PKCδ is required for Jagged-1 induction of human mesenchymal stem cell osteogenic differentiation.

JAG1, the gene for the Jagged-1 ligand (Jag1) in the Notch signaling pathway, is variably mutated in Alagille Syndrome (ALGS). ALGS patients have skeletal defects, and additionally JAG1 has been shown to be associated with low bone mass through genome-wide association studies. Plating human osteoblast precursors (human mesenchymal stem cells-hMSCs) on Jag1 is sufficient to induce osteoblast differentiation; however, exposure of mouse MSC (mMSC) to Jag1 actually inhibits osteoblastogenesis. Overexpression of the notch-2 intracellular domain (NICD2) is sufficient to mimic the effect of Jag1 on hMSC osteoblastogenesis, while blocking Notch signaling with a γ-secretase inhibitor or with dominant-negative mastermind inhibits Jag1-induced hMSC osteoblastogenesis. In pursuit of interacting signaling pathways, we discovered that treatment with a protein kinase C δ (PKCδ) inhibitor abrogates Jag1-induced hMSC osteoblastogenesis. Jag1 results in rapid PKCδ nuclear translocation and kinase activation. Furthermore, Jag1 stimulates the physical interaction of PKCδ with NICD. Collectively, these results suggest that Jag1 induces hMSC osteoblast differentiation through canonical Notch signaling and requires concomitant PKCδ signaling. This research also demonstrates potential deficiencies in using mouse models to study ALGS bone abnormalities.

Zhu F ，Sweetwyne MT ，Hankenson KD 《-》

MicroRNA-34a inhibits osteoblast differentiation and in vivo bone formation of human stromal stem cells.

Osteoblast differentiation and bone formation (osteogenesis) are regulated by transcriptional and post-transcriptional mechanisms. Recently, microRNAs (miRNAs) were identified as novel key regulators of human stromal (skeletal, mesenchymal) stem cells (hMSC) differentiation. Here, we identified miRNA-34a (miR-34a) and its target protein networks as modulator of osteoblastic (OB) differentiation of hMSC. miRNA array profiling and further validation by quantitative RT-PCR revealed that miR-34a was upregulated during OB differentiation of hMSC, and in situ hybridization confirmed its OB expression in vivo. Overexpression of miR-34a inhibited early commitment and late OB differentiation of hMSC in vitro, whereas inhibition of miR-34a by anti-miR-34a enhanced these processes. Target prediction analysis and experimental validation confirmed Jagged1 (JAG1), a ligand for Notch 1, as a bona fide target of miR-34a. siRNA-mediated reduction of JAG1 expression inhibited OB differentiation. Moreover, a number of known cell cycle regulator and cell proliferation proteins, such as cyclin D1, cyclin-dependent kinase 4 and 6 (CDK4 and CDK6), E2F transcription factor three, and cell division cycle 25 homolog A were among miR-34a targets. Furthermore, in a preclinical model of in vivo bone formation, overexpression of miR-34a in hMSC reduced heterotopic bone formation by 60%, and conversely, in vivo bone formation was increased by 200% in miR-34a-deficient hMSC. miRNA-34a exhibited unique dual regulatory effects controlling both hMSC proliferation and OB differentiation. Tissue-specific inhibition of miR-34a might be a potential novel therapeutic strategy for enhancing in vivo bone formation.

Chen L ，Holmstrøm K ，Qiu W ，Ditzel N ，Shi K ，Hokland L ，Kassem M ... -  《-》

Notch ligand, JAG1, is evolutionarily conserved target of canonical WNT signaling pathway in progenitor cells.

Katoh M ，Katoh M 《INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE》

Notch signaling during chondrogenesis of human bone marrow stem cells.

Notch signaling is an important mechanism involved in early development which helps to determine the differentiation and fate of cells destined to form different tissues in the body. Its role in the differentiation of adult stem cells, such as those found in bone marrow is much less clear. As there is great interest in the potential of human bone marrow stem cells (hMSC) as a source of cells for the repair of articular cartilage and other tissues, it is important to understand if Notch signaling promotes or suppresses differentiation. Using primary human bone marrow stem cells (hMSC) in 3D cell aggregate culture a new study has investigated the expression of the canonical Notch pathway genes during chondrogenesis and showed that the Notch ligand, Jagged1 (JAG1) sharply increased in expression peaking early in differentiation. A Notch target gene, HEY1, was also expressed with a temporal profile, which closely followed the expression of JAG1 and this preceded the rise in type II collagen expression that characterized chondrogenesis. The JAG1 mediated Notch signaling was shown with a Notch inhibitor (DAPT) to be necessary for chondrogenesis, as inhibition days 0-14, or just days 0-5, blocked chondrogenesis, whereas Notch inhibition days 5-14 did not. In further experiments Notch signaling was shown to be critical for full chondrogenesis, as adenoviral hJAG1 transduction of hMSCs, which caused continuous expression of JAG1 and sustained Notch signaling, completely blocked chondrogenesis. In these cultures there was inhibited production of extracellular matrix and failure to differentiate was interpreted as the retention of the hMSC in a pre-chondrogenic state. The results in this study thus showed that JAG1 mediated Notch signaling in hMSC was necessary to initiate chondrogenesis, but must be switched off for chondrogenesis to proceed and it will be important to establish if this is a mechanism common to all chondrocyte differentiation.

Oldershaw RA ，Hardingham TE 《-》

Jagged1 immobilization to an osteoconductive polymer activates the Notch signaling pathway and induces osteogenesis.

Treatment of nonunion fractures is a significant problem. Common therapeutics, including autologous bone grafts and bone morphogenetic proteins, show well-established limitations. Therefore, a need persists for the identification of novel clinical therapies to promote healing. The Notch signaling pathway regulates bone development. Clinically, loss-of-function mutations to the Notch ligand Jagged1 decrease bone mass and increase fracture risk. Jagged1 is also the most highly upregulated ligand during fracture repair, identifying it as a potential target to promote bone formation. Therefore, the objective of this study was to develop a clinically translatable construct comprised of Jagged1 and an osteoconductive scaffold, and characterize its activity in human mesenchymal stem cells (hMSC). We first evaluated the effects of Jagged1 directly immobilized to a novel poly(β-amino ester) relative to indirect coupling via antibody. Direct was more effective at activating hMSC Notch target gene expression and osteogenic activity. We then found that directly immobilized Jagged1 constructs induced osteoblast differentiation. This is the first study to demonstrate that Jagged1 delivery transiently activates Notch signaling and increases osteogenesis. A positive correlation was found between Jagged1-induced Notch and osteogenic expression. Collectively, these results indicate that Jagged1 coupled to an osteogenic biomaterial could promote bone tissue formation during fracture healing.

Dishowitz MI ，Zhu F ，Sundararaghavan HG ，Ifkovits JL ，Burdick JA ，Hankenson KD ... -  《-》