Peter A. Bell

Armet/Manf and Creld2 are components of a specialized ER stress response provoked by inappropriate formation of disulphide bonds: implications for genetic skeletal diseases

Mutant matrilin-3 (V194D) forms non-native disulphide bonded aggregates in the rER of chondrocytes from cell and mouse models of multiple epiphyseal dysplasia (MED). Intracellular retention of mutant matrilin-3 causes endoplasmic reticulum (ER) stress and induces an unfolded protein response (UPR) including the upregulation of two genes recently implicated in ER stress: Armet and Creld2. Nothing is known about the role of Armet and Creld2 in human genetic diseases. In this study, we used a variety of cell and mouse models of chondrodysplasia to determine the genotype-specific expression profiles of Armet and Creld2. We also studied their interactions with various mutant proteins and investigated their potential roles as protein disulphide isomerases (PDIs). Armet and Creld2 were up-regulated in cell and/or mouse models of chondrodysplasias caused by mutations in Matn3 and Col10a1, but not Comp. Intriguingly, both Armet and Creld2 were also secreted into the ECM of these disease models following ER stress. Armet and Creld2 interacted with mutant matrilin-3, but not with COMP, thereby validating the genotype-specific expression. Substrate-trapping experiments confirmed Creld2 processed PDI-like activity, thus identifying a putative functional role. Finally, alanine substitution of the two terminal cysteine residues from the A-domain of V194D matrilin-3 prevented aggregation, promoted mutant protein secretion and reduced the levels of Armet and Creld2 in a cell culture model. We demonstrate that Armet and Creld2 are genotype-specific ER stress response proteins with substrate specificities, and that aggregation of mutant matrilin-3 is a key disease trigger in MED that could be exploited as a potential therapeutic target.

Genotype to phenotype correlations in cartilage oligomeric matrix protein associated chondrodysplasias

Pseudoachondroplasia (PSACH) and autosomal dominant multiple epiphyseal dysplasia (MED) are chondrodysplasias resulting in short-limbed dwarfism, joint pain and stiffness and early onset osteoarthritis. All PSACH, and the largest proportion of MED, result from mutations in cartilage oligomeric matrix protein (COMP). The first mutations in COMP were identified in 1995 in patients with both PSACH and MED and subsequently there has been over 30 publications describing COMP mutations in at least 250 PSACH–MED patients. However, despite these discoveries, a methodical analysis of the relationship between COMP mutations and phenotypes has not been undertaken. In particular, there has, to date, been little correlation between the type and location of a COMP mutation and the resulting phenotype of PSACH or MED. To determine if genotype to phenotype correlations could be derived for COMP, we collated 300 COMP mutations, including 25 recently identified novel mutations. The results of this analysis demonstrate that mutations in specific residues and/or regions of the type III repeats of COMP are significantly associated with either PSACH or MED. This newly derived genotype to phenotype correlation may aid in determining the prognosis of PSACH and MED, including the prediction of disease severity, and in the long term guide genetic counselling and contribute to the clinical management of patients with these diseases.

Abnormal Chondrocyte Apoptosis in the Cartilage Growth Plate is Influenced by Genetic Background and Deletion of CHOP in a Targeted Mouse Model of Pseudoachondroplasia

Pseudoachondroplasia (PSACH) is an autosomal dominant skeletal dysplasia caused by mutations in cartilage oligomeric matrix protein (COMP) and characterised by short limbed dwarfism and early onset osteoarthritis. Mouse models of PSACH show variable retention of mutant COMP in the ER of chondrocytes, however, in each case a different stress pathway is activated and the underlying disease mechanisms remain largely unknown. T585M COMP mutant mice are a model of moderate PSACH and demonstrate a mild ER stress response. Although mutant COMP is not retained in significant quantities within the ER of chondrocytes, both BiP and the pro-apoptotic ER stress-related transcription factor CHOP are mildly elevated, whilst bcl-2 levels are decreased, resulting in increased and spatially dysregulated chondrocyte apoptosis. To determine whether the abnormal chondrocyte apoptosis observed in the growth plate of mutant mice is CHOP-mediated, we bred T585M COMP mutant mice with CHOP-null mice to homozygosity, and analysed the resulting phenotype. Although abnormal apoptosis was alleviated in the resting zone following CHOP deletion, the mutant growth plates were generally more disorganised. Furthermore, the bone lengths of COMP mutant CHOP null mice were significantly shorter at 9 weeks of age when compared to the COMP mutant mice, including a significant difference in the skull length. Overall, these data demonstrate that CHOP-mediated apoptosis is an early event in the pathobiology of PSACH and suggest that the lack of CHOP, in conjunction with a COMP mutation, may lead to aggravation of the skeletal phenotype via a potentially synergistic effect on endochondral ossification.

