Increased intracellular proteolysis reduces disease severity in an ER stress–associated dwarfism
Lorna A. Mullan, … , Michael D. Briggs, Raymond P. Boot-Handford
Lorna A. Mullan, … , Michael D. Briggs, Raymond P. Boot-Handford
Published September 18, 2017
Citation Information: J Clin Invest. 2017;127(10):3861-3865. https://doi.org/10.1172/JCI93094.
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Brief Report Bone Biology Development

Increased intracellular proteolysis reduces disease severity in an ER stress–associated dwarfism

Abstract

The short-limbed dwarfism metaphyseal chondrodysplasia type Schmid (MCDS) is linked to mutations in type X collagen, which increase ER stress by inducing misfolding of the mutant protein and subsequently disrupting hypertrophic chondrocyte differentiation. Here, we show that carbamazepine (CBZ), an autophagy-stimulating drug that is clinically approved for the treatment of seizures and bipolar disease, reduced the ER stress induced by 4 different MCDS-causing mutant forms of collagen X in human cell culture. Depending on the nature of the mutation, CBZ application stimulated proteolysis of misfolded collagen X by either autophagy or proteasomal degradation, thereby reducing intracellular accumulation of mutant collagen. In MCDS mice expressing the Col10a1.pN617K mutation, CBZ reduced the MCDS-associated expansion of the growth plate hypertrophic zone, attenuated enhanced expression of ER stress markers such as Bip and Atf4, increased bone growth, and reduced skeletal dysplasia. CBZ produced these beneficial effects by reducing the MCDS-associated abnormalities in hypertrophic chondrocyte differentiation. Stimulation of intracellular proteolysis using CBZ treatment may therefore be a clinically viable way of treating the ER stress–associated dwarfism MCDS.

Authors

Lorna A. Mullan, Ewa J. Mularczyk, Louise H. Kung, Mitra Forouhan, Jordan M.A. Wragg, Royston Goodacre, John F. Bateman, Eileithyia Swanton, Michael D. Briggs, Raymond P. Boot-Handford

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Figure 1

Effects of CBZ treatment on ER stress induced by 4 different MCDS-causing mutations in collagen X.

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Effects of CBZ treatment on ER stress induced by 4 different MCDS-causin...
(A) BiP, (B) CHOP, and (C) spliced XBP1 (sXBP1) mRNA (relative to β actin mRNA) in cells transiently expressing 1 of 4 mutant collagen X constructs and treated for 24 hours with CBZ (20 μM). Mean ± SEM (n = 4). UTF, untransfected control. (D) Western blot of intracellular collagen X (Col X) protein levels with and without 24-hour CBZ treatment and (E) quantification of 3 independent experiments (mean ± SEM). (F–I) Quantification of 3 independent Western blot experiments for intracellular N617K, G618V, Nc1del10, and Y598D collagen X protein levels (75 kD) in the presence or absence of 20 μM CBZ for 24 hours (see Supplemental Figure 2D for representative blots). Inhibitors of the proteasome (PSII) or lysosome (CQ) were added at 16 hours after transfection for a further 8 hours. Collagen is expressed relative to its GAPDH level and was normalized to the retained collagen X level at the 16-hour untreated time point. Mean ± SEM. *P < 0.05; **P < 0.005; ***P < 0.0005. All statistical analyses by ANOVA.