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Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells
Published on: September 1, 2019
Nell-1, a key functional mediator of Runx2, partially rescues calvarial defects in Runx2(+/-) mice
Xinli Zhang1, Kang Ting, Catherine M Bessette
1Dental and Craniofacial Research Institute, University of California Los Angeles, Los Angeles, CA, USA.
Abstract:
Mesenchymal stem cell commitment to an osteoprogenitor lineage requires the activity of Runx2, a molecule implicated in the etiopathology of multiple congenital craniofacial anomalies. Through promoter analyses, we have recently identified a new direct transcriptional target of Runx2, Nell-1, a craniosynostosis (CS)-associated molecule with potent osteogenic properties. This study investigated the mechanistic and functional relationship between Nell-1 and Runx2 in regulating osteoblast differentiation. The results showed that spatiotemporal distribution and expression levels of Nell-1 correlated closely with those of endogenous Runx2 during craniofacial development. Phenotypically, cross-mating Nell-1 overexpression transgenic (CMV-Nell-1) mice with Runx2 haploinsufficient (Runx2(+/-)) mice partially rescued the calvarial defects in the cleidocranial dysplasia (CCD)-like phenotype of Runx2(+/-) mice, whereas Nell-1 protein induced mineralization and bone formation in Runx2(+/-) but not Runx2(-/-) calvarial explants. Runx2-mediated osteoblastic gene expression and/or mineralization was severely reduced by Nell-1 siRNA oligos transfection into Runx2(+/+) newborn mouse calvarial cells (NMCCs) or in N-ethyl-N-nitrosourea (ENU)-induced Nell-1(-/-) NMCCs. Meanwhile, Nell-1 overexpression partially rescued osteoblastic gene expression but not mineralization in Runx2 null (Runx2(-/-)) NMCCs. Mechanistically, irrespective of Runx2 genotype, Nell-1 signaling activates ERK1/2 and JNK1 mitogen-activated protein kinase (MAPK) pathways in NMCCs and enhances Runx2 phosphorylation and activity when Runx2 is present. Collectively, these data demonstrate that Nell-1 is a critical downstream Runx2 functional mediator insofar as Runx2-regulated Nell-1 promotes osteoblastic differentiation through, in part, activation of MAPK and enhanced phosphorylation of Runx2, and Runx2 activity is significantly reduced when Nell-1 is blocked or absent.
Insights
Nerve-expedited لیگاند-1 (Nell-1) is a crucial downstream mediator of Runx2, promoting osteoblast differentiation by activating MAPK pathways and enhancing Runx2 phosphorylation. This interaction is vital for craniofacial development and bone formation.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Mesenchymal stem cell differentiation into osteoprogenitors relies on Runx2.
- Runx2 is linked to congenital craniofacial anomalies.
- Nell-1 is a direct Runx2 transcriptional target with osteogenic properties.
Purpose of the Study:
- To investigate the mechanistic and functional relationship between Nell-1 and Runx2 in osteoblast differentiation.
- To elucidate Nell-1's role as a downstream mediator of Runx2.
- To understand the impact of Nell-1 and Runx2 interactions on craniofacial development.
Main Methods:
- Promoter analysis to identify Nell-1 as a Runx2 target.
- Generation of Nell-1 overexpression and Runx2 haploinsufficient mouse models.
- Cross-mating experiments and analysis of calvarial explants.
- siRNA-mediated knockdown of Nell-1 in mouse calvarial cells.
- Western blot analysis to assess MAPK pathway activation and Runx2 phosphorylation.
Main Results:
- Nell-1 and Runx2 expression patterns correlate during craniofacial development.
- Nell-1 overexpression partially rescued calvarial defects in Runx2 haploinsufficient mice.
- Nell-1 induced mineralization in Runx2(+/-) but not Runx2(-/-) calvarial explants.
- Nell-1 knockdown reduced Runx2-mediated osteoblastic gene expression and mineralization.
- Nell-1 activated MAPK pathways (ERK1/2, JNK1) and enhanced Runx2 phosphorylation.
Conclusions:
- Nell-1 is a critical downstream mediator of Runx2 in osteoblastic differentiation.
- Runx2-regulated Nell-1 promotes osteoblast differentiation via MAPK activation and Runx2 phosphorylation.
- Runx2 activity is significantly impaired by the absence or blockade of Nell-1.
