RESEARCH ARTICLE 17-DMAG regulates p21 expression to induce chondrogenesis in vitro and in vivo Karri L. Bertram 1,2 , Nadia Narendran 1 , Pankaj Tailor 1,3 , Christina Jablonski 1,2 , Catherine Leonard 1,4 , Edward Irvine 1 , Ricarda Hess 1 , Anand O. Masson 1,2 , Saleem Abubacker 1 , Kristina Rinker 5,6 , Jeff Biernaskie 4,7,8 , Robin M. Yates 7 , Paul Salo 1,4 , Aru Narendran 9 and Roman J. Krawetz 1,3,4, * ABSTRACT Cartilage degeneration after injury affects a significant percentage of the population, including those that will go on to develop osteoarthritis (OA). Like humans, most mammals, including mice, are incapable of regenerating injured cartilage. Interestingly, it has previously been shown that p21 (Cdkn1a) knockout (p21 -/- ) mice demonstrate auricular (ear) cartilage regeneration. However, the loss of p21 expression is highly correlated with the development of numerous types of cancer and autoimmune diseases, limiting the therapeutic translation of these findings. Therefore, in this study, we employed a screening approach to identify an inhibitor (17-DMAG) that negatively regulates the expression of p21. We also validated that this compound can induce chondrogenesis in vitro (in adult mesenchymal stem cells) and in vivo (auricular cartilage injury model). Furthermore, our results suggest that 17-DMAG can induce the proliferation of terminally differentiated chondrocytes (in vitro and in vivo), while maintaining their chondrogenic phenotype. This study provides new insights into the regulation of chondrogenesis that might ultimately lead to new therapies for cartilage injury and/or OA. KEY WORDS: Cartilage, p21, Mesenchymal stem cells, Regeneration, Macrophage INTRODUCTION Approximately one in eight individuals suffer from osteoarthritis (OA), a disease characterized by degeneration of the cartilage surfaces of the joints with resulting pain and disability. Within a generation (30 years), there will be a new diagnosis of OA every 60 s (http://www.arthritisalliance.ca/images/PDF/eng/Initiatives/ 20111022_2200_impact_of_arthritis.pdf ). As early as the 1700s, it was observed that the intrinsic regeneration capacity of articular cartilage is minimal (Hunter, 1995), and there is still a lack of approved therapeutic approaches proven to induce cartilage repair. Therefore, regenerative medicine approaches for cartilage repair offer a paradigm shift, which could fundamentally change health care delivery for patients suffering from cartilage injuries and/or OA. Unlike most other tissues in the body, it is largely believed that articular cartilage does not contain a stem and/or progenitor cell population(s). Recent publications have challenged this dogma and suggested that such a population does exist in the superficial zone of articular cartilage and possesses the ability to undergo chondrogenesis (Jiang and Tuan, 2015; Yu et al., 2014). Nevertheless, it is clear that cartilage demonstrates an ineffective repair response after injury (Hunziker, 2002). It has long been assumed that the collagen matrix within the articular cartilage is static, with very little turnover occurring throughout adulthood, and a recent study by Heinemeier et al. supports this hypothesis (Heinemeier et al., 2016). Interestingly, however, in many mammals including humans, mesenchymal stem cell (MSC) populations exist both within the synovial membrane and synovial fluid (De Bari et al., 2001; Jones et al., 2004; Mochizuki et al., 2006; Yoshimura et al., 2007). In mouse and rabbit model systems, endogenous synovial MSCs can migrate to the site of cartilage injury and undergo chondrogenic differentiation in vivo (Hunziker and Rosenberg, 1996; Kurth et al., 2011). However, in these model systems, minimal repair of the cartilage is observed after injury. Recently, our group investigated the use of exogenous synovial MSCs to treat focal cartilage defects in mice, and observed that injection of these cells into an injured joint did confer some level of therapeutic benefit (Mak et al., 2016). Additionally, in that study, we also injected synovial MSCs derived from Murphy Roth’s Large (MRL) mice [demonstrated to have an increased level of spontaneous injury repair (Clark et al., 1998; Diekman et al., 2013)], and found that MRL synovial MSCs display superior cartilage repair capacity compared with C57BL/6 synovial MSCs (Mak et al., 2016). Mammals typically do not demonstrate cartilage repair after injury, although there are a few notable exceptions, such as the African Spiny mouse, which can almost completely regenerate ear cartilage injuries (Seifert et al., 2012). Although mouse pinna/ auricular cartilage is elastic cartilage, it is similar to articular cartilage in the sense that ear cartilage does not spontaneously heal after injury (Clark et al., 1998). Interestingly, it has also been observed that MRL mice also have the capacity to regenerate articular cartilage after a focal defect (Fitzgerald et al., 2008). While the Spiny mouse and MRL mouse both demonstrate increased wound healing (including cartilage) after injury, these mice have a number of differences at the genetic and epigenetic levels compared with nonhealing strains (such as C57BL/6 mice) (Gawriluk et al., 2016). This makes it difficult to determine which gene(s) is responsible for the healer phenotype. Although a number of Received 11 January 2018; Accepted 3 August 2018 1 McCaig Institute for Bone and Joint Health, University of Calgary, Calgary, AB T2N 4N1, Canada. 2 Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB T2N 4N1, Canada. 3 Department Cell Biology and Anatomy, University of Calgary, Calgary, AB T2N 4N1, Canada. 4 Department of Surgery, University of Calgary, Calgary, AB T2N 4N1, Canada. 5 Department of Chemical and Petroleum Engineering, University of Calgary, Calgary, AB T2N 4N1, Canada. 6 Centre for Bioengineering Research and Education, University of Calgary, Calgary, AB T2N 4N1, Canada. 7 Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada. 8 Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, AB T2N 4N1, Canada. 9 Division of Pediatric Oncology, Alberta Children’s Hospital, Calgary, AB T3B 6A8, Canada. *Author for correspondence (rkrawetz@ucalgary.ca) R.J.K., 0000-0002-2576-4504 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 © 2018. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2018) 11, dmm033662. doi:10.1242/dmm.033662 Disease Models & Mechanisms