The role of autophagy in cartilage physiology and metabolism : implications for growth and ageing

Sammanfattning: Cartilage is the main constituent of the embryonic skeleton. At the ends of long bones cartilage forms a growth plate consisting of chondrocytes in distinct stages of differentiation and arranged into three zones. These chondrocytes mediate linear bone growth through synchronized proliferation, differentiation, and production of matrix. The cartilage lining the articulating surfaces of bones also contains chondrocytes arranged in different layers that secrete extracellular matrix and preserve cartilage integrity. Articular cartilage is relatively permanent, whereas the growth plate is transient. Although each of these cartilaginous structures has a unique structure and function, one fundamental similarity is that the chondrocytes in both are exposed to little blood and, thereby, low levels of oxygen and nutrients. Autophagy is an intracellular pathway of lysosomal degradation that protects cells from both internal and external stressors and promotes cell viability when nutrition is limited. The protein kinase mTORC1 is a negative regulator of autophagy and its activity is, in turn, governed by various stimuli such as nutrition and growth factors, depletion of which inhibits mTORC1 and activates autophagy. Attenuated autophagy leads to various developmental and ageing-associated degenerative diseases. Therefore, our primary hypothesis was that autophagy promotes chondrocyte survival, so, that inhibition of this process may impair the linear growth of bones and promote the development of agerelated osteoarthritis. Our second hypothesis was that autophagy improves metabolic parameters during long-term intermittent caloric restriction. First, we studied the role of autophagy in the chondrocytes of mouse metatarsal bones and in C5.18 cells by blocking this process with the lysosomal inhibitors bafilomycin A1 and chloroquine. We found that mTORC1 activity in chondrocytes was increased by blocking lysosomal V-ATPase enzymes. This effect is chondrocyte-specific and in contrast to well-accepted dogma. At the same time, inhibition of lysosomal activity stimulated the linear growth of mouse metatarsal bones by enhancing chondrocyte hypertrophy. Moreover, chondrocytes with impaired autophagy showed similar responses (Paper I). Subsequently, to investigate the effects of autophagy on linear bone growth (Paper II) and ageassociated osteoarthritis (Paper III) directly we abrogated autophagy in chondrocytes by conditional deletion of the autophagy related Atg5 or Atg7 gene. We observed reduced axial and appendicular bone growth due to attenuated chondrocyte proliferation and elevated cell death in both cases. Moreover, chondrocyte viability in the human growth plate and mouse metatarsal bones was reduced by treatment with 3-methyladenine or bafilomycin A1, inhibitors of autophagy (Paper II). Fibrillations and proteoglycan loss in the articular cartilage of aged mice without a functional Atg5 gene was elevated indicating the development of osteoarthritis (Paper III). These impaired bone growth and degenerative changes in articular cartilage are the consequences of enhanced apoptosis mediated by activation of caspases-3 and -9 (Paper II and III). Furthermore, release of cytochrome C initiated the cleavage of caspases even in the absence of autophagy (Paper II). Finally, we examined the role of autophagy in metabolism during intermittent caloric restriction (according to a 5:2 diet) in obese individuals and with and without type II diabetes. We observed improvements in anthropometric and metabolic parameters in both our diabetic and non-diabetic subjects. Moreover, in diabetic subjects whose insulin sensitivity was improved by caloric restriction, autophagy also increased (Paper IV). In conclusion, the observations from our in vitro and in vivo studies confirm that autophagy is essential for the survival and homeostasis of chondrocytes in the growth plate and articular cartilage. At the same time, mTORC1 activation is chondrocyte-specific and independent of autophagy. In addition, autophagy improves metabolic parameters during intermittent caloric restriction in humans. Elucidating mTOR induced autophagy in greater detail will provide further insights in to disorders of linear growth, cartilage degeneration and metabolism, there by opening up novel approaches to treatment.