MAML1 Enhances the Transcriptional Activity of Runx2 and Plays a Role in Bone Development

Takashi Watanabe,Masayo Inagawa,Daisuke Harada,Yoshiaki Ito,Gerard Karsenty,Sumio Sugano,Ken-Ichi Katsube,Toshihisa Komori,Yutaka Suzuki,Motoo Kitagawa,Hiroshi Asahara,Toshinao Oyama,Maki Asada

doi:10.1371/journal.pgen.1003132

Abstract

Mastermind-like 1 (MAML1) is a transcriptional co-activator in the Notch signaling pathway. Recently, however, several reports revealed novel and unique roles for MAML1 that are independent of the Notch signaling pathway. We found that MAML1 enhances the transcriptional activity of runt-related transcription factor 2 (Runx2), a transcription factor essential for osteoblastic differentiation and chondrocyte proliferation and maturation. MAML1 significantly enhanced the Runx2-mediated transcription of the p6OSE2-Luc reporter, in which luciferase expression was controlled by six copies of the osteoblast specific element 2 (OSE2) from the Runx2-regulated osteocalcin gene promoter. Interestingly, a deletion mutant of MAML1 lacking the N-terminal Notch-binding domain also enhanced Runx2-mediated transcription. Moreover, inhibition of Notch signaling did not affect the action of MAML1 on Runx2, suggesting that the activation of Runx2 by MAML1 may be caused in a Notch-independent manner. Overexpression of MAML1 transiently enhanced the Runx2-mediated expression of alkaline phosphatase, an early marker of osteoblast differentiation, in the murine pluripotent mesenchymal cell line C3H10T1/2. MAML1−/− embryos at embryonic day 16.5 (E16.5) had shorter bone lengths than wild-type embryos. The area of primary spongiosa of the femoral diaphysis was narrowed. At E14.5, extended zone of collagen type II alpha 1 (Col2a1) and Sox9 expression, markers of chondrocyte differentiation, and decreased zone of collagen type X alpha 1 (Col10a1) expression, a marker of hypertrophic chondrocyte, were observed. These observations suggest that chondrocyte maturation was impaired in MAML1−/− mice. MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development.

Highlights

Runt-related transcription factor 2 (Runx2) is a transcription factor belonging to the Runx gene family, which is homologous to Drosophila runt, a pair-rule gene involved in somitogenesis [1]
To identify new molecules involved in bone and cartilage development and/or homeostasis, we utilized approximately 10,000 arrayed and addressable cDNA clones, which allowed systematic, efficient, and unbiased screening of cDNAs encoding factors that could activate critical bone differentiation activity via activation of Runx2, master regulator of bone development
We analyzed MAML12/2 mice to investigate the role of MAML1 in bone development

Summary

Introduction

Runt-related transcription factor 2 (Runx2) is a transcription factor belonging to the Runx gene family, which is homologous to Drosophila runt, a pair-rule gene involved in somitogenesis [1]. It has been reported that several transcription factors and cofactors, such as TAZ [3], Grg5 [4], Rb [5], and HDAC4 [6], interact with Runx and positively or negatively regulate its function. To further elucidate the function of Runx, we performed luciferase assaybased screening of additional factors regulating the transcriptional activity of Runx using a full-length cDNA library containing approximately 10,000 clones. The screening system identified the mastermind-like (MAML) family of proteins showed especially strong potential for regulating Runx transcriptional activity. The area of primary spongiosa of the femoral diaphysis was narrowed, indicated that chondrocyte maturation was impaired. These data suggest that MAML1 enhanced the transcriptional activity of Runx and plays a role in bone development

Methods

Results

Discussion

Conclusion