Chondrocyte-Specific Pathology During Skeletal Growth and Therapeutics in a Murine Model of Pseudoachondroplasia

Abstract

Mutations in the gene encoding cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia (PSACH), a severe dwarfing condition. Pain, a significant complication, has generally been attributed to joint abnormalities and erosion and early onset osteoarthritis. Previously, we found that the Inflammatory-related transcripts were elevated In growth plate and articular cartilages, Indicating that Inflammation plays an important role in the chondrocyte disease pathology and may contribute to the overall pain sequelae. Here, we describe the effects of D469-delCOMP expression on the skeleton and growth plate chondrocytes with the aim to define a treatment window and thereby reduce pain. Consistent with the human PSACH phenotype, skeletal development of D469del-COMP mice was normal and similar to controls at birth. By postnatal day 7 (P7), the D469del-COMP skeleton, limbs, skull and snout were reduced and this reduction was progressive during postnatal growth, resulting in a short-limbed dwarfed mouse. Modulation of prenatal and postnatal expression of D469del-COMP showed minimal retention/cell death at P7 with some retention/cell death by P14, suggesting that earlier treatment intervention at the time of PSACH diagnosis may produce optimal results. Important and novel findings were an increase In Inflammatory proteins generally starting at P21 and that exercise exacerbates Inflammation. These observations suggest that pain in PSACH may be related to an intrinsic inflammatory process that can be treated symptomatically and is not related to early joint erosion. We also show that genetic ablation of CHOP dampens the inflammatory response. observed in mice expressing D469del-COMP. Toward identifying potential treatments, drugs known to decrease cellular stress (lithlum, phenyl butyric add, and valproate) were assessed. Interestingly, all diminished the chondrocyte pathology but had untoward outcomes on mouse growth, development, and longevity. Collectively, these results define an early treatment window in which chondrocytes can be salvaged, thereby potentially Increasing skeletal growth and decreasing pain.

Antisense Reduction of Mutant COMP Reduces Growth Plate Chondrocyte Pathology

“…To circumvent this problem, we generated the mutant (MT)-COMP mouse with the common D469del PSACH mutation that expresses human MT-COMP in chondrocytes in the presence of the inducing agent doxycycline (DOX). [16][17][18][19] MT-COMP mice recapitulate the PSACH clinical findings of reduced growth and cellular abnormalities, including massive intracellular retention of COMP and other ECM proteins. 16,17,19 The intracellular retention of MT-COMP activates the protein kinase RNA-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response, which is the cellular response mechanism that is activated by misfolded proteins.…”

“…[17][18][19][41][42][43] This is a new and exciting approach not previously considered for cartilage-specific disorders because the growth plate has been considered to be an inaccessible avascular tissue. Here and in our previous antioxidant/anti-inflammatory therapeutic approaches, we show that the growth plate and articular cartilage are accessible and amenable to different therapies.…”

“…[16][17][18][19] COMP pentamers often include both wild-type and misfolded mutant subunits, with an estimated 97% of the protein stalling and getting trapped in the ER. 21,30 This suggests that, in the presence of MT-COMP, wild-type (WT) COMP subunits may contribute to the cytotoxic intracellular retention.…”

“…29 In contrast to tissue culture experiments, the growth plate cartilage has long been considered difficult to target with drugs or antisense molecules because of its relative avascularity compared with the highly vascular liver and kidney. 31 However, our success with drug therapy in the growth plate 16,18 led us to ask whether ASOs with high-affinity nucleoside modifications 32 could be delivered to growth plate chondrocytes and reduce MT-COMP intracellular retention. Recent studies have shown that chemically modified ASOs can reduce mRNA targets efficiently in extra-hepatic tissues and show pharmacological benefits in disease models.…”

“…21,30,47,48 Misfolded COMP causes massive retention of COMP and other extracellular proteins in the ER, which forms an inappropriate intracellular matrix eliciting oxidative and inflammatory stress, which is cytotoxic to the chondrocyte. 3,5,14,[16][17][18]43,49 We have shown that treatments that target inflammation (aspirin), oxidative stress (resveratrol), and ER stress (absence of C/EBP homologous protein [CHOP] in MT-COMP mice, valproate) reduce the MT-COMP chondrocyte pathology. 16,17 Moreover, we have shown that shRNAs in vitro reduce COMP expression, ER stress, and intracellular retention.…”