. Michael Underhill, Lisa M. Hoffman and Andrea D Weston, Department of Physiology and School of Dentistry, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada.

Development of the appendicular skeleton relies on a complex interplay between multiple signaling pathways to coordinate condensation and differentiation of chondroprogenitors. Although the pheonotypic changes associated with chondroblast differentiation have been well characterized, much less is known about the mechanisms underlying these changes. Retinoid excess has been associated with congenital malformations of the skeleton, premature closure of the growth plate and reduced bone mass, indicating that many of the stages within the skeletogenic program appear to be affected by retinoids. To better understand the role of retinoids in skeletogenesis we have used a variety of approaches to assess retinoic acid receptor function in chondrogenesis. Overexpression of a weak constitutively form of the retinoic acid receptor, RSR, in the limbs of transgenic mice causes appendicular skeletal defects due to an interference of chondrogenesis (J. Cell Biol. 136:445-457). Analysis of these animals shows that transgene expression prevents chondroblast differentiation, maintaining a prechondrogenic phenotype, even in response to bone morphogenetic proteins (BMPs) (Weston et al., J. Cell Biol. 148:679-690). Subsequently, we have established a close association between RAR activity and the transcriptional activity of SOX9, a transcription factor required for cartilage formation. Specifically, inhibition of RAR-mediated signaling in primary cultures of mouse limb mesenchyme, results Sox9 expression and activity. This induction is attenuated by the histone deacetylase inhibitor, TSA and by co-expression of a dominant-negative N-CoR-1, indicating an unexpected requirement for RAR-mediated repression in skeletal progenitor differentiation. These results will be presented along with additional findings that provide a molecular framework for understanding the processes underlying the commitment and differentiation of chondrocytes.

This research was supported by grants to T.NM.U. from the Canadian Institutes of Health Research and the Canadian Arthritis Network.

Yoshihiko Yamada, BS, MS, PhD, Chief, Molecular Biology Section, Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental Research, National Institutes of Health

“Perlecan mutations in mice and humans: critical role of perlecan in skeletal development and diseases”

Perlecan has been identified as a major heparan sulfate proteoglycan in basement membrane and in some other extracellular matrices. The perlecan gene (HSPG) is more than 100 kb in size with at least 94 exons and encodes a ~400-kDa protein core with three glycosaminoglycan chains covalently attached. Perlecan has various biological activities, such as influencing cell growth and differentiation by modulating cellular signaling and by interacting with matrix and with growth factors. We and other groups previously created gene knockout mice for the perlecan gene in order to determine the role of perlecan in development. The perlecan-null mice showed chondrodysplasia, with micromelia, a narrow thorax, dyssegmental ossification of the spine, craniofacial abnormalities, and, in 6% of mice exencephaly. The radiographic, clinical, and chondro-osseous morphology of the mice are remarkably similar to a lethal autosomal rexcessive disorder in humans, dyssegmental dysplasia, Silverman-Handmaker type (DDSH). Individuals have a flat face, micrognathia, cleft palate, reduced joint mobility, and frequently have a encephalocoele. We identified a homozygous 89-bp duplication in exon 34 of HSPG2 in a pair of siblings with DDSH born to consanguineous parents and heterozygous point mutations in the 5’ donor site of intron 52 and in the middle of exon 73 in the third unrelated patient, causing skipping of the entire exons 52 and 73 of the HSPG2 transcript, respectively. These mutations are predicted to cause a frameshift, resulting in a truncated protein core. The cartilage matrix from the patients stained poorly with antibody perlecan. Truncated perlecan was not secreted by the patient fibroblasts. Thus DDSH is caused by a functional null mutation of perlecan. These findings demonstrate the critical role of perlecan in cartilage development.

