[Frontiers in Bioscience E2, 1065-1072, June 1, 2010]

IkappaBalpha regulates Hes1 in osteoclast differentiation and resorption

Li Duan1, 2, Paul de Vos3, Marijke Faas3, Mingwen Fan4, Yijin Ren1

1 Department of Orthodontics, University Medical Centre Groningen, University of Groningen, The Netherlands, 2 School of Stomatology, Hainan Medical College, Hainan, China, 3Pathology and Medical Biology, University Medical Centre Groningen, University of Groningen, The Netherlands, 4Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School and Hospital of Stomatology, Wuhan University, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Cell culture, transfection, and infection
3.2. Osteoclast formation assays
3.3. Resorption assays
3.4. Real-time PCR
3.5. Cell extracts preparation and Western blot analysis
3.6. Electrophoretic mobility shift assay(EMSA)
3.7. Chromatin immunoprecipitation (ChIP)
3.8. Data analysis
4. Results
4.1. Stable expression of IkappaBalphaM in RAW 264.7 .cells
4.2. NF-kappaB mediates RANKL-induced differentiation and resorption
4.3. NF-kappaB inactivation down-regulates MMP-9 and DC-STAMP expression
4.4. NF-kappaB inactivation up-regulates Notch-dependent Hes1 gene expression
4.5. NF-kappaB inactivation exerts a positive effect on the transcriptional activation of the Hes1 promoter
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

During osteoclast differentiation and resorption, both NF-kappaB and Notch signalling are activated. This study defines the mechanism about the influence of NFkappaB on Notch. To this end, IkappaBalphaM and Wild-type-IkappaBalpha were transfected into RAW 264.7 cells. The number of cells that differentiated into osteoclasts was quantified. The resorption area was measured. NF-kappaB transcriptional activity was determined by EMSA. Hes1, DC-STAMP and MMP-9 mRNA expression levels were determined by RT-PCR. Hes1 protein expression was determined by western blots. ChIP was used to study binding of IkappaBalpha to the Hes1 promoter. NF-kappaB inactivation inhibited the differentiation and resorption ability of osteoclasts. Compared with Wild-type cells, NF-kappaB inactivation resulted in an up-regulation of Hes1 expression, and a down-regulation of DC-STAMP and MMP-9 expression. Moreover, in response to RANKL, NF-kappaB inactivation resulted in a down-regulation of DC-STAMP and MMP-9 expression compared with Wild-type cells. The Hes1 promoter was detected by ChIP using IkappaBalpha antibody. In conclusion, our data suggest IkappaBalpha regulates Hes1-mediated activity in osteoclast differentiation and resorption, which support a cross-talk between NF-kappaB and Notch in osteoclast activity.