OSX-CRE directed ablation of site-1 protease in mice results in kyphosis and scoliosis

Abstract

s / Osteoarthritis and Cartilage 23 (2015) A26eA81 A80 95 OSX-CRE DIRECTED ABLATION OF SITE-1 PROTEASE IN MICE RESULTS IN KYPHOSIS AND SCOLIOSIS D. Patra, L. Sandell. Washington Univ. Sch. of Med., St. Louis, MO, USA Purpose: Site-1 protease (S1P) is a proprotein convertase that is primarily known for processing of latent, endoplasmic reticulum (ER) membrane-bound transcription factors SREBPs and ATF6 to their free and active form. SREBPs are involved in cholesterol and fatty acid homeostasis; ATF6 is involved in ER stress response. Cartilage-specific ablation of S1P in mice (S1Pcko) using Col2-Cre results in poor cartilage with a drastic decrease of collagen type IIB (Col IIB) in the matrix and a complete lack of endochondral bone formation, even though the molecular program for chondrocyte maturation is not disrupted. In a postnatal model using Col2-CreERT mice, ablation of S1P in postnatal chondrocytes gradually eliminates the primary growth plate with a gradual abolition of Ihh and type X collagen expression in the growth plate indicating a cessation of hypertrophic chondrocyte differentiation. The goal of this study is to understand the importance of S1P in chondrocyte maturation in the growth plate. Methods: The osterix protein is expressed in prehypertrophic chondrocytes (as well as in osteoblasts). S1Pf/f mice (homozygous for the S1Pflox allele inwhich exon 2 of S1P is floxed) were crossed with Osx-Cre mice (inwhich the Cre recombinase is under the influence of the Osterix (Osx) promoter) to obtain mice with S1P ablation in prehypertrophic chondrocytes (S1POsx mice). S1POsx mice and wild type littermates were studied by histology, immunofluorescence (IF), and micro-computed tomography (mCT) techniques. Results: Mice with a homozygous deletion for S1P (S1Pf/f;Osx-Cre mice or S1Pcko-Osx) suffer from severe chondrodysplasia that is visible in their gross morphology beginning from postnatal day 7. Mice which are heterozygous for S1P deletion (S1Pþ/f-Osx) have a size intermediate to the S1Pcko-Osx mice and their wild type littermates; additionally, these mice are also hunchbacks (kyphosis) which gets more pronounced with age. Interestingly, mCT analysis not only confirmed the kyphosis in S1Pþ/ f-Osx mice, but also revealed scoliosis in the S1Pcko-Osx mice. Micro-CT analysis also showed decreased bone mineralization in both mutant mice with the S1Pcko-Osx mice showing a more drastic decrease than the heterozygote resulting in fragile bones. In embryonic studies, S1Pcko-Osx mice exhibited a delay in the onset for endochondral bone formation, while the S1Pþ/f-Osx showed normal onset of endochondral bone formation as the wild type. In immunofluorescence studies for type II collagen (Col II) distribution, while the Col2-Cre-directed S1Pcko mice exhibited Col II entrapment in all zones of the growth plate including the resting and proliferative zone, S1Pcko-Osx mice showed Col II entrapment only in the hypertrophic zone, but not the resting and proliferative zones; negligible Col II entrapment was observed in the growth plate of S1Pþ/f-Osx mice. Conclusions: These results indicate the indispensable nature of S1P for cartilage and overall skeletal development. S1P is essential for normal growth plate activity and cartilage formation. The current study also endorses the importance of S1P in normal skeletal development and is important for both axial and appendicular skeletal development. For the first time, we demonstrate an important functional link between S1P and diseases of the vertebral column. These studies also suggest an indispensable role for S1P in osteoblasts. Thus understanding the contributions of S1P in cartilage, bone and vertebrae development will contribute to the understanding of overall skeletal homeostasis. 96 SOX9 DIRECTLY REGULATES CTGF/CCN2 TRANSCRIPTION IN GROWTH PLATE CHONDROCYTES AND IN NUCLEUS PULPOSUS CELLS OF INTERVERTEBRAL DISC C. Oh y, H. Yasuda z, S. Henry z, Z. Zhang z, M. Xue y, B. de Crombrugghe z, D. Chen y. yRush Univ. Med. Ctr., Chicago, IL, USA; z The Univ. of Texas, M.D. Anderson Cancer Ctr., Houston, TX, USA Purpose: Several lines of evidence demonstrated that connective tissue growth factor (CTGF/CCN2) stimulates chondrocyte proliferation and maturation and is required for cartilage development. Since SOX9 is a key transcription factor controlling several steps of the chondrocyte differentiation pathway, in the present studies, we have determined whether SOX9 directly regulates Ctgf (Ccn2) expression in chondrocytes Methods: Primary sternal chondrocytes derived from Sox9 flox/flox mice were infected with