Nuclear αNAC Influences Bone Matrix Mineralization and Osteoblast Maturation In Vivo

Abstract

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 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 ␣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.

The PTH-Gαs-Protein Kinase A Cascade Controls αNAC Localization To Regulate Bone Mass

“…Accordingly, the expression of the osteocyte differentiation markers E11 and Sost was increased. Osteoblasts isolated from the calvaria of mutant ␣NAC S43A/S43A mice mineralized their matrix faster than wild-type cells (17). Thus, inhibition of ␣NAC nuclear translocation in vivo mimics most of the phenotype of mice in which G␣ s has been specifically deleted from osteoblasts (13).…”

“…and Osx-CRE; G␣ s fl/fl mutant mice is an increased number of osteocytes measured in cortical bone (13,17). We thus counted the osteocytes present in cortical bone at the femoral midshaft following alteration in gene dosage for G␣ s and ␣NAC (Fig.…”

“…B009) was from Biolog (Bremen, Germany), while PD98059 was procured through Cell Signaling Technology (Danvers, MA). Anti-␣NAC antibodies have been described previously (17,24). The affinity-purified rabbit polyclonal anti-␣NAC-phospho-S99 antibody (pS99), raised against the peptide NH 2 -TGVTRVTIRK-phospho-S-KNILFVITKP-C OOH, was produced by and purchased from PhosphoSolutions (Aurora, CO).…”

“…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 nuclear ␣NAC with reduced Ocn promoter occupancy, leading to a significant decrease in Ocn gene transcription (17). The bones of the S43A mutant mice had a significantly reduced bone volume.…”

“…One of the molecules affecting maximal Ocn expression is the transcriptional coregulator ␣NAC (nascent-polypeptide-associated complex and coregulator alpha) (16,17). ␣NAC shuttles between the cytoplasm and the nucleus, and one of the signals that induce its nuclear accumulation is phosphorylation by integrinlinked kinase (ILK) (18) at residue Ser43 (19).…”