Cellular stress due to impairment of collagen prolyl hydroxylation complex is rescued by the chaperone 4-phenylbutyrate

Abstract

Osteogenesis imperfecta (OI) types VII, VIII and IX, caused by recessive mutations in cartilage-associated protein ( CRTAP ), prolyl-3-hydroxylase 1 ( P3H1 ) and cyclophilin B ( PPIB ), respectively, are characterized by the synthesis of overmodified collagen. The genes encode for the components of the endoplasmic reticulum (ER) complex responsible for the 3-hydroxylation of specific proline residues in type I collagen. Our study dissects the effects of mutations in the proteins of the complex on cellular homeostasis, using primary fibroblasts from seven recessive OI patients. In all cell lines, the intracellular retention of overmodified type I collagen molecules causes ER enlargement associated with the presence of protein aggregates, activation of the PERK branch of the unfolded protein response and apoptotic death. The administration of 4-phenylbutyrate (4-PBA) alleviates cellular stress by restoring ER cisternae size, and normalizing the phosphorylated PERK (p-PERK):PERK ratio and the expression of apoptotic marker. The drug also has a stimulatory effect on autophagy. We proved that the rescue of cellular homeostasis following 4-PBA treatment is associated with its chaperone activity, since it increases protein secretion, restoring ER proteostasis and reducing PERK activation and cell survival also in the presence of pharmacological inhibition of autophagy. Our results provide a novel insight into the mechanism of 4-PBA action and demonstrate that intracellular stress in recessive OI can be alleviated by 4-PBA therapy, similarly to what we recently reported for dominant OI, thus allowing a common target for OI forms characterized by overmodified collagen. .

Diversity and bioactivity of fungi associated with the marine sea cucumber Holothuria poli : disclosing the strains potential for biomedical applications

“…The isolates biotechnological potential was investigated as their ability to synthetize metabolites for pharmaceutical applications. Investigated targets were the cytotoxicity against an hepatic cancer cell line (HepG2) and the bioactivity towards stem cell to promote their differentiation into bone or cartilage cell progenitors (Alves et al 2011;Besio et al 2019aBesio et al , 2019b.…”

Zebrafish: A Resourceful Vertebrate Model to Investigate Skeletal Disorders

“…For instance, an altered distribution of bone collagen fiber diameter, a frequently described feature in various skeletal pathological conditions, was detected in the crtap and p3h1 knock-out models of OI type VII and VIII by TEM, revealing the crucial role of the collagen post translational modification complex in bone organization (17). TEM was also used to show enlarged endoplasmic reticulum cisterna in these models, reinforcing ER stress as a key element in the OI phenotype and a potential target for new therapeutic approaches (17,226,227).…”

“…Finally, the unfolded protein response (UPR) was shown to play an important role in the modulation of the phenotype in rare skeletal diseases ( 226 , 227 ). Interestingly, transgenic zebrafish lines allowing different branches of this pathway to be followed are already available ( 216 , 217 , 228 , 229 ).…”

4-Phenylbutyric acid enhances the mineralization of osteogenesis imperfecta iPSC-derived osteoblasts

“…Regarding OI, amelioration of bone mineralization and skeletal deformities of zebrafish OI models by 4-PBA has been reported in 2017 (19). Furthermore, 4-PBA suppressed ER enlargement and improved cellular homeostasis of human dermal fibroblasts from collagenous and non-collagenous OI patients (20,21). We independently searched for a suitable drug for treating OI and discovered 4-PBA.…”