Mutation in the Collagen-Specific Molecular Chaperone Heat-Shock Protein-47 (Hsp47) Causes Endoplasmic Reticulum Stress in Osteoblast Cells of Osteogenesis Imperfecta (OI) Patients
Presentation Number: SAT 353
Date of Presentation: April 1st, 2017
Panjab University, Chandigarh, India
Introduction: OI is a heritable disorder of abnormal collagen and predisposes to increased bone deformity and fragility. The underlying molecular mechanism of abnormal collagen is poorly understood. The Hsp47 is involved in controlling the quality of procollagen folding in the endoplasmic reticulum(ER) and attenuates misfolding of triple helix collagen, suggesting that Hsp47 might be involved inthe pathogenesis of OI. However, Hsp47 cannot prevent the export of incompletely folded chains from the ER by preferentially binding and tagging them. Instead, Hsp47 preferentially binds to, and is co-transported with, natively folded procollagen from the ER to Golgi.
Objective: In the present study, we investigated the mechanisms underlying the apoptosis of Hsp47-/- cells and involvement of autophagy and unfolded protein response (UPR) in the clearance of misfolded type I procollagen in the absence of Hsp47. We hypothesized that misfolded type I procollagen in the ER might cause ER stress, resulting in apoptosis of Hsp47-disrupted triple helix of collagens when autophagy isinhibited.
Methodology: Peripheral blood mononuclear cell (PBMC) derived osteoblasts were cultured. RNA and proteins were isolated from bone. Mutation analysis of Hsp47 gene was done through sequencing followed by restriction digestion. Hsp47 control- and mutant-gene were cloned in expression vector pEGFP-C1.Further,in-vitro analysis was performed to study the interaction between triple helix and mutant Hsp47 under confocal microscope. Immunoblot for mutant protein analysis was done, RT-PCR and quantitative RT-PCR were performed for expression and functional analyses.
Results and Conclusion: ER stress-induced apoptosis may underlie the clearance of collagen-producing osteoblast cells. Experimental findings suggest that Hsp47 plays a pivotal role in ER/cis-Golgi transport. The changes in triple helical structure could affect fibril assembly in extra cellular matrix and lead to irregular mineralization and an OI phenotype.
Disclosure: VD: , AMBRX.