Abstract
Osteogenesis imperfecta (OI) is a heritable brittle bone disease mainly caused by mutations in the two type I collagen genes. Collagen synthesis is a complex process including trimer formation, glycosylation, secretion, extracellular matrix (ECM) formation, and mineralization. Using OI patient-derived fibroblasts and induced pluripotent stem cells (iPSCs), we investigated the effect of 4-phenylbutyric acid (4-PBA) on collagen synthesis to test its potential as a new treatment for OI. Endoplasmic reticulum (ER) retention of type I collagen was observed by immunofluorescence staining in OI patient-derived fibroblasts with glycine substitution and exon skipping mutations. Liquid chromatography–mass spectrometry analysis revealed excessive glycosylation of secreted type I collagen at the specific sites in OI cells. The misfolding of the type I collagen triple helix in the ECM was demonstrated by the incorporation of heat-dissociated collagen hybridizing peptide in OI cells. Type I collagen was produced excessively by OI fibroblasts with a glycine mutation, but this excessive production was normalized when OI fibroblasts were cultured on control fibroblast-derived ECM. We also found that mineralization was impaired in osteoblasts differentiated from OI iPSCs. In summary, treatment with 4-PBA normalizes the excessive production of type I collagen, reduces ER retention, partially improves misfolding of the type I collagen helix in ECM, and improves osteoblast mineralization. Thus, 4-PBA may improve not only ER retention, but also type I collagen synthesis and mineralization in human cells from OI patients.
Highlights
Alter type I collagen structure such as glycine substitution mutations usually result in severe Osteogenesis imperfecta (OI) type III or moderate OI type IV [3,4,5]
The current study examined these complex type I collagen syntheses in a step-by-step manner using OI patient-derived dermal fibroblasts, as well as osteoblasts differentiated from OI patientspecific induced pluripotent stem cells
Because type I collagen secreted from OI fibroblasts was efficiently deposited on the culture dish by 4-phenylbutyric acid (4-PBA), we investigated its structure in extracellular matrix (ECM) derived from fibroblasts treated with 4-PBA. 4-PBA decreased the normal type I collagen chains to some extent, but it drastically decreased the misfolded type I collagen chains stained by collagen hybridizing peptide (CHP) in OI #3 and 5 (Fig. S8)
Summary
Alter type I collagen structure such as glycine substitution mutations usually result in severe OI type III or moderate OI type IV [3,4,5]. These results showed that treatment with 4-PBA reduced ER retention but did not much improve overglycosylation of type I collagen in OI fibroblasts.
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