MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development

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.

Figure 1. MAML1 enhances the transcriptional activity of Runx2.

A, 293T cells were transiently transfected with p6OSE2-Luc reporter, Runx2 expression plasmid, and about 10,000 FLJ expression plasmids. B, 293T cells were transiently transfected with a p6OSE2-Luc reporter or p6OSE2-mutant-Luc together with MAML1, MAML2, MAML3, and Runx2 expression plasmids. Luciferase levels were normalized to the Renilla luciferase activity of a cotransfected phRL-TK-Luc reporter and presented as fold activation relative to the luciferase level of the p6OSE2-Luc reporter construct alone. Error bars represent the standard deviation of triplicate transfections.

Figure 2. MAML1 enhances Runx2 activity in a Notch-independent manner in vitro.

A, The structure of MAML1 and its truncated forms. B, 293T cells were transiently transfected with a p6OSE2-Luc reporter alone or together with truncated forms of MAML1 Error bars represent the standard deviation of triplicate transfections. C, 293T cells were transiently transfected with a pTP1-Luc reporter and NotchΔE expression plasmid in the presence of γ-secretase inhibitor (DAPT). Error bars represent the standard deviation of triplicate transfections. D, 293T cells were transiently transfected with a p6OSE2-Luc reporter with Runx2, MAML1 and NotchΔE expression plasmid in the presence of γ-secretase inhibitor (DAPT). Error bars represent the standard deviation of triplicate transfections.

Figure 3. MAML1 promotes Runx2-mediated osteoblastic differentiation.

A, C3H10T1/2 cells were transiently transfected with a Runx2 and/or MAML1 expression plasmids and cultured for 2 days. B, Total RNA was isolated and reverse-transcribed and TaqMan real-time PCR was performed to investigate the expression level of alkaline phosphatase gene, a marker of osteoblast.

Figure 4. Analysis of skeletal defects in MAML1^−/− mice.

A, Whole mounted embryos at E16.5 were stained with Alcian Blue and Alizarin Red. B, The femoral sections at E16.5 were stained with Alcian Blue (upper panel). Arrow bar indicates the area of primary spongiosa. The expression of collagen, type 10 alpha 1, a marker of hypertrophic chondrocyte, was shown by in situ hybridization (lower panel). Green bars, primary spongiosa; red bars, hypertrophic zone; blue bars, proliferating zone. Black bars, 200 µm. C, The primary spongiosa length of MAML1 null (KO) embryos and wild type (WT) littermates. D. Femoral sections at E14.5. The expression of type 10 alpha 1 collagen, type 2 alpha 1 collagen and sox9 was shown by in situ hybridization. Bars, 100 µm.