The transcription factor mohawk plays an important role for maintaining human ACL homeostasis and ligament/tendon differentiation of mesenchymal stem cells

Abstract

s / Osteoarthritis and Cartilage 21 (2013) S9–S62 S48 markers measured (Figure 1C). This phenotype represented the sole AF cell type identified thus far. Conclusions: We generated the first human annulus fibrosis cell lines and present an detailed characterization of their phenotype in comparison to our novel nucleus pulposus cell lines. The distinct migratory behavior of isolated immortal AF clones, in contrast to that of cell pools, suggests that distinct phenotype may be preferentially immortalized and, by inference, that functionally different subtypes may exist in AF cell population. Our future investigations will focus on integrin expression, in relation to culturing them on defined (e.g. fibronectin, collagen) surfaces, as such conditions were suggested to mimic AF-like conditions. ĂThis research forms part of the Project P2.01 IDiDAS of the research program of the BioMedical Materials institute, cofunded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation. 77 THE TRANSCRIPTION FACTOR MOHAWK PLAYS AN IMPORTANT ROLE FOR MAINTAINING HUMAN ACL HOMEOSTASIS AND LIGAMENT/ TENDON DIFFERENTIATION OF MESENCHYMAL STEM CELLS K. Otabe, H. Nakahara, A. Hasegawa, F. Ayabe, T. Matsukawa, M.K. Lotz, H. Asahara. The Scripps Res. Inst., La Jolla, CA, USA Background/Purpose: Mohawk homeobox (MKX) has been demonstrated as a tendon and ligament specific transcriptional factor. MKX knock-out mice showed hypoplastic tendons throughout the body, due to reduced type I collagen production in tendon cells. However, in mature human tendon or ligament tissues, the function of MKX has not been fully understood. The aims of this study are to characterize the expression of MKX in normal human knee joints and osteoarthritis (OA) affected knee joints and to investigate the role of MKX in human ligament homeostasis and in ligament/tenogenic differentiation of mesenchymal stem cells. Methods: Human ACL specimens were obtained from the knee joints at autopsy within 24-48 hours postmortem with approval of the Scripps Human Subjects Committee. 7 normal donors (mean SD age 33.6 11.7 years) and 8 donors with OA (mean SD age 77.5 11.5 years) were analyzed. All cartilage surfaces were graded macroscopically. ACL degeneration was assessed macroscopically and histologically using quantitative scoring systems. ACL tissues were analyzed for the expression of MKX by immunohistochemistry and quantitative RT-PCR assays. Primary cultured human ACL cells were stimulated with IL-1b to examine whether this pro-inflammatory cytokine modulates MKX expression. Moreover, in order to examine the function of MKX in ECM production and differentiation in these cells, we performedMKX knockdown by MKX specific siRNA. To examine the function of MKX in ECM production and differentiation in human bone marrow mesenchymal stem cells (BMMSC) (n1⁄46 from 2 donors), we performed over-expression of MKX by adenoviral vector and analyzed gene and protein levels of COL1a1, and tenascin b (TNXb), important ECM components of ACL. Results: The expression of MKX mRNA was remarkably decreased during OA development. In addition, the expression of COL1a1 was also decreased in the OA group. Immunohistochemistry revealed that the percentage of MKX positive cells was significantly reduced in OA group. In primary cultured human ACL cells, the expression of MKX was significantly reduced by the treatment of IL-1b. The expression of ECM genes COL1a1 and TNXb and transcription factor Scleraxis (SCX), which is a specific regulator of the tendon/ligament lineage, were downregulated by IL-1b treatment. On the other hand, SOX9, which is involved in the modulation of chondrocyte like phenotype, was upregulated by IL-1b treatment. The expression of COL1a1 and TNXbwere decreased by MKX specific siRNA treatment, while SOX9 was increased. By contrast, the expression of COL1a1 and TNXb were increased by overexpression of MKX in BMMSCs. Conclusion: To our knowledge, this is the first report to investigate the MKX expression and function in human ligaments. The present findings demonstrate that the ACL in OA-affected joints are deficient in MKX expression and this is associated with reduced ECM production. Reduced MKX in OA may be due to inflammatory stimuli such as IL-1b. In cultured ACL cells, and in mesenchymal stem cells MKX promotes the maintenance and induction of tenocyte phenotype. Thus, MKX is a key factor that is required for maintenance of ligament structure and