Exploring the role of endoplasmic reticulum stress in skeletal dysplasia

Abstract

The term skeletal dysplasia comprises a range of diseases that lead to varying degrees of skeletal deformities, causing pain and reduction in quality of life. Whilst each disease is typically rare, together they are estimated to occur in up to 1 of 5000 people. The intracellular retention of secreted proteins has been identified as one common disease mechanism in several skeletal dysplasias, including pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). Yet, the specific pathways that are involved may be mutation-, gene- or disease-specific, or subject to disease modifiers and thus require specific treatments. Several approaches to study skeletal dysplasia and the resulting endoplasmic reticulum stress in vitro, including the use of human induced pluripotent stem cells (hiPSCs), were evaluated. HT1080 fibrosarcoma cells overexpressing wild type and mutant p.D469del COMP were validated as an in vitro model system for PSACH before performing RNA sequencing and DNA methylation analysis. Additionally, several mutant COMP constructs were overexpressed in HT1080 cells to study a potential common mechanism of COMP-MED and PSACH. Whilst hiPSCs offer exciting opportunities for the in vitro study of skeletal diseases, high variation during differentiation protocols rendered them unsuitable for this study. DNA methylation analysis revealed an unexpectedly large number of differentially methylated CpG sites, whilst RNA sequencing of D469del COMP cells revealed an increase in inflammatory signalling, and a marked upregulation of MMP9 and GALNT18. Elevated MMP9 expression correlated with increased extracellular MMP9 activity. This was also found in cell lines overexpressing other disease-causing COMP proteins, but not in a cell model of MATN3-MED. The data presented in this thesis indicates that COMP mutations act via a common mechanism that is distinct from MED-causing mutations in MATN3. It also provides evidence that MMP9 could be a marker for COMP-caused stress in vitro and thus facilitate future drug screenings.