Selective Deficiency of the “Bone-related” Runx2-II Unexpectedly Preserves Osteoblast-mediated Skeletogenesis

Abstract

Runx2 (runt-related transcription factor 2) is a master regulator of skeletogenesis. Distinct promoters in the Runx2 gene transcribe the "bone-related" Runx2-II and non-osseous Runx2-I isoforms that differ only in their respective N termini. Existing mutant mouse models with both isoforms deleted exhibit an arrest of osteoblast and chondrocyte maturation and the complete absence of mineralized bone, but they do not distinguish the separate functions of the two N-terminal isoforms. To elucidate the function of the bone-related isoform, we generated selective Runx2-II-deficient mice by the targeted deletion of the distal promoter and exon 1. Homozygous Runx2-II-deficient (Runx2-II ؊/؊ ) mice unexpectedly formed axial, appendicular, and craniofacial bones derived from either intramembranous ossification or mesenchymal cells of the bone collar, but they failed to form the posterior cranium and other bones derived from endochondral ossification. Heterozygous Runx2-II-deficient mice had grossly normal skeletons, but were osteopenic. The commitment of mesenchymal cells ex vivo to the osteoblast lineage occurred in Runx2-II ؊/؊ mice, but osteoblastic gene expression was impaired. Chondrocyte maturation appeared normal, but the zone of hypertrophic chondrocytes was not transformed into metaphyseal bone, leading to widened growth plates in Runx2-II ؊/؊ mice. Compensatory increments in Runx2-I expression occurred in Runx2-II ؊/؊ mice but were not sufficient to normalize osteoblastic maturation or transcriptional activity. Our findings support distinct functions of Runx2-II and -I in the control of skeletogenesis. Runx2-I is sufficient for early osteoblastogenesis and intramembranous bone formation, whereas Runx2-II is necessary for complete osteoblastic maturation and endochondral bone formation.The Runx 1 family of transcription factors consists of three highly conserved mammalian genes, designated Runx1, Runx2, and Runx3 (1-5), that regulate, respectively, hematopoiesis (6), skeletogenesis (7-10), and neuronal development (11)(12). RUNX2 is the master transcription factor essential for skeletogenesis. Targeted disruption of the Runt domain of Runx2 results in the complete lack of both intramembranous and endochondral bone formation due to an arrest in osteoblast and chondrocyte maturation (8,10,(13)(14)(15)(16)(17)(18)(19). Haploinsufficiency of RUNX2 causes cleidocranial dysplasia in humans, and heterozygous Runx2 deficient mice show a similar phenotype, suggesting that the expression level of Runx2 influences the skeletal phenotype (10).Separate promoters control the expression of Runx2 isoforms with different 5Ј-UTRs and N-terminal sequences that bind to the same consensus DNA cis-acting elements, interact with similar co-factors, and demonstrate overlapping transactivation potential in vitro (17-24). The Runx2-II isoform (also called PEBP2␣A, major til-1 and Cbfa1.iso, Osf2) is derived from the distal "bone-related" promoter (P1) and encodes a 528-amino acid protein that begins with the 19 amino acids MASNSLFSAVTPCQQ...

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