Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research · 2007
Craniosynostosis-associated gene nell-1 is regulated by runx2.
СтатьяПоддержка NIH: внешняя программаПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Truong Thien, Zhang Xinli, Pathmanathan Dharmini, Soo Chia, Ting Kang
Аннотация
UNLABELLED: We studied the transcriptional regulation of NELL-1, a craniosynostosis-related gene. We identitifed three OSE2 elements in the NELL-1 promoter that are directly bound and transactivated by Runx2. Forced expression of Runx2 induces NELL-1 expression in rat calvarial cells.
INTRODUCTION: We previously reported the upregulation of NELL-1 in human craniosynostosis and the overexpression of Nell-1 in transgenic animals that induced premature suture closure associated with increased osteoblast differentiation. To study the transcriptional regulation of NELL-1, we analyzed the 5' flanking region of the human NELL-1 gene. We identified three osteoblast specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of the transcription start site and further studied the functionality of these sites.
MATERIALS AND METHODS: An area of 2.2 kb and a truncated 325 bp, which lacked the three OSE sites, were cloned into a luciferase reporter gene, and co-transfected with Runx2 expression plasmid. The three OSE2 sites were individually mutated and co-transfected with Runx2 expression plasmid into Saos2 cells. Gel shifts and supershifts with Runx2 antibodies were used to determine specific binding to OSE2 sites. CHIP assays were used to study in vivo binding of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into wildtype and Runx2(-/-) calvarial cells. Nell-1, osteocalcin, and Runx2 expression levels were measured using RT-PCR.
RESULTS: Addition of Runx2 dose-dependently increased the luciferase activity in the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct showed significantly lower basal level of activity. Nuclear extract from Saos2 cells formed complexes with site A, B, and C probes and were supershifted with Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal promoter activity. Furthermore, mutation of sites B and C had a blunted response to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn expression were rescued.
CONCLUSIONS: Runx2 directly binds to the OSE2 elements and transactivates the human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream target of Runx2. These findings may also extend our understanding of the molecular mechanisms governing the pathogenesis of craniosynostosis.
INTRODUCTION: We previously reported the upregulation of NELL-1 in human craniosynostosis and the overexpression of Nell-1 in transgenic animals that induced premature suture closure associated with increased osteoblast differentiation. To study the transcriptional regulation of NELL-1, we analyzed the 5' flanking region of the human NELL-1 gene. We identified three osteoblast specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of the transcription start site and further studied the functionality of these sites.
MATERIALS AND METHODS: An area of 2.2 kb and a truncated 325 bp, which lacked the three OSE sites, were cloned into a luciferase reporter gene, and co-transfected with Runx2 expression plasmid. The three OSE2 sites were individually mutated and co-transfected with Runx2 expression plasmid into Saos2 cells. Gel shifts and supershifts with Runx2 antibodies were used to determine specific binding to OSE2 sites. CHIP assays were used to study in vivo binding of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into wildtype and Runx2(-/-) calvarial cells. Nell-1, osteocalcin, and Runx2 expression levels were measured using RT-PCR.
RESULTS: Addition of Runx2 dose-dependently increased the luciferase activity in the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct showed significantly lower basal level of activity. Nuclear extract from Saos2 cells formed complexes with site A, B, and C probes and were supershifted with Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal promoter activity. Furthermore, mutation of sites B and C had a blunted response to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn expression were rescued.
CONCLUSIONS: Runx2 directly binds to the OSE2 elements and transactivates the human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream target of Runx2. These findings may also extend our understanding of the molecular mechanisms governing the pathogenesis of craniosynostosis.