A novel transgenic mouse model of growth plate dysplasia reveals that decreased chondrocyte proliferation due to chronic ER stress is a key factor in reduced bone growth

SUMMARY Disease mechanisms leading to different forms of chondrodysplasia include extracellular matrix (ECM) alterations and intracellular stress resulting in abnormal changes to chondrocyte proliferation and survival. Delineating the relative contribution of these two disease mechanisms is a major challenge in understanding disease pathophysiology in genetic skeletal diseases and a prerequisite for developing effective therapies. To determine the influence of intracellular stress and changes in chondrocyte phenotype to the development of chondrodysplasia, we targeted the expression of the G2320R mutant form of thyroglobulin to the endoplasmic reticulum (ER) of resting and proliferating chondrocytes. Previous studies on this mutant protein have shown that it induces intracellular aggregates and causes cell stress and death in the thyroid gland. The expression and retention of this exogenous mutant protein in resting and proliferating chondrocytes resulted in a chronic cell stress response, growth plate dysplasia and reduced bone growth, without inducing any alterations to the architecture and organization of the cartilage ECM. More significantly, the decreased bone growth seemed to be the direct result of reduced chondrocyte proliferation in the proliferative zone of growth plates in transgenic mice, without transcriptional activation of a classical unfolded protein response (UPR) or apoptosis. Overall, these data show that mutant protein retention in the ER of resting and proliferative zone chondrocytes is sufficient to cause disrupted bone growth. The specific disease pathways triggered by mutant protein retention do not necessarily involve a prototypic UPR, but all pathways impact upon chondrocyte proliferation in the cartilage growth plate.

New therapeutic targets in rare genetic skeletal diseases

Introduction: Genetic skeletal diseases (GSDs) are a diverse and complex group of rare genetic conditions that affect the development and homeostasis of the skeleton. Although individually rare, as a group of related diseases, GSDs have an overall prevalence of at least 1 per 4,000 children. There are currently very few specific therapeutic interventions to prevent, halt or modify skeletal disease progression and therefore the generation of new and effective treatments requires novel and innovative research that can identify tractable therapeutic targets and biomarkers of these diseases. Areas covered: Remarkable progress has been made in identifying the genetic basis of the majority of GSDs and in developing relevant model systems that have delivered new knowledge on disease mechanisms and are now starting to identify novel therapeutic targets. This review will provide an overview of disease mechanisms that are shared amongst groups of different GSDs and describe potential therapeutic approaches that are under investigation. Expert opinion: The extensive clinical variability and genetic heterogeneity of GSDs renders this broad group of rare diseases a bench to bedside challenge. However, the evolving hypothesis that clinically different diseases might share common disease mechanisms is a powerful concept that will generate critical mass for the identification and validation of novel therapeutic targets and biomarkers.

The utility of mouse models to provide information regarding the pathomolecular mechanisms in human genetic skeletal diseases: The emerging role of endoplasmic reticulum stress (Review)

Genetic skeletal diseases (GSDs) are an extremely diverse and complex group of rare genetic diseases that primarily affect the development and homeostasis of the osseous skeleton. There are more than 450 unique and well-characterised phenotypes that range in severity from relatively mild to severe and lethal forms. Although individually rare, as a group of related genetic diseases, GSDs have an overall prevalence of at least 1 per 4,000 children. Qualitative defects in cartilage structural proteins result in a broad spectrum of both recessive and dominant GSDs. This review focused on a disease spectrum resulting from mutations in the non-collagenous glycoproteins, cartilage oligomeric matrix protein (COMP) and matrilin-3, which together cause a continuum of phenotypes that are amongst the most common autosomal dominant GSDs. Pseudoachondroplasia (PSACH) and autosomal dominant multiple epiphyseal dysplasia (MED) comprise a disease spectrum characterised by varying degrees of disproportionate short stature, joint pain and stiffness and early-onset osteoarthritis. Over the past decade, the generation and deep phenotyping of a range of genetic mouse models of the PSACH and MED disease spectrum has allowed the disease mechanisms to be characterised in detail. Moreover, the generation of novel phenocopies to model specific disease mechanisms has confirmed the importance of endoplasmic reticulum (ER) stress and reduced chondrocyte proliferation as key modulators of growth plate dysplasia and reduced bone growth. Finally, new insight into related musculoskeletal complications (such as myopathy and tendinopathy) has also been gained through the in-depth analysis of targeted mouse models of the PSACH-MED disease spectrum.

