Mechanical stretch increases Smad3‐dependent CCN2 expression in inner meniscus cells

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.

Cellular and molecular actions of CCN2/CTGF and its role under physiological and pathological conditions

“…and fluid flow shear stress, cause CCN2 expression in different types of the cells [27,28]. Application of low-intensity pulsed ultrasound (LIPUS) is a therapeutic intervention already utilized in orthopaedic clinics, and it also increases CCN2 expression in gingival epithelial cells [29].…”

Connective tissue growth factor, matrix regulation, and diabetic kidney disease

“…The most well recognized function of CCN family members is their role in adhesion and regulation of cellular proliferation, survival and apoptosis. CCN-2 expression is sensitive to cell environmental changes, including the presence of some growth factors, in particular TGF-b and VEGF, cytokines, exposure to oxygen deprivation and mechanical forces [17][18][19]. CCN-2 was originally identified as a protein secreted by human umbilical endothelial cells [16] and was described as having PDGF-like mitogenic and chemotactic activity.…”

Tensile strain increases expression of CCN2 and COL2A1 by activating TGF-β-Smad2/3 pathway in chondrocytic cells

“…In addition, excessive CTS (10% strain) stimulates expression of MMP-3/13 and ADAMTS-5 in human articular chondrocytes (Saito et al, 2013). On the other hand, appropriate stretching force (5-6% strain) increases expression of anabolic factors, such as COL2A1 and multifunctional growth factor CYR61/CTGF/NOV (CCN) 2, in human fibrochondrocytes derived from the inner meniscus and chondrocytic HCS-2/8 cells (Nishida et al, 2008;Kanazawa et al, 2012;Furumatsu et al, 2012).…”