Lrp6 is a target of the PTH-activated αNAC transcriptional coregulator.

In the nucleus of differentiated osteoblasts, the alpha chain of nascent polypeptide associated complex (αNAC) interacts with cJUN transcription factors to regulate the expression of target genes, including Osteocalcin (Bglap2). PTH induces the phosphorylation of αNAC on serine 99 through a Gαs-PKA dependent pathway. This leads to activation of αNAC and expression of Bglap2. To identify additional target genes regulated by PTH-activated αNAC, we performed ChIP-Seq against αNAC in PTH-treated MC3T3-E1 cells. This identified Low density lipoprotein receptor-Related Protein 6 (Lrp6) as a potential αNAC target. LRP6 acts as a co-receptor for the PTH receptor to allow optimal activation of PTH signaling. PTH increased Lrp6 mRNA levels in primary osteoblasts. Conventional quantitative ChIP confirmed the ChIP-Seq results. To assess whether αNAC plays a critical role in PTH-stimulated Lrp6 expression, we knocked-down Naca expression in MC3T3-E1 cells. Reduction of αNAC levels decreased basal expression of Lrp6 by 30% and blocked the stimulation of Lrp6 expression by PTH. We cloned the proximal mouse Lrp6 promoter (-2523/+120 bp) upstream of the luciferase reporter. Deletion and point mutations analysis in electrophoretic mobility shift assays and transient transfections identified a functional αNAC binding site centered around -343 bp. ChIP and ChIP-reChIP against JUND and αNAC showed that they cohabit on the proximal Lrp6 promoter. Luciferase assays confirmed that PTH-activated αNAC potentiated JUND-mediated Lrp6 transcription and Jund knockdown abolished this response. This study identified a novel αNAC target gene induced downstream of PTH signaling and represents the first characterization of the regulation of Lrp6 transcription in osteoblasts.