Mechanical Stretch Increases Smad3-Dependent CCN2 Expression in Inner Meniscus Cells Takayuki Furumatsu, 1 Tomoko Kanazawa, 1 Yoshiaki Miyake, 1,2 Satoshi Kubota, 2 Masaharu Takigawa, 2 Toshifumi Ozaki 1 1 Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, Japan, 2 Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, Japan Received 13 February 2012; accepted 20 April 2012 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22142 ABSTRACT: The intrinsic zone-specific properties of the menisci are determined by biomechanical environments. In this study, we examined mechanical stretch-dependent expression of multifunctional growth factor CYR61/CTGF/NOV (CCN) 2, and investigated the role of CCN2 in meniscus cells. Uni-axial cyclic tensile strain (CTS) was applied using a STB-140 system. CTS-induced expression of CCN2 and a1(I) collagen (COL1A1) was assessed by quantitative real-time PCR analysis. The distribution of CCN2 and Smad2/3 in stretched cells was investigated by immunohistochemical analysis. Smad2/3-dependent CCN2 transactivation was measured by luciferase reporter assay. The relationship between Smad2/3 and CTS-induced CCN2 transcription was investigated by chromatin immunoprecipitation. CTS stimulated gene expression of CCN2 and COL1A1 in inner meniscus cells, but not in outer meniscus cells. Recombinant CCN2 increased COL1A1 expression only in inner meniscus cells. CCN2 synthesis and nuclear translocalization of phosphorylated Smad2/3 in inner meniscus cells were stimulated by CTS. The CCN2 promoter activity was synergistically enhanced by overexpressed Smad3 in stretched inner meniscus cells, but was not by Smad2. Chromatin immunoprecipitation revealed that CTS increased the association between Smad3 and the Smad-binding element on the CCN2 proximal promoter in inner meniscus cells. Our results suggest that stretch-induced CCN2 may have a crucial role in regulating COL1A1 expression in the inner meniscus. ß 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res Keywords: CCN2; meniscus; cyclic tensile strain; collagen, Smad3 The meniscus is a fibrocartilaginous tissue that plays an important role in controlling complex biomechanics of the knee, such as tension, compression, and shear stress. 1 Proper functioning of the meniscus depends on the composition and organization of its extracellular matrix (ECM). 2 Type I collagen accounts for more than 90% of the total meniscal collagen and is present throughout the entire meniscus. On the other hand, type II collagen is restricted to the inner region of the meniscus. 2–4 However, the distributions of types I and II collagen in human menisci are different from those in rabbit and sheep menisci. 5 In the adult human meniscus, a perimeniscal capillary plexus supplies the outer 10–25% of the meniscus, whereas the inner 70– 80% of the meniscus is composed of avascular tissue. 6 The avascular inner meniscus has a more chondrocytic phenotype compared with the outer meniscus. 7,8 Human inner meniscus cells derived from the inner region of the menisci have chondrocytic morphology and an ability to produce type II collagen, a cartilage- specific ECM component. 8 On the other hand, outer meniscus cells show fibroblastic morphology and mainly synthesize type I collagen that can resist a circumferential tensile stress. 8 In humans, axial knee joint contact forces in the stance phase reach a magni- tude of À2,100 N (2.8 body weight) during normal gait. 9 Larger tensile strains (>7%) are computed in inner regions of the lateral menisci compared with the outer regions. 10 Previous studies demonstrate that mechanical stimuli regulate the expression of growth factors, ECM proteins, and catabolic molecules in the menisci. 7,11–14 However, the distinct regulation of growth factor and ECM production in response to mechanical stretch remains unclear in human inner and outer meniscus cells. CCN2, also known as connective tissue growth factor, is a classical member of the CYR61/CTGF/NOV (CCN) family that has a multifunctional role in cellu- lar proliferation, differentiation, ECM synthesis, and tissue regeneration. 15 Ccn2-null mice are perinatal lethal, showing severe chondrodisplasia characterized by deficient ECM production, impaired endochondral ossification, and reduced growth plate angiogenesis. 16 These phenotypes indicate that CCN2 plays an essen- tial role in skeletal development during embryogene- sis. In the adult, the expression of CCN2 is associated with injury repair, fibrosis, angiogenesis, and tumori- genesis. 17 We have previously reported that CCN2 promotes the proliferation of chondrocytic cells, 18,19 osteoblastic cells, 20 anterior cruciate ligament (ACL)- derived cells, 21 and vascular endothelial cells. 22 In addition, CCN2 stimulates the expression of several types of collagen genes in a cellular phenotype- dependent manner. In chondrocytic HCS-2/8 cells and rabbit growth cartilage cells, CCN2 activates not only a1(II) collagen (COL2A1) expression but also the expression of a1(X) collagen (COL10A1), a marker of hypertrophic chondrocytes. 18,19 However, COL10A1 is not induced by CCN2 treatment in rabbit articular chondrocytes despite CCN2 promotes COL2A1 expression. 19 On the other hand, CCN2 increases the Additional supporting information may be found in the online version of this article. This work was supported by Japan Society for the Promotion of Science (No. 20791040 and 23592216), JSPS Fujita Memorial Fund for Medical Research, Japan Orthopaedics and Traumatol- ogy Foundation (No. 225), and the Nakatomi Foundation. Correspondence to: Takayuki Furumatsu (T: 81-86-235-7273; F: 81-86-223-9727; E-mail: matino@md.okayama-u.ac.jp) ß 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2012 1