A Novel Form of Chondrocyte Stress is Triggered by a COMP Mutation Causing Pseudoachondroplasia

Pseudoachondroplasia (PSACH) results from mutations in cartilage oligomeric matrix protein (COMP) and the p.D469del mutation within the type III repeats of COMP accounts for approximately 30% of PSACH. To determine disease mechanisms of PSACH in vivo, we introduced the Comp D469del mutation into the mouse genome. Mutant animals were normal at birth but grew slower than their wild-type littermates and developed short-limb dwarfism. In the growth plates of mutant mice chondrocyte columns were reduced in number and poorly organized, while mutant COMP was retained within the endoplasmic reticulum (ER) of cells. Chondrocyte proliferation was reduced and apoptosis was both increased and spatially dysregulated. Previous studies on COMP mutations have shown mutant COMP is co-localized with chaperone proteins, and we have reported an unfolded protein response (UPR) in mouse models of PSACH-MED (multiple epiphyseal dysplasia) harboring mutations in Comp (T585M) and Matn3, Comp etc (V194D). However, we found no evidence of UPR in this mouse model of PSACH. In contrast, microarray analysis identified expression changes in groups of genes implicated in oxidative stress, cell cycle regulation, and apoptosis, which is consistent with the chondrocyte pathology. Overall, these data suggest that a novel form of chondrocyte stress triggered by the expression of mutant COMP is central to the pathogenesis of PSACH. Hum Mutat 33:218–231, 2012. © 2011 Wiley Periodicals, Inc.

wild-type (wt) and recombinant (HR) ES clones following Hind III digestion. C) DNA sequencing confirming that the mutation is present in the recombinant alleles. D) Agarose gel electrophoresis of PCR amplified cDNA confirming a lack of genomic DNA contamination. The correct cDNA fragments at ~350 bp were observed whilst there were no bands at ~700 bp corresponding to genomic DNA. E) SDS-PAGE and Western blot showing comparable levels of COMP protein in wild-type and mutant cartilage at 3 weeks of age. Key:-wt (wildtype), m/m (mice homozygous for the mutation).
Supp. Figure S2. Growth curves for mice over a 9-week time period. Male and female mice were weighed at 3, 6 and 9 weeks after birth and their weights charted. A) Male and female mice homozygous for Comp:∆D469 mutation had slower growth curves than wild type and heterozygous mutant animals. Separate growth charts for males and females are shown at 3, 6 and 9 weeks for mice of all three genotypes. B) Homozygous Comp p.D469del males were significantly smaller than their litter-mates at 6 weeks and 9 weeks of age. C) Homozygous Comp p.D469del females were significantly smaller than their littermates by 9 weeks of age. N=7 mice per sex per genotype, One-Way ANOVA. Supp. Figure S3. Measurement of tibia length from male mice between 3 and 9 weeks of age. Tibias lengths of male mice were measured at 3, 6 and 9 weeks of age for wild type, heterozygous and homozygous Comp p.D469del littermates. Tibias from mice homozygous for Comp p.D469del were significantly shorter that their littermates at 6 and 9 weeks of age. N>7 mice per genotype, One-way ANOVA . Tibia length during growth 5%** 6%* Supp. Figure S4. Representative images of wild type (wt) and mutant (mut/mut) growth plates showing the localisation of cartilage collagen proteins. A & B) IHC using a type IX collagen antibody showed some intracellular retention in mutant chondrocytes (insert), whilst staining to type IX collagen was more pronounced in the resting zone (white arrow) and in the pericellular region of the early proliferative zones of both wild type and mutant growth plates. C & D) IHC using a type II collagen antibody demonstrated patchy mislocalisation in the mutant growth plate, whilst type X collagen (E & F) was also diffuse in the growth plate of mice homozygous for p.D469del. Supp. Figure S5. Time of onset and progression of apoptosis in the growth plate of Comp D469del mice. A) TUNEL positive cells (white circles) were detected in the proliferative zones of growth plates from mice homozygous for the p.D469del COMP mutation, but not in their wild-type littermates. B) Quantification of the TUNEL assay results showed a significant increase in apoptosis in the proliferative zone of growth plates from mice homozygous for the mutation (n=36 section from 3 mice per genotype, independent samples t-test, * p<0.05, ***p<0.001). May play a role in regulation of cell proliferation and Sdpr induction appears to be coupled to growth arrest. SDPR is a substrate for protein kinase C (PKC) phosphorylation. AK004781 SRY-box containing gene 17 Sox17 8.15 Sox17 is a transcription factor that regulates cell cycle and proliferation.

BB039247
CD93 antigen Cd93 8.13 CD93 antigen plays a role in intercellular adhesion and in clearance of apoptotic cells.

