The transcriptional cofactor Jab1/Cops5 is crucial for BMP-mediated mouse chondrocyte differentiation by repressing p53 activity.

We previously reported that the evolutionary conserved transcriptional cofactor Jab1/Cops5 is critical for mouse chondrocyte differentiation by selectively repressing BMP signaling. In this study, we first uncovered that the endogenous Jab1 interacts with endogenous Smad1/5/8. Furthermore, although Jab1 did not directly interact with Acvr1 (Alk2), a key Type I BMP receptor, the interaction between endogenous Smad1/5/8 and Acvr1 was increased in Jab1-null chondrocytes. Thus, Jab1 might negatively regulate BMP signaling during chondrocyte differentiation in part by sequestering Smad1/5/8 away from Acvr1. Next, to identity Jab1 downstream targets in chondrocytes, we performed RNA-sequencing analysis of Jab1-null chondrocytes and discovered a total of 1993 differentially expressed genes. Gene set enrichment analysis revealed that key targets inhibited by Jab1 includes p53, BMP/transforming growth factor beta, and apoptosis pathways. We confirmed that endogenous Jab1 interacts with endogenous p53. There was significantly elevated p53 reporter activity, an enhanced expression of phospho-p53, and an increased expression of a key p53 downstream target, Puma, in Jab1-null chondrocytes. Moreover, treatments with a p53-specific inhibitor and/or a BMP Type I receptor-specific inhibitor reversed the elevated p53 and BMP signaling activities in Jab1-null chondrocytes and partially restored columnar growth plate structure in E17.5 Jab1-null mouse tibia explant cultures. Finally, we demonstrated that the chondrocyte-specific Jab1 overexpression in mice resulted in smaller-sized embryos with disorganized growth plates. In conclusion, our data showed that the delicate Jab1-mediated crosstalk between BMP and p53 pathways is crucial to maintain proper chondrocyte survival and differentiation. Moreover, the appropriate Jab1 expression level is essential for proper skeletal development.

The transcriptional co-regulator Jab1 is crucial for chondrocyte differentiation in vivo.

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 Jab1(flox/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. In Jab1 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.

Loss of jab1 in osteochondral progenitor cells severely impairs embryonic limb development in mice.

The transcriptional cofactor Jab1 controls cell proliferation, apoptosis, and differentiation in diverse developmental processes by regulating the activity of various transcription factors. To determine the role of Jab1 during early limb development, we developed a novel Jab1(flox/flox) ; Prx1-Cre conditional Knockout (cKO) mutant mouse model in which Jab1 was deleted in the osteochondral progenitor cells of the limb buds. Jab1 cKO mutant mice displayed drastically shortened limbs at birth. The short-limb defect became apparent in Jab1 cKO mutants at E15.5 and increasingly worsened thereafter. By E18.5, Jab1 cKO mutant mice exhibited significantly shorter limbs with: very few hypertrophic chondrocytes, disorganized chondrocyte columns, much smaller primary ossification centers, and significantly increased apoptosis. Real-time RT-PCR analysis showed decreased expression of Sox9, Col2a1, Ihh, and Col10a1 in Jab1 cKO mutant long bones, indicating impaired chondrogenesis. Furthermore, in a micromass culture model of early limb mesenchyme cells, alcian blue staining showed a significant decrease in chondrogenesis in Jab1 cKO limb bud cells. The expression of Sox9 and its downstream targets Col2a1 and Aggrecan, as well as BMP signaling downstream targets, Noggin, Id1, and Ihh, were significantly decreased in Jab1 cKO micromass cultures. Moreover, over-expression of SOX9 in Jab1 cKO micromass cultures partially restored Col2a1and Aggrecan expression. Jab1-deficient micromass cultures also exhibited decreased BMP signaling response and reduced BMP-specific reporter activity ex vivo. In summary, our study demonstrates that Jab1 is an essential regulator of early embryonic limb development in vivo, likely in part by co-activating Sox9 and BMP signaling.

The master developmental regulator Jab1/Cops5/Csn5 is essential for proper bone growth and survival in mice.

Jab1, also known as Csn5/Cops5, is a key subunit of the COP9 Signalosome, a highly conserved macromolecular complex. We previously reported that the conditional knockout of Jab1 in mouse limb buds and chondrocytes results in severely shortened limbs and neonatal lethal chondrodysplasia, respectively. In this study, we further investigated the specific role of Jab1 in osteoblast differentiation and postnatal bone growth by characterizing a novel mouse model, the Osx-cre; Jab1flox/flox conditional knockout (Jab1 cKO) mouse, in which Jab1 is deleted in osteoblast precursor cells. Jab1 cKO mutant mice appeared normal at birth, but developed progressive dwarfism. Inevitably, all mutant mice died prior to weaning age. The histological and micro-computed tomography analysis of mutant long bones revealed severely altered bone microarchitecture, with a significant reduction in trabecular thickness. Moreover, Jab1 cKO mouse tibiae had a drastic decrease in mineralization near the epiphyseal growth plates, and Jab1 cKO mice also developed spontaneous fractures near the tibiofibular junction. Additionally, our cell culture studies demonstrated that Jab1 deletion in osteoblast precursors led to decreased mineralization and a reduced response to TGFß and BMP signaling. Moreover, an unbiased reporter screen also identified decreased TGFß activity in Jab1-knockdown osteoblasts. Thus, Jab1 is necessary for proper osteoblast differentiation and postnatal bone growth, likely in part through its positive regulation of the TGFß and BMP signaling pathways in osteoblast progenitor cells.

The crucial p53-dependent oncogenic role of JAB1 in osteosarcoma in vivo.

Osteosarcoma (OS) is the most common primary bone cancer and ranks amongst the leading causes of cancer mortality in young adults. Jun activation domain-binding protein 1 (JAB1) is overexpressed in many cancers and has recently emerged as a novel target for cancer treatment. However, the role of JAB1 in osteosarcoma was virtually unknown. In this study, we demonstrate that JAB1-knockdown in malignant osteosarcoma cell lines significantly reduced their oncogenic properties, including proliferation, colony formation, and motility. We also performed RNA-sequencing analysis in JAB1-knockdown OS cells and identified 4110 genes that are significantly differentially expressed. This demonstrated for the first time that JAB1 regulates a large and specific transcriptome in cancer. We also found that JAB1 is overexpressed in human OS and correlates with a poor prognosis. Moreover, we generated a novel mouse model that overexpresses Jab1 specifically in osteoblasts upon a TP53 heterozygous sensitizing background. Interestingly, by 13 months of age, a significant proportion of these mice spontaneously developed conventional OS. Finally, we demonstrate that a novel, highly specific small molecule inhibitor of JAB1, CSN5i-3, reduces osteosarcoma cell viability, and has specific effects on the ubiquitin-proteasome system in OS. Thus, we show for the first time that the overexpression of JAB1 in vivo can result in accelerated spontaneous tumor formation in a p53-dependent manner. In summary, JAB1 might be a unique target for the treatment of osteosarcoma and other cancers.