Cre recombinase-expressing adenovirus (Ad-CMVCre). We performed ChIP-on-chip assay using anti-SOX9 antibody, covering the Ctgf gene from 15 kb upstream of its 5’-end to 10 kb downstream of its 3’-end to identify SOX9 interaction site. The SOX9 binding site was then verified by the Ctgf reporter assays, electrophoretic mobility shift assay and the ChIP-qPCR assay. In addition, we also generated chondrocyte-specific Sox9 knockout (KO)mice to further determine the role of SOX9 in regulation of Ctgf in vivo Results:We found that expression of CtgfmRNA and proteinwas downregulated in primary sternal chondrocytes from Sox9 flox/flox mice infected with Ad-CMV-Cre. One high-affinity interaction site was identified in the Ctgf proximal promoter by ChIP-on-chip assay which is located in -70/-64 region of the Ctgf promoter. We found the same SOX9 binding site in the Ctgf promoter in nucleus pulposus (NP) cells. The function of this binding site was determined by a serious of reporter assays, an electrophoretic mobility shift assay and a ChIP-qPCR assay. Deletion of Sox9 in growth plate and articular chondrocytes in long bone or in NP cells in intervertebral disc tissue significantly downregulated CTGF expression in 2-month-old Sox9 KO mice Conclusions: Our studies suggest that Ctgf is a direct target of SOX9 in growth plate chondrocytes and in NP cells in disc tissues. CTGF expression is regulated by SOX9 during chondrogenesis and homeostasis of disc tissue in adult mice. Our findings establish a strong link between two regulatory molecules that have a major role in cartilage and disc tissue 97 BAPX1/NKX3.2; A NOVEL CHONDROCYTE HYPERTROPHY MOLECULAR SWITCH IN OSTEOARTHRITIS M.M. Caron y, P.J. Emans y, D.A. Surtel y, P.M. van der Kraan z, L.W. van Rhijn y, T.J. Welting y. yMaastricht Univ. Med. Ctr., Maastricht, Netherlands; zRadboud Univ. Med. Ctr., Nijmegen, Netherlands Purpose: Osteoarthritis (OA) is the most common degenerative joint disorder and presents with degradation of articular cartilage, leading to loss of joint mobility and function, accompanied by chronic pain. Recently we showed that Bone Morphogenetic Protein 7 (BMP-7) is able to suppress chondrocyte hypertrophic differentiation from progenitor cells and that the transcriptional repressor Bapx1/Nkx3.2 plays a key role in this action. As OA involves a shift of the articular chondrocyte phenotype towards hypertrophic differentiation andmineralization, we hypothesized that impaired Bapx1/Nkx3.2 function and concomitant loss of hypertrophic protection of the articular chondrocyte is central in the changing chondrocyte phenotype that drives OA. Methods: Healthy and OA human articular chondrocytes (HACs) were isolated from healthy knee cartilage or from OA cartilage from total knee arthroplasty. HACs were exposed to BMP-7, IL-1b, TNFa or OA synovial fluid. Lossand gain-of-function experiments were performed using siRNA duplexes targeting Bapx1/Nkx3.2 and by transiently overexpressing FLAG-Bapx1/Nkx3.2 by transfection of cloned vectors. Mouse experimental models for OAwere used (DMMand collagenaseinduced OA) and (immuno)histochemistry was applied on knee joint tissue sections. Geneand protein expression of Bapx1/Nkx3.2 and important chondrogenic-, hypertrophic-, cartilage degradingand inflammatory mediators were determined by RT-qPCR and immunoblotting. ALP activity was determined using a colorimetric assay and PGE2 levels were measured by EIA. Results: Exposure of healthy HACs to BMP-7 lead to a pronounced increased expression of Sox9, Col2a1 and Acan expression. At the same time, expression of hypertrophy associated genes Runx2, Col10a1 and ALP was suppressed, suggesting the existence of a general pro-chondrogenic and hypertrophy-suppressive action of BMP-7. Interestingly, BMP-7 significantly increased Bapx1/Nkx3.2 mRNA levels. Knockingdown Bapx1/Nkx3.2 in healthy HACs did not significantly influence the expression levels of chondrogenic genes, but led to a major hypertrophic shift in the HAC phenotype. Overexpression of FLAG-Bapx1/ Nkx3.2 again did not change expression levels of Sox9, Col2a1 and Acan. However, it decreased the expression of Runx2, Col10a1 and ALP in healthy HACs suggesting that Bapx1/Nkx3.2 fulfills a central regulating role in balancing the chondrocyte phenotype with specific regulatory functions in controlling the chondrocytes’ hypertrophic phenotype. In OA(-induced) chondrocytes, Bapx1/Nkx3.2 expression negatively correlated with expression of chondrocyte hypertrophic markers, cartilage degrading enzymes (MMP13, ADAMTS5) and inflammatory mediators (COX-2, IL-6). Furthermore, Bapx1/Nkx3.2 positivity was diminished in articular chondrocytes in knee joints from mouse