AKAP11 is a positive regulator of osteoblast extracellular matrix formation and mineralization

Abstract

Objectives: Bone matrix formation and mineralization are vital processes for skeletal health. Previous GWAS has identified AKAP11, in close proximity with RANKL, as a susceptibility locus of bone mineral density(BMD). AKAP11 is a signaling-related molecule that facilitates signal compartmentation and transduction in various cells. However, its role in bone and relationship with RANKL is unknown. In this study, we investigated the role of AKAP11 during osteoblast differentiation and matrix mineralization. Methods: To evaluate the function of AKAP11 in osteoblast, AKAP11 knockout(KO) cells were generated using CRISPR/Cas9 gene editing in the mouse pre-osteoblastic MC3T3-E1 cells. Cell proliferation and osteogenic differentiation ability were evaluated. Expression of major bone signaling molecules was measured using qPCR. To better elucidate the molecular role of AKAP11, RNA-seq was performed to evaluate the transcriptome of WT and AKAP11-KO cells throughout differentiation. Results: In the AKAP11-KO lines, Alizarin red S(ARS) staining of differentiated MC3T3-E1 revealed significant reduction of calcium mineral deposition (AKAP11 ± : - 44.1%, AKAP11-/-: - 94.5%; P< 0.0001). Alkaline phosphatase (ALP) activity was significantly inhibited (AKAP11-/-: - 41.1%; P< 0.001) since differentiation phase(Day7). Furthermore, significant suppression of bone matrix genes expression were observed (OC: - 58.9%; IBSP: - 27.9%; SPP1: - 85.1%; P<0.001) during the mineralization phase(Day16). In contrast, cell proliferation rate was not altered as demonstrated by MTT assay. There were also no significant expression alternations in major bone differentiation markers (RUNX2; OSX) and signaling molecules during osteogenesis. Notably, RANKL was not expressed in both WT and KO cells. Pathways enrichment analysis compared the transcriptomes of AKAP11 KO lines line to WT controls revealed significant enrichment of differentially expressed genes in pathways associated with matrix organization, including collagen trimerizations (P = 0.014) and collagen fibrils assembly(P = 0.025) pathways. Conclusions: This study suggested that AKAP11 plays a pivotal role in the bone matrix formation process, which is independent of RANKL and significantly contributes to proper matrix mineralization.