The regulation of collagen (I) homotrimer synthesis and its role in musculoskeletal dysfunction

Musculoskeletal diseases such as osteoarthritis, osteoporosis and soft-tissue injuries together with tissue and organ fibrosis impose a huge healthcare burden, particularly in the ageing population. Therefore understanding the regulation of abnormal collagen (I) synthesis and its role in bone and joint function is of critical importance in order to develop strategies to target these debilitating diseases.

Collagen (type I) is the most abundant structural protein in the body and is the major component of bone and joint tissues. Type I collagen forms fibres that surround cells and make tissues resilient to mechanical loading. The natural form of type I collagen can be degraded and reformed by cells using biological enzymes and this process allows skeletal tissues to adapt to changes in mechanical loading. When the tissue structure is inadequate to resist external loads, tissue injury including fractures and ruptures can occur. In the general population such problems are manifested as diseases including osteoporosis (weak bone), osteoarthritis (cartilage loss and bone overgrowth) and soft tissue injuries. Over-production of type I collagen (termed fibrosis) furthermore restricts tissue function leading to disability and increased morbidity and mortality.

Genetic and biochemical studies have found that an abnormal form of type I collagen, termed collagen (I) homotrimer, is present in both degenerative and fibrotic diseases. This abnormal collagen alters the biophysical properties of collagen fibrils and is resistant to enzymatic breakdown. Collagen (I) homotrimer may therefore affect the ability of tissues to respond to changing mechanical loads and to counteract fibrosis. The aim of this project is to determine whether relative collagen (I) mRNA (COL1A1 and COL1A2) levels direct collagen (I) homotrimer synthesis and if collagen (I) homotrimer produces an inadequate but persistent fibrillar matrix that leads to age-related musculoskeletal disease and fibrosis, or whether pathology could be accounted for by cellular stress caused by over-production of the collagen alpha-1(I) chain.

A complex series of cellular interactions normally results in collagen (I) heterotrimer but this project will test the hypothesis that increased levels of the COL1A1 mRNA overwhelms the ability of the cells to control heterotrimer synthesis and results in the concurrent production of abnormal collagen (I) homotrimer. Cells contain an elaborate system of controls that regulate gene activity and protein production and small RNA molecules termed microRNAs appear to be particularly important. MicroRNAs bind to the mRNA intermediates (between gene activity and protein production) often decreasing their effectiveness. miR-133 is known to target COL1A1 rather than COL1A2 and is less abundant in several fibroses. This project will test how increasing or decreasing miR-133 activity affects collagen (I) homotrimer synthesis and will determine whether it could be a novel target for musculoskeletal and fibrotic diseases.

To reveal how collagen fibrils containing collagen (I) homotrimer affect musculoskeletal tissues a comprehensive analysis of the structural and biomechanical alterations in hard and soft collagenous tissues will be performed in mice lacking the COL1A2 gene. Preliminary phenotyping data (IMPC) indicates that these mice have abnormal bone morphology and defects in soft collagenous tissues. A well-characterised osteogenesis imperfecta mouse model ('oim') that produces collagen (I) homotrimer along with truncated alpha-2(I) chains will be used as a control. The apparently more severe oim phenotype may result from cellular stress, therefore cellular stress will be evaluated in genetically manipulated cell cultures and mouse tissues. Cellular stress can be targeted by several pharmaceuticals so could potentially be reduced to help treat these diseases.

Type I collagen is the major structural component of vertebrate tissues. The COL1A1 and COL1A2 genes encode the alpha-1(I) and alpha-2(I) polypeptides which form collagen heterotrimers. Abnormal homotrimeric collagen (I) derived from COL1A1 alone is associated with age-related musculoskeletal diseases and fibrosis which are major contributors to morbidity and pose huge clinical and societal challenges. This project aims to determine how COL1A1 and COL1A2 mRNA levels influence collagen (I) homotrimer synthesis, the direct effect of collagen (I) homotrimer on the structure and biomechanical properties of musculoskeletal tissues and whether synthesis causes detrimental cellular stress.

