The Effects of Expression of Thermolabile R789C and R992C Collagen II Mutants on Cell Behavior and Extracellular Matrix Structure and Function
The hallmark feature of mutations in COL2A1 is alterations of skeletal development with highly variable clinical severity. The resulting spectrum of chondrodysplasia phenotypes ranges from perinatal lethality to milder disturbances such as premature osteoarthritis. There has been a great deal of time and effort put into the development of gene and cell therapies targeted at overcoming the devastating effects of disorders due to mutations in collagenous genes, with particular emphasis on osteogenesis imperfecta; however, the majority of these previous endeavors have been relatively ineffective. Very little has been done to determine the minimal therapeutic conditions which would be necessary in order to achieve clinical efficacy, i.e. restore normal function to affected tissues. In order to design effective therapies, there remains a need to gain an understanding of what these minimal conditions are, which likely vary significantly between tissues and mutation types.
Here, I examined the molecular and cellular effects of expression of the thermolabile R789C and R992C collagen II mutants on extracellular matrix organization and cell behavior. Through recombinant expression of these mutants in a cell-based, inducible experimental system, we are able to effectively modulate the expression levels of exogenous collagens, thereby altering their contribution from 0% to 30% of the total collagen pool. By expressing mutant collagens at 100% capacity at the onset of the experiment, we are able to promote the incorporation of mutant collagens into cell/matrix systems and then monitor remodeling and recovery after reducing their expression to 75, 50, 25, or 0% of the original 100%. Quantitative studies of PDI and BiP, indicators of intracellular stress and the unfolded protein response, as well as analysis of apoptosis, suggest that these processes are attenuated during the recovery period only in the complete absence of expression of mutant molecules. Under conditions where expression of R789C and R992C is only partially reduced, these processes continue with no relief from intracellular stress or apoptosis. My studies suggest that therapies targeted at remodeling tissues affected by thermolabile collagen mutants may only be achieved by completely suppressing expression of mutant molecules rather than through their partial elimination.
0379: Cellular biology