Osteoblast Malfunction Caused by Cell Stress Response to Procollagen Misfolding in α2(I)-G610C Mouse Model of Osteogenesis Imperfecta

Abstract

Glycine (Gly) substitutions in collagen Gly-X-Y repeats disrupt folding of type I procollagen triple helix and cause severe bone fragility and malformations (osteogenesis imperfecta, aka OI). However, these mutations do not elicit the expected Endoplasmic Reticulum (ER) stress response, in contrast to other protein folding diseases. Thus, it has remained unclear whether cell stress and osteoblast malfunction contribute to the bone pathology caused by Gly substitutions. Here we used a mouse with a Gly610 to cysteine (Cys) substitution in the procollagen α2(I) chain to show that misfolded procollagen accumulation in the ER leads to an unusual form of cell stress, which is neither a conventional unfolded protein response stress nor ER overload. Despite pronounced ER dilation, there is no upregulation of BIP expected in the former and no activation of NFκB signaling expected in the latter. Altered expression of ER chaperones αB crystalline and HSP47, phosphorylation of EIF2α, activation of autophagy, upregulation of general stress response protein CHOP, and osteoblast malfunction reveal some other adaptive response to the ER disruption. We show how this response alters differentiation and function of osteoblasts in culture and in vivo. We demonstrate that bone matrix deposition by cultured osteoblasts is rescued by activation of misfolded procollagen autophagy, suggesting a new therapeutic strategy for OI.

Makings of a brittle bone: Unexpected lessons from a low protein diet study of a mouse OI model

“…Altered structure, composition and deposition pattern of bone matrix as well as the LP diet effect on matrix mineralization indicated abnormal differentiation and/or function of Het osteoblasts, consistent with our study of G610C neonates and calvarial osteoblasts [7]. Electron microscopy (EM) of lumbar (L2) vertebrae confirmed severe dilation of ER cisternae in Het osteoblasts, apparently caused by accumulation of misfolded mutant collagen molecules inside the ER (Fig.…”

“…In the G610C mouse model, separate analysis of neonates [7] and the present study of adult animals suggest that osteoblast malfunction plays a major role in bone pathology. Delayed folding and intracellular retention of procollagen [7] combined with severe ER dilation ([7], Fig.…”

“…Delayed folding and intracellular retention of procollagen [7] combined with severe ER dilation ([7], Fig. 6A) suggest accumulation of misfolded procollagen in osteoblast ER.…”

“…It is commercially available (Jackson Laboratories, Stock No: 007248) and has been utilized in several OI studies [6-10]. G610C mice mimic the mutation and OI phenotype found in a large group of related patients from an Old Order Amish community in Lancaster County, PA. As expected for most Gly substitutions [11], the G610C mutation disrupts folding of the procollagen precursor of type I collagen and causes accumulation of misfolded procollagen in the Endoplasmic Reticulum (ER), resulting in cell stress and osteoblast malfunction [7]. LP diet was selected as a simple approach to activating autophagy [12] with an expected additional effect of reducing overall procollagen synthesis (and therefore misfolding) by osteoblasts.…”

“…LP diet was selected as a simple approach to activating autophagy [12] with an expected additional effect of reducing overall procollagen synthesis (and therefore misfolding) by osteoblasts. Since earlier studies suggested that autophagy might play a key role in degradation of misfolded procollagen in OI [7, 13, 14], our objective was to see how an LP diet would affect the bone material before initiating more complex studies of more precise autophagy targeting. We combined multiple approaches to analyzing bone material properties in order to distinguish different LP diet effects on bone synthesis, structure and mineralization.…”