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- Troeberg Group | Metalloprotease Regulation Research Group
In my DPhil studies to date, I have identified an age-dependent change in expression of ADAM15 in human cartilage. ADAM15 expression was highest in cartilage from adults, with lower expression observed in cartilage from young donors (aged below 20) and old donors (older than 55 years old). A similar age-dependence in Adam15 expression was observed in mouse joints: Adam15 expression was significantly lower in joints of young (6-day old) and old (18-month old) mice than in adult (3-, 4.5- and 5.5-months old) mice. To our knowledge, this is the first time an ADAM has been found to have this age-dependent pattern of expression. I am currently validating that ADAM15 expression is also altered at the protein level.
I searched for the potential regulators of ADAM15 expression by treating HTB-94 chondrosarcoma cells with cytokines, growth factors and vitamins, etc. I identified three cytokines, IL-13, GMC-SF and IFN-γ, that increase ADAM15 expression in these cells. Interestingly, these three cytokines all act through a common signalling adaptor- Janus kinase 2 (JAK2). IL-13 has been linked to chondroprotection in a bovine OA model, where it prevents IL-1-induced collagen release, a hallmark of cartilage damage in OA. To investigate whether IL-13 may contribute to the age-dependent decrease in Adam15 expression, I quantified Il-13 expression in mouse joints with age. As with Adam15, I found that Il-13 expression decreased in mouse joints with age. This correlation raises the possibility that age-dependent changes in IL-13 expression may contribute to the age-dependent reduction of ADAM15 expression. I am currently investigating the signalling pathways downstream of IL-13 (JAK/STAT signalling), and how these contribute to changes in ADAM15 expression.
In the second part of this project, I aimed to identify substrates of ADAM15 using a label-free quantitative secretomic approach in collaboration with Professor Stefan Lichtenthaler (Technical University of Munich, Germany). Several ADAMs have been identified as sheddases, meaning they are able to cleave their substrates on the plasma membrane, and release them to the cell surroundings. Therefore, when the expression of a particular ADAM is changed, the abundance of its substrate in the CM will also change, and can be detected using secretome mass spectrometry analysis. The advantage of secretome analysis is that it can identify and compare the relative abundance of all proteins in the conditioned media (CM) in an agnostic manner. To manipulate ADAM15 expression, both transient over-expression and knock down strategies were optimised in HTB-94 cells. I found that knock down with siRNA gave better results, with a greater number of differentially expressed proteins identified in the mass spectrometry analysis. I have also optimised a method to isolate immature murine costal chondrocytes from 5 to 6 day-old mice to allow secretome analysis in wild-type and Adam15-null mice, in collaboration with Professor Mark Day (University of Michigan, Ann Arbor).
It is known that some ADAMs are more proteolytically active in the presence of serum. However, the bovine serum albumin (BSA) in the serum-containing medium I use to culture cells is known to mask the signals of other less abundant proteins in mass spectrometry analyses. To circumvent this problem, I established a method to label endogenously-produced proteins using the secretome protein enrichment with click sugar (SPECS). Through this method, I identified that ADAM15 promotes IL-1 signalling.