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.

SignificanceType I collagen, a major component of bone, skin, and other connective tissues, is synthesized in the endoplasmic reticulum (ER) and passes through the secretory pathway. Rerouting of its procollagen precursor to a degradative pathway is crucial for reducing intracellular buildup in pathologies caused by defects in procollagen folding and trafficking. Here, we identify an autophagy pathway initiated at ER exit sites (ERESs). Procollagen proteins following this pathway accumulate at ERESs modified with ubiquitin, LC3, p62, and other autophagy machinery. Modified ERESs carrying procollagen are then engulfed by lysosomes through a microautophagy-like mechanism, not involving conventional, double-membrane autophagosomes. Procollagen homeostasis thus involves a noncanonical mode of autophagy initiated at ERESs, which might also be important in degradation of other secretory proteins.

Glycine substitutions in type I collagen appear to cause osteogenesis imperfecta (OI) by disrupting folding of the triple helix, the structure of which requires Gly in every third position. It is less clear, however, whether the resulting bone malformations and fragility are caused by effects of intracellular accumulation of misfolded collagen on differentiation and function of osteoblasts, effects of secreted misfolded collagen on the function of bone matrix, or both. Here we describe a study originally conceived for testing how reducing intracellular accumulation of misfolded collagen would affect mice with a Gly610 to Cys substitution in the triple helical region of the α2(I) chain. To stimulate degradation of misfolded collagen by autophagy, we utilized a low protein diet. The diet had beneficial effects on osteoblast differentiation and bone matrix mineralization, but it also affected bone modeling and suppressed overall animal growth. Our more important observations, however, were not related to the diet. They revealed how altered osteoblast function and deficient bone formation by each cell caused by the G610C mutation combined with increased osteoblastogenesis might make the bone more brittle, all of which are common OI features. In G610C mice, increased bone formation surface compensated for reduced mineral apposition rate, resulting in normal cortical area and thickness at the cost of altering cortical modeling process, retaining woven bone, and reducing the ability of bone to absorb energy through plastic deformation. Reduced collagen and increased mineral density in extracellular matrix of lamellar bone compounded the problem, further reducing bone toughness. The latter observations might have particularly important implications for understanding OI pathophysiology and designing more effective therapeutic interventions.