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Runx2 protein expression utilizes the Runx2 P1 promoter to establish osteoprogenitor cell number for normal bone formation.

ABSTRACT: The Runt-related transcription factor, Runx2, is essential for osteogenesis and is controlled by both distal (P1) and proximal (P2) promoters. To understand Runx2 function requires determination of the spatiotemporal activity of P1 and P2 to Runx2 protein production. We generated a mouse model in which the P1-derived transcript was replaced with a lacZ reporter allele, resulting in loss of P1-derived protein while simultaneously allowing discrimination between the activities of the two promoters. Loss of P1-driven expression causes developmental defects with cleidocranial dysplasia-like syndromes that persist in the postnatal skeleton. P1 activity is robust in preosteogenic mesenchyme and at the onset of bone formation but decreases as bone matures. Homozygous Runx2-P1(lacZ/lacZ) mice have a normal life span but exhibit severe osteopenia and compromised bone repair in adult mice because of osteoblastic defects and not increased osteoclastic resorption. Gene expression profiles of bone, immunohistochemical studies, and ex vivo differentiation using calvarial osteoblasts and marrow stromal cells identified mechanisms for the skeletal phenotype. The findings indicate that P1 promoter activity is necessary for generating a threshold level of Runx2 protein to commit sufficient osteoprogenitor numbers for normal bone formation. P1 promoter function is not compensated via the P2 promoter. However, the P2 transcript with compensatory mechanisms from bone morphogenetic protein (BMP) and Wnt signaling is adequate for mineralization of the bone tissue that does form. We conclude that selective utilization of the P1 and P2 promoters enables the precise spatiotemporal expression of Runx2 necessary for normal skeletogenesis and the maintenance of bone mass in the adult.

SUBMITTER: Liu JC

PROVIDER: S-EPMC3191046 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Runx2 protein expression utilizes the Runx2 P1 promoter to establish osteoprogenitor cell number for normal bone formation.

Liu Julie C JC Lengner Christopher J CJ Gaur Tripti T Lou Yang Y Hussain Sadiq S Jones Marci D MD Borodic Brent B Colby Jennifer L JL Steinman Heather A HA van Wijnen Andre J AJ Stein Janet L JL Jones Stephen N SN Stein Gary S GS Lian Jane B JB

The Journal of biological chemistry 20110615 34

The Runt-related transcription factor, Runx2, is essential for osteogenesis and is controlled by both distal (P1) and proximal (P2) promoters. To understand Runx2 function requires determination of the spatiotemporal activity of P1 and P2 to Runx2 protein production. We generated a mouse model in which the P1-derived transcript was replaced with a lacZ reporter allele, resulting in loss of P1-derived protein while simultaneously allowing discrimination between the activities of the two promoters ...[more]

PMID: 21676869

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Project description:Rationale: The migration of mesenchymal osteoprogenitor cells (OPCs) to bone formation surface is the initial step of osteoblastogenesis before they undergo osteoblast differentiation and maturation for governing bone formation. However, whether the migration capacity of OPCs is compromised during aging and how it contributes to the aging-related bone formation reduction remain unexplored. In the present study, we identified a migration inhibitory factor (i.e., long noncoding RNA PMIF) and examined whether targeting lnc-PMIF could facilitate osteoprogenitor cells migrating to bone formation surface to promote bone formation during aging. Methods: Primary OPCs from young (6-momth-old) and aged (18-momth-old) C57BL/6 mice and stable lnc-PMIF knockdown/overexpression cell lines were used for in vitro and in vivo cell migration assay (i.e., wound healing assay, transwell assay and cell intratibial injection assay). RNA pulldown-MS/WB and RIP-qPCR were performed to identify the RNA binding proteins (RBPs) of lnc-PMIF. Truncations of lnc-PMIF and the identified RBP were engaged to determine the interaction motif between them by RNA pulldown-WB and EMSA. By cell-based therapy approach and by pharmacological approach, small interfering RNA (siRNA)-mediated lnc-PMIF knockdown were used in aged mice. The cell migration ability was evaluated by transwell assay and cell intratibial injection assay. The bone formation was evaluated by microCT analysis and bone morphometry analysis. Results: We reported that the decreased bone formation was accompanied by the reduced migration capacity of the bone marrow mesenchymal stem cells (BMSCs, the unique source of OPCs in bone marrow) in aged mice. We further identified that the long non-coding RNA PMIF (postulated migration inhibitory factor) (i.e., lnc-PMIF) was highly expressed in BMSCs from aged mice and responsible for the reduced migration capacity of aged OPCs to bone formation surface. Mechanistically, we found that lnc-PMIF could bind to human antigen R (HuR) for interrupting the HuR-β-actin mRNA interaction, therefore inhibit the expression of β-actin for suppressing the migration of aged OPCs. We also authenticated a functionally conserved human lncRNA ortholog of the murine lnc-PMIF. By cell-based therapy approach, we demonstrated that replenishing the aged BMSCs with small interfering RNA (siRNA)-mediated lnc-PMIF knockdown could promote bone formation in aged mice. By pharmacological approach, we showed that targeted delivery of lnc-PMIF siRNA approaching the OPCs around the bone formation surface could also promote bone formation in aged mice. Conclusion: Toward translational medicine, this study hints that targeting lnc-PMIF to facilitate aged OPCs migrating to bone formation surface could be a brand-new anabolic strategy for aging-related osteoporosis.

Dataset Information

Runx2 protein expression utilizes the Runx2 P1 promoter to establish osteoprogenitor cell number for normal bone formation.

Publications

Runx2 protein expression utilizes the Runx2 P1 promoter to establish osteoprogenitor cell number for normal bone formation.

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