The first objective will test the hypothesis that there is a finite cellular buffering mechanism controlling collagen (I) heterotrimer synthesis and determine whether miR-133 or miR-129 are negative regulators of collagen (I) homotrimer synthesis. Over-expression of COL1A1 and knock-down of COL1A2 will be carried out in osteoblasts and tendon fibroblasts. Collagen (I) mRNA and polypeptide chain ratios will be measured using qRT-PCR and metabolic labelling respectively. Transfection of osteoblasts and tendon fibroblasts with miR mimics and antagomirs will be used to determine whether miR-133/-129 are negative regulators of COL1A1, but not COL1A2 synthesis and may be suitable pharmacological targets. The second objective will examine how the structure and biomechanical properties of bone, joints and soft-tissues are altered by collagen (I) homotrimer. Biomechanical properties of bone and tendon will be assessed by 3-point bending, nanoindentation and tensile testing. Osteoporosis and osteoarthritis will be assessed by micro-computer tomography and histological scoring and correlated to collagen fibril structure and organisation. Cellular stress will be evaluated in the third objective in cell cultures and tissues by Western blotting, imaging and qRT-PCR for stress markers.

Musculoskeletal conditions as well as tissue and organ fibrosis impose a large burden on public health services. Musculoskeletal conditions in the UK account for 41% of work-related illnesses with an incidence of 0.55% and a prevalence of 1.6% in 2015/16. This resulted in a total loss of 8.8 million working days (source HSE) with associated costs to employers in sick pay (£9 billion per year) and to the state in benefits payments (£13 billion per year). In the general population the prevalence of musculoskeletal disease is much higher at 15.6% in adults, but over 30% in those over 65. (Source Arthritis Research UK Epidemiology Unit, 2011). Costs to the NHS are substantial at £260 million for consultation and hospital costs and £186 million for prescriptions.

The development of effective future treatment strategies is likely to benefit the NHS through reduced numbers of repeat patient appointments and operations and increased efficiency in service delivery. Future effective treatments are therefore likely to benefit clinicians and patients by providing increased quality of life and health span, reduced pain and disability and reduced hospital visits. Potential beneficiaries include the ageing population and those participating in sporting activities. This has the potential to lead to increased independent living in older age along with increased family and community involvement, indirectly contributing to improved cardiovascular and mental health.

This project aims to identify mechanisms by which collagen (I) homotrimer contributes to bone, joint and soft-tissue dysfunction, and to infer whether particular therapeutic strategies to target collagen (I) homotrimer may be of benefit in osteoporosis, osteoarthritis, soft-tissue injury and fibrosis. The research is therefore likely to provide commercialisation opportunities to UK industry, reduce the public health burden of musculoskeletal and fibrotic diseases and provide personal benefits of increased health span to an increasingly aged population.

The project is likely to benefit the commercial private sector through opportunities to translate the research findings. Specifically the project will determine whether microRNAs targeting COL1A1 could be a suitable pharmaceutical treatment strategy for osteoporosis, osteoarthritis and fibrosis. Through a greater understanding of the effect of collagen (I) homotrimer on tissue structure and biomechanical properties, additional targets for pharmaceutical intervention could be identified. We anticipate that this project would lead to preclinical testing of microRNA mimics or antagomirs in osteoarthritis, osteoporosis or fibrosis. Together these opportunities would benefit the UK economy either through the creation of new spin-out companies or by adding value to existing SMEs. Uptake of this technology by large pharmaceutical companies would be expected to indirectly benefit the UK through license or sale of intellectual property rights, further investment and/or job creation.

The commercial private sector will also benefit from this research through staff development and technological transfer. A previous MRC-funded post-doctoral research associate (MR/J002909/1: New Investigator Research Grant to the PI) led a team to the UK final of the Biotechnology YES competition, developed a keen interest in research commercialization and is now employed by a UK Biotechology Company. Complementary multi-disciplinary training by the project's academic co-investigators will benefit UK industry by an increased awareness of commercialisation opportunities resulting from the research and potentially by facilitating further transfer of a highly skilled individual to the biotechnology workforce.