Nuclear αNAC Influences Bone Matrix Mineralization and Osteoblast Maturation In Vivo

Nascent-polypeptide-associated complex and coactivator alpha (αNAC) is a protein shuttling between the nucleus and the cytoplasm. Upon phosphorylation at residue serine 43 by integrin-linked kinase, αNAC is translocated to the nuclei of osteoblasts, where it acts as an AP-1 coactivator to increase osteocalcin gene transcription. To determine the physiological role of nuclear αNAC, we engineered a knock-in mouse model with a serine-to-alanine mutation at position 43 (S43A). The S43A mutation resulted in a decrease in the amount of nuclear αNAC with reduced osteocalcin gene promoter occupancy, leading to a significant decrease in osteocalcin gene transcription. The S43A mutant bones also expressed decreased levels of α1(I) collagen mRNA and as a consequence had significantly less osteoid tissue. Transient transfection assays and chromatin immunoprecipitation confirmed the α1(I) collagen gene as a novel αNAC target. The reduced quantity of bone matrix in S43A mutant bones was mineralized faster, as demonstrated by the significantly reduced mineralization lag time, producing a lower volume of immature, woven-type bone characterized by poor lamellation and an increase in the number of osteocytes. Accordingly, the expression of the osteocyte differentiation marker genes DMP-1 (dentin matrix protein 1), E11, and SOST (sclerostin) was increased. The accelerated mineralization phenotype was cell autonomous, as osteoblasts isolated from the calvaria of S43A mutant mice mineralized their matrix faster than did wild-type cells. Thus, inhibition of αNAC nuclear translocation results in an osteopenic phenotype caused by reduced expression of osteocalcin and type I collagen, accelerated mineralization, and immature woven-bone formation.