Endocrine Abstracts

JOINT3340

Background: Spondyloepimetaphyseal dysplasia (SEMD) is a rare developmental disorder of bone and cartilage with short stature and skeletal deformities affecting the extracellular matrix of cartilage. MMP13 (collagenase-3) has been considered to have an important role in skeletal biology in view of its exclusive presence in the skeleton during embryonic development in cartilaginous growth plates and primary centers of ossification. MMP13 takes part in catalyzing ECM components in the growth plate and at the same time cleaving and releasing biologically active molecules stored in the ECM, such as VEGFA, essential for chondrogenic differentiation, apoptosis, and matrix remodeling. Although previous studies had identified the association of SEMD_MO with a missense MMP13 mutation, the mechanism by which SEMD_MO is associated with MMP13 is poorly understood.

Patient and Methods: The proband was a 5. 5years old male exhibiting short stature(-3. 27SD). His X-ray examination revealed ulnar and radial bones with widened metaphysis, “brush and cup-like”changes, irregular edges. The WES analysis showed the probably pathogenic variant c. 124T>G in heterozygosity in exon2 of the MMP13 gene, described in the literature in patients with SEMD. The proteins which possibly could interact with MMP13 were preliminarial identified through CoIP-MS method and a bioinformatic analysis has been made as well. CoIP-WB experiment was used to verify the in vivo interaction. We generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) via nucleofection with episomal plasmids. These iPSCs were further differentiated into chondrogenic via mesenchymal stem cells (MSCs). RT-PCR and Western blot methods were used to substantiate whether both transcription level and protein level of PERK, ATF6, IRE1a are upregulated by BiP.

Results: Cell lines harboring the variant displayed decreased amount of MMP13 proteins in culture medium and degraded misfolded protein fragments of approximately 30kDa of the mutant protein intracellular through the ubiquitin-proteasome system. The results of CoIP-MS experiments and related bioinformatics analysis showed that the MMP13 Y42D variant increased the interaction between MMP13 and Bip and these proteins were mainly involved in several biological processes, including protein folding, protein processing in ER, response to ER stress. In patient-iPSC-derived chondrogenic, both transcription level and protein level of PERK, ATF6, IRE1a are upregulated by BiP.

Conclusion: Our study indicate that the variant in MMP13 lead to increasing MMP13-Bip protein interaction, activating endoplasmic reticulum stress. Under ER stress, Bip could upregulate transcription and expression level of PERK, ATF6, IRE1a and promote cell death.