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Medicine, SMBD- Jewish General Hospital, Mc. Gill University, Montreal, PQ, Canada. View Megan Mather’s professional profile on LinkedIn. Baylor College of Medicine/VAMC. We previously reported that neonatal mice with targeted disruption of the PTH gene (PTH. At 4 months of age, however, PTH. Haploinsufficient PTHr. P+/. To determine whether PTHr. P plays any role in maintaining higher bone mass in postnatal PTH. At 4 months of age, PTH. Trabecular bone volumes of tibiae, femora and vertebrae were increased by 1. PTH. Alkaline phosphatase positive osteoblast numbers and bone formation rates (assessed by double- calcein labeling) were decreased in PTH. The number and size of TRAP positive osteoclasts were also diminished in PTH. This reduction was associated with a decrease in RANKL positive osteoblasts as demonstrated by immunohistochemical staining. Bone levels of PTHr. P m. RNA as determined by RT- PCR and immunohistochemical levels of PTHr. P in osteoblasts were enhanced in PTH. These studies show that PTHr. P haploinsufficiency can reduce further the decreased bone turnover in PTH deficient animals and that the increased postnatal trabecular bone volume observed in PTH deficient animals is dependent on the action of PTHr. P. PTHr. P therefore is an important mediator of postnatal bone growth. Disclosures: D. Miao, None. Relief of Inhibition by Twist Proteins Determines the Onset of Osteoblast Differentiation. 41 0 Encontro Anual da Sociedade Americana de. Neurology, Baylor College of Medicine, VAMC 2002 Holcombe. Initiative Competitive Grants program.P. 1. Moleuclar and Human Genetics, Baylor College of Medicine, Houston, TX, USA, 2. Molecular Biology, U T Southwestern Medical Center, Dallas, TX, USA, 3. Molecular Biology and Pharmacology, Washington University Medical School, St Louis, MO, USA, 4. Molecular Biology, UT Southwestern Medical Center, Dallas, TX, USA. The transcription factor Runx. That Runx. 2 expression precedes osteoblast differentiation by three to four days suggests that its function may be transiently inhibited. The craniosynostosis of Twist+/. Here we show that, in the developing skull, Twist is expressed at higher levels than Runx. Runx. 2- expressing cells before osteoblast differentiation occurs. Twist inhibits osteoblast gene expression and Runx. Runx. 2's expression. Compound heterozygotes for Twist and Runx. Runx. 2+/. Transgenic mice expressing Twist in osteoblast progenitors mimic the Runx. Runx. 2 expression. Twenty residues in Twist's C- terminus form a unique antiosteogenic domain, the Twist box, that interacts with Runx. DNA- binding domain to inhibit its DNA- binding function. These findings reveal that osteoblast differentiation only begins when an inhibition is relieved and provide a molecular explanation for Saethre- Chotzen syndrome. Twist's broad expression during development suggests that this may be a general mechanism to control Runx- dependent cell differentiation programs. Disclosures: P. Bialek, None. GADD3. 4/PP1c Recruited by Smad. Dephosphorylates TGF. Pathology, University of Alabama at Birmingham, Birmingham, AL, USA, 2. Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL, USA. Transforming growth factor . The cascade of phosphorrylation from receptors to effectors is a well- characterized, pivotal event in TGF- . Smad. 7, induced by TGF- . However, the possible inhibitory mechanism by dephosphorylation in the TGF- . Screening a human chondrocyte c. DNA library in yeast two- hybrid system with Smad. Smad. 7 interacted with the growth arrest and DNA damage protein, GADD3. PP1) holoenzyme. Immunoprecipitation (IP) experiments demonstrated that Smad. GADD3. 4 and PP1 on TGF- . The complex was shown to dephosphorylate T. Blockage of Smad. RNA inhibited association of the GADD3. PP1c complex with T. Overexpression of either dominant- negative Smad. GADD3. 4, absent of their binding domain, inhibited Smad. T. Moreover, SARA, as a Smad anchor for receptor activation, also acts as anchor for PP1c via its PP1c- binding motif. Most importantly, dominant- negative SARA, absent of its PP1c- binding motif, inhibited involvement of PP1c in this proposed complex and dephosphorylation of T. Furthermore, transcriptional response assay and FACS- DNA profiling analysis demonstrated that Smad. GADD3. 4/PP1c complex inhibited TGF- . These results indicate that Smad. PP1 holoenzyme that targets T. Thus, we have characterized a novel T. Dephosphorylation of T. This complex could potentially be used as a drug target to modulate TGF- . Shi, None. 1. 