The growth arrest and DNA damage-inducible 45␤ (GADD45␤) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of GADD45␤ in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45␤ as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45␤ mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45␤ protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45␤ ؊/؊ mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45␤ in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45␤ stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45␤ plays an essential role during chondrocyte terminal differentiation.Growth arrest and DNA damage-inducible (GADD) 4 45␤ is a member of the GADD45 family of small (18 kDa) proteins, also including GADD45␣ and GADD45␥. The GADD45 family is known to be associated with cell growth control, apoptotic cell death, and the cellular response to DNA damage (1, 2). Initially, GADD45␤, encoded by MyD118, was identified as a myeloid differentiation primary response gene activated by IL-6 in murine myeloid leukemia cells upon induction of terminal differentiation (1, 3). More recently, GADD45␤, which is induced by TGF-␤ in a SMAD-dependent manner, has been identified as a positive regulator of TGF-␤-induced apoptosis (4). Although GADD45␣ has been identified on DNA microarrays as prominently expressed genes in chondrocytes from adult articular cartilage and in chondrosarcoma or immortalized chondrocyte cell lines (5, 6), a role for GADD45 family members, including GADD45␤, during cartilage development has not been reported previously.Formation of the vertebrate skeleton through endochondrial ossification, involving progressive differentiation of proliferating chondrocytes to growth-arrested hypertrophic cells, is one of the most complex processes in biology. In the embryonic or postnatal growth plate, terminal chondrocyte differentiation occurs during conversion of cartilage to a vascularized tissue that supports matrix remodeling, cartilage calcification, and recruitment of osteogenic precursors. Cascades of growth and differentiation factors act through positive and negative signaling kinases and transcription factors to t...

The therapeutic potential of administering stem cells to promote angiogenesis and myocardial tissue regeneration after infarction has recently been demonstrated. Given the advantages of using embryonic stem cells and mouse models of myocardial infarction for furthering the development of this therapeutic approach, the purpose of this study was to determine if embryonic stem cells could be loaded with superparamagnetic iron oxide (SPIO) particles and imaged in a mouse model of myocardial infarction over time using MRI. Mouse embryonic stem cells were labeled with SPIO particles. When incubated with 11.2, 22.4, and 44.8 g Fe/ml of SPIO particles, cells took up increasing amounts of iron oxide. Embryonic stem cells loaded with SPIO compared to unlabeled cells had similar viability and proliferation profiles for up to 14 days. Free SPIO injected into infarcted myocardium was not observable within 12 hr after injection. After injection of three 10-l aliquots of 10 7 SPIO-loaded cells/ml into infarcted myocardium, MRI demonstrated that the mouse embryonic stem cells were observable and could be seen for at least 5 weeks after injection. The therapeutic potential of administration of adult stem cells in animal models of ischemic injury has recently been demonstrated by several investigators (1-3). These studies demonstrated that regional blood flow and capillary density was significantly higher and cardiac function was improved compared to control animals. The ability to track stem cells in vivo will greatly facilitate the development of this therapeutic modality. Two recent studies have demonstrated the ability to use MRI to track iron-labeled adult stem cells in pigs in the setting of myocardial infarction (MI) (4,5). In one study, mesenchymal cells were harvested from the bone marrow of swine, labeled with dextran-coated superparamagnetic iron oxide (SPIO) particles, and injected locally in a pig model of MI. In the second study, adult stem cells with myogenic potential were harvested from skeletal muscle of swine, labeled with SPIO particles, and injected into myocardial tissue using a percutaneous catheter.The previous studies used adult mesenchymal stem cells; however, there are advantages in using embryonic stem (ES) cells for research and eventual clinical application. Unlike primary adult stem cells, ES cells can be engineered to express other factors such as angiogenic growth factors or other proteins that might promote myocardial tissue regeneration. In clinical settings such as MI, the availability of sufficient numbers of cells at or near the onset of MI seems more likely to be feasible with ES cells than with adult stem cells. Several recent studies specifically demonstrated this therapeutic potential of using ES cells for transplantation in animal models for a variety of human diseases (6 -8), including their ability to limit myocardial tissue injury after infarction (9,10).Some of the advantages of performing these studies in mice, as opposed to other, larger animals, include the ability to perform stud...

Dissecting the molecular mechanisms that guide the proper development of epicardial cell lineages is critical for understanding the etiology of both congenital and adult forms of human cardiovascular disease. In this study, we describe the function of BAF180, a polybromo protein in ATP-dependent SWI/SNF chromatin remodeling complexes, in coronary development. Ablation of BAF180 leads to impaired epithelial-to-mesenchymal-transition (EMT) and arrested maturation of epicardium around E11.5. Three-dimensional collagen gel assays revealed that the BAF180 mutant epicardial cells indeed possess significantly compromised migrating and EMT potentials. Consequently, the mutant hearts form abnormal surface nodules and fail to develop the fine and continuous plexus of coronary vessels that cover the entire ventricle around E14. PECAM and *-SMA staining assays indicate that these nodules are defective structures resulting from the failure of endothelial and smooth muscle cells within them to form coronary vessels. PECAM staining also reveal that there are very few coronary vessels inside the myocardium of mutant hearts. Consistent with this, quantitative RT-PCR analysis indicate that the expression of genes involved in FGF, TGF, and VEGF pathways essential for coronary development are down-regulated in mutant hearts. Together, these data reveal for the first time that BAF180 is critical for coronary vessel formation.

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