Research Article: RNAi Reduces Expression and Intracellular Retention of Mutant Cartilage Oligomeric Matrix Protein

Date Published: April 22, 2010

Publisher: Public Library of Science

Author(s): Karen L. Posey, Peiman Liu, Huiqiu R. Wang, Alka C. Veerisetty, Joseph L. Alcorn, Jacqueline T. Hecht, Alfred Lewin.

Abstract: Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

Partial Text: Cartilage oligomeric matrix protein (COMP), also known as thrombospondin 5 (TSP-5), is a low abundance glycoprotein that is found in the extracellular matrix (ECM) of cartilage, tendon, ligament and smooth muscle [1], [2], [3]. Serum and synovial levels of COMP are now used to assess cartilage erosion in osteoarthritis and joint injury [4], [5]. Interest in this extracellular matrix protein increased when it was recognized that mutations in COMP caused pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1) [6], [7]. It had long been recognized that both PSACH and MED chondrocytes retained lamellar appearing material in large rough endoplasmic reticulum (rER) cisternae [8]. This material was subsequently shown to be composed of COMP and other ECM proteins including types II and IX collagens and matrilin-3 (MATN3) [9], [10]. Recently, using flouresence deconvolution analysis, these retained proteins were shown to be organized into a matrix network suggesting that the stalled mutant COMP inappropriately interacts intracellularly with matrix protein partners [9], [10], [11]. The massive retention of intracellular matrix is toxic to the chondrocytes and prolonged rER stress induces apoptosis causing chondrocyte death [12]. The resulting loss of chondrocytes in the growth plate translates into decreased long bone growth and the disproportionate short stature found in PSACH. Intracellular retention and death of chondrocytes causes the loss of these proteins in the ECM [13], [14]. The resulting downstream effect is a disorganized type II collagen network most likely due to the absence of type IX collagen which is needed to crosslink type II collagen [15]. Altogether, the loss of these proteins from the matrix and the poorly organized ECM structure of the articular cartilage contribute to joint abnormalities and early onset osteoarthritis characteristic of both PSACH and MED/EDM1 [14], [16].

PSACH is a severe dwarfing condition caused by mutations in COMP that specifically affects growth plate chondrocytes. These mutations interfere with protein folding which initiates the ER stress response causing retention of COMP and other ECM proteins that assemble prematurely into ordered intracellular matrix [9], [10], [11], [13], [14]. The massive intracellular retention of this matrix network causes chondrocyte death and results in diminished limb growth and joint abnormalities. The objective of this study was to determine whether suppression/reduction of COMP expression could resolve the cellular chondrocyte pathology. We show that COMP mRNA and protein levels can be significantly decreased by shRNAs directed against the COMP type 3 repeat domain. Specifically, shRNA 3B was effective at reducing endogenous and recombinant COMP expression and maintaining this reduction for up to ten weeks (data not shown). The shRNA-mediated reduction of MT-COMP expression prevented the development of the PSACH cellular phenotype as demonstrated by the absence of retained COMP and other ECM proteins. This is the first study to show that the PSACH cellular phenotype can be mitigated by RNAi reduction of COMP expression.