The proprotein convertase Site-1 protease (S1P) has emerged as a critical regulator of mammalian skeletal development. It is an indispensable component of the regulated intramembrane proteolysis systems that govern lipid metabolism and endoplasmic reticulum stress response. Our studies implicate S1P as a mandatory component of the protein secretory apparatus that defines all connective tissues. We have studied the functions of S1P in a progressive set of S1P knock-out mouse models using Col2-Cre, Col2-CreER(T) and Osx-Cre mice. In addition to a lipid phenotype, studies on S1P ablation in chondroprogenitor cells (S1Pcko model) have demonstrated chondrodysplasia characterized by the specific endoplasmic reticulum (ER) entrapment of type IIB procollagen, poor cartilage matrix development, and loss of endochondral bone formation in mice. Our recent studies demonstrated that along with type IIB procollagen, chondrocytes are also unable to secrete Adamts3, the type II procollagen N-proteinase. We have discovered a well-conserved S1P consensus sequence in the C-terminus of Adamts3, which suggests mandatory C-terminal processing of Adamts3 by S1P. Post-natal S1P ablation in chondrocytes (using Col2-CreER(T)) demonstrated complete disruption of hypertrophic chondrocyte differentiation. When we followed this observation by ablating S1P in prehypertrophic chondrocytes and osteoblasts using Osx-Cre mice, adolescent mice exhibited kyphosis and scoliosis coupled to chondrodysplasia and fragile long bones. These observations confirm the requirement of S1P to overall skeletal development and suggest potential cell-specific functions for S1P. Our mechanistic characterization of the S1Pcko model has demonstrated that lipid metabolism is down-regulated in S1Pcko chondrocytes. Disrupted ER membrane functions induced by changes in lipid composition may trap the type IIB procollagen in the ER. In light of these findings, in Specific Aim 1 we will investigate deviations in S1Pcko ER membrane lipid composition by mass spectrometry and analyze disrupted ER membrane functions by calcium imaging.
In Specific Aim 2 we will investigate Adamts3 as a novel S1P substrate and analyze whether type IIB procollagen trafficking is regulated by a physical association with Adamts3. We will also study cartilage-specific ablation of Adamts3 in mice to analyze its role in cartilage development and maintenance.
In Specific Aim 3, taking the lead from our previous knock-out models, we will characterize how disruptions in chondrocyte and osteoblast functions due to S1P ablation could lead to spine abnormalities in order to identify the biological trigger required for kyphosis and scoliosis development. This approach will allow investigation of multiple novel functions of S1P; additionally it will also provide novel insights into the regulation of protein secretory pathways and how their disruptions lead to the clinical phenotypes of chondrodysplasia and spine deformities.
Skeletal development is dependent on normal cartilage development as cartilage is the template on which future skeletal development is shaped. Defects in skeletal development are often traced to defective cartilage development that can be attributed to abnormal programming of chondrocytes, the cells that produce the cartilage. In this proposal we have designed several new approaches to investigate the contribution of an enzyme called Site-1 protease (S1P) to normal cartilage and skeletal development. Our laboratory has shown that lack of S1P activity in chondrocytes in mice results in dwarfism with abnormal cartilage and no bone formation. Our observations indicate that S1P plays vital roles during cartilage development in a manner that has not been investigated before, namely in the maturation of other enzymes required to process the type II collagen, a major cartilage component. Our recent studies also indicate a role for S1P in bone growth and development. This proposal will lay the foundation for advancing our knowledge of S1P-directed regulatory mechanisms in bone and cartilage that could help correct defective skeletal and cartilage abnormalities in the future such as that seen in kyphosis/scoliosis or osteoarthritis, respectively
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