The Transcriptional Co-Regulator Jab1 Is Crucial for Chondrocyte

234 Research Article The transcriptional co-regulator Jab1 is crucial for chondrocyte differentiation in vivo Dongxing Chen1, Lindsay A. Bashur1, Bojian Liang1,*, Martina Panattoni2, Keiko Tamai3,`, Ruggero Pardi2 and Guang Zhou1,3,4,§ 1Department of Orthopaedics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA 2San Raffaele University, School of Medicine and Scientific Institute San Raffaele, Milan, Italy 3Department of Genetics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA 4Case Comprehensive Cancer Center, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA *Present address: Department of Orthopaedics, China-Japan Union Hospital, Jilin University, Changchun, Jilin Province, People’s Republic of China `Present address: Oncology Research Laboratories, Daiichi Sankyo Co., Ltd.., Tokyo, Japan §Author for correspondence ([email protected]) Accepted 11 November 2012 Journal of Cell Science 126, 234–243 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.113795 Summary The evolutionarily conserved transcriptional cofactor Jab1 plays critical roles in cell differentiation, proliferation, and apoptosis by modulating the activity of diverse factors and regulating the output of various signaling pathways. Although Jab1 can interact with the bone morphogenetic protein (BMP) downstream effector Smad5 to repress BMP signaling in vitro, the role of Jab1 in BMP-mediated skeletogenesis in vivo is still poorly understood. As a key regulator of skeletogenesis, BMP signaling regulates the critical Ihh-Pthrp feedback loop to promote chondrocyte hypertrophy. In this study, we utilized the loxP/Cre system to delineate the specific role of Jab1 in cartilage formation. Strikingly, Jab1 chondrocyte-specific knockout Jab1flox/flox; Col2a1-Cre (cKO) mutants exhibited neonatal lethal chondrodysplasia with severe dwarfism. In the mutant embryos, all the skeletal elements developed via endochondral ossification were extremely small with severely disorganized chondrocyte columns. Jab1 cKO chondrocytes exhibited increased apoptosis, G2 phase cell cycle arrest, and increased expression of hypertrophic chondrocyte markers Col10a1 and Runx2. Jab1 can also inhibit the transcriptional activity of Runx2, a key regulator of chondrocyte hypertrophy. Notably, our study reveals that Jab1 is likely a novel inhibitor of BMP signaling in chondrocytes in vivo.InJab1 cKO chondrocytes, there was heightened expression of BMP signaling components including Gdf10/Bmp3b and of BMP targets during chondrocyte hypertrophy such as Ihh. Furthermore, Jab1 cKO chondrocytes exhibited an enhanced response to exogenous BMP treatment. Together, our study demonstrates that Jab1 represses chondrocyte hypertrophy in vivo, likely in part by downregulating BMP signaling and Runx2 activity. Journal of Cell Science Key words: Jab1/Csn5, Chondrodysplasia, BMP, Runx2, COP9 signalosome Introduction (Kronenberg, 2003). During endochondral bone development, During development, endochondral ossification forms most of Ihh is synthesized by prehypertrophic chondrocytes and early the skeleton, including vertebrae, ribs and long bones. It involves hypertrophic chondrocytes. Ihh binds to its receptor Patched-1 an initial cartilage anlagen within which chondrocytes undergo (Ptc-1), which triggers a cascade that leads to the activation of an elaborate and well-controlled differentiation process target genes including Pthrp under a mechanism still poorly (Kronenberg, 2003). The mesenchymal precursors first understood. Pthrp acts on its receptor to keep the chondrocyte condense and commit to chondrocyte lineage under master proliferating and inhibits the production of Ihh. This Ihh/Pthrp regulator Sox9, followed by the robust secretion of cartilage- negative-feedback loop controls the length of proliferating specific extracellular matrix including type II collagen and rapid columns during cartilage growth (Kronenberg, 2003). proliferation to form orderly columns. Chondrocytes then stop The intracellular factor Jab1 (c-Jun activation domain-binding dividing, exit the cell cycle, and start prehypertrophy with the protein-1) interacts with numerous proteins to regulate diverse expression of type X collagen under key regulators Runx2, signaling pathways and cell differentiation processes (Shackleford Runx3 and Indian Hedgehog (Ihh). Subsequently, chondrocytes and Claret, 2010). Jab1 is also the fifth subunit of the evolutionarily undergo overt hypertrophy and endochondral ossification occurs conserved proteolysis regulator COP9 signalosome (Csn5) when hypertrophic chondrocytes within the cartilaginous model complex (Bech-Otschir et al., 2002; Olma et al., 2009). CSN is begin to undergo apoptosis in the center of the