Cysteine rich with EGF-like domains 2 (Creld2) is a novel endoplasmic reticulum (ER) resident molecular chaperone that has been recently implicated in the ER stress signalling response (ERSS) and the unfolded protein response (UPR). Global transcriptomic data derived from in vivo mouse models of rare chondrodysplasias; Multiple Epiphyseal Dysplasia (MED Matn3 p.V194D) and Metaphyseal chondrodysplasia type Schmid (MCDS Col10a1 p.N617K), identified a significant upregulation in Creld2 expression in mutant chondrocytes. These chondrodysplasias share a common disease signature consisting of aberrant folding of a matrix component often as a result of inappropriate alignment of intramolecular disulphide bonds. This in turn culminates in toxic protein aggregation, intracellular retention mutant polypeptides and a classical ER stress response. The aim of this study was to further analyse the function of Creld2 in cartilage development and chondrodysplasias in which endochondral bone growth is perturbed. Protein disulphide isomerases (PDIAs) were amongst the most up-regulated genes in the MED and MCDS mouse models, consistent with the prolonged exposure of normally 'buried' cysteine residues. This led to the hypothesis that Creld2 was functioning as a novel PDI-like oxidoreductase to assist in the correct folding and maturation of aggregated misfolded polypeptide chains through REDOX regulated thiol disulphide exchange. A series of Creld2-CXXA substrate trapping mutants were generated in order to determine whether Creld2 possessed inherent isomerase activity. Here potential substrates interacting with Creld2 were 'trapped' as mixed disulphide intermediates, then isolated by immunoprecipitation and identified by mass spectrometry analysis. It was demonstrated that Creld2 possessed a catalytic active CXXC motif in its N-terminus that enabled the molecular chaperone to participate in REDOX regulated thiol disulphide exchange with at least 20 potential substrates including; laminin (alpha3,β3,γ2), thrombospondin 1, integrin alpha3 and type VI collagen. There was also numerous co-chaperones and foldases thought to be part of a specialised protein-protein interactome (PPI) for folding nascent polypeptides translocating the ER lumen. Moreover, co-immunoprecipitation experiments supported a protein-protein interaction between Creld2 and mutant matrilin-3, thereby inferring a potential chondro-protective role in resolving non-native disulphide bonded aggregates in MED. An established biochemical approach was employed to test the hypothesis that all MATN3-MED disease causing mutations have a generic cellular response to the β-sheet V194D mutation, consisting of intracellular retention, protein aggregation and ER stress induction. Several missense mutations were selected for analyses which encompassed a spectrum of disease severity and included examples of both β-sheet and alpha helical mutations. It was possible to define a reliable and reproducible assay for categorising MATN3 missense mutations into pathological or benign based on these basic parameters. This study was extended further to determine whether there were common pathological mechanisms behind MED and Bethlem myopathy (BM) caused by missense mutations in von Willebrand Factor A domain (vWF-A) containing proteins (matrilin-3 and type VI collagen respectively). We chose to compare and contrast the effects of an archetypal MATN3-MED causing mutation (R121W) with the equivalent COL6A2-BM causing mutation (R876H). These mutations compromised protein folding and maturation, resulting in the familiar disease profile of intracellular retention, protein aggregation and an ER stress response in an artificial overexpression system. However, the mutant C2 domain was efficiently targeted for degradation whilst mutant matrilin-3 vWF-A domain appeared to be resistant to these molecular processes.Molecular genetics was employed to study the role of Creld2 in vivo. Creld2-/- null mice (both global and conditional) were generated to directly examine the role of Creld2 in endochondral bone growth. Global knock-out mice were viable with no overt phenotype at birth. However, female Creld2-/- null mice showed a significant reduction in body weight and tibia bone length at 3 weeks of age. A cartilage specific knock-out was generated to determine whether these skeletal abnormalities were attributed to a systemic or a direct effect on cartilage development. [Creld2Flox/Flox Col2Cre (+)] demonstrated a severe chondrodysplasia with significantly reduced body weight and long bone growth compared to control littermates. Morphological and histochemical analysis of mutant growth plates revealed gross disorganisation of the chondrocyte columns with extensive regions of hypocellularity. These pathological features were confirmed to be the result of reduced chondrocyte proliferation and increased/spatially dysregulated apoptosis throughout all zones of differentiation. Taken together, these data provide evidence that Creld2 possesses isomerase activity and exhibits distinct substrate specificity. Furthermore, Creld2 has a fundamental role in post-natal cartilage development and chondrocyte differentiation in the growth plate.

