Nuclear NAC Influences Bone Matrix Mineralization and Osteoblast Maturation In Vivo

Nascent-polypeptide-associated complex and coactivator alpha (NAC) is a protein shuttling between the nucleus and the cytoplasm. Upon phosphorylation at residue serine 43 by integrin-linked kinase, NAC 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 NAC, 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 NAC with reduced osteocalcin gene promoter occupancy, leading to a significant decrease in osteocalcin gene transcription. The S43A mutant bones also expressed decreased levels of 1(I) collagen mRNA and as a consequence had significantly less osteoid tissue. Transient transfection assays and chromatin immunoprecipitation confirmed the 1(I) collagen gene as a novel NAC target. The reduced quantity of bone matrix in S43A mutant bones was mineralized faster, as demonstrated by the significantly reduced mineral-ization 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 mineral-ization 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 NAC nuclear translocation result