MCB Accepts, published online ahead of print on 2 November 2009

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Mol. Cell. Biol. doi:10.1128/MCB.00378-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Nuclear NAC influences bone matrix mineralization and osteoblast maturation in vivo

Thomas Meury, Omar Akhouayri, Toghrul Jafarov, Vice Mandic, and René St-Arnaud^*

Genetics Unit, Shriners Hospital for Children, Montreal (Quebec) Canada H3G 1A6; Department of Human Genetics, McGill University, Montreal, (Quebec) Canada H3A 2T5; Departments of Medicine and Surgery, McGill University, Montreal (Quebec) Canada H3A 2T5

^* To whom correspondence should be addressed. Email: rst-arnaud{at}shriners.mcgill.ca.

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 nucleus 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 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 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 markers, 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 mutant S43A mice mineralized their matrix faster than wild-type cells. Thus inhibition of NAC nuclear translocation results in an osteopenic phenotype, caused by reduced expression of osteocalcin and type I collagen, accelerated mineralization, and immature woven bone formation.