00. Evidence that JNK/c- Jun Signaling Promotes Osteoclastogenesis using Transgenic Mice Overexpressing Dominant- Negative c- Jun Selectively in Osteoclasts. F. 1. Dept Biochem, Osaka Univ Grad Sch Dent, Osaka, Japan, 2. Kaken Phram Co., Inc, Kyoto, Japan, 3. Sec Oral Mol Biol, Facult Dent Kyushu Univ, Fukuoka, Japan, 4. Div Hematol Oncol, Univ Pittsburgh Cancer Inst, Pittsburgh, PA, USA, 5. Cancer Res Inst, Kanazawa Univ, Kanazawa, Japan. RANKL is a central cytokine that promotes osteoclastogenesis. Upon RANKL binding to RANK, cytoplasmic molecules involving JNK, c- Jun, c- Fos and NF- . This notion is supported by genetic studies which demonstrated that c- Fos and NF- . On the other hand, the role of JNK and its substrate c- Jun, which forms the AP- 1 complex with c- Fos, still needs to be elucidated. In the present study, we investigated the role of JNK/c- Jun signaling in osteoclast differentiation by generating transgenic mice (DN- c- Jun Tg) carrying DN- c- Jun driven by the TRAP gene promoter. The DN- c- Jun Tg manifested severe phenotype of osteopetrosis including defect of tooth eruption, smaller body size and increased radiodensity in long bones. Histological examination showed that bone marrow cavity was markedly reduced due to reduced trabecular bone resorption and the number of osteoclasts was markedly diminished. To examine whether the osteopetrosis was due to the abnormality of osteoclast development, we determined multinucleated TRAP- positive osteoclast- like cell (OC) formation in spleen cells isolated from DN- c- Jun Tg or wild- type littermate mice. Number of OC formed in spleen cells of DN- c- Jun Tg was significantly reduced compared with that of wild type mice, indicating that the osteopetrosis seen in DN- c- Jun Tg is due to impaired osteoclastogenesis. We then examined the relationship between RANKL and JNK/c- Jun activation in vitro. Soluble RANKL (s. RANKL) activated JNK and c- Jun in the BMM. Consistent with the in vivo results, OC formation in the presence of s. RANKL and M- CSF in mouse bone marrow cultures was significantly inhibited by a specific inhibitor of JNK, SP6. Furthermore, overexpression of dominant- negative (DN)- JNK1 or DN- c- Jun using adenovirus system suppressed OC formation and increased apoptosis. In conclusion, these results collectively suggest that activation of JNK/c- Jun signaling pathway is essential to osteoclastogenesis that regulated by RANKL both in vivo and in vitro. Thus, our data provide the new insight into the molecular mechanisms by which RANKL regulates the osteoclast differentiation. Disclosures: F. Ikeda, None. The Role of the PERK e. IF2alpha Kinase in Regulating Skeletal Growth and Development. D. Biology, Penn State University, University Park, PA, USA. The orchestration of skeletal growth is regulated by a complex repertoire of differentiation factors and hormonal signals that control cell proliferation and secretion of the extracellular matrix (ECM). Inasmuch as the ECM comprises the major organic fraction of bone and cartilage, the growth and development of the skeletal system is more dependent upon protein secretion than any other major tissue in the body. Recently, a new e. IF2alpha kinase denoted PERK was discovered that is highly expressed in secretory tissues, predominately in bone tissue and the endocrine and exocrine pancreas. We have generated a knockout mutation of the mouse Perk gene (Zhang, et al., Mol. Biol 2. 2: 3. 86. Wolcott- Rallison syndrome (WRS). The skeletal dysplasias exhibited by Perk- /- mice include severe osteopenia, reduced cortical bone, delayed mineralization, and a marked deficiency of ECM in the hypertrophic region of the growth plate. The long bones of Perk- /- mice display reduced trabecular bone (2. BMD (2. 3 fold compared to controls, femur) and reduced growth plates. Bone collagen- I in Perk- /- mice is reduced approximately 2- fold, whereas intracellular procollagen- I in osteoblasts is increased 8fold. Ultrastructural analysis of osteoblasts indicated that procollagen- I processing and secretion are impaired in the endoplasmic reticulum. These defects are similar to that seen in osteogenesis imperfecta associated with mutations in the procollagen gene that result in abnormal procollagen retention in the ER. In addition to the cortical defects, histomorphic analyses of long bone growth plates in Perk- /- mice identified an array of defects including a reduction in the height of the hypertrophic and proliferative zones. The reduction in the hypertrophic zone is due to a 2. In addition, an abrupt transition from the proliferative to the hypertrophic zone in the Perk- /- growth plates was seen, as indicated by a deficiency in chondrocytes exhibiting intermediate width- height ratios. Perk- /- mice display a 3. ECM in the hypertrophic zone. In Perk- /- mice, the parallel ECM tracts are diminished in number and width giving rise to abnormally thin trabeculae. We are examining the cell- autonomous nature of these defects by generating tissue- specific knockout mutations of Perk in the skeletal system using the Cre/lox. P system. Disclosures: D. R. Cavener, None. Fra- 2: A Novel Regulator of Bone Remodeling. A. 1. Research Institute of Molecular Pathology, Vienna, Austria, 2. Hamburg University School of Medicine, Hamburg, Germany. The three Fos proteins c- Fos, Fos. B, Fra- 1 have been shown to play crucial roles in bone biology, but little is known about Fra- 2, the fourth Fos protein. Bunun nedeni, denizcilik tarihimizle ilgili fazla ara. Spikerlik kursu olarak, CNNT Visa, Master ve Dinners kartlar
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