cartilage. similar, both in size and composition, to the lid of the 19S Osteoblasts are recruited here from adjacent perichondrium and proteasome regulatory particle (Wei and Deng, 2003). CSN has secrete bone matrix, forming a primary ossification center deneddylase activity to regulate the stability of complex Skip1– (Kronenberg, 2003). Ihh is a master regulator of skeletal Cullin–F-box proteins (SCF), a major group of cullin ring E3 development by coordinating chondrocyte proliferation, ubiquitin ligase (CRL). SCF core component cullin is covalently chondrocyte differentiation, and osteoblast differentiation modified with the ubiquitin-like protein NEDD8. Repeated cycles Jab1 and chondrocyte differentiation 235 of NEDD8 conjugation (neddylation) and NEDD8 removal in chondrocytes results in severe chondrodysplasia with reduced (deneddylation) maintain SCF E3 ubiquitin ligase activity (Wei Col10a1 expression, a phenotype very similar to chondrocyte- et al., 2008). By regulating SCF activity, CSN can regulate the specific BMP receptors null mice (Retting et al., 2009). Thus, the stability and activity of numerous cell-cycle proteins such as p27, effect of BMP signaling in embryonic cartilage formation largely p53 and transcriptional regulators including IkB-a (Wei et al., depends on Smad 1/5/8. Further analysis reveals that Smad1 and 2008; Chamovitz, 2009). Within the whole COP9 signalosome, Smad5 are positive and redundant regulators of chondrocyte Jab1/Csn5 is the only subunit possessing a catalytic metal binding differentiation, whereas Smad8 is mostly dispensable for metalloprotease motif that is required for removing NEDD8 from cartilage formation (Retting et al., 2009). the cullin subunit of SCF (Shackleford and Claret, 2010). Thus, The effect of BMP signaling during skeletogenesis ultimately Jab1 is required for COP9 signalosome activity. impinges on a complex transcriptional network in which the Jab1 is involved in signaling transduction, cell cycle control, transcription factors Sox9 and Runx2 play essential roles apoptosis, DNA repair, and a vast array of developmental (Kronenberg, 2003). Runt domain transcription factor Runx2 processes by regulating the stability and activity of various regulates all the major genes expressed by osteoblasts in tissue transcription factors (Wei et al., 2008; Chamovitz, 2009; Kato culture (Ducy et al., 1997). Runx2 null mice display a complete and Yoneda-Kato, 2009; Shackleford and Claret, 2010). lack of osteoblast differentiation (Komori et al., 1997; Otto et al., Furthermore, Jab1 overexpression has been implicated in the 1997). Moreover, mutations in RUNX2 result in cleidocranial initiation and progression of various cancers, suggesting an dysplasia (CCD), a dominantly inherited skeletal dysplasia with oncogenic role (Shackleford and Claret, 2010). The constitutive generalized bone defects (Lee et al., 1997; Mundlos et al., 1997; deletion of Jab1 in mice results in early embryonic lethality by Zhou et al., 1999). Besides its essential role in osteoblast E8.5 with impaired proliferation and accelerated apoptosis differentiation, Runx2 is also important for chondrocyte (Tomoda et al., 2004; Tian et al., 2010), whereas cell type- maturation. Continuous expression of Runx2 in mouse specific deletions of Jab1 in T-cell, B-cell, or myeloid cells all proliferating chondrocytes accelerated the hypertrophy (Takeda lead to severe postnatal cell differentiation defects and increased et al., 2001; Ueta et al., 2001). Conversely, chondrocyte apoptosis (Panattoni et al., 2008; Deng et al., 2011; Sitte et al., maturation was delayed in some skeletal elements in Runx22/2 2012). Thus, Jab1 plays essential roles both in general mice (Inada et al., 1999; Kim et al., 1999). Runx2 directly binds embryogenesis and in the differentiation of specific organs and to the conserved cis-elements in Col10a1 and Ihh promoters, and tissues. However, the specific function of Jab1 in skeletogenesis contributes to their activity in prehypertrophic and hypertrophic in vivo was completely unknown prior to this study. chondrocytes (Yoshida et al., 2004; Zheng et al., 2003). Bone morphogenetic protein (BMP) signaling coordinates all Moreover, Runx2 and related Runx3 play an essential and steps of skeletal growth and differentiation during endochondral redundant role during chondrocyte hypertrophy. Runx22/2; ossification (Chen et al., 2004; Yoon and Lyons, 2004; Wan and Runx32/2 mice show the complete absence of chondrocyte Cao, 2005). BMPs are members of the TGF-b superfamily that hypertrophy with reduced chondrocyte proliferation and reduced activate heterodimeric receptors with serine/threonine kinase cell size in the embryonic limbs (Yoshida et al., 2004). activity (Chen et

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