Mice with global deletion of mitogen inducible gene 6 display rapid and severe cartilage and subchondral bone damage after ligament and meniscus injury

Abstract

s / Osteoarthritis and Cartilage 21 (2013) S9–S62 S13 protein subunits was investigated in growth plates of 6 week old mice by immunohistochemistry. Expression of RMRP, its protein subunits and important chondrogenic differentiation markers (Col2a1, Acan, Col10a1, Sox9, Runx2) was also determined by RT-qPCR and immunoblotting in chondrogenically differentiating ATDC5 cells and human bone marrow stem cells (hBMSCs). In addition, RMRP was functionally investigated during chondrogenic differentiation by targeting RMRP expression using a specific siRNA duplex and analysing the outcome on chondrogenic gene expression. Furthermore, chondrogenic induction of dermal fibroblasts was used as a model to test the effect of CHH-associated mutations in RMRP on chondrogenic differentiation of CHH patient-derived fibroblasts. Results: To determine expression of RNase MRP during chondrogenic differentiation in vivo, mouse growth plate sections were immunohistochemically stained for RNaseMRP protein subunits.Without exception, all proteins showed identical growth plate distribution patterns: Resting zone chondrocytes express RNaseMRPproteins,whereas nooronlyweak expression levelswere observed inproliferative chondrocytes. In contrast, expression of RNase MRP proteins was clearly detectable in hypertrophic chondrocytes in the growth plate. This differentiation phase-specific expression of RNase MRP protein subunits was confirmed for Rpp25, Rpp30 and Rpp40 on ATDC5 and hBMSC differentiation models (mRNA); their expressionwas upregulated from day 14 in differentiation onwards, simultaneously with hypertrophic genes. Knockdown of RMRP expression by RNAi resulted in upregulation of RNase MRP mRNA substrates Clb2 and viperin and decreased chondrogenic marker expression (Sox9, Col2a1, Runx2, Col10a1), suggesting a role for RMRP in chondrogenic differentiation. In addition, knockdown of RMRP also resulted in altered Bapx1/Nkx3.2 expression, further substantiating a role in chondrocyte hypertrophy. To verify our results we transdifferentiated CHH patientderived fibroblasts and matched controls to acquire a chondrocyte-like phenotype. Chondrogenic capacity of the CHH patients was clearly disrupted and showed a more than 50% reduced Col10a1 expression as compared to matched control cultures. Conclusions: Our data show that expression of RNase MRP and its subunits is regulated during chondrogenic differentiation, is predominantly associated with chondrocyte hypertrophy and shows functional cross-talk with chondrogenic pathways. These data are the first to show the association of RNase MRP with skeletal development and may provide an explanation for the CHH-associated skeletal phenotype as well as unveiling a potential novel regulator of chondrocyte hypertrophic differentiation in skeletal development. 10 MICE WITH GLOBAL DELETION OF MITOGEN INDUCIBLE GENE 6 DISPLAY RAPID AND SEVERE CARTILAGE AND SUBCHONDRAL BONE DAMAGE AFTER LIGAMENT AND MENISCUS INJURY D.M. Joiner, K.D. Less, E. VanWieren, B.O. Williams. Van Andel Res. Inst., Grand Rapids, MI, USA Purpose: The Epidermal Growth Factor Receptor (EGFR) Signaling pathway plays important roles in osteochondral metabolism and joint disease, which can be triggered by damage to ligaments andmenisci. The goal of this study was to examine the early effect of global deletion of mitogen inducible gene 6 (MIG6), a negative regulator of EGFR, on cartilage and subchondral bone in mice after ligament and meniscus injury. Methods: Global loss of function MIG6 knock-out mice (MIG6-/-) and wild-type (WT) littermates were anesthetized and ligament transections and meniscus removal were performed on the right knee using an established surgical destabilization method. Subchondral bone from surgery knees and untouched contralateral control knees was assessed with x-ray. HE arrows point to bone erosion). Bone damage in surgery knees was greater for MIG6-/animals compared to the control knee and to surgery knees from WT littermates (Figure 2). Ectopic cartilaginous growth pockets were present inMIG6-/mice 4 weeks after ligament and meniscus damage (Figure 1). MIG6-/mice also had increased EGFR expression 4 weeks after surgery (Figure 3) and increased osteoclasts (Figure 4; arrows point to osteoclasts) in the knee joint compared to WT animals. Conclusions: MIG6-/mice displayed rapid and more severe bone and cartilage damage after knee injury compared to WT mice. MIG6 and EGFR could be potential therapeutic targets for preventing cartilage and bone damage and subsequent joint disease development after ligament and meniscus injury.