Through linkage mapping and a positional candidate approach, Nichole et al. identified homozygous mutations in HSPG2 of two SJS (Schwartz-Jampel syndrome) patients. SJS is a rare autosomal recessive skeletal dysplasia associated with myotonia. Patients with SJS survive and show much milder phenotypes compared to DDSH. However, the mechanisms causing the phenotype are unknown. We identified five different mutations that resulted in various forms of perlecan in three unrelated SJS patients. Heterozygous mutations in two SJS patients produced either truncated perlecan that lacked domain V or significantly reduced levels of wild type perlecan. The third patient has a homozygous 7kb-deletion hat resulted in producing reduced amounts of nearly full-length perlecan. Thus partial functional mutations of HSPG2 cause SJS.

Perlecan is present in the muscle BM and enriched at the neuromuscular junction (NMJ). To define the role of perlecan in NMJ function in vivo, we examined expression of molecules clustering at the NMJ of perlecan-null mice, which we previously created. We observed that most of molecules localized at normal NMJ, such as acetylcholine receptor (AchR), _- and_-dystroglycans, utrophin, rapsin, and agrin, were concentrated at the NMJ of perlecan-null mice. However, acethylcholinesterase (AchE) was completely absent at the perlecan-null NMJ, whereas it co-localized with perlecan and other clustered molecules at normal NMJ. Thus, it is likely perlecan is the unique acceptor molecule for AchE at the NMJ and that the enrichment of AchE at the synapse depends entirely on the presence of its binding partner, perlecan. Our results confirm that perlecan binds AchE and may explain the poor movement of newborn perlecan-null mice and the myotonia phenotype of SJS.

Fred S. Kaplan, MD, Professor of Orthopaedic Molecular Medicine, Chief, Division of Metabolic Bone Diseases and Molecular Orthopaedics, Director of the Center for Research in FOP and Related Diseases, University of Pennsylvania School of Medicine, University of Pennsylvania Medical Center, Hospital of the University of Pennsylvania

“MHS: Multiple Hereditary Skeletons - An Endochondral Conundrum”

Frederick S. Kaplan, MD; Lourdes Serrano de la Pena, PhD; Jaimo Ahn, PhD; Paul C. Bilings, PhD; and Eilen M. Shore, PhD Departments of Orthopaedic Surgery, Medicine and Genetics, The University of Pennsylvania School of Medicine, Philadelphia, PA

Fibrodysplasia ossificans progressiva (FOP) is an autosomal dominant disorder that is characterized by congenital malformation of the great toes and by post-natal episodes of heterotopic endochondral ossification that lead to the formation of a heterotopic skeleton. In FOP patients, the process of heterotopic endochondral ossification and the resulting ectopic bone appear normal; the timing and location of induction of the bone formation are abnormal.

Given the role of BPM signaling in the induction of bone formation, alterations in the BMP signaling pathway were hypothesized to be involved in the pathogenesis of FOP. The first molecular insights into FOP arose from observations of increased BMP4 mRNA and protein expression in cells obtained from FOP patients, suggesting the presence of altered BMP4 regulation and/or signal transduction.

Our recent data indicates that the BMP4 signaling pathway is dysregulated in the cells of patients who have FOP. We have found that FOP cells fail to properly regulate ambient concentrations of BMP4 and fail to appropriately regulate the transcription of BMP pathway genes such as for BMP4 antagonists. Recent preliminary data indicates that the Type IA BMP receptor (BMPRIA) is six to 10 fold more abundant on the surface of FOP cells than control cells and may be constitutively active in FOP cells, while the expression of the Type IB BMP receptor (BMPRIB) is reciprocally downregulated in these cells. These data are consistent with developmental studies that show that postnatal over- expression of BMPRIA can lead to heterotopic ossification and that embryonic under-expression of BMPRIB can lead to digital malformations that closely mimic those seen in patients who have FOP. There are no mutations in the genes encoding BMP4, multiple BMP4 antatgonists, or the BMP receptors in FOP patients. Taken together, these data allow us to hypothesize that a primary defect may exist in the BMP4 signaling pathway in FOP cells that leads to the overabundance of BMPRIA on the cell membrane, to constitutive activation of BMPRIA and to the post-natal molecular and cellular pathology of FOP.