BG917242
Lipoma HMGIC fusion partner-like 2 Lhfpl2 8.10 A member of the lipoma HMGIC fusion partner (LHFP) gene family, which is a subset of the superfamily of tetraspan transmembrane protein encoding genes. NM_133654 CD34 antigen Cd34 7.90 Possible adhesion molecule with a role in early hematopoiesis by mediating the attachment of stem cells to the bone marrow extracellular matrix or directly to stromal cells. Could act as a scaffold for the attachment of lineage specific glycans, allowing stem cells to bind to lectins expressed by stromal cells or other marrow components. Presents carbohydrate ligands to selectins. BF100813 Endothelin receptor type B Ednrb 7.10 Non-specific receptor for endothelin 1, 2, and 3. Ednrb-/mice exhibit neurodegeneration suggesting Ednrb is important for cell survival; however this did not involve canonical apoptosis signals.

NM_011582
Thrombospondin 4 Thbs4 7.01 Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. C-terminal peptide can stimulate cell proliferation.

NM_133736
Regulator of G-protein signaling 5 Rgs5 6.92 Inhibits signal transduction and acts as a hypoxia-inducible apoptotic stimulator. Plays a role in mediating responses to oxidative stress through the MEK-ERK1/2 signalling pathway. AW543698 Cadherin 5 Cdh5 6.70 A calcium-dependent cell-cell adhesion glycoprotein that plays an important role in cell polarity through control of the cohesion and organization of the intercellular junctions. It associates with alphacatenin forming a link to the cytoskeleton. Oxidative stress can modulate Wnt/b-catenin and CDH5 in diabetes.

BC020152
Integrin, beta-like 1 Itgbl1 6.62 Proposed to participate in cell adhesion, cell-matrix interactions and integrin-mediated signalling pathways. Along with Gpha2 it is up regulated by dexamethasone of murine cochlear explants NM_019397 EGF-like-domain, multiple 6 Egfl6 6.55 Mediates a paracrine mechanism of cross-talk between vascular endothelial cells and osteoblasts, promoting cell migration and angiogenesis during bone development, grow and repair. Supp.

Ddx3y
-7.93 DEAD-box RNA helicases have been implicated a variety of processes that regulate gene expression, such as transcription, splicing, mRNA export and translation. It is also proposed to be involved in cell cycle control and the regulation of apoptosis.

AV348246
Neuronal PAS domain protein 4 Npas4 -4.48 Transcription factor that is implicated in adaptation to cellular and oxidative stresses.

BB656631
SRY-box containing gene 11 Sox11 -3.53 Probably important in the developing nervous system. May also have a role in lymphocyte proliferation.

Eif2s3y
-3.15 Along with Ddx3y it is induced following angiotensin II treatment of mice. Eif2s3y functions in the early steps of protein synthesis by forming a ternary complex with GTP and initiator tRNA. AK011963 Peroxiredoxin 2 Prdx2 -2.91 An anti-oxidant enzyme involved in redox regulation of the cell and reduces peroxides through the thioredoxin system. Peroxiredoxin 2 can inhibit cell apoptosis through the NF-kappa-B pathway. BB218576 calcium/calmodulin-dependent protein kinase II, delta

Camk2d
-2.74 Angiotensin II-induced oxidative stress resets the Ca2+ dependence of Camk2d and promotes cell death. Inhibition of Camk2d prevents an increase in p53 and apoptosis. NM_133743 Ly6/Plaur domain containing 3 Lypd3 -2.67 Supports cell migration and may be involved in cell-matrix interactions. NM_008127 Gap junction protein, beta 4 Gjb4 -2.44 Gap junction protein that is also known as connexin Cx30.3 and though to mediate cell-cell communication.

AU046270
Acireductone dioxygenase 1 Adi1 -2.38 Adi1 is a member of the Cupin superfamily, which binds to and inhibits the activities of membrane-type 1 matrix metalloproteinase (MMP14). Increased Adi1 expression can cause apoptosis.

AV128350
Block of proliferation 1 Bop1 -2.14 Component of the Pes1-Bop1-WDR12 (PeBoW) complex, which is required for maturation of 28S and 5.8S ribosomal RNAs and formation of the 60S ribosome. May play a role in cytoskeletal remodelling. PeBoW complex is required for ribosome biogenesis and cell proliferation.

BF148215
Strawberry notch homolog 2 (Drosophila) Sbno2 -2.12 Sbno2 has transcriptional repression activity and has been shown to repress NF-kappa-B signalling. NM_009864 Cadherin 1 Cdh1 -2.11 Involved in mechanisms regulating cell-cell adhesions, mobility and proliferation of epithelial cells. Has a potent invasive suppressor role and is a ligand for integrin alpha-E/beta-7. L07264 Heparin-binding EGF-like growth factor Hbegf -2.09 Cell migration can be mediated by MMP7-mediated release of HBEGF that activates ERBB1 and downstream ERK and PI3K signalling.

NM_053015 Melanophilin
Mlph -2.07 Rab effector protein involved in melanosome transport. The actin-activated MgATPase activity of myosin 5a is activated by binding to melanophilin. Myosin 5 is a motor protein that moves cargoes along actin filaments. BB486599 ST8 alpha-N-acetyl-neuraminide alpha-2,8sialyltransferase 6 Promotes the exchange of GDP by GTP. Together with leukocyte antigen-related (LAR) protein, it could play a role in coordinating cell-matrix and cytoskeletal rearrangements necessary for cell migration and cell growth.