An innate protector of ageing cartilage: FSTL3

Summary During life, cartilage tissues must serve two very contrasting, almost contradictory roles. During early life, cartilage tissues facilitate increases in the length of long bone and, thus function as a transient tissue in which the resident cells, named chondrocytes, have very high rates of growth. This could hardly contrast more with the role cartilage must serve in adult joints, where it has a permanent, lifetime role in protecting joints from any possible friction and damage during movement. This permanent function is intimately coupled to very low, if any, turnover of resident chondrocytes. Ageing predisposes to an inevitable loss of cartilage that results in osteoarthritis; one of the most common ageing-related disorders. This crippling, painful and debilitating disorder has dramatic affects on the quality of life and is the leading cause of chronic disability in the US - affecting nearly 27 million people (together with 8 million in the UK). It is also the most common joint disease in cats, dogs and horses. Cartilage damage and loss is also a feature of sports injury and degeneration caused by repetitive wear and tear, particularly in the obese. Conventional treatment generally only treats the painful symptoms, so effective therapeutic interventions are desperately needed. It has been known for some time, however, that chondrocytes in adult osteoarthritic cartilage from patients and animals re-display the high rates of growth normally characteristic of early life, and that this is intimately linked to disease progression. Our pilot studies have found that a natural cell product (follistatin-like 3, FSTL3), known to regulate cell turnover in other adult tissues, might normally protect against age-related onset of OA by limiting chondrocyte growth. If this were the case, one might expect that deficiency in FSTL3 would be deleterious to the long-term, permanent function of cartilage. Our preliminary data in genetically modified mice lacking FSTL3 have shown a loss of cartilage in the joints of adults which develop an OA-like syndrome very rapidly. Thus, the FSTL3 'brake function' on chondrocyte growth will not be apparent during early life, when increased turnover is absolutely necessary, but only becomes apparent in the adult when growth has slowed. Loss of FSTL3, therefore, only impacts on cartilage integrity in the adult during ageing. These data prompt us to hypothesise: that that FSTL3 restrains chondrocyte turnover to maintain normal adult cartilage and prevent its progressive loss during ageing. To further investigate the mechanisms by which FSTL3 protects against cartilage loss and helps to maintain cartilage upon ageing we propose the following experiments in this research proposal. We will first identify when the first signs of cartilage loss are seen in FSTL3 KO mice and whether this is exacerbated when the knee cartilage is experimentally damaged, as it might be by sporting-type injury in life. We will then investigate whether the lack of FSTL3 increases the rate at which chondrocytes, multiply and die and which genes FSTL3 may influence to achieve this cartilage protection. Our research will, therefore identify how FSTL3 regulate chondrocyte behaviour and how it acts as an innate protector of cartilage loss during ageing. Finally, we will make new genetic mouse mutants that will allow us to establish if FSTL3 achieves this protection through its expression solely in cartilage and therefore whether this makes FSTL3 a good selective target for therapy. Greater appreciation of how chondrocyte growth is controlled will offer routes towards treatment for problems both of cartilage degeneration in disease and ageing and of damage in injury. Hence, the contributions from this work to our understanding of the processes underlying cartilage integrity may support development of preventative and therapeutic approaches to dealing more effectively with major and debilitating illnesses.

Follistatin-like 3 (FSTL3) is a secreted glycoprotein that binds and inhibits a subset of transforming growth factor beta (TGFb) ligands, including activin. To reveal its physiological roles we generated FSTL3 gene-deleted mice (FSTL3 KO). Emerging evidence pinpoints a crucial role for TGFb ligands, and particularly activin, in the maintenance of articular cartilage. Our pilot studies using FSTL3 KO mice reveal the pathological outcomes of unrestrained activin activity. We found that ageing FSTL3 KO mice suffer a series of severe joint phenotypes underscoring the importance of FSTL3 action in maintaining cartilage health, including: aggressive cartilage lesions, osteophyte formation, meniscal and cruciate ligament deformations and more rapid closure of growth plate upon cessation of growth, compared to age-matched controls. Herein we will test the hypothesis that FSTL3 restrains activin-induced chondrocyte turnover to maintain normal adult cartilage and prevent its progressive loss during ageing. Using tissue-specific, inducible or global knockout mice, in vivo mechanical challenge and cell culture models we will elucidate the roles of FSTL3 in chondrocyte turnover, a key determinant in cartilage maintenance. Specifically, we will investigate whether: 1) the effects of FSTL3 as an innate protector of cartilage are evidenced only after cessation of growth or during repair of cartilage after injury; 2) FSTL3 expression in chondrocytes alone is sufficient for cartilage maintenance after cessation of growth or whether there is a requirement of a systemic FSTL3 effect. We will also discover the signalling pathways and gene transcription events, influenced by FSTL3, that are crucial for maintaining chondrocyte number and homeostasis. Our findings will define the processes underpinning the maintenance of cartilage integrity during adulthood and ageing, and support development of preventative and therapeutic strategies for cartilage disorders such as osteoarthritis.

Impact Summary One of the most common disorders associated with ageing, osteoarthritis, is characterised by damage to and loss of articular cartilage. This crippling disorder is the leading cause of chronic disability in the US - affecting nearly 27 million people (together with 8 million in the UK). The condition is also the most common joint disease in cats, dogs and horses. Conventional treatment is generally only palliative, with no treatment available to address the underlying causes or progression of the disease. Major surgery, such as hip replacement, can improve quality of life in severe cases, and is now an option for dogs as well as human patients, but effective therapeutic interventions are desperately needed. Cartilage damage and loss is also a feature of sports injury and degeneration caused by repetitive wear and tear, particularly in the obese. Again, animals, especially performance or working horses are affected as well as human patients. The proposed work is focused on elucidating the mechanism of action of follistatin-like 3 (FSTL3) and transforming growth factor beta (TGFb) ligand signalling pathways in protecting cartilage against progressive loss. A greater understanding of the operation of chondrocytes during development and, particularly, after attainment of skeletal maturity, will offer routes towards treatment for problems both of cartilage degeneration in disease and ageing and of damage in injury. Hence, the contributions from this work to our understanding of the processes underlying cartilage integrity may support development of preventative and therapeutic approaches to dealing more effectively with major and debilitating illnesses. The following groups will benefit: 1. Industry: These results will clearly be valuable to the pharmaceutical and biotechnology industries with interests in novel therapeutics for osteoarthritis and other cartilage problems. 2. Policymakers and government agencies: Indirectly, The NHS, DEFRA and animal health and welfare charities will benefit from insight to cartilage health revealed by our studies since cartilage degeneration is responsible for huge healthcare costs and loss of economic activity, as well as lowering of quality of life. The RVC's active involvement with public engagement in the field of animal welfare will also benefit from this research which will feed into reports, commissions and recommendations made by these and similar societies 3. Society: In the long term, the ultimate beneficiaries of the research will be human, and companion and performance animal patients along with their carers, dependents or owners. 4. Academics: Academics researching in a number of fields across biology and ageing will benefit, as outlined in the 'academic beneficiaries' section of this proposal. The most immediate impact may be for skeletal biology researchers who will gain new insight into control of tissue remodelling. A further beneficiary of the project will be the post-doctoral researcher engaged to undertake the study. He/she will receive training in a range of laboratory techniques and work within a multi-disciplinary team, exploring different aspects of developmental and musculoskeletal biology. RVC also offers opportunities for its researchers to develop complementary skills and pursue involvement in social impact resulting from research, through training and participation in media relations and public engagement programmes.