Analysis of the cartilage proteome from three different mouse models of genetic skeletal diseases reveals common and discrete disease signatures

Summary Pseudoachondroplasia and multiple epiphyseal dysplasia are genetic skeletal diseases resulting from mutations in cartilage structural proteins. Electron microscopy and immunohistochemistry previously showed that the appearance of the cartilage extracellular matrix ECM in targeted mouse models of these diseases is disrupted; however, the precise changes in ECM organization and the pathological consequences remain unknown. Our aim was to determine the effects of matrilin-3 and COMP mutations on the composition and extractability of ECM components to inform how these detrimental changes might influence cartilage organization and degeneration. Cartilage was sequentially extracted using increasing denaturants and the extraction profiles of specific proteins determined using SDS-PAGE/Western blotting. Furthermore, the relative composition of protein pools was determined using mass spectrometry for a non-biased semi-quantitative analysis. Western blotting revealed changes in the extraction of matrilins, COMP and collagen IX in mutant cartilage. Mass spectrometry confirmed quantitative changes in the extraction of structural and non-structural ECM proteins, including proteins with roles in cellular processes such as protein folding and trafficking. In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis. We demonstrated that mutation of matrilin-3 and COMP caused changes in the extractability of other cartilage proteins and that proteomic analyses of Matn3 V194D, Comp T585M and Comp DelD469 mouse models revealed both common and discrete disease signatures that provide novel insight into skeletal disease mechanisms and cartilage degradation.

Pseudoachondroplasia and Multiple Epiphyseal Dysplasia: Molecular Genetics, Disease Mechanisms and Therapeutic Targets.

Genetic skeletal diseases (GSDs) are a diverse and complex group of over 450 rare diseases that affect the development and homoeostasis of the skeleton. Although individually rare, as a group of related genetic skeletal diseases, they have an overall prevalence of at least 1 per 4000 children, which extrapolates to a minimum of 225,000 people in the European Union, and this extensive burden in pain and disability leads to poor quality of life and high healthcare costs.

Animal models and systems biology approaches for the functional validation of genetic determinants of skeletal diseases

Rare skeletal diseases are a diverse group of diseases that primarily affect development of the skeleton. There are more than 450 unique phenotypes that, although individually rare, have an overall prevalence of at least 1 per 4,000 children. Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are skeletal diseases caused by missense mutations/deletions in the genes encoding important cartilage extracellular matrix proteins (ECM), and are characterized by disproportionate short stature, joint pain and early-onset osteoarthritis. In-depth characterization of MED and PSACH mouse models has revealed that endoplasmic reticulum (ER) stress, reduced cell proliferation and abnormal ECM assembly are important pathological consequences of mutant protein expression. Ongoing work aims to consolidate data from other models of skeletal disorders using a systems biology approach as part of the EU FP7 SYBIL (Systems biology for the functional validation of genetic determinants of skeletal diseases) project, in order to gain a mechanistic understanding of disease processes and to deliver new and validated therapeutic targets. Key to delivery of new targets and therapies is the identification of relevant disease biomarkers, which will allow the monitoring of responses to therapeutic interventions. This is particularly critical for skeletal diseases, for which biopsy material is not readily accessible. We have identified differences in the extractability of a number of ECM components from the cartilage of MED and PSACH mouse models, relative to controls. The differences in extractability of these proteins (which include FACIT collagens (types XII and XIV), tenascins (C and X), and fetuin A) may represent differences in the stability of these proteins within the cartilage ECM, which might potentially be exploited for use as biomarkers of disease progression. We are currently using biochemical and mass spectrometry analysis of easily obtained biological samples such as blood, urine and cell culture medium in order to identify and validate novel biomarkers for skeletal diseases.