Height is a complex physical trait that displays strong heritability. Adult height is related to length of the long bones, which is determined by growth at the epiphyseal growth plate. Longitudinal bone growth occurs via the process of endochondral ossification, where bone forms over the differentiating cartilage template at the growth plate. Estrogen plays a major role in regulating longitudinal bone growth and is responsible for inducing the pubertal growth spurt and fusion of the epiphyseal growth plate. However, the mechanism by which estrogen promotes epiphyseal fusion is poorly understood. It has been hypothesised that estrogen functions to regulate growth plate fusion by stimulating chondrocyte apoptosis, angiogenesis and bone cell invasion in the growth plate. Another theory has suggested that estrogen exposure exhausts the proliferative capacity of growth plate chondrocytes, which accelerates the process of chondrocyte senescence, leading to growth plate fusion. The overall objective of this study was to gain a greater understanding of the molecular mechanisms behind estrogen-mediated growth and height attainment by examining gene regulation in chondrocytes and the role of some of these genes in normal height inheritance. With the heritability of height so well established, the initial hypothesis was that genetic variation in candidate genes associated with longitudinal bone growth would be involved in normal adult height variation. The height-related genes FGFR3, CBFA1, ER and CBFA1 were screened for novel polymorphisms using denaturing HPLC and RFLP analysis. In total, 24 polymorphisms were identified. Two SNPs in ER (rs3757323 C>T and rs1801132 G>C) were strongly associated with adult male height and displayed an 8 cm and 9 cm height difference between homozygous genotypes, respectively. The TC haplotype of these SNPs was associated with a 6 cm decrease in height and remarkably, no homozygous carriers of the TC haplotype were identified in tall subjects. No significant associations with height were found for polymorphisms in the FGFR3, CBFA1 or VDR genes. In the epiphyseal growth plate, chondrocyte proliferation, matrix synthesis and chondrocyte hypertrophy are all major contributors to long bone growth. As estrogen plays such a significant role in both growth and final height attainment, another hypothesis of this study was that estrogen exerted its effects in the growth plate by influencing chondrocyte proliferation and mediating the expression of chondrocyte marker genes. The examination of genes regulated by estrogen in chondrocyte-like cells aimed to identify potential regulators of growth plate fusion, which may further elucidate mechanisms involved in the cessation of linear growth. While estrogen did not dramatically alter the proliferation of the SW1353 cell line, gene expression experiments identified several estrogen regulated genes. Sixteen chondrocyte marker genes were examined in response to estrogen concentrations ranging from 10-12 M to 10-8 M over varying time points. Of the genes analysed, IHH, FGFR3, collagen II and collagen X were not readily detectable and PTHrP, GHR, ER, BMP6, SOX9 and TGF1 mRNAs showed no significant response to estrogen treatments. However, the expression of MMP13, CBFA1, BCL-2 and BAX genes were significantly decreased. Interestingly, the majority of estrogen regulated genes in SW1353 cells are expressed in the hypertrophic zone of the growth plate. Estrogen is also known to regulate systemic GH secretion and local GH action. At the molecular level, estrogen functions to inhibit GH action by negatively regulating GH signalling. GH treated SW1353 cells displayed increases in MMP9 mRNA expression (4.4-fold) and MMP13 mRNA expression (64-fold) in SW1353 cells. Increases were also detected in their respective proteins. Treatment with AG490, an established JAK2 inhibitor, blocked the GH mediated stimulation of both MMP9 and MMP13 mRNA expression. The application of estrogen and GH to SW1353 cells attenuated GH-stimulated MMP13 levels, but did not affect MMP9 levels. Investigation of GH signalling revealed that SW1353 cells have high levels of activated JAK2 and exposure to GH, estrogen, AG490 and other signalling inhibitors did not affect JAK2 phosphorylation. Interestingly, AG490 treatment dramatically decreased ERK2 signalling, although GH did stimulate ERK2 phosphorylation above control levels. AG490 also decreased CBFA1 expression, a transcription factor known to activate MMP9 and MMP13. Finally, GH and estrogen treatment increased expression of SOCS3 mRNA, suggesting that SOCS3 may regulate JAK/STAT signalling in SW1353 cells. The modulation of GH-mediated MMP expression by estrogen in SW1353 cells represents a potentially novel mechanism by which estrogen may regulate longitudinal bone growth. However, further investigation is required in order to elucidate the precise mechanisms behind estrogen and GH regulation of MMP13 expression in SW1353 cells. This study has provided additional evidence that estrogen and the ER gene are major factors in the regulation of growth and the determination of adult height. Newly identified polymorphisms in the ER gene not only contribute to our understanding of the genetic basis of human height, but may also be useful in association studies examining other complex traits. This study also identified several estrogen regulated genes and indicated that estrogen modifies the expression of genes which are primarily expressed in the hypertrophic region of the epiphyseal growth plate. Furthermore, synergistic studies incorporating GH and estrogen have revealed the ability of estrogen to attenuate the effects of GH on MMP13 expression, revealing potential pathways by which estrogen may modulate growth plate fusion, longitudinal bone growth and even arthritis.

There is an understanding among biological anthropologists that long bone epiphyseal shape is highly regulated by genetic and biomechanical factors. Conversely, long bone diaphyseal geometry and robusticity have been shown to respond to activity in life. The current study examined the assumption of epiphyseal consistency by exploring the relationship between a well established bony response to activity (Cross-Sectional Geometry) and shape change among the proximal humerus and femur. Long bone samples were taken from four hunter-gatherer populations: the Andaman Islanders, the Indian Knoll, Point Hope Alaskans, and the Sadlermiut. Shape was measured through landmark configurations placed on the proximal end of a total of 91 humeri and 84 femora. Cross-sectional Geometry measures (J) were taken from each specimen, as well. Principal Component Analyses were conducted on the landmark shape data to determine where the shape variation was occurring among the sample. These Principal Components were then compared via Bivariate Regression to the J values taken from the diaphysis. Significant relationships occurred between the development of the lesser tubercle and an increase in J among the humerus sample. Significant relationships were also found among the femur sample; as when J increased the proximal epiphyses were more likely to be more gracile, and the space between the femoral head and the greater trochanter increased. The humerus results indicated a more robust proximal epiphysis in groups with activities that rely heavily on the upper body, such as rowing, swimming, and harpooning. The femur results were more complex, as the relationship between activity and proximal shape is likely heavily influenced by a genetically predetermined body shape. These results indicated that there is a relationship between activity and proximal epiphyseal shape, but that it, like all relationships, is complex, and comprised of multiple factors. Ultimately, proximal long bone shape is the result of multiple influences including, activity, genetics, population adaptation, health, and many more. Future research should focus on determining if the relationship between activity and shape exists among other populations, and when and where it begins during growth and